i -fifth mmim ,:* Sheffield Laboratory of Yale University PAPERS ON PYRIMIDINES EDITED BY HENRY L. WHEELER o PROFESSOR OF ORGANIC CHEMISTRY AND TREAT B. JOHNSON ASSISTANT PROFESSOR OF ORGANIC CHEMISTRY. NEW HAVEN, CONNECTICUT, U. S. A. PREFACE In the present volume the Authors have incorporated all the Papers on Pyrimidines, which have appeared from the Sheffield Laboratory prior to the year 1910. A bibliography of publications on pyrimidines, arranged chronologically, has also been appended to this book, but it must be understood that it is not intended to be complete. The aim of the authors has been to furnish a guide to readers, who are unacquainted with pyrimidine literature, rather than to give a complete list of publications in this field. H. L. W. and T. B. J. Sheffield Laboratory, New Haven. January, 1910. ^58663 CONTENTS RESEARCHES ON THE CYCLOAMIDINES : PYRIMIDINE DERIVA- TIVES. By Henry L. Wheeler. (American Chemical Journal, 1898, XX, 481). ON SOME CONDENSATION-PRODUCTS OF THE PSEUDOTHIOUREAS : SYNTHESIS OF URACIL, THYMINE AND SIMILAR COM- POUNDS. By Henry L. Wheeler and Henry F. Merriam. (American Chemical Journal, 1903, XXIX, 478). SYNTHESES OF AMINOOXYPRIMIDINES HAVING THE COMPOSI- TION OF CYTOSINE: 2-AMiNO-6-OxYPYRiMmiNE AND 2-OxY-6-AMiNOPYRiMiDiNE. By Henry L. Wheeler and Treat B. Johnson. (American Chemical Journal, 1903, XXIX, 492). ON CYTOSINE OR 2-OxY-6-AMiNOPYRiMiDiNE FROM TRITICO- NUCLEIC ACID. By Henry L. Wheeler and Treat B. Johnson. (American Chemical Journal, 1903, XXIX, 505). 5-METHYLCYTOSiNE. By Henry L. Wheeler and Treat B. ^ Johnson. (American Chemical Journal, 1904, XXXI, 591). SYNTHESIS OF 2-AMiNO-5-METHYL-6-OxYPYRiMiDiNE. By Treat B. Johnson and Samuel H. Clapp. (American Chemical Journal, 1904, XXXII, 130). 2-OxY-4, 6-DiAMiNOPYRiMiDiNE. By Henry L. Wheeler and George S. lamieson. (American Chemical Journal, 1904, XXXII, 342). THE STRUCTURE OF SOME SUBSTITUTION PRODUCTS. By Henry L. W'heeler and H. Stanley Bristol. (American Chemical Journal, 1905, XXXIII, 437). THE ACTION OF POTASSIUM THIOCYANATE UPON SOME IMIDE- CHLORIDES. By Henry L. Wheeler and H. Stanley Bristol. (American Chemical Journal, 1905, XXXIII, 448). THE ACTION OF AQUEOUS AND ALCOHOLIC AMMONIA AND ANILINE ON SOME HALOGEN AND MERCAPTO PYRIMI- DINES. By Treat B. Johnson and Carl O. Johns. (American Chemical Journal, 1905, XXXIV, 175). ^s>W CONTENTS 2-ETHYLMERCAPTO-5-AMiNO-6-OxYPYRiMiDiNE. By Treat B. Johnson. (American Chemical Journal, 1905 XXXIV, 191). ON 2, 5-DiAMiNO-6-OxYPYRiMiDiNE. By Treat B. Johnson and Carl O. Johns. (American Chemical Journal, 1905, XXXIV, 554). SOME 5-IoDOPYRiMiDiN DERIVATIVES: S-IODOCYTOSINE. By Treat B. Johnson and Carl O. Johns. (Journal of Biological Chemistry, 1906, I, 305). ON METHODS OF SYNTHESIZING ISOBARBITURIC ACID AND 5- OXYCYTOSIN. By Treat B. Johnson and Elmer V. McCollum. (Journal of Biological Chemistry, 1906, I, 437). THE ACTION OF POTASSIUM THIOCYANATE UPON IMIDE CHLORIDES. By Treat B. Johnson and Elmer V. Mc- Collum. (American Chemical Journal, 1906, XXXVI, 136). ON THE FORMATION OF PURINES FROM UREAPYRIMIDINES. By Treat B. Johnson and Elmer V. McCollum. (Ameri- can Chemical Journal, 1906, XXXVI, 149). ON 5-NlTROCYTOSINE AND ITS REDUCTION TO 2-OXY-5, 6- DIAMINOPYRIMIDINE. By Treat B. Johnson, Carl O. Johns and Frederick W. Heyl. (American Chemical Journal, 1906, XXXVI, 160). 5-ETHYLCYTOSiN. By Treat B. Johnson and George A. Menge. (Journal of Biological Chemistry, 1906, II, 105). SYNTHESIS OF URACIL-S-CARBOXYLIC ACID. By Henry L. Wheeler, Treat B. Johnson and Carl O. Johns. (Ameri- can Chemical Journal, 1907, XXXVII, 392. ON A COLOR TEST FOR URACIL AND CYTOSIN. By Henry L. Wheeler and Treat B. Johnson. (Journal of Biologi- cal Chemistry, 1907, III, 183). ON SOME SALTS OF CYTOSIN, ISOCYTOSIN, 6-AMiNOPYRiMioiN AND 6-OxYpYRiMiDiN. By Henry L. Wheeler. (Jour- nal of Biological Chemistry, 1907, III, 285). URACiL-4-CARBOXYLic ACID. By Henry L. Wheeler. (Ameri- can Chemical Journal, 1907, XXXVIII, 358). THE ACTION OF METHYL IODIDE ON 2-ANiLiNO-6-OxYpRiMi- DINE, AND THE SYNTHESIS OF 2-ANILINOPYRIMIDINE. By Treat B. Johnson and Frederick W. Heyl. (Ameri- can Chemical Journal, 1907, XXXVIII. 237). CONTENTS 7 SYNTHESIS OF THYMiN-4-CARBOXYLic ACID ^ By Treat B. Johnson. (Journal of Biological Chemistry, 1907, 111. 299). SYNTHESIS OF CYTOSINE-S-CARBOXYLIC ACID. By Henry L. Wheeler and Carl O. Johns. (American Chemical Journal, 1907, XXXVIII, 594). SYNTHESIS OF THYMINE-S-CARBOXYLIC ACID. By Treat B. Johnson and Carl Frank Speh. (American Chemical Journal, 1907, XXXVIII, 602). SYNTHESIS OF 4-METHYLURACiL-5-AcETic ACID. By Treat B. Johnson and Frederick W. Heyl. (American Chemical Journal, 1907, XXXVIII, 659). A METHOD OF SEPARATING THYMIN FROM URACIL. By Treat B. Johnson. (Journal of Biological Chemistry, 1908, IV, 407). THE ACTION OF NITRIC ACID ON 2, 6-DioxYPYRiMmiNES. OXY- NITROHYDROTHYMINE. By Treat B. Johnson. (Ameri- can Chemical Journal, 1908, XL, 19). SYNTHESIS OF URAciL-3-AcETic ACID. By Henry L. Wheeler and Leonard M. Liddle. (Journal American Chemical Society, 1908, XXX, 1152). SYNTHESIS OF URACiL-4-AcETic ACID. By Henry L. Wheeler and Leonard M. Liddle. (Journal American Chemical Society, 1908, XXX, 1156). SYNTHESIS OF SOME NITROGEN-ALKYL DERIVATIVES OP- CYTOSIN, THYMIN AND URACIL. By Treat B. Johnson and Samuel H. Clapp. (Journal of Biological Chemis- try, 1908, V, 49). THE ACTION OF DIAZO-BENZENE SULFONIC ACID ON THYMIN, URACIL AND CYTOSIN. By Treat B. Johnson and Sam- uel H. Clapp. (Journal of Biological Chemistry, 1908, V, 163). THE ACTION OF POTASSIUM THIOCYANATE UPON SOME IMIDE CHLORIDES. By Treat B. Johnson and Walter F. Storey. (American Chemical Journal, 1908, XL, 131). SYNTHESIS OF CYTOSINE-S-CARBOXAMIDE. . By Henry L. Wheeler and Carl O. Johns. (American Chemical Journal, 1908, XL, 233). SYNTHESIS OF SOME BENZYL DERIVATIVES OF URACIL AND THYMINE. By Treat B. Johnson and John H. Derby, Jr. (American Chemical Journal, 1908, XL, 444). SYNTHESIS OF NEW DERIVATIVES OF S-HYDROXYURACIL (ISO- BARBITURIC ACID. By Treat B. Johnson and D. Breese Jones. (American Chemical Journal, 1908, XL, 538). 8 CONTENTS THE THIO DERIVATIVES OF URACIL AND THE PREPARATION OF URACIL IN QUANTITY. By Henry L. Wheeler and Leonard M. Liddle. (American Chemical Journal, 1908, XL, 548). ON THE FORMATION OF PURINE DERIVATIVES FROM 4-METHYL- CYTOSINE. By Carl O. Johns. (American Chemical Journal, 1909, XLI, 58). SYNTHESIS OF I-METHYL-S-HYDROXYURACIL. By Treat B. Johnson and D. Bresse Jones. (Journal American Chemical Society, 1909, XXXI, 591). THE PREPARATION OF S-METHYL- AND S-BENZYLURACIL. By Henry L. Wheeler and Treat B, Johnson. (American Chemical Journal, 1909, XLII, 30). THE PREPARATION OF 1, 4-DiMETHYLURACiL AND OF THE MONOBENZYL DERIVATIVES OF 4-METHYLURACIL. By Henry L. Wheeler and David F. McFarland. (Ameri- can Chemical Journal, 1909, XLII, 101). SULPHUR DERIVATIVES OF S-HYDROXYURACIL : PREPARATION OF 5-BENZYLMERCAPTOURACIL AND 5-BENZYLMERCAP- TOCYTOSINE. By Treat B. Johnson and Herbert H. Guest. (American Chemical Journal, 1909, XLII, 271). DIMETHYL DERIVATIVES OF 2-AMiNOPYRiMiDiNE. PREPARATION OF 2-METHYLAMINO-5-METHYLPYRIMIDINE. By Treat B. Johnson and Kenneth G. Mackenzie. (American Chemical Journal, 1909, XLII, 353). THE ACTION OF METHYL IODIDE AND OF BENZYL CHLORIDE UPON 2-OXY-4-METHYL-6-METHYLMERCAPTOPYRIMID- INE. By Henry L. Wheeler and David F. McFarland. (American Chemical Journal, 1909, XLII, 431). SYNTHESIS OF S-CYANURACIL. By Treat B. Johnson. (Ameri- can Chemical Journal, 1909, XLII, 505). THE THIO DERIVATIVES OF THYMINE AND THE PREPARATION OF THYMINE. By Henry L. Wheeler and David F. McFarland. (American Chemical Journal, 1910, XLIII, 19). ON SOME PICROLONATES. By Henry L. Wheeler and George S. Jamieson. (Journal of Biological Chemistry, 1908, IV, 111). APPENDIX Bibliography of Pyrimidine Literature. [REPRINTED FROM THE AMERICAN CHEMICAL JOURNAL. VOL. XX. No. 6. JUNE, 1898.] Contributions from the Sheffield Laboratory of Yale University. LX. RESEARCHES ON THE CYCLOAMIDINES : PYRIMIDINE DERIVATIVES. BY H. I,. WHEELER. For convenience of reference, in this work, those com- pounds which have the amidine formation in NXR' -1 i ___ i in in which two of the radicals R, R', and R" are replaced by a ring structure or a bivalent grouping, are referred to as cyclo- amidines. The dotted lines in the above formula show the three possible ways of replacing the radicals, and they indicate three forms of cycloamidines. It will be noticed, however, that III is the tautomeric form of I. The tautomeric form of II being obtained when the position of the hydrogen X is changed to the opposite nitrogen atom. There are therefore only two types of cycloamidines, each type having a pseudomeric or tautomeric form as in the case of the simple amidines. The first type, which contains only one of the nitrogen atoms in the ring is represented by such compounds as a-aminoquinoline, the <*-aminobenzoxazines, etc. The second type, with both nitrogen atoms in the ring struc- ture, is found in the anhydro bases, glyoxalines, lophin, etc. Then, further, combinations of these types and their tauto- meric forms occur : The cyanalkines, <*-amino- and anilido- pyrimidines, aminoquinoxalines, cyanuramide, dicyanortho- phenylenediamines, and the like, are examples of the com- bination types. 482 Wheeler. The object of the work, of which this paper gives a pre- liminary account, is primarily to investigate the action of alkyl halides on some of these cycloamidines which have not yet been examined in this respect, and also to compare this action in general with that of the simple amidines. Pechmann 1 assumes that when a simple amidine, repre- sented by the general formula above, is acted on with alkyl iodides, the hydrogen X is directly replaced, and hence this reaction serves to determine structure. It seems to the writer that alkyl iodides may be added either to the amido group or to the imido group or, when the substituents are similar,* to both and perhaps to the atoms joined by double union. In two of the latter cases an ami- dine would result of a different structure from that obtained by direct substitution, so that this method of determining structure seems to be not without objection. The cycloamidines, in general, unite with alkyl iodides and a number of cases have been described among those of the first type, where no direct substitution or replacement of hy- drogen in the amino group takes place, at least not as the first step of the reaction. To illustrate this the following may be cited : Thiele and Ingle 3 obtained dialkylaminotetrazols by the action of alkyl iodides on aminotetrazol. They be- lieve that in these only one of the alkyl groups replaces hy- drogen of the amino group. Claus 4 shows that a-amino- quinoline takes up alkyl iodides in the same manner as the quinoline and pyridine bases in general, and that the amino- hydrogen is not substituted. Again, K. v. Meyer 5 has shown in several cases that the cyanalkines form similar addition- products, the amino group not being substituted. Even acyl chlorides add to the cyanalkines without substi- tuting the amino group. Herfeldt 6 found that cyanbenzylin or aminophenyldibenzylmiazin unites with acetyl and benzoyl 1 Ber. d. chem. Ges., 28, 2362 and 869. 2 Pechmann (loc. cit.) found that when the substituents are similar radicals, the amidine gave two isomeric alkylated products. This is precisely what would be ex- pected if the action is one of addition. There being in that case no decided tendency to addition entirely to either one of the nitrogen atoms. 8 Ann. Chem. (Liebig), 287, 249. * J. prakt. Chem., 17, 209. * Ibid. 22, *66, and 39, 265. 6 ibid. [2], 53, 249. Researches on the Cycloamidines . 483 chlorides to form well crystallized addition- products, and that an acetyl or benzoylcyanbenzylin was not obtained. In these cases it is a tertiary nitrogen which is the point of attack or addition. The action of alkyl iodides on the cycloamides of the second type is that of addition also, and, as in the case of the simple amidines, this action has generally been considered to be a direct replacement of hydrogen, since by treating the addi- tion-product with alkali alkyl compounds result. The same question arises here as in the case of the simple amidines. That the alkyl group invariably replaces the hydrogen, how- ever, has not been proved. It is not improbable that, like the cycloamidines of the first class, it is the tertiary nitrogen that unites with the alkyl halide. The writer has found that phenylmethylanilidopyrimidine acts with methyl and ethyl iodides forming stable addition- products, and that no substitution takes place, that the alkyl iodide unites with one of the tertiary nitrogen atoms of the ring and not with the anilido group follows in all probability from the behavior of these products with alkali : C 17 H 1B N 8 .CH 8 I + NaOH = C 1T H 16 N S + CH 3 OH + Nal. That is, methyl alcohol and sodium iodide are quantitatively formed, and unaltered anilidopyrimidine is regenerated. The formula that appears most probable for these addition- products is therefore either I or II, and from analogy a simi- lar structure might be expected in the case of the alkyl halo- gen addition-products of the simple amidines. 1 i The fact that the alkyl iodide addition-products of the simple amidines give up hydrogen iodide and not the alkyl group as above cannot be considered as showing that the addition takes place to the NH group of the amidine. To be sure, the H grouping N would be expected to give up hydrogen iodide, and those CH/ \ alkyl iodide addition-products of the cyloamidiues which do not separate hydrogen iodide most probably do not have this grouping. But it cannot be maintained that because an addition-product gives up hydrogen iodide, it has this grouping. 484 Wheeler. CH, CH S 1C C \ / % /A CH 3 N CH N CH II I II I C 6 H 5 C C NHC ft H B C 6 H 5 C C NHC 6 H 5 \ S \// N N / \ CH 3 I I II R R"N=:C NHR' CH 3 I III This being the case, the structure of the simple amidines must be completely revised, and the opposite or so-called tau- tomeric structure must be assigned to them. At any rate, no method has yet been devised for determining the structure of the amidines in question that is without objection. It will be shown below that phenylmethylpyrimidon, a cycloamidine of the second type, unites with one molecule of methyl iodide to form a beautiful crystalline addition-product of formula IV or V. CH, CH 9 I C / ^ \ / \ N CH CH 3 N CH II I or || | C 6 H 5 C CO C 6 H 6 C CO N N CH 3 I H H IV V This compound, on treatment with alkali, gives up hydro- gen iodide and not the alkyl group, and an alkylated pyrimi- Researches on the Cycloamidines. 485 don results. This alkyl derivative differs decidedly from the product obtained by heating phenylmethylchlorpyrimidine with sodium methylate. The alkyl iodide therefore is added to one of the nitrogen atoms of the pyrimidon ring, while Pinner 1 found that, if the oxypyrimidine is heated with ethyl iodide in the presence of alkali, the ethyl group attaches itself to oxygen. In order to determine to which nitrogen the alkyl halide is added, a synthesis of the methylpyrimidon was attempted as follows : Methylbenzamidine hydrochloride was prepared. This, with acetoacetic ester, according to Pinner's explana- tion of the formation of the pyrimidines, might be expected to give the base corresponding to formula IV. It was found, however, that methylbenzamidine does not give a pyrimidine derivative with acetoacetic ester, at least not under the same conditions that benzamidine does, so that for the present the question of the position of the alkyl group must be left unde- cided. Nef has shown that carbostyril is not acted on by ethyl iodide. The above pyrimidon differs from carbostyril, in its ring structure, in having a tertiary nitrogen. It would seem most probable therefore that it is due to this that the sub- stance takes up methyl iodide and that the addition-product is perhaps best represented by formula V. Experimental Part. N C.CH, Phenylmethylanilidopyrimidine , C 6 H 6 C CH . N C NHC 6 H 5 This was prepared according to Pinner's 3 directions by warm- ing phenylmethylchlorpyrimidine with aniline. Instead of purifying the material by dissolving the reaction-product in alcohol and precipitating it with ether, it was found best to crystallize it directly from water, precipitate the base from the hot aqueous solution and then crystallize this from ben- zene and alcohol. The material thus obtained crystallizes in 1 Die Imidoather und ihre Derivate, p. 343. [2 Ann. Chem. (Liebig), 276, 242. * Die Imidoather und ihre Derivate, p. 248. 486 Wheeler. clusters of small prisms and melts at i6o-i6i. Pinner gives its melting-point as i5O-i53. The hydrochloride was obtained in colorless needles melting at 240 as described by Pinner. The hydrobromide was obtained by precipitating the base from a benzene solution with hydrobromic acid gas. It forms minute needles when crystallized from alcohol and it melts at 250. The hydroiodide forms colorless needles melting at 231. Phenylmethylanilidopyrimidine methyl iodide. Six grams of the above cycloamidine were heated with an excess of methyl iodide in methyl alcohol solution for seven hours from ioo- 105. The material was then washed with alcohol and crys- tallized from water and alcohol. Thus obtained, the sub- stance melts at about 2io-2i3 with strong effervescence. If suddenly heated it melts much lower. The material dried in the air contains 2 molecules of water of crystallization. [Analysis I. and II.] When dried at a temperature of 130 it was obtained anhydrous [Analysis III.]. Determinations I. and III. were made by dissolving the substance in water and precipitating with silver nitrate. Determination II. was made by the method of Carius. Calculated for Found. Ci 8 H 18 N 3 I.2H 3 0. Ci 8 H 18 N,I. I. II. III. I 28.9 31.5 28.8 28,7 31.5 This addition-product is readily soluble in warm water and in alcohol, from which it crystallizes in fine, colorless needles. Ammonia or alkali immediately precipitates a substance melt- ing at i6o-i6i, which has all the properties of phenyl- methy lanilidopy rimidine . Phenylmethylmethylanilidopyrimidine, N C CH 3 C 6 H 5 C GH . This was prepared by warming N C NCH 8 C 6 H 5 phenylmethylchlorpy rimidine with methyl aniline. It is the compound that should result by treating the above addition- product with alkali, if the action of methyl iodide on the Researches on the Cycloamidines. 487 cycloamidine takes place by substitution. A comparison of the above with the following properties show that these com- pounds are different. This compound crystallizes from alco- hol in colorless prisms which melt at 113. It dissolves readily in ether, alcohol, and benzene, but is insoluble in water. A nitrogen determination gave : Calculated for C 18 H 17 N S . Found. N 15.2 15.0 This compound dissolves readily in dilute hydrochloric acid, and platinum chloride gives a bright yellow, granular precipitate, which melts at about 228, with effervescence. The hydroiodide is readily soluble in warm alcohol, less readily in water. It melts when slowly heated at about 198. An iodine determination gave : Calculated for C 18 H 18 N 3 I.2H 3 O. Found. I 28.9 28.9 The nitrate separates from dilute nitric acid in long, color- less needles or prisms, which melt with violent effervescence at about 170. Phenylmethylanilidopyrimidine ethyl iodide. This was ob- tained when 5 grams of phenylmethylanilidopyrimidine were heated with 10 grams of ethyl iodide for twelve hours at 120- 130. The reaction -product was extracted with hot water and then crystallized from alcohol, when it separated in color- less needles melting at about 215. When crystallized from water the material shrivels at about 115, then melts with effervescence at or near 215. An iodine determination of air-dried material indicates that the substance separates with i molecule of water of crystallization. [Analysis I.] The material dried at I2o-i3o becomes anhydrous. [Analysis no Calculated for Found. Ci 9 H 20 N 8 I.H 2 0. C, 9 H ao N 3 I. I. II. I 29.19 30.4 29.24 30.3 This addition-product and the analogous methyl compound give off the methyl iodides when heated to their melting- points. Ammonia or alkali precipitate phenylmethylanilido- pyrimidine from aqueous or alcoholic solutions. 488 Wheeler. Phenylmethylethylanilidopyrimidine, N C.CH S / \ C 6 H t C CH . This was prepared like the cor- \ / N C N responding methylanilidopyrimidine for the sake of compari- son with the base obtained from the above addition-product. It is an entirely different base. This melts at 87 when crys- tallized from alcohol. It is readily soluble in hot alcohol, dif- ficultly in cold, and insoluble in water. It separates in beau- tiful, long, colorless prisms. A nitrogen determination gave : Calculated for C I9 H 1(> N3. Found. N 14.53 I4-56 The hydrochloride is difficultly soluble in cold water, readily in hot, and crystallizes in colorless needles which appear to be hydrous. On heating the material shrivels at about 100, then melts at about 210. The platinum chloride salt separates as a bulky, light-yellow precipitate. It is insoluble in hot water, and melts at about 218. The hydroiodide crystallizes in prisms. An attempt to prepare an ethyl bromide addition-product of phenylmethylanilidopyrimidine was unsuccessful. 3.8 grams of anilidopyrimidine were heated with an excess of ethyl bromide from io2-iO4 for five hours, when the material was found to be unaltered. It was heated again, this time with the addition of some alcohol, the temperature being kept between 120 130 for seventeen hours. On opening the tube then, the products were found to be the hydrobromide of phenylmethylanilidopyrimidine described above, and ether. Ethyl bromide, therefore, neither effects substitution nor ad- dition . Phenylmethylpyrimidon methyl iodide. Phenylmethyloxy- pyrimidine and methyl iodide, 4 grams of the former and 10 of the latter, were heated for six hours at 100. No reaction was evident. The material was then heated for nine hours at Researches on the Cycloamidines. 489 170, when addition took place. The material dissolved al- most entirely in water and, on concentrating the aqueous solution to a syrup, a mass of needles was obtained. This was crystallized from alcohol when beautiful, large, colorless prisms separated. These did not melt sharply, but decom- posed with effervescence at 230. An iodine determination gave: Calculated for C n Hi N 3 O.CH 3 I. Found. I 38.7 38.6 n-Methylphenylmethylpyrimidon. When the above com- pound with methyl iodide was dissolved in a little water and dilute alkali was added, this compound separated as a mass of colorless needles. It was purified by crystallizing from water, when it separated in the form of beautiful, large prisms, which melt at about gi-g2. A nitrogen determination gave : Calculated for Ci 3 H I3 N,O. Found. N 14.0 13.6 This compound has basic properties ; it dissolves readily in dilute acids, the ordinary salts being all quite soluble. It is readily soluble in alcohol and ether, less readily in water, and is not dissolved by a small amount of alkali. The nitrate and platinum chloride double salt are the least soluble of the ordinary salts. These were obtained by spontaneous evapora- tion of the solutions. The nitrate crystallizes in colorless, flattened prisms which melt with effervescence at about 195. The platinum chloride double salt separates in form of very large prisms with the color of azobenzene. It gave no definite melting-point below 275, but turned dark at about 235. Methylbenzamidine hydrochloride, C 6 H 5 C(NH)NHCH 8 .HC1. This was prepared by dissolving benzimidoethylester hy- drochloride in a 33 per cent, solution of methylamine. On adding the hydrochloride to the solution of the amine the free ester separates ; on shaking this dissolves, and in a short time a mass of colorless needles separates. This material was crystallized from water in which it is not extremely soluble. A chlorine determination gave : 49 Wheeler. Calculated for C 8 H 10 N a .HCl. Found. Cl 20.82 20.75 The properties of this amidine hydrochloride apparently differ decidedly from those of the corresponding ethylami- dine. Lessen 1 prepared ethylamidine hydrochloride in a state of purity only with some difficulty, while Pinner* ob- tained the hydroiodide as an oil which would not crystallize. NEW HAVEN, March, 1898. Ann. Chem. (Uebig) 265, 159. 2 Ber. d. chem. Ges.ji, 7. I REPRINTED FROM THE AMERICAN CHEMICAL JOURNAL, VOL. XXIX, No. 5, MAY, 1903.] Contributions from the Sheffield Laboratory of Yale University. CII. ON SOME CONDENSATION-PRODUCTS OF THE PSEUDOTHIOUREAS : SYNTHESIS OF URA- CIL, THYMINE, AND SIMILAR COM- POUNDS. BY HENRY I*. WHEELER AND HENRY F. MERRIAM. Urea and thiourea have been shown to undergo condensa- tion with a variety of substances, but apparently no one has attempted to use the simple pseudothioureas in any synthetic process whatever. 1 The pseudothioureas in question, HN=C(SR) NH,, (R=rCH 8 or C 2 H 6 ) , have not been isolated. Their halogen hydride salts result from the addition of alkyl halides to thio- urea. These are described as extremely unstable compounds, and Claus 2 states that when the ethyl bromide addition-prod- 1 Rathke has shown that their salts give guanidine when boiled with ammonia. Ber. d. chem. Ges., 17, 309 (1884). Ann. Chem. (Liebig), 179, 146 (1875). Condensation- Products of the Pseudothioureas. 479 uct is treated with alkali it decomposes into ethyl rhodanide, ammonia, and hydrogen bromide. In the case of the methyl iodide addition- prod uct, Bernth- sen and Klinger 1 found that, with silver oxide, a solution containing a strong base resulted, which gave a blue color to red litmus. This base was supposed to be a hydroxyl com- pound, H 2 NCSNH 2 .CH,OH, which, on evaporating in a desic- cator, decomposed. The ethyl iodide addition-product, which Claus describes as an exceptionally unstable compound, 2 un- der similar treatment with silver oxide gave a strong alka- line solution which, on evaporation, contained cyanamide (di- cyandiamide). A similar decomposition was observed when the compounds were treated with mercuric oxide. We have made no attempts to isolate these free pseudothio- ureas, but, at first, in order to obtain an idea of their stability, we treated the methyl iodide addition-product in aqueous solu- tion with an excess of alkali and benzoyl chloride, and ob- tained an excellent yield of the corresponding benzoylpseudo- thiourea, 3 C 6 H 5 CON-C(SCH,) NH 2 . The ethyl bromide addition-product, on shaking with alkali and phenyl mustard oil, gave a good yield of the phenylpseudodithiobiuret, C.H 6 NHCSN=C(SC 2 H 6 )NH 2 . The free pseudothioureas are, therefore, not so unstable as one might be led to believe from previous work, and, owing to their strong basic nature, it seemed probable that they would undergo condensation with aldehyde and ketone esters more readily, and prove more reactive in general, than the normal ureas. This we have found to be the case. Ernert* states that urea does not condense with the substi- tuted acetoacetic esters or with benzoylacetic ester. Warm- ington, 5 also, was unable to obtain a condensation of urea and the latter ester, in aqueous solution, but found that they could be condensed in the dry state at a high temperature. We have readily obtained such condensations with the 1 Ber. d. chem. Ges., II, 493 (1878). * Ibid., 8, 41 (1875). 8 J. Am. Chem. Soc., aj, 293 (1901). Ann. Chem. (Uebig), 358, 362 (1890). * J. prakt. Chem., [a], 47, 202 (1893). 480 Wheeler and Merriam. pseudothioureas at ordinary temperatures. These pyrimidine condensations, which are analogous to Pinner's, 1 take place in alkaline solution in a similar manner to that observed by Michael in the case of urea with acetoacetic and malonic es- ters.* They are also similar to Traube's synthesis of pyrimi- dine derivatives in which guanidine and ethyl cyanacetate are employed. 8 A method similar to that of preparing methyluracil by condensing urea with acetoacetic ester, by means of hydro- chloric acid, 4 has not been applied to the preparation of ura- cil, probably on account of the ease with which free formyl- acetic ester passes into trimesic ester. On the other hand, the sodium salt of formylacetic ester is not only easily prepared, 5 but is also quite stable, and we have found that it readily con- denses, in aqueous solution, with both methyl- and ethyl- pseudothioureas, giving a good yield of the corresponding methyl- and ethyl-mercaptouracils. For example, with methyl- pseudothiourea, as follows : 6 NH 2 C 2 H 6 OCO NH CO C 2 H 5 OH CH 3 S C + CH = CH 3 SC CH + II II II II NH NaOCH N CH NaOH In order to obtain a practically quantitative yield of uracil from these compounds, heating with acids in a sealed tube is not necessary. 7 When they are boiled with concentrated hy- drochloric acid, or better with hydrobromic acid, they evolve mercaptan and yield uracil as follows : * NHCO NH CO II II RS C CH + H,O = OC CH + RSH. II II I II N CH NH CH 1 "Die Imidoather u. ihrc Derivate," p. 209. 2 J. prakt. Chem., 35, 456 (1887). Ber. d. chem. Ges., 33, 1371 (1900). * Behrend : Ann. Chem. (Liebig), 339, 9 ; 251, 238. 6 Wislicenus : Ber. d. chem. Ges., ao, 2933 (1887). 8 An attempt to condense urea with this salt gave a negative result. 1 1ist has shown that 2-methylmercapto-4-methyl uracil gives 4-methyluracil when heated with concentrated hydrochloric acid at 180*. Ann. Chem. (lyiebig), 336, 12 (1886). Condensation- Products of the Pseudothioureas . 481 Emil Fischer and Georg Roeder 1 have recently prepared | uracil, thymine, and other pyrimidine derivatives by means of an interesting synthesis. Their method consists in heating urea with unsaturated acids and brominating the hydroura- cils thus formed. The bromine derivatives are then con- verted into uracils by treatment with alkali or pyridine. This synthesis has the disadvantage that the unsaturated acids are not in all cases easily accessible compounds, and the yields, especially in the case of uracil, are below those ob-j tained by our method. Uracil prepared by our method agreed in all respects with the description given by Fischer and Roeder. We have found that sodium acetoacetic ester and pseudomethylthiourea give 2-methylmercapto-4-methyluracil, which L,ist 2 has prepared by a different method. This, on boiling with hydrobromic acid, gave Behrend's 3 4-methyluracil. The isomeric 2-methylmercapto-5-tnethyluracil and 5- methyluracil, or thymine, were obtained in a similar manner. Free ethyl formylpropionate directly condensed with pseudo- methylthiourea, and the sodium salt was also caused to react with the pseudothiourea : NH 2 C 2 H 5 OCO NH CO C,H 5 OH CH 3 SC + C.CH 3 = CH 3 SC CCH 8 + II II II ' II NH HOCH N CH H 2 O On boiling this mercapto derivative with hydrochloric acid we obtained thymine, which was identical in all respects with the natural product obtained from the nucleic acid of the spleen, a sample of which, through the kindness of Dr. P. A. Levene, was sent to us for comparison. When the samples were mixed the melting-point was not altered. With the sodium salt of ethyl benzoylacetate and pseudo- methylthiourea we obtained 2-methylmercapto-4-phenyluracil, which was converted by acids into 4-phenyluracil, identical with the phenyluracil described by Fischer and Roeder. 1 Her. d. chem. Ges., 34, 3751 (1901). 9 Ann. Chem. (Uebig), 336, 12 (1886). s Ibid., 339, 8. 482 Wheeler and Merriam. With ethyl methylacetoacetate and pseudoethylthiourea we prepared 2-ethylmercapto-4,5-dimethyl-6-oxypyrimidirie, which gave a new dimethyluracil (4,5-dimethyluracil) : NH CO NH CO II II C 3 H 5 SC CCH 3 OC CCH $ . II II I II N CCH 3 NH CCH S With ethyl ethyl-acetoacetate we prepared 2-methyl- mercapto-4-methyl-5-ethyl-6-oxypyrimidine and 4-methyl-5- ethyluracil. L,ike urea, 1 pseudomethylthiourea readily con- denses with acetylacetone and two products are formed. It condenses also with ethyl cyanacetate. Beside the above condensations, which involve an attach- ment to the two nitrogen atoms of the pseudothiourea, an- other type occurs in which only one nitrogen and the mer- capto group take part. For example, amino acids react with the pseudothioureas in aqueous solution to form guanidine acids : HN=C (NH 2 ) SR + H 2 NCH 3 COOH = HN=C (NH 2 ) NHCH 2 COOH + RSH. Since by appropriate treatment these compounds can be con- verted by loss of water into the corresponding rings, it seems probable that the isocreatinine obtained by Thesen 2 from fish flesh (haddock) may be prepared by this method, using N-methylpseudomethylthiourea and aminoacetic acid. We wish to reserve the study of the condensation-products of the pseudothioureas for this laboratory. EXPERIMENTAL PART. Our experiments with the pseudothioureas have been con- fined to pseudomethyl- and pseudoethylthiourea. Pseudomethylthiourea hydriodide was prepared by moisten- ing finely powdered thiourea with alcohol and then adding an excess of methyl iodide. Heat was evolved so that a con- denser was necessary. After standing twelve hours, ether 1 Evans : J. prakt. Chem., 48, 489 (1893). 8 Hoppe-Seyler : Ztschr. physiol. Chem., 34, 4 (1898). Condensation- Products of the Pseudothiourcas . 483 was added and the residue filtered, washed with ether, and dried. The crude addition-product thus prepared was used in the following experiments. Pseudoethylthiourea hydrobromide, for use in these condensa- tions, can be conveniently prepared from thiourea containing considerable ammonium thiocyanate ; that is to say, from the product obtained by heating ammonium thiocyanate at 170 for forty-five minutes 1 and then crystallizing once or twice from water. The thiourea is powdered, moistened with alco- hol, and an excess of ethyl bromide added. On boiling this mixture for four or five hours the greater part of the mass dis- solves, leaving ammonium bromide as an insoluble residue. The solution is then filtered and most of the alcohol evapora- ted. The oil thus obtained is transferred to a distilling-bulb and the ethyl rhodanide distilled off under diminished pres- sure on the water-bath. The residue, on cooling, solidifies to a crystalline mass, which, after drying on paper in the air, melts at about 88 and is pure enough for use in the following experiments : NHCO 2-Methylmcrcapto-6-oxypyrimidinc> CH 3 SC CH. In this II II N CH preparation the crude sodium salt of formylacetic ester* was considered as 70 per cent pure. 3 Seven grams of sodium hydrate were dissolved in 70 cc. of water and 38 grams of the methyl iodide addition-product of thiourea were added, and then 29 grams of sodium formyl- acetic ester. The mixture was allowed to stand for twelve hours at ordinary temperature, then warmed on the water- bath for a few minutes, cooled, and acidified with acetic acid. The crystalline precipitate thus produced was filtered, washed, and dried. It weighed 11.3 grams, a yield of 46 per cent, calculating from the methyl iodide addition-product. It crys- tallized from water in long prisms, or short, lozenge-shaped 1 Reynolds and Werner : J. Chetn. Soc. (Condon), 83, i (1903). 2 Wislicenug : Ber. d. chem. Ges., ao, 2933. Von Pechmann : Ibid. % as, 1047. 4 84 Wheeler and Merriam . tablets, and the surfaces of the crystals were generally etched. It is quite soluble in alcohol and acetone, and it melts at 198* to 199. A nitrogen determination gave : Calculated for C 6 H 6 ON 2 S. Found. N 19.72 19.86 NH CO I I 2-Ethvlmercapto-6-oxypyrimidine, C 2 H 5 SC CH. Fifty- It II N CH nine grams of the ethyl bromide addition-product of thiourea were treated with one molecular proportion of sodium hydrate in 10 per cent aqueous solution, and then 40 grams of formyl- acetic ester were added. After standing for twenty-four hours the solution was divided into two equal portions. One, after warming on the water-bath until effervescence stopped, was cooled and immediately acidified with acetic acid. This gave 6.3 grams of the mercapto derivative. The other por- tion was allowed to stand for a week. On acidifying then, 6.1 grams of the mercapto compound were obtained, a yield of 32 per cent of the calculated. In another experiment 25 grams of the ethyl bromide addition-product were dissolved in more dilute alkali, namely, 5.5 grams of sodium hydrate in 95 cc. of water, and 27 grams of sodium formylacetic ester were added. After two hours' standing, one-half of the solu- tion was warmed, cooled, and acidified with acetic acid, whereupon 5 grams of the mercapto compound separated. After standing two days the remaining portion gave 4.9 grams of the condensation-product, a yield of 47 per cent of the cal- culated. It is evident from this that the materials react in a short time, and that long standing does not influence the yield. The reaction possibly involves an addition similar to that in the case of acetoacetic ester and urea 1 , since it frequently hap- pens that these mixtures can stand for several days, and then, if the solution is not warmed before acidifying, no precipita- tion whatever occurs. Moreover, the fact that usually a yield 1 Behrend and Ernert : Ann. Chem. (I,iebig), 258, 360 (1890). Condensation- Products of the Pseudothioureas . 485 approximating only one-half the calculated is obtained may also possibly be accounted for by the assumption that, like urea and acetoacetic ester in alkaline solution, 2 molecules of the ester react with only one of the pseudothiourea. In one experiment, however, 5 grams of the ethyl bromide addition-product, i.i grams of sodium hydrate, and 6 grams of the sodium salt in 10 cc. of water, on standing over night, gave 2.9 grams of the condensation-product, a yield of 68.8 per cent of the calculated. On crystallizing from 10 cc. of alcohol, 1.75 grams of pure ethylmercaptooxypyrimidine sepa- rated at once. This substance is easily soluble in warm alco- hol and hot water, difficultly soluble in cold, and forms beau- tiful, colorless, small, stout prisms and pyramids melting at 152. A nitrogen determination gave: Calculated for C4H 8 ON 2 S. Found. N 17.94 17.93 NH CO Uracil, OC CH. Fifteen grams of the above methyl- / I II NH CH mercaptouracil and 150 cc. of concentrated hydrochloric acid were boiled for four hours, when mercaptan had ceased being evolved. The solution was evaporated to dry ness and the residue, after drying, weighed 11.9 grams (94.4 per cent yield). It melted with effervescence at 335 (on an Anschiitz thermometer in a paraffin-bath), and, on crystallizing from water, it formed balls of minute, pure- white needles, which, on rapidly heating, melted at 338. By holding the tempera- ture a few degrees below this the material could be made to melt. It agreed in properties with Fischer and Roeder's 1 preparation. When the ethylmercapto derivative (1.5 grams) was heated in a closed tube with concentrated hydrochloric acid (5 cc.) for four and one-half hours, about 0.05 gram of an insoluble by-product was formed, and 0.9 gram of pure uracil was obtained. A nitrogen determination gave : i Loc. tit. 486 Wheeler and Mertiam. Calculated for Found. N 25.00 25.24 One hundred parts of water at 25 dissolve 0.358 part of uracil. Uracil may be boiled with alkali and recovered unaltered by acidifying the solution. The alkaline solution probably contains the substance as a salt of /?-uramidoacrylic acid, just as Behrend's methyluracil gives /?-uramidocrotonic acid. When uracil is dissolved in ammonia and most of this re- moved by boiling, then silver nitrate -is added and the solu- tion is boiled, an amorphous silver salt is precipitated, which is soluble in an excess of ammonia and nitric acid. The composition of the salt prepared in this manner varied. One preparation gave 63.8 and 64 per cent silver, another 59.9 and 60. i per cent silver, while the calculated for silver yff-urami- doacrylate is 62.79 per cent. Uracil does not form a picrate. 5-Bromuradl. Two and one-half grams of uracil were suspended in carbon disulphide and 3.8 grams of bromine were added. Hydrogen bromide was slowly evolved, and after two days, on evaporating to dryness, a red residue was ob- tained which, on warming on the water-bath, became color- less. When crystallized from water it formed small, stout, colorless prisms, and when heated turned dark above 200, and melted with some effervescence at about 293. A nitro- gen determination gave : Calculated for C 4 HsOaN a Br. Found. N 14.66 14.60 2-Methylmercapto-4. methyl-6-oxypyrimidine, NH CO I I CH 3 SC CH . Sixteen grams of the methyl iodide addi- II II N - CCH, tion-product of thiourea were dissolved in 20 cc. of water and added to a cold 30 per cent solution of 4.5 grams of potassium hydrate. Nine and six-tenths grams of acetoacetic ester were then added and the mixture allowed to stand. The Condensation- Products of the Pscudothioureas . 487 next day long prisms had formed. After two days these were filtered and found to weigh 5 grams. This material was sol- uble in acids and alkali, and it melted at 219, the melting- point assigned to this compound by List. When 2 grams of this were boiled with 10 cc. of hydrobromic acid for one hour mercaptan was given off and 1.2 grams of Behrend's4-methyl- uracil were obtained. 2-Methylmercapto-^-methyl-6-oxypyrimidine, NH CO CH 8 SC CCH 3 . Sixty-five grams of the methyl iodide ad- I! II. N CH dition-product of thiourea were treated with 17 grams of potassium hydrate in 150 cc. of water, and 45 grams of sodium formylpropionic ester were then added. The mixture, on standing two and a half days, was filtered from some sedi- ment and neutralized with acetic acid. A fine crystalline precipitate of methylmercaptothymine came down, which weighed 9.2 grams, or 20 per cent of the calculated. It crys- tallized in strings of small plates from water, in which it is difficultly soluble when hot and nearly insoluble when cold. On heating, it sintered at about 225 and melted at 233. A nitrogen determination gave : Calculated for CH 8 ON 2 S. Found. N 17.94 17-95 NH CO I I 5-Methyluracil ( Thymine) , OC CCH 3 . The above mer- I II NH CH captothymine reacted less readily with hydrochloric acid than in the other cases. Nine and two-tenths grams were boiled with 100 cc. of concentrated hydrochloric acid for ten hours, whereupon no more mercaptan was given off. The solution, on evaporating, then gave 7.3 grams of crude thymine (melting at 319), which is 99 per cent of the calculated. This crystal- lized from water in small, apparently rectangular, colorless 488 Wheeler and Merriam. plates, and on rapidly heating melted at 326 (on an An- schiitz thermometer) . Fischer and Roeder state that their preparation sintered at 318 and melted at 321. A sample of thymine which Dr. P. A. L,evene, of the Pathological Institute at Ward's Island, N. Y., kindly gave us, proved to be identi- cal with our compound. This sample was obtained from the nucleic acid of the spleen, and on mixing it with some of our substance the melting-point was unchanged. A nitrogen de- termination in the case of the synthetic product gave : Calculated for C 5 HeO 2 N 2 . Found. N 22.22 22.36 One hundred parts of water at 25 dissolve 0.404 part of thymine. 2-EthylmercaptO'4. ^-dimethyl- 6-oxypyrimidinc , NH CO I I CjH 5 SC CCH 3 . Six grams of potassium hydrate were II II N CCH 8 dissolved in 50 cc. of water and cooled. To this was added 13 grams of the ethyl bromide addition- product of thiourea and then 10 grams of methyl acetoacetic ester. The oil grad- ually disappeared and a white solid was formed. After stand- ing for two days the solution was made slightly acid with hy- drochloric acid and filtered. The precipitate in this case proved to be a mixture. On boiling with about 30 cc. of alcohol and filtering while hot, a separation was effected. The solu- ble portion was crystallized from a mixture of alcohol and ether. It formed stout prisms melting at about 156, sinter- ing at about 151, and a nitrogen determination gave : Calculated for C 8 H la ON 2 S. Found. N 15.2 15.6 The compound that was not readily dissolved by boiling alcohol was purified by crystallizing it from a large amount of alcohol. It was found to be free from sulphur, and on heat- ing it began to decompose at about 260, and effervesced at 269" to 270, turning reddish-brown. It consisted of minute, Condensation- Products of the Pseudothioureas . 489 microscopic crystals that formed twins having the form of a cross. We did not stop to determine the structure of this compound, but we believe that possibly it is acetomethyl- acetylcyanamide, CH 8 CO(CH S )CHCONHCN. A nitrogen determination agreed with the calculated for this formula. Calculated for CH 8 2 N,. Found. N 20.00 20.03 NH CO I I 4.,5-Dimcthyluracil, OC CCH 8 . About i gram of the I II NH CCH, above ethylmercaptodimethyluracil was boiled for four hours with 10 cc. of concentrated hydrochloric acid. Mercaptan was given off and there was a smooth decomposition into the oxygen derivative. The solution was evaporated to dry ness and the residue was crystallized twice from alcohol. It crys- tallized in microscopic needles which melted to a clear liquid, with partial decomposition, at 292. A nitrogen determina- tion gave : Calculated for CjH 8 2 N 2 . Found. N 20.00 20.34 2-Methylmcrc&pto-4.-methvl-5-cthyl-6-oxypyrimidine, NH CO I I CH.SC C.C,H 6 . Ten grams of the methyl iodide addi- II II N C.CH, tion-product of thiourea were dissolved in a solution of 2.6 grams of potassium hydrate (i molecule) in 20 cc. of water, and to this 7 grams of ethyl ethylacetoacetate were added. A little alcohol was also added, and finally some more alkali, and the mixture was allowed to stand for a week. Solid ma- terial gradually separated, which was filtered after neutral- izing the solution. The yield of crude material was 3.2 grams. It was crystallized from alcohol, from which it sepa- rated in long, flat, colorless prisms with branching, brush-like 49 2 Wheeler and Merriam. drate, and precipitated with carbon dioxide, it gave 0-benz- glycocyamidine. 1 Pseudomethylthiourea Chloracetate , H 2 NC(SCH 8 ) NH.C1CH,COOH. Thirty-three grams of the methyl iodide addition-product of thiourea were dissolved in a small quantity of cool alcohol, and a concentrated alco- holic solution of 8.5 grams of potassium hydrate was added. The solution was filtered from potassium iodide and then 14.5 grams of chloracetic acid were slowly added. Crystalline ma- terial soon separated which, on drying, weighed 17 grams. When crystallized from alcohol it formed transparent, rec- tangular plates, melting at 162. It was very soluble in water. A nitrogen determination gave : Calculated for C 4 H 9 OjN 2 SCl. Found. N 15.17 15.26 NEW HAVEN, CONN., Feb. 26, 1903. 1 Griess : Ber. d. chem. Ges., 13, 977. [REPRINTED FROM THB AMERICAN CHEMICAL JOURNAL, VOL. XXIX, No. 5, MAY, 1903.] Contributions from the Sheffield Laboratory of Yale University. CIIL SYNTHESES OF AMINOOXYPYRIMIDINES HAVING THE COMPOSITION OF CYTOSINE : 2-AMINO 6-OXYPYRIMIDINE AND 2-OXY- 6-AMINOPYRIMIDINE. BY HENRY L,. WHEELER AND TREAT B. JOHNSON. The interest which in recent years has been attached to the study of pyrimidine derivatives has been increased by a new discovery of Kossel and Steudel. Working with a large quantity of sturgeons' testicles they obtained, in the histidine fraction, a basic substance agreeing in composition with the formula C 4 H 5 ON 3 . Kossel and Steudel state their belief that this compound is an aminooxypyrimidine, 2 since hitherto all of the nitrogen compounds obtained from nucleic acid have been found to contain the pyrimidine ring. This new sub- stance was found to closely resemble cytosine, which Kossel and Neumann 3 obtained in the year 1894 as a cleavage-prod- 2 Ztschr. physiol. Chem., 37, 179 (1902). 3 Ber. d. chem. Ges., 37, 2215 (1894). Syntheses of Aminooxypyrimidines . 493 uct of thymus nucleic acid. Kossel and Steudel have recently undertaken the reinvestigation of thymus cytosine and they now 1 conclude that the product is identical with sturgeon cytosine. They state that it can also be prepared from her- rings' testicles and call attention to the fact of its wide occur- rence in animal organs. Dr. P. A. Levene has kindly sent us the proof of his article on " Darstellung und Analyse einiger Nucleinsauren," in which he describes the preparation of this base from the nucleic acid of the spleen and pancreas. 2 Kossel and Steudel 3 conclude that material yielding uracil is widely disseminated in animal organs, and it seemed probable that perhaps uracil results from the cleavage of cytosine by treatment with sulphuric acid at high temperatures. More- over, they state in their last paper 4 that cytosine, by the action of nitrous acid, gives a substance having the properties of uracil. From this it appears that of the seven theoretically possible 5 aminooxypyrimidines, the first to be considered are those that would be expected to yield uracil by the above treatment. An examination of the following formulas will show that for- mulas I. and II. are the only ones that agree with this be- havior : N=C NH, CO CH I II NH CH I. NH CO ! I HC C NH 2 NH CO NHCH CO I III. N==CH C NH, HC CH Hi COH N CH N C NH 2 N C NH, IV. V. VI. 1 Ztschr. physiol. Chera., 37, 379 (1903). 8 To appear in Ztschr. physiol. chein., 38, (1903). See also Am. J. Physiol., 8, XII (1903); J. Am. Chem. Soc., 25, R. 188 (1903). 3 7*f., 37, 347 (1903). 4 Ibid., 37, 380 (1903). 6 Tautomeric forms are not considered. 494 Wheeler and Johnson. N CH II II H,N C COH I I N=CH VII. We have now prepared the two aminooxypyrimidines rep- resented by formulas I. and II. and we find that both products agree closely in properties with the descriptions of cytosine. Of the two compounds, 2-oxy-6-amino-pyrimidine, formula I., agrees better than the isomer, 2-amino-6-oxypyrimidine. Formula II., in fact, seems to be excluded as representing cytosine chiefly because this compound crystallizes from water in anhydrous crystals. Its picrate melts a little higher than the melting-point given for cytosine picrate, but in other re- spects the salts are closely similar. On the other hand, 2-oxy-6-aminopyrimidine crystallizes with i molecule of water of crystallization, like cytosine, and its properties agree in practically every respect, as far as can be judged, with the descriptions of the natural substance. The picrate, when prepared from the pure base, has a higher decomposing-point than that given for the picrate from natural cytosine. However, we are inclined to believe that our base and cytosine are identical and, therefore, the structure of cytosine is to be represented by formula I. 1 It will be necessary to compare our material with the natural before this can be definitely settled, and we hope soon to obtain a sample of the natural substance. When 2-oxy-6-aminopyrimidine was heated with 20 per cent sulphuric acid for 3 hours at 150- 170 it was found that 15 per cent of the material was converted into uracil. Dr. Osborne informs us that to obtain uracil it was necessary to heat triticonucleic acid with acids for a considerable time. 2 Since uracil results from the cleavage- cytosine, the natural 1 The substance can naturally be viewed as a-oxy-4-aminopyrimidine, and, on ac- count of the usual nomenclature, this aspect better illustrates its character as a sort of hydrated fragment of uric acid. Its relation to uracil, however, is better shown by representing the substance as a 6-amino derivative or the tautomeric 6-imino form. 2 See Report Conn. Agr. Expt. Sta. for 1901 p. 409. Syntheses of Aminooxypyrimidines . 495 assumption is that the analogous thy mine, which until re- cently has been more widely obtained as a cleavage-product, results from the decomposition of a corresponding base, the as yet undiscovered 5-methylcytosine, this base undergoing cleavage more readily than cytosine. We intend to give an ac- count of this new base later, the preparation of these bases being one of the first steps toward the synthesis of the nucleic acids. We prepared the synthetic cytosine as follows : The ethyl bromide addition-product of thiourea was treated with i molecule of alkali in aqueous solution and the sodium salt of ethyl formylacetate added. On standing a number of hours, then warming for a few minutes, cooling, and acidifying with acetic acid, 2-ethylmercapto-6-oxypyrimidine was obtained : NH 2 C 2 H 5 OCO NH CO C 2 H 5 OH C 2 H 5 SC + CH = C 2 H 6 SC CH + II II II II NH NaOCH N CH NaOH The 2-ethylmercapto-6-oxypyrimidine was converted into 2-ethylmercapto-6-chlorpyrimidine by means of phosphorus pentachloride and the phosphorus oxychloride was removed by evaporation in a vacuum. NH CO Nr=CCl I I I CH + PC1 6 = C 2 H 5 SC CH + POC1, + HC1. II II II II N CH N CH ,1.1 when dry, is stable at 150. When boiled with hydrochloric acid it gives uracil. The chlorpyrimidine, for conversion into the amino derivative, was prepared as follows : 6.3 grams of 2-ethylmercapto-6-oxypyrimidine and 8.5 grams of phosphorus pentachloride were heated on the steam-bath until the action was complete. The phosphorus oxychloride was removed by heating in an oil-bath at 140, under a pres- sure of 12 mm., for about fifteen minutes. The crude oil thus obtained weighed 6.5 grams, a yield of 92 per cent of the cal- culated. In another preparation 15.5 grams of the 2-ethyl- mercapto-6-oxypyrimidine gave 15.5 grams of chlorine com- pound, or 90 per cent of the calculated. N=:C NH 2 2-Ethylmercaplo-6-aminopyrimidint, C 2 H 6 SC CH . II II N CH When 2-ethylmercapto-6-chlorpyrimidine (6.5 grams) was heated in a closed tube with alcoholic ammonia at 140 to 150, for six hours, the action was complete. An odor of mercaptan was apparent on opening the tube, showing too energetic treatment, and ammonium chloride had separated. The alcohol was removed by evaporation, after filtering from ammonium chloride, and the dark varnish which was obtained was washed with a few cubic centimeters of cold water. By this treatment the material completely solidified to a hard 498 Wheeler and Johnson. cake. It was very soluble in alcohol and in benzene, and fairly soluble in hot water. The weight of the crude product was 4.7 grams, a yield of 82 per cent of the calculated. The base dissolved immediately in dilute hydrochloric acid and was precipitated by ammonia in stout tables. It was purified by boiling in alcohol with animal charcoal and then crystal- lizing from 50 per cent alcohol. It separated in the form of colorless plates and melted at 85 to 86. A nitrogen deter- mination gave : ' Calculated for C 6 H 9 N 8 S. Found. N 27.1 27.6 In another preparation the chlorpyrimidine was heated with alcoholic ammonia at 115 to 120 for five and a half hours. From 15.5 grams of the chlorine compound n.6 grams of the base were obtained, which is a yield of 84 per cent of the calculated. NzmzC NH 2 Cytosincor2-Oxy-6-aminopyrimidine, CO CH .H 2 O. I II NH CH The above 2-ethylmercapto-6-aminopyrimidine (3 grams) was boiled with hydrobromic acid of boiling-point 125 (i5cc.) for two hours, when mercaptan had ceased being evolved. Qn evaporating the free hydrobromic acid on the steam-bath a beautiful crystalline mass of the hydrobromide was obtained. It crystallizes in the form of glistening prisms from a little water. The free base was obtained by dissolving the hydrobromide in water and precipitating with ammonia. The weight of the crude base was 1.9 grams, which is a yield of 79 per cent of the calculated. In another experiment 7.5 grams of 2-ethyl- mercapto-6-aminopyrimidine gave 5.6 grams of 2-oxy-6- aminopyrimidine, or 90 per cent of the calculated. The base for analysis was boiled with animal charcoal and crystallized from water. It was dried in the air and the water of crystal- lization determined by heating in an oven at 100 to 1 10. 0.5166 gram substance lost 0.0727 gram on heating. Syntheses of Aminooxypyrimidines. 499 Calculated for C 4 H 6 ON 8 .H,O. Found. H 3 O 13.95 14-07 The analysis of the dried material gave the following re- sults : 0.2188 gram substance gave 0.3498 gram CO 2 and 0.0835 gram H 2 O. 0.0939 gram substance gave 31 cc. moist N at 25 and 773 mm. pressure. Calculated for C4H 6 ON 8 . Found. C 43-25 43.6o 4.50 4.25 N 37.84 37.72 Properties of 2-Oxy-6-aminopyrimidine. The free base, when first obtained, crystallized from hot water in the form of needle-like prisms. It was then somewhat colored (pale brownish), and melted, or rather decomposed, at 320 to 325 with violent effervescence, first becoming dark-colored a little below 300. After boiling in aqueous solution with animal charcoal it crystallized in the form of beautiful, colorless, transparent plates. Ttie edge of one of these crystals, which perhaps was made up of a number of plates, measured three- quarters of an inch in length. The faces were so badly etched, or striated, or covered with vicinal growths, that crys- tallographic measurements were impossible. They probably belong to the mono- or tr iclinic system . The plates had the same effervescing-point on heating as the needle-like prisms. These decomposing-points were taken in capillary tubes, in a paraffin- bath, on an Anschiitz thermometer 13.5 centimeters in length, and are uncorrected. The plates dissolve in about 129 parts of water at 25. The base is precipitated by phosphotungstic acid. It would, therefore, be found in the histidine fraction. When the acidified solution is treated with potassium bismuth iodide it gives a brick-red precipitate. It gives the murexide reaction. With sulphuric and hydrochloric acids it gives readily soluble salts. The chloroplatinate, and espe- cially the picrate, are difficultly soluble in water. The gold double salt is also not very soluble. Kossel and Steudel give 500 Wheeler and fohnson. no melting- or decomposing-point for their cytosine, but state that the picrate turned brown on heating to 255 and melted at 270 with decomposition. 2-Oxy-6-aminopyrimidine Picrate. Picric acid produced an immediate precipitate, which crystallized in bright-yellow needles or very slender, needle- like prisms. When made from material that had not been decolorized with animal charcoal, and therefore containing a slight impurity, it decomposed with violent effervescence quite sharply at about 264. When prepared from the pure oxyaminopyrimidine the crystals had a more pronounced prismatic habit and they showed no melt- ing-point, but sintered, became black, and decomposed at about 300 to 305. One hundred parts of water dissolve 0.076 parts of the salt at 25. Our experience with these picrates has shown that the melting-points are rather indefinite. They can be made to effervesce at a somewhat higher or lower tem- perature, according to the rate of heating. They are difficult to analyze on account of the ease with which oxides of nitro- gen pass over. A nitrogen determination of a sample pre- pared from the pure base, for example, gave : Calculated for C 4 H 6 ON3.C 6 Hj(N0 2 ) 8 OH. Found. N 24.7 26.4 The chloroplatinate separates as a precipitate in yellow flakes. A platinum determination gave : Calculated for Found. Pt 30.83 30.60 2-Oxy-6-acetaminopyrimidine was prepared by heating 0.5 gram of the amino derivative with acetic anhydride. It proved to be practically insoluble in alcohol and difficultly soluble in water. It crystallized from hot water in colorless, microscopic prisms, with rough faces, frequently occurring in crosses. It showed no signs of melting at 300. A nitrogen determination gave : Calculated for C8H 7 O 8 N 8 . Found. N 27.44 27.82 Syntheses of Aminooxypyrimidines. 501 The phenyl isocyanate derivative was prepared by heating 0.5 gram of the base with 0.55 gram of phenylisocyanate on the steam-bath for six hours. The white crystalline product which resulted proved to be very insoluble. It was boiled re- peatedly with alcohol and then digested with water. The in- soluble material thus obtained began to sinter on heating at about 255, and then melted with violent effervescence at 260. A nitrogen determination agreed with the calculated for an ad- dition of 2 molecules of phenyl isocyanate to one of the base. Calculated for (i : i) (2 : i) CnHioOjN*. Ci 8 Hi 5 8 N5. Found. N 24.34 20.05 20.4 Action of Bromine. Three-tenths gram of 2-oxy-6-amino- pyrimidine was treated with i gram of glacial acetic acid, and 0.5 gram of bromine was added. An immediate reaction took place, heat was evolved, and an orange powder separated, which dissolved on warming. Some water was added to the cooled solution and the precipitate produced was crystallized from water. It formed well-crystallized, colorless needles, and it melted sharply at 247 with effervescence. When dis- solved in water and made alkaline with ammonia it gave a strong, wine-red color, and, on cooling, a brick-red substance separated. This had no definite melting-point. NH CO 2-Amino-6-oxypyrimidine, H,N C CH. Thirty grams II II N CH of guanidine carbonate were dissolved in 100 cc. of water and mixed with 54 grams of crystallized barium hydrate in 400 cc. of water. The solution was filtered and 55 grams of sodium ethyl formylacetate were added. The whole dissolved except a slight sediment. After standing for three hours the solu- tion was divided into two equal portions. One was allowed to stand over night, the other was warmed on the steam-bath for a few minutes, and then just acidified with sulphuric acid. The filtered solution on evaporation, and adding ammonia in excess, gave a crystalline, brown precipitate, weigh- 502 Wheeler and Johnson. ing 6.7 grams, or 36 per cent of the calculated. The portion that stood over night, on similar treatment, gave practically the same yield, so that long standing does not better the yield. In another experiment 10 grams of guanidine carbon- ate and 22 grams of sodium ethyl formylacetate gave 4.8 grams of the base, which is a yield of 39 per cent of the cal- culated. On boiling the crude products with animal char- coal, in aqueous solution, the color is slowly removed and, on cooling, small, flat, colorless, transparent prisms generally separate. These crystals, when air-dried, contained less than 0.5 per cent of water. A nitrogen determination (I.) gave : I. 0.0900 gram substance gave 30.6 cc. of moist N at 26. 5 and 762 mm. pressure. II. 0.1357 gram substance gave 44.8 cc. of moist N at 24 and 768 mm. pressure. Calculated for Found. C 4 H 5 ON 3 . I. II. N 37-83 37.98 37-45 In another experiment, as above, the base was crystallized from water, purified with animal charcoal, and when a por- tion had separated from the still warm solution, in the form of prisms, the mother- liquor was decanted. It then became al- most semi-solid from the separation of a mass of long, slender, colorless, silky needles which, on drying in the air, formed a bulky, felt-like mass. This material contained, or occluded, water, but the determinations were not constant : 0.8567 gram substance lost 0.1036 gram on heating to 100 to 110, equiva- lent to 12.0 per cent water. The needle form appears to re- sult also from dilute solutions or on rapidly cooling. 2.0527 grams of substance prepared in this manner, on standing in the air for eight or nine hours and then heating, lost 0.1657 gram, equal to 8.7 per cent of water. The calculated for i molecule of water is 13.9 per cent. A nitrogen determination of the dried material is given above (Analysis II.). Properties of 2-Amino-6-oxypyrimidine. It was found that the needles gave the stout prisms and the prisms could be ob- tained in needle form under slight changes in the crystalliza- tion. The crystals were not suitable for crystallographic Syntheses of A minooxypyrimidincs, 503 measurements, the faces being highly etched, especially the end faces of the prisms. The material melts, or rather de- composes, with violent effervescence, at about 276. This effervescing-point may vary several degrees, according to the rate of heating. The base is precipitated by phosphotungstic acid. It is not precipitated by potassium bismuth iodide in hydrochloric acid solution, but it gives a brick-red precipitate in solutions of the base in sulphuric acid. It gives the murexide reaction. Its sulphate and hydrochloride are readily soluble in water and the former crystallizes in flat prisms. The picrate is very difficultly soluble and the gold and platinum double salts are also not very soluble. When heated with 20 per cent sul- phuric acid for two hours, at 145 to 150, a portion of the ma- terial is converted into uracil. 2-Amino-6-oxypyrimidine Picrate. When picric acid is added to a hot solution of the base in water a mass of bright- yellow needles separate. If slowly cooled slender prisms are formed. On heating, the crystals become less transparent at about 1 80, showing evidence of darkening at about 255, and signs of melting at 270. Between this point and 280 they generally effervesce. They dissolve in about 1200 to 1300 parts of water at 25. A nitrogen determination gave : Calculated for C 4 H 6 ON3.C 8 H 2 (N02)30H. Found. N 24.70 24.72 The chloroplatinate separates in the form 'of small yellow needles, which form acicular crystals or spikes on slowly cooling the hot solutions. On heating, it decomposes above 200, becoming brown, but not melting below 286. A platinum determination gave : Calculated for (C 4 H60N3) 2 H 2 PtCl 6 . Found. Pt(=i94.8) 30.83 30.56 The gold chloride double salt, which seems to be more solu- ble than the platinum double salt, forms bright-yellow, stout prisms, which easily form a supersaturated solution. A gold determination gave : 504 Wheeler and Johnson. Calculated for C 4 H 6 ON8.HCl.AuCl3. Found. Au(=i95.7) 43-51 43-57 2-Acetamino-6-oxypyrimidine. One-half gram of the base required about 7.5 grams of boiling acetic anhydride to effect solution. On cooling, a colorless mass of crystals separated, so bulky that the test-tube could be inverted without loss of the contents. This material crystallizes in bunches of minute mother-of-pearl scales from alcohol, and it melts at 247 to a clear liquid. A nitrogen determination gave : Calculated for C 6 H 7 O2N 3 . Found. N 27.45 27.69 2-Amino-5-brom-6-oxypyrimidine. Three grams of the base were suspended in 20 cc. of glacial acetic acid, in which it is difficultly soluble, and 4.5 grams of bromine were dropped in. The action took place smoothly and an orange powder sepa- rated. It weighed 6.6 grams, while the calculated amount for a hydrobromide of 2-amino-5-brom-6-oxypyrimidine is 7.2 grams. The yield obtained was 83 per cent of the theoretical. This hydrobromic acid salt forms long, slender, needle-like prisms, when crystallized from water, and it melts at about 273 with strong effervescence. When the aqueous solution of this is treated with ammonia the brom-base separates as a compact, granular precipitate, insoluble in alcohol and ex- tremely difficultly soluble in water. It crystallizes from strong acetic acid in radiating masses of pointed plates, and it melts with decomposition at about the same temperature as the hydrobromide or the bromine-free base. It has acid properties and it dissolves in strong ammonia, but separates again unaltered on boiling off the ammonia. A nitrogen de- termination gave : / . Calculated for C 4 H 4 ON 3 Br. Found. N 22.10 22.13 NEW HAVEN, CONN., March 20, 1903. I Reprinted from the American Chemical Journal, Vol. XXIX. No. 5. May, 1903.1 Contributions from the Sheffield Laboratory of Yale University. Civ. ON CYTOSINE OR 2-OXY-6-AMINOPYRIMI- DINE FROM TRITICO-NUCLEIC ACID. BY HENRY L. WHEELER AND TREAT B. JOHNSON. It has been shown by Osborne and Harris 1 that uracil can be obtained from the nucleic acid of the wheat embryo. 2 As we wished to compare our synthetic uracil with some of the natural we asked Dr. Osborne for a sample. Not having the pure substance at hand, he very kindly supplied us with 1.35 grams of material obtained by evaporating the mother-liquors from the crystallization of his uracil. We found that the material, after crystallizing six times from water, gave a solution which, on slowly evaporating, de- posited flat, needle-like crystals, about a centimeter in length. This material was obviously not uracil, and, at Dr. Osborne' s suggestion, we then examined the material for cytosine. We found, in fact, that it consisted of a mixture of apparently about equal parts of uracil and cytosine. When a solution of 3 grams of picric acid, dissolved in 200 cc. of hot water, was added to the 1.35 grams of material dis- solved in 50 cc. of hot water and the mixture was allowed to stand for twelve hours, beautiful, long, silky, bright-yellow needles separated. The weight of the picrate obtained was 1.7 grams. The needles, which had every appearance of purity, melted with effervescence, as Kossel and Steudel 3 state in the case of thymus cytosine at about 270, turning brown a number of degrees below this temperature. 4 We took exactly i gram of this picrate for the preparation of the free base. The picrate was treated with an excess of sulphuric acid and 1 Ztschr. physiol. Chem., 36, 85 ; Annual Report for 1901 of the Connecticut Agri- cultural Experiment Station, page 408. Osborne and Campbell : J. Am. Chem. Soc., 22, 379. * Ztschr. physiol. Chem., 37, 378 (1903). * Owing to the ease with which this picrate gives off oxides of nitrogen, the analyt- ical results came high both for carbon and nitrogen, so that the results were with- out value. In this connection it may be stated that a jnitrogen determination gave 36.5 per cent nitrogen, while a precisely similar result was obtained in the case of the picrate from the synthetic base, namely 26.4 per cent nitrogen. The calculated for C4H 6 ON 8 .CcH a (NOa) 8 OH is 24.7 per cent nitrogen. 506 Wheeler and Johnson . the picric acid was removed by shaking with ether. The sulphuric acid was mostly removed by barium hydrate, and, on concentrating and adding ammonia, colorless, well-crys- tallized, slender, needle-like prisms of the free base separated. The amount of this material was 0.1641 gram. This, after washing with water, was dried by standing in the air, on paper, for twelve hours. It then lost 0.0181 gram on heating to constant weight at 110 to 120, equivalent to 11.30 per cent of water, while the calculated for C 4 H 5 ON 3 .H 2 O is 13.9 per cent water. This base melted with strong effervescence at about 323, precisely like the synthetical 2-oxy-6-arninopyrimidine, and when a portion was mixed with the latter substance the melt- ing- or effervescing-point was not lowered. After determining the solubility in water (see below) the base was then converted into the chloroplatinate. A platinum determination gave : 0.0830 gram substance gave 0.0253 gram platinum. Calculated for (C 4 H5ON 8 ) 2 H 2 PtCl6. Found. Pt(=i94.8) 30.83 30.48 The remaining material was given to Professor S. L. Penfield for a crystallographic examination and comparison with the corresponding platinum double salt prepared from 2-oxy-6- aminopyrimidine. Kossel and Steudel 1 state, in regard to the platinum chloride double salts prepared by them, that 11 Das Platindoppelsalz des Storcytosins zeigte namlich fast durchweg doppelbrechende Zwillingsformen, welche in der Weise aneinander gelagert waren,dass die Ausloschungsrich- tung in dem einen Krystall einen Winkel von 53 mit der Ausloschungsrichtung des anderen Krystalls bildete. Diese Winkel wurden beim Cytosin aus Herings-und Stb'rtestikeln gleich befunden, beim Cytosin aus Thymusnucleinsaure haben wir bisher solche Zwillingsformen uberhaupt tiicht beobachtet." 1 Ztschr. physiol. Chem., 37, 379 (1903)- Cytosine, Etc.^Jrom Tritico- Nucleic Acid. 507 Crystallography of 2~Oxy~6-aminopyrimidine Chloroplatinate {Synthetic Cytosine Chloroplatinate}. The crystals submitted for study were thin plates and for examination with the microscope they were embedded in oil, and over them a cover glass was placed. Fig. I. Fig. II. Fig. III. Figure I. represents the habit of one of the crystals which was unbroken and of almost ideal symmetry. It is a twin, presumably belonging to the monoclinic system, the large flat face b being the clinopinacoid (oio). The edges c, a, and e appear as sharp lines under the microscope, and represent the direction of faces at right angles to b\ It may be assumed that c is the base (ooi) and a the orthopinacoid (100), the in- clination of the axes, ft, being about 60. Some beveling form, 5, appears at the upper corners, which may be taken as the monoclinic pyramid (T 1 1 ) , its intersection with b making an angle of about 41 with the vertical axis (the trace of a), as indicated in the figure. The faces e at the top are ortho- domes, inclined about 75 to the vertical axis, and they have the symbol (Io2). The twinning plane, uniting the two in- dividuals, is the orthopinacoid a. The extinction, indicated in the figure by the arrows, is inclined about 2. 5 either side of the twinning plane. A second crystal, Fig. II., was like the one just described, except for the faces d at the top, which make an angle of about 91 with the vertical axis. The form d is evidently an orthodome, and has the symbol (103). A third crystal, Fig. III., showed two kinds of twinning. Crystals I. and II. are twinned about the orthopinacoid as in 508 Wheeler and Johnson . Fig. I., while II. and III. have the base c as the twinning plane. The extinction is indicated by the arrows. In crys- tals II. and III. the extinction directions make an angle of 55 with one another. Crystallography of Wheat Cytosine Chloroplatinate . The crystals submitted for study were much thinner than those prepared from synthetic cytosine, hence when mounted in oil for examination they were much more broken. Fig. IV. Fig. V. Fig. VI. Fig. IV. represents a fragment of a twin crystal, the part to the right of the twinning plane not being shown. The ar- rangement of the faces and the angles is like Fig. II., except /", at the top, which makes an angle of 106 with the vertical axis and corresponds to an orthodome (106). Fig. V. represents a part of another twin crystal, like Fig. IV., except that/ and c terminate the crystal instead of d and /. The extinction was 2. 5 from the vertical axis as in the case of the synthetic preparation. Among the broken fragments two kinds of twinning were observed, some with a as the twinning plane and others like Fig. VI., with the base c as the twinning plane. In the latter the twinning law is the same as that of crystals II. and III. of Fig. III. The extinction is nearly parallel to , and makes an angle with the twinning plane of 62. 5, or the two extinc- tion directions make an angle of 55 with one another. Aside from the extinction directions the optical properties of the crystals were not very decisive. When examined in convergent light an indistinct interference figure was seen, presumably the center of the cross of an obtuse bisectrix. Cytosien, Etc. , from Tritico- Nucleic Arid. 509 The identity of the synthetic cytosine and wheat cytosine is established beyond question by the crystallographic similarity of the two chloroplatinates. The measurements given were made under the microscope, and only approximate accuracy is claimed for them. The symbols of the domes e (102), d (103), and/ (106) were de- termined by plotting. Dr. P. A. Levene has very kindly sent us a pure sam- ple of his base from the spleen which has the properties and composition of Kossel and Steudel's cytosine. The amount of this at our disposal was about 0.12 gram. It formed thin, flat, colorless crystals or prismatic plates, and it melted or effervesced, side by side with the synthetic material at 323, and, when portions of these substances were mixed, the melting-point was not altered in the slightest. After de- termining the solubility we converted this material into the chloroplatinate. Professor Penfield then reported on the crys- tallography as follows : Crystallography of Spleen Cytosine Chloroplatinate. The crystals are like the ones previously described, having the same angles, though presenting some variation in habit. Fig. VII. is typical, showing a, b, c, and s forms, but with Fig. IX. 5 1 o Wheeler and Johnson . the reentrant angle at the top having a curved contour so that no definite symbol can be assigned. The extinction, as in the other preparations, is 2. 5 either side of the twinning plane, as indicated by the arrows in the figure. Fig. VIII. represents a penetration twin, drawn with camera lucida. The two opposite parts I. and I. extinguish simul- taneously, as also II. and II. Many crystals of the crop sub- mitted for examination were long, lath-shaped individuals, Fig. IX. Several having this habit were twinned about the*: face. The three chloroplatinates prepared from synthetic cytosine, wheat cytosine, and spleen cytosine are crystallographically identical and show no greater variation in habit than is gen- erally observed on crystals of any substance formed under varying conditions. The solubility of the three samples of cytosine in water at 25 was determined with the following results : loo parts water dissolve 0.83 part wheat cytosine. 100 parts water dissolve 0.78 part spleen cytosine. loo parts water dissolve 0.79 part synthetic cytosine. The high result in the case of wheat cytosine is probably due to the fact that it was not as pure as the others. Owing to lack of material the determination was made with the above-mentioned sample, which had been washed but not re- crystallized from water. We conclude, from these results, that the products obtained from the three different sources are identical, and that cyto- sine, is as Kossel and Steudel have predicted, an oxyamino- pyrimidine, viz., 2-oxy-6-aminopyrimidine. Its structure is to be represented by the following formula, or a tautomeric form : N=rC NH 2 OC CH .H 2 O. I II HN CH Hitherto cytosine has been found only in animal organs. The present results offer further evidence of the similarity of nucleic acids of animal and vegetable origin. Cytosine, Etc. , from Tritico- Nucleic Add. 511 We take pleasure in thanking Dr. Osborne and Dr. I,evene for the material placed at our disposal and Professor Penfield for the crystallographic descriptions. NEW HAVEN, CONN., March 25, 1903. L Reprinted from the American Chemical Journal, Vol. XXXI, No. 6. June, 1904.] Contributions from the She meld Laboratory of Yale University. CXVI. RESEARCHES ON PYRIMIDINE DERIVA- TIVES : 5-METHYLCYTOSINE. [FIFTH PAPER.] BY HENRY I*. WHEELER AND TREAT B. JOHNSON. In a previous paper we 1 showed that cytosine gives uracil on heating with acids. If uracil is to be reckoned as cytosine in the nucleic acids then 5-methyluracil or thymine possibly results by the cleavage of a corresponding base, namely, , 5-methylcytosine (formula I.) or tfre isomeric 2-amino-5- r methyl- 6-oxypyrimidine (III.) : / N=C NH, HN CO HN CO I 1,-N - II II OC CQH/ -~ OC CCH, H,N C CCH S I II I II II II HN CH HN CH N CH I. II. III. We therefore decided to prepare these new bases and to compare their properties with those of cytosine. In this paper we describe 5-methylcytosine and in a later paper by Dr. Johnson and Mr. S. H. Clapp, the compound represented by formula III. will be discussed. We prepared the compound represented by formula I. as follows : The ethyl bromide ad- dition-product of thiourea was treated with one molecular 1 This JOURNAL, 29, 494 (1903). 59 2 Wheeler and Johnson. proportion of alkali in aqueous solution and the sodium salt of ethyl formylpropionate added. On standing, then warm- ing for a few minutes, cooling, and acidifying with acetic acid, 2-ethylmercapto-5-tnethyl-6-oxypyrimidine was obtained. NH 2 C 2 H 5 OCO NH CO C 2 H 5 OH I I II C 2 H 5 SC + CCH 3 = C 2 H 5 SC CCH 3 + II II II II NH NaOCH N CH NaOH The 2-ethylmercapto-5-methyl-6-oxypyrimidine was con- verted into 2-ethylmercapto-5-methyl-6-chlorpyrimidine by means of phosphorus pentachloride : NH CO N=CC1 II II C 2 H 5 SC CCH 3 + PC1 5 = C 2 H 5 SC CCH 3 + POC1 3 . II li II I + HC1 N CH N CH When 2-ethylmercapto-5-methyl-6-chlorpyrimidine was heated with strong^a,lcgholic_ammonia it gave 2-ethylmercap- to-5-methyl-6-aminopyrimidine. ppl xr p TSTTT v^^i i> v^^J-N -C1 9 II II C 2 H 5 SC CCH 3 + 2NH 3 = C 2 H 6 SC CCH 8 + NH 4 C1. II II II II N CH N CH Finally the 2-ethylmercapto-5-methyl-6-aminopyrimidine was converted into the halogen hydride salt of 2-oxy-5-methyl-6- aminopyrimidine by boiling with either Jiydrochloric or hy- N=C NH 2 N=C NH 2 ,HX II II C 2 H 5 SC CCH 3 + H 2 O = OC CCH 8 + C 2 H 5 SH. II II I II N CH HN CH 5-Methylcytosine separates from water with a half molecule of water of crystallization while cytosine crystallizes with i molecule of water. 5-Methykyiosinc. 593 5-Methylcytosine is about five times as soluble in water as cytosine and ten or eleven times as soluble as thymine or uracil and it is readily decomposed into thymine by 20 per cent sulphuric acid at 150. It is probable that in the decom- position of the nucleic acids by the prolonged action of hot acids this base would, if present, be converted entirely into thymine. One of the most characteristic properties of 5-methylcyto- sine is its tendency to form the so-called abnormal or basic 2 : i salts with hydrochloric and hydrobromic acids. When am- monia in excess is added to a solution of cytosine in these acids, free cytosine usually separates. In the case of 5-methyl- cytosine in the halogen hydride acids the precipitate gener- ally consists of the salt (C 6 H 7 ON S ) 2 HX.H 2 O. Cytosine gives a i : 2 salt with concentrated hydrochloric acid ; 5-methylcytosine gives a i : i salt under the same con- ditions. The picrate of 5-methylcytosine, which forms yellow, long, slender, needle-like prisms, has the same solubility in water as cytosine picrate. 5-Methylcytosine is precipitated by phosphotungstic acid ; it therefore differs from the new base prepared by Kutscher 1 from thymus nucleic acid. An attempt was made to prepare 2-oxy-5,6-diaminopyrimi- dine by brominating 2-ethylmercapto-6-oxypyrimidine 2 (IV.), chlorinating in the 6-position by means of phosphorus penta- chloride, and then attempting to replace both of the halogens by the amino group in this compound (VI.) by means of al- coholic ammonia at a high temperature. It was found that the chlorine could be replaced by the amino group with ease, but that the bromine was far more firmly bound, the compound VII. being almost quantitatively obtained. This was con- verted into 5-bromcytosine (VIII.) and this compound then heated with aqueous ammonia, but here again the desired di- amino derivative was not obtained. These results recall the similar behavior of 4-methyl-6-amino-5-brompyrimidine de- 1 Ztschr. physiol. Chem., 38, 173 (1903). 2 Wheeler and Merriam : This JOURNAL, 29, 484 (1903). 594 Wheeler and Johnson. scribed by Gabriel and Colman. 1 This substance did not re- act with ammonia without complete decomposition. HN CO HN CO N=CC1 C 3 H 5 SC CH C 2 H 5 SC CBr - C a H 5 SC CBr. II H II II II II N CH N CH N CH IV. V. VI. N=C NH, N=C NH 2 N=C-NH a C 2 H 5 SC CBr -~- OC CBr OC C NH 2 . II II I II | II N CH HN CH HN CH VII. VIII. IX. The action of nitric acid was next tried on cytosine, where- upon it was found that nitration took place ; the compound formed was not a 5-nitro derivative, but rather the nitramide whose structure is, in all probability, shown by formula X. N=C NH a N=C NHNO, II II OC CH OC CH I II I II HN CH HN CH x. The formation of similar nitramide derivatives in the pyrimi- dine series has been observed by Gabriel and Colman. 8 Work on the preparation of oxydiaminopyrimidines will be continued here. These particular pyrimidine compounds are of special interest not only because Kutscher 8 believes that he has isolated such a compound, in the form of its picrate, from yeast, but also on account of the fact that if an oxydi- aminopyrimidine is found to crystallize or combine with 3 molecules of water it would then have the same percentage composition as Drechsel's so-called diaminoacetic acid* and perhaps the substances would prove to be identical. Will- statter 6 has shown that diaminoacetic acid, if capable of ex- 1 Ber. d. chem. Ges., 34, 1239 (1901). 2 Ibid., 34, 1240-1241 (1901). * Ztschr. physiol. Chem., 38, 176 (1903). * Beilstein : Handbuch I., 1194. 6 Ber. d. chem. Ges., 35, 1378 (1901). 5-Methylcytosinc. 595 istence, would not agree in properties with Drechsel's com- pound. In other words, Drechsel's compound might be a pyrimidine derivative as follows : CHCOOH = C 4 H 12 0,N 4 = C 4 H 6 ON 4 + 3 H 2 O. n = J EXPERIMENTAL PART. 2-Ethylmercapto-5-methyl-6-oxypyrimidine, HN CO I I C,H 5 SC CCH,. When a mixture of 150 grams of ethyl pro- II II HN CH pionate and 135 grams of ethyl formate, in somewhat over 2 volumes of dry ether, were slowly dropped upon 34 grams of sodium, about 125 grams of crude sodium ethylformylpro- pionate 1 were obtained. In using this salt and also sodium ethylformyl acetate to condense with the alkyl halide addi- tion-products of thiourea we 2 employed the substances in molecular proportions. It has since been found in both cases that only about one-half, or less, of the quantity of the salt taken reacts in the desired manner, and therefore that one- half the calculated quantity of pseudoethylthiourea hydrobro- mide gives about the same, if not a better, yield of pyrimidine as when the substances are used in molecular proportions. From 125 grams of the salt and 150 grams of the ethyl bro- mide addition-product of thiourea we obtained, in the manner already described, 39.5 grams of this mercaptopyrimidine. This is 28 per cent of the calculated. In another experiment 90 grams of the sodium salt with 87 grams of the thiourea addition-product gave 20.5 grams of the pyrimidine deriva- tive, or 26 per cent of the theoretical amount. 2-Ethylmercapto-5-methyl-6-oxypyritnidine crystallizes from hot water in the form of long, slender prisms. It is very sol- 1 Wislicenus : Ber. d. chem. Ges., 20, 2933 (1887). 2 This JOURNAL, 29, 487 ; Ibid., 29, 496 (1903). 596 Wheeler and Johnson. uble in alcohol and it melts at 158 to 159. A nitrogen de- termination gave : Calculated for C 7 H 10 ON 2 S. Found. N 16.47 16.78 2-Ethylmercapto-5-methyl-6-chlorpyrimidine, I I C 2 H S SC CCH 3 . Phosphorus pentachloride reacts less II II N CH smoothly with the above mercapto derivative than it does with 2-ethylmercapto-6-oxypyrimidine. When the mixture of the substances in molecular proportions is warmed on the steam- bath, hydrogen chloride is evolved, and, on cooling, the ma- terial solidifies to a deliquescent, crystalline cake. This is apparently a double compound of the chlorpyrimidine and phosphorus oxychloride. If an attempt is made to distil this directly in a vacuum, decomposition takes place and the ma- terial turns black. If, however, the cake is transferred to crushed ice it liquefies, and on shaking out with ether and drying, the resulting oil can then be distilled under dimin- ished pressure. In one experiment, 35 grams of the mercapto- oxy derivative and 43.5 grams of phosphorus pentachloride gave 24 grams of the distilled chloride, which is a yield of 61.8 per cent of the calculated. In another, 20 grams gave 14.5 grams of the chloride or 65 per cent of the calculated. 2-Kthylmercapto-5-methyl-6-chlorpyrimidine boils at 146 to 147 at 17 mm. pressure, and at 157 to 159 at 25 to 26 mm. pressure. It was thus obtained as a colorless, almost odorless, oil, which did not solidify in a freezing- mixture. 2-Ethylmercapto-6-chlorpyrimidine, prepared and purified in a similar manner, was found to boil at 135 at 24 mm. pressure. A nitrogen determination in the case of 2-ethylmercapto-5- methyl-6-chlorpyrimidine, boiling at 157 to 159, gave : Calculated for C 7 H 9 N 2 SC1. Found. N 14.85 15.00 5-Mcthylcytosinc. 597 2-Ethylmercapto-5-methyl-6-aminopyrimidinc, N=C NH, I I CjH 5 SC CCH S . . In the preparation of this amino deriva- II II N-CH tive, and also in the case of mercaptocytosine, the alcoholic ammonia used should be thoroughly saturated (in the cold), as otherwise an ethoxy derivative is formed instead of an amino compound. We found that 13 grams of the above chloride and about 75 cc. of alcoholic ammonia, when heated for three hours at 127, gave 10 grams of amino derivative, or 86.9 per cent of the calculated, and also that 10.1 grams of the chloride, when heated for two hours at 124 to 130, gave 6.6 grams of the amino base after crystallizing from 50 per cent alcohol, or 74 per cent of the theoretical amount. 2-Bthylmercapto-5-methyl-6-aminopyrimidine is very solu- ble in alcohol and quite difficultly soluble in water. It crys- tallizes from 50 per cent alcohol in the form of colorless, small, stout, six-sided tables and prisms, which melt at 96 to 97. It has a tendency to separate from its solutions as an oil, which finally crystallizes. A nitrogen determination gave : Calculated for C 7 H U N 8 S. Found. N 24.86 25.20 2-Ethylmercapto-6-ethoxypyrimidine, C 2 H 5 SC CH . 2-Kthylmercapto-6-chlorpyrimidine was II II N CH dissolved in alcohol and ammonia gas was passed in, the whole being heated on the steam-bath. After two hours, 7.5 grams of ammonium chloride had separated, the calculated quantity being 7.6 grams. On evaporating the alcoholic solution an oil was obtained which distilled at 137 to 138 at 18 mm. 598 Wheeler and Johnson. pressure. A nitrogen determination agreed with the calcula- ted for an ethoxy derivative : Calculated for C 8 H 12 ON 2 S. Found. N 15.21 15.38 That this ethoxy derivative is not an intermediate product in the preparation of the amino compound was shown when on heating the oil with alcoholic ammonia for four or five hours at 150 to 175 it still remained unaltered. This oil dissolves in dilute hydrochloric acid and is precipi- tated unaltered by ammonia. On boiling for several hours with strong acid it is converted quantitatively into uracil, melting at 338. A nitrogen determination gave 25.4 per cent nitrogen, while the calculated is 25.0. Cytosine Dihydro chloride. When 2-ethylmercapto-6-amino- pyrimidine is warmed on the water- bath for three or four hours with concentrated hydrochloric acid, then cooled, beautiful, stout, brittle, colorless, flat prisms separate. The following analysis shows that this material is an acid salt : Calculated for C 4 H 5 ON 3 .2HC1. Found. Cl 38.58 39.09 This salt is analogous to the acid sulphate, C 4 H 5 ON 3 . H 2 SO 4 , described by Kossel and Steudel. 1 5-Methylcytosine Monohydro chloride (Anhydrous}. When 2-ethylmercapto-5-methyl-6-aminopyrimidine was warmed with hydrochloric acid, in a similar manner to the above, mercaptan was evolved and the crystals obtained were color- less tables and flat prisms, which, when dried over potassium hydroxide, showed signs of melting at about 270, and melted with effervescence at 288. The material was readily soluble in water. A chlorine determination showed that this was an anhydrous i : i salt. Calculated for C 6 H 7 ON 3 .HC1. Found. Cl 21.98 21.99 When 6.6 grains of the crude mercapto base were warmed 1 Ztschr. physiol. Chem., 38, 53 (1903). 5-Methylcytosine. 599 on the steam-bath with hydrochloric acid until mercaptan ceased being evolved, the solution evaporated to dryness, and the residue dissolved in a little water, 0.7 gram thymine was obtained, the formation of this being due to the presence of an ethoxy derivative. When this i : i hydrochloride was dis- solved in water and to the warm solution an excess of ammo- nia was added, microscopic colorless needles slowly separated. These were not very soluble in hot water and difficultly in cold. The material was recrystallized from water. The curious result was then observed that the material still con- tained chlorine and that we had to deal with a basic salt or, more probably, a mixture of free base and a hydrous basic salt. The analytical results agreed fairly with the calculated for (C 6 H 7 ON 8 ) 6 2HC1. 3 H 3 0. Calculated. Found. Cl 9.4 9.09 H a O 7.1 7.53 The water determination was made by heating the material to constant weight at 150. When the i : i hydrochloride is boiled with water a basic salt also is obtained. N=C NH 2 5-Methylcytosine, OC CCH. . In order to obtain the I II HN CH free base the above material was dissolved in water, the chlo- rine was removed by means of silver sulphate, the excess of silver was precipitated with hydrogen sulphide and the sul- phuric acid by barium hydrate, the excess of the latter by carbon dioxide. The clear, colorless solution was then con- centrated to a small volume, whereupon, on cooling, beauti- ful, flat, colorless, prismatic, brittle crystals separated that formed a sandy powder. When heated, the crystals gave off water above 100 and showed signs of melting at 240, melt- ing with effervescence at 2^0 ; this is 50 lower than the melting-point of cytosine. 100 parts of water dissolved 4.5 parts of the base at 25. 6oo Wheeler and Johnson. 2.387 gram substance lost 0.1544 gram on heating to con- stant weight at 135 to 145' (I.). Calculated for Found. (C 5 H 7 ON 8 )2.H 2 0. I. II. . H 2 O 6.71 6.46 6.30 Calculated for C 6 H 7 ON 3 . Found. N 33-60 33.44 Silver nitrate, added to an aqueous solution of this base, gives a gelatinous white precipitate soluble in an excess of ammonia and in nitric acid. The ammonia solution can be boiled without reduction. Mercuric chloride gives a white precipitate in dilute solu- tions of the base ; this dissolves on heating and separates again, on cooling, possibly in crystalline form. Phosphotungstic acid gives a white precipitate insoluble in dilute hydrochloric or sulphuric acids. 5-Methylcytosine Monohydrochloride {Hydrous} . An attempt was made to prepare a dihydrochloride of the methyl base by dissolving the above i : i salt in water and then saturating the solution with hydrogen chloride. The solution was then allowed to evaporate spontaneously in a desiccator over sul- phuric acid, whereupon small, stout, transparent prisms or rhombohedrons separated. The analysis agreed with the cal- culated for the formula C 5 H 7 ON 3 .HC1.2H 2 O. Calculated. Found. H 2 O 18.2 18.0 Cl 21.98 22. On keeping these crystals in a closed tube they became opaque. 5-Methylcytosine Basic Hydrochloride. Bxperiments were made to determine whether or not 5-methylcytosine gives a 2 : i and a 3 : i salt by mixing solutions of the base and the above hydrochloride in the required proportions. It was found that when a strong solution of the base (0.12 gram) was mixed with a warm solution of the hydrochloride (0.153 gram) in a little water a precipitate was formed immediately. This dissolved on boiling, and on cooling, little balls of mi- 5-Methylcytosinc. 601 nute needles or prisms separated. When dried over calcium chloride the salt was found to have the composition repre- sented by Analysis I. When two molecular proportions of the base to one of the hydrous hydrochloride were used the same basic salt was obtained. For example : 0.15 gram of the hydrochloride was dissolved in 2.5 cc. of water and 2.2 grams of the base were dissolved in 5 cc. of water. On mix- ing these solutions at ordinary temperature a gelatinous mass separated, so bulky that the test-tube could be inverted with- out loss of the contents. On warming, the material dissolved and on cooling small prisms separated. Analysis II.: Calculated for Found. (C 6 H T ON 8 ) 2 HC1.H 2 0. I. II. H a O 5-9 6.2 5.9 Cl n.6 11.3 This hydrous basic salt is analogous to the hydrous basic cytosine sulphate frequently obtained by Levene. 1 This salt has the formula (C,H 5 ON 3 ) 4 H 2 SO 4 .2H 2 O. The corresponding anhydrous basic sulphate has been de- scribed by Kossel and Steudel. 2 A neutral sulphate of cytosine has also been described by Levene. 3 5-Methylcytosine Basic Hydrobromide. Ten grams of 2-ethyl- mercapto-5-methyl-6-aminopyrimidine were boiled with i5cc. of strong hydrobromic acid for four hours. The acid was then evaporated, whereupon beautiful, large, stout prisms sepa- rated. These crystals were very soluble in water. Upon adding strong ammonia to the warm aqueous solution an im- mediate precipitate in the form of a fine, crystalline powder was obtained. The yield was 7 grams. This precipitate was difficultly soluble in water. When heated it sintered at about 295 and then decomposed with effervescence at 3i9-32O. This material was boiled for some time with strong aqueous ammonia ; it nevertheless still contained bromine, and on analysis the following results were obtained : The air- dried salt lost water very slowly below 150, dried at i75-i85. 1 Ztschr. physiol. Chem., 39, 7 ; 39, 135, 481 (1903). /**., 38,53(1903). ., 37, 405(1903). 602 Wheeler and Johnson . 0.4838 gram lost 0.0290 gram water or 5.9 per cent. The calculated for (C 5 H 7 ON 3 ) 2 HBr.H 2 O is 5.1 per cent. The dried residue then gave the following results : Calculated for (C 6 H 7 ON 3 )2HBr. Found. C 36.25 36.20 H 4-53 4-85 N 25.37 25.68 This basic salt was then treated with silver sulphate as de- scribed above in the case of the basic hydrochloride. The free base obtained agreed in every particular with the prep- aration from the basic hydrochloride. A water determination is given above (II.). When 0.7 gram of this basic salt was heated at I5o-i6o for three hours with 20 per cent sulphuric acid, 0.20 gram of thymine, melting at 325-326, was obtained. Forty per cent of the material was therefore hydrolyzed. Acetyl-5-methylcytosine, prepared by dissolving the base in acetic anhydride, forms needles from water. The material did not melt or effervesce below 290 although it showed signs of decomposition at about 255. 5-Methylcyto$ine Picrate. When 0.238 gram of the base in a little water was mixed with 40 cc. of a saturated aqueous solution of picric acid, an immediate precipitate was formed. This dissolved on boiling and adding more water; on cooling, long, slender, bright yellow, needle-like prisms separated. On heating, this material showed signs of change at about 250- 255 and from then on got darker until at 286 it decomposed with effervescence. A certain mixture of this material with pure cytosine picrate began to change color, on heating, at 255 and melted with effervescence at 273. It is noteworthy that this picrate has the same solubility in water as cytosine picrate. 1 One hundred parts of water dissolve 0.07 part of the salt. The chloroplatinate is quite soluble in water. The aqueous solution, on standing in a desiccator, deposits small orange- colored rosettes. 1 Wheeler and Johnson : Loc. cit. 5-Methylcytosine. 603 2-Ethylmercapto-5-brom-6-oxypyrimidine y HN CO CjH 6 SC CBr. When 2-ethylmercapto-6-oxypyrimidine ' II II N CH (5.55 grams) was dissolved in three parts of glacial acetic acid and one molecular proportion of bromine was added (6 grams) , a mass of needles separated ; these were washed with water and dried ; the weight was 7.45 grams or 89 per cent of the calcu- lated. In another experiment 16 grams of the mercapto com- pound gave 22.1 grams of the brom-derivative or 91.7 per cent of the theoretical amount. This material was difficultly sol- uble in water, but more readily soluble in alcohol, from which solution beautiful, colorless, needle-like prisms were obtained. These, on heating, showed signs of change at about 184 and melted completely without effervescence at 189. A nitrogen determination gave : Calculated for C6H 7 ON 2 SBr. Found. N 11.9 12. i An excess of bromine does not effect further substitution in the cold. When the substance is boiled with hydrochloric acid, 5-bromuracil is obtained. 1 Dr. Merriam found that when this latter substance was heated with aqueous ammonia to 1 80, 5-aminouracil resulted, the compound being identified by means of its picrate. 8 The action of alcoholic ammonia on the brommercapto compound at temperatures between 140 and 200 did not lead to a smooth result. 2-Ethylmcrcapto-5-brom-6-chlorpyrimidine, N^CCl I I C.H 5 SC CBr. 2-Ethylmercapto-5-brom-6-oxy p y r i m i di n e II II N CH (21 grams) and phosphorus pentachloride (18.6 grams) were mixed and warmed on the steam-bath. After standing over 1 Wheeler and Merriam : Loc. cit. 2 Behrend and Grunwald : Ann. Chem. (I,iebig), 309, 258 (1899). 604 Wheeler and Johnson. night the phosphorus oxychloride was removed by distilling under diminished pressure and the remaining oil poured off from a small amount of resin ; this was washed with a little ether. The weight of crude oil then obtained, on evaporating the ether, was 20.5 grams or 92 per cent of the calculated. On standing, this oil solidified in the form of soft, talc-like, colorless plates which, when pressed on paper, melted at about 27. A nitrogen determination then gave : Calculated for C,H 6 N 2 SClBr. Found. N 1 1 .04 ii .06 2-Elhylmercapto-5-brom-6-aminopyrimidtne> N=C NH, C 2 H 6 SC CBr . The above chlorobromide (19 grams) II II N CH was heated with alcoholic ammonia (40 cc. cold saturated solution) to 1 60 for four hours. On cooling, a crystalline mass separated with little or no color. Water was added to the contents of the tube ; the material that separated, on dry- ing, weighed 15.7 grams or 89.7 per cent of the calculated. The substance was readily soluble in hot alcohol and diffi- cultly in cold, and very difficultly soluble in hot water. On crystallizing from alcohol it formed beautiful, colorless prisms which melted sharply at 123 to 124. The material con- tained sulphur and bromine, and a nitrogen determination gave the following result : Calculated for CH 8 NSBr. Found. N 17.9 17.8 N=C NH 2 5-Bromcytosine, OC CBr . Twelve grams of the I II HN CH above mercapto compound were boiled with 50 cc. of con- centrated hydrochloric acid for four or five hours. The acid was then evaporated and the residue crystallized from water, 5-Meihylcytosinc. 605 whereupon minute prisms of the hydrochloric acid salt were obtained. To the hot solution of this material strong ammo- nia was added in excess, and then, on cooling, bunches of needle-like prisms of the free base separated. The yield was 7.5 grams, or 77.3 per cent of the calculated, more being ob- tained from the mother-liquor. When the pure base, crys- tallized from water, was heated it gave no definite melting- point but decomposed rapidly above 235. A nitrogen deter- mination gave : Calculated for C 4 H 4 ON 8 Br. Found. N 22.10 22.11 When this material was heated at 155 to 190 with con- centrated aqueous ammonia for four hours, the products were a black resin and a brick-red amorphous powder, the material having undergone decomposition. N C NH NO 2 Nitrocytosine, OC CH . Two grams of anhy- I II HN CH drous cytosine (synthetic), 4. cc. of concentrated nitric acid, and 4 cc. of concentrated sulphuric acid were mixed and warmed for a few minutes at 85. The mixture was then poured upon crushed ice and neutralized (slight excess of ammonia added). The crystalline material that separated weighed 1.8 grams, or 64 per cent of the calculated. In an- other experiment 2 grams of cytosine and 6 cc. of each acid gave 1.7 grams of the crude nitro compound. This nitro compound is very difficultly soluble in water, from which it separates, in the form of colorless, minute needles. These are readily soluble in strong ammonia. When heated, the ma- terial begins to turn brown at about 280, decomposes rapidly above 300, but does not melt or effervesce at 350. A nitro- gen determination gave the following result : Calculated for C4H 4 O 3 N 4 . Found. N 35.89 35.80 When an attempt was made to reduce this material with 606 5-Methylcytosine. tin and hydrochloric acid the only crystalline material ob- tained was ammonium chloride. According to Behrend and Griinwald 1 5-nitrouracil gives 80 per cent of the calculated of aminouracil, when reduced in ammoniacal solution by means of aluminium amalgam. An attempt to reduce our nitrocyto- sine in a similar manner gave nothing but amorphous yellow and blue-black decomposition-products. For this reason the above nitramide structure is assigned to the compound. NEW HAVBN, CONN., Feb. i, 1904. i Loc. cit. [Reprinted from the American Chemical Journal, Vol. XXXII, No. 2. August, 1904.] Contributions from the Sheffield Laboratory of Yale University. CXV. RESEARCHES ON PYRIMIDINES. SYNTHE- SIS OF 2-AMINO-5-METHYIv-6-OXYPYRIMI- DINE. [SIXTH PAPER.] BY TRKAT B. JOHNSON AND SAMUEL H. CLAPP. In a previous paper from this laboratory, Wheeler and Syn thesis of 2- A mino-^ -methyl- 6-oxypyrimidine. 131 Johnson 1 described the two monoaminooxypyrimidines which can give uracil on hydrolysis, viz., 2-aminooxypyrimidine (Formula I.), and 2-oxy-6-aminopyrimidine or cytosine (For- mula II.): NH CO Nz=zC.NH 2 II II H 2 NC CH OC CH II II I II N CH NH CH I. II. In a similar manner the two monoaminomethylpyrimidines which could give thymine on hydrolysis are, viz., 2-oxy-6- amino-5-methylpyrimidine (Formula III.) and 2-amino-6- oxy-5-methylpyrimidine (Formula IV.) : NH CO H 2 NC C.CH, II II NH-CH N CH III. IV. Wheeler and Johnson 2 in a recent paper have described 2-oxy-6-amino-5-methylpyrimidine (Formula III.) and have shown that it is more or less readily converted into thymiue on hydrolysis. We will describe in this paper the second monoamino- methylpyrimidine (Formula IV.), which theoretically could give thymine on hydrolysis, viz., 2-amino-5-methyl-6-oxy- pyrimidine. We have synthesized this aminopyrimidine by condensing guanidine with the sodium salt of formylethyl- propionate. We have not only been able to obtain the pyrimidine base which we desired, but have also succeeded in isolating intermediate products in the reaction. These have given us a more thorough insight into the mechanism of these pyrimidine condensations. We find, namely, that in this condensation there are three steps in the reaction first a condensation of guanidine with 1 This JOURNAL, 29,492 (1903). 2 Ibid., 3 1, 592 (1904). 132 Johnson and Clapp. the formyl group of the sodium formylethylpropionate giving an acrylic ester (Formula V.), then a saponification of the acrylic ester to the free acid (Formula VI.), and finally a condensation to the pyrimidine with loss of a molecule of water : NH 2 COOC 2 H 5 H 2 N.C + C.CH 3 = II II NH NaOCH NH 2 COOC 2 H 5 NH, COOH NH CO II II II H,NC CH 3 H 2 NC CH 3 ~ H 2 NC CH S N CH N CH N CH V. Under the conditions by which we worked we did not ob- serve the formation of the intermediate guanidineacrylic es- ter, but have evidence of its existence because of the fact that we have been able to isolate the intermediate guanidine- methylacrylic acid. Miiller 1 has described two substituted acrylic acids that are analogous to our intermediate acid. He found that phenylurea and methylurea readily condensed with oxalethylacetate, giving a phenyluracilcarboxylic ester and methyluracilcarboxylic ester. By dissolving these two esters in alkali and treating with strong hydrochloric acid they were decomposed into uramidoacrylic acids (Formulas VII. and VIII.) : NH 2 COOH NH 2 COOH II II OC CH OC CH I II I II C 6 H 5 N CH CH 3 N CH VII. VIII. Behrend, 2 in his synthesis of methyluracil from acetoacetic ester and urea, was able to isolate an intermediate uramido- crotonic ester, but was unable to isolate the free acid from its 1 J. prakt. Chem., 56, 475. a Ann. Chem. (lyiebig), 329, 8. Synthesis of 2-Amino-5-methyl-6-oxypyrimidinc. 133 sodium or potassium salt on account of the ease with which it condensed to the pyrimidine. We, furthermore, have made the interesting observation that our intermediate a-methyl-y#-guanidineacrylic acid is capable of existing in two isomeric modifications. One acid melts at 331 to 332 C. with violent effervescence ; the other at 318 to 320 C. to a clear oil. Both acids can be converted by treatment with alkali into the 2-amido-5-methyl-6-oxy- pyrimidine. It appears to us that the explanation for this interesting case of isomerism is that we are dealing with an ethylene linking, and have another case of cis and trans isomerism : CH 3 C COOH CH 8 C COOH II II H C N=C(NH 1 ) J (H 2 N) S C=N C H IX. X. Cis. Trans. Wislicenus 1 was able to isolate three isomeric modifications of formylphenylethylacetate. He represented their structure .as follows : C 6 H 5 .CH.COOC 2 H 5 C 6 H 5 .C.COOC 2 H 5 C 6 H 5 C COOC 2 H 5 CHO H-C OH HO C H XI. XII. XIII. He furthermore showed that the enol form was capable of forming isomeric salts, which gave isomeric acyl modifica- tions when treated with acyl chlorides. In a later paper Wislicenus and Bindemann 2 showed that analogous acyl modifications could be obtained from the sodium salt of formylethylacetate. Finally, Wislicenus 3 extended his investigations to the sodium salt of formylethyl- propionate, which we used in our condensation, and observed that this also gave stereochemical isomers when treated with -acyl chlorides. The close analogy between Wislicenus' iso- 1 Ann. Chem. (I^iebig), 313, 33. s Ibid., 316, 18. 3 Ibid., 316, 333- 134 Johnson and Clapp. mers and our modifications is shown by the following formu- las : CH, C COOC 2 H 8 CH 3 C COOC 2 H 5 II II H C O (OCR) (RCO)-O C H i i CH 3 C COOH CH S C COOH II II H C N=C(NH 8 ) Z (H 2 N) 2 C=N C H It seems probable to the writer that Pechmann's 1 isomeric derivatives which he obtained by treating the sodium salt of formylethylacetate with aniline and paratoluidine are to be explained in a similar manner : H C COOC 2 H 5 H C COOC 2 H 5 II it H C NHR RHN C H XIV. XV. Erlenmeyer 2 has shown that ethyl formate can be condensed with hippuric ethyl ester by means of sodium ethylate to the sodium salt of oxymethylenehippuric ester. We have pre- pared this salt and find that it condenses very smoothly with pseudoethylthiourea, giving 2-ethylmercapto-5-benzoylamino- 6-oxypyrimidine. We, furthermore, have condensed ethyl formate with phthal- imidoethylacetateto the sodium salt of formylphthalimidoethyl- acetate. We find that this salt condenses with pseudoethyl- thiourea with the formation of 2-ethylmercapto-5-phthalimido- 6-oxypyrimidme, and an intermediate acrylic acid. The two above pyrimidines show a most striking behavior towards alkali. 2-Kthylmercapto-5-benzoylamino-6-oxypyrim- idine dissolves in alkali and is precipitated unaltered by acids, while, on the other hand, the analogous 2-ethylmercapto-5- phthalimido-6-oxypyrimidine is converted under the same con- ditions into the corresponding pseudoureaphthalimidoacrylic acid. 3 1 Ber. d. chem. Ges., 35, 1052. Ibid., 36, 3769- 8 Since the above was written we have found that 2-ethylmercapto-5-benzoyl- atnino-6-oxypyrimidine is converted by alkali into the sodium salt of the correspond- ing pseudoureabenzoylaminoacrylic acid. We have not succeeded in isolating the free acid. Synthesis of 2-Amino-5-methyl-6~oxypyrimidinc. 135 NH CO | I /COv NaOH C 2 H $ S.C CN< >C 6 H< II || ^CO/ N CH NH 2 COOH I I /COv C,H 5 SC CN< >C 6 H 4 . II II ^CO/ N CH EXPERIMENTAL PART. NH CO I I 2-Amino-5-mcthyl-6~oxypyrimidinc, H 2 N C C CH S . II II N CH Seventy-two grams of guanidine carbonate were dissolved in the least possible quantity of cold water and mixed with 54 grams of barium hydroxide dissolved in 400 cc. of water. To this solution, filtered clear from the precipitated barium car- bonate, 283 grams of crude sodium formylethylpropionate were added. After standing at ordinary temperature for about eight hours the clear solution was divided into two equal parts. One part was heated on the steam-bath for about half an hour and then neutralized with dilute sulphuric acid. A brown precipitate separated immediately and was filtered off and saved (see tran s acid). The clear solution that remained was evaporated to dryness, then redissolved in a little warm water, and filtered from a small amount of insolu- ble material that was present. The pyrimidine base was then precipitated with phosphotungstic acid, the tungstic acid pre- cipitate decomposed with barium hydroxide, and the excess of barium hydroxide removed in the usual way with carbon dioxide. On concentrating the solution that remained, and cooling, the base separated in the form of stout prisms and melted with effervescence at 320 to 321. The second part of the original solution was allowed to stand at ordinary temperature for two weeks. It was then neutralized with dilute sulphuric acid as before. An insolu- 136 Johnson and Clapp. ble brown precipitate was obtained as in the first part. After filtering off this insoluble precipitate we precipitated the pyrimidine base with mercuric chloride instead of phospho- tungstic acid. The mercury was removed from the precipi- tate with hydrogen sulphide and then the base allowed to crystallize from the concentrated solution. The yield of pure base by the two different methods of treatment was about equal (4 grams), showing that long standing does not increase to any extent the yield. The same observation was made by Wheeler and Johnson 1 in their preparation of isocytosine or 2-amino-6-oxypyrimidine. 2-Amido-5-methyl-6-oxypyrimidine crystallized from hot water in two distinct crystalline forms, stout prisms, and long, slender, needle-like prisms. It melts at 320 to 321 with vio- lent effervescence. It does not contain water of crystalliza- tion. One hundred parts of water dissolve 0.43 part base at 25 C. A nitrogen determination gave : Calculated for Found. C 5 H 7 ON 3 . I. II. N 33.60 33.68 33.91 The base was not hydrolyzed to thymine after heating in a sealed tube with 25 per cent sulphuric acid for five hours. In a second condensation we modified our experiment in the following manner : After adding the sodium formyl- ethylpropionate to the guanidine solution, we added a molec- ular proportion of sodium hydroxide (29 grams). The alka- line solution was then allowed to stand at a temperature of 40 to 50 for about ten hours, and then heated on the steam - bath for three hours. The solution was then divided into two equal parts and one part neutralized with acetic acid, the other with dilute sulphuric acid. An immediate precipitate of the trans acid resulted in both cases. The weight of in- soluble acid obtained here was 12.2 grams. The two filtrates were combined and precipitated with an excess of mercuric chloride. The mercury was removed in the usual way with hydrogen sulphide and the filtrate con- centrated. On cooling the concentrated solution we obtained 1 Loc, cit. Synthesis of 2-Amino-5-methyl-6-oxypyrimidine. 137 a mass of needle-shaped prisms mixed with stout prisms. Some of these needle- formed prisms melted at 319 to 320 to a clear oil, followed by a slow effervescence. That this body was not the same product as we obtained in our first conden- sation was shown by the fact that when it was mixed with the free pyrimidine the melting-point was lowered to 308- 310 (see cis acid). The yield of this crystalline product was 18.2 grams. Behavior with Alkali. The base dissolves very readily in the cold in both dilute (2 per cent) and strong (9 per cent) solutions of sodium hydroxide, and is precipitated unaltered when the alkaline solution is neutralized with carbon dioxide or dilute sulphuric acid. It separates in the form of needle- formed prisms and melts at 320 to 321 with violent efferves- cence. In appearance this crystalline form of the base re- sembles very much that of the cis acid. That the ring is not opened by the treatment with alkali is shown by the fact that when this body was mixed with the cis acid the melting-point was lowered to 307. Furthermore, a nitrogen determination gave: Calculated for C 5 H 7 ON 3 . Found. N 33.60 33.90 The Hydro chloride. Some of the base was dissolved in con- centrated hydrochloric acid and the solution concentrated in a vacuum. The hydrochloride separated in stout prisms. A chlorine determination agreed with the calculated for mono- hydrochloride : Calculated for C 6 H 7 ON 3 .HC1. Found. Cl 21.98 21.73 The Picrate. The picrate separated immediately from water in yellow, microscopic crystals. They were practically insoluble in boiling alcohol and water. After prolonged boil- ing with water the insoluble picrate was filtered off and ana- lyzed for nitrogen : Calculated for C 5 H 7 ON3.C6H 3 O 7 N3. Found. N 23.73 23.92 138 Johnson and Clapp. The Sulphate. The sulphate crystallized from water in the form of flattened prisms or plates. A sulphur determination agreed with the calculated for an acid salt. It is interesting to note that Jaeger 1 obtained analogous acid sulphates from the isomeric 2-amido-4-methyl-6-oxypyrimidine, and from 2-atnido-4,5-dimethyl-6-oxypyrimidine : Calculated for CsH^ONs.HaSO^ Found. S 14.34 14-62 Platinum Chloride Double Salts. Two characteristic plati- num chloride double salts were obtained. The first was pre- pared by adding hydrogen platinic chloride to a cold, dilute hydrochloric acid solution of the base. It analyzed for a salt with 4 molecules of water of crystallization : Calculated for Found. (C 6 H 7 ON 3 .HCl)2PtCl4.4HsO. I. II. Pt 26.56 26.51 26.6l When hydrogen platinic chloride was added to a hot hydro- chloric acid solution of the base and then the solution allowed to cool slowly, the platinum salt separated in thick tables and melted at 254 to 255. A water determination and an analy- sis for platinum agreed with the calculated for a double plat- inum salt containing two molecules of water of crystallization : 0.1995 gram salt lost 0.0106 gram H 2 O at 120 to 130 C. Calculated for (C 6 H7ON3.HC1) 2 PtCl4.2H 2 O. Found. Pt 27.99 27.76 H,O 5.17 5.30 Trans- a- Methyl- fi-guanidineacrylic A cid, CH 8 C COOH || . This acid was always obtained (H 2 N),C=N C H in the condensation described above, when we neutralized the alkaline solution, preliminary to precipitating the pyrimidine base with mercuric chloride. It separated as an amorphous brown precipitate. One of the most characteristic physical properties that distinguished this acid from its cis modification and the pyrimidine was its insolubility in water. One hun- 1 Ann. Chem. (Liebig), a6a, 371. Synthesis of 2-Amino-5-methyl->-oxypyrimidine. 139 dred parts of water dissolve 0.08 part acid at 25 C. It crys- tallized from boiling water in poorly developed prisms, and melted according to the rate of heating from 329 to 332 with violent effervescence. It possesses both acid and basic prop- erties. A nitrogen determination gave : Calculated for C 5 H 9 2 N 3 . Found. N 29.37 29.32 In our second condensation the trans acid came down less pure and was mixed with a considerable amount of stout prisms. These were separated by boiling with water. After filtering and cooling, the prisms separated again and melted at 320 to 321 with effervescence. That this was the pyrimi- dine base was shown by the fact that when mixed with the pure pyrimidine the melting-point was not lowered. The trans acid was not changed to the cis modification or the pyrimidine after boiling in water for six hours. A nitrogen determination in the above pyrimidine, melting at 320 to 321, gave : Calculated for C 6 H 7 ON 3 . Found. N 33.60 33.68 Behavior with 9 Per Cent Sodium Hydroxide. When the trans acid was dissolved either in the cold or warm sodium hydroxide solution, and the solution neutralized with carbon dioxide it separated in the form of stout prisms and melted at 320 to 321 with violent effervescence. That this was the pyrimidine base was confirmed by a nitrogen determination : Calculated for C 5 H 7 ON 8 . C5H 9 O 2 N 8 . Found. N 33.60 29.37 33-52 The formation of the pyrimidine from the trans acid was furthermore confirmed by the formation and analysis of its platinum chloride salt. It separated in the form of stout prisms and melted at 254 to 255. When mixed with the double salt previously described as melting at 254 to 255 the melting-point was not lowered. A platinum determination gave : 140 Johnson and Clapp. Calculated for (C5H 7 ON3.HCl) a PtCl4.2H 2 O. Found. Pt 27.99 27.93 Behavior with 2 Per Cent Sodium Hydroxide. Some of the trans acid melting at 331 was dissolved in the cold, dilute sodium hydroxide solution. On neutralizing with dilute sul- phuric acid it separated again as a brown, poorly crystallized precipitate. It was extremely insoluble in water and melted at 330 to 332 with violent effervescence. The trans acid was precipitated again unaltered after this treatment with di- lute alkali. When mixed with the pure analyzed acid the melting-point was not lowered. This experiment is of inter- est because it enabled us to determine the cis and trans forms of our acid. The isomeric modification could not be precipi- tated unaltered from an alkaline solution. Cis- a- Methyl- ft-guanidineacrylic Acid, CH 8 C COOH || . This acid was obtained in our sec- H C N=C(NH a ) 2 ond condensation when we precipitated with mercuric chlo- ride to obtain the pyrimidine base. After removing the mer- cury from the precipitate with hydrogen sulphide and concen- trating the solution it separated with the pyrimidine base in slender, needle-like prisms. It was obtained pure by recrys- tallizing from hot water. It was approximately about twice as soluble in water as the pyrimidine. One hundred parts of water dissolve 0.84 part acid at 25. The most noticeable physical property which this acid possesses is its behavior on melting. It melts at 319 to 320 to a clear oil, followed by a slight effervescence and slow decomposition. A nitrogen de- termination gave : Calculated for Found. C 6 H 9 2 N3. I. II. N 29.37 29.05 29.6 In order to be certain that we were dealing here with a sec- ond modification of the guanidineacrylic acid, and not with a modification of our pyrimidine containing water of crystalliza- tion, some of the acid was heated for one hour at 115 to 120, and again at 140 for one hour. The body lost no weight by Synthesis of 2-Amino-^-methyl-6-oxypyrimidine. 141 this treatment, thus confirming our assumption that we had in hand an isomeric acid. Behavior with 9 Per Cent Sodium hydroxide. The cis acid dissolved immediately in the sodium hydroxide solution. When the alkaline solution was neutralized with carbon di- oxide or with dilute hydrochloric acid an asbestos-like pre- cipitate of needle-like prisms were obtained. They melted at 319 to 321 with effervescence. When mixed with the pyrimi- dine (m. p. 320) the melting-point was not lowered. When mixed with the cis acid (m. p. 319 to 320) the melting-point was lowered to from 302 to 303. That the cis acid had been converted into the pyriinidine was confirmed by a nitrogen de- termination : Calculated for C 5 H 7 ON 8 . Found. N 33.60 33.23 Behavior with 2 Per Cent Sodium Hydroxide. The cis acid that was used in this experiment melted sharply at 320 to a clear oil and analyzed for 29.05 per cent of nitrogen, calcula- ted 29.37 P er cent. It dissolved immediately in the cold, di- lute sodium hydroxide solution. When the alkaline solution was neutralized with carbon dioxide or sulphuric acid the base was precipitated in needle-like prisms melting at 320 with effervescence. A nitrogen determination agreed with the calculated for the pyrimidine : Calculated for C 5 H 7 ON 3 . Found. N 33.60 33.43 Acetic acid also precipitated from an alkaline solution of the cis acid, the pyrimidine melting at 320. Condensation of Ethyl Formate with Phthalimidoethylacetate . This condensation was effected by suspending the phthalimido- ethyl acetate in benzene or ether with the calculated amount of sodium and adding slowly the ethyl formate. The con- densation was not smooth, and after standing for several hours, with occasional shaking, much phthalimidoethylacetate remained unaltered. From 50 grams of phthalimidoethylace- tate about 50 grams of crude sodium salt were obtained. 142 Johnson and Clapp. This sodium salt is exceedingly unstable in aqueous solu- tion. Some of the salt was dissolved in water and then fil- tered. When this clear salt solution was allowed to stand for several hours at ordinary temperature it gradually deposited a crystalline solid. This crystallized from alcohol in needle- like prisms and melted at 111. It proved to be unaltered phthalimidoethylacetate. When the filtered solution was acidi- fied with hydrochloric acid no further precipitation took place. On the other hand, when a solution of the sodium salt was immediately acidified with hydrochloric acid a semisolid pre- cipitate was obtained. It was insoluble in hot water. It dissolved in cold alcohol with the exception of a small amount of white, crystalline material. This insoluble product crys- tallized from hot alcohol in needles and melted at 193 to 194. It was soluble in alkali to a yellow solution. This body was identified as 4-oxyisoquinoline-3-carboxylic ethyl ester, which was obtained by Gabriel and Colman 1 by heating phthalimido- ethylacetate with sodium ethylate and alcohol at 100. A nitrogen determination gave : Calculated for Ci 2 H n O 4 N. Found. N 6.0O 6.21 When the above alcoholic extract was evaporated to dry- ness a thick syrup was obtained which immediately solidified on cooling. It crystallized from alcohol in prisms and melted at ni to 112. It was identified as phthalimidoethylacetate. 2- Ethylmercapto-^-phthalimido-6-oxypyrimidine , NH CO I I /co v C 2 H 5 S.C C.N< J>C 6 H 4 . Tbispyrimidine wasobtained II II X CO' N=CH by condensing pseudoethylthiourea in aqueous solution with the sodium salt of formylphthalimidoethy lacetate. Six and two- tenths grams of the crude salt were treated with 160 cc. of water and filtered from the unaltered phthalimidoethylacetate. To the clear solution was then added an aqueous solution of 1 Ber. d. chem. Ges., 33, 983. Synthesis of 2-Amino-^-methyl-6-oxypyrimidine. 143 the pseudourea, that was prepared by mixing a solution of 36 grams of the hydrobromide with 1 1 grams of potassium hydrox- ide. The resulting mixture was then allowed to stand at ordi- nary temperature for one hour, and then heated on the steam- bath. Mercaptan was evolved in large amount, and on prolonged heating the solution assumed a dark-green color. A crystal- line precipitate finally began to separate from the hot solution when the mixture was removed from the bath and allowed to cool. This crystalline body proved to be phthalimidoethyl- acetate. On acidifying the solution with sulphuric acid a thick, gummy precipitate was obtained, which was purified by boiling with water and animal charcoal. It crystallized from alcohol in rhombic plates and melted at 230 to 231 to a yellow oil. It gave a strong test for sulphur. A nitrogen determination gave : Calculated for Found. N 13-95 14-3 When the above pyrimidine was dissolved in alkali and then acidified with dilute hydrochloric or sulphuric acid a white, crystalline precipitate was immediately obtained. It crystallized from 50 per cent alcohol and melted with violent effervescence at 130 to 131, then solidified again in the hot bath, and did not melt again until 229 to a clear, yellow oil. It gave a strong test for sulphur. A nitrogen determination agreed with the calculated for -phthalimido-/?-pseudoethyl- thioureaacrylic acid, C 6 H 4 (CO),N C(COOH)=CH.N=C(SC 2 H 5 ) NH, : Calculated for CHH 18 O 4 N 8 S. Found. N 13.16 13.26 This same acid was obtained in the above condensation when the acid filtrates were allowed to stand. It separated in small prisms and was purified by crystallizing from alcohol. When the acid was heated above its melting-point in an oil- bath it immediately decomposed with loss of a molecule of water, giving the pyrimidine derivative melting at 231. 144 Johnson and Clapp. When mixed with the pyrimidine the melting-point was not lowered. Sodium Salt of Oxymethylenehippuric Ethyl Ester, y NH.COC 6 H 6 NaO CH C^ .This salt was used by Erlen- N COOC 2 H 5 meyer 1 in his synthesis of serine and cystine. He gives no directions for preparing the salt, but states that he obtained it by condensing ethyl formate with hippuric ethyl ester in presence of sodium ethylate. We have prepared the salt by condensing hippuric ethyl ester with ethyl formate in benzene in the presence of metallic sodium. The condensation is very slow. From 25 grams of hippuric ester we obtained 35 grams of the crude salt. 2-Ethylmercapto-5-benzoylamino-6-oxypyrimidine, NH CO I I C 2 H 5 S.C CNH.COC 6 H 5 . Thirty-five grams of the above II II N CH sodium salt were dissolved in cold water and combined with an aqueous solution of pseudoethylthiourea. Only 0.5 molec- ular proportion of pseudourea was used in this reaction, or 13 grams of the hydrobromide, which were neutralized with 4 grams of potassium hydroxide. The mixture was allowed to stand at ordinary temperature for forty-eight hours. No odor of mercaptan was noticeable. When the mixture was ex- amined a large amount of crystalline material had separated from the aqueous solution. This crystallized from alcohol in needles and melted at 238 to 239* to a yellow oil. It gave a strong test for sulphur and did not decompose when heated to 260. A nitrogen determination gave : Calculated for Ci3H 13 O2N 3 S. Found. N 15.27 15.50 The pyrimidine was soluble in alkali and was precipitated unaltered by hydrochloric acid. 1 Ber. d. chem. Ges., 36, 3769; 35, 2720. Synthesis of 2-Amino-^-methyl-^-oxypyrimidine. 145 When the alcoholic filtrates from the above purification were combined and evaporated to dryness an oil was obtained which finally solidified. It was identified as unaltered hip- puric ester. More of the above pyrimidine was obtained by acidifying the original solution after filtering off the crystalline material that had separated. It was precipitated as a semisolid, which quickly solidified on cooling. It crystallized from alco- hol in needles and melted at 238 to 239. NEW HAVEN, CONN., Feb. 24, 1904. [Reprinted from the American Chemical Journal, Vol. XXXII, No. 4. October, 1904.] Contributions from the Sheffield Laboratory of Yale University. CXIX. RESEARCHES ON PYRIMIDINES : 2-OXY- 4,6-DIAMINOPYRIMIDINE. 1 [SEVENTH PAPER.] BY HENRY I,. WHEELER AND GEORGE S. JAMIESON. In this paper we describe a number of pyrimidines derived from 2-thiobarbituric acid. Our chief object in investigating 1 Part of a thesis presented by Mr. G. S. Jamieson for the degree of Doctor of Philosophy, Yale University, 1904. 2- Oxy-j. , 6-dia minopyri midine. 343 these compounds was to prepare 2-0x7-4, 6-diaminopyrimi- dine. Kutscher 1 believes lie obtained an oxydiaminopyrimidine from yeast nucleic acid, the acid having been decomposed by sulphuric acid at a high temperature and under pressure. He considers the substance as differing from cytosine inas- much as a hydrogen atom is replaced by an amino group. It may be added that since the pyrimidine derivatives hitherto obtained from nucleic acids have an oxygen atom in the 2-position, for example, uracil, thymine, and cytosine, it is probable that a diamino derivative would be similarly con- stituted. This being the case, there are only two oxydiamino- pyrimidines that fulfil these conditions, namely, 2-oxy-4,6- diaminopyrimidine (I.) and 2-oxy-5,6-diaminopyrimidine (II.) : N C NH 2 N C NH 2 OC C NH 2 I II HN CH II. Our first attempts to prepare the oxydiaminopyrimidine represented by Formula I. were as follows : 2-Thiobarbituric acid (III.) was prepared by the method of Michael. 2 It was found that this substance could be converted into 2-methyl- mercapto-4,6-dioxypyrimidine (IV.) by means of sodium, ethylate and methyl iodide in alcoholic solution : HN CO HN CO N= =CC1 II II II SC CH 2 ~ CH,SC CH 2 ~ CH 8 SC CH II II I II II HN CO N CO N CC1 III. IV. V. When the mercapto derivative (IV.) was heated on the steam-bath with 2 molecular proportions of phosphorus penta- chloride, it was converted into 2-methylmercapto-4,6-dichlor- pyrimidine (V.). The reaction was not smooth and a con- 1 Ztschr. physiol. Chem., 38, 176 (1903). 2 J. prakt. Chem., 35, 456 (1887) ; 49, 38 (1894). 344 Wheeler and Jamieson. siderable amount of decomposition-products was always ob- tained. It was later observed that the dichloride could be obtained more smoothly by using phosphorus oxychloride in- stead of the pentachloride. When the dichloride was heated with alcoholic ammonia it was found that only one chlorine atom was easily removed and 2-mercapto-5-chlorcytosine (VI.) was formed. This took place at temperatures ranging from 125 to about 190. Even below the latter temperature mercaptan was evolved, and above 190 2,4,6-triaminopyrimidine (VIII.), which has been described by Gabriel, 1 was obtained. There seemed to be little tendency for the two chlorine atoms to react without also displacing the mercapto group. The 2-mercapto-4-chlorcytosine was then converted into 4-chlorcytosine (VII.) by boiling with hydrochloric acid. When this was heated with alcoholic ammonia a reaction took place, but the chief product was not the desired 2-oxy-4,6- aminopyrimidine : N C NH 2 N C-NH 2 CH.SC CH II II N CC1 VI. - OC HN VII. CH II -CC1 N H 2 N C II =:C NH 2 1 CH ij II N II C NH 2 VIII. It was finally found that 2-oxy-4,6-diaminopyrimidine could easily be prepared from 2-thio-4,6-diaminopyrimidine (IX.) which Traube 2 has recently obtained by condensing malonic nitrile with thiourea. This substance united smoothly with methyl iodide and the resulting mercapto derivative (X.) formed on treating the addition-product with ammonia, on carefully boiling with hydrochloric acid, gave the 2-0x3^-4,6- diaminopyrimidine (I.) : 1 Ber. d. chem. Ges., 34, 3364. 8 Ann. Chem. (Mebig), 331, 80 (1904). 2-Oxy~4,6-diaminopyrimidine. 345 N=C NH 2 N C NH 2 HN II II I SC CH CH 3 SC CH ~ OC I II II II I HN C NH 2 N C NH 2 HN IX. X. XL It was then found that if the warming with hydrochloric acid was continued for more than a short time on the steam-bath the 2-oxy-4,6-diaminopyrimidine was completely converted into barbituric acid (XI.)- It is therefore obvious, from the readiness with which this compound is decomposed by hydro- chloric acid, that it is not identical with Kutscher's new base. If the latter is an oxydiaminopyrimidine it probably is 2-oxy- 5,6-diaminopyrimidine. EXPERIMENTAL, PART. 2- Thiobarbituric add was prepared according to Michael's 1 directions, by the condensation of sodium ethyl malonate with thiourea in absolute alcoholic solution. Twenty-five grams of ethyl malonate gave 20 grams of crude sodium thiobarbitu- rate. From this a yield of 12 grams of acid was obtained. It was found that if instead of the calculated quantity of sodium ethylate two molecular proportions were used a larger yield of the acid resulted. For example, in two experiments, using 25 grams of ethyl malonate, 27 grams of sodium thiobarbitu- rate were obtained ; in three other cases, using the same pro- portions, 28 grams of salt separated, the yield of thiobar- bituric acid in each case being 16.5 grams. HN CO I I 2-Methylmercaptobarbituric Acid, CH 3 SC CH 2 . The II I N CO best yield of this substance was obtained when 12 grams of thiobarbituric acid were added to a cold, absolute alcoholic solution of 1.9 grams of sodium, then 15 grams of methyl iodide were added and the solution was not allowed to warm. The reaction was complete in two days. The alcohol and i J. prakt. Chem., 35, 456 (1887) ; 49, 38 (1894). 346 Wheeler and Jamie son. excess of methyl iodide were then evaporated and the residue was treated with water and acidified with acetic acid. The precipitate was filtered, washed with water, absolute alcohol, and ether. A yield of 8.6 grams of sodium-free material was obtained. In another experiment, 40 grams of acid gave 2.5 grams of the mercapto derivative, the usual yield being be- tween 53 and 66 per cent of the calculated. Attempts to methylate thiobarbituric acid by other methods, such as by direct addition of methyl iodide, or by the action in alcoholic ammonia, or in aqueous solution and in the presence of alkali, either failed entirely or did not give as satisfactory results as the above method. The 2-methylmercaptobarbituric acid crystallized from water in needles. It is sparingly soluble in hot water and less soluble in alcohol. It did not melt at 300, but gradually turned a light-brown. A nitrogen determination gave : Calculated for CsH^OaNaS. Found. N 17.72 17.87 N=CC1 2-Methylmercapto-4,6-dichlorpyrimidine, CH 3 SC CH. II II N CC1 Four grams of the above 2-methylmercaptobarbituric acid were heated with 9 grams of phosphorus pentachloride on the steam- bath. When the evolution of hydrogen chloride ceased the dark-red solution was cooled and treated with crushed ice. The dichloride was then shaken out with ether, the ethereal solution dried over calcium chloride, evaporated, and the residue distilled at 28 mm. pressure, whereupon 2.5 grams of colorless oil were obtained, boiling at 154, this being a yield of 50.2 per cent of the calculated. In two other experi- ments with phosphorus pentachloride, using in each case 8 grams of the methylmercaptobarbituric acid, 3 grams and 3.7 grams of dichloride were obtained. Both specimens boiled at 135 to 136 at 14 mm. pressure. The oil thus obtained solidified in the form of table-like prisms, which melted at 41 to 42. When phosphorus oxychloride was used instead 347 of the pentachloride, the remaining treatment being the same, the dichloride was directly obtained in the crystalline condi- tion, it being unnecessary to distil under reduced pressure. The material from the ethereal solution was purified by recrys- tallizing from a little alcohol. It then formed stout, pointed prisms and the melting-point was not altered. It is very sol- uble in ether, alcohol, and petroleum ether. In two experi- ments, using 5 grams of the methylmercaptobarbituric acid, 3.3 grams and 3.4 grams of the dichloride were obtained. The yield by the action of either the pentachloride or the oxy- chloride of phosphorus, especially in large quantities, was found to be very irregular. A nitrogen determination gave : Calculated for C5H 4 NsSCl 2 . Found. N I4-3 6 I4-76 2-Methylmercapto-4.-chlor~6-aminopyrimidine, N=C NH, I I CH 3 SC CH .Two and a half grams of the 2-methyl- II il N CC1 mercapto-4,6-dichlorpyrimidine were heated in a sealed tube with about 75 cc. of alcoholic ammonia for four hours at 125 to 126. On cooling, the ammonium chloride which had separated was filtered and the filtrate evaporated. The resi- due was purified by recrystallizing from water, in which it was difficultly soluble. It crystallized in colorless, needle- like prisms, which melted at 127 to 128 to a colorless liquid. A yield of 1.5 grams of purified material was obtained. Four other experiments gave the following yields : Chloride. Monoamine. Grams. Grains. 3-7 2.6 3-0 2.2 3-2 2.7 3-3 2.6 A nitrogen determination gave : Calculated for C 6 H 6 N 3 SC1. Found. N 23.93 24.04 348 Wheeler and Jamie son. Action of Alcoholic Ammonia. This chloraminopyrimidine (i.i grams) was heated with about 75 cc. of alcoholic ammo- nia (alcohol saturated with dry ammonia at o) at 145 for three hours and then for two hours at 150, whereupon the material was recovered completely unaltered. The i.i grams of substance were reheated with the same volume of fresh alco- holic ammonia at 170 for two hours, and then at 190 for a half hour ; 0.7 gram of unaltered material was obtained. Two and four-tenths grams of the chloraminopyrimidine were heated with alcoholic ammonia for two hours at 185 to 190, and four hours at 215 to 220. On opening the tube the alcohol was found to be saturated with mercaptan. A considerable amount of a light-yellow precipitate had separa- ted from the alcohol. This was filtered, washed with alco- hol and then with water. It melted at about 234 to 240 and it gave an alkaline reaction to turmeric paper. It contained neither sulphur nor chlorine and was found to be 2,4,6-tri- aminopyrimidine, which has been described by Gabriel. 1 N=C NH 2 4-Chlorcytosine, OC CH . This was obtained by I I! HN CC1 boiling 0.5 gram of the above mercapto derivative with con- centrated hydrochloric acid. Mercaptan was readily evolved and upon evaporating the solution, 4-chlorcytosine hydrochlo- ride was obtained. This crystallized from hot water in balls of very small, slender, hard prisms or needles. From dilute aqueous solutions of this salt, ammonia precipitates a crop of flat, colorless prisms ; from more concentrated solutions the base separates as a jelly resembling thick starch paste, which is difficult to filter. When dried on paper it adheres strongly to the fiber. It is fairly soluble in water and it does not melt at 300, although it sinters and gradually turns red. A nitro- gen determination gave : Calculated for C4H 4 ON 3 C1. Found. N 28.86 28.73 1 Ber. d. chera. Ges., 34, 3364. 2- Oxy-4,6-diaminopyrimidine. 349 The chlorine in this compound is very firmly bound. It was not removed by tin and hydrochloric acid, or by red phos- phorus and hydriodic acid. When 1.5 grams of this material were heated with alcoholic ammonia at 183 for two hours 0.95 gram of substance insol- uble in alcoholic ammonia was formed. It was difficultly sol- uble in water ; if the solution was cooled rapidly, very small crystals were obtained ; if slowly cooled, little balls were formed. This substance did not melt at 307. It was free from chlorine and a nitrogen determination gave only 24.9 per cent nitrogen. It was not identified, "since two attempts to prepare the compound failed on account of the explosion of the sealed tubes and no time was available to repeat the work. 2-Methylmercapto-4.,6-diaminopyrimidine, Ni=z:C NH 2 I I CH S SC CH . Malonic nitrile and thiourea were con- II II N C NH 8 densed according to Traube's 1 directions in an alcoholic solu- tion with sodium ethylate. Two grams of the 2-thio-4,6-di- aminopyrimidine were moistened with alcohol and 2.2 grams of methyl iodide were added. The mixture stood two hours without change. Suddenly, heat was evolved and the solution solidified. The alcohol was evaporated and the residue was dissolved in water. Ammonia then precipitated 2.2 grams of colorless crystalline material. This was quite soluble in hot water, from which, on cooling, it separated in the form of slender prisms. It melted at 185 to 186 with a little effer- vescence, and a nitrogen determination gave : Calculated for C 6 H 8 N 4 S. N 35-89 2~Oxv-4,6-diaminopynmidinc, OC CH . One gram HN 1 Ann. Chem. (I II II N CH NH CO I I C 2 H 5 SC CNH 2 + C 2 H 5 OH + CO 2 . II II N CH V. This interesting saponifi cation and condensation of the trans- acid to the mercaptoaminopyrimidine, (V.) led me to examine its behavior when warmed with a dilute solution of sodium i Loc. cit. Researches on Pyrimidines. 195 hydroxide. The experimental data at hand seem to indicate that the Zratw-acid is saponified by dilute alkali to an intermediate 2-ethylmercapto-5-carboxylamrr -oxypyrimidine, (VI.). NH 2 COOH I I Na C 2 H 5 SC CNF^X)C 2 H 5 II H N CH NH CO CNHCOOH + C 2 H 5 OH. II -OH VI. existence ? ( ^is intermediate carbamic acid is particu- interestin " oecause of the fact that it contains a cyclic ide linking. Analogous acids in the acyclic series >.-en obtained by Fisher, 1 in his important researches on .ypeptides. The relationship between the above pyrimidine, (VI.) and a true polypeptide linking is made evident by the following comparison : Acyclic series. HN CO CH 2 NH COOH. Cyclic series. HN CO C NH COOH I II C 2 H 6 SC=N CH It has been the usual experience, during the investigation of pyrimidines in this laboratory, to find that a mercaptooxy- pyrimidine reacts normally with phosphorus oxychloride, or phosphorus pentachloride, to give a chlorpyrimidine deriva- tive. It has also been observed repeatedly that the resulting chlorpyrimidines are obtained combined with phosphorus oxy- chloride. These phosphorus compounds are very unstable and 1 Ber. d. chem. Ges., 36, 2094 (1903). 196 Johnson. are decomposed by water, with formation of the free chlor- pyrimidine. I now find that 2-ethylmercapto-5-amino-6-oxy- pyrimidine reacts with phosphorus oxychloride to form a phosphorus compound which is abnormally stable. The analytical determinations agree with the calculated for a phos- phoryldichloride derivative (VII.) : NH CO N CC1 I 1 POC1, I I C 2 H 5 SC CNH 2 ^ C 2 H 5 SC CNHPOC1 2 . ' II II ' II II N CH N CH VII. This phosphorus derivative could be warmed with water without decomposition. When it was heated with alcoholic ammonia 2 chlorine atoms were removed and a stable com- pound was obtained, which I have, provisionally, represented as a phosphoryltriamide derivative, 1 (VIII.) : ; 2 H 5 SC N- CC1 I NH 3 CNHPOC1 2 1 II N CH The fact that this amino radical should show such pro- nounced affinity for phosphorus, is of interest. In a recent com- munication from this laboratory 2 it has been shown that the iso- meric2-ethylmercapto-4-oxy-6-aminopyrimidine reacts perfectly normally with phosphorus oxychloride, to form 2-ethylmercapto- 4-chlor-6-aminopyrimidine, (IX.). Furthermore, Gabriel and 1 These phosphoryl pyrimidine derivatives will receive further attention in this laboratory. J Johnson and Johns : Preceding paper. Researches on Pyrimidines. 197 Colman 1 have shown that 2-aniino-4-oxy-6-methylpyrimidine is converted, by phosphorus oxychloride, into 2-amino-4-chlor- 6-methylpyrimidine. Later, 2 they observed that 2-amino-6- oxypyrimidine (isocytosine) als r 'eacts normally with phos- phorus oxychloride, to give 2-amino 6-chlorpyrimidine, (XI.). IN i^;rsi M 2 i 3 i NH,C CH . "NT JN VJVJ1 I C 2 H 5 SC II N- ! CF jj !~~ CC1 IX. 1 NH,.C II N- C II PTT X XL i a previous pub! Nuc! .^saure des Weisen- ,c produced evidence to that, in triticoni, etc acid, there is a direct union be- the nitrogen of the purine bases. at _ ^olication, has produced more evidence _ tact of this union in the nucleic acid mole- Fur Uiermore, his experiments tend to show that it is the ^_ii atom occupying position 7 in the purine molecule that probably joined to the phosphorus. It is of interest to note, in this connection, that the nitrogen atom in the aminopyrimi- dine, (V.) which shows such affinity for phosphorus, corre- sponds to the nitrogen atom occupying position 7 in the purine molecule. iN -- C6 2 C sC N 7 | | 3 N -- C EXPERIMENTAL. Sodium Salt of Formykarbethoxyaminoethylacetate, C 2 H 5 OOCNHC : (CHONa)COOC 2 H 6 . The carbethoxyamino- ethylacetate, used for the preparation of this sodium salt, was 1 Ber. d. chem. Ges., 32, 2921. 2 Ibid., 36, 3383. 3 Z. physiol. Chem., 36, 85 (1902). 4 Ber. d. chem. Ges., 37, 708. 198 Johnson. prepared by Fisher's 1 method, from the hydrochloride of aminoethylacetate and chlorethylformate. The hydrochloride of aminoethylacetate was prepared according to Klages' 2 direc- tions, from methyleneaminoacetonitrile. The method is ex- tremely suitable for preparing large quantities of this hydro- chloride. Ethyl formate condenses very smoothly with carb- ethoxy aminoethylacetate, in the presence of sodium, in either benzene or ether ; but it has been my experience that the con- densation is much smoother and more rapid when benzene is used as the diluent. This sodium salt is much more stable in contact with the air than the sodium salt of formylethylace- tate. I also found that the condensation could safely be ac- celerated by gently warming the benzene solution on the steam-bath. The purity of the salt obtained was not affected by this treatment. In all my condensations the yield of crude sodium salt has been about equal to the weight of carbetlioxy- aminoethylacetate used. Trans-a-carbethoxyamino-fi-pseiidoethylthiourea Acrylic Acid. C 2 H 5 OOC.NH C COOH || . Twenty-five grams of the H 2 N. (C 2 H 5 S)C=N CH sodium salt of formylcarbethoxyaminoethylacetate were dis- solved in water and added to an aqueous solution of pseudo- ethylthiourea, which was previously prepared by treating 17 grams of pseudoethylthiourea hydrobromide with 6 grams of potassium hydroxide. The clear solution, which resulted, was then allowed to stand for a few hours, at the ordinary temper- ature. Upon neutralizing with acetic acid a beautiful, white, crystalline compound separated. It was insoluble in alcohol and cold water. It could be dissolved, repeatedly, in cold solutions of sodium hydroxide and be precipitated, unaltered, by acids. It was purified for analysis by recrystallizing from hot water. It separated as a colorless powder, consisting of microscopic prisms. It showed no sharp melting-point but decomposed, with effervescence, at 259. A yield of 1 1 . 5 grams of crude material was obtained. For analysts it was dried at 100. 1 Ber. d. chem. Ges., 36, 2107. Ibid., 36, 1508. Researches on Pyrimidines. 199 0.1574 gram substance gave 0.1341 gram BaSO 4 (Carius). Calculated for Found. N 16.00 15.75 S 12 a 11.70 The following table i* ;-en to show the comparative yields obtained in four di^ m preparation- of this trans-acid : Table. Weight of carbeth- oxyatn ; ac *- , 1e used. Weig> on -product 'It Of trans-Add. Percentage of theoretical. Grams. Grams. Grams. Per cent. 30 40 20 25 *i-5 4 6 45 - 20.0 45 40 45 19-5 44 :dibethoxyamino-6-oxypyrimidine, ' , CNHCOOC 2 H 5 . This compound was easily ob- ,i I! N CH tained when the trans-acid, above described, was dissolved in warm acetic anhydride. On adding water the pyrimidine separated in prisms. It was easily purified by recrystallizing from 15 per cent acetic acid and separated, on cooling, in well- developed prisms, which melted, without effervescence, at 189- 190, to a clear oil. The pyrimidine dissolves in sodium hy- droxide solution and is reprecipitated, unaltered, by acids. It was also obtained, in an impure condition, when the trans-acid was boiled with glacial acetic acid. Analysis : Calculated for C9H 13 O 3 N 3 S. Found. N 17.28 17.35 2-Ethylmercapto-5-amino-6-oxypyrimidine, NH CO C 2 H 5 SC CNH 2 . Ten grams of the trans-acid were dis- II II N CH solved in 50 cc. of water, containing 5 grams of sodium hydrox- 2OO Johnson. ide. The alkaline solution was boiled for about 20 minutes and then allowed to stand for 30 minutes. When the alkaline solution was made slightly acid with acetic acid the pyrimidine separated in beautiful, needle-like crystals, which melted, to a clear oil, at about 155. It was purified by recrystallizing from hot water. It separated in clusters of radiating needles and, without effervescence, melted at 160, to a clear oil. The yield was practically quantitative. This pyrimidine was also formed when 2-ethylmercapto-5-carbethoxyarnino-6-oxypyrimi- dine was boiled with sodium hydroxide. Analysis (Kjeldahl) : Calculated for C6H 9 ON 3 S. Found. N 24.56 24.34 Disilver Salt of 2-Ethylmercapto-5-carboxylamino-6-oxypyrimi- N COAg I I dine, C 2 H 5 SC CNHCOOAg. The acid of this salt was ob- it II N CH tained, in solution, from two different preparations of the trans- acid. It was formed by boiling the latter with a dilute solution of sodium hydroxide. It was very soluble in water and was not precipitated from the alkali solution by mineral acids or car- bon dioxide. The disilver salt was obtained, after acidifying the alkaline solution with nitric acid, by adding a slight ex- cess of 20 per cent silver nitrate solution. The salt was diffi- cultly soluble in water and showed no crystalline form. Analysis of the substance, thoroughly dried over sulphuric acid : o. 1399 gram silver salt gave 0.0707 gram metallic silver. Calculated for C T H 7 O 3 N 3 SAg 2 . Found. Ag 5- 35 50-53 2-Ethylmercapto-5-phosphoryldichloramino-6-chlorpyrimidine, N > 2 H 5 SC CNH.POC1,. Four and eight- tenths grams of II II N CH Researches on Pyrimidines. 201 2-ethylmercapto-5-amino-6-oxypyrimidine were warmed, on a sand-bath, with an excess of phosphorus oxychloride. A re- action set in at once, with f 'ution of hydrochloric acid gas. After this ceased the excess of phosphorus oxychloride was removed by distillation under '.Uminished pressure. I obtained a yellow, amorphou residue. Wh. n this was treated with water very litt 1 vas evolved and a yellow, crystalline de- posit remai- >d suspended in the water. This residue was fil- tered a*"d then warmed with water on the steam-bath, to de- strov any phosphorus oxychloric' A thick, oily product, in- soluble in water, was obtained by this treatment ; it imme- iately solidified, on coolr _; to a hard, crystalline cake. It was insoluble in the . d nary organic solvents and also in ammonium hydroxide. When treated with a solu- tion of sodium lr. oxide it showed a very peculiar be- .uct was insoluble in sodium hydroxide, but r unc 5 .rwent decomposition and the solution assumed k green color. It gave strong tests for sulphur and phos- arus. It had no sharp melting-point, but decomposed at about 247-25o. Analysis : Calculated for C 6 H 7 ON 3 SC1 3 P. Found. N 13.70 13.41 2-Ethylmercapto-5-phosphoryltriamino-6-chlorpyrimidine, N CC1 | I /NH, C 2 H 5 SC CNHPOC/ + 2 NH(CH 3 ) 2 =Br N(CH 3 ) 2 + NO 2 COOC 2 H 5 COOC 2 H S NO 2 CH/ NH(CH 3 ) COOC 2 H S The ease with which the iodine in 2-ethylmercapto-5-iodo-6- oxypyrimidin (IV) could be replaced by hydrogen in the pres- ence of amines suggested that this iodopyrimidin, under certain conditions, might be used for syntheses in reactions involving double decomposition. We now find that under the conditions employed in our experiments the iodine was very firmly bound and could not be replaced. For example: It could be heated with the potassium salt of phthalimid in alcohol, or with the sodium salt of urethane in benzene at 160 C. and be recovered unaltered. In these experiments it shows the stability of the corresponding i l J3er. d. deutsch. chem. Gescellsch., xxxvii, p. 1775, 1904. 2 Beilstein, Handbuch, i, p. 1194. 3io Researches on Pyrimidins bromine derivative 2-ethylmercapto-5-brom-6-oxypyrimidin. This compound and also 2-ethylmercapto-5-brom-6-amino-pyr- imidin * could be heated with the potassium salt of phthalimid at 200 C. without any evidence of the formation of potassium bromide. EXPERIMENTAL PART. NH-CO I I 2-Eihylmercapto-5-iodo-6-oxypyrimidin, C,H 5 SC CI (by N - CH Dr. J. G. Statiropulos) . Ten grams of 2-ethylmercapto- 6-oxypyrimidin , 2 were dissolved in 50 c.c. of water containing 3 grams of sodium hydroxide. To this solution was then slowly added 1 7 grams of pulverized iodine. After all the iodine had been added the solution was warmed on the steambath until the free iodine dissolved. The iodopyrimidin separated from the hot solution. After acidifying with acetic acid the solution was filtered and the pyrimidin purified by crystallization from alcohol. It deposited in slender prisms, arranged in crosses, and melted at i96C. to a clear oil. It was insoluble in water, and readily soluble in benzene. The yield was practically quanti- tative. Analysis: For C 6 H 7 ON 2 SI Calculated: N=9-93 per cent. Found: N. = 10.02 per cent. NH-CO 2, 6 -Dioxy- 5 -iodopyrimidin (^-iodouracil) , CO CI (by I II NH-CH Dr. J. G. Statiropulos). This compound was easily obtained when uracil was dissolved in alkali and treated with iodine as in the preparation of the above 2-ethylmercapto-5-iodo-6-oxypyr- imidin. It crystallized from hot water in glistening scales, and decomposed at 272 C. It was soluble in hot alcohol. Analysis: 1 Wheeler and Johnson, loc. cit. 2 Wheeler and Merriam, loc. cit. Treat B. Johnson and Carl O. Johns 311 For C 4 H 3 O 2 NJ Calculated: N = 11.86 per cent. Found: N = n.77 per cent. N = C. NH 2 ^-Iodocytosin, CO CI. Six and three-tenths grams NH-CH of synthetical cytosin 1 were dissolved in about 50 c.c. of water containing 3.3 grams of potassium hydroxide. To this solu- tion were slowly added, with frequent shaking, 14.5 grams of finely pulverized iodine. The reaction was smooth and the iodocytosin began to separate immediately. After all the iodine had been added the solution was warmed on the steambath for a few minutes to complete the reaction. After neutralizing the free alkali with acetic acid the base was filtered off and re- crystallized from boiling water. It separated on cooling in characteristic, branched crystals (Figure i), which decomposed between 225 and 245 C. without effervescence, giving off iodine vapors. It was practically insoluble in alcohol and benzene. The yield was quantitative. Analysis (Kjeldahl): 0.1188 gm. of substance gave 0.0210 gm. of nitrogen = 15 c.c. of TIT HC1. For C 4 H 4 ON 3 I Calculated: N = i7-72 per cent. Found: 1^ = 17.67 per cent. Solubility of iodocytosin in water : One liter of water at 25 C. dissolved (i) 0.936 gram, (2) 0.988 gram. Iodocytosin is very stable in the presence of boiling hydro- chloric acid. One gram of the base was boiled with concentrated hydrochloric acid for two hours. The acid solution was then evaporated to dryness and the residue treated with a little dilute ammonia. We obtained the unaltered base which decomposed at from 220 to 235C. Analysis (Kjeldahl) : 0.12 10 gm. of substance gave 0.02 142 gm. of nitrogen = 15. 3 c.c. ^ HCI. For C 4 H 4 ON 3 I Calculated: N = i7.72 per cent. Found: N =17.70 per cent. Pier ate of iodocytosin: The picrate crystallized from hot water in long needles. It had no definite melting point but 1 Wheeler and Johnson, loc. cit. 312 Researches on Pyrimidins decomposed with effervescence at from 247 to 257C. according to the rate of heating. Analysis (Kjeldahl): 0.1705 gm. of substance gave o. 03066 grm. of nitrogen = 2 1.9 c.c. T ^ HC1 For C 4 H 4 ON 3 I.C 6 H 3 O 7 N 3 Calculated: N = 18.02 p^r cent. Found: N = 17.98 per cent. Acetic acid salt of iodocytosin: This salt is of special interest on account of its dissociation in a hot solution of acetic acid. Some iodocytosin was dissolved in hot acetic acid and the solution divided into two equal parts. The first part was then cooled quickly, when the unaltered base immediately separated in the form of irregular prisms. Analysis (Kjeldahl) : 0.1253 S 00 " of substance gave 0.02198 gm. of nitrogen = 15.70.0. T ^ HC1. For C 4 H 4 ON 3 I Calculated: N = i7-72 per cent. Found: N = i7.54 p^r cent. The second part was allowed to stand for several hours. The unaltered base separated at first in distorted prisms. On stand- ing these prisms slowly assumed a new form and were trans- formed into large well developed prisms with distinct faces (Figure 2). They became opaque at about noC.,and decom- posed at from 220 to 2 40 C. according to the rate of heating. A nitrogen determination agreed with the calculated percentage in the acetic acid salt (Kjeldahl): o.i32ogm.of substance gave 0.0189 gm. of nitrogen = 13. 5 c.c.^HCl 0.1029 gm. of substance gave 0.01456 gm. of nitrogen = 10.4. c.C'nrHCl. For C 6 H 8 O 3 N 3 I Calculated: N = i4.T4 per cent. Found: N = (i) 14.31; (2) 14.15 per cent. In order to decide whether the salt would dissociate again, it was dissolved in hot acetic acid and the solution cooled. The base was again obtained in the form of irregular prisms. Analysis (Kjeldahl): 0.0811 gm. of substance gave 0.01428 gm.of nitrogen =10. 2 c.c. T ^ For C 4 H 4 ONJ Calculated: N = i7-72 per cent , , Found: N = i7.6o per cent. Treat B. Johnson and Carl O. Johns 313 N=CC1 2-Ethylmercapto-5-iodo-6-chlorpyrimidin, C 2 H S SC CI !! II N-CH This compound was prepared by warming on the steambath 20 grams of 2-ethylmercapto-5-iodo-6-oxypyrimidin with 50 c.c. of phosphorus oxy chloride. After the evolution of hydrochloric acid ceased the excess of phosphorus oxychloride was removed by heating at 100 C. under a pressure of 50 millimeters of mer- cury. We obtained a thick, brown varnish which immediately solidified when triturated with ice water. This contained iodine and melted at 68 C. to a turbid oil. It was purified by crystallizing from petroleum ether. It deposited in large prisms and melted at 69 C. to a clear oil. The yield was 20 grams, or 95 per cent, of the theoretical. Analysis (Kjeldahl): 0.2050 gm. of substance gave 0.01918 gm. of nitrogen = i3.7 c.c. y^HCl. For C 6 H 6 N 2 SC1I Calculated: N=9-32 p.r cent. Found: N=9-35 per cent. N=C.NH 2 2-Ethylmercapto-5-iodo-6-aminopyrimidin, C 2 H 5 SC CI II II N-CH This compound was obtained when 10 grams of 2-ethylmercapto- 5-iodo-6-chlorpyrimidin were heated with alcoholic ammonia for three hours at i28-i3o C. When the tube was examined the base had crystallized from the alcoholic solution in long, slender prisms. The crude material melted at 126 C. When allowed to crystallize slowly from alcohol it deposited in slender prisms that arranged themselves in clusters radiating from a common center. It melted at 127 C. The yield was quantitative. Analysis (Kjeldahl): 0.1368 gm. of substance gave 0.02044 gm. of nitrogen =14. 6 c.c. T ^ HC1. For C 6 H 8 N 3 SI Calculated: N = i4-94 per cent. Found: N = i4-94 per cent. When 5 grams of this amino-pyrimidin were boiled with con- centrated hydrochloric acid for one hour mercaptan was evolved 314 Researches on Pyrimidins and it was converted practically quantitatively into 5-iodo- cytosin. Analysis (Kjeldahl): 0.1386 gm. of substance gave 0.02422 gm. of nitrogen = 17.3 c.c. T ^ HC1. For C 4 H 4 ON 3 I Calculated: N = i7.72 per cent. Found: N = i7-47 per cent. N=C.NHC 6 H 5 2-Ethylmercapto-^-iodo-6-anilino-pyrimidin, C 2 H S SC CI II II N-CH was obtained by warming a benzene solution of 2 grams of 2-ethylmercapto-5-iodo-6-chlorpyrimidin and 1.4 grams of aniline. After evaporating the excess of benzene and washing with water to dissolve aniline hydro chloride, the base was obtained as an oil which would not solidify. It was purified by converting it, into its sulphuric acid salt. This crystallized from alcohol, which contained sulphuric acid, in well developed prisms. The sulphate had no definite melting point but decomposed above 200 C. A nitrogen determination (Kjeldahl) agreed with the calculated value for a normal sulphate: 0.0923 gm. of substance gave 0.00938 gm. of nitrogen = 6. 7 c.c. HC1. For (C 12 H 12 N 3 SI) 2 H 2 S0 4 Calculated: N = io.34 per cent. Found: N = 10.17 P er cent. NH-CO 2-Anilino-6-oxypyrimidin, C 6 H S NH.C CH. This compound N-CH was prepared by warming on the steambath 2-ethylmer- capto-6-oxypyrimidin with the calculated quantity of aniline. It was also formed when an alcoholic solution of the mercapto- pyrimidin and aniline were digested for several hours. It was moderately soluble in alcohol, and insoluble in benzene and water. It was purified for analysis by crystallization from alcohol. It deposited in well developed plates and melted at 23o-23i C. to a yellow oil. Analysis: For C 10 H 9 ON 3 Calculated: N = 22.46 per cent. Found: N = 22.54 per cent. Action of Aniline on 2-Ethylmercapto-^-iodo-6-oxypyrimidin. Fifteen grams of the iodopyrimidin were heated on the steambath Treat B. Johnson and Carl O. Johns 315 with 2 5 grams of aniline for six hours. It dissolved in the warm aniline to give a clear solution which gradually assumed a dark blue color. After the evolution of mercaptan ceased, the excess of aniline was removed by distillation with steam. We obtained a dark crystalline product that was insoluble in water. It was washed with cold alcohol to remove the coloring matter and puri- fied by repeated crystallizations from alcohol and acetic acid. It failed to respond to tests for iodine and sulphur. It melted at 23o-23i C. to a yellow oil. It agreed in all its properties with 2 -anilino-6-oxy pyrimidin (M. P. 23o-23i C.). A mixture of the two compounds melted sharply at 23o-23i C. Analyses for carbon, hydrogen, and nitrogen gave the following results: 0.0845 S 01 - f substance gave 16.7 c.c. nitrogen gas at 20 C. and 768 mm. 0.2491 gm. of substance gave o.iiQ3gm. of H 2 O and 0.5881 gm. of CO 2 . Calculated for Ci H 9 ON 3 Found C 64.2 percent 64.4 percent H 4.8 " " 5.3 " " N 22.46 " " 22.8 " " Action of Aniline on 2-Ethylmercapto-^-iodo-6-aminopyrimidin. This pyrimidin was recovered unaltered after heating with aniline for nine hours at iooC. It melted at i27C. and when mixed with the original material the melting point was not lowered. Action of Aniline on $-Iodocytosin. This base was recovered unaltered after heating with aniline, in benzene, for one hour at i5o-i6oC. The temperature was then raised to i90-i93C., and maintained for two hours. When the tube was examined there was no evidence that any reaction had taken place. The iodocytosin was again recovered unaltered. Action of Aqueous Ammonia on 2-Ethylmercapto-$-iodo-6-oxy- pyrimidin. Five grams of the pyrimidin were heated with concentrated aqueous ammonia for three hours at 156 to 163 C. When the tube was examined the solution had assumed a red color and drops of mercaptan were floating on the surface of the liquid. The solution gave a strong test for iodine. The am- moniacal solution was evaporated to dry ness. We obtained a crystalline residue that was soluble in water except a small amount of white crystalline material. This was identified as 316 Researches on Pyrimidins the unaltered 2-ethylmercapto-5-iodo-6-oxypyrimidin. It melted at 196 C. and gave a strong test for iodine. The aqueous fil- trate was concentrated on the steambath and combined with a strong solution of picric acid. We obtained a beautiful yellow picrate that crystallized from hot water in prisms. It had no definite melting point but decomposed above 26oC. and effer- vesced violently at about 285C. It did not contain water of crystallization. It contained neither iodine nor sulphur. A nitrogen determination agreed with the calculated value for 2-amino-6-oxypyrimidin (XIV) (Isocytosin) . Analysis: 0.1229 gm. of substance gave 0.03052 gm.of nitrogen = 2 1.8 c.c. T ^ HC1. For C 4 H 5 ON3.C 6 H 3 O 7 N 3 Calculated : N = 24 . 70 per cent. Found: N = 24.83 per cent. Action of Alcoholic Ammonia on 2-Ethylmercapto-$-iodo-6-oxy- pyrimidin. After heating the pyrimidin with alcoholic ammonia for six hours at 141 to 148 C. it was recovered unaltered. It was then heated for six hours at 150 to i55C. Under these conditions only a trace of mercaptan was detected. The con- tents of the tube were evaporated to dry ness. We obtained a crystalline residue. This was insoluble in water and after one crystallization from alcohol it melted at 195 C. It contained both sulphur and iodine. When mixed with the original 2- ethylmercapto-5-iodo-6-oxypyrimidin the melting point was not altered. We did not detect the presence of any isocytosin. Action of Alcoholic Ammonia on j-Iodocytosin. Five grams of iodocytosin were heated with strong alcoholic ammonia for six hours at 170 to 180 C. When the tube was opened considerable decomposition had taken place, and a well crystallized product was suspended in the alcohol. It crystallized from hot water in plates. It did not melt below 300 C. and contained no iodine. It contained one molecule of water of crystallization and agreed in all its properties with cytosin. Analysis: 0.2711 gm.of substance lost 0.0372 gm. of H 2 O at no-i2oC. For C 4 H 5 ON 3 .H 2 O Calculated: H 2 O = i3.95 percent. Found: H 2 O = i3.72 per cent. o 0909 gm. of anhydrous base gave 0.0343 gm. of nitrogen = 24. 5 c.c. T NHC1. Treat B. Johnson and Carl O. Johns 317 For C 4 H S ON 3 Calculated : N = 3 7 . 84 per cent. Found: N = 37-73 per cent. Action of Aqueous and Alcoholic Ammonia on j-Iodouracil. When iodouracil was heated with concentrated aqueous am- monia at i7o-i8oC. for four hours it was recovered unaltered. Some of the pyrimidin was then heated with strong alcoholic ammonia under the same conditions. When the tube was ex- amined slight decomposition had taken place and a colorless compound was suspended in the alcohol. It crystallized from hot water in balls of microscopic prisms and did not melt below 3ooC. More of the same material was obtained when the al- coholic solution was concentrated. It did not contain iodine and was identified as uracil. The yield was practically quanti- tative. We did not detect the presence of any amino-uracil. Analysis (Kjeldahl): 0.1192 gm, of substance gave 0.02982 gm. of nitrogen = 21. 3 c.c. ^ 0.0757 SP*- f substance gave 0.0189 g 111 - f nitrogen = 13. 5 c.c. T^ For C 4 H 4 (XN 2 Calculated : N = 25 . oo per cent. Found: N= (i) 25.01; (2) 24.96 per cent. a-Cyanbutyrylurea, CH 3 . CH 2 . CH (CN). CO. NH. CO. NH 2 . This compound was obtained by condensing a-cyanbutyric acid with urea. We proceeded according to the directions given by Traube 1 for the preparation of cyanacetylurea from cyanacetic acid and urea. It crystallized from water in arborescent crystals and melted at i8iC. with slight effervescence. Analysis: For C 6 H 9 2 N 3 Calculated : N = 2 7 . i per cent. Found : N = 2 7 . 5 per cent. N=C.NH 2 2, 4-Dioxy-5-ethyl-6-aminopyrimidin, \ \ CO CHC 2 H 5 .- a-Cyanbu- NH-CO tyrylurea dissolved in 33 per cent, sodium hydroxide solu- tion with 'absorption of heat. Fourteen grams of the urea were dissolved in 42 grams of the alkali solution and allowed to stand for several hours at ordinary temperature. The solution was then neutralized with hydrochloric acid. We obtained a 1 Ber. d. deutsch. chem. Gesellsch., xxxiii, p. 1371, 1900. 318 Researches on Pyrimidins beautiful crystalline product that decomposed at with violent effervescence. It crystallized from hot water in needle-like prisms and decomposed at 339 C. with effervescence (using an Anschutz thermometer). It was soluble in hydro- chloric acid, and was reprecipitated unaltered with ammonia. Analysis : For C H 9 2 N 3 Calculated : N = 2 7 . i per cent. Found: N = 26.9 per cent. DESCRIPTION OF PLATE I. Figure i. 5-Iodocytosin, crystallized from hot water. Magnified 60 times. Figure 2. The acetic acid salt of 5-iodocytosin. Magnified 60 times. THE JOURNAL OF BIOLOGICAL CHEMISTRY. VOL. I. PLATE I. FIGURE I. FIGURE 2. II. RESEARCHES ON PYRIMIDINS: ON METHODS OF SYN- THESIZING ISOBARBITURIC ACID, AND 5-OXY- CYTOSIN. (Fourteenth Paper.) BY TREAT B. JOHNSON AND ELMER V. McCOLLUM. (From the Sheffield Laboratory of Yale University.} (Received for publication, January 2, 1906.) In this paper we describe a new synthesis of isobarbituric acid, I. In the course of the work we have also prepared several new derivatives of this acid and of cytosin, II, which are of especial interest. NH CO N = C.NH 2 II II CO COH CO CH I II I II NH-CH NH-CH I II Wislicenus and Scheidt 1 have shown that diethyloxalate condenses with the ethyl ester of ethylglycollic acid, in presence of sodium ethylate, to form diethyloxalethylgly collate, III. COOC 2 H S + C 2 H 5 O. CH 2 . COOC 2 H S - COOC 2 H 5 C 2 H 5 OH + C 2 H 5 OOC. CO.CH(OC 2 H 5 ).COOC 2 H S . Ill It was also observed by Conrad 2 that diethylglycollate undergoes an inner condensation, in benzene, in the presence of metallic sodium to form the sodium salt of ethyl a, >/-diethoxyacetoace- tate 3 ,IV. 2 C 2 H 5 O.CH 2 .COOC 2 H s +Na = C 2 H 5 O.CH 2 .CO.C.Na.(OC 2 H 5 ).COOC 2 H s + C 2 H S OH. IV It might be expected that ethylformate would also condense. 1 Ber. d. deutsck. chem. Gesellsch., xxiv, p. 432, 1891. 2 Ibid., xi, p. 58, 1878. 3 Ann. d. Chem. (Liebig), cclxix, p. 28, 1892. 437 438 Researches on Pyrimidins with diethylglycollate in the presence of metallic sodium to form the sodium salt of ethyl-tf-ethoxy-0-oxyacrylate, 1 V. We find that this condensation takes place in the normal manner according to the following equation: C 2 H S O.CH 2 .COOC 2 H 5 +H.COOC 2 H 5 +Na = C 2 H s OH + C 2 H s O.C:(CHONa)COOC 2 H 5 V When this sodium salt, V., was dissolved in water with the calculated quantity of pseudoethylthiourea they reacted to form 2-ethylmercapto-5-ethoxy-6-oxypyrimidin, VI., as follows : NH 2 COOC 2 H 5 NH CO C 2 H S SC + COC 2 H S = C 2 H $ SC COC 2 H S +C 2 H s OH + NaOH II II II II NH CHONa N CH VI This mercaptopyrimidin, VI., was converted practically quantitatively into isobarbituric acid, I., when heated with hydrochloric acid. This change involves two distinct reactions, as follows: The 2-ethylmercapto-5-ethoxy-6-oxypyrimidin, VI., is first converted into 2,6-dioxy-5-ethoxypyrimidin, VII., with evolution of ethylmercaptan. This intermediate ethoxy- NH CO NH CO C 2 H 5 SC COC 2 H S +H 2 O = CO COC 2 H S + C 2 H 5 SH N CH NH CH VI VII pyrimidin, VII., then reacts with hydrochloric acid to give isobarbituric acid, I., with formation of ethylchloride. NH CO NH CO CO COC 2 H S +HC1= CO COH+C 2 H 5 C1 NH CH NH CH VII I The only method, given in the literature, for preparing isobar- bituric acid is that described by Behrend. 2 He prepared it by reducing nitrouracil, VIII., with tin and hydrochloric acid. We find that ethylformate condenses with ethylphenoxyacetate C 6 H5O.CH 2 .COOC 2 H S to give a quantitative yield of the sodium salt of ethyl -a- phenoxy-/?-oxyacrylate C 6 H 5 O.C:(CHONa).COOC 2 H b . We shall condense ethylformate with other glycollic esters and study some of the reactions of this new class of substituted acrylic esters. (T. B. J.) 2 Ann. d. Chem. (Liebig), ccxxix, p. 39; Behrend and Roosen, ibid., ccli, P- 239. Treat B. Johnson and Elmer V. McCollum 439 This method of preparation is not suitable for preparing large quantities of the acid. Part of the nitrouracil is recovered in the form of a minouracil, IX., and the yield of isobarbituric acid, I., corresponds to only about thirty to forty-five per cent, of the theoretical. NH CO CO COH (Yield, 30-45 per cent.) NH CO II 7\ NH CH CO CNO 2 I I II NH CH NH CO VIII ^ I I CO CNH 2 NH CH IX When 2-ethylmercapto-5-ethoxy-6-oxypyrimidin, VI., was warmed with phosphorus oxychloride, it was converted smoothly into 2-ethylmercapto-5-ethoxy-6-chlorpyrimidin, X. NH CO N = CC1 C 2 H 5 SC COC 2 H 5 + (POC1 3 ) = C 2 H S SC COC 2 H 5 N CH N CH VI X This chlorpyrimidin, X., gave a theoretical yield of 2-ethyl- mercapto-5-ethoxy-6-aminopyrimidin, XI., when heated with strong alcoholic ammonia: N = CC1 N = CNH 2 C 2 H 5 SC COC 2 H S + 2 NH 3 = C 2 H S SC COC 2 H S +NH 4 C1. N CH N CH XI Especially interesting was the behavior of this aminopyrimi- din, XL, towards hydrochloric acid. When heated with this reagent, it was converted into isobarbituric acid. We have investigated the mechanism of this reaction and find that it involves the formation of several intermediate compounds. We have succeeded in isolating each intermediate product of the reaction. 2-Ethylmercapto-5-ethoxy-6-aminopyrimidin, XI., is first 440 Researches on Pyrimidins converted into 2-oxy-5-ethoxy-6-aminopyrimidin or $-ethoxycy- tosin, XII. N = CNH 2 N = CNH 2 C 2 H S SC COC 2 H 5 + H 2 O = CO COC 2 H S +C 2 H S SH II II II N CH NH CH XII 5-Ethoxycytosin, XII., then reacts in two ways, according to the strength of the hydrochloric acid used, giving 2,5-dioxy-6- aminopyrimidin or $-oxycytosw, XIII., and 2,6-dioxy~5- ethoxypyrimidin, XIV., as follows: N = CNH 2 N = CNH 2 CO COC 2 H S +HC1 (concentrated) = CO COH + C 2 H 5 C1 NH CH NH CH XII XIII N = CNH 2 NH CO CO COC 2 H 5 +HC1 (dilute) = CO COC 2 H S +NH 4 C1 NH CH NH CH XIV 5-Oxycytosin, XIII., and 2, 6-dioxy-5-ethoxypyrimidin, XIV., then react with an excess of hydrochloric acid to form isobar- bituric acid according to the following equations: N = CNH 2 CO COH + H 2 + HC1 II x NH CO + NH 4 C1 NH CH | I XIII CO COH I II NH CO /j NH CH + C 2 H S C1 CO COC 2 H S +HC1 NH CH XIV The sodium salt of ethyl-or-ethoxy-/3-oxyacrylate, V., can also be used for preparing 5-oxy-derivatives of isocytosin. 1 Dr. C. O. Johns finds that this salt condenses with guanidin to 1 Wheeler and Johnson, A mer. Chem. Jour., xxix, p. 492, 1903. Treat B. Johnson and Elmer V. McCollum 441 give 2-amino-5-ethoxy-6-oxypyrimidin or $-eihoxyisocytosin, XV. NH 2 ;_COOC 2 H 5 NH CO H 2 NC + COC 2 H 5 = NH 2 .C COC 2 H S +NaOH + C 2 H s OH NH CHONa N CH XV We shall continue the study of these interesting pyrimidin derivatives in this laboratory. EXPERIMENTAL PART. Sodium Salt of Ethyl-a-ethoxy-p-oocyacrylate, NaO.CH:C(OC 2 H5).COOC 2 H5. This salt was prepared by slowly adding to ether, in which was suspended the calculated quantity of sodium, a mixture of molecular proportions of ethylformate and the ethyl ester of ethylglycollic acid. The condensation proceeded smoothly with evolution of hydrogen gas. In the course of 8 to 12 hours, the sodium all dissolved. We made no attempts to isolate the sodium salt. It was hydroscopic and very soluble in ether. After the condensation was complete, the excess of ether was evaporated in a partial vacuum and the crude salt dissolved in water. This aqueous solution was used for experiments that we describe in this paper. We have assumed, for the purpose of calculation, that the yield of sodium salt corresponds to about oo per cent, of the theoretical. NH CO 2 -Ethylmercapto-$-ethoxy-6-oxy pyrimidin, C 2 H 5 SC COC 2 H N CH This pyrimidin was prepared as follows: 80 grams of the hydro- bromic acid salt of pseudoethylthiourea were dissolved in about 150 c.c. of cold water and added to a strong aqueous solution containing two molecular proportions of the sodium salt of ethyl- #-ethoxy-/3-oxyacrylate. This solution was then combined with 50 c.c. of water containing a molecular proportion of potassium hydroxide (24.5 grams). The mixture was then thoroughly shaken; allowed to stand one hour at ordinary temperature, and then heated on the steam-bath for another hour. It was then thoroughly cooled and acidified with acetic acid. An excess of acetic acid should be avoided as the pyrim- 44 2 Researches on Pyrimidins idin is somewhat soluble in this reagent. The mercaptopyrim- idin separated at once as a brown, crystalline product. The crude material melted at i6o-i65 C. The yield was 50 grams, or 6 1 per cent, of the theoretical, calculating from the weight of pseudothiourea used. This yield of pyrimidin was not increased when we used more than a one-half molecular proportion of the hydrobromic acid salt of the pseudothiourea for the condensa- tion. Long standing of the alkaline solution also does not increase the yield of pyrimidin. In one experiment the solution was divided into three equal parts and treated as follows: one part was warmed immediately on the steam-bath for one hour and then acidified with acetic acid; the second part was allowed to stand two hours, and the third twenty-four hours before warming on the steam-bath and acidifying with acetic acid. The yields of pyrimidin from the three equal portions were practically the same. The pyrimidin is difficultly soluble in hot water and practically insoluble in the cold. It deposited from hot alcohol in rhombic-shaped prisms and melted at 169 C. to a clear oil. Analysis (Kjeldahl) : 0.4170 gram of substance gave 0.05712 gram of nitrogen = 40.8 c.c. j^ normal HC1. Calculated for C pyrimidine, N=:CNH a CH S SC C : NOH. Twenty-eight grams of 2-methylmer- II I N CO capto-4-oxy-6-aminopyrimidine 1 and 7.2 grams of sodium hy- droxide were dissolved in 450 cc. of water. To this solution was added a concentrated solution of 14 grams of sodium ni- trite and then, slowly, 24 grams of glacial acetic acid. The mixture was allowed to stand for 2 hours, when the nitroso de- rivative deposited as a yellow, crystalline precipitate. After thoroughly washing with cold water, alcohol and ether the crude material was used for the following preparation : 1 Johnson and Johns : Loc. cit. Researches on Pyrimidines. 173 2-Methylmercapto-4-oxy-5,6-diaminopyrimidine, N CNH, II II CH 3 SC CNH 2 . This compound was prepared by redu- NH CO cing the above nitroso derivative with ammonium sulphide. The base deposited from water in needle-like prisms that melted at 2i5-2i6, with effervescence. It was very soluble in alcohol. It was unstable and gradually assumed a red color when exposed to the air. Analysis (Kjeldahl) : Calculated for C 6 H 8 ON 4 S. Found. N 32.55 32.15 2-Ethylmercapto-6-paratoluidinopyrimidine, N= -CNHC 6 H 4 CH 3 C 2 H 5 SC CH . This compound was prepared ' II II N CH by dissolving 2-ethylmercapto-6-chlorpyrimidine and para- toluidine in dry benzene, in the proper proportions, and warm- ing the solution for several hours on the steam-bath. The ex- cess of benzene was then evaporated on the steam-bath and the pyrimidine purified in the form of its hydrochloride. This deposited from dilute hydrochloric acid in needles that decom- posed at i98-2O7, with effervescence. The salt was soluble in hot alcohol and deposited, on cooling, in needles. Analysis (Kjeldahl) : Calculated for C 13 Hi 6 N 3 S.HCl. Found. N 14.92 14.87 The free base was obtained when the hydrochloride was treated with aqueous ammonia. It separated as a pasty mass, which finally solidified. It deposited from petroleum ether in prismatic crystals that melted, at 104, to a clear oil. They were extremely soluble in alcohol and benzene. Analysis (Kjeldahl) : 174 Johnson, Johns and Heyl. Calculated for Ci 8 H 16 N 3 S. Found. N 17.14 16.93 N=CNHC 6 H 4 CH 3 2-Oxy-6-paratoluidinopyrimidine, CO CH , was I II NH - CH obtained in the form of its hydrochloride by boiling 2-ethyl- mercapto-6-paratoluidinopyrimidine with concentrated hydro- chloric acid until ethylmercaptan ceased to be evolved. It de- posited from alcohol in microscopic prisms that decomposed between i8o-i9o, with effervescence. It was soluble in hot water. Analysis (Kjeldahl) : Calculated for Found. N 17.68 17.81 When an aqueous solution of this hydrochloric acid salt was treated with ammonia the free base separated as a crystalline precipitate. It was insoluble in hot water and the ordinary organic solvents, but was purified for analysis by prolonged extraction with hot water. It decomposed slowly when heated above 280 and then melted, to a dark colored oil, at 288- 289, with violent effervescence. Analysis (Kjeldahl) : Calculated for CnH n ON 3 . Found. N 20.89 20.71 2-Ethylmercapto-6-orthotoluidinopyrimidine, N==CNHC 6 H 4 .CH $ C.H 5 SC CH . - From 2-ethylmercapto-6-chlor- II II N - CH pyrimidine and orthotoluidine. Its hydrochloride deposited from dilute hydrochloric acid in granular crystals that melted at 23o-232, with effervescence. Analysis (Kjeldahl) : Calculated for Ci 3 H 16 N 3 S.HCl. Found. N 14.92 M- 6 3 Researches on Pyrimidines. 175 When an aqueous solution of the hydrochloride was treated with ammonia the base separated as an oil which solidified on standing. It deposited from alcohol in prisms that melted, at 87, to a clear oil. Analysis (Kjeldahl) : Calculated for Ci 3 H 15 N 3 S. Found. N 17.14 l 7-4 N CNHC 8 H 4 CH 3 2-Oxy-6-orthotoluidinopyrimidine y CO CH ,was I II NH -- CH prepared by boiling 2-ethylmercapto-6-orthotoluidinopyrimidine with hydrochloric acid. The hydrochloride was very soluble in water but insoluble in benzene and acetone. It was puri- fied for analysis by crystallizing from 95 per cent alcohol. It deposited in lenticular shaped crystals that decomposed at 227- 231, with effervescence. Analysis (Kjeldahl) : Calculated for C U H U ON 8 .HC1. Found. N 17.68 17.35 The pyrimidine base was insoluble in cold water. It de- posited from hot water in radiating prisms that decomposed at 262. Analysis (Kjeldahl) : Calculated for Found. N 20.89 21.05 2-Ethylmercapto-6-paraanisidinopyrimidine, N=CNHC 6 H 4 6CH 8 C 3 H 5 SC CH .From 2-ethylmercapto-6-chlor- II II N - CH pyrimidine and paraanisidine. The hydrochloride of this base deposited from hot water in needles that melted at 2Oo-2Oi, with effervescence, to an oil. Analysis (Kjeldahl) : Calculated for C 13 H 16 ON 3 S.HC1. Pound. N 14.11 14.11 176 Johnson, Johns and Heyl. When the salt was treated with ammonia the base was ob- tained as an oil, that did not solidify after standing for several weeks. N=CNHC 6 H 4 OCH 3 2-Oxy-6-paraanisidinopyrimidine, CO CH . I II NH - CH The hydrochloride deposited from water in small, radiating needles. It was purified for analysis by recrystallizing from alcohol that was acidified with hydrochloric acid. It decom- posed between 145 and 165, with effervescence. Analysis (Kjeldahl) : Calculated for Found. I. II. N 16.56 16.72 16.65 The free base was difficultly soluble in all ordinary media and showed no crystalline form. It was purified for analysis by drying at 110, for 2 hours. It melted at 262. Analysis (Kjeldahl) : Calculated for Found. C u Hii0 2 N 3 . I. II. N 19.35 19.68 19.53 2'Ethylmercapto-6-metanitroanilinopyrimidine y - 2 H 5 SC Ni=CNHC 6 H 4 NO 3 C 2 H,SC CH . From 2-ethylmercapto-6-chlor- II II N CH pyrimidine and metanitroaniline. In this experiment we ob- served that the free base could be more easily purified than its hydrochloric acid salt. It was insoluble in water and soluble in alcohol. It melted at 175. Analysis (Kjeldahl) : Calculated for Found. C 12 H 12 2 N 4 S. I. II. N 20.29 20-6 20.13 The hydrochloride was prepared by dissolving the base in dilute hydrochloric acid. It deposited in needles that decom- Researches on Pyrimidines. 177 posed at I4o-i55, with effervescence. Analysis (Kjeldahl): Calculated for Found. Ci2H 12 O 2 N 4 S. HC1. I. II. N 17.92 17.5 17-67 N CNHC 6 H 4 NO 2 2-Oxy-6-metanitroanilinopyrimidine y CO CH . I II NH CH This compound could not be obtained in the usual manner by boiling the above 2-ethylmercapto-6-metanitroanilinopyrimi- dine with hydrochloric acid. The mercaptopyrimidine was recovered unaltered after boiling with concentrated hydro- chloric acid for 15 hours. In order to remove the mercapto radical it was necessary to boil with hydrobromic acid. The hydrobromide was purified by crystallization from 95 per cent alcohol. It decomposed between 160 and 180, with effer- vescence. When the salt was treated with alcoholic ammonia the pyrimidine base separated in needle-like prisms. They decomposed when heated above 275. Analysis (Kjeldahl) : Calculated for C 10 H 8 3 N 4 . Found. N 24.14 24.35 NEW HAVEN, CONN M June i, 1906. Reprinted from THE JOURNAL OF BIOLOGICAL CHEMISTRY, 1906, ii, pages 105-115 III. RESEARCHES ON PYRIMIDINS: 5-ETHYLCYTOSIN. (Eighteenth Paper.) BY TREAT B. JOHNSON AND GEORGE A. MENGE. (From the Sheffield Laboratory of Yale University.) (Received for publication, May 13, 1906.) Methods of synthesizing uracil, I, thymin, II, cytosin, IV, and 5-methylcytosin, V, have been described in previous papers from this laboratory. 1 Thymin, II, and 5-methylcytosin, V, are the simplest mono- 5-alkyl derivatives of uracil, I, and cytosin, IV, respectively. They may be considered as the second members of two homolo- gous series of which uracil, I, and cytosin, IV, are the primary substances. NH CO NH CO NH CO CO CH -> CO C.CH, H> CO C.CH 2 CH 3 NH CH NH CH NH CH I II III N = CNH 2 N - CNH, N = CNH 2 CO CH -> CO C.CH 3 -> CO C.CH,.CH 3 NH CH NH CH NH CH IV V VI The work described in this paper was undertaken with the object of preparing 5-ethyluracil, III, and 5-ethylcytosin, VI, the third homologues of these two series. We find that ethylformate condenses with normal ethylbuty- rate in the presence of sodium ethylate, or metallic sodium, to form the sodium salt of ethyl formylbutyrate, VII: CH 3 .CH 2 .CH 2 COOC 2 H S CH 3 .CH 2 .C:(CHONa)COOC,H, + C 2 H 5 OH VII When this sodium salt, VII, was dissolved in water with the Wheeler and Merriam, Amer. Chem. Jour., xxix, p. 478, 1903; Wheeler and Johnson, ibid., xxix, p. 492, 1903; ibid,, xxxi, p. 591, 1904. 105 io6 Researches on Pyrimidins : 5-Ethylcytosin calculated quantity of pseudoethylthiourea they condensed to form 2-ethylmercapto-5-ethyl-6-oxypyrimidin, IX. This con- densation involves the intermediate formation of a-eihyl-/3- pseudoethylthioureaacrylic acid, VIII. We have succeeded in isolating this compound. NH 2 COOC 2 H $ NH 2 COOH C 2 H 5 SC + CC 2 H S - C 2 H S SC CC 2 H 5 +NaOH-f C 2 H 5 OH NH CH.ONa N CH VIII NH 2 COOH NH CO C 2 H S SC CC 2 H 5 = C 2 H 5 SC CC 2 Hs + H 2 O IX Practically a quantitative yield of 5-ethyluracil, III, was ob- tained when this mercaptopyrimidin, IX, was boiled with hydro- bromic acid: NH CO NH CO C 2 H 5 SC CC 2 H S + H 2 O = CO C C 2 H S + C 2 H 5 SH N CH NH CH IX III When 2-ethylmercapto-5-ethyl-6-oxypyrimidin, IX, was heated on the steam-bath with phosphorus pentachloride it was converted into 2-ethylmercapto-5-ethyl-6-chlorpyrimidin, X: NH CO N = CC1 CjHjSC CC 2 H 5 +PC1 5 = C 2 H S SC CC 2 H $ +POC1 3 + HC1 Jl-CH A-CH X This mercaptochlorpyrimidin, X, then gave a quantitative yield of 2-ethylmercapto-5-ethyl-6-aminopyrimidin, XI, when lieated with alcoholic ammonia: N=CC1 N jSC CC 2 H $ + 2 NH,= C 2 H S SC CC 2 H S + NH 4 C1 II II II li N CH N CH XI Finally this 2-ethylmercapto-5-ethyl-6-aminopyrimidin, XI Treat B. Johnson and George A. Menge 107 was converted into the hydrobromide of 5-ethylcytosin, VI, by boiling with hydrobromic acid. N = CNH ? N = CNH, C 2 H S SC CC 2 H $ + HBr + H 2 O = C 2 H S SH + CO CC 2 H,.HBr N CH NH CH VI 5-Ethylcytosin, VI, separates from water without water of crystallization. This property is of especial interest. Cytosin, IV, and 5-methylcytosin, V, the preceding members of the same series, crystallize with one molecule and a half molecule of water of crystallization respectively. C 4 H 5 ON 3 .H 2 0, C,H 7 ON 3 .* H 2 p, C 6 H 9 ON 3 . (Cytosin) (5-Methylcytosin) (5-Ethylcytosin) 5-Ethylcytosin is more soluble in water than cytosin, and less soluble than 5-methylcytosin. It forms normal salts (1:1) with hydrochloric, hydrobromic, and nitric acids. One of the most characteristic properties of 5-methylcytosin was its tendency to form basic salts 1 with hydrobromic and hydrochloric acids. We now find that 5-ethylcytosin likewise possesses this interesting property. When ammonia was added to an aqueous solution of the hydrobromide we obtained a mixture of two hydrous, basic salts. Our analytical determina- tions indicated that we were dealing with a 2:1, XII, and a 3:1 hydrobromide, XIII. On account of the small amount of 5- ethylcy tosin at our disposal we were unable to make as thorough a study of these basic salts as we desired. (C 6 H 9 ON 3 ) 2 . HBr. H 3 O, (C d H 9 ON 3 ) 3 . HBr. H,O. XII XIII 5-Ethylcytosin is precipitated by phosphotungstic acid. The- amino radical is more firmly linked in this base than in cytosin and 5-methylcytosin. While the latter two bases are converted into uracil and thymin when heated with 20 per cent, sulphuric acid, 5-ethylcytosin was recovered unaltered when treated under the same conditions. i Wheeler and Johnson, loc. cit. o < o s o . ii I' O H ffi o ** fe a o O Q H fc B if >< :2 I" 2 * lg *% js 13 ^ P. Solubili Water a OJDOO HHBM . w & - ffi 5 o-o =o 000 i 00=0- is- lift **. 1 08 Treat B. Johnson and George A. Menge 109 EXPERIMENTAL PART. Sodium Salt of Ethyl Formylbutyrate , C,H S .C: (CHONa)COOC 2 H s . This salt can be prepared very easily by proceeding in the following manner: A known weight of alcohol-free sodium ethylate was suspended in dry ether. A mixture of the calcu- lated quantities of ethylformate and ethylbutyrate was then added to the ether and the condensation allowed to proceed at ordinary temperature. Sufficient heat was evolved to cause the ether to boil. The mixture was allowed to stand for four days when the excess of ether was removed by sucking a current of dry air through the solution. We obtained a thick oil which immediately solidified to a solid cake when treated with a little water. No further purification was attempted. This salt was- dissolved in cold water and the solution washed with ether to re- move any unaltered esters. This solution was then used for the condensations described in this paper. In our calculations, we have assumed that the sodium salt is formed in practically theoretical amount. NH-CO 2-Ethylmercapto-$-ethyl-6-oxypyrimidm, C 2 H S SC CC 2 H, N-CH One hundred and fifty grams of normal ethylbutyrate were con- densed with ethylformate in presence of sodium ethylate as- described in the preceding experiment. The resulting sodium salt of ethyl formylbutyrate was then dissolved in water, and treated with an aqueous solution of 0.5 molecular proportion of pseudoethylthiourea. This was prepared by dissolving 119 grams of the hydrobromide in ice water and neutralizing the hydro- bromic acid with 3 6 grams of potassium hydroxide. The alkaline solution was allowed to stand over night at ordinary temper- ature and then heated on the steam-bath for about two hours. The solution was then filtered and acidified with acetic acid. The mercaptopyrimidin separated at once as a flocculent pre- cipitate. After drying over sulphuric acid the crude material weighed 40 grams, corresponding to 38 per cent, of the theoretical,. 1 10 Researches on Pyrimidins : 5-Ethylcytosin calculating from the weight of pseudourea used. It was ex- tremely soluble in alcohol and benzene, and moderately soluble in ether. It was difficultly soluble in water. It deposited from hot water or dilute alcohol in well developed prisms that melted at 1 1 9-1 20 C. to a clear oil. Analysis (Kjeldahl): Calculated for C 8 H, 2 ON a S: Found: N -15 . 17 per cent. 15 . 12 per cent. a-Ethyl-p-pseudoethylihioureaacrylic acid , H 2 N(C 2 H S S):N.CH:C(C 2 H S ).COOH. This was obtained as an intermediate product in the preceding condensation. After filtering from the 2-ethylmercapto-5-ethyl- 6-oxypyrimidin the acetic acid filtrate was concentrated on the steam-bath and then allowed to stand at ordinary temperature for several hours. A heavy, flocculent precipitate separated on cooling. The substance was very soluble in alcohol. When heated above its decomposition point it was converted into the mercaptopyrimidin above. It deposited from hot alcohol in plates that melted at 148 149 C., with effervescence, to a clear oil. When this oil was allowed to cool below 100 C. it solidified. Upon heating again in the capillary tube it melted at 117-! 18 C. to a clear oil. Analysis (Kjeldahl): Calculated for C 8 H, 4 O 2 N 2 S: Found: N -13.86 per cent. 13 .70 per cent. N- CC1 2-Ethylmercapto-$-ethyl-6-chlorpyrimidin, C 2 H $ SC CC 2 H S Twenty-five grams of 2-ethylmercapto-5-ethyl-6-oxypyrimidin and a slight excess over one molecular proportion of phosphorus pentachloride were mixed in a flask and gently heated on the steam-bath. A violent reaction took place with evolution of hydrochloric acid gas. We obtained a dark colored liquid that was heated for several minutes at 100 C. to complete the reaction. The liquid was then poured upon crushed ice to de- compose the phosphorus halides. The chlorpyrimidin was ex- tracted with ether, dried over calcium chloride, and purified by distillation under diminished pressure. It boiled at i6o-i63 G. Treat B. Johnson and George A. Menge in under 24 mm. pressure. It would not solidify in a freezing mixture. The yield of pure distilled material was 14 grams, or about 54 per cent, of the theoretical. When boiled with water it was converted into the original 2-ethylmercapto-5-ethyl-6-oxy- P3'rimidin. Analysis (Kjeldahl): Calculated for C,H U N 2 SC1: Found: N -=13 8 per cent. 13-43 per cent. N=CNH 2 2-Ethylmercapto-$-ethyl-6-ammopyrimidin, C 2 H 5 SC CC 2 H S N- CH This aminopyrimidin was obtained when the chlorpyrimidin above was heated in a sealed tube with alcoholic ammonia at 130 140 C. for two hours. The excess of ammonia and alcohol was then removed by evaporation on the steam-bath. The residue was then treated with water to remove ammonium chloride. We obtained an oil that was very soluble in ether. When the ether solution was allowed to evaporate spontaneously the aminopyrimidin deposited in prismatic crystals. It was insoluble in ligroin but extremely soluble in benzene. It deposited from a mixture of these two solvents in stout prisms that melted at 74-76 C. to an oil. Analysis (Kjeldahl): Calculated for C 8 H 13 N 3 S: Found: N=22.95 per cent. 22.88 per cent NH-CO 2, 6-Dioxy-$-ethylpyrimidin ($-Ethyluracil) , co CC 2 H $ NH-CH This pyrimidin was prepared by boiling 2-ethylmercapto-5- ethyl-6-oxypyrimidin with hydrobromic acid. It was difficultly soluble in water. One part of the pyrimidin dissolved in about 625 parts of water at 25 C. It deposited from hot water in balls of microscopic prisms that melted at about 300 C. with decom- position. Analysis (Kjeldahl): Calculated for C 6 H 8 O 2 N 2 : Found: N=2o.o per cent 19.8 per cent. H2 Researches on Pyrimidins : 5-Ethylcytosin $-Eihylcytosin Monohydrobromide (Anhydrous). This salt was obtained when 2-ethylmercapto-5-ethyl-6-amino- pyrimidin was boiled with hydrobromic acid. When the acid solution was allowed to stand, the hydrobromide deposited in large prisms. They did not contain water of crystallization. When heated in a capillary tube the salt began to darken at about 265 and melted at 284-286 C. with effervescence. Analysis (Kjeldahl): Calculated for C 6 H 9 ON 3 .HBr: Found: N=i9.i3 per cent. 19.2 per cent. N = CNH 2 2-Oxy-$-ethyl-6-aminopyrimidin ($-Ethylcytosiri) , co CC Z H S NH CH In order to obtain this base, the monohydrobromide, described above, was dissolved in water; the bromide was removed by means of silver sulphate; the excess of silver was precipitated with hydrogen sulphide, the sulphuric acid by barium hydroxide, and then the excess of barium with carbon dioxide. The clear solution was then concentrated to a small volume, whereupon on cooling the base deposited in beautiful, slender prisms. They did not contain water of crystallization. One part of the base dissolved in about 75 parts of water at 25 C. When heated in a capillary tube it melted with effervescence at 282-283 C. Analysis (Kjeldahl): Calculated for CH 9 ON 3 : Found: N =30.21 per cent. 30.03 per cent. The base was precipitated from its aqueous solution by mercuric chloride and phosphotungstic acid. The phospho- tungstate was soluble in boiling water and deposited again on cooling. When a solution of the pyrimidin base was treated with a solution of potassio-bismuth iodide a red precipitate was ob- tained. It was insoluble in hot water. 5-Ethylcytosin was heated with 20 per cent, sulphuric acid for six hours at i4o-i5oC. When the pressure tube was exam- ined there was no apparent decomposition. Upon concentrating on the steam-bath we did not obtain a deposit of 5-ethyluracil. The sulphuric acid was removed with barium hydroxide, and the Treat B. Johnson and George A. Menge 113 excess of alkali with carbon dioxide. When the neutral solution was concentrated to a small volume, the unaltered base deposited in characteristic, slender prisms that melted at 282-283C. with effervescence. When mixed with the original base the melting-point was not lowered. Platinum Chloride Double Salt. This salt was extremely soluble in water. It deposited from hot water in radiating, transparent prisms. A nitrogen deter- mination gave the following result: Calculated for (C 6 H 9 ON 3 ) 2 .H 2 PtCl 6 .2H a O: Found: N 11.62 per cent. 11.56 per cent The Pier ate of $-Ethylcytosin. This salt was prepared by treating a solution of the base with picric acid. It was difficultly soluble in water. It deposited from hot water in yellow, opaque crystals. When heated in a capillary tube they decomposed at 277-278C. $-Ethylcytosin Monohydrochloride (Anhydrous). This salt was prepared by dissolving 5-ethylcytosin in dilute hydrochloric acid and allowing the solution to slowly evaporate in a desiccator over sulphuric acid. It deposited in flat prisms. They were extremely soluble in cold water and melted at 238- 240 C. with slight effervescence. Analysis (Kjeldahl): Calculated for C 6 H 9 ON 3 . HC1 : Found : N-23-93 per cent. 23.90 per cent. Nitrate of $-Ethylcytosin. Obtained by allowing a nitric acid solution of the base to slowly evaporate in a desiccator over sulphuric acid. It de- posited in stout prisms. They decomposed at about 1 70-! 7 2 C. The salt was very soluble in cold water. Analysis (Kjeldahl) : Calculated for C 6 H 9 ON 3 .HNO 3 : Found: N 27.72 per cent. 2 7-39 P 6 *" cent. Basic Hydrobromides of $-Ethylcytosin. Some of the 2-ethylmercapto-5-ethyl-6-aminopyrimidin was boiled with hydrobromic acid until ethyl mercaptan ceased to be evolved. The solution was then evaporated to dry ness to remove the excess of acid. Upon adding ammonia to an aqueous solution H4 Researches on Pyrimidins : 5-Ethylcytosin of the hydrobromide we obtained an immediate precipitate in the form of well developed prisms. This precipitate was'difficultly soluble in water. It deposited from hot water in clusters of radiating prisms associated with diamond-shaped crystals. They decomposed at 258-26o C. with violent effervescence. We were unable to raise this decomposition point by repeated recrystallizations from hot water. The material gave a strong test for bromine. The analytical determinations indicated that we were not dealing with a homogeneous salt, but a mixture of two hydrous, basic salts. Our determinations agreed with the calculated values for a mixture of about equal proportions of a 2:1 and a 3:1 hydrobromide. 0.3358 gram of substance lost 0.0127 gram on heating to constant weight at ioo-iio C. For (C 6 H 9 ON 3 ) 2 .HBr.H 2 O Calculated: H 2 O =4.8 per cent. For (C 6 H 9 ON 3 ) 3 HBr.H 2 O Calculated: H 2 O = 3.4 per cent. Average: H 2 O 4.1 per cent. Found: H 2 O - 3.78 per cent. Nitrogen determinations in the hydrous material (Kjeldahl) : For (C 6 H 9 ON 3 ) 2 .HBr.H 2 O Calculated : N =22.31 per cent. For (C 6 H 9 ON 3 ) 3 .HBr.H 2 O Calculated: N= 24.41 per cent. Average : N =23.36 per cent. Found : N (i) 23.29; (2) 23.31 per cent. Nitrogen determinations in the anhydrous material (Kjeldahl) : For (C 6 H 9 ONj) a .HBr Calculated : N =23.39 per cent. For (C,H 9 ON 3 ) 3 .HBr Calculated : N = 25 .30 per cent. Average : N =24.34 per cent. Found : N = (i) 24.48; (2) 24.07 per cent. N = CC1 2, 6-Dichlorpyrimidin, C1C CH N CH This compound has been described by Gabriel. 1 He prepared iBer, d. deutsch. chem. Gesellsch., xxxviii, p. 1690, 1905. Treat B. Johnson and George A. Menge 115 it by heating uracil with phosphorus oxy chloride at 140 C. He states that it melted at 61 C. and boiled at 2o8.5-209.5 C. at 773 mm. We had prepared the same chloride, previous to this publication, by warming 2-thiouracil 1 on the steam-bath with phosphorus pentachloride. From 35 grams of 2-thiouracil we obtained 19.2 grams of the dichlorpyrimidin boiling at 101 C. at 23 mm. In another experiment we obtained 13 grams from 20 grams of 2-thiouracil. It boiled at 198 C. at 760 mm. Our compound melted at 63 C. to a clear oil. It was soluble in alcohol, benzene, and ether. Its vapors attacked the eyes. Analysis (Kjeldahl): Calculated for C 4 H 2 NjCl,; Found: N 18.79 P er cent. 18.78 per cent. Wheeler and Bristol, Amer. Ghent. Jour, xxxiii, p. 458, 1905. [Reprinted from the American Chemical Journal, Vol. XXXVII. No. 4. April, 1907.] Contributions from the Sheffield Laboratory of Yale University. CXLII. RESEARCHES ON PYRIMIDINES: SYNTHESIS OF URACIL-5-CARBOXYUC ACID. [NINETEENTH PAPER.] BY HENRY I,. WHEELER, TREAT B. JOHNSON, AND CARL O. JOHNS. A new substance, having acid properties, called orotic acid (Acido orotico) has been isolated by Biscaro and Belloni 1 from milk. They assign to this compound the empirical formula, C 5 H 4 O 4 N 2 .H 2 O, and believe that its structure is to be represented by one of the following formulas: NH.CH 2 .CO /NH .CO .CH | NH . CO.CO OC | . \NH. CO .CO 1 Annuario della Soc. Chimica di Milano, XI, fasc. I (1905); /dzYf.,XI, fasc. II (1905) Vide also Centrabl., 1905, I, 63, 64. We take this occasion to thank the above authors for sending us reprints of these two articles. Researches on Pyrimidines. 393 It seemed to us that instead of a ring with 7 members, one with 5 or 6 would most probably prove to be correct. The properties of the substance and the fact that it gives urea on oxidation, suggested that it might be a pyrimidine. The above empirical formula is that of a uracil carboxylic acid having a molecule of water of crystallization. There are 2 uracil carboxylic acids which have the acid group at- tached to carbon, namely, the 4- (I.) and 5- (II.) derivatives, HN CO HN CO I I I CH , OC CCOOH. I II I II HN CCOOH HN CH I. II. As neither of these acids have hitherto been prepared, a de- scription is now given of the preparation and properties of the latter acid, while an account of uracil-4-carboxylic acid will appear in a later article. The study of these acids, and also the carboxyl derivatives of thymine and cytosine, is of in- terest on account of the possibility that in the nucleic acids uracil, thymine and cytosine may exist as carboxyl deriva- tives, being held together not alone by means of phosphorus atoms, as suggested by Bang 1 and Osborne and Harris, 2 but also by an acid amide or polypeptide grouping as in the case of the proteids. In such an event the means hitherto used to dis- rupt the nucleic acid molecule must also cause a loss of the carboxyl group from the pyrimidines. In this connection it is interesting to note that uracil-4-carboxylic acid can be heated with 20 per cent sulphuric acid, at i6o-i 70, without alteration while, by such treatment, uracil- 5 -carboxylic acid is quantitatively converted into uracil. In fact, simply on prolonged boiling of this acid with hydrochloric acid it is con- verted into uracil. Uracil may, therefore, exist in the nucleic acids as a 5-carboxyl compound. We have shown that the pseudothioureas, H 2 NC(SR) : NH, will react with aldehyde 3 1 Z. physiol. Chem., 31, 425 (1900). 2 Conn. Exper. Sta. Rep., 1901, p. 418. 3 THIS JOURNAL, 29, 480 (1903). 394 Wheeler, Johnson and Johns. and ketone esters to form pyrimidines in cases where urea re- fuses to act. According to Behrend the alkyl acetoacetic esters do not condense with urea. 1 We found that the methyl and ethyl derivatives give pyrimidines with the pseudothio- ureas. In that article we described 4,5-dimethyluracil as new, having missed the fact that Schlenker had obtained this sub- stance by a different method. 2 Another striking example of the difference between urea and a pseudothiourea was found when we tried the behavior of Claisen's ethoxymethylenemalonic ester 3 toward these compounds. Urea showed no sign of reacting at 140 and a condensation was not effected on standing in alkaline solu- tions. On the other hand, when ethoxymethylenemalonic ester was added to an alkaline, aqueous solution of ethylpseudothiourea hydrobromide, 4 an immediate reaction took place and a salt of 2-ethylmercapto-5-carbethoxy-6-oxypyrimidine separated. This condensation can be represented as follows : HNH C 2 H 5 OCO HN CO C 2 H 5 OH i CC( C 2 H 5 SC + CC0 2 C 2 H 5 ==C 2 H 5 SC CCO 2 C 2 H 5 + II II II II NH C 2 H 5 OCH N CH C 2 H 6 OH 2-Kthylmercapto-5-carbethoxy-6-oxypyrimidine (I. see be- low) can be converted directly into uracil-5-carboxylic acid (IV.) by simply boiling with aqueous hydrochloric acid. When the mercapto ester (I.) is boiled, in alcoholic solution, with a small quantity of hydrochloric acid, uracil-5-ethylcarboxylate (II.) results. And this, on saponification, gives uracil-5- carboxylic acid. On the other hand, warm alkalies saponify the mercapto ester (I.) and give 2-ethylmercapto-5-carboxyl- 6-oxypyrimidine (III.), which, with hot hydrochloric acid, gives uracil-5-carboxylic acid (IV.). When the acid (IV.) is melted it gives uracil (VI.). 1 Ann. Chem. (Uebig), 229, 16 (1885). 2 Ber. d. chem. Ges., 34, 2812 (1901). a Ann. Chem. (I,iebig), 297, 75 (1897). 4 THIS JOURNAL, 29, 483 (1903). Researches on Pyrimidines. 395 The formation of uracil, in this manner and also on heating with acids, shows that the condensation took place as repre- sented in the above equation and that the product (I.) is not a barbituric acid derivative. In fact, we have not yet observed a satisfactory condensation of malonic ester, or any of its derivatives, with the pseudothioureas to form barbituric acid derivatives. The substance represented by Formula V., uramidomethylenmalonic ester, was occasionally found as a by-product in the mother liquors of the condensation. On saponification it gave uracil- 5 -carboxy lie acid. These compounds and their transformations can be represented as follows: HN CO 6 SC HN CO HNH COOC 2 H 6 C C 2 H 6 SC CC0 2 C 2 H 6 *^C 2 H 5 SC CCO 2 H OC CCO 2 C 2 H 6 N CH N CH III. HN CH V. HN CO OC CC0 2 C 2 H 5 I II HN CH II. HN-CO OC CCO 2 H I II HN CH IV. HN CO OC CH I II HN CH VI. In some respects the properties of uracil-5-carboxylic acid agree closely with those of orotic acid. It is difficultly solu- in water, difficultly or insoluble in organic solvents and it crys- tallizes with the required i molecule of water of crystalliza- tion. Orotic acid is said to decompose at 260. Our acid melts, with decomposition, at 278. The potassium salt that crystallizes from solutions containing an excess of alkali is a monopotassium salt, C 5 H 3 O 4 N 2 K. Like orotic acid, our acid is dibasic and forms 2 series of salts. The potassium salt gives precipitates with barium chloride (crystalline), lead acetate and silver nitrate. On the other hand, uracil-5-carboxylic acid differs from 396 Wheeler, Johnson and Johns. orotic acid as follows: It does not give a monosilver salt hav- ing the composition C 5 H 3 O 4 N 2 Ag.H 2 O, which Biscaro and Belloni state is formed when silver nitrate is added to a solu- tion of the monopotassium salt. In our case, when the mono- potassium salt or alkali, acid and silver nitrate, in the pro- portion i : i : i were used, the silver salt formed was a disilver salt, a corresponding amount of acid remaining in the filtrate. When methyl iodide was allowed to react, under pressure, on either mono or disilver orotate, Biscaro and Belloni obtained a monomethyl derivative melting at 248-25o. When we heated our silver salt with methyl iodide, in a closed tube at 100, we obtained a mixture of substances from which a dimethyl derivative, melting about 254-256, was isolated. Their monoethyl ester melted at 200; our product, obtained in like manner from the silver salt, melted at i62-i63, and the analysis showed that it was a diethyl derivative. The free uracil-5-carboxylic acid does not give a precipi- tate with barium chloride, and we were unable to obtain a di- chloride, which could be crystallized from boiling water with- out reverting to the acid, when the potassium salt was heated with phosphorus oxychloride. EXPERIMENTAL PART. 2-Ethylmercapto-5-carbethoxy-6-oxypyrimidine, HN CO CC0 2 C 2 H 5 . C 2 H 5 SC CCO 2 C 2 H 5 . The best results in the prepara- N CH tion of this substance were obtained as follows: Fifty-four grams of ethoxymethylenemalonic ester were added to a solu- tion of 50 grams of ethylpseudothiourea hydrobromide in 50 cc. of water. To this mixture 30 grams of potassium hydrox- ide were slowly added and the solution was kept cold. The addition of alkali produced a yellow color and the solution became semisolid from the separation of a bulky, yellow precipitate. This impure potassium salt, when dry, weighed 58 grams, the yield being 87 per cent of the calculated, theory Researches on Pyrimidines. 397 requiring 66.5 grams. It crystallized from water in the form of colorless prisms and from alcohol in needles. When a strong aqueous solution of the potassium salt was treated with dilute hydrochloric acid, avoiding an excess of acid, 2-ethylmercapto-5-carbethoxy-6-oxypyrimidine separated as a white, crystalline precipitate. This substance is readily soluble in hot alcohol and it separates in the form of long, slender, colorless prisms; from water it forms long, bulky, asbestos-like needles. It melts at 131 to a clear oil and it gave the following results on analysis (Kjeldahl) : Calculated for Found. C 9 Hi2O 8 N 2 S. I. II. N 12.23 12.02 12.28 This ester dissolves in cold hydrochloric acid and is repre- cipitated by ammonia. It has both acid and basic proper- ties. Uramidomethylenemalonic Ester, H 2 NCONHCH : C(CO 2 C 2 H 5 ) 2 . This substance was obtained, in 2 experiments, by pre- cipitating with hydrochloric acid, the filtrate from which the potassium salt of 2-ethylmercapto-5-carbethoxy-6-oxypyrimidine had separated. In the above case the amount obtained was 4.8 grams. It is very difficultly soluble in boiling water, readily soluble in hot alcohol; when crystallized from 50 per cent alcohol it forms bunches of colorless prisms melting at 206, with effervescence. Nitrogen determinations gave (Kjeldahl) : Calculated for Found. C 9 H 14 6 N2. I. II. N 12.17 12.22 12. O6 This material has acid properties. When warmed with alkali, saponification takes place and hydrochloric acid then precipitates uracil-5-carboxylic acid. 2-Ethylmercapto-5-carboxyl-6-oxypyrimidine, HN CO I I C 2 H 5 SC CCO 2 H. Ten grams of the potassium salt of II II N CH 2-ethylmercapto-5-carbethoxy-6-oxypyrimidme were dissolved 398 Wheeler, Johnson and Johns. in hot alcohol and a hot solution of 4.3 grams of potassium hydroxide, in a little water, were added. Saponification took place immediately and the solution became a thick jelly. The alcohol was then evaporated and the residue taken up in a little water. On adding an excess of hydrochloric acid a white precipitate of the mercapto acid separated. It weighed 7.1 grams, while the calculated is 7.5 grams, the yield being 94.6 per cent of theory. This acid dissolves readily in hot alcohol, less readily in hot water, from which solvent it forms bunches of colorless, sharp- cornered plates. It melts to a clear oil at 167 and effer- vesces at a higher temperature. Nitrogen determination: Calculated for C 7 H 8 3 N 2 S. Found. N 14-00 13.72 This acid appears to be stronger than acetic acid, since it was not liberated from its potassium salt by means of an excess of acetic acid. It gives a bulky, white, gelatinous precipitate with alkali and silver nitrate. When warmed with hydrochloric acid, it readily evolves mercaptan and gives uracil-5-carboxylic acid. HN CO I I Uracil-5-ethykarboxylate, OC CCO^Hg. Ten grams HN CH of the potassium salt of 2-ethylmercapto-5-carbethoxy 6 oxypyrimidine were dissolved, or suspended, in about 75 cc. of strong alcohol, and about 5 cc. of concentrated hydrochloric acid were added. The whole was then boiled with a return condenser for several hours; the alcohol was evaporated and the residue was taken up in about 60 cc. of warm water. The hydrochloric acid was neutralized with ammonia and the solution was then filtered hot. On adding an excess of strong ammonia to the filtrate the ammonium salt of uracil- 5 -ethyl- carboxylate separated as a sponge-like mass of long, slender, colorless needles. This was dissolved in about 50 cc. of water by warming and then a slight excess of acetic acid was added. Researches on Pyrimidines. 399 On cooling, a granular, crystalline precipitate of the free ester separated. This was found to be very soluble in hot water and moderately so in cold. It was difficultly soluble in alco- hol. When crystallized from water it formed flat, sharp- pointed prisms, melting at 236-237. Analysis: Calculated for Found. C 7 H 8 4 N 2 . I. II. N 15.21 15.09 15.21 The ammonium salt of uracil-5-ethylcarboxylate is charac- terized by some peculiar properties. When a not too dilute solution of the ester is treated with a little strong ammonia, long, slender needles of the ammonium salt form at once. The vapor from a rod dipped in ammonia and held above the surface of an aqueous solution of the ester will cause crys- tals to form. When a solution of the salt is boiled, ammonia escapes and, on cooling, nothing separates until more ammo- nia is added. If the solution is concentrated by heating, the free ester separates. The ammonium salt is insoluble, or difficultly soluble in alcohol. An aqueous solution of the ester was precipitated with ammonia and the precipitate was dried over calcium chloride. The needles thus obtained melted at about 220 to an oil that effervesced. Nitrogen determina- tion (Kjeldahl) : Calculated for C 7 H 8 4 N 2 NH 3 .H 2 0. Found. N 19.13 19.33 When uracil-5-carboxylic ethyl ester is warmed with aqueous hydrochloric acid it is readily saponified and uracil-5-carboxylic acid is obtained. HN CO I I Uracil-5-carboxylic Acid, OC CCO 2 H.H 2 O. This acid HN CH can be prepared directly from the condensation product of ethoxymethylenemalonic ester and ethylpseudothiourea hy- drobromide, or, in other words, from the potassium salt of 2-ethylmercapto-5-carbethoxy-6-oxypyrimidine, without the 400 Wheeler, Johnson and Johns. isolation of the above intermediate products, by simply dissolv- ing the salt in a moderate amount of strong hydrochloric acid and evaporating to dryness on the steam-bath. If the prod- uct contains sulphur, the operation is repeated. The resi- due is then crystallized from water as the acid is practically insoluble in alcohol. It is difficultly soluble in water and sepa- rates in the form of colorless, characteristic, minute pyramids with rough faces. Or small prisms form, the faces sometimes alternating with those of a pyramid. The crystals contain iH 2 O, which is given off on heating above 100. The acid melts partially, but quite sharply, at 278, with energetic effervescence. The aqueous solution reddens litmus and it gives no immediate precipitate with lead or copper acetate, or barium chloride. Analysis : I. 0.5002 gram substance, dried at about 50, lost 0.0502 gram of water when heated for i hour at 122 II. 0.5486 gram substance lost 0.0561 gram water when heated for 2 hours at io5-uo. Calculated for Found. C 6 H 4 04N2.H 2 0. I. II. III. H 2 O IO.34 10.03 10.22 10.02 N 16.09 16.15 16.03 16.26 Nitrogen determination of the anhydrous acid : Calculated for Found. C5H 4 4 N2. I. II. III. N 17.94 17-88 17.99 17.91 Nitrogen was determined by Kjeldahl's method except in Analysis III. The acid used for Analysis III. (water) and Analysis VI. was obtained by the saponification of uramido- methylenemalonic ester. The other samples were obtained from the potassium salt described above. Behavior on Heating. About 0.5 gram of the acid was heated in a test tube, immersed in a sulphuric acid bath, at a temperature a little above its melting point until efferves- cence ceased. The dark-colored product was boiled with water and animal charcoal and the solution concentrated. On cooling, the characteristic crystals of uracil separated, melting or effervescing at 335. Analysis (Kjeldahl) : Researches on Pyrimidines. 401 Calculated for Found. N 25.00 24.89 Action of Acids: Formation of Uracil. The action of 20 per cent sulphuric acid was tried as follows: Fifty-five-hun- dredths gram of the acid potassium salt of uracil-5-carboxylic acid was sealed in a tube with 5 cc. of sulphuric acid, sp. gr. = 1.15, and heated at i6o-i69 for 2 hours. On opening the tube, carbon dioxide escaped and the minute microscopic needles that had separated, after washing with water, were found to melt at 336 without further purification. A nitrogen determination agreed with the calculated for uracil : Calculated for C 4 H 4 O 2 N 2 . Found. N 25.00 24.97 The needles weighed 0.25 gram, which amount is 71 per cent of the calculated, or, allowing for solubility, it is prac- tically a theoretical yield. The Action of Concentrated Hydrochloric Acid. One gram of uracil-5-carboxylic acid was boiled with about 50 cc. of concentrated hydrochloric acid, using a return condenser, for 1 8 hours. The acid was then evaporated and the residue was crystallized from water, whereupon over 0.3 gram of uracil separated. Esterification: Methyl Uracil- 5-carboxylate, HN CO I CCO 2 CH 3 . When uracil-5-carboxylic acid is warmed I II HN CH with acids in methyl or ethyl alcohol, it is smoothly esterified. One gram of the anhydrous acid was suspended in 75 cc. of methyl alcohol, to which 5 drops of concentrated sulphuric acid had been added. On boiling for 8 hours, all of the acid went into solution and on evaporation to a small volume a gelatinous mass was formed. This gave a white powder when stirred with cold water. The material thus obtained was readily soluble in hot water and on cooling, short, stout, color- 402 Wheeler, Johnson and Johns. less prisms separated. These sintered at about 2 2 5 and decom- posed at 233. Nitrogen determination (Kjeldahl): Calculated for Pound. C 6 H 6 4 N 2 . I. II. N 16.47 16.62 16.58 When uracil-5-carboxylic acid was warmed with ethyl alcohol and sulphuric acid, the ethyl ester described above was obtained. It was identified by the melting point 236 and by its behavior towards ammonia. The Acid Potassium Salt, C 5 H 3 O 4 N 2 K.C 5 H 4 O 4 N 2 . When uracil- 5-carboxylic acid and either sodium or potassium hydroxide were mixed, in molecular proportions, the hot aqueous solu- tion, on cooling, deposited an acid salt. For example, 8 grams of the hydrous acid and 2.5 grams of potassium hy- droxide were dissolved in about 500 cc. of boiling water. On cooling, 2 grams of slender, needle-like crystals separated. These effervesced at about 270. Analysis: Calculated for Found. C 10 H 7 8 N 4 K. I. II. III. N 16.00 15.89 15.98 K 11.14 10.87 The Monopotassium Salt, u. C 5 H 3 O 4 N 2 K. Three grams of uracil-5-carboxylic acid were dissolved in a solution of 2 grams of potassium hydroxide, 1.9 grams = 2 mols., in about 20 cc. of water ; on concentrating on the steam-bath small needles separated. These were dried at about 55. Nitrogen deter- minations (Kjeldahl) : Calculated for Found. C 6 H 2 O 4 N 2 .K2. C 6 H 3 O 4 NaK. I. II. N 12.06 14.43 14.72 14.71 The analyzed material showed no signs of melting at 295. On dissolving the dried salt in water the solution was found to be neutral to litmus and turmeric. It gave a white, insolu- ble precipitate with lead acetate and the hot solution gave a crystalline precipitate with barium chloride. The Barium Salt, (C 5 H 3 O 4 N 2 ) 2 Ba. Barium chloride was added, in excess, to a hot, dilute, neutral solution of the above Researches on Pyrimidines. 403 potassium salt. Stout, microscopic prisms, pointed at both ends, crystallized out. These were almost insoluble in hot water. They were dried at no-i2o but did not give off water. Calculated for Ci H 6 O 8 N4Ba. Found. Ba 30.64 28.83 The Sillier Salt. Two grams of the monopotassium salt were dissolved in 100 cc. of water and 1.7 grams of silver nitrate in 50 cc. of water were slowly added, with stirring, in order to prevent a local excess of the nitrate. A white, bulky, gelatin- ous precipitate separated which did not alter or become granu- lar on warming. The solution was diluted with 100 cc. of water and allowed to settle; the precipitate was washed by decan- tation and then filtered on cheese cloth. It was dried at 100 for analysis. One molecular proportion more of silver nitrate was then added to the filtrate. This gave a slight precipitate. On adding 0.5 gram of potassium hydroxide, in a little water, a quantity of white salt was again precipitated, apparently equal in amount to that which was obtained at first. The analytical results did not agree with those calculated for a pure salt. They indicate that the precipitate was a mixture of a mono- and a disilver salt. A sample prepared by taking molecular proportions of acid, sodium hydroxide and silver nitrate gave no better results (Analyses III. and IV.). Calculated for Calculated for Found. C5H 3 O 4 N 2 Ag. CsHgO^NaAga. I. II. III. IV. N 10.64 7.56 8.86 8.54 ........ Ag 41.64 58.37 ........ 49.0 51.5 The following shows that uracil-5-carboxylic acid gives a disilver salt: Eleven grams of the acid, dissolved in water and treated with 2 molecules of sodium hydroxide and 2 mole- cules of silver nitrate, gave a pure white precipitate which, when dry, weighed 2 1 grams. The calculated for a mono salt is 1 6 grams, that for a disilver salt is 23.4 grams. Dimethyl Derivative. Ten grams of the disilver salt were suspended in ether and 15 grams of methyl iodide were added. 404 Wheeler, Johnson and Johns. The mixture was heated in a closed tube at 98- 103 for 1.5 hours. The ether and excess of methyl iodide were evaporated and the residue extracted with alcohol. This gave a gummy extract which, when recrystallized from alcohol, yielded small prisms, melting at 25^-2 56 , with slight effervescence. The substance dissolved with difficulty in hot alcohol and was moderately soluble in hot water. It had a very bitter taste. A nitrogen determination (Kjeldahl), made after drying at uo-i2o, gave: Calculated for C 7 H 8 O 4 N 2 . Found. N 15.21 15.16 A diethyl derivative was prepared in a similar manner. It crystallized from alcohol, in which it is moderately soluble when cold, in the form of clusters of blunt prisms, melting at i62-i63. In both of these reactions of the silver salt the chief product isolated was the free, unaltered acid which was obtained by extracting the residue with water. A nitro- gen determination (Kjeldahl), in the case of the material melting at 162-! 63, showed that it was a diethyl deriva- tive: Calculated for Found. N 13.20 13.45 Ethoxymethylenemalonic ester and methyl pseudothio- urea hydriodide were condensed, using i molecular propor- tion of potassium hydroxide. Under these conditions | the free 2-methylmercapto-5-carbethoxy-6-oxypyrimidine did .not separate but, instead, a poor yield of a basic hydriodide of the ester was obtained. A nitrogen determination (Kjel- dahl) agreed with the calculated for a 2: i salt: Calculated for ,:.;., (C 8 HioO 8 N 2 S)2HL Found. N 10. o 10.1 This salt forms needles from alcohol; when boiled with water, mercaptan was given off. It had no definite melting point. Researches on Pyrimidines. 405 2-Methylmercapto-5-carboxyl- 6 -oxypyrimidine, HN CO I I CH 3 SC CCO 2 H. This was obtained by warming the I! II N CH above salt with potassium hydroxide and then precipitating with hydrochloric acid. It crystallized from hot water in well developed, colorless prisms, melting, to a clear oil, at 235. Nitrogen determination (Kjeldahl) : Calculated for C 6 H 6 O 3 N 2 S. Found. N 15.05 14.71 Our chief experiments were performed with the ethyl- mercapto derivatives, since they are more insoluble than the methyl compounds. NEW HAVEN, CONN., January, 1907. [Reprinted from the American Chemical Journal, Vol. XXXVII, No. 6. June, 1907.] Contributions from the Sheffield Laboratory of Yale University. CXUV. RESEARCHES ON PYRIMIDINES: SOME CON- DENSATION PRODUCTS OF A SUBSTITUTED PSEUDOTHIOUREA: SYNTHESIS OF i-METHYLURACIL. BY TREAT B. JOHNSON AND F. W. HEYL. (TWENTIETH PAPER.) Substituted ureas apparently do not condense with /2-ketone esters to form pyrimidines. While urea reacted with ethyl acetoacetate to form 4-methyluracil, II., Behrend 1 obtained no condensation products corresponding to methyluracil when phenylurea, diphenylurea and dimethylurea were treated with this ketone ester. The alkyl derivatives of 4-methyluracil, II., in which the alkyl groups are linked to nitrogen, have all been prepared by heating this pyrimidine with alkyl halides 2 in presence of alkalis, or by condensing ^-alkyluraminocrotonic esters, III., to pyrimidines, by treatment with alkali. 3 The corre- sponding alkyl derivatives of uracil, I., have not been de- scribed. 1 Ann. Chem. (Liebig), 229, 15; 233, i. 1 Behrend: Ann. Chem. (I^iebig), 229, 23; 231, 256 ; 253, 67. Hagen : Ibid., 244, 2. Hoffman: Ibid., 253, 73. Behrend and Dietrich : Ibid., 309, 260. Behrend and Meyer : Ber. d. chem. Ges., 33, 624. 3 Behrend and Meyer : Ber. d. chem. Ges., 33, 621. Behrend and Buchholz : Ann. Chem. (I^iebig), 314, 209. Behrend and Thurm : Ibid., 323, 160. Behrend and Hesse: Ibid., 329, 341. Researches on Pyrimidines. 629 NH CO NH CO RNH COOC 2 H 6 III II CO CH CO CH , CO CH I II I II I II NH CH NH CCH 3 NH C.CH 3 I. II. III. It has been shown in papers from this laboratory 1 that elementary pseudothioureas condense more readily with aldehyde and ketone esters than the normal oxygen ureas. In this paper we describe the behavior of an aliphatic, mono- substituted pseudothiourea towards some ketone esters. We find that they condense to give substituted mercapto- pyrimidines, which are easily converted to uracil derivatives by digesting with hydrochloric acid. Theoretically, 2 isomeric mercaptopyrimidines i-methyl- 2-ethylmercapto-6-oxypyrimidine, V., or 3-methyl-2-ethyl- mercapto-6-oxypyrimidine, VI., might be formed by con- densing ethyl formylacetate with methyl pseudoethylthio- urea, IV. We find that they condense to give i-methyl-2- CH S N CO N CO II II I HN:C(SC 2 H 5 )NHCH 3 , C 2 H 5 SC CH , C 2 H 5 SC CH IV. II || || II . N CH CH 3 N CH V. VI. ethylmercapto-6-oxypyrimidine, V. We obtained no evi- dence of the formation of the isomeric derivative, VI.: CH 3 NH COOC 2 H 5 C,H 5 SC + CH = II II NH HOCH CH 3 N CO C 2 H 5 SC CH + C 2 H 5 OH + H,O. II II N CH v. 1 Wheeler and Merriam : THIS JOURNAL, 29, 478 (1903). Wheeler and Johnson : Ibid., 31, 591 (1904). Wheeler and Bristol : Ibid., 33, 437 (1905). 630 Johnson and Heyl. The same mercaptopyrimidine, V., was also obtained when 2-ethylmercapto-6-oxypyrimidine 1 was warmed in alcohol with molecular proportions of potassium hydroxide and methyl iodide. NH CO C 2 H B SC CH + KOH + CH 3 I = II II N CH CH 3 N CO C 2 H 5 SC CH + KI + H 2 O. II II N CH v. When the mercaptopyrimidine, V., was digested with con- centrated hydrochloric acid, it was converted quantita- tively into i-methyluracil, VII., with evolution of ethyl^mer- captan. CH 3 N CO CH S N CO II II C 2 H 5 SC CH + H 2 == C,H 6 SH + CO CH II II I II N CH NH CH. VII. The structure of the methyluracil, VII., and the corre- sponding mercapto derivative, V., was shown in the fol- lowing manner: It was converted smoothly into i-methyl- 2,6-dioxy-5-nitropyrimidine, 2 VIII., by treatment with fum- ing nitric and sulphuric acids. Our nitromethyluracil, VIII., CH S N CO CH 3 N CO CO CH + HNO 3 = CO CNO 2 + H 2 O, 'I II I II NH CH NH CH VIII. was anhydrous and melted at 264-265. The isomeric 1 Wheeler and Merriam : Loc. cit. 2 Behrend and Thurm : Loc. cit. Researches on Pyrimidines. 631 3-methyl-2,6-dioxy-5-nitropyrimidine 1 contains i molecule of water of crystallization and melts at 255-256. Methyl pseudoethylthiourea condensed with ethyl aceto- acetate to give i,4-dimethyl-2-ethylmercapto-6-oxypyrimi- dine, IX. Its structure was confirmed by the fact that it was converted into Behrend's /3-dimethyluracil or i,4-di- methyl-2,6-dioxypyrimidine, 2 X., by treatment with hydro- chloric acid. CH 3 NH COOC 2 H 5 C 2 H 5 SC + CH II II NH HOCCH 3 CH 3 N CO I I C 2 H 5 SC CH II II N CCH 3 NH CCH 3 . IX. X. It is interesting to note that methylurea and methylthio- urea condense with ethyl cyanacetate to give 2,6-dioxy-3- methyl-4-aminopyrimidine, 3 XI., and 2-thio-3-methyl-4-amino- 6-oxypyrimidine, 4 XII., respectively, NH CO NH CO I I I CH , CS CH I II CH 3 N CNH 2 XI. It was shown in a previous paper 5 that pseudoethylthio- urea condenses with diethyl formylgly collate to form 2-ethyl- rnercapto-5-ethoxy-6-oxypyrimidine, XIII. We find that ethyl formylphenoxy acetate 6 also condenses with this pseudothiourea to give 2-ethylmercapto-5-phenoxy-6-oxy- pyrimidme, XIV. : 1 Behrend and Thurm : Loc. cit. 2 Behrend and Dietrich : Loc. cit. Behrend and Thurm : Loc. cit. 8 Conrad : Ann. Chem. (Liebig), 340, 314. 4 Trauber and Winter: Archiv.'Pharm., 244, n (1906). 5 Johnson and McCollum : J. Biolog. Chem., i, 437. 6 Johnson and McCollum : Loc. cit. 632 Johnson and Heyl. NH CO NH CO II II C 2 H 6 SC COC 2 H 5 , C 2 H 6 SC COC 6 H 5 II II II II N -CH N CH. XIII. XIV. EXPERIMENTAL PART. Methylpseudoethylthiourea Hydriodide, CHgNHCCSCjHg) : NH.HI, was prepared by treating mono- methylthiourea with ethyl iodide. It was obtained as a heavy oil, which finally solidified in a vacuum over sulphuric acid. The salt was extremely hydroscopic and on prolonged ex- posure to the atmosphere changed to an oil. i -Methyl-2-ethylmercapto-6-oxypyrimidine, CH 3 N CO C 2 H 5 SC CH. Molecular proportions of the above pseudo- II II N CH urea hydriodide and the sodium salt of ethyl formylacetate were separately dissolved in the least possible quantities of cold water. The 2 solutions were then combined and treated with an aqueous solution containing i molecular proportion of potassium hydroxide. The mixture was allowed to stand at ordinary temperatures for about 12 hours and then heated for a few hours on the steam bath. No crystalline material deposited on cooling. The mercaptopyrimidine was ex- tracted with ether and the ether allowed to evaporate spon- taneously, when the pyrimidine deposited in prismatic crys- tals. They were extremely soluble in hot alcohol and in ether, but soluble with difficulty in cold water. The pyrimidine de- posited from alcohol in stout prisms, that melted at 79- 80, without effervescence, to a clear oil. It was soluble in dilute hydrochloric acid and was precipitated from the acid solution by phosphotungstic acid. Analysis (Kjeldahl) : Calculated for C 7 H 10 ON 2 S. Found. N [16.47 16.6 Researches on Pyrimidines. 633 The same mercaptomethylpyrimidine was also obtained when 2-ethylmercapto-6-oxypyrimidine 1 was treated with methyl iodide under the following conditions : One molecu- lar proportion of potassium hydroxide and 9.8 grams of 2-ethylmercapto-6-oxypyrimidine were dissolved in 50 cc. of 95 per cent alcohol. Twelve grams of methyl iodide were then added and the solution digested on the steam bath until it gave no alkaline reaction with turmeric. It was then cooled, ^the insoluble potassium iodide separated by filtration and the excess of alcohol removed by evaporation on the steam bath. We obtained a syrup that would not solidify on cooling. After treatment with a small volume of water, to remove any potassium iodide, the mercaptopyrimidine deposited in well-developed prisms. They were crystallized from alcohol and melted at 79-8o, to a clear oil. When mixed with the above mercaptopyrimidine the melting point was not changed. Analysis (Kjeldahl): Found. Calculated for . > I. II. N 16.47 16.33 16.6 2-Ethylmercapto-6-oxypyrimidine was recovered unaltered after heating with i molecular proportion of methyl iodide for 2 hours, at i55-i65. i -Methyl-2,6-dioxypyrimidine (i -Methyluracil) , CH 3 N CO CO CH. One and five-tenths grams of i -methyl- I II NH CH 2-ethylmercapto-6-oxypyrimidine were digested with 15 cc. of 20 per cent hydrochloric acid until the evolution of ethyl mercaptan ceased. When the acid solution was evaporated to dryness the methyluracil deposited in microscopic, pris- matic crystals that melted sharply at i74-i75, without effervescence, to a clear oil. It was extremely soluble in cold water and alcohol but insoluble in benzene. It was not 1 Wheeler and Merriam. 634 Johnson and Heyl. precipitated from its aqueous solution with picric acid. It was dried for analysis at 110 (Kjeldahl): Calculated for C 5 H 6 O 2 N 2 . Found. N 22.22 22.21 When uracil was heated with 2 molecular proportions of methyl iodide at i86-2oo, for 2 hours, it was recovered unaltered. Calculated for C 4 H 4 2 N 2 . N 25. o CH 8 N i-Methyl-^-nitro-6-oxypyrimidine, 1 CO CNO 2 . Four NH CH and three-tenths grams of i -methyluracil were dissolved in a mixture of 1 5 cc. of fuming nitric acid and 1 5 cc. of concen- trated sulphuric acid, and the solution heated on the steam bath until effervescence practically ceased. The acid solu- tion was then poured upon crushed ice, when a heavy, granular precipitate deposited. The compound was soluble with difficulty in cold water, but deposited from hot water in stout, prismatic crystals that melted at 264-265, with effervescence, to a clear oil. It deposited from hot alcohol in hexagonal plates. When the compound was mixed with some nitrouracil the melting point was lowered to 240. It did not contain water of crystallization. Analysis (Kjeldahl): Calculated for C 5 H 5 4 N 3 . Found. N 24.56 24.8 CH 3 N CO i-Methyl-2,6-dioxy-5-brompyrimidine, CO CBr. One I II NH CH gram of methyluracil was dissolved in 8 cc. of glacial acetic acid and 1.3 grams of bromine added. Hydrobromic acid was evolved at once and the bromine derivative deposited 1 Behrend and Thurm : Loc. cit. Researches on Pyrimidines. 635 in prismatic crystals. The compound was soluble in hot water but insoluble in cold. It deposited from 95 per cent alcohol in well-developed, transparent prisms that melted to a clear oil, at 228-229. Analysis (Kjeldahl): Calculated for C 6 H 6 OjN a Br. N 13-65 i~Ethyl-2 t 6-dioxypyrimidine (i-Ethyluracif) , CO CH. NH CH This compound was prepared from 2-ethylmercapto-6- oxypyrimidine as follows: The mercaptopyrimidine was first converted into i-ethyl-2-ethylmercapto-6-oxypyrimidine by treatment with potassium hydroxide and ethyl iodide, as in the preparation of i-methyl-2-ethylmercapto-6-oxy- pyrimidine. The,;, diethylmercaptopyrimidine was obtained as an oil, which, without purification, was digested with con- centrated hydrochloric acid until the evolution of ethyl mercaptan ceased. When the hydrochloric acid was removed by evaporation the ethyluracil was obtained as a crystalline deposit. The yield was practically quantitative. It was very soluble in hot water and alcohol. It deposited from benzene in foliated prisms that melted at I73-I74 to a clear oil. Analysis (Kjeldahl): Calculated for C 6 H 8 2 N 2 . Found. N 20.00 19.75 CH 3 N CO i,4-Dimethyl-2,6-dioxypyrimidine, CO CH. This I II NH CCH 3 compound has been prepared by Behrend and his co-workers. 1 We obtained it by condensing methylpseudoethylthiourea with ethyl acetoacetate to ^form i,4-dimethyl-2-ethylmercapto- pyrimidine. When this mercaptopyrimidine was boiled with concentrated hydrochloric acid the dime thy luracil was formed Loc. cit. 636 Johnson and Heyl. with evolution of ethyl mercaptan. It deposited from hot water in prismatic crystals, that melted at 260 to an oil. The compound agreed in its properties with Behrend's 1,4- dimethyluracil. Analysis (Kjeldahl): Calculated for C 6 H 8 O 2 N2. Found. N 2O . OO 2O . OO Sodium Salt of Ethyl * III HN CO I I OC CBr, I I HN CHOH II HN CO I I /OH OC C< I ! X OH HN CHOH IV HN CO I I OC CHOH I I HN CO HN CO OC CH I II HN CH I That the present test involves the formation of dialuric acid was shown as follows: The freshly prepared barium precipitate was dissolved in hydrochloric acid and the barium was removed by means of dilute sulphuric acid. On evaporating this solution then in a desiccator we obtained crystals of alloxantin (VI). Dialuric acid undergoes oxidation in the air to alloxantin, 1 while such behavior was never observed in the case of isodialuricacid. 2 HN CO OC CHOH HN CO HN CO CO NH OC CH O COH CO II II HN CO CO NH VI Behrend was the first to show that certain pyrimidins give bromoxyhydro-derivatives. For example, he prepared dibrom- oxyhydromethyluracil (VII) 3 from 4-methyluracil. 1 Baeyer: Ann. d. Chem. (Liebig), cxxvii, p. 12, 1863. 2 Behrend and Roosen: Loc. cit. * Ann. d. Chem. (Liebig), ccxxix, p. 18, 1885. Henry L. Wheeler and Treat B. Johnson 185 This compound gives no color with barium hydroxide. Of more interest in connection with the test, however, is the fact that the similar compound from thymin, bromoxyhydrothymin (VIII) , described by Walter Jones, 1 also gives no color with barium hydroxide. Obviously these compounds would not be expected to yield dialuric acid on treating with baryta water. HN CO HN CO II II OC CBr OC C (CH 3 ) Br II II HN C (CH 3 ) OH HN CHOH VII VIII In Richard Burians' interesting work 2 on the question whether cytosin is a primary product or whether it results by secondary decomposition of some other substance when the nucleic acids are submitted to hydrolysis, he boiled guanin and adenin mixed with various carbohydrates in 30-40 per cent sulphuric acid. He did not obtain cytosin by this treatment, but instead, from guanin 2-amino-6-oxypyrimidin (isocytosin) was formed (IX). 3 The synthesis of this pyrimidin has been described by us. 4 On the other hand, adenin (5 grams) gave 6-aminopyrimidin (X), 5 (0.5 gram). These pyrimidins are therefore to be considered in the test. HN CO N=C-NH 2 I I I H 2 N-C CH HC CH II II II II N CH N CH IX X When isocytosin is treated with bromine water it yields a bromine derivative that is not identical with dibromoxyhydro- uracil. This substance gives an intense blue color on carefully add- ing a solution of barium hydroxide. It is a more decided blue 1 Zeitschr. f. physiol. Chem., xxix, p. 20, 1900. 2 Ergeb. d. Physiol. (Asher-Spiro), v, p. 794, 1905. 3 Amer. Chem. Journ., xxix, p. 492, 1903. 4 Buttner: Ber. d. deutsch. chem. Gesellsch., xxxvi, p. 2232, 1903. 8 Wheeler and Bristol: Amer. Chem. Journ., xxxiii, p. 458, 1905. i86 Color Test for Uracil and Cytosin than that which results from dibromoxyhydrouracil and, what is more important, it is readily distinguished from the latter by immediately disappearing on adding an excess of the barium hydroxide solution. This behavior serves as a delicate test for isocytosin. Finally 6-aminopyrimidin was prepared by a new method; starting with 2-thiouracil, 1 which can readily be obtained in quantity, 2,6-dichlorpyrimidin was prepared by means of phos- phorous pentachloride. 2 The dichlorpyrimidin then gave 2-chlor- 6-aminopyrimidin with alcoholic ammonia, and this was found to reduce smoothly to 6-aminopyrimidin when warmed with con- centrated hydriodic acid. The material thus obtained gave no color whatever with bro- mine water and barium hydroxide. THE TEST. Bromine water is added to about 5 cc. of the solution to be examined until the color is permanent. Too much bromine is to be avoided since a large excess interferes with the test. It is advisable, especially when only small quantities of cytosin or uracil are present to remove the excess of bromine by passing a stream of air through the solution. Then on adding barium hydroxide in excess the purple color is almost immediately pro- duced. Very dilute solutions do not give the test. In such cases on evaporating to dryness and then taking up the material in a little bromine water, removing the excess of bromine, etc., a quantity as small as o.ooi gram of uracil gives a decided bluish-pink or lavender color. In applying the test in the case of cytosin it is advisable to warm or boil the solution with bromine water, cool, and then apply the test as above, being sure to have a slight excess of bro- mine present before adding barium hydroxide. Dibromoxyhy- drouracil is decomposed by prolonged boiling with water into 1 Gabriel: Ber. d. deutsch.chem. Gesellsch.,x'x.'x.vin, p. 1690, 1905; Johnson and Menge: This Journal, ii, p. 115, 1906. 2 Gabriel: Ber. d. deutsch. chem. Gesellsch., xxxviii, p. 1690, 1905. THE JOURNAL OF BIOLOGICAL CHEMISTRY. VOL. III. PLATE II. Henry L. Wheeler and Treat B. Johnson 187 5-bromuracil, 1 which gives no color with barium hydroxide. If, however, 5-bromuracil is treated with bromine water it is con- verted back again into dibromoxyhydrouracil. Picric acid inter- feres with the color and should be removed before applying the test. . Dibromoxyhydrouracil, HN CO HN CHOH In preparing this compound for use in further experiments we usually took 5 grams of uracil, suspended in 20 cc. of water, and added a little over 15 grams of bromine. The uracil dissolved completely on warming, and, on cooling, a crystalline mass sepa- rated. The material thus obtained had a yellow color from excess of bromine, and the yield that first separated was almost 90 per cent of the calculated. On crystallizing once from water colorless, large, flat prisms or blocks separated. The habit of these crystals is shown in the microphotographs (magnified 60 times) Plate II. The same substance was obtained when 0.6 gram of cytosin sul- phate was suspended in water and bromine added until the salt dissolved. The solution was then concentrated to a small volume and cooled. The prisms obtained melted at 205-6 C. (Analy- sis (III). The analytical results were as follows: Calculated for Found: C4H 4 O 3 N 2 Br 2 : I. II. III. IV. N 9.72 9.59 9.63 9.46 Br 55.55 56.00 Dibromoxyhydrouracil melts with effervescence at 203-6. It shows signs of decomposition below this temperature. It is more soluble in water than uracil. The solution of the pure white crystals is neutral to litmus, but on boiling it has an acid reaction and finally 5-bromuracil separates. In accordance with this silver nitrate gives no precipitate in the cold but on warming with this reagent silver bromide separates. 1 Wheeler and Merriam: Amer. Chem. Journ., xxix, p. 486, 1903. 1 88 Color Test for Uracil and Cytosin Dibromoxyhydrouracil dissolves readily in alcohol. If boiled with alcohol 5-bromuracil separates. If the alcoholic solution is treated with a solution of sodium in alcohol a purple percipitate is at once produced similar to the barium hydroxide percipitate. Alcoholic potassium hydroxide also produces a similar colored precipitate. These colored alkali salts differ from the barium hydroxide precipitate by being instantly decomposed and decol- orized by treatment with water. The aqueous solution then turns green and finally orange on standing. Aqueous ammonia immediately dissolves the dibrom-deriva- tive, and removes bromine; the solution slowly takes on yellow, then a garnet color and if sufficient material is present a reddish- brown precipitate separates. Dibromoxyhydrouracil is almost insoluble in ether. THE PURPLE PRECIPITATE. The precipitate produced by adding barium hydroxide to an aqueous solution of dibromoxyhydrouracil when exposed on paper to dry in the air turned red. When treated with acetic acid it changed to a bright red powder, while the precipitate when freshly precipitated dissolved completely in acetic acid. The analysis of the precipitate was therefore abandoned. It was shown that the substance yields alloxantin on treatment with acids as follows: Four and a half grams of dibromoxyhydro- uracil were dissolved in 40 cc. of water and added to 1 1 grams of crystallized barium hydroxide in 100 cc. of water. The purple precipitate was rapidly filtered but no attempt was made to wash it. It was immediately dissolved in dilute hydrochloric acid and the barium was removed by adding 20 per cent of sulphuric acid. The colorless solution, which on testing a portion with barium hydroxide again gave a purple precipitate, was allowed to evapo- rate in a desiccator. On standing over night it gave small, stout, colorless transparent prisms. These melted at 243 C. with effervescence. Behrendand Friedrich 1 state that alloxantin melts at 2435. This material was not dialuric acid since its aqueous solution failed to decompose carbonate of sodium and it was not 1 Ann. d. Chem. (Liebig), cccxliv, p. n, 1906. Henry L. Wheeler and Treat B. Johnson 189 isodialuric acid because it melted over 100 higher. A nitrogen determination agreed with the calculated for alloxantin : Calculated for C 8 H 6 O 8 N 4 + 2H 2 0: Found: N 17.40 percent, 17 . n per cent. 6-Aminopyrimidin, N=C NH 2 I I HC CH II II N CH One gram of 2-chlor-6-aminopyrimidin was dissolved in 20 cc. of colorless, concentrated hydriodic acid and then evaporated to dryness on the water bath. Iodine separated in abundance. The residue was evaporated several times with a solution of sul- phur dioxide. The colorless solution was then treated with an excess of silver sulphate, filtered and the silver was then removed with hydrogen sulphide. On concentrating the solution a syrup was obtained, which, when taken up in boiling alcohol, gave well- crystallized colorless prisms. The yield was over 0.8 gram. The crystals melted at 143 to clear oil, and nitrogen determinations agreed with the calculated for an acid sulphate of 6-amino- pyrimidin. Calculated for Found: C 4 H S N3.H 2 S0 4 : I. II. N 21.76 2I -55 21. ii The solution of this material was freed from sulphuric acid by means of barium hydroxide and the excess of barium hydroxide was removed with carbonic acid. The free base proved to be extremely soluble in water. The aqueous solution was precipi- tated by phosphotungstic and picric acid, and it gave a precipi- tate of silver salt when treated with silver nitrate in neutral solu- tion. This precipitate was soluble in ammonia. With bromine water and barium hydroxide it gave no color. V. RESEARCHES ON PYRIMIDINS: ON SOME SALTS OF CYTOSIN, ISOCYTOSIN, 6-AMINOPYRIMIDIN AND 6-OXYPYRIMIDIN. (Twenty-second Paper.) BY HENRY L. WHEELER. (From the Sheffield Laboratory of Yale University.) (Received for publication, June 10, 1907.) It has been shown by Richard Burian 1 when guanin (I) , mixed with carbohydrates, is heated with 30-40 per cent sulphuric acid that hydrolysis and reduction take place at the same time, the imidazole group is removed and isocytosin 2 (II) and uracil (IV) result. It may be added that the probable formation and decomposition of xanthin (III) would also give uracil. This decomposition of guanin may be represented as follows: HN CO I I H 2 N C C NH< N C I 1 HN CO I I N CH II CH HN CO I I OC C NH\ I II HN C III I HN CO I OC CH I II HN CH IV CH When adenin (V) was treated in a similar manner, Burian obtained 6-aminopyrimidin (VI), while 6-oxypyrimidin (VIII), which would be expected to result in this case both by the 1 Asher-Spiro : Ergeb. d. Physiol., v, p. 795, 1905. 2 Wheeler and Johnson: Anter. Chem. Journ., xxix, p. 492, 1903. 285 286 Researches on Pyrimidins decomposition of hypoxanthin (VII) and by the hydrolysis of 6-aminopyrimidin, escaped detection. 1 N=C NH 2 HN CO II II HC C NHx > HC C NH\ II II /CH || || )CH N C N/ N C N/ V VII 1 1 N=C NH 2 HN CO II II HC CH > HC CH II II II II N CH N CH VI VIII The three pyrimidin bases, isocytosin, 6-aminopyrimidin and 6-oxypyrimidin, are probably formed in the energetic hydrolysis of the nucleic acids by sulphuric acid and, since it has now been found that the general reagents which precipitate cytosin also precipitate these bases, an examination of the properties of the compounds and some of their salts was undertaken, along with the similar ones of cytosin. The statement of Burian that 6- aminopyrimidin and isocytosin according to their entire behavior must obstinately adhere to cytosin ("dem sie ihrem ganzen Ver- halten nach hartnackig anhaften mussten") has been found to be more especially true in the case of isocytosin. Of the three bases 6-oxypyrimidin is new. Isocytosin was first prepared synthetically in this laboratory 2 and later it was obtained in a different manner by Gabriel and Colman. 3 In the case of 6-aminopyrimidin, Burian states that the base was isolated in the form of the silver salt, by precipitating in neutral solution with silver nitrate. This was decomposed by means of hydrogen sulphide and the base precipitated by phos- photungstic acid. A solution of the free base was then obtained in the usual manner from which he prepared and analyzed the 1 In an article published while this paper was in press (Zeitschr. f. physiol. Ghent., li, p. 444, 1907), Burian describes the isolation of 6-oxy- pyrimidin. 2 Loc. cit. 3 Ber. d. deutsch. chem. Gesellsch., xxxvi, p. 3382, 1903. Henry L. Wheeler 287 picrate and chloroplatinate. He says nothing further in regard to the base or its salts. 6-Aminopyrimidin, however, was first prepared by Ernst Biitt- ner, who obtained it from barbituric acid by means of a series of operations. 1 This author described the free base and he pre- pared the hydrochloride, easily soluble rhombic tables; the chloro- platinate and picrate, needles, difficultly soluble. Our knowl- edge of these pyrimidins stood at this point when the following work was begun. It has now been found that 6-oxypyrimidin is formed when 2-thiouracil (IX), which can be easily prepared 2 in any desired quantity, is treated with hydrogen dioxide. HN CO HN CO II II SC CH+3O+H 2 O = HC CH + H 2 SO 4 I II II II HN CH N CH IX The writer finds, however, that 6-oxypyrimidin is more smoothly obtained by warming 2, 6-dichlorpyrimidin (X), pre- pared from uracil 3 or 2-thiouracil, 4 with hydriodic acid and red phosphorus. The hydrogen iodide salt results from which the pure base can be obtained in the usual manner. N=CC1 HN CO I I I C1C CH+3HI+H 2 0= HC CH.HI+I 2 + 2 HC1 II II II II N CH N CH ~ X The 6-aminopyrimidin used in this work was prepared by a shorter and more convenient method than the one employed by Biittner mentioned above. 2, 6-Dichlorpyrimidin 5 was heated 1 Ber. d. deutsch. chem. Gesellsch., xxxvi, p. 2232, 1903. 2 Wheeler and Bristol: Amer. Chem. Journ., xxxiii, p. 458, 1905. 3 Gabriel: Ber. d. deutsch. chem. Gesellsch., xxxviii, p. 1690, 1905. 4 Johnson and Menge: This Journal, ii, p. 114, 1906. 5 Care should be taken in working with 2, 6-dichlorpyrimidin not to expose the material to the skin, since it has a very corrosive action. In one case deep and painful blisters were formed on the hands, resembling those produced by hydrofluoric acid. 288 Researches on Pyrimidins with alcoholic ammonia. This gave a mixture of 2-amino-6- chlorpyrimidin (XI) and 2-chlor-6-aminopyrimidin (XII). 1 It was found, on boiling this mixture with water and zinc dust, that 2-amino-6-chlorpyrimidin was reduced to the very soluble 2-aminopyrimidin 2 which could easily be removed, while 2-chlor- 6-ammopyrimidin remained unaltered. The pure 2-chlor-6- aminopyrimidin then on warming on the steam-bath with hydri- odic acid was smoothly reduced and the hydrogen iodide salt of 6-aminopyrimidin was obtained. Although Biittner worked with small quantities of this base (0.25 gram) his results and the writer's agree in every respect. N=CC1 N=C NH 2 N=C NH 2 II II H 2 N C CH C1C CH > HC CH II II II II II II N CH N CH N CH XI XII EXPERIMENTAL PART. 6-Oxypyrimidin, HN CO I I HC CH II II N CH Thirteen grams of 2, 6-dichlorpyrimidin were slowly added to 50 cc. of concentrated hydriodic acid and 6 grams of red phosphorus on the steam -bath. As soon as all was added the mixture was boiled for a few minutes and then the hydrogen iodide was removed, as far as possible, by evaporation in a vacuum at 100. The residue on taking up in hot water and filtering from red phosphorus formed a syrup, which deposited well crystallized needles, decomposing with effervescence when kept at 300 (6-oxypyrimidin hydriodide). The whole was dis- solved in water and an excess of silver sulphate was added and filtered, the filtrate was precipitated with hydrogen sulphide, the phosphoric and sulphuric acids removed by means of barium 1 Gabriel : Loc. tit. 2 Bftttner: Loc. tit. Henry L. Wheeler 289 hydroxide, and then, on removing the excess of barium with carbon dioxide and evaporating to dryness, a very soluble crystalline cake was obtained. This weighed 5.6 grams. On crystallizing from ethyl acetate beautiful, long, thin, prismatic needles separated melting to a clear oil at 164- 165. The per cent of nitrogen in this material agreed with the calculated for 6-oxypyrimidin (Analysis I and II). The same compound was also obtained as follows: Twenty grams of 2-thiouracil were suspended in a liter of hot water and about 530 cc. of commercial hydrogen dioxide solution were added in portions. The thiouracil dissolved and after boiling a few minutes, sulphur dioxide was added and the solution was evaporated to a convenient volume. The sulphuric acid, which had been formed in the reaction, was removed by means of an excess of barium hydroxide and the excess of the latter was pre- cipitated with carbon dioxide. The solution was then evapor- ated to dryness and the residue was extracted with boiling alco- hol. This dissolved the 6-oxypyrimidin, leaving a mixture weigh- ing about 7 . 7 grams of uracil and a barium salt of an organic acid that was not further examined. The barium salt was insoluble in alcohol but readily soluble in water, it had the peculiar prop- erty of swelling up to many times its original volume when heated on platinum foil. The alcoholic solution was evaporated to dryness and the residue extracted with ethyl acetate. This gave about 6 grams of crude 6-oxypyrimidin. When crystallized from a large amount of benzene it formed colorless needles melting less sharply than the above preparation (Analysis III). All the nitrogen determinations in this paper were made by Kjeldahl's method. Calculated for Found: C 4 H 4 ON 2 : I. II. III. N .... 29.16 29.02 28.78 28.85 6-Oxypyrimidin is insoluble in petroleum ether, very diffi- cultly soluble in ether, somewhat more soluble in hot benzene and still more soluble in ethyl acetate. This solvent is the best to use to extract and crystallize the material. It is extremely soluble in water and alcohol, and, in this respect, like 6-amino- pyrimidin could not possibly be mistaken for the far more insolu- ble cytosin and isocytosin. 290 Researches on Pyrimidins This monooxypyrimidin unlike the dioxypyrimidin (uracil) gives no color with bromine and barium hydroxide. It is precipitated by phosphotungstic acid, and, in neutral solution, by silver nitrate or mercuric chloride. A strong solu- tion is precipitated by picric acid and also by hydrochloro- platinic acid. The platinum chloride double salt separates slowly and forms prisms. It did not have a definite melting or decomposing point. 6-Aminopyrimidin, N=C NH 2 I I HC CH II II N CH 2-Chlor-6-aminopyrimidin reduces smoothly to 6-aminopyri- midin when warmed on the water-bath with concentrated hydriodic acid and red phosphorous. 1 The residue, after the removal of hydrogen iodide by evaporation was treated with silver sulphate, barium hydroxide, etc., as in the case of 6-oxy- pyrimidin, for the preparation of the free base. It was found that 2 . 7 grams of 2-chlor-6-aminopyrimidin, 27 cc.of concentrated hydriodic acid and an excess of red phosphorus gave 1.6 gram of crude base while the calculated is 1.9 gram. When crystal- lized from ethyl acetate, snow white clusters of thin, leaf -like crystals separated. The appearance of these clusters was similar to those of 6-oxypyrimidin. The material melted at i5i-i52. Buttner 2 gives the same melting point for this base. It is ex- tremely soluble in water and alcohol and it is precipitated in more dilute solutions by the same reagents which precipitate 6-oxypyrimidin. The Acetyl Compounds. A peculiar acetyl derivative of 6-oxypyrimidin was formed when the base was dissolved in acetic anhydride and evaporated to dryness on the steam-bath. When the residue was crystal- lized from alcohol, in which it is quite soluble, it formed colorless needles or spikes. It is sharply distinguished from the other 1 Wheeler and Johnson: This Journal, iii, p. 186, 1907. 2 Loc. cit. Henry L. Wheeler 291 acetyl compounds by having two melting points. When heated rapidly it melted to a clear oil at 180, or a little below, then if the temperature was kept at this point it solidified and on further heating it remelted with effervescence at 2i5-22o. (Analysis I and II.) Another sample of the acetyl compound was prepared and crys- tallized from water. It then separated in the form of prismatic scales which had the same behavior on heating as the above. It was dried at 5o-55. (Analysis III.) The analytical results are low for a simple acetyl derivative, but they agree with the calculated for the expected acetyl compound with a molecule of water of crystalliza- tion, or, if water of constitution, equally well for acetylformami- dine acrylic acid, CH 3 CONH-CH =N-CH =CHCOOH, or sim- ply an acetic acid salt of 6-oxypyrimidin. The latter, however, is excluded since the free base dissolves in glacial acetic acid and on evaporation is recovered unaltered. In order to determine whether the compound has water of crystallization or water of constitution, a portion of the material, which had been crystallized from water, was heated at 109 115 for three hours. It then lost 4 per cent in weight This was due, not to the fact that water was given off but that the substance volatilized, since a nitrogen determination, after heating, gave the same result as in the case of the previous determinations (Analysis IV) . This result makes it appear improbable that the substance has water of crystallization. The view that the com- pound is acetylformamidine acrylic acid, therefore, remains at present as most probable. This, however, must be left for future work to decide. Calculated for Found: C 6 H 8 O 3 N 2 : I. II. III. IV. N 17.94 17.75 17.64 17.88 17.86 Acetyl-6-aminopyrimidin. A quarter of a gram of the pure base was dissolved in acetic anhydride and heated to boiling, then evaporated to dryness on the steam-bath When crystallized from about 5 cc. of water it formed an asbestos-like mass of fine needles. About 0.2 gram separated. It melted at 202, to a clear oil, without effervescence. Analysis: Calculated for C 6 H 7 ON 3 : Found: N 30.43 30.35 292 Researches on Pyrimidins The Pier ate s. The picrate of 6-oxypyrimidin is far more soluble than the picrates of cytosin and isocytosin. It also differs decidedly in appearance from these salts. 6-Aminopyrimidin picrate, on the other hand, closely resembles cytosin picrate both in regard to solubility and crystalline form. The presence of this picrate is possibly the cause of the picrate of cytosin from natural sources invariably melting lower than that of synthetic cytosin. 1 6-Oxypyrimidin Picrate. A saturated aqueous solution of picric acid was mixed with a moderately strong solution of 6- oxypyrimidin ; as no precipitate was formed the solution was con- centrated to almost the volume of picric acid solution employed. On standing a long, flat, fern-like growth of crystals separated. It melted to a clear oil at 190. Analysis: Calculated for CioH 7 O 9 N 5 : Found: N 21.53 21.55 6-Aminopyrimidin Picrate. Forty cc. of picric acid solution were added to 0.2 gram of 6-aminopyrimidin in a little water. A bulky precipitate was formed at once which dissolved on adding 40 cc. of water and then boiling. On cooling, long bright, yel- low, hair-like needles separated. On heating these showed evi- dence of change a little below 200 and then suddenly melted at 226 to a clear oil. This then turned brown and vigorously effervesced at 270 280. Analysis: Calculated for Found: C 10 H 8 7 N 6 : I. II. N 25.92 25.76 25.92 The Hydrochlorides. The hydrochlorides of 6-oxypyrimidin, 6-aminopyrimidin and of isocytosin are even more soluble than the easily soluble cyto- sin hydrochloride. The latter and 6-oxypyrimidin hydrochlor- ide separate with water of crystallization. The hydrochlorides are difficultly soluble in alcohol. 6-Oxypyrimidin Hydrochloride, C 4 H 4 ON 2 .HC1.H 2 O. Pure 6-oxy- pyrmidin was dissolved in dilute hydrochloric acid and evapo- rated to dryness on the steam -bath. The residue formed a syrup 1 Amer. Chem. Journ., xxix, pp. 494, 500, 505, 1903. Henry L. Wheeler 293 which solidified on cooling. It was dissolved in water and allowed to crystallize by standing over sulphuric acid. Thick transparent prisms or oblong blocks separated some 10-15 millimeters in length. These melted partially below 100, finally melting to an oil at about 205-2io. Analysis: Calculated for Found: C 4 H 7 O 2 N 2 C1: I. II. N 18.60 18.54 18.64 A water determination was made by heating the material at 114 for one hour. This gave 13.1 per cent while the calculated is 11.96. When reheated at 120 it was found that the material slowly volatilized, which explains the high result. 6-Aminopyrimidin Hydro chloride, C 4 H 5 N 3 .HC1. The base was evaporated to dry ness with hydrochloric acid. The material was then taken up in a little water and left to crystallize in a desiccator. Transparent prisms or tables separated from the syrupy solution. When dried over calcium chloride they melted to an oil at 257 and then effervesced. A nitrogen determination showed that the salt was anhydrous. Calculated for C 4 H 5 N 3 .HC1: Found: X 31.93 31.49 Isocytosin Hydrochloride , C 4 H 5 ON 3 .HCL This salt has a de- cided tendency to crawl up the sides of a dish when left to crystallize. The crystals which separate in this manner are prisms, when precipitated from an aqueous solution by the addi- tion of alcohol it forms little square tables or blocks. When heated it begins to change in appearance at about 250 and then effervesces about 270. Analysis: Calculated for C 4 H 5 ON 3 .HC1: Found: N.. 28.47 28.27 Cytosin Hydrochloride, C 4 H 5 ON 3 .HC1.H 2 O. If cytosin is dis- solved in strong hydrochloric acid and left to crystallize in a des- iccator cytosin dihydrochloride is obtained, C^CN^HCl. 1 If the acid solution is evaporated to dryness and the residue is taken up in water and left to crystallize spontaneously, large, transparent plates separate of the hydrous, i : i salt. This salt 1 Wheeler and Johnson: Amer. Chem. Jottrn., xxxi, p. 598, 1904. 294 Researches on Pyrimidins loses its water rapidly at 50 and in a few hours on exposure to the air. It differs from the hydrous 6-oxypyrimidin hydro- chloride since on standing over night the crystals become entirely opaque. It is more soluble than the dihydrochloride. The latter and also the hydrous cytosin hydrochloride both melt at 275- 279. Analysis: Calculated for C 4 H 5 ON 3 .HC1.H 2 O: Found: N 25.37 25.52 Some of the material which had stood for about 3-4 hours was dried to a constant weight at a little above 100. It lost 9.5 per cent of water while the calculated for one molecule of water is 10.8 per cent. The Sulphates. The sulphates of 6-oxypyrimidin and 6-aminopyrimidin are very soluble in water. The neutral sulphate of isocytosin is less soluble and it resembles the neutral sulphate of cytosin except that it does not crystallize with water. The three sulphates which cytosin forms have been prepared and some new facts are given for their identification. The sulphates were prepared from the hydrochlorides by treating the latter with an excess of silver sulphate, removing the excess of silver by means of hydrogen sulphide and then evaporating. Owing to their solubility, the sulphates of 6-oxypyrimidin and 6-aminopyrimidin were crys- tallized by means of alcohol. The sulphates of isocytosin and cytosin were allowed to crystallize at ordinary temperatures in order to determine their degree of hydration. 6-Oxypyrimidin Sulphate, (C 4 H 4 ON 2 ) 2 H 2 SO 4 . This salt was purified by precipitating the strong aqueous solution with alcohol. After the fourth precipitation it came down in the form of micro- scopic prisms and melted with effervescence about 218. Analy- sis: Calculated for Found: C 8 H 8 2 N 4 .H 2 S0 4 : I. II. N 19.31 19.29 19.32 This neutral sulphate also separates from alcoholic solutions containing a considerable excess of sulphuric acid. Henry L. Wheeler 295 6-Aminopyrimidin Sulphate, C 4 H 5 N 3 .H 2 SO 4 , was described in a previous paper. 1 Isocytosin Sulphate, (C 4 H 5 ON 3 ) 2 H 2 SO 4 . This salt separated from the aqueous solution containing a slight excess of sulphuric acid, on standing, in the form of balls composed of radiating clusters of small prisms. It melted at 276 with effervescence. Analysis : Calculated for Found: C 8 HioO2N e .H 2 SO 4 : I. II. N 26.25 26.30 25.98 Basic Cytosin Sulphate, (C 4 H 5 ON 3 ) 4 H 2 SO 4 .2H 2 O. This hy- drous salt has frequently been obtained by Levene. 2 It was obtained in the anhydrous condition by Kossel and Steudel. 3 It is the least soluble of the sulphates mentioned in this paper. It has the highest decomposing point, the same as that of cyto- sin itself, namely, 323. It was obtained, in the present work, when cytosin monohydrochloride was treated with a slight excess of silver sulphate, and the silver then removed by means of hydro- gen sulphide. On concentrating the solution the salt separated in the form of sharply defined, long, needle-like prisms. Neutral Cytosin Sulphate, (C 4 H 5 ON 3 ) 2 H 2 SO 4 .2H 2 O. The anhy- drous form of this salt was first obtained by Levene, 4 having dried the material in a toluol bath. The hydrous form, here described, separated on allowing the mother liquor from the above basic salt to evaporate in the air. Stouter, more compact masses of prisms formed which, on drying in the air, melted with effervescence at 287. Levene found 290. The analyses now show that this salt crystallizes with two molecules of water. Calculated for Found: C 8 Hi O 2 N 3 .H 2 SO 4 .2H 2 0: I. II. III. N 23 . 59 23 . 66 23 . 45 H 2 O .... 10.11 10.13 The water determination was made by heating the salt at 1 18- 120 for an hour. Acid Cytosin Sulphate, C 4 H 5 ON 3 .H 2 SO 4 . This salt has been obtained by Kossel and Steudel ; they simply mention that it is 1 Wheeler and Johnson: This Journal, iii, p. 189, 1907. 2 Zeitschr. f. physiol. Chem., xxxix, pp. 7, 135, 481, 1903. 3 Ibid., xxxviii, p. 52, 1903. 4 Ibid., xxxviii, p. 81, 1903. 296 Researches on Pyrimidins easily soluble. 1 It is easily obtained by dissolving the neutral sulphate in 20 per cent sulphuric acid and allowing the material to crystallize in a desiccator. The stout, transparent crystals thus obtained appear to be rhombohedrous. These have the characteristic property of becoming opaque when an attempt is made to wash them with water. This is the most soluble sulphate of cytosin. After pressing on paper and drying over calcium chloride the material melted at 197 to a colorless oil. Analysis : Calculated for Found: C 4 H 5 ON3.H 2 S0 4 : I. II. N 20.09 19.86 19.89 Acid Cytosin Phosphate, C 4 H 5 ON 3 .H 3 P0 4 . This salt was obtained when cytosin monohydrochloride (7 grams) was boiled with phosphorous oxychloride (50 cc.) for two hours. The phos- phorous oxychloride was then evaporated and the residue treated with ice. The material dissolved and, evaporating, a syrup was obtained from which dilute alcohol precipitated well crystallized, long, flat prisms. When crystallized from water, in which the salt is very soluble, it melted at 236 with effervescence. Analy- sis: Calculated for Found: C 4 H 5 ON3.H 3 P04: I. II. 20.09 20.09 19.95 The melting or effervescing points of the substances com- pared in this work are given in the following table. The effer- vescing points even of the pure compounds may vary to a cer- tain amount, perhaps several degrees, according to the rate of heating, amount of substance used, etc. No especial accuracy is claimed. It is believed, however, that the melting points given will be found to be sufficiently constant to serve as an aid for the identification of the substances. There is in general a wide difference in the melting points in each series. This should be especially serviceable in the case of the sulphates of cytosin. The picrolonates mentioned in the table are the most insoluble salts prepared. Since some new facts have been observed in the case of picrolonates these salts will be described in a later paper. 1 Zeitschr. f. physiol. Chem., xxxviii, p. 52, 1903. Henry L. Wheeler 297 "5 t>. o "5 ^ ^H o 1 ^ Tf o V ^^ (M M oo H >J_ fi 1 1 1 o 1 So r^ O X "? O o" .. 9 ^^ -1 0*0 , M- M- N.C CH. r PT / ii ii ^6^5 NPTT V^Xl X. We find that 2-anilino-6-oxypyrimidine, V., reacts with methyl iodide, in the presence of potassium hydroxide, giving two monomethyl derivatives 2-anilino-6-methoxypyrimidine, VII., and i-methyl-2-anilino-6-oxypyrimidine, VIII. The structure of these two pyrimidines was shown in the following manner: The same methoxypyrimidine (m. p. 119) was ob- tained when 2-anilino-6-chlorpyrimidine was warmed with sodium methylate. A quantitative yield of the pyrimidine was also formed when methyl iodide was allowed to act on the silver salt of 2-anilino-6-oxypyrimidine at ordinary tem- perature. Researches on Pyrimidines. 239 N=C.OCH, C.H 5 NH.C CH -f NaCl. II II N CH The second isomer was identical with the methyl pyrimidine obtained by heating i-methyl-2-ethylmercapto-6-oxypyrimi- dine, 1 I., with aniline at 150. It was soluble in alkali and C.H 6 SH. melted at 149- 150. The isomeric 2-methylanilino-6-dKy* pyrimidine, X., which was prepared by heating 2-ethylmer* capto-6-oxypyrimidine \vith methyl aniline, melted at 187. NH CO I I C 2 H 5 SC CH + CH 8 NH.C 6 H 6 = N - CH C 2 H 5 SH. 2-Amlino-6-axypyrimidine reacts in a smooth manner with phosphorus oxyehloride to give 2-anilino-6-chlorpyriinidine, XI. When this was digested with water and zinc dust it was reduced practically quantitatively to 2-anilinopyrimidine, XII. 1 Johnson and Heyl: Loc. cit. Johnson and Heyl. Cl N CH CH -*> CeH 5 NH.C CH. All attempts to hydrolize 2-anilinopyrimidine, XII., and 2-anilino-6-oxypyrimidine to 2-oxypyrimidine and uracil, respectively, were unsuccessful. They could be digested for hours with concentrated hydrochloric acid without altera- tion. 2-Anilinopyrimidine was completely decomposed when heated with hydrochloric acid at 168. On the other hand, 2-anilino-6-oxypyrimidine was not changed by this treat- ment, and was also recovered unaltered after heating with 20 per cent sulphuric acid at 230. The above pyrimidines might be expected to react with carbon bisulphide, giving 2-thiopyrimidine, XIII., and 2-thio- uracil, XIV., with formation of phenylisocyanate. We find, N==CH NH CO I I h* I I CS CH CS CH I II I II NH CH NH CH XIII. XIV. on the other hand, that they are extremely stable in presence of this reagent, and were recovered ualtered after heating with carbon bisulphide at 230. We examined, in the course of our work, the action of ani- line and ammonia on 2-ethylmercapto-5-ethoxy-6-oxypyr- imidine, 1 XV. We find that this mercaptopyrimidine shows no tendency to react with aniline at 100. In order to intro- duce the anilino group it was necessary to heat for several hours at 200. This behavior is of interest since 2-ethylmer- capto-6-oxypyrimidine 2 and 2-ethylmercapto-5-brom-6-oxy- pyrimidine 3 react with aniline at 100 to give quantitative 1 Johnson and McCollum: J. Biol. Chem., 1, 437. * Johnson and Johns: Loc. ctt. Wheeler and Bristol: THIS JOURNAL, 33, 437. Researches on Pyrimidines. 241 yields of the corresponding 2-anilinopyrimidines. 2-Ethyl- mercapto-5-ethoxy-6-oxypyrimidine was also recovered un- altered after heating with ammonia at 230. NH CO NH CO C 2 H 5 S.C COC S H 5 ->- C 6 H 5 NH.C COC,H 5 -* N CH XVI. N= =CH I I COC,H 5 - C 6 H 5 NHC COC 2 H 6 . II II N CH XVIII. 2-Anilino-5-ethoxy-6-oxypyrimidine, XVI., reacted with phosphorus oxychloride to give a good yield of 2-anilino-5- ethoxy-6-chlorpyrimidine, XVII. When this chloride was heated with ammonia a quantitative yield of 2-anilino-5- ethoxy-6-aminopyrimidine was obtained. 2-Anilino-5-ethoxy- pyrimidine, XVIII., was prepared by reducing the chlorpyr- imidine, XVII., with zinc dust. EXPERIMENTAL. N= =C.OCH S I I 2-Anilino-6-methoxypyrimidine, C 6 H 5 HN.C CH . N CH This pyrimidine was obtained when methyl iodide was allowed to act on the silver salt of 2-anilino-6-oxypyrimidine 1 at ordi- nary temperature. It was also prepared by treating 2-ariilino- 6-chlorpyrimidine with sodium me thy late. It deposited from 95 per cent alcohol in transparent prisms that melted at 119 to a clear oil. It was converted into 2-anilino-6-oxypyrimidine (m. p. 231) when digested with 20 per cent hydrochloric acid. Analysis (Kjeldahl): 1 Johnson and Johns: Loc. cit. 242 . Johnson and Heyl. Calculated for Found. N 20.89 20.93 i-Methyl-2-anilino-6-oxypyrimidine, CH.N - CO I I , C B H 5 HN .C CH .H,O Molecular proportions of 2 -anilino- II II N - CH 6-oxypyrimidine (20 grams) and potassium hydroxide (6 grams) were dissolved in 95 per cent alcohol. Twenty grams of methyl iodide were then added and the solution heated on the steam-bath until it failed to give an alkaline reaction with turmeric. The solution was then filtered from insoluble potassium iodide and the excess of alcohol evaporated on the steam-bath. We obtained a syrup which deposited a crys- talline precipitate when treated with cold water. This was separated and triturated with a 10 per cent sodium hydroxide solution and the insoluble portion purified by crystallization from 95 per cent alcohol. It separated in prismatic crystals that melted at 1 19 to a clear oil. It was identified as 2-anilino- 6-methoxypyrimidine. A mixture of the 2 compounds melted sharp at 119. Analysis (Kjeldahl): Calculated for CnHiiON 8 . Found. N 20.89 21.2 When the sodium hydroxide filtrate (above) was acidified with acetic acid a mixture of 2-anilino-6-oxypyrimidine and i-methyl-2-anilino-6-oxypyrimidine separated. After a frac- tional crystallization from alcohol and repeated recrystalli- zations from benzene and water the methylpyrimidine was obtained pure. It deposited from hot water in slender prisms, that melted at i49-i5o to a clear oil. It contained i mole- cule of water of crystallization, which was determined by heating at ioo-no. 0.8187 gram substance lost 0.0686 gram. Calculated for CiiHnON 8 .H 2 O. Found. H 2 O 8.21 8.37 Researches on Pyrimidines. 243 Analysis of anhydrous material : 0.2333 gram substance gave 0.5597 gram CO 2 and 0.1130 gram H 2 O. Nitrogen determined by Kjeldahl method : Calculated for CiiHnON 3 . Found. C 65.43 65.67 H 5.38 5-47 N 20.89 20.55 Action of Aniline on i-Methyl-2-ethylmercapto-6-oxypyr- imidine. 1 The mercaptopyrimidine was recovered unaltered after heating with aniline for several hours at 100. One and two-tenths grams of the mercaptopyrimidine and 0.7 gram of aniline were heated for 7 hours at i25-i55. The excess of aniline was then removed by distillation with steam and the insoluble residue treated with hot benzene. A crys- talline compound separated from the benzene on cooling. It was purified by recrystallization from water and deposited in prisms that melted at i49-i5O. When this derivative was mixed with some i-methyl-2-anilino-6-oxypyrimidine (m. p. i49-i5o), the melting point was not changed. Analysis (Kjeldahl): Calculated for CiiH u ON s . Found. N 20 . 89 2O . 6 NH CO & 2 - Methylanilino - 6- oxypyrimidine, ^>N .C N CH This pyrimidine was prepared by heating 7 grams of 2-ethylmer- capto-6-oxypyrimidine 2 with 6 grains of methyl aniline for 14 hours at 100. It was soluble in warm benzene and alcohol. It separated from hot alcohol in large, prismatic crystals that melted at 187 to a clear oil. The yield corresponded to about 74 per cent of the theoretical. Analysis (Kjeldahl) : 1 Johnson and Heyl: Loc. tit. * Wheeler and Merriam: THIS JOURNAL, 29, 478 (1903). 244 Johnson and Heyl. Calculated for Found. C u HuON 8 . I. II. N 20.89 20.55 20.66 N CC1. 2-Anilino-6-chlorpyrimidine, C 6 H 5 NH.C CH. The hy- N CH drochloride of this pyrimidine was obtained when 2-anilino-6- oxypyrimidine was warmed with an excess of phosphorus oxychloride. After the evolution of hydrochloric acid had ceased the solution was poured upon crushed ice to destroy the excess of phosphorus oxychloride. The hydrochloride of 2-anilino-6-chlorpyrimidine separated as a thick varnish, which soon solidified. This salt was triturated with a dilute solution of sodium hydroxide or ammonia when the chlorpyr- imidine deposited. It crystallized from alcohol in plates that melted at 134 to an oil. Analysis (Kjeldahl): Calculated for Found. CioH 8 N 3 Cl. I. II. N 20.43 20.09 20.3 Nz= =:CH I I 2-Anilinopyrimidine, C 6 H 5 NH.C CH. Five and five- N CH tenths grams of 2-anilino-6-chlorpyrimidine and 30 grams of zinc dust were suspended in 400 cc. of water and the mixture boiled for 2.5 hours. The solution was then filtered and the residue of zinc dust extracted twice with 300 cc. of boiling water. The aqueous filtrates were combined and concentra- ted to one-half their volume, when the anilinopyrimidine separated, on cooling, in characteristic, slender prisms. The yield was 3.3 grams, or 78 per cent of the theoretical. The base was practically insoluble in cold and difficultly soluble in hot water. It crystallized from 95 per cent alcohol in pris- matic crystals that melted at 116 to a clear oil. Analysis (Kjeldahl) : Researches on Pyrimidines. 245 Calculated for C 10 H 9 N 3 . Found. N 24.56 24.22 2-Anilinopyrimidine could be boiled with concentrated hy- drochloric acid without change. One gram of the base was heated with about 5 cc. of concentrated hydrochloric acid for 2 hours at i48-i68. Under these conditions it was de- composed into tarry products, from which nothing definite was isolated. An attempt was made to convert i-anilino- pyrimidine into 2-thiopyrimidine by heating with carbon bi- sulphide. One gram of the pyrimidine was heated with 6 cc. of carbon bisulphide, for 4 hours at I9o-i95. We obtained a clear solution which deposited beautiful, needle-like prisms that melted at 116. The compound gave no test for sulphur and was identified as unaltered material. The Hydrochloric Acid Salt. This salt was extremely solu- ble in water and alcohol. It had no definite melting point, but decomposed when heated above 125. Analysis (Kjel- dahl) : Calculated for Ci H 9 N 8 .HCl. Found. N 20.24 19-77 The Sulphuric Acid Salt. This salt separated from 20 per cent sulphuric acid in elongated prisms that melted at 140- 142. The salt dissociates when warmed in aqueous solu- tions. Analysis : Calculated for CioH 9 N 3 .H 2 SO 4 . (Ci H9N 3 )2H 2 SO 4 . Found. N 15.5 19.09 15.10 The Platinum Chloride Double Salt. This salt separated in yellow rosettes when a hydrochloric acid solution of the base was treated with hydrogen -platinic chloride. The salt was slowly decomposed in boiling water and metallic platinum deposited. It melted with decomposition at 2i8-22i. Calculated for (C 10 H 9 N3.HCl) 2 .PtCl4. Found. Pt 25.91 26.6 246 Johnson and Heyl. Behavior of 2-Anilino-6-oxypyrimidine towards Hydro- chloric and Sulphuric Acids. This pyrimidine is more stable in presence of acids than 2-anilinopyrimidine. It was recovered unaltered after heating with concentrated hydrochloric acid for 4 hours at i5O-i6o. It was not decomposed after heat- ing with 25 per cent sulphuric acid at 230 for 2 hours. Behavior of 2-Anilino-6-oxypyrimidine toivards Carbon Bi- sulphide. There were no indications of any reaction after heating 2 grams of the pyrimidine with 5 cc. of carbon bisul- phide for 5 hours, at 150- 1 60. The mixture was then heated for 3 hours at 1 85 -2 15. When the tube was opened there was no pressure and the ualtered pyrimidine was recovered melt- ing at 229. In a third experiment i gram of the pyrimidine was heated with 5 cc. of carbon bisulphide for 6 hours, at 230. When the tube was opened there was some pressure due to the presence of hydrogen sulphide and a crystalline residue was suspended in the carbon bisulphide. This was identified as the unaltered pyrimidine; it melted at 228- 229, and when mixed with 2-anilino-6-oxypyrimidine the melting point was not changed. Analysis (Kjeldahl) : Calculated for C 10 H 9 ON 3 . Found. N 22.46 22.17 t Hydrochloride of 2-Ethylmercapto- 5 ,6 -diethoxy pyrimidine, N= =COC 3 H 5 C 7 H 5 SC COC 2 H 5 .HC1. This pyrimidine was obtained as II II N CH an oil by treatment of 2-ethylmercapto-5-ethoxy-6-chlorpyr- imidine 1 with the calculated quantity of sodium ethylate. When an ether solution of the pyrimidine was saturated with dry hydrochloric acid gas the salt deposited in prismatic crystals that melted with decomposition at i29-i3i. The salt was hydroscopic. Analysis (Kjeldahl) : Calculated for CioH 6 O 2 N 2 S.HCl. Found. N 10.58 10.55 1 Johnson and McCollum: J. Biol. Chem., 1, 443 (1906). Researches on Pyrimidines. 247 Action of Ammonia on 2-Methylmercapto-^-ethoxy-6-oxy- pyrimidine. This mercapto pyrimidine was recovered unal- tered after heating with concentrated alcoholic ammonia for 2 hours at I4o-i6o and again for 1.5 hours at 2io-235. 2- A nilino-5-eihoxy-6-oxy pyrimidine, NH - CO I I C 6 H 5 H N.C C.OC 2 H 5 . 2-Ethylmercapto-5-ethoxy-6-oxy- N --- CH pyrimidine 1 was recovered unaltered after heating with ani- line on the steam-bath for several hours. Four grams of the mercaptopyrimidine and 2 grams of .aniline were dissolved in 10 cc. of alcohol and heated for 2 hours at i85-2i6. When the tube was examined the presence of ethyl rnercaptan was apparent, and a crystalline residue had deposited. This was a mixture of unaltered mercaptopyrimidine and the anilino derivative that melted above 160. The anilino compound was separated from the mercapto derivative by dissolving in cold dilute hydrochloric acid and making alkaline with am- monia. The anilino derivative deposited in plates and was purified for analysis by recrystallization from alcohol. It melted at 23i-232. In a second experiment we heated 10 grams of the mercaptopyrimidine and 5.1 grams of aniline for 6 hours at i9o-2Oo and obtained 6.8 grams of crude anilino compound melting at 2i4-22o. We did not observe the formation of any 2,5-dianilino-6-oxypyrimidine under the conditions employed. Analysis (Kjeldahl) : Calculated for Ci2Hi 3 O 2 N 3 . Found. N 18.18 18.10 2-Anilino-_5-ethoxy-6-chlorpyrimidine, C 6 H 5 HNC COC 2 H 5 Ten and five-tenths grams of 2-ani- II II N -- CH lino-5-ethoxy-6-oxypyrimidine were warmed with 30 cc. of 1 Johnson and McCollum: Loc. cit. 248 Johnson and Heyl. phosphorus oxychloride until the evolution of hydrochloric acid ceased. The solution was then poured upon crushed ice, when the chlorpyrimidine separated as a semi-solid. The chloride was dissolved in ether and thoroughly dried with calcium chloride. When the ether was evaporated the chlor- ide deposited in prismatic crystals that were extremely solu- ble in organic solvents. The compound crystallized from alcohol in stout prisms, that melted at ni-ii2 to an oil. Analysis (Kjeldahl) : Calculated for Ci 2 H 12 ON 3 Cl. Found. N 16.82 16.55 N= =CH . I I 2- Anilino-5-eihoxy pyrimidine, C 8 H 5 NHC COO 2 H 5 . One N CH gram of 2-anilino-5-ethoxy-6-chlorpyrimidine was digested for i hour with to grams of zinc dust and 150 cc. of water. When the hot mixture was filtered the pyrimidine deposited in prismatic crystals. It was difficultly soluble in water and more was obtained by repeatedly extracting the zinc resi- due with hot water. The pyrimidine dissolves in concentra- ted sulphuric acid to give a yellow solution. It crystallized from 95 per cent alcohol in short prisms that melted to a clear oil at I30-i3i. Analysis (Kjeldahl): Calculated for Found. C 12 H 18 ON 8 . I. II. N 19.53 18.98 19.33 2-Anilino-5-etho%y-6-aminopyrimidine, N=C.NH 2 C 6 H 5 NH.C COC 2 H 5 This pyrimidine was obtained when II II N CH 2-anilino-5-ethoxy-6-chlorpyrimidine was heated with alco- holic ammonia at i8o-i88. When i gram of the chloride was heated with alcoholic ammonia, for 2 hours, at 150, Researches on Pyrimidines. 249 the reaction was not complete, and the product obtained gave a pronounced test for chlorine after crystallization from alco- hol. The pyrimidine crystallized from 95 per cent alcohol in prisms that melted at 133 134 to a clear oil. Analysis (Kjeldahl): Calculated for Ci a H 14 ON 4 . Found. N 24.34 24.06 NBW HAVEN, CONN., June 15. 1907. vnoi 'i> -to! Jrf^J bsO [ju-tevio onibin.hvq yrIT -loii ,.'//ioD .HJIVA .70Q1 ?-I VI. RESEARCHES ON PYRIMIDINS: SYNTHESIS OF THYMIN-4-CARBOXYLIC ACID. BY TREAT B. JOHNSON. (From the Sheffield Laboratory of Yale University.) (Received for publication, June 27, 1907.) I shall describe, in this paper, the preparation and properties of thymin-4-carboxylic acid, NH CO I I CO CCH 3 I II NH CCOOH The study of the carboxylic acids of uracil, cytosin and thymin is of interest on account of the possibility that these pyrimidins may be linked in nucleic acids by acid amide groupings, CO.NH. It has already been shown 1 that uracil may exist in nucleic acids as a 5-carboxyl compound since uracil- 5 -carboxylic acid can be quantitatively converted into uracil if heated with 20 per cent sulphuric acid at 160-170. I now find that thymin-4-carbox- ylic acid can be heated with 20 per cent sulphuric acid, under the same conditions, without alteration. Thymin therefore can- not exist in the nucleic acids as a ^.-carboxyl compound. Wislicenus observed that diethyl oxalacetate and its homo- logues show a wide difference in their behavior towards ammonia and amines. Diethyl oxalacetate, for example, combines with ammonia to form an unstable addition product, which is changed to an ammonium salt of the lactone ester of oxalcitric acid (I) 2 when warmed with alcohol. Diethyl methyloxalacetate, on the other hand, does not form 1 Amer. Chem. Journ., xxxvii, p. 392. 2 Wislicenus and Beckh: Ber. d. deutsch. chem. Gesellsch., xxviii, p. 789; Ann. d. Chem. (Liebig), ccxcv, p. 339. 299 300 Researches on Pyrimidins an addition product with ammonia but reacts at 110, giving aminomethylmaleinimide (II). 1 C 2 H 6 2 C-CH C0 2 C 2 H 6 II Another striking example of this difference between diethyl oxalacetate and diethyl methyloxalacetate was found when we investigated the behavior of pseudothioureas towards these esters. Pseudoethylthiourea combines with diethyl oxalacetate, giving a stable addition product (Professor Wheeler) . On the other hand, diethyl methyloxalacetate reacts with pseudomethylthiourea, in presence of potassium hydroxide, giv- ing a good yield of the potassium salt of 2-methylmercapto-4- carboxyl-5-methyl-6-oxypyrimidin (III). The condensation can be represented as follows: NH, COOC 2 H 5 NH CO I ! II C 2 H 5 SC + C-CH 3 CH 3 SC C'CH 3 + 2C 2 H 5 OH. II II II il NH HO-C'COOC 2 H 5 N C'COOH III 2-Methylmercapto-4-carbethoxy - 5 - methyl - 6-oxypyrimidin (V) was obtained, in one experiment, as a secondary product of the condensation. 2-Methylmercapto-4 - carboxyl-5-methyl-6-oxypyrimidin (III) can be converted into thymin-4-carboxylic acid (VII), by digest- ing with concentrated hydrochloric acid. When this acid was boiled, in ethyl alcohol solution, with a small quantity of sul- phuric acid it was converted into thymin-4-ethylcarboxylate (VIII). This same ester was also obtained when 2-methylmer- capto-4-carbethoxy-5-methyl-6-oxypyrimidin (V), was boiled in alcoholic solution with sulphuric acid. When thymin-4-car- boxylic acid (VII), is melted it undergoes complete decom- 1 Wislicenus and Kiesewetter: Ber. d. deutsch. chem. Gesellsch., xxxi, p. 194. Treat B. Johnson 301 position. On the other hand, 2-methylmercapto-4-carboxyl-5- methyl-6-oxypyrimidin (III), melts with evolution of carbon- dioxide and is converted quantitatively into 2-methylmercapto- 5-methyl-6-oxypyrimidin (IV). This mercaptopyrimidin can then be changed to thymin (VI) by boiling with hydrochloric acid. 1 The formation of thymin in this manner from 2-methylmer- capto-4-carboxyl-5-methyl-6-oxypyrimidin (III), shows that the condensation takes place as represented in the preceding equa- tion and that the product is not a hydantoin derivative. The above compounds and their various transformations are repre- sented as follows: NH CO NH CO NH CO II II II CH 3 SC CCH 3 < II II N CH r NH CO I I CO CCH, I II NH CH VI Thymin-4-carboxylic acid is characterized by its insolubility in cold water and by its property of crystallizing from aqueous solution with and without water of crystallization. It gives very insoluble barium and lead salts, and is not precipitated by phos- photungstic acid. Thymin-4-carboxylic acid reacts in the normal manner with bromine water giving oxybromhydrothymin-4-carboxylic acid (IX). NH CO NH CO II I I CH CO C-CH,, + Br, + H 2 O = CO C < ~" 3 + HBr II I I NH CCOOH NH C'OH COOH IX 1 Amer. Chem. Journ., xxix, p. 487. 3C CCH + II II CH 3 SC CCH 3 II II II II N CCOOH I 1 " NH CO 1 1 II II N CCO 2 C 2 H, r NH CO 1 1 1 1 CO CCH 3 <=> \ II NH CCOOH VII 1 1 CO C-CH 3 1 II NH CCO 2 C 2 H S VIII 302 Researches on Pyrimidins EXPERIMENTAL. 2-Methylmercapto-4.-carboxyl-5-methyl-6-oxypyrimidin, NH CO I I CH 3 S-C C-CH 3 II II N C-COOH Thirty grams of pseudornethylthiourea hydriodide and 50 grams of the sodium salt of diethyl oxalpropionate were dissolved in about 500 cc. of water and two molecular proportions of potassium hydroxide (16 grams) added to the solution. The mixture was allowed to stand on the steam-oven for 8-10 hours and then concentrated to a volume of 150 cc. After thorough cooling, and acidifying with hydrochloric acid, the mercapto-pyrimidin deposited in prismatic crystals. It was practically insoluble in cold water and difficultly soluble in boiling water and alcohol. It separated from water or alcohol in rectangular prisms that melted at 243-244 with effervescence to a clear oil. It deposited from glacial acetic acid in stout prismatic crystals. The yield of acid corresponded to about 80 per cent of the theoretical. Analysis (Kjeldahl) : Calculated for C 7 H 8 O 3 N 2 S: Found: N ................... 14. 00 per cent. 13.88 per cent. 2-Methylmercapto-4.-carbethoxy-5-methyl-6-oxypyrimidin, NH CO I I CH 3 SC CCH 3 II II N CCOOC 2 H 5 This ester was obtained, associated with 2-methylmercapto- 4-carboxyl-5-methyl-6-oxypyrimidin, when I used one instead of two molecular proportions of potassium hydroxide in the above condensation. It was difficultly soluble in cold water and alcohol, but deposited from hot alcohol or water in slender needles that melted at 201202 to a clear oil without effervescence. An- alysis (Kjeldahl): Calculated for Found: N ................... 12. 30 per cent. 12.37 per cent. Treat B. Johnson 303 Potassium Salt of 2-Methylmercapto-^-carboxyl-^-methyl-6-oxy- pyrimidin, NH CO I I CH 3 SC C-CH 3 '6H 2 O ' II II N CCOOK A good yield of this salt was obtained under the following con- ditions: Pseudomethylthiourea and diethyl oxalpropionate were condensed as in the above experiment, in presence of two mole- cular proportions of potassium hydroxide. After standing for about twelve hours at 40-45 the solution was then acidified with acetic acid and concentrated to a volume of about 150 cc. On cooling the potassium salt deposited in distorted needles that decomposed with effervescence when heated above 230. When this acetic acid filtrate was treated with hydrochloric acid the mercapto-acid separated melting at 243. The potassium salt was very soluble in hot water and deposited, on cooling, in needles. They contained water of crystallization which was determined by heating at 100-110 for two hours. 1.2424 gram substance lost o . 4032 gram water. Calculated for C 7 H 7 O3N 2 SK.6H 2 O: H 2 O 31.21 per cent. 32 . 4 per cent. Nitrogen determination in the anhydrous salt (Kjeldahl) : 4 Calculated for C 7 H 7 03N 2 SK: Found: N 1 1 . 76 per cent. 11.91 per cent. Behavior of 2-Metkylmercapto-^.-carboxyl-^-methyl-6-oxypyrimi- din on Heating. About two grams of the mercapto-acid were heated in a sulphuric acid bath at 245 until all effervescence ceased. I obtained a clear oil that crystallized, on cooling, in large, prismatic crystals. When these prisms were heated they melted sharply at 230-231 without effervescence to a clear oil. The compound deposited from water in flat prisms that melted at 233 and was identified as 2-methylmercapto-5-methyl-6-oxy- 304 Researches on Pyrimidins pyrimidin. 1 When mixed with this pyrimidin the melting point was not lowered. Analysis (Kjeldahl) : Calculated for _, C 6 H 8 ON 2 S: Found: N 17.94 percent. 17.80 per cent. Thymin- ^.-carboocylic Acid, NH CO I I CO CCH 3 -H 2 I II NH CCOOH A quantitative yield of this acid was obtained when 2-methyl- mercapto-4-carboxyl-5-methyl-6-oxypyrimidin was digested with concentrated hydrochloric acid. The oxygen acid separated from the acid solution as a granular powder that was difficultly soluble in boiling water and practically insoluble in alcohol. A most characteristic behavior of this acid is its property of crystal- lizing from hot water in anhydrous condition and with one mole- cule of water of crystallization. When a hot, saturated aqueous solution of the acid was allowed to cool slowly the anhydrous acid first deposited in balls of microscopic prisms resembling very much the crystalline form of uracil. They decomposed at 328-330 (Anschutz thermometer) and did not lose weight when heated at 120. Analysis (Kjeldahl): Calculated for C 6 H 6 O 4 N 2 : Found: N 16 . 47 per cent. 16 . 37 per cent. After filtering from the anhydrous acid and allowing the fil- trate to stand, transparent, rectangular prisms of the hydrous acid deposited. They decomposed at the same temperature as the anhydrous acid (3 2 8-3 3 o) . The acid was not precipitated by phosphotungstic acid. Water determination: 0.6742 gram substance lost 0.0687 gram water after heating one hour at 1 10-120. Calculated for C 6 H 6 4 N 2 .H 2 0: Found: H 2 O 9 . 60 per cent. 10.1 per cent. l Amer. Chem. Journ., xix, p. 478. Treat B. Johnson 305 Analysis of the anhydrous acid: 0.2592 gram substance gave 0.4002 gram CO 2 and o. 0854 gram H 2 O. Nitrogen determination (Kjeldahl) : C H N.. Calculated for C 6 H 6 4 N 2 : 42 . 35 per cent. 3 . 52 " " 16.47 " " Found : 42.11 per cent. 3.66 " " 16.23 " " Action of 20 per cent Sulphuric Acid. One-half a gram of the thymin acid was heated with 5 cc. of 20 per cent sulphuric acid for two hours at 160-170. When the tube was opened there was no pressure and the unaltered acid was suspended in the sulphuric acid. I recovered 0.45 gram of acid melting at 326- 329. Analysis (Kjeldahl): Calculated for ,-, , CH 6 4 N 2 : Found: N 16. 47 per cent. 16. 15 per cent. Potassium Salt, C 6 H 5 O 4 N 2 K.2H 2 O. This salt was prepared by dissolving molecular proportions of potassium hydroxide and thymin-4-carboxylic acid in water. When the solution was concentrated and allowed to stand for a few hours the salt deposited in radiating prisms. It was dried for analysis in a desiccator over sulphuric acid (Kjeldahl) : Calculated for Calculated for C 6 H 5 O 4 N 2 K: C 6 H.,O 4 N 2 K.2H 2 O: Found: N 13 . 45 per cent. 1 1 . 06 per cent. 10.97 per cent. Lead Salt (C 6 H 5 O 4 N 2 ) 2 Pb. This salt deposited in well devel- oped prisms when a solution of lead acetate was added to a hot, saturated, aqueous solution of thymin-4-carboxylic acid. It was practically insoluble in cold water. Analysis (Kjeldahl) : Calculated for , (C 6 H 5 O4N 2 ) 2 Pb: Found: N 10 . 27 per cent. 10 . 00 per cent. Barium Salt (C 6 H 5 O 4 N 2 ) 2 Ba. Thymin-4-carboxylic acid gives no precipitate when treated with a solution of barium chloride. The barium salt was prepared by dissolving the acid in potas- sium hydroxide and then adding the calculated amount of barium chloride. It separated from water in corpuscular crystals. Analysis (Kjeldahl): Calculated for (C 6 H 6 O 4 N 2 1 2 Ba: Found: N 11.77 per cent. 11 .30 per cent. 306 Researches on Pyrimidins Thymin-f-ethylcarboxylate, NH CO I I CO CCH 3 I II NH CCOOC 2 H 5 was prepared by esterifying the acid with ethyl alcohol and sulphuric acid. It was also obtained when 2-methylmercapto- 4-carbethoxy-5-methyl-6-oxypyrimidin was boiled in alcoholic solution with a small amount of hydrochloric acid. The ester deposited from hot water in distorted prisms that melted at 255 to a clear oil without effervescence. Analysis (Kjeldahl) : Calculated for C 8 H 10 4 N 2 : Found: N ................... 14. 14 per cent. 14.07 per cent. Oxybromhydrothymin-4.-carboxylic A cid, NH CO I I CH CO C < utl * I I Br NH C'OH COOH Three and five-tenths grams of finely pulverized thymin-4- carboxylic acid were suspended in about 40 cc. of bromine water and bromine slowly added until the acid had completely dissolved. There was no evolution of carbon dioxide and when the solution was allowed to evaporate spontaneously in the atmosphere well-developed, prismatic crystals of the hydro- derivative separated. It was purified for analysis by recrys- tallization from bromine water. It crystallized in small, pris- matic crystals that charred when heated above 270 and then decomposed with violent effervescence from 295-300 accord- ing to the rate of heating. When thymin-4-carboxylic acid was heated with bromine water at 146-152 it was completely decom- posed with formation of bromoform. Analysis (Kjeldahl) : Calculated for Calculated for Found: C 5 H5O 3 N 2 Br: I. II. N ..... 10. 48 per cent. 12.66 per cent. 10.45 10.45 per cent. [Reprinted from the American Chemical Journal, Vol. XXXVIII. No. 5. November, 1907.] Contributions from the Sheffield Laboratory of Yale University. CXLIX. RESEARCHES ON PYRIMIDINES: SYN- THESIS OF CYTOSINE-5-CARBOXYLIC ACID. (TWENTY-SIXTH PAPER.) BY HENRY I,. WHEKLER AND CARL O. JOHNS. The present work was undertaken with the obj'ect of making a study of some material to be used for further syntheses, and also in order to examine the properties of cytosine-5-carboxylic acid and some of its derivatives. The acid has now been pre- pared by means of the following reactions: In a previous paper 1 we showed that ethoxymethylenmalonic ester, C 2 H 5 OCH = C(C0 2 C 2 H 5 ) 2 , in alkaline solution, condenses normally with ethylpseudo- thiourea, H 2 N C(SC 2 H 5 )=NH, giving 2-ethylmercapto-5- 1 THIS JOURNAL, 37, 392 (1907). Researches on Pyrimidines. 595 carbethoxy-6-oxypyrimidine (I.). We now find that this cyclo amide reacts smoothly with phosphorus oxychloride r yielding an oil which is the imide chloride, 2-ethylmercapto- 5-carbethoxy-6-chlorpyrimidine (!!.) The transformation of this into 2-ethylmercapto-5-carbethoxy-6-aminopyrimidine (III.), on standing with alcoholic ammonia at ordinary tem- perature, is also a perfectly smooth reaction. The imide chloride, therefore, in this case, is far more reactive than 2-ethyl- mercapto-6-chlorpyrimidine. The latter does not react on standing overnight with alcoholic ammonia, but it does react in a short time at 120. When the mercaptoamino ester (III.) is heated with alcoholic ammonia the mercapto group is the first to be removed (at about 170) and 2,6-diamino-5-carbethoxypyrimidine (IV.) results. When the mercaptoamino ester (III.) is carefully warmed with dilute alcoholic potassium hydroxide it is readily saponi- fied and a potassium salt of 2-ethylmercapto-5-carboxyl-6- amino-pyrimidine (V.) is formed. If the treatment with potassium hydroxide is more energetic, mercaptan is also re- moved and the potassium salt of cytosine-5-carboxylic acid is obtained (VI.). The moderate action of strong hydrochloric acid on 2-ethyl- mercapto-5-carbethoxy-6-aminopyrimidine (III.) first removes ethylmercaptan, giving 5-carbethoxycytosine (VII.), which with aqueous ammonia at 150 is partially converted into cy- tosine-5-carboxamide (VIII.). Longer boiling with hydrochloric acid saponifies the ester (III.), both mercaptan and alcohol separate, and cytosine-5- carboxylic acid (VI.) results. The synthesis of the acid and the transformations of the new pyrimidines can be illustrated by the formulas on page 596. The action of acids on cytosine-5-carboxylic acid (VI.) developed the interesting fact that the presence of the carboxyl radical in the 5 -position rendered the amino group apparently more easily subject to hydrolysis than in the case of unsub- stituted cytosine. It was found also that the 6-amino group was removed before the carboxyl radical when cytosine-5- 596 Wheeler and Johns. a 5- fc x- tf W o 8 00 = o O o W l ri w fc rf w o 8- I o W tf 5 W 1*4 5- o o=< o" ffl 1 _=s O O w I r<" W 5 o 80=0 00=9 0^0=0 I I- If wow Researches on Pyrimidines. 597 carboxylic acid was boiled with 20 per cent sulphuric acid, the chief product obtained being uracil-5-carboxylic acid (IX.), while only a small amount of cytosine was formed. Unless groups attached to cytosin-5-carboxylic acid would alter the course of hydrolysis, these results exclude the possibility that cytosin exists in the nucleic acids in the form of a 5-carboxyl derivative, or, in other words, it is not a grouping of this sort which is the source of cytosine. Further action of acids, either at a high temperature, or on more prolonged boiling, strips all of the groups from the above compounds, leaving uracil (X.) as the last pyrimidine to be obtained. EXPERIMENTAL PART. 2- Eihylmercapto-5-carbethoxy-6-chlor pyrimidine, N=CCl I | C,H 6 SC CCO a C 2 H 6 . Thirteen grams of 2-ethyl-mercapto- N CH 5-carbethoxy-6-oxypyrimidine were boiled for two and a half hours with 40 cc. of phosphorus oxychloride. The phosphorus oxychloride was then removed by evaporation at 100 under diminished pressure. This left a syrup which was treated with cracked ice and the resulting oil, which did not solidify, was extracted with ether and dried over calcium chloride. It boiled at 203 at 20 mm. pressure. The yield of oil is almost quantitative and the crude product is pure enough to use for the following preparations without distilling. Nitrogen de- terminations in the case of the distilled product gave: Calculated for Found. C 9 H ai 2 N s SCl. I. II. N 11.35 11.33 11.31 2-Ethylmercapto-5-carbethoxy-6-aminopyrimidine, N :C NH 2 I I C 2 H 5 SC CCO 2 C,H 6 . Fourteen grams of 2-ethylmercapto- N CH 5-carbethoxy-6-chlorpyrimidine were added to a cold saturated 59 8 Wheeler and Johns. solution of alcoholic ammonia in a bottle. A reaction set in almost at once and a mass of crystals separated. The bottle was kept cool by means of ice- water. The material was allowed to stand overnight and then the alcohol and ammonia were evaporated and the residue treated with water. The yield was 90 per cent of the calculated. The resulting product melted to an oil in hot water but did not dissolve. It dis- solved readily in hydrochloric acid. It was easily soluble in hot and moderately soluble in cold alcohol from which solvent it crystallized in rectangular plates melting at 102. Analysis: Calculated for Found. I. II. N 18.50 18.41 18.42 When this substance was boiled with strong hydrochloric acid for about one hour, the solution evaporated to dryness and the residue dissolved in sodium hydroxide and precipitated with acetic acid, cytosine-5-carboxylic acid was obtained. N= =C NH 2 I I 2,6-Diamino-5-carbethoxypyrimidine, H 2 N C CCO 2 C 2 H 5 . N - CH This was obtained by heating the above 2-ethylmercapto-5- carbethoxy-6-aminopyrimidine with a saturated solution of alcoholic ammonia at 1 68- 178 for one and a half hours. When the tube became cool, a solid separated. The material was difficultly soluble in cold alcohol and water and it crystal- lized from alcohol in the form of needles melting to a clear oil at 205-2O7. Analysis: Calculated for Found. C 7 H 10 02N 4 . I. II. N 30.76 30.62 30.76 This substance is soluble in hydrochloric acid from which solu- tion it is reprecipitated by ammonia. The material which re- mained in the alcoholic mother-liquor from the above was found to be unaltered mercapto-derivative. Under the con- ditions of the experiment it appears that the carbethoxy group was not attacked. Researches on Pyrimidines. 599 2-Ethylmercapto-5-carboxyl-6-aminopyrimidine, N=C NH 2 I | C 2 H 5 SC CCOOH .When 2-ethylmercapto-5-carbethoxy- N CH 6-aminopyrimidine in alcoholic solution was treated with an equal weight of potassium hydroxide in a small amount of water it was readily saponified. On evaporating to dryness the resulting residue dissolved easily in water. When this solution was made acid with acetic acid a white crystalline substance slowly separated. This was slightly soluble in hot water and difficultly soluble in hot alcohol, from which it crys- tallized in microscopic prisms. It decomposed with efferves- cence at 230. It was very soluble in ammonia and also in an excess of hydrochloric acid. Analysis : Calculated for Found. C 7 H 9 OjN 3 S. I. II. N 21.10 2095 21.04 During the above hydrolysis there was a decided odor of es- caping mercaptan. In order to determine whether mercaptan could be conveniently split off by means of potassium hydrox- ide, another portion of 2-ethylmercapto-5-carbethoxy-6-amino- pyrimidine was heated a little longer on the steam-bath with somewhat more than 2 molecular proportions of potassium hy- droxide. On acidifying the residue, a substance was obtained which did not contain sulphur and agreed in properties with cytosine-5-carboxylic acid. (Analysis III. below.) N- C NH, Cytosine-5-carboxylic Acid,OC CCOOH . This amino acid I! II HN CH was first prepared by evaporating the above mercaptoamino acid with concentrated hydrochloric acid on the steam-bath. After evaporating once with a moderate amount of acid, the residue was taken up in water and evaporated again to remove hydrochloric acid. It was found that the acid was almost in- soluble in hot water, alcohol and acetic acid. The specimen 6oo Wheeler and Johns. used for analysis was purified by dissolving in ammonia and precipitating with acetic acid. It came down in a very finely divided state and was hard to filter. It decomposed with effervescence at 2 56 '-257. Analysis: Calculated for Found. C 6 H 5 8 lsr 8 . I. II. in. N 27.09 27.07 27.07 26.80 To prepare this acid it is not necessary to isolate the 2-ethyl- mercapto-5-carboxyl-6-aminopyrimidine. The easiest way is to start with 2-ethylmercapto-5-carbethoxy-6-aminopyrimidine and evaporate this once with an equal weight of potassium hy- droxide in dilute alcohol. Concentrated hydrochloric acid is then added to the residue and the evaporation carried on again to dryness. The mixture is taken up in water, to remove potassium chloride, and, on filtering and washing with water, the acid is obtained in an almost pure condition. The aqueous solution of cytosine-5-carboxylic acid does not give a precip- itate with barium chloride, picric acid or lead acetate. It gives but a slight turbidity with silver nitrate. Action of Twenty Per Cent Sulphuric Acid: Formation of UracU-5'Carboxylic Acid. One gram of cytosine-5-car- boxylic acid was boiled on a return condenser for 20 hours with 40 cc. of sulphuric acid. A solid separated on cooling. It weighed 0.5 gram. This was crystallized from water and came down in clusters of pointed, microscopic prisms which decomposed at 277. They were indentified as uracil-5-car- boxylic acid. Analysis: 0.3133 gram substance lost 0.0329 gram water at I2O-I3O. Calculated for C 6 H 4 04N 2 .HsO. Found. H,O 10.34 10.50 N 17-94 l8.22 A duplicate experiment performed as above gave 0.45 gram of uracil-5-carboxylic acid, which separated from the solution. After filtering from this, the sulphuric acid was removed and the solution concentrated to 10 cc., then precipitated with picric acid. This gave 0.17 gram picrate, crystallizing in needles, equal to 0.06 gram cytosin. Formation of Uracil. One half gram of cytosine-5-carbox- Researches on Pyrimidines. 60 1 ylic acid was heated with 5 cc. of 20 per cent sulphuric acid at i62-i67 for 2 hours. On opening the sealed tube, consid- erable pressure was noticed and a deposit which had the ap- pearance of uracil had separated. This was recrystallized from water and identified as uracil. Analysis: Calculated for C 4 H 4 OjN 2 . Found. N 25.00 24.97 Action of Concentrated Hydrochloric Acid: Formation of Uracil. One gram of cytosine-5-carboxylic acid was boiled with 50 cc. of concentrated hydrochloric acid, using a return condenser, for 22 hours. The acid was then evaporated and the residue was treated with boiling water. A small portion was insoluble in hot water. This was unaltered cytosine-5- carboxylic acid; an analysis gave 26.80 per cent of nitrogen while the calculated is 27.09 per cent. The portion which was soluble in water crystallized in balls like uracil. Analysis gave 24.90 per cent nitrogen. Cytosine-j-carboxylic Acid Hydrochloride was prepared by crystallizing some of the acid from 20 per cent hydrochloric acid. It formed ragged pointed prisms which melted at 275- 276 and it contained a molecule of water of crystallization. Analysis : 0.1327 gram lost 0.0117 gram of water at 130- 140. Calculated for C 6 H 6 O 8 N8.HC1.H,O. Found. H a O 8.59 8-82 Calculated for C 6 H 6 O3N 8 .HC1. Found. N 21.93 21.71 N C NH, I I 5-Carbethoxycytosine, OC CCO 2 C a H 6 , was formed by HN CH evaporating 2-ethylmercapto-5-carbethoxy-6-aminopyrimidine with concentrated hydrochloric acid on the steam bath. The product was dissolved in hot water and the solution after making slightly alkaline with ammonia gave needles which 602 Wheeler and Johns. browned and decomposed slowly at about 26o-275. This ester was only slightly soluble in hot alcohol. Analysis: Calculated for Found. C 7 H 9 O 3 N 8 . I. II. N 22.95 22.97 23.00 N= =C NH. I I Cytosine-5-carboxamide, OC CCONH,. The action of HN CH concentrated aqueous ammonia on the above ester was not at all smooth. In one experiment in which the ester was heated with concentrated ammonia for 2 hours at 140- 150, about 25 per cent of the material remained unaltered but on evapora- ting the mother-liquor far more soluble material was obtained. This crystallized in tufts of hair-like needles. It was recrystal- lized from water. (Analysis I.) On heating, no definite melt- ing or decomposition point was observed. Analyses of two different samples gave the following results which show that the expected amide had been obtained. Calculated for Found. C 6 H 6 2 N 4 . I. II. N 36.36 35-94 35-00 NEW HAVBN, COWN., May, 1907. [Reprinted from the American Chemical Journal, Vol. XXXVIII. No. 5. November, 1907.] CL. RESEARCHES ON PYRIMIDINES: SYNTHESIS OF THYMINE-5'-CARBOXYLIC ACID. [TWENTY- SEVENTH PAPER.] BY TREAT B. JOHNSON AND CARL FRANK SPEH. The possibilty that uracil, cytosine and thymine might be linked in nucleic acids by acid amide or polypeptide group- ings was an incentive to make a thorough study of the car- boxyl derivatives of these pyrimidines. Descriptions of uracil-5-carboxylic acid, 1 I., thymine-4-carboxylic acid, 2 IV., cytosine-5-carboxylic acid, 3 III., and uracil-4-carboxylic acid, 4 1 Wheeler, Johnson and Johns: THIS JOURNAL, 37, 392. 2 Johnson: Jour. Biolog. Chem., 3, 299. 3 Wheeler and Johns: THIS JOURNAL, 38, 594. 4 Wheeler: Ibid., 38, 358. Researches j)n Pyrimidines. 603 II., have already been published in papers from this laboratory. NH CO NH CO N= :=CNH, II I J II, CO CCOOH, CO CH , CO CCOOH II! I II I II NH CH NH CCOOH NH CH I. II. III. NH CO NH CO I I I --II CO CCH 3 , CO .* CCH 2 COOH. I II I Ml NH CCOOH NH CH IV. V. We shall describe in this paper the preparation and proper- ties of thymine-5'-carboxylic acid, V. The carbon atom in the methyl radical of thymine is designated 5' in order to avoid confusion and to distinguish from position 7 in the purine molecule. We find that this acid, V., L* JL^I ' \ &/* Pyrimidine. Purine. can be heated with 20 per cent sulphuric acid at i6o-i7O without alteration. Thymine, therefore, cannot exist in nucleic acids as a j'-carboxyl compound. The information acquired by investigating these pyrimidine acids has advanced decidedly our knowledge of the structure of the nucleic acid "molecule. The results of our work justify us in concluding that uracil is the only one of the three pyr- imidines uracil, cytosine and thymine that can be linked in nucleic acids by an acid amide grouping CO.NH. Uracil might exist as a $-carboxyl compound. This conclusion is sup- ported by the fact that uracil-5-carboxylic acid, 1 I., is quan- titatively converted into uracil when heated with 20 per cent sulphuric acid at i6o-i7o. Uracil-4-carboxylic acid, 1 IL, and thymine-4-carboxylic acid, 1 IV., could be heated under i Loc. cti. 604 Johnson and Speh. the same conditions without alteration. The possibility of cytosine being linked as a 5-carboxyl compound is excluded, since cytosine-5-carboxylic acid, 1 III., is changed to uracil- 5-carboxylic acid by hydrolysis with dilute sulphuric acid. Every a-derivative of ethyl /?-oxyacrylate (et hylf ormyl- acetate) that we have examined in this laboratory has con- densed with a pseudothiourea, in presence of alkali, giving a 2-mercapto pyrimidine, viz. : 1. Ethyl /?-oxyacrylate, 2 HOCH:CH.COOC 2 H 5 . 2. Ethyl a-methyl-/?-oxyacrylate, 8 HOCH : C(CH 3 )COOC 2 H 5 . 3. Ethyl a-phthalimido-/?-oxyacrylate, 4 / C HOCH: C(N< X CO' 4. Ethyl a-benzoylamino-/?-oxyacrylate, 5 HOCH: C(NHCOC 8 H 5 )COOC 2 H 6 . 5. Ethyl a-phenyl-/9-oxyacrylate, fl HOCH : C(C 8 H 5 )COOC 2 H 6 . 6. Ethyl a-urethane-/?-oxyacrylate, 7 HOCH: C(NHCOOC 2 H 5 )COOC 2 H 5 . 7. Ethyl a-ethoxy-/?-oxyacrylate, 8 HOCH : C(OC 2 H 5 )COOC 2 H 5 . 8. Ethyl a-ethyl-/?-oxyacrylate, 9 HOCH : C(C 2 H 5 )COOC 2 H 5 . 9. Ethyl a-phenoxy-/?-oxyacrylate, 10 HOCH : C(OC 8 H 5 )COOC 2 H 5 . 1 Loc. ctt. * Wheeler and Merriam: THIS JOURNAL, 29, 478. Wheeler and Merriam: Loc. cit. 4 Johnson and Clapp: THIS JOURNAL, 32, 130. * Johnson and Clapp: Loc. eft. 6 Wheeler and Bristol: THIS JOURNAL, 33, 448. 7 Johnson: Ibid., 34, 191. Johnson and McCollum: Jour. Biolog. Chem., 1, 437. THIS JOURNAL, 36, 149; 9 Johnson and Menge: Jour. Biolog. Chem., 2, 105. M Johnson and Heyl: THIS JOURNAL, 37, 628. Researches on Pyrimidines. 605 We now find that the sodium salt of diethyl formylsuccin- ate condenses in a smooth manner with pseudoethylthio- urea, giving ethyl 2-ethylmercapto-6-oxypyrimidine-5-ace- tate, VI. The condensation is represented by the following equation : NH 2 COOC a H 6 C + CCH 3 COOC 8 H 6 = II II NH HOCH NH CO I H,0 + C a H 6 OH + C 3 H 6 SC CCH 3 COOC,H ft . II II N CH VI. When ethyl 2-ethylmercapto-6-oxypyrimidine-5-acetate, VI., was digested with concentrated hydrochloric acid, or with alcohol and hydrochloric acid, it was converted quantitatively into thymine-5'-carboxylic acid, VIII. Esterification of thymine- 5'-carboxylic acid with ethyl alcohol and sulphuric acid gavethy- mine-5'-ethylcarboxylate, IX. 2-Ethylmercapto-6-oxypyrimi- dine-5-acetic acid , VII., was obtained when ethyl 2-ethylmercap- to-6-oxypyrimidine-5-acetate, VI., was saponified with an alco- holic solution of potassium hydroxide. When this mercapto acid was digested with hydrochloric acid it was converted into thymine-5'-carboxylic acid. Ethyl 2-ethylmercapto-6-oxypyrimidine-5-acetate, VI., re- acts in a smooth manner with phosphorus oxychloride, giving 2-ethylmercapto-6-chlorpyrimidine-5-acetic acid, X. When the mercapto ester, VI., was heated with ammonia it was con- verted into 2-ethylmercapto-6-oxypyrimidine-5-acetamide, XL, and 2-amino-6-oxypyrimidine-5-acetamide, XII. These differ- ent transformations are represented by the formulas on the following page. Thymine-5'-carboxylic acid is difficultly soluble in water and melts at about the same temperature as thymine (315- 320). It crystallizes from water in anhydrous condition. It is a strong acid and gives an insoluble, crystalline lead salt, 6o6 Johnson and Speh. 8-8 =g I Id B-8-g ffi o q w o o f evolving carbon dioxide and ethylmercaptan. Analysis (Kjeldahl) : Calculated for Found. CgHjaOaNoS. I. II. N 12.28 12.55 12.30 Potassium Salt, C 9 H n O 3 N 2 SK. This salt is extremely soluble in cold water. It crystallized from 50 per cent alcohol in mi- croscopic needles. Analysis (Kjeldahl): 664 Johnson and Heyl. Calculated for Found. I. II. N 10.52 10.67 IO -45 Ethyl 2- Ethylmercapto-4-methyl-6-oxypyrimidine- ^-acetate, NH - CO I I C 2 H 6 SC CCH 2 COOC 2 H 6 . II II N - CCH 8 This ester was not obtained as a product of the preceding con- ! densation. This is explained by the fact that it was necessary to use an excess of potassium hydroxide to keep the diethyl acetosuccinate in solution and the ester, if formed, was always saponified. It was prepared by boiling the mercapto acid in alcoholic solution with a few drops of sulphuric acid. Two grams of the mercapto acid, 25 cc. of absolute alcohol, and 2 cc. of concentrated sulphuric acid were digested on the steam bath for 5 hours. The excess of alcohol was then evaporated at ordinary temperature, when we obtained an oily residue with some colorless, prismatic crystals in suspension. These were filtered by suction and the syrup again allowed to stand for a few hours, when a second crop was obtained. The yield was 0.25 gram and the compound was identified as 4-methyluracil-5- ethylacetate (see below). The syrup was treated with 20-30 cc. of cold water, when a crystalline compound deposited which melted at 1 5 1 -i 63 to a clear oil. It deposited from 95 per cent alcohol in hairlike crystals that melted at i63-i65 to a clear oil. It gave a test for sulphur. Analysis (Kjeldahl) : Calculated for Found. C n H 16 8 N 2 S. I. II. N 10.93 I][ -4 11.08 4-Methyluracil- ^-acetic Acid, NH - CO I I CO CCH 2 COOH. I II NH - CCH S 2-Ethylmercapto-4-methyl-6-oxypyrimidine-5-acetic acid dis- solves at once in cold, concentrated hydrochloric acid and the Researches on Pyrimidines. 665 solution can be evaporated to dryness on the steam bath and the acid recovered unaltered. A quantitative yield of the oxy- gen acid was obtained, nevertheless, by boiling the mercapto- jyrimidine with concentrated hydrochloric acid until the evo- .ution of ethylmercaptan ceased. It deposited from the [boiling acid solution as a heavy, granular powder. The acid I is practically insoluble in boiling alcohol and insoluble in cold (water. It crystallized from hot water in long, transparent | prisms that sintered at about 329 and decomposed at 340 (Anschiitz thermometer) with violent effervescence. Bromine j does not react with the acid in acetic acid solution at ordinary temperature. Analysis : I. o. 1950 gram of substance gave 0.3274 gram of CO 2 and 10.0787 gram of H 2 O. II. 0.2352 gram of substance gave 0.3935 gram of CO 2 and 0.0955 gram of H 2 O. Nitrogen (Kjeldahl): Calculated for Found. C 7 H 8 O 4 N,. I. II. III. IV. C 45.65 45-77 45-62 4-34 4 4 8 4-50 N 15.21 l 5-<>9 I5-!0 Solubility in Water at 25. One hundred grams of water dissolved : i. ii. 0.0901 gram acid 0.0894 gram acid Potassium Salt of 4- Methyluracil- ^-acetic Acid, C 7 H ? O 4 N 2 K 3H 2 O, was prepared by dissolving the acid in water with one molecular proportion of potassium hydroxide. The salt was extremely soluble in water but crystallized from very- concentrated solutions in prismatic crystals. They contained water of crystallization which was determined by heating the salt at io5-i25 for 5 hours. o. 8455 gram of the salt lost o. 1544 gram H 2 O. Calculated for C 7 H 7 O 4 N 2 K.3H a O. Found. H 2 O 19.5 18.26 Nitrogen determination for anhydrous salt (Kjeldahl) : Calculated for C 7 H 7 O 4 N 2 K. Found. N 12.60 12.42 666 Johnson and Heyl. Silver Salt of 4- Methyluracil- ^-acetic Acid, C 7 H ? O 4 N 2 Ag. When the potassium salt described above was dissolved in water and a molecular proportion of silver nitrate added, a gelatinous silver salt deposited. It was insoluble in boiling water and did not assume a crystalline form. Analysis for silver : Calculated for C 7 H 7 O 4 N 2 Ag. Found. Ag 37.11 40.00 Barium Salt of ^Methyluracil-^-acetic Acid, (C 7 H ? O 4 N 2 ) 2 Ba.H 2 O. When the calculated proportions of the acid and barium chloride were dissolved in hot water and the solution cooled, the unaltered acid separated. The salt was prepared by dissolving the potassium salt in water and adding the cal- culated amount of barium chloride. The salt separated, on cooling, in small prisms which were dried for analysis in a des- iccator over sulphuric acid. It contained one molecule of water of crystallization which was determined by heating for 2 hours at uo-i36. 0.8114 gram of salt lost 0.0300 gram of H 2 O. Calculated for (C 7 H 7 O 4 N 2 ) 2 Ba.H 2 O. Found. H 2 O 3.45 3-69 Nitrogen determination for anhydrous salt (Kjeldahl) : Calculated for (C 7 H 7 O 4 N 2 ) 2 Ba. Found. N 11.12 11.05 Lead Salt of 4- Methyluracil- ^-acetic Acid, (C 7 H 7 O 4 N 2 ) 2 Pb. H 2 O. This salt was obtained as an insoluble crystalline powder when lead acetate was added to a solution of the acid. It crystallized from water in glistening plates or flat prisms which were dried for analysis in a desiccator over sulphuric acid. It contained one molecule of water of crystallization which was determined by heating at 117- 125 for 4 hours. 1.2847 grams of salt lost 0.0377 gram H 2 O. Calculated for (C 7 H 7 O 4 N2)2Pb.H 2 O. Found. H 2 O 3.04 2.93 Researches on Pynmidines. 667 Analysis of the anhydrous salt : 0.2594 gram substance gave 0.2769 gram CO 2 and 0.0583 gram H 2 O. Nitrogen (Kjeldahl) : Calculated for Found. I. II. III. C 29.32 29.12 .... .... H 2.44 2.49 ........ N 9.77 10.00 9.73 Action of 20 Per Cent Sulphuric Acid. The acid was recovered unaltered after heating with sulphuric acid for 2 hours at I53-I73. When crystallized from water it decomposed at 340. Analysis (Kjeldahl) : Calculated for Found C 7 H 8 O 4 N2. I. II. III. N 15.21 15.31 15.32 15.17 Ethyl Ester of 4.- Methyluracil- ^-acetic Acid, NH - CO CO C.CH 2 COOC 2 H 5 . I II NH - CCH 3 This ester was obtained as a secondary product when we ester- ified 2-ethylmercapto-4-methyl-6-oxypyrimidine-5-acetic acid. It was easily obtained in pure condition by esterifying the oxy- gen acid in the usual manner. It crystallized from 95 per cent alcohol in needles that melted at 2 2 1 -2 2 2 to a clear oil. Anal- ysis (Kjeldahl): Calculated for a. Found. N 13.20 13.12 Methyl Ester of 4- Methyluracil- 5- acetic Acid, NH - CO I I CO CCH 2 COOCH 3 . I II NH -- CCH 3 This ester deposited from methyl alcohol in fine needles that decomposed at 28o-282. Analysis (Kjeldahl): 668 Johnson and Heyl. Calculated for Found. C 8 Hio0 4 N 2 . i. II. N H-H 14.10 14.4 2- A mino-4-methyl- 6-oxypyrimidine- ^-acetic A cid, NH CO I I NH,C CCH 7 COOH. Four grams of 2-ethylmercapto-4-methyl-6-oxypyrimidine-5- acetic acid were heated with alcoholic ammonia for 4 hours at 1 70- 1 80. When the tube was opened there was some pressure, and the odor of ethylmercaptan was apparent. The solution was evaporated to dryness and the crystalline residue purified by recrystallization from water. It deposited in needles that decomposed at 322 with effervescence. Analysis (Kjel- dahl) : Calculated for Found. C 7 H 9 O 3 N 3 . I. II. N 22.95 23.3 23.2 2-EthylmercaptO"4-methyl- 6-chlorpyrimidine- ^-acetic A cid , N CC1 C 2 H 6 SC CCH,COOH. Ten grams of 2-ethylmercapto-4-methyl-6-oxypyrimidine-5- acetic acid and 50 cc. of phosphorus oxychloride were warmed on the steam bath until the evolution of hydrochloric acid gas practically ceased (2 hours). A dark colored solution was ob- tained. This was slowly poured upon crushed ice to decompose the excess of phosphorus oxychloride. No insoluble material deposited after this treatment, but when the acid solution was neutralized with sodium hydroxide the chloride separated in slender needles which dissolved again when an excess of alkali was added. The chloride was soluble in boiling benzene but insoluble in cold benzene ; very soluble in alcohol. It was puri- fied for analysis by recrystallization from hot water and depos- Researches on Pyrimidines. 669 ited in needles that melted at ii8-ii9 with evolution of carbon dioxide. The yield was good. Analysis (Kjeldahl) : Calculated for Found. . II. N 11.35 n-53 11-56 2-Ethylmercapto-4-methyl-6-chlorpyrimidine-5-acetamide, N - CC1 C,H 5 SC CCH 2 CONH 2 . II II N - CCH 8 2-Ethylmercapto-4-methyl-6-oxypyrimidine-5-acetic acid was digested with an excess of phosphorus oxychloride until the evolution of hydrochloric acid gas ceased. Instead of being poured into water as in the previous experiment, this solution was poured slowly into a large volume of cold, alcoholic ammonia. The excess of alcohol and ammonia was then evaporated and the residue triturated with cold water to dissolve ammonium chloride. We obtained a crystalline compound that was insol- uble in cold water but crystallized from hot water in sheaves that decomposed at 167. The compound was very soluble in hot alcohol but can be crystallized from 50 per cent alcohol and from benzene. It was insoluble in dilute sodium hydroxide solution. Analysis (Kjeldahl) : Calculated for Found. C 9 H 12 ON 3 SC1. I. II. N 17.10 17.5 17.4 Action of Alcoholic Ammonia on 2-Ethylmercapto-4-methyl-6- chlorpyrimidine- ^-acetic Acid. The mercaptopyrimidine was recovered unaltered after heating with strong, alcoholic am- monia for 2 hours at 85 95 and again for 3 hours at i io 120. Action at 12 5-i35 ' Formation of 2-Ethylmercapto-4-methyl-6- aminopyrimidine- $-acetic Acid, N- - CNH 2 I I C 2 H 5 SC CCH 2 COOH. II II N - CCH 3 6 7o Johnson and Heyl. Two and five-tenths grams of the chloride were heated with alcoholic ammonia for 2.5 hours at 125 -i 35 . When the tube was opened there was some pressure and a small amount of colorless, crystalline material was suspended in the alcohol. This was identified as the ammonium salt of 2-ethylmercapto- 4 - methyl-6-aminopyrimidine-5-acetic acid. The free acid was obtained by triturating the salt with dilute acetic acid. It de- posited from 95 per cent alcohol in beautiful needles that melted sharply at 221. They gave tests for sulphur and chlorine. This reaction was not smooth and the yield of the amino deriv- ative was poor. When the alcoholic filtrates were evaporated to dryness a crystalline residue deposited. This proved to be a mixture from which nothing sufficiently pure for analysis was isolated. Analysis (Kjeldahl) : Calculated for CgHjgOsNgS. Found. 18.50 18.64 2 , 6-Diamino-4-methylpyrimidine-5-acetic A rid, N=:CNH 2 NH 2 C CCH 2 COOH. This base was prepared by heating 2-ethylmercapto- 4 -methyl-6- chlorpyrimidine-5-acetic acid with alcoholic ammonia for 2 hours at i67-i8o. When the tube was opened there was much pressure, and a crystalline compound had deposited from the alcohol. This crystallized from hot water in small prismatic crystals that melted at 27 9 -28o with effervescence, turning brown at about 270. It did not give tests for sulphur and chlorine, and dissolved in alkali and acids. Analysis (Kjeldahl) : Calculated for C 7 HioO 2 N 4 . Found. N 30.77 30.61 NEW HAVEN, CONN., July 6, 1907. VIII. RESEARCHES ON PYRIMIDINS : A METHOD OF SEPARATING THYMIN FROM URACIL. (Twenty-ninth Paper.) BY TREAT B. JOHNSON. (From the Sheffield Laboratory of Yale University.) (Received for publication, March 28, 1908). New data about the cleavage products of nucleic acids con- tribute to our knowledge of the constitution of these acids. Practical analytical methods for the quantitative determination of the products of hydrolysis are of the greatest importance. Kossel and his co-workers have developed excellent methods for determining the purin bases xanthin, guanin, adenin and hypoxanthin. On the other hand, methods for separating quan- titatively the pyrimidins are lacking. A delicate qualitative test for uracil and cytosin, in presence of thymin, has been described in a publication from this laboratory, 1 but at present there is no test, or characteristic derivative known, which serves for the identification of thymin in presence of uracil. For its detection we make use of the sublimation, the behavior towards silver nitrate and its elementary analysis. A careful review of the nucleic acid literature reveals the fact that several investigations, in this field, signify the necessity of a practical method of separating thymin from uracil. Kut- scher 2 in an investigation on the autolysis of thymus glands isolated 0.6 gram of a crystalline substance whose properties corresponded with those of thymin, but whose content of nitro- gen did not agree with the theoretical value. He found 23.4 and 24.1 per cent of nitrogen while the calculated value for thy- min is 22.22 per cent. His views are summarized in his own words: "Diese Reactionen der Krystalle sowie ihr Verhalten gegen ammoniakalische Silberlosung sprachen fur Thymin, doch 1 Wheeler and Johnson: This Journal, iii, p. 183. 2 Zeitschr. f. physiol. Chem., xxxiv, p. 114. 407 408 Separation of Thymin from Uracil bestatigte die Analyse diese Voraussetzung nicht vollig. Auch durch vielfache Umkrystallization und andere Reinigungsver- suche Hess sich dieser Sticks t off werth nicht herunterdrucken. Es musste also dem Thymin eine stickstoff reichere Substanz viel- leicht Uracil beigemengt sein." Steudel 1 in an investigation on the hydrolysis of thymus nucleic acid with hydriodic acid isolated 5.9330 grams of a substance which gave 23.38 per cent of nitrogen on analysis. He says: "Als die Flussigkeit jetzt eingeengt wurde, schied sich ein Ge- menge von Thymin und Uracil aus, das ich in Ermangelung einer guten Trennungsmethode nicht weiter aufgeteilt, sondern als solches analysiert habe." He 2 also obtained a mixture of thymin and uracil in a later investigation on the oxidation of nucleic acid with nitric acid. In a recent paper, entitled "Die Zusammensetzung der Nucleinsauren aus Thymus und aus Heringssperma," Steudel 3 wrote: "Der nach der Krystallization der alloxurbasen noch bleibende Rest lieferte weiterer Behand- lung Thymin und Uracil. Diese beiden Korper liessen sich durch fraktionierte Krystallization zwar nicht quantitativ aber doch qualitativ gut trennen." I shall describe in this paper a new, characteristic derivative of thymin, and a method of separating, practically quantitatively thymin from uracil. Uracil and thymin do not react, below 100, with nitric acid of density 1.41. On the other hand uracil dissolves, at ordinary temperature, in fuming nitric acid of density 1.5 giving practically a quantitative yield of 5 -nitro uracil I. Thymin reacts, under the same conditions, giving a quantitative yield of the addition product oxynitrohydrothymin* 1 1 . NH CO NH CO I I HN0 3 | | CO CH > CO CNO 2 I I! I II NH CH NH CH I 1 Zeitschr. f. physiol Chem., xlii, p. 169. *Ibid. t xlviii, p. 425. 8 Ibid., liii, p. 14. 4 Johnson: Amer. Chem. Journ. (To be published in vol. xl.) Treat B. Johnson 409 X CH 3 NH CO NH CO | | HN0 3 | CO CCH 3 -- > CO NH CH NH CHOH II In a paper, entitled "Die Constitution des Thymins," 1 Steudel has described the action of concentrated nitric acid on thymin. He obtained a compound to which he assigned the empirical for- mula, C 4 H 4 O 3 N 4 . He gave no melting point for the compound, but states that it did not contain water of crystallization ; that it was soluble in warm water and ammonia and gave, on reduc- tion, a derivative which responded to Weidel's alloxan reaction. The data which I have obtained 2 seem to indicate that Steu- del's nitrothymin was a secondary decomposition product and not a simple thymin derivative. The formation of the hydropyrimidin II, involves a direct addition of nitric acid to the double bond between the four and five positions of the pyrimidin ring. Oxynitrohydrothymin II, exists in two modifications which I have designated by the Greek letters a and /?. The two isomers are formed under practically the same conditions. The a-derivative is the stable modification and melts at 183 to 185. The /^-derivative melts at 230 to 235 and rearranges to the a-form at the ordinary temperature. These isomeric oxynitrohydrothymins are especially characterized by their crystalline habit, and are converted quantitatively into thymin by reduction with tin and hydrochloric acid. 5-Nitrouracil and Oxynitrohydrothymin show a remarkable difference in solubility in cold, absolute ethyl alcohol. The hydro- pyrimidin is extremely soluble in this reagent, while 5-nitrouracil requires approximately 800 to 900 parts of cold alcohol for com- plete solution. I have devised a simple method of separating thymin from uracil by the use of this difference in solubility in alcohol. The mixture of uracil and thymin is treated with the proper proportions of fuming nitric acid (sp. gr. 1.5) and con- verted into 5-nitrouracil and Oxynitrohydrothymin respectively. 1 Zeitschr. /. physiol. Chem., xxxii, p. 241. 2 Loc. cit. 4io Separation of Thymin from Uracil The conditions of this operation are described in detail in the experimental part of this paper. The hydrothymin is then separated from nitrouracil by trituration with absolute alcohol. The hydrothymin can be identified by its melting point and crys- talline habit. Furthermore the thymin can be recovered by reduction of the hydropyrimidin with tin and hydrochloric acid. The nitrouracil is identified by elementary analysis and reduction to 5-aminouracil 1 with aluminum amalgam. This base gives a characteristic picrate which melts at 24y. 2 The Action of Nitric Acid on Thymin. Nitric Acid (sp. gr. 1.415): Five-tenths of a gram of thymin was dissolved in 2 cc. of concentrated nitric acid and the solution evaporated to dryness on the steam bath. There was no evidence of any reaction and the thymin was recovered unaltered. It deposited from hot water in plates that decomposed at about 321. When mixed with pure thymin this decomposition point was not lowered. Analysis (Kjeldahl) : Calculated for C.,HO 2 N 2 : Found : N 22.22 22.33 Nitric Acid (sp. gr. 1.5): The Formation of a-Oxynitrohydro- thymin : NH CO I I /N0 2 CO C< I X CH 3 NH CH.OH Thymin dissolves in cold, fuming nitric acid with slight evolution of heat. If red fumes are evolved by this treatment it is an indication that the thymin is not pure. The hydropyrimidin was obtained perfectly pure, and the yield was quantitative, when thymin was treated with fuming nitric acid under the following conditions: One gram of thymin was dissolved in 6 cc. of nitric acid and the solution allowed to evaporate at ordinary temperature. The nitropyrimidin deposited in large, well-de- 1 Behrend and Griinwald: Ann. d. Chem., cccix, p. 256. 2 Wheeler and Bristol: Anter. Chem. Journ., xxxiii, p. 437. Treat B. Johnson 411 veloped prisms or blocks (see figure) which decrepitated above 130 and melted at 183 with violent effervescence. The com- pound was soluble in hot water, extremely soluble in alcohol, and separated from both these solvents in blocks that melted at 183 to 185. It was insoluble in benzene. The pyrimidin did not lose weight after drying for 1.5 hours at 100 to 110 and again for one-half hour at 1 10 to 115. When heated above 120 it slowly underwent decomposition. The composition of the compound was not altered by recrystallization from absolute alcohol (Analysis III) : 0.2086 gram substance gave 0.2454 gram CO 2 and 0.0695 gram H 2 O. Nitrogen determinations (Kjeldahl) : Calculated for Calculated for Found: C 5 H 7 O 5 N 3 : C 5 H 6 O 2 N 2 (Thymin): I. II. III. C 31.74 47.61 32.08 H 3.70 4.76 3.70 N ' 22.22 22.22 22.00 21.95 a-Oxynitrohydrothymin dissolves in water giving an acid reaction. The pyrimidin undergoes decomposition when its aqueous solution is boiled. The addition of barium hydroxide to its aqueous solution produces no precipitate or color. No thymin deposited when an alcoholic solution of the pyrimidin was treated with ammonia. When the nitropyrimidin was dis- solved in concentrated sulphuric acid, and a few drops of ferrous sulphate solution added, the characteristic test for nitric acid was obtained. a-Oxynitrohydrothymin can also be prepared by dissolving thymin in fuming nitric acid (1.5) and evaporating the solution to dryness, at once, on the steam bath. This method of nitration can be recommended for preparing quickly small quantities of the hydropyrimidin. The yields are not quantitative since part of the thymin undergoes oxidation. The best results are obtained by this method, when i gram portions of thymin are nitrated under the following conditions: One gram of thymin is dissolved in 4 cc. of nitric acid (1.5) and the solution evaporated to dryness, at 100, as quickly as possible. The nitrothymin is obtained as a colorless, crystalline residue which crystallizes from water in blocks decomposing at 183 to 185. It is possible, in this manner, to prepare several grams of the hydropyrimidin 412 Separation of Thymin from Uracil in a few minutes. The yields obtained by this method of nitra- tion were very unifrom and are given in the table below: Nitric acid, sp. gr. 1.5 Weight of thymin. Weight of crude oxyni trohydro- thymin. Weight of hydropyrimidin after crystalliza- tion from water. Percentage of theoretical. cc. grams. grams. gram. per cent. 1 4 1.0 1.1 0.80 57.1 2 4 1. 1.1 0.82 58.5 3 4 1. 1.1 0.79 56.4 4 4 1. 1.1 0.81 57.8 5 8 2. 2.15 1.60 55.5 6 4 1. 1.2 0.90 64.4 7 8 2. 2.1 1.50 53.5 Crystallography of Oxynitrohydrothymin by W. E. Ford. The pyrimidin crystallizes in the triclinic system, showing a combination of 6 (oio), c (ooi), a (100), M (no), d (034) and x (in). The crystals were small, averaging about 2 mm. broad by i mm. thick. In habit they present the appearance of dia- mond shaped tables with beveled edges, as is illustrated in the figure. The face b (oio) is always the most prominent, while the prism with the pinacoid a, and the base with the dome d form the beveling faces. Frequently one of these latter faces is much subordinated in size, or may be entirely wanting. The negative pyramid x is always small and often not present. The crystal Treat B. Johnson 413 faces, although distinct to the eye, were very poorly adapted for measurement with the reflection goniometer. They were usually quite rough or curved and gave indistinct and broad signals on the goniometer. A series of the crystals were measured on the two circle goniometer and the average of the best readings obtained were taken for the fundamental angles, but the angles as given and the crystallographic constants calculated from them can be considered only as approximate. The measured angles were as follows : (010) : (110) = 63 54'. (010) : (100) = 96 15'. (010) : (001) = 73 38'. Zone (010). (110) : zone (010). (001) = 79 35'. Zone (010). (110) : zone (010). (101) = 50 5'. (010) : (034) = 71 33'; calc. = 71 46'. Using the first five measurements as fundamentals the crystal constants were calculated to be: a: b: c = 0.578 : 1.000 : 0.420. a = 107 35', /? = 100 25', r = 80 59'. The crystals show a good cleavage parallel to c (ooi). On account of the nature of the material only a few of the optical facts concerning the compound could be determined. It pos- sesses a strong double refraction. The extinction direction on b (oio) is inclined to the edge between b and a at 31. The crystals when looked at in the polariscope in a direction perpen- dicular to c (ooi), the cleavage face, show the emergence of an optic axis nearly in the center of the field. Reduction of a-Oxynitrohydrothymin with Aluminum- Amalgam. Two and five-tenths grams of the hydropyrimidin were dis- solved in cold water and reduced for 1.5 hours with an excess of aluminium-amalgam. The temperature was not allowed to rise above 40 during the reduction. After filtering from aluminium- amalgam and aluminium hydroxide the filtrate was evaporated to dryness. I obtained a crystalline deposit which was difficultly soluble in cold water, but separated from hot water in plates melting at 320. When mixed with thymin the melting point 414 Separation of Thymin from Uracil was not lowered. It dissolved in fuming nitric acid giving the original a-oxynitrohydrothymin melting at 183 to 185. Calculated for CsHeOaNa: Found: N .................................. 22.22 22.01 Reduction of a-Oxynitrohydrothymin with Tin and Hydrochloric Acid. Two and two-tenths grams of the pyrimidin were reduced for one hour with an excess of tin and concentrated hydrochloric acid. The acid solution was then evaporated to dryness, the residue dissolved in water, and the tin removed by precipitation with hydrogen sulphide. When the aqueous nitrate was con- centrated and cooled, thymin separated in glistening plates which decomposed at 320. Analysis (Kjeldahl) : Calculated for C 5 H G O 2 N 2 : Found: N ......................... ......... 22.22 22.10 ft-Oxynitrohydrothymin : NH CO CO C I I N0 2 NH CHOH I have performed fifteen experiments, during this research, to determine the behavior of fuming nitric acid towards thymin under different conditions. I have taken different amounts of thymin (0.5 to 5.0 grams) and have varied the proportions of nitric acid. I have also allowed the acid solutions to evaporate under different conditions at room temperature, on the steam oven, in a vacuum over sulphuric acid and at 100 but in only two experiments have I observed the formation of /?-oxynitro- hydrothymin. The conditions, under which I obtained this isomer were as follows: One and six-tenths grams of thymin were dissolved in 8 cc. of cold, fuming nitric acid (1.5) and the solution allowed to evaporate, in the air, over night. The next morning large, transparent blocks had deposited which showed no signs of melting at 183 to 185 but decomposed at 230 to Treat B. Johnson 415 235 according to the rate of heating. One of the crystals selected for analysis weighed 0.2600 gram. The isomer did not revert to the a-derivative when crystallized from water or abso- lute alcohol, but separated on cooling in well developed prisms decomposing at 230 to 236. In another experiment 0.5 gram of thymin was dissolved in 10 cc. of fuming nitric acid and the solution allowed to evaporate over sulphuric acid, in a vacuum desiccator. I obtained practically a quantitative yield of the /^-derivative melting at 230 to 235. The compound did not lose weight when heated for one-half hour at 90 to 100. Analyses (Kjeldahl) : Calculated for Found : C 5 H 7 O 5 N 3 : I. II. N 22.22 22.00 22.1 Reduction of fi-Oxynitrohydrothymin with Tin and Hydrochloric Acid. This compound was reduced in the same manner as the a-derivative. The excess of tin was removed with hydrogen sulphide and the nitrate evaporated to dryness. I obtained a crystalline residue which separated from water in plates melting at 315 to 320. The compound sublimed when heated in a test- tube and when mixed with thymin the melting point was not lowered. It dissolved in fuming nitric acid giving a-oxynitro- hydrothymin melting at 183 to 185. Analysis (Kjeldahl) : Calculated for C 5 H r ,O 2 N 2 : Found: 22.22 22.31 Rearrangement of ft-Oxynitrohydrothymin into a-Oxynitrohydro- thymin. Some of the a-pyrimidin, melting at 183 to 185, was pre- served in a desiccator from May 29, 1907, until January 17, 1908. It apparently underwent no change and melted at 181 to 183 with effervescence. A sample of the /?-pyrimidin, melting at 230 to 235 was preserved from June 19, 1907, to October 7, 1907. It then decomposed at 227 to 235, and a nitrogen determi- nation (Kjeldahl) gave 22 per cent nitrogen; calculated 22.22 per cent. This material was not examined again until January 4i 6 Separation of Thymin from Uracil 1 6, 1908. It then had completely rearranged to the a-pyri- midin and melted at 183 to 1 88 with effervescence. A mixture of this material and pure a-oxynitrohydrothymin melted 183 to 186. Analysis (Kjeldahl) : Calculated for C 5 H 7 O 5 N 3 : Found: N 22.22 21.9 Method of Analysis. The mixture of thymin and uracil, which is to be analyzed, is dried at 100, then pulverized finely, and dissolved, at ordinary temperature (20), in fuming nitric acid. It is essential for the success of this separation that the nitric acid have a density of 1.5 and that enough be taken to react with all the uracil and thymin present. It has been my experience that the best pro- portions are about 10 cc. of fuming nitric acid for i gram of the pyrimidin mixture. The operation is performed best in a glass, crystallizing dish of about 5.5 cm. diameter, when working with i gram portions. When the pyrimidins have completely dissolved the solution is evaporated to complete dryness at a temperature of 50 to 60. If impurities are present, which are oxidized by nitric acid at this temperature, the excess of acid can be removed by drying in a vacuum over sulphuric acid and potash. The mixture of nitrouracil and oxynitrohydrothymin is then pulverized in a mortar and triturated thoroughly with cold, absolute alcohol, using 15 cc. of alcohol for each gram of the original mixture. The insoluble nitrouracil is filtered off with suction, washed with 5 cc. of cold alcohol and purified by recrystallization from hot alcohol or water. It is identified in the manner described above. In order to obtain the oxynitrohydrothymin the alcohol wash- ings are allowed to evaporate spontaneously in a good draught. It is not advisable to remove the alcohol by heating on the steam bath. After removal of the alcohol the oxynitrodrothy- min is recrystallized from the least possible quantity of hot water and identified by its characteristic melting point and crystalline habit. The results of the analyses of three different mixtures of uracil and thymin are given in the following table: Treat B. Johnson 417 "L J to oo O II e rf O 01 rH rH H ill! STf tO O rt< to to I* rH O -H ||H1 1 IS S O CO fe> d d d o ( 1 13 .S I* c I 3 o O fc 1 C "S % . ^ S t * 1 ~ 2 _, a S o> 'Y ^ o' S . ii II C * O *=8 00 *3 |g 9 I' 00 ^ J | S"! J| S .1 3 i 5 "el "08 CO N ON c8 J^"J* J Q oo s co is (N ^3 ^-^ H d d d 1 go oo O "3 B 6 So o i i 1 *s "o c 1~ 3 S 32 8 ^ o MO (N fe co ,0 P 2 d ^ . ^ * -s 5 -2 i o P 8 o T 1 >O (N .S .2 q 3 I ill 11 ^ 's T * tO tO C^ C^J O C 4 H 3 O 2 N 2 (NO 2 ), I. ( 5 -Nitro- uracil). 5-Bromuracil, C 4 H 3 O 2 N 2 (Br) -> C 4 H 3 O 2 N 2 (Br)HNO 3 , II. 5-Chloruracil, C 4 H 3 O 2 N 2 (C1) -^ C 4 H 3 O 2 N 2 (C1)HNO 3 , III. Thymine, C 4 H 3 O 2 N 2 (CH 3 )-> C 4 H 3 O 2 N 2 (CH 3 )HNO 3 , IV. 1 Z. physiol. Chem., 32, 241. 2 Loc. tit. 20 Johnson. I now find that the compound II., prepared from 5-bromuracil and nitric acid, is identical with bromnitrooxyhydrouracil, V., which Behrend 1 obtained by treatment of nitrouracil with bromine water. This interesting result, and the fact that the analogous compounds, III. and IV., are prepared under the same conditions, indicate that they have a similar structure and are to be considered as oxynitrohydropyrimidines ; viz., chlornitrooxyhydrouracil, VI., and oxynitrohydrothymine, VII., respectively: NH CO NH CO NH CO I I /Br | | /Cl | A CO C< , CO C< , CO C< I I X NO 2 | | X N0 2 | | \N0 2 NN CHOH NH CHOH NH CHOH V. VI. VII. The formation of these hydropyrimidines involves a direct addition of nitric acid to the double bond between the 4 and 5 positions of the pyrimidine ring. The preparation of brom- nitrooxyhydrouracil, V., from 5-nitrouracil and 5-bromuracil shows that the nitro group and bromine atom are linked to the same carbon atom. It also proves Behrend' s 2 original con- clusion that the formation of oxyhydropyrimidines, from cer- tain pyrimidines, by the action of chlorine and bromine water involves an addition of hypochlorous and hypobromous acids at the double bond. NH CO NH CO NH CO CO CBr + HN0 3 -> CO C / <- H 2 O + Br 2 + CO CNO | II II X N0 2 | || NH CH NH CHOH NH CH v. I NH - CO NH -- CO I I /Br | | /NO, CO C< CO C< I I X>H | | X OH NH -- CHNO 2 NH -- CHBr VIII. IX. 1 Ann. Chem. (Liebig), 240, 11. 2 Ann. Chem. (Liebig), 229, 20. Researches on Pyrimidines. 21 I also find that 5-bromuracil and 5-chloruracil react with chlorine water and bromine water, respectively, giving the same chlorbromoxyhydrouracil, X. When this hydropyrimidine was digested with alcohol it was converted into 5-chloruracil. NH CO NH CO NH CO I I I /Cl || CO CBr + H 2 O + Cl, -> CO C< T> Br, -f H 2 O + CO CC II I I X Br | || NH CH NH CHOH NH CE X. Thymine apparently shows no tendency to form salts with acids. It can be crystallized from concentrated hydriodic and hydrobromic acids without alteration. The possibility of the nitro derivative IV. being a nitric acid salt, XI. is excluded since thymine is not formed by treatment with alkalis, and also because it does not react with bromine water to give oxybrom- hydro thy mine. 1 The compound is not a substituted nitro derivative, as represented by formulas XII., XIII., and XIV., because it does not contain water of crystallization. It can be crystallized repeatedly from absolute alcohol without alteration. NH CO N0 2 N CO NH CO II II II CO CCH 3 .HN0 3 CO CCH 3 .H 2 CO CCH 3 .H 2 O I II I II I II NH CH NH CH NO 2 N CH XI. XII. XIII. NH CO I I CO CCH 2 N0 2 .H 2 0. I II NH CH XIV. I have made the interesting observation that oxynitrohy- drothymine, VII., can exist in two modifications which I have designated by the Greek letters a and ft. The two isomers are obtained under practically the same conditions. The a. deriva- tive is the stable modification and melts at i83-i85. The ft derivative melts at 23o-235 and rearranges to the a form at 1 Jones: Z. physiol. Chem., 29, 20. 22 Johnson. ordinary temperature. The two isomers are especially char- acterized by their crystalline habit and are converted quantita- tively into thymine when reduced with tin and hydrochloric acid. This interesting case of isomerism is possibly similar in nature to that recently observed by Osten. 1 He found, for example, that methylisodialuric acid, XV., and its diethyl ether, XVI., occur in two isomeric modifications which undergo re- versible transformations in the presence of acids and alkalis. NH - CO NH - CO I I /OH | /OC,H 5 CO C< CO C< I I X OH | | X)C 2 H 5 NH - COH NH - COH CH S CH 3 XV. XVI. Oxynitrohydropyrimidines are probably formed in every case when 5-nitropyrimidines are obtained by the action of nitric acid. For example: the formation of nitrouracil from uracil probably involves an addition of nitric acid, giving the unstable hydropyrimidine XVII., which then breaks down with loss of a molecule of water, giving nitrouracil. In the case of thymine, on the other hand, the molecule cannot lose water in the normal manner and therefore the intermediate hydropyrimidine, VII., is capable of isolation. NH CO NH CO NH CO CO CH + HNO 3 CO C< -> CO CNO 2 -fH 2 O. H | || NH CH NH CHOH NH CH XVII. It is interesting to note here the behavior of nitric acid to- wards hydrouracil. 2 This compound contains no double bond between the 4 and 5 positions and the 5 position is not the point of attack. It reacts with nitric acid, giving a nitro derivative, XVIII., in which the nitro group is linked to the 3 position of the pyrimidine ring. 1 Ann. Chem. (Liebig), 343, 133. 2 Franchimont and Friedmann: Rec. trav. chim., 26, 218. Researches on Pyrimidines. NH CO NH- I I I CO CH 2 + HNO 3 F* CO NH CH, NO 2 N- XVIII. -CO CH 2 + H 2 O. CH, The isolation of the above oxynitrohydropyrimidines lends new interest to some early observations on the action of nitric acid on certain benzene derivatives. For example: nitric acid reacts with toluene, in presence of sulphuric acid, giving 3, 5-dinitroparacresol, XIX., and 3,5-dinitroorthocresol, XX. 1 Orthoxylene gives a mixture of 3,5-dinitroparaxylenol, XXI., and 3,5-dinitroorthoxylenol, 2 XXII. Orthocresolsulphonic acid reacts with nitric acid, giving 3,5-dinitroorthocresol, 8 XX. Similar observations have also been made in the naphthalene series. 4 Armstrong explains these abnormal reactions by as- suming an addition of nitric acid to a double bond in the benzene ring, giving unstable addition products. These then break down in two ways, either with separation of nitrous acid, giving a phenol, or with loss of water, giving a nitro derivative. ' ;': CH S \ NO. NO 2 N0 2 NO 2 OH XIX. \/ XX. CH 3 OH N0 2 \ NO 2 XXII. 1 Nolting and Forel: Ber. d. chetn. Ges., 18, 2670. 2 Nolting and Pick: Ibid., 21, 3158. 3 Claus and Jackson: J. prakt. Chem. (2), 38, 333. 4 Armstong and Rossiter: P. Chem. Soc., 1891, 87-89; Ber. d. chera. Ges., 24, R. 721. Johnson. The action of nitric acid on orthocresolsulphonic acid, XXIII., may be represented by the following equation. The writer is not aware that such intermediate addition products with nitric acid have been isolated in the benzene series. CH 3 CH, \S0 3 H H 2 O -f H 2 S0 4 . EXPERIMENTAL PART. The Action of Nitric Acid (sp. gr. 1.5) on s-Bromuracil. NH CO 1 l/Br Oxynitrobromhydrouracil, CO C<( . This compound |\N0 2 NH CHOH was first described by Behrend. 1 I prepared it in the following manner: Three grams of finely pulverized 5-bromuracil were dissolved in 19 cc. of nitric acid and the solution allowed to evaporate in a vacuum over sulphuric acid. I obtained 3.3 grams of the pure hydropyrimidine which crystallized in large blocks and rectangular prisms. The pyrimidine had no definite melting point, but decomposed from 150 to 165 according to the rate of heating. It was difficultly soluble in cold water but could not be purified by recrystallization from this solvent. When warmed with water above 40 it was decomposed with 1 Loc. ctt. Researches on Pyrimidines. 25 formation of brompicrin, which was recognized by its disa- greeable odor. The pyrimidine did not contain water of crystal- lization. It agreed in its chemical behavior, so far as I was able to judge, with the hydropyrimidine described by Behrend. 0.3006 gram of substance gave 0.2204 gram CO 2 and 0.0481 gram H 2 O. Nitrogen (Kjeldahl). Calculated for Found. C 4 H 4 5 N 8 Br. I. II. C IQ.OO 20.00 H 1.57 1.77 N 16.53 16.35 ReductionofOxynitrobromhydrouracilwithTin and Hydrochloric Acid. Two grams of the hydropyrimidine were reduced, on the steam bath, for one hour with an excess of tin and hydrochloric acid. The solution was then evaporated to dryness to remove the excess of hydrochloric acid and the residue redissolved in water. The tin was then removed by precipitation with hy- drogen sulphide and the filtrate concentrated to a volume of 20 cc. Five grams of potassium cyanate were then added to the solution when I obtained an insoluble, granular precipitate. The compound was difficultly soluble in hot water and prac- tically insoluble in the common organic solvents and dilute sulphuric acid. It did not contain halogens, and left no in- organic residue when burned on a platinum foil. When the compound was warmed with bromine water and then treated with an excess of barium hydroxide solution, a purple precipitate was obtained. Its characteristic properties and nitrogen determinations identified the compound as Behrend's 1 hy- NH CO I I droxyxanthine, CO CNHCONH 2 . I II NH CH Nitrogen (Kjeldahl) : Calculated for Found. C 5 H 6 O 3 N4. I. II. N 32.94 3 2 -8o 32.95 1 Ann. Chem. (Liebig), 229, 40; 240, 6. 26 Johnson. NH CO Oxydichlorhydrouracil, CO C <^ . H 2 O. This compound NH CHOH was prepared by dissolving uracil in an excess of strong chlorine water, or by oxidizing uracil with potassium chlorate and dilute hydrochloric acid. Five grams of finely pulverized uracil were suspended in about 100 cc. of chlorine water and chlorine gas conducted into the solution until the pyrimidine had dissolved. The excess of chlorine was then removed with a current of air and the solution concentrated on the steam bath. On cooling, prismatic crystals of chloruracil (see below) deposited. These were removed by filtration and the filtrate concentrated further. The hydropyrimidine then separated, on cooling, in the form of tables melting at 205^-208 with effervescence. In a second experiment 2 grams of uracil were dissolved in 80 cc. of 20 per cent hydrochloric acid and the solution heated to 7o-9O. Potassium chlorate was then occasionally added in small portions and the oxidation continued at 5o-6o for four hours. The excess of chlorine was then removed in the usual manner and the solution allowed to stand. The oxydichlorhydrouracil finally separated in stout prisms which decomposed at 208- 210. The hydropyrimidine crystallized from water in large, well- developed prisms melting at 2i2-2i5 with effervescence. Aqueous solutions of the pyrimidine gave a purple precipitate when warmed with a solution of barium hydroxide. The pyrimidine was recovered unaltered after boiling with alcohol for 24 hours. The corresponding dibrom derivative 1 is con- verted into 5-bromuracil by the same treatment. Oxydichlor- hydrouracil contained one molecule of water of crystallization which was determined by heating for one hour at ioo-no. 0.9157 gram of substance lost 0.0778 gram of H 2 O. Calculated for C^^sNaClg-HjO. Found. H 2 O 8.30 8.49 Nitrogen determination for hydrous pyrimidine (Kjeldahl) : Calculated for aO. Found. N 12.90 12.67 i Wheeler and Johnson: J. Biol. Chem., 3, 183. Researches on Pyrimidines. 27 NH CO 5-Chloruracil, CO CC1- This compound was always ob- ! I! NH CH tained associated with oxydichlorhydrouracil when uracil was treated with chlorine water. It can be prepared by reducing the hydropyrimidine with tin and hydrochloric acid. The pyrimi- dine separated from the acid solution in the form of rectangular prisms and square tables. It was practically insoluble in alco- hol and cold water. It crystallized from hot water in prisms melting at 3oo-3O5 according to the rate of heating. The pyrimidine did not contain water of crystallization. Nitro- gen (Kjeldahl) : Calculated for Found. C 4 H 3 2 N 2 C1. I. II. III. N 19.11 19.03 19.5 19.00 NH CO I /Cl Oxynitrochlorhydrouracil, CO C( . iJH 2 O. This I X N0 2 NH CHOH pyrimidine was prepared by dissolving i . o gram of 5-chlor- uracil in 10 cc. of fuming nitric acid (sp. gr. 1.5). When the solution was allowed to evaporate spontaneously, in the at- mosphere, the pyrimidine separated in hard, prismatic crys- tals. The substance had no definite melting point but de- composed with effervescence from 150 to 160, according to the rate of heating. It slowly underwent decomposition when heated at ioo-iio and was also decomposed by boil- ing water. The compound contained water of crystalliza- tion (Analysis I.), which it slowly lost when exposed to the atmosphere. After drying over sulphuric acid, in a desicca- tor, for 6 days, the analytical determination agreed with the calculated value for the anhydrous material (Analyses II. and III.). (Kjeldahl): Calculated for Found. C 4 H 4 5 N 3 Cl.iiHjO. I. N 17.75 17-7 Calculated for Found. C 4 H 4 06N 3 C1. II. III. N 20.04 J 9-57 19-6 28 Johnson. NH CO I I /Cl Oxychlorbromhydrouracil, CO C<( .H 2 O. This pyr- I | X Br NH CHOH imidine can be prepared from 5-bromuracil or 5-chloruracil by dissolving them in an excess of chlorine water and bromine water, respectively. It was very soluble in water and crystallized from bromine water in prismatic crys- tals, decomposing at i95-2oo, with effervescence. It re- acted with barium hydroxide, in aqueous solution, giving a purple precipitate. The pyrimidine contained one molecule of water of crystallization, which was determined by heating for one hour at 9o-no. The compound slowly loses its water of crystallization in the atmosphere and becomes anhy- drous after standing for several hours in a desiccator over sulphuric acid. I. 0.7413 gram substance lost 0.0516 gram H 2 O. II. 0.2029 gram substance lost 0.0123 gram H 2 O. Calculated for Found. C 4 H 4 O 3 NClBr.H 2 O. I. II. H 2 O 6.88 6.95 6.50 Nitrogen determination for hydrous pyrimidine (Kjeldahl) : Calculated for C 4 H 4 O 3 N 2 ClBr.H,O. Fo v.nd. N 10.70 10.66 Nitrogen determination for anhydrous pyrimidine (Kjel- dahl) : Calculated for Found. C 4 H 4 O 8 N 2 ClBr. I. II. N n-49 11.31 ii. 4 Action of Boiling Alcohol on Oxychlorbromhydrouracil. Five-tenths of a gram of the hydropyrimidine, melting at i95-2oo, was dissolved in 20 cc. of absolute alcohol and the solution boiled for ten hours. The alcohol was then re- moved by evaporation and the product obtained was crys- tallized from hot water. It separated in stout prisms, decom- posing at 3OO-305. It gave a test for chlorine and a mix- Researches on Pyrimidines. 29 ture of the compound and 5-chloruracil decomposed sharply at 300-305. Analysis (Kjeldahl) : Calculated for Calculated for C 4 H 3 O7N 2 C1. C 4 H 3 O2N 2 Br. Found. N 19.11 14.66 19-04 Action of Bromine Water on j-Iodouracil. 5-Iodouracil was decomposed by bromine water with liberation of iodine. About i gram of the pyrimidine was dissolved in bromine water, and the solution allowed to evaporate spontaneously in the atmos- phere. Transparent prisms finally separated, which decom- posed sharply at 2oo-205. The compound did not con- tain iodine, but gave a strong test for bromine. It was con- verted into 5-bromuracil when heated with absolute alcohol. A nitrogen determination agreed with the calculated value for oxydibromhydrouracil 1 (Kjeldahl) : Calculated for C4H 4 O3N 2 Br 2 . Found. N 9.72 9.6 The Action of Nitric Acid on Thy mine. Nitric Acid (sp. gr. 1.415). Five- tenths of a gram of thy- mine was dissolved in 2 cc. of concentrated nitric acid and the solution evaporated to dry ness on the steam bath. There was no evidence of any reaction and the thymine was recov- ered unaltered. It deposited from hot water in plates that decomposed at about 321. When mixed with pure thymine this decomposition point was not lowered. Analysis (Kjeldahl) : Calculated for Found. N 22.22 22.23 Nitric Acid (sp. gr. J-5). The Formation of a-Oxynitrohy- NH - CO I I /CH S drothymine, CO C^ Thymine dissolves in cold, I I X NO, NH - CHOH fuming nitric acid with slight evolution of heat. If red fumes are evolved by this treatment it is a safe indication that the 1 Wheeler and Johnson: Loc. cit. 30 Johnson. thy mine is not pure. The hydropyrimidine was obtained perfectly pure, and the yield was quantitative, when thy- mine was treated with fuming nitric acid under the following conditions: One gram of thy mine was dissolved in 6 cc. of nitric acid and the solution allowed to evaporate at ordi- nary temperature. The nitro derivative deposited in large, well-developed prisms or blocks, which decrepitated above 130 and melted at 183 with violent effervescence. The compound was soluble in hot water, extremely soluble in alco- hol, and separated from both these solvents in blocks that melted at i83-i85. It was practically insoluble in ben- zene. The pyrimidine did not lose weight after heating for 1.5 hours at ioo-no and again for one-half hour at no- 115. When heated above 120 it slowly underwent decom- position. The composition of the compound was not altered by recrystallization from absolute alcohol (Analysis III.). 0.2086 gram substance gave 0.2454 gram CO 2 and 0.0695 gram H 2 O. Nitrogen (Kjeldahl) : Calculated for Calculated for Found. C 6 H 7 O 5 N 3 . C 6 H 6 O2N 2 . I. II. III. C 31-74 47-6i 32.08 I ... 3.70 4.76 3.70 N 22.22 22.22 ... 22. OO 21. 95 a-Oxynitrohydrothymine dissolves in water, giving an acid reaction. The pyrimidine undergoes decomposition when its aqueous solution is boiled. The addition of barium hy- droxide to its aqueous solution produces no precipitate or color; also no thymine deposited when an alcoholic solution of the pyrimidine was treated with ammonia. When the nitropyrimidine was dissolved in concentrated sulphuric acid, and a few drops of ferrous sulphate solution were added, the characteristic test for nitric acid was obtained. a-Oxy- nitrohydrothymine can also be prepared by dissolving thy- mine in fuming nitric acid (sp. gr. i . 5) and evaporating the solution to dryness, at once, on the steam bath. This method of nitration can be recommended for preparing quickly small quantities of the hydropyrimidine. The yields are not quan- Researches on Pyrimidines. 31 titative, since part of the thymine undergoes oxidation. The best results are obtained when i gram portions are nitrated under the following conditions: One gram of thymine is dissolved in 4 cc. of nitric acid (sp. gr. 1.5) and the solution evaporated to dry ness as quickly as possible. The nitrothy- mine is obtained as a colorless, crystalline residue which separates from water in blocks decomposing at i83-i85< It is possible, in this manner, to prepare several grams of the hydropyrimidine in a few minutes. The yields obtained by this method of nitration were very uniform and are given in the table below. 5* O. *- S uT3 E >>"> * rt v >>o " JJ -2 ** D * C jj ~ bo B ** >O "*^ *S *ti M 6 & & P 1 4 i 2 4 i 3 4 i 4 4 i 5 8 2' 641 7 8 2 Crystallography of Oxynitrohydrothymine. By W. E. FORD. The pyrimidine crystallizes in the triclinic system, show- ing a combination of b (oio), c (ooi), a (100), M (no), d (034) and X (in). The crystals were small, averaging about 2 mm. broad by i mm. thick. In habit they present the appear- ance of diamond- shaped tables with beveled edges, as is illus- trated in the figure. The face, b (oio), is always the most prominent, while the prism with the pinacoid, a, and the base with the dome, d, form the beveling faces. Frequently one of these latter faces is much subordinated in size, or may be entirely wanting. The negative pyramid, X, is always small, and often not present. The crystal faces, although distinct to the eye, were very poorly adapted for measurement with bfi gf fEft I P I . I 0.80 57-i I . I 0.82 58.5 I . I 0.79 56.4 I . I 0.81 57-8 2 15 i. 60 55-5 I .2 0.90 64.4 2 .1 1.50 53-5 Johnson. the reflection goniometer. They were usually quite rough or curved and gave indistinct and broad signals on the goniom- eter. A series of the crystals was measured on the two- circle goniometer, and the average of the best readings ob- tained was taken for the fundamental angles, but the angles as given and the crystallographic constants calculated from them can be considered only as proximate. The measured angles were as follows : (oio) : (no) = 6354'. (oio) : (100) = 96i5'- (oio) : (ooi) = 7338'- Zone (oio) . (no) : Zone (oio) Zone (oio) . (no) : Zone (oio) (ooi) (101) 7935'. 5o5'. (oio) : (034) = 7i33'; calc. = 7i46'- Using the first five measurements as fundamentals, the crystal constants were calculated to be a:b:c = 0.578:1.000:0.420. a = io735', /? looks', T = 8o59'. The crystals show a good cleavage parallel to c (ooi). On account of the nature of the material only a few of the optical facts concerning the compound could be determined. It possesses a strong double refraction. The extinction direc- tion on 6 (oio) is inclined to the edge between b and a at 31. The crystals, when looked at in the polariscope in a direction Researches on Pyrimidines. 33 perpendicular to c (ooi), the cleavage face, show the emer- gence of an optic axis nearly in the center of the field. Action of Nitric Acid (sp. gr. 1.5) in Presence of Concentra- ted Sulphuric Acid. There was an immediate reaction, with evolution of red fumes, when o . 5 gram of thymine was dis- solved in a mixture of 3 cc. of nitric and 3 cc. of sulphuric acids. When the mixture was allowed to stand, without cooling, the reaction continued to increase in violence, with evolution of heat, and the thymine was completely decom- posed. In a second experiment, i gram of thymine was dis- solved in a cold mixture of 3 cc. nitric and 2 cc. concentrated sulphuric acids. This solution was held at a temperature of 7o-75 for about three-quarters of an hour, when gas bub- bles had practically ceased to be formed. The acid solution was then cooled and poured into 15 cc. of ice water. After standing for several hours, stout, well-developed prisms de- posited from the solution. They melted at i83-i85, with effervescence, and were identified as oxynitrohydrothymine. They were soluble in alcohol and warm water. Analysis (Kjeldahl) : Calculated for C 5 H 7 O 6 N 3 . Found. N 22.22 21.66 Action of Bromine Water on a-Oxynitrohydrothymine. One gram of the hydropyrimidine was dissolved in an excess of bromine water and the solution allowed to evaporate spon- taneously. There was no evidence of a reaction and oxy- nitrohydrothymine separated in large, transparent blocks melting at i83-i85. Analysis (Kjeldahl) : Calculated for C 6 H 7 O 5 N3. Found. N 22.22 22.42 Reduction of a-Oxynitrohydrothymine with Aluminum Amal- gam. Two and five- tenths grams of the hydrothymine were dissolved in cold water and reduced for 1.5 hours with an excess of aluminum amalgam. The temperature was not allowed to rise above 40 during the reduction. After fil- 34 Johnson. tering from aluminum amalgam and aluminum hydroxide the filtrate was evaporated to dryness. I obtained a crys- talline deposit which was difficultly soluble in cold water, but separated from hot water in plates melting at 320. When mixed with thy mine the melting point w r as not lowered. It dissolved in fuming nitric acid, giving the original a-oxyni- trohydrothymine melting at i83-i85. Analysis (Kjeldahl) : Calculated for C 6 H 6 OeN 2 . Found. N 22.22 22.01 Reduction of a-Oxynitrohydrothymine with Tin and Hydro- chloric Acid. Two and two- tenths grams of the hydropyrimi- dine were reduced for one hour with an excess of tin and con- centrated hydrochloric acid. The acid solution was then evapo- rated to dryness, the residue dissolved in water, and the tin removed by precipitation with hydrogen sulphide. When the aqueous filtrate was concentrated and cooled, thy mine separated in glistening plates, decomposing at 320. Analy- sis (Kjeldahl) : Calculated for C 5 H 6 O 2 Na. Found. N 22.22 22.10 HH CO I I /CH 3 ft-Oxynitrohydrothymine, CO C <^ I have per- I I X N0 2 NH CHOH formed fifteen experiments, during this research, to determine the behavior of fuming nitric acid towards thymine under different conditions. I have taken different amounts of thymine (o . 55 . o grams) , and have varied the proportions of nitric acid. I have also allowed the acid solution to evapo- rate under different conditions at room temperature, on the steam oven, in a vacuum over sulphuric acid, and at 100 but in only two experiments have I observed the formation of fi-oxynitrohydrothymine. The conditions under which I obtained this isomer were as follows: One and six- tenths grams of thymine were dissolved in 8 cc. of cold, fuming nitric acid (sp. gr. i . 5) and the solution was allowed to evaporate Researches on Pyrimidines. 35 in the atmosphere overnight. The following morning large, transparent blocks had deposited, which showed no signs of melting at i83-i85, but decomposed at 2 30 -2 3 5, accord- ing to the rate of heating. One of the crystals selected for analysis weighed 0.2600 gram. The isomer did not revert to the a derivative when crystallized from water or absolute alcohol, but separated, on cooling, in well-developed prisms decomposing at 23o-236. In another experiment, 0.5 gram of thymine was dissolved in 10 cc. of fuming nitric acid and the solution allowed to evaporate over sulphuric acid in a vacuum desiccator. I obtained practically a quantitative yield of the /? derivative melting at 23o-235. The com- pound did not lose weight when heated for one-half hour at 9o-ioo. Analysis (Kjeldahl) : N Calculated for C 5 H 7 6 N 8 . 22.22 Found. I. 22.00 II. 22. I Reduction of [3-Qxynitrohydrothymine with Tin and Hydro- chloric Acid. This compound was reduced in the same man- ner as the a derivative. The excess of tin was removed with hydrogen sulphide and the filtrate evaporated to dryness. I obtained a crystalline residue which separated from water in plates melting at 3i5-32o. The compound sublimed when heated in a test tube, and when mixed with thymine, the melting point was not lowered. It dissolved in fuming nitric acid, giving a-oxynitrohydrothymine melting at 183 -i85 . Analysis (Kjeldahl) : N Calculated for C 6 H 6 2 N 2 . 22.22 Found. 22.31 Rearrangement of fi-Oxynitrohydrothymine into a-Oxynitro- hydrothymine. Some of the a-pyrimidine, melting at 183- 185, was preserved in a desiccator from May 29, 1907, until January 17, 1908. It apparently underwent no change, and melted at i8i-i83 with effervescence. A sample of the /?-pyrimidine, melting at 23o-235, was preserved from June 19, 1907, to October 7, 1907. It then decomposed at 227- 235, and a nitrogen determination (Kjeldahl) gave 22.00 36 Researches on Pyrimidines. per cent; calculated, 22.22 per cent nitrogen. This material was not examined again until January 16, 1908. It then had completely rearranged to the a-pyrimidine and melted at i83-i88 with effervescence. A mixture of this material and pure a-oxynitrohydrothymine melted at i83-i86 with effervescence. Analysis (Kjeldahl) : Calculated for CsH 7 O 5 N3. Found. N 22.22 21.9 I take pleasure in thanking Professor W. E. Ford for the crystallographic description of oxynitrohydrothymine and also Mr. D. B. Jones for his help in this investigation. NEW HAVEN, CONN, Jan. 18, 1908. 1 1 52 HENRY L. WHEELER AND LEONARD M. LIDDLE. [CONTRIBUTIONS FROM THE SHEFFIELD LABORATORY OF YALE UNIVERSITY.] RESEARCHES ON PYR1MIDINES : SYNTHESIS OF URACIL- 3 -ACETTC ACID. [THIRTY-FIRST PAPER.] BY HENRY L. WMKRLER AND LEONAI-D M. LIDDI.K. Received May 8, 1908. Johnson and Heyl found when methyl iodide and 2-ethylmercapto-6- oxypyrimidine (for nula I) are warmed in alcoholic solution in the presence of potassium hydroxide, that the methyl group substitutes in the i-position of the pyrimidine and that i -methyl- 2-ethylmercapto-6-oxypyrimidine, II, results. 1 The formation of a 3-methyl derivative has not yet been observed in this reaction. When the mercapto derivative was warmed with hydrochloric acid, i-methyl uracil, III, was obtained. The struc- ture of this methyl uracil was settled by converting it into i-methyl- 5-nitro-6-oxypyrimidme which had been obtained previously, in a different manner by Behrend and Thurm. 2 We have obtained an entirely different result when 2-ethylmercapto- 6-oxypyrimidine is treated with ethyl chloracetate and alkali in alcoholic solution. The alkylation in this case, although under similar condi- tions, takes place in the 3-position and 2-ethylmercapto-6-oxypyrim- idine-3-ethyl acetate, IV, is formed. When this ester is warmed with alkali it dissolves and hydrochloric acid then precipitates 2-ethylmercapto-6-oxypyrimidine-3-acetic acid, V. If this acid is warmed with hydrochloric acid, rnercaptan is evolved and uracil-3-acetic acid results, VI. The mercapto acid, V, was also obtained by heating 2-ethylmercapto- 6- oxy pyrimidine in alkaline solution with potassium chloracetate and uracil-3-acetic acid can be directly prepared by treating uracil in alkaline solution with chloracetic acid At this point it seemed possible that alkyl iodides might be found to regularly give i-alkyl derivatives. We therefore examined the action of ethyliodoacetate on uracil and it was found that this gave the same acid as the chlorine compound. It is evident from these results that the structure of each new alkyl derivative must be determined separately and uracil-3-acetic acid should be of service in this direction. 3 The proof of the structure of the acetic acid compounds was obtained as follows: Johnson and Heyl's i-methyl- uracil, III, was treated with chloracetic acid and an excess of alkali; 1 THIS JOURNAL, 37, 628 (1907). 2 Ann. (Liebig), 323, 160. 3 The above results also show that alkylation does not give any clue to the posi- tion of hydrogen in the substance alkylated. Since 2-ethyl-mercapto-6-oxypyrimidine is soluble in alkali it is represented as having the NH-CO group with hydrogen in the i -position. RESEARCHES ON PYRIMIDINES. 1 1 53 this gave i-methyluracil-3-acetic acid, VII, and the same methyl acid was formed when our uracil acetic acid, VI, was methylated with methyl iodide and potassium hydroxide in methyl alcohol. The structure of these compounds and their relations are expressed in the following for- mulas : HN CO CH 3 N CO CH 3 N CO II II II C 2 H 5 SC CH -> C 2 H 5 SC CH -> OC CH I! li II II II N CH N CH HN CH L IL N CO N CO HN CO CH 3 N CO II I I! I II II C 2 H 5 SC CH -> C 2 H 5 SC CH -> OC CH -^ OC CH I II I II II I II 2 H 5 OCOCH 2 N CH HOCOCH 2 N CH HOCOCH 2 N CH HOCOCH 2 N CH IV. V. VI. VII. Experimental Part. 2-Ethylmercapto-6-oxypyrim'idine-3-ethyl Acetate (formula IV, above). Molecular proportions of sodium, 2-ethylmercapto-6-oxypyrimidine and ethylchloracetate were dissolved in alcohol, in the order named, and the mixture was warmed until it no longer gave an alkaline reaction (2-3 hours). The solution, filtered from sodium chloride, gave a thick oil on evaporating to dryness. When this was shaken with ether and water it deposited prismatic crystals. The material was crystallized from water; 5 grams dissolved in about 20 cc. of hot water and on cool- ing bunches of beautiful, long, colorless prisms separated which melted to a clear oil, without effervescence, at 129. Analysis: Calculated for C 10 H 14 O 3 N 2 S; N 2 11.57; found, 11.87. This ester is readily soluble in alcohol and in dilute alkali. 2-Ethylmercapto-6-oxypyrimidine-3-acetic Acid (formula V). The solu- tion of the above ester in dilute alkali gave no precipitate when acidified with acetic acid. On adding hydrochloric acid in slight excess a granular precipitate was formed. This was found to be very soluble in hot alcohol and moderately so in cold. It is difficultly soluble in water. The long, slender prisms from alcohol had a fern-like growth and melted at 208-209 with some effervescence. Calculated for C 8 H 10 O 3 N 2 S: N, 13.08; found, 13.12. Five grams of 2-ethylmercapto-6-oxypyrimidine were dissolved in two molecular proportions (3.6 grams) of potassium hydroxide and three grams of chloracetic acid in 5 cc. of water were added. When this solu- tion was evaporated nearly to dryness on the steam bath, 1.5 grams of unaltered 2-ethylmercapto-6-oxypyrimidine separated. The solution 1 1 54 HENRY L. WHEELER AND LEONARD M. was filtered, made alkaline with sodium hydroxide and reheated a short time on the steam bath. On cooling and adding hydrochloric acid 3.1 grams of the above mercapto acid were obtained. When this material was warmed with strong hydrochloric acid, on the steam bath, mercaptan was evolved and uracil-3-acetic acid was obtained. Uracil-3-acetic Acid (formula VI). The above mercapto acid and ester are quantitatively converted into uracil-3-acetic acid by means of hydrochloric acid. It was found, however, that uracil-3-acetic acid could be more conveniently prepared directly from uracil as follows: An aqueous solution of uracil (10.2 grams) in two molecular proportions of potassium hydroxide was boiled with the calculated quantity of chloracetic acid. When the solution gave an acid reaction it was made alkaline by the addition of a few drops of potassium hydroxide. When it finally remained alkaline after 10 minutes boiling, it was cooled and acidified with hydrochloric acid. The yield of uracil-3-acetic acid (10.8 grams) was 70 per cent, of the theoret- ical. The acid was readily soluble in hot water and moderately soluble in cold. It formed small blocks or tables which melted with efferves- cence at 285. It was nearly insoluble in methyl or ethyl alcohol. Calculated for C 6 H 6 O 4 N 2 : N, 16.47; found, 16.57, 16.69. This acid showed no tendency to form a hydantoin. It was found to be very stable when heated with acids. It was obtained unaltered when boiled for 10 hours with hydrochloric acid. It dissolved in boiling acetic anhydride and on evaporating and crystallizing the residue from 85 per cent, alcohol, or water, unaltered acid was obtained. It was also obtained unaltered when heated with 20 per cent, sulphuric acid for one and a half hours at 200. It gave no color with bromine water and barium hydroxide. The potassium salt of uracil-3-acetic acid, prepared by dissolving the acid in the calculated quantity of potassium hydroxide, crystallized in long, flat prisms which were very soluble in cold water. The copper salt was obtained as a minutely crystalline light blue salt on mixing a solution of the potassium salt with one of copper sulphate. Silver and zinc sulphates also gave crystalline salts while the precipitates obtained with mercuric chloride and lead acetate were amorphous. The barium salt obtained by dissolving the acid in a solution of barium hydroxide and then removing the excess of barium by carbon dioxide and concentrating, formed balls of needles like uracil. It was readily soluble in hot water. It was not decomposed by acetic acid. Calculated for (C 6 H 3 O 4 N 2 ) 2 Ba: N, 11.77; found, 11.46. RESE ARCHES ON PYRIMIDINKS. 1 1 55 HN CO I I Uracil-j-methylacetate, OC CH. The acid (1.75 grams) I II CH 3 OCOCH 2 N CH was esterified in methyl alcohol (50 cc.) with a few drops of concen- trated sulphuric acid. On evaporating to about 15 cc. and cooling, 1.2 grams or 63 per cent, of the ester separated. It was readily soluble in hot water and hot alcohol. It formed beautiful color- less needles and although it was repeatedly crystallized from water it melted as if it was a mixture, partially melting at about 177, then melting to a clear oil at about 216. Its appearance and the following nitrogen determination indicated that it was pure. Calculated for C 7 H 8 O 4 N 2 : N, 15.29; found, 15.13. HN CO I I 5-Bromuracil- ^-acetic Acid, OC CBr. Two grams of uracil- I II HOCOCH 2 N CH 3-acetic acid dissolved in water were treated with an excess of bromine and the solution was then allowed to evaporate in the air. On drying the residue over sulphuric acid, it weighed 2.8 grams or 96 per cent, of the calculated yield for a monobrom substitution product. It was readily soluble in hot water, difficultly so in cold and nearly insoluble in alcohol. It formed bunches of colorless needles which melted with effervescence at 244. Calculated for C 6 H 3 O 4 N 2 Br: N, 11.24; found, 11.58. HN CO I I $-Nitr our acil- 3-acetic Acid, OC CNO 2 . Two grams of uracil- I II HOCOCH 2 N CH 3-acetic acid were treated with 10 cc. of concentrated nitric acid and 5 cc. of concentrated sulphuric acid on the steam bath. In twenty minutes a crop of colorless crystals had separated, weighing 1.75 grams or 70 per cent, of the calculated amount. This material was readily soluble in hot water and difficultly soluble in alcohol. It formed colorless prisms which melted with effervescence at 264-265. Calculated for C 6 H 5 O 6 N 3 : N, 19.55; found, 19.66, 19.28. CH 3 N CO i -Methyluracil- 3-acetic Acid, OC CH. From i-methyluracil'. I II HOCOCH 2 N CH A mixture of 2.6 grams methyl uracil, 3 grams of chloracetic acid 1156 HENRY L. WHEELER AND LEONARD M. LIDDLE. and 3.2 grams of potassium hydroxide in 20 cc. of water were warmed on the steam bath. In a half hour the solution was acid to litmus. It was made alkaline and boiled for a short time. On acidifying the solution with hydrochloric acid and cooling, crystals sepa- rated; this material weighed 2.7 grams. It crystallized from water in needle-like prisms or in prismatic plates, and melted constantly at 2 39~~ 2 4 O to a clear oil without effervescence. It forms beautiful, trans- parent prisms from alcohol. Analysis I. From Uracil-3-acetic Acid: Uracil acetic acid (1.2 grams) and four molecular proportions of potassium hydroxide were dissolved in a little water. An excess of methyl iodide (5 grams) was added and sufficient methyl alcohol to effect solution. The mixture was allowed to stand in a warm place for two days when the solution was acid to litmus. It was evaporated to dryness and the residue was crystallized from water containing a little sulphur dioxide. This gave 0.6 gram of material crystallizing in thin prismatic plates and having properties identical with the acid from i-methyl uracil. When these substances were mixed, the melting point was not lowered. Analysis II. Calculated for C 7 H 8 O 4 N 2 : N, 15.21; found: I, 15.18; II, 15.41. NEW HAVEN, CONN., May, 1908. [Reprinted from the Journal of the American Chemical Society. Vol. XXX. No. 7. July, 1908.] [CONTRIBUTIONS FROM THE SHEFFIELD LABORATORY OF YALE UNIVERSITY.] RESEARCHES ON P YRIMIDINES : SYNTHESIS OF URACIL-4- ACETIC ACID. [THIRTY-SECOND PAPER.] BY HENRY L- WHEELER AND LEONARD M. LIDDLE. Received May 8, 1908. Pinner has shown that acetone dicarboxylic ester condenses with benzamidine giving phenyloxypyrimidine acetic ester. 1 He also found that the ester condenses with other aromatic amidines of the general formula, R C(NH 2 )NH. Acetamidine and phenylacetamidine, he states did not give crystalline products. The pseudothioureas may be viewed as amidines derived from the iminothiocarbonic esters. 2-Kthylpseudothiourea is ethylthiocarbam- idine or mercaptof ormamidine : C 2 H 5 S C(OR)NH -> C 2 H 5 S C(NH 2 )NH. In accordance with this we have found that 2-ethylpseudothiourea con- denses very smoothly, in alkaline solution, with acetonediethylcarboxyl- ate, yielding 2-ethylmercapto-6-oxypyrimidine-4-ethyl acetate (I). The condensation may be represented as follows: 1 Ber., 28, 480 (1895). RESEARCHES ON PYRIMIDINES. 1 1 57 HNH C 2 H 5 OCO HN CO C,H 5 OH II II C 2 H 5 SC H- CH == C 2 H 5 SC CH + II I! ' II II NH HOCCH 2 C0 2 C 2 H 5 N CCH 2 CO 2 C 2 H 5 H 2 O I. When this pyrimidine ester, I, is warmed with aqueous alkali it is readily saponified, and on acidifying the solution with hydrochloric acid, 2- ethylmercapto-6-oxypyrimidine-4-acetic acid, II, is obtained. When this mercapto acid is digested on the steam bath with concentrated hydrochloric acid, mercaptan is given off and uracil-4-acetic acid, III, results. HN CO HN CO I I I C 2 H 5 SC CH -> OC CH II II I II N CCH 2 COOH HN CCH 2 COOH TI - HN CO HN CO II II C 2 H 5 SC CN SC CH II II I II N CCH 3 HN-CCH 3 IV. V. On melting 2-ethylmercapto-6-oxypyrimidine-4-acetic acid, carbon diox- ide escapes and the material is converted into 2-ethylmercapto-4-methyl- 6- oxy pyrimidine, IV. If melted in a stream of dry hydrogen chloride ethyl chloride is given off, in addition to carbon dioxide, and 2-thio- 4-methyluracil, V, is formed. These last two pyrimidines have been described by List 1 who obtained them in a different manner. The action of dry hydrogen chloride on the mercaptopyrimidines, leading to the formation of 2-thiopyrimidines with loss of alkyl halide, is a new reaction in the pyrimidine series. We have found that it can be applied in other cases. Taken in connection with the action of dilute nitric acid (Marckwald 2 ) or hydrogen peroxide (Traube 3 ) we are now able to replace a 2-alkylmercapto group by hydrogen. This is of im- portance for synthetic work since formamidine has not yet been shown to lend itself to pyrimidine condensations. Experimental Part. HN CO I I 2-Ethylmercapto-6-oxypyrimidine-4-ethylacetate, C 2 H 5 SC CH . Twelve II II N CCH 2 C0 2 C 2 H 6 1 Ann. chem. (Liebig), 236, 3 (1886). 2 Ber., 25, 3112 (1892). a Ann. chem. (Liebig), 331, 71 (1904). 1158 HENRY L. WHEELER AND LEONARD M. LIDDLE. grams of potassium hydroxide were dissolved in 20 cc. of water and to this cold solution 18.3 grams of the ethyl bromide addition prod- uct of thiourea were added. As soon as solution took place, 20 grams of acetone diethylcarboxylate were slowly added and the mixture was allowed to stand for about an hour, then warmed for a short time on the steam bath. On cooling, and acidifying with acetic acid, 17 grams of the condensation product separated. This was found to be very soluble in hot alcohol, hot water and acetic acid, less soluble in cold alcohol and nearly insoluble in cold water. On crystallizing from hot alcohol long, colorless, silky needles were obtained which melted sharply to an oil at 131. Calculated for C 10 H U O 3 N 2 S: N, 11.57; found, 11.60, 11.67. HNCO I I 2-Ethylmercapto-6-oxypyrimidine-4-acetic acid, C 2 H 5 SC CH . N CCH 2 COOH Two grams of the above ester were dissolved in 25 cc. of water containing 1.5 grams of potassium hydroxide. The solution was heated on the steam bath for a half hour, then cooled and the acid precipitated with hydro- chloric acid. The crystalline precipitate weighed 1.5 grams or 86 per cent, of the calculated. It was readily soluble in hot alcohol and it gave burrs of colorless needles. It was less soluble in water and it melted at 155 with vigorous effervescence. Calculated for C 8 H 10 O 3 N.jS: N, 13.08; found, 13.10, 13.24. This acid is not precipitated from alkaline solution by acetic acid. One gram of 2-ethylmercapto-6-oxypyrimidine-4-acetic acid was heated in an oil bath at 170 until effervescence ceased. The residue was de- colorized with charcoal and recrystallized from water. It melted at 145-147, and when mixed with 2-ethylmercapto-4-methyl-6-oxy- pyrimidine the melting point was not altered. One gram of the acid was melted in an oil bath at 170 and a stream of dry hydrogen chlor- ide was then allowed to pass over the substance. It immediately solidi- fied. On crystallizing from water, it formed colorless prisms which gave a strong test for sulphur. This material had all the properties of 2-thio- 4-methyluracil. Analysis : Calculated for C 5 H 6 ON 2 S: N, 19.71; found, 19.78. HN CO I I Uracil-4-acetic acid, OC CH.H 2 O. When the above mercapto acid I II HN CCH 2 COOH. was warmed on the steam bath with concentrated hydrochloric acid, it readily evolved mercaptan and gave a quantitative yield of uracil- RESEARCHES ON PYRIMIDINES. 1 1 59 4- ace tic acid. The acid was crystallized from water. It formed flat prisms which sintered above 300 and melted with effervescence at 340. In concentrated aqueous solution, it separates at first in an anhydrous condition then in crystals containing one molecule of water. Calculated for C 6 H 6 O 4 N 2 .H 2 O : H 2 O = 9.57; found, 9.97. Calculated for C 6 H 6 O 4 N 2 : N, 16.47; found, 16.95. Uracil-4-acetic acid is soluble in about 8 parts of boiling water and in 30 parts of cold. It is much less soluble in alcohol. It is a strong acid and the aqueous solution reddens litmus. It does not give a pre- cipitate with barium chloride or mercuric chloride. It forms an amor- phous silver salt soluble in ammonia. The Potassium Salt, C C H 5 O 4 N 2 K. Two grams of the acid were dis- solved in a little water containing 0.7 gram of potassium hydroxide. On adding alcohol, balls of a minute, colorless crystal separated. The salt was very soluble in water and insoluble in alcohol. It was dried in a desiccator for analysis. Calculated for C 6 H 5 O 4 N 2 K : N, 13.46; found, 13.40. HN CO I I Uracil-4-methylacetate, OC CH . Bight- tenths gram of II HN-CCH 2 COOCH 3 anhydrous acid was esterified by dissolving in 15 cc. of methyl alcohol containing two drops of concentrated sulphuric acid and warming the solution for one hour. On concentrating the solution to about 7 cc., 0.5 gram of ester separated. The material was washed with water and recrystallized from water. It then formed large, elon- gated, six-sided prisms which melted to an oil at 216-218 with slight effervescence. It was very soluble in hot water and hot alcohol. It was dried over sulphuric acid for analysis. Calculated for C 7 H 8 O 4 N 2 : N, 15.21; found, 15.39. HN CO I Uracil-^-ethylacetate, OC CH.H 2 O. Two grams of uracil-4- acetic I II HN -- CCH 2 COOC 2 H 5 acid were dissolved in 25 cc. of absolute alcohol containing several drops of concentrated hydrochloric acid and the solution was boiled for three hours. It was then evaporated to dryness and the residue crystal- lized three times from about 90 per cent, alcohol. There was then ob- tained about 0.8 gram of ester. It was very soluble in hot alcohol and it separated, on cooling, in colorless prisms, which curiously contained a molecule of water of crystallization. This is the first pyrimidine ester Il6o RESEARCHES ON PYRIMIDINES. that has been found to have water of crystallization. It melted to an oil at 187-189. It was dried over sulphuric acid for analysis. Calculated for C 8 H 10 O 4 N 2 .H a O: H 2 O = 8.33; found, 8.25; N = 12.96; found, 12.97. HN CO I I 5-Nitrouracil-4-acetic acid, OC CNO 2 . Uracil-4-acetic acid I II HN CCH 2 COOH was nitrated by dissolving i gram of the acid in 4 cc. of a mixture of two parts concentrated nitric acid and one of sulphuric acid. The solu- tion was then heated on the steam bath. The product was insoluble in alcohol and difficultly soluble in hot water. It formed minute cubes which melted with effervescence at 153. Calculated for C 6 H 5 O 6 N 3 : N, 19.53; found, 19.39. HN CO I Dibromoxyhydrouracil-4-acetic acid, OC CBr 2 . One gram of uracil- HN C(OH)CH 2 COOH 4-acetic acid was dissolved in water and a large excess of bromine was added. On spontaneous evaporation colorless, elongated plates sepa- rated. They became dark colored above 180 and melted with effervescence at 240. The substance appears to crystallize with a half molecule of water of crystallization. Calculated for C 6 H 6 O 5 N 2 Br r V,,H 2 O : H 2 O = 2.54; found, 2.89. N = 7.87, found, 7-55- A nitrogen determination in the case of the substance dried at 106 gave 8.43 per cent, nitrogen while the calculated is 8.10 per cent. NEW HAVEN, CONN., May, 1908. IX. RESEARCHES ON PYRIMIDINS: SYNTHESES OF SOME NITROGEN- ALKYL DERIVATIVES OF CYTOSIN, THYMIN AND URACIL. (Thirty- third Paper.) BY TREAT B. JOHNSON AND SAMUEL H. CLAPP. (From the Sheffield Laboratory of Yale University.) (Received for publication, May 28, 1908.) The nitrogen-methyl derivatives of cytosin, thymin and uracil are of especial interest because of the occurrence of methyl- purins in nature. Purins may be considered as compound rings in which imidazol has been grafted into pyrimidins. A similar relationship exists between 3-methyluracil, IV, 1,3 -dimethyl - uracil, VI, and the purins, theobromin, III, theophyllin, V, and caffein, VII, as exists between uracil, II, and xanthin, I. NH CO I I CO CNH \ CH NH CN ' I NH CO I I CO CNCH 3 NH CO I I CO CH ! II NH CH II NH CO I 1 CO CH CH 3 N CN/ III on 3 i\ Lxti IV CH 3 N- CO CH 3 N CO i i CH 3 N C CO CNHv CO CH CO C ^ i CH 3 N- I / CH 3 N CH -CN V VI 1 CH 3 N C VU 49 50 Researches on Pyrimidins These alkyl derivatives are also of interest because of the possibility that future investigations may show the presence of methylpyrimidins in animal or vegetable organisms. It is interesting to note here that Suzuki, Aso and Mitarai, 1 in a paper titled "Ueber die chemische Zusammensetzung der japanischen Sojasauce oder Schoyu," have described a decom- position product of Schoyu to which they have assigned the empirical formula C 6 H 9 N 3 . They state that the compound is probably an isomer of aminodimethyl pyrimidin. 2 We shall describe in this paper the syntheses and properties of 3-methylcytosin, VIII, i-methylthymin, IX, 3 -methyl thy min, X, i,3-dimethylthymin, XI, and i,3-dimethyluracil, VI. N=C.NH 2 CH 3 N CO NH CO I I CO CH I II CH 3 .N CH VIII CH 3 N CO I I CO CCH 3 CO CH I II I II CH 3 N CH CH 3 N CH XI VI Two nitrogen-methyl derivatives of thymin and uracil have been described in the literature, viz: 1,3 -dimethyl thy min, XI, and i-methyluracil, XII. Dimethylthymin 3 was prepared by heating the mono-potassium salt of thymin with methyliodide at 150. We prepared this compound and also i,3-dimethyl- uracil, VI, by warming thymin and uracil respectively in alco- holic solution with the required proportions of potassium hydroxide and methyliodide. i-Methyluracil, XII, was described in a paper from this laboratory 4 and was prepared in the following manner: 1 C. Blatt: ii, 1649, I 97- Bull. College Agr., Tokio, vii, 477. 2 Schwarze: Journ. f. prakt. Chem., xlii, p. i; Schlenker: Ber. d. deutsch, chem. Gesellsch., xxxiv, p. 2819; Schmidt: ibid., xxxv, p. 1577. 3 Steudel: Zeitschr. physiol. Chem., xxx, p. 539. 4 Johnson and Heyl: Amer. Chem. Journ., xxxvii, p. 628. Treat B. Johnson and Samuel H. Clapp 51 i-methyl-2-pseudoethylthiourea was condensed with the sodium salt of ethyl formylacetate giving i -methyl- 2 -ethylmercapto-6- oxypyrimidin, XIV. This same mercaptopyrimidin was also obtained by treatment of 2-ethylmercapto-6-oxypyrimidin, XIII, with methyliodide in presence of alkali. Hydrolysis of this mer- captopyrimidin with hydrochloric acid gave i-methyluracil, XII. NH CO CH 3 N CO CH 3 N CO II II II C 2 H 5 SC CH -> C 2 H 6 SC CH -> CO CH II II II II I II N CH N CH NH CH XIII XIV XII We now find that 2-ethylmercapto-5-methyl-6-oxypyrimidin XV, reacts with methyliodide in presence of potassium hydroxide giving about equal proportions of the two isomeric pyrimidins i,5-dimethyl-2-ethylmercapto-6-oxypyrimidin, XVI (m. 65) and 3,5-dimethyl-2-ethylmercapto-6-oxypyrimidin, XVII (m. 156). Hydrolysis of these mercaptopyrimidins with concentrated hydrochloric acid gave i-methylthymin, XVIII (m. 202 to 205) and 3 -methyl thymin, XIX (m. 280 to 282), respectively. NH CO C 2 H 5 SC CCH 3 II II N CH XV CH 3 N CO 1 C 2 H 5 SC CCH 3 II II N CO II C 2 H 5 SC CCH 3 1 II II II N CH XVI 1 II CH 3 N CH XVII 1 PM N PO 1 NH CO CO CCH 3 CO CCH 3 NH CH CH 3 N CH XVIII XIX 52 Researches on Pyrimidins The structures of the isomeric methylthymins and incidentally the corresponding mercaptopyrimidins were established in the following manner : Wheeler and Johnson 1 have shown that cyto- sin reacts smoothly with bromine water giving oxydibromhydro- uracil, XX. When this hydropyrimidin was digested with alcohol it was converted quantitatively into 5-bromuracil, XXI. Since the 6-amino radical is removed by this treatment, N CNH a NH CO NH CO I I II II CO CH -> CO CBr 2 - CO CBr I II II I II NH CH NH CHOH NH CH XX XXI it seemed probable to the writers that substituted cytosin derivatives would behave in a similar manner giving substituted uracils. Our experimental data confirms this assumption. We find that cytosin reacts with methyliodide in presence of po- tassium hydroxide giving 3-methylcytosin, VIII. This pyri- midin reacted with bromine water giving a quantitative yield of 3-methyloxydibromhydrouracil, XXII. When this hydropyri- midin was warmed with alcohol it was converted into 3 -methyl - 5-bromuracil, XXIII, melting at 255 to 260. The isomeric i-methyl-5-bromuracil 2 XXIV, melts at 2 2 8 to 229. 5-Methyl- cytosin apparently reacts with methyliodide in a similar man- ner as cytosin giving 3, 5-dimethylcytosin, XXV. When this N CNH 2 NH CO NH CO CH 3 N CO II II I I I I CO CH > CO CBr 2 > CO CBr CO CBr I II I II I II CH 3 N CH CH 3 N CHOH CH 3 N CH NH CH VIII XXII XXIII XXIV pyrimidin was treated successively with bromine water and alcohol, it was converted quantitatively into 3-methylthymin, XIX, melting at 280 to 282. This result also proves that the 1 This Journal, iii, p. 183. 2 Johnson and Heyl : Loc. cit. Treat B. Johnson and Samuel H. Clapp 53 N=CNH 2 I I CO CCH 3 I II CH 3 N CH XXV NH CO NH CO I I CO CCH 3 I II CH 3 N CH XIX mercaptopyrimidin, XVII, which melts at 156, is a 3-methyl derivative since it gives 3-methylthymin on hydrolysis. The introduction of methyl groups into uracil, thymin, and cytosin, has a similar influence on their physical properties as in the case of purins. They increase the solubility and lower the melting points. For example: while uracil and thymin are difficultly soluble in alcohol, the methyl derivatives of these pyrimidins dissolve easily in this solvent and are moderately soluble in cold water. The 3-methyl derivatives of 2 -ethyl - mercapto-5-methyl-6-oxypyrimidin, thymin and 5-bromuracil are more soluble in water than the isomeric i-methylpyrimidins. It is also interesting to note that the 3-methylpyrimidins melted higher, in every series examined, than the isomeric i-methylpyrimidins : 2- E thy Imercap to- 1 , 5- dimethy 1- 6-oxypyrimidin. (65) 1-Methylthymin. (202 to 205) 1 - Methy loxynitrohy dro thymin. (135 to 136) l-Methyl-5-bromuracil. (228 to 229) 2-Ethylmercapto-3,5-dimethyl- 6-oxypyrimidin. (156) 3-Methylthymin. (280 to 282) 3-Methyloxynitrohydrothymin. (178 to 181) 3- Methyl- 5-bromuracil . (255 to 260) i-Methylthymin and 3-methylthymin reacted in a similar manner with fuming nitric acid as thymin giving characteristic oxynitrohydrothymins, 1 XXVIII and XXIX. CH 3 N CO NH CO I I otr I I otr I /CH 3 ,CH 3 CO C< and CO C/ I | X N0 2 | | \N0 2 NH CHOH CH,N CHOH XXVIII XXIX 1 Johnson: Atner. Ghent. Journ., xl; This Journal, iv, p. 407, 54 Researches on Pyrimidins Conductivity measurements on thymin and its methyl deriva- tives disclosed the interesting facts that thymin and i,3-dimeth- ylthymin gave practically constant conductivities at 25. On the other hand, i -methyl thymin and 3-methylthymin gave abnormal conductivities which increased with the length of time these pyrimidins were kept in solution (see Appendix) . This inter- esting behavior is possibly due to a slow hydrolysis of the pyrimidin ring giving, in solution, /?-uraminoacrylic acids, XXVI and XXVII. N(CH 3 ).CO.NH.CH:C(CH 3 ).CO-^CH 3 NH.CO.NH.CH:C(CH 3 ).COOH. J I XXVI 123 456 NH.CO.N(CH 3 )CH:CH.CO > H 2 N.CO.N(CH 3 ).CH:C(CH 3 ).COOH ! I XXVII The methyl derivatives of thymin, described in this paper, should be of interest to the pharmacologist. It is a well known fact that the methylated dioxypurins possess a pronounced diuretic action. Sweet and Levene 1 have recently shown that the administration of thymin to a dog also caused a most pro- nounced diuresis. Whether the methylated thymin will possess a higher diuretic action than thymin must be decided by further study. EXPERIMENTAL PART. 2-Ethylmercapto-i,5-dimethyl-6-oxypyrimidin: CH 3 N CO I I C 2 H 5 S.C C.CH 3 II II N CH Five grams of 2-ethylmercapto-5-methyl-6-oxypyrimidin and a molecular proportion of potassium hydroxide (1.6 grams) were dissolved in boiling 95 per cent alcohol. An excess of 1 Journ. of Eocper. Med., ix, p. 229. Treat B. Johnson and Samuel H. Clapp 55 methyliodide was then added and the solution boiled for about one hour when it no longer reacted alkaline to turmeric. The undissolved potassium iodide was filtered off and the filtrate heated on the steam-bath to remove the excess of alcohol. We obtained an oily residue, which deposited a mixture of 2 -ethyl - mercapto-i,5-dimethyl-6-oxypyrimidin and unaltered 2 -ethyl - mercapto-5-methyl-6-oxypyrimidin, when triturated with cold water. The filtrate contained the isomeric 2-ethylmercapto- 3,5-dimethyl-6-oxypyrimidin (see below). 2-Ethylmercapto- i,5-dimethyl-6-oxypyrimidin was freed from the unaltered material by treatment with a small volume of a cold, dilute solution of sodium hydroxide. The weight of the crude pyri- midin was 2.1 grams or 30 per cent of the theoretical. It was purified for analysis by crystallization from hot water, and separated, on slow cooling, in long, slender prisms which melted at 65 to a clear oil without effervescence. It did not contain water of crystallization. It was dried for analysis over sulphuric acid (Kjeldahl) : Calculated for C 8 H I2 ON 2 S: Found: N 15.22 15.17 2-Ethylmercapto-3 , f -dimethyl- 6-oxypyrimidin : N CO II I C 2 H 5 SC CCH 3 I II CH 3 N CH In order to isolate this pyrimidin from the above filtrate, the solution was evaporated to dryness and the residue extracted several times with cold chloroform. When the chloroform was evaporated, at ordinary temperature, 1.8 gram of the crude pyrimidin were obtained, or 33.4 per cent of the theo- retical. The compound crystallized from benzene in prisms which melted at 156 to a clear oil without effervescence. It was dried for analysis over sulphuric acid (Kjeldahl) : Calculated for C 8 H 12 ON 2 S: Found: N. 15.22 15.20 56 Researches on Pyrimidins j , ^-Dimethyl- 2 , 6-dioxy pyrimidin ( i-methylthymin) : CH 3 N -- CO I I CO CCH 3 I II NH CH A quantitative yield of this pyrimidin was obtained when 2-ethylmercapto-i,5-dimethyl-6-oxypyrimidin was digested with hydrobromic acid until the evolution of ethylmercaptan ceased (8 hours). The acid solution was then evaporated to dryness and the pyrimidin crystallized from water. It separated in aggregates of stout prisms which melted at 202 to 205, with effervescence, to a clear oil. The compound was readily soluble in boiling alcohol and acetone. It did not contain water of crystallization. Analysis : 0.2680 gram of substance gave 0.5081 gram of CO 2 andO.1379 gram H 2 O. Nitrogen determination (Kjeldahl) : Calculated for Found: C e H 8 O 2 N 2 : I. II. III. C ........................ 51.43 51.71 H ........................ 5.71 5.71 N ........................ 20.00 20.07 20.07 5 , ^-Dimethyl- 2 , 6-dioxypyrimidin (^-methylthymiri) : I I CO CCH 3 I II CH 3 N - CH This pyrimidin was not the only product formed when 2-ethylmercapto-3,5-dimethyl-6-oxypyrimidin was digested with hydrobromic acid for 5 hours. When the acid solution was evaporated to dryness, we obtained a mixture of 3 -methyl - thymin and a compound which was difficultly soluble in cold water. The latter was soluble in warm alcohol and crystallized from hot water in needles melting at 229 to 230, without effervescence to a clear oil, It was soluble in alkalies and gave a strong test for sulphur. The analytical determinations indi- Treat B. Johnson and Samuel H. Clapp 57 cated that the compound was 2-ihio-^ ^-dimethyl- 6-oxypyrimidin. Analysis (Kjeldahl) : NH CO i I CS CCH S I II CH 3 N CH Calculated for Found: C 6 H 8 ON 2 S: I. II. N 17.95 18.4 18.3 3-Methylthymin is more soluble in cold water than this 2-thiopyrimidin and separated, nearly pure, when the aqueous nitrates (above) were concentrated and cooled. It was purified for analysis by recrystallization from water and melted, when heated slowly, at 280 to 282 to a clear oil. This pyrimidin showed a very characteristic behavior when crystallized from hot water. When the hot, saturated, aqueous solution was quickly cooled, the pyrimidin separated immediately as a bulky, homogeneous mass of long, prismatic needles. On standing, these prisms soon disintegrated, apparently redissolved, and were replaced by characteristic octahedral prisms. The trans- formation was complete in a few minutes. The compound did not contain water of crystallization. Analysis (Kjeldahl) : Calculated for C 6 H 8 O 2 N 2 : Found: N 20.00 19.91 i-Methyl-5-brom-4.-oxyhydrothymin: CH 3 N CO I I /CH 3 CO C< I l X Br NH CHOH This compound was obtained when i-methylthymin was dis- solved in an excess of bromine water and the solution allowed to evaporate in a vacuum over sulphuric acid. It crystallized from bromine water in stout prisms and melted, on slow heating, 58 Researches on Pyrimidins at about 123 to 125 to a clear oil. It was dried for analysis over sulphuric acid (Kjeldahl) : Calculated for Found: N ................................ 11.81 11.92 i-Methyl-^-nitro-^-oxykydrothymin: CH 3 N - CO I I/CH, CO C< I | X N0 2 NH CHOH This compound was prepared by dissolving i -methyl thymin in a small volume of fuming nitric acid (sp. gr. 1.5) and allowing the solution to evaporate spontaneously in the air. It deposited in well-developed prisms melting at about 135 to 136 with effervescence. Analysis : I. 0.0844 gram substance gave 15.5 cc. moist N 2 at 21 and 728 mm. II. Nitrogen determination (Kjeldahl): Calculated for Found: C 6 H 9 O 5 N 3 : I. II. N ........................ 20.69 20.84 21.1 j-Methyl-j- nitr o- ^.-oxyhydrothymin : NH CO I I/CH, CO C< .H 2 O I | X N0 2 CH 3 N - CHOH This pyrimidin was prepared in the same manner as its isomer. It separated in large prisms which decomposed at about 178 to 181 with effervescence. This decomposition point varies according to the rate of heating. A nitrogen determination indicated that the compound contained one molecule of water of crystallization. It slowly underwent decomposition when heated at 100. Analysis (Kjeldahl) : Calculated for Calculated for C 6 H S 5 N 3 .H 2 0: CcH^Nv Found: N.. 19.00 20.69 18.74 Treat B. Johnson and Samuel H. Clapp 59 Mono-potassium salt of tkymin: KN CO I I CO C.CH 3 I II NH CH Finely pulverized thymin and a molecular proportion of potassium hydroxide were dissolved in boiling absolute alcohol and the solution boiled for four hours. The potassium salt separated, on cooling, in long needles. It was purified for analy- sis by recrystallization from 95 per cent alcohol. Nitrogen determinations in the salt, dried to a constant weight at 110, agreed with the calculated value in a mono-potassium salt of the closed ring (Kjeldahl) : Calculated for Found: C 5 H 5 O 2 N 2 K: I. II. N 17.07 17.22 17.32 /, 3-Dimethylthymin: CH 3 N CO I I CO C.CH 3 I II CH 8 N CH This pyrimidin was first described by Steudel. 1 We obtained the same derivative, in a smooth manner, under the following conditions: Five grams of thymin and 4.6 grams of potassium hydroxide were dissolved in 90 cc. of 95 per cent alcohol and an excess of methyl iodide added to the warm solution. The solution was boiled for twenty minutes, then evaporated to dryness, and the crystalline residue extracted several times with cold chloroform When the chloroform was evaporated we obtained 2 . 7 grams of the dimethyl pyrimidin. It was very soluble in water and chloroform and difficultly soluble in ether and petroleum ether. It crystallized from alcohol in 1 Loc. cit. 60 Researches on Pyrimidins long needles melting sharply at 153 to a clear oil. Analysis (Kjeldahl) : Calculated for C 7 Hi O 2 N 2 : Found: N ................................ 18.18 18.32 / ij-Dimethyl-f-brom- ^.-oxyhydrothymin : CH 3 X CO I/CH, CO C< I \Br CH 3 N CHOH Was prepared by dissolving i, 3-dimethylthymin in bromine water. When the aqueous solution was concentrated in a vacuum, the pyrimidin separated in prisms melting at 132 to 133 to a clear oil. Analysis (Kjeldahl): Calculated for Found : N ................................ 11.16 11.25 Mono-potassium salt of uracil: KX -- CO I I CO CH.H 2 I II NH CH One molecular proportion of potassium hydroxide was dissolved in 450 cc. of absolute alcohol and 13 grams of finely pulverized uracil suspended in the solution. After digesting for eight hours the uracil was completely changed into the potassium salt. It was difficultly soluble in absolute alcohol and very soluble in cold water. It separated from dilute alcohol in balls of long needles. The yield was quantitative. The salt contained one molecule of water of crystallization which was determined by heating at 120. I. 0.3365 gram substance lost 0.0351 gram H 2 O. II. 0.2871 " " 0.0302 " Calculated for Found: C4H 3 O 2 N 2 K.H 2 O: I. II. H 2 O .................... 10.70 10.43 10.52 Treat B. Johnson and Samuel H. Clapp 61 Nitrogen determination in salt dried at 160 (Kjeldahl): N. Calculated for C 4 H 3 2 N 2 K: 18.67 Found : I. II. 18.51 18.55 Potassium determination in the hydrous salt: 0.1727 gram substance gave 0.0775 gram KC1. K. Calculated for C 4 H 3 2 N 2 K.H 2 0: 23.21 Calculated for C 4 H 3 2 N 2 K: 25.98 Found : 23.55 Nitrogen determination in hydrous salt (Kjeldahl) : X. Calculated for C 4 H 3 O 2 N 2 K.H 2 O: 16.67 Calculated for C 4 H 3 2 N 2 K: 18.65 Found : 16.81 i ,3-Dimethyluracil : CH 3 N CO ' I I CO CH I II CH 8 N CH This compound was prepared by warming, in alcoholic solution, molecular proportions of potassium hydroxide and the potas- sium salt of uracil with an excess of methyliodide. After boiling for three hours the solution was evaporated to dryness and the pyrimidin extracted with chloroform. It crystallized from a mixture of alcohol and ether in long, slender prisms which melted at 121 to 122. It was extremely soluble in cold water, alcohol and chloroform, but insoluble in ether and petroleum ether. Nitrogen (Kjeldahl) : Calculated for I. Found : II. III. N. 20.00 20.05 20.06 20.24 i,3-Dimeihyl-dibromoxyhydrouracil: CH 3 .N CO I I CO CBr 2 I I CH 3 .N CHOH 62 Researches on Pyrimidins This pyrimidin crystallized from bromine water in micro- scopic prisms with curved outline. It melted at 135 to 136 to a clear oil. Analysis (Kjeldahl) : Calculated for Found: N ................................ 8.86 9.0 /, ^-Dimethyl- ^-bromuracil: CH 3 N CO I I CO CBr I II CH 3 N CH This compound was prepared by digesting the above hydro- uracil derivative with absolute alcohol. It was purified for analysis by recrystallization from water and melted at 181 to 182 to a clear oil. Analysis (Kjeldahl) : Calculated for C 6 H 7 O2N 2 Br : Fou nd : N ................................ 12.79 13.0 2-Oxy- ^-methyl- 6-aminopyrimidin (^-methylcytosin) : N=C.NH 2 I I CO CH I II CH 3 .N CH Six and eight-tenths grams of anhydrous cytosin and 3.4 grams of potassium hydroxide were dissolved in 60 cc. of boil- ing, absolute alcohol, and 16 grams of methyl iodide added to the solution. After boiling for two hours the solution was evaporated to dryness and the residue of pyrimidin and potas- sium iodide dissolved in cold water. The iodine was removed with silver sulphate and the excess of silver by precipitation with hydrogen sulphide. The sulphuric acid was then quantitatively removed with barium hydroxide and the filtrate from barium sulphate concentrated to a small volume. The base was pre- cipitated from this solution with a hot, saturated solution of mercuric chloride and the mercury precipitate decomposed in Treat B. Johnson and Samuel H. Clapp 63 the usual way with hydrogen sulphide; the chlorine removed with silver sulphate, the excess of silver with hydrogen sulphide, and the sulphuric acid with barium hydroxide. When this solution was evaporated to dryness we obtained about i . i gram of the pyrimidin associated with a small amount of unaltered cytosin. The small yield is partly explained by the fact that the base volatilizes with aqueous vapors. It was purified by recrys- tallization from methylalcohol. It separated in beautiful, distinct prisms which decomposed at about 278 to 279 to a dark oil. This decomposition point varies according to the rate of heating. The base was extremely soluble in water and did not contain water of crystallization. Analysis (Kjeldahl) : Calculated for Found: C 5 H 7 ON 3 : I. H. N 33.60 33.57 33.9 The chloroplatinate of ^-methylcytosin: Was prepared by adding a solution of platinum chloride to a hydrochloric acid solution of the base. It crystallized from water in long, slender prisms. The salt contained two molecules of water of crystallization which was determined by heating at I. . 0513 gram salt lost 0. 0028 gram H 2 O. II. 0.2103 " " " 0.0111 " " III. 0.0841 " " " 0.0045 " Calculated for Found : (C s H 7 ON 3 .HCl) 2 .Pt C1 4 .2H 2 O: I. II. III. H 9 O.. 5.18 5.45 5.28 5.35 Platinum determination in anhydrous salt : I. 0.04 78 gram salt gave 0.0142 gram Pt. II. 0.1991 " " " 0.0591 " III. 0.0793 " " " 0.232 Calculated for Found : (C 5 H 7 ON 3 .HCl) 2 .PtCl 4 : I. II. III. Pt.. 29.54 29.71 29.68 29.26 Pier ate of j-methylcytosin: This salt was very insoluble in cold water, and crystallized from hot water in long prisms. The decomposition point varies 64 Researches on Pyrimidins greatly according to the rate of heating. When heated slowly it decomposed at about 280 with effervescence. Analysis: 0.1351 gram substance gave 28.7 cc. N 2 at 25 and 770 mm. Calculated for C 5 H 7 ON3.C 6 H3O 7 N 3 : Found: N .............................. 23.73 24.0 $-M ethyl- 5-bromuracil: NH CO I I CO CBr I II CH 8 N -- CH When 3-methylcytosin was dissolved in a little cold water and liquid bromine added to the solution, a heavy precipitate was obtained. This dissolved immediately, on warming, and after heating about ten minutes on the steambath, the solution was evaporated to dryness under diminished pressure. The -residue obtained was then dissolved in a small amount of absolute alcohol and the solution boiled for five hours. On cooling, the brompyrimidin separated in needles. It crystallized from hot water in long, slender, distorted needles. They decomposed at about 255 to 260 to a clear oil with practically no effer- vescence. The compound was soluble in cold, dilute sodium hydroxide solution and was precipitated again by acetic acid. It gave a strong test for bromine. A mixture of the pyrimidin and the isomeric i -methyl- 5-bromuracil 1 melted at 175 to 195. A mixture of the pyrimidin and 5-bromuracil melted from 230 to 270. It did not contain water of crystallization. Analysis (Kjeldahl) : Calculated for Found : I. II. N ........................ 13.66 13.53 13.68 2-Oxy- 6-methylphenylamino pyrimidin : N C.N(CH 8 ) (C 6 H S ) I I CO CH I II NH CH 1 Johnson and Heyl : Loc. cit. Treat B. Johnson and Samuel H. Clapp 65 Ten grams of 2-ethylmercapto-6-chlorpyrimidin and two molecular proportions of monomethylaniline (twelve grams) were dissolved in dry benzene and the solution boiled for eight hours. The benzene was then evaporated, the residue dissolved in ammonia and the excess of monomethylaniline removed by distillation with steam. When this solution was concentrated we obtained the mercaptopyrimidin as an oil which did not solidify on standing. The crude mercapto derivative was con- verted into the oxygen derivative by digesting with hydrobromic acid. The yield was 80 per cent of the theoretical. The com- pound is very insoluble in water and chloroform and crystallizes from alcohol in beautiful hexagonal tables which do not decom- pose below 285. Analysis (Kjeldahl) : Calculated for CnH u ON 3 : ' Found: N 20.90 20.71 2-Oxy- 3-methyl- 6-methylphenylaminopyrimidin : N=CN(CH 3 ) (C 6 H 6 ) I I CO CH I II CH 3 N CH Seven and five-tenths grams of 2-oxy-6-methylphenylamino- pyrimidin, 2.1 grams of potassium hydroxide, and 12 grams of methyliodide were dissolved in 150 cc. of absolute alcohol and the solution boiled for two hours. The solution was then evaporated to dryness and the residue extracted with chloroform. When the chloroform was evaporated we obtained the hydriodide of the pyrimidin base. The base was obtained by decomposing the salt with sodium hydroxide and weighed 5.5 grams or 69 per cent of theoretical. It crystallized from water in long, striated prisms which melted sharply at 186 to 187 to a clear oil without effervescence. Analysis (Kjeldahl): Calculated for CuHuONjt: Found: N 19.53 19.78 2-Oxy-j , ^-dimethyl- 6-aminopyrimidin (3 , $-Dimethyl-cytosiri) : N=C.NH 2 I I CO CCH 8 I II CH 3 .N CH 66 Researches on Pyrimidins 5-Methylcytosin 1 was converted into this base by alkylation with potassium hydroxide and methyliodide in the usual manner, and the new pyrimidin isolated in the same way as 3-methyl- cytosin. The compound was extremely soluble in water and volatilized with aqueous vapors. It separated from methyl- alcohol in prisms. It had no definite melting point, but decom- posed from 300 to 310, according to the rate of heating, with effervescence. Analysis (Kjeldahl) : Calculated for Found; C 6 H 9 ON a : I. II. N 30.22 30.09 29.76 Conversion of 3,$-dimethylcytosin into ^-methylthymin: Some 3,5-dimethylcytosin was dissolved in strong bromine water, the solution warmed on the steambath for ten minutes, and then evaporated to dryness in a vacuum. We obtained a crystalline deposit which was digested with absolute alcohol for five hours. The alcohol was then removed by evaporation and the residue redissolved in hot water. Needle-like prisms separated, on cooling, which melted at 160 with effervescence to a colorless oil. This oil immediately solidified on cooling and then melted at 280 to 282 to an oil without effervescence. They did not contain bromine and were soluble in alkalies. We did not obtain enough of this material for analysis but it was probably ^-methyluramino-a-methylacrylic acid: NH 2 COOH I CO C.CH 3 I! CH,.N CH When the filtrate (above) was concentrated and cooled the characteristic crystals of 3 -methyl thymin separated. They melted at 280 to 282 to an oil. A mixture of pure 3 -methyl thymin and this compound melted at the same temperature. Analysis (Kjeldahl) : Calculated for C 6 H 8 O 2 N 2 : Found; N.. 20.00 20.3 Wheeler and Johnson: Amer. Chem. Journ., xxxi, p. 591. Treat B. Johnson and Samuel H. Clapp 67 CONDUCTIVITY MEASUREMENTS ON THYMIN, i-METHYL- THYMIN, 3-METHYLTHYMIN, i, 3 -DIMETHYLTHYMIN AND 4-METHYLURACIL. BY N. A. MARTIN. In a series of conductivity measurements on thymin and its nitrogen-methyl derivatives it was found that the conductivities of thymin and 1,3 -dimethyl thymin remained nearly constant. The very slight increase in conductivity observed was probably due to small amounts of impurities absorbed from the glass and air. The conductivity of water was found to increase at about the same rate. On the other hand, duplicate determinations on i -methyl - thymin and 3 -methyl thymin did not give agreeing results nor could dissociation constants be calculated. The investigation showed that the conductivity of these two pyrimidins rose with the length of time they were kept in solution. The greatest rise in conductivity was observed in the determinations on 3- methyl thymin. The measurements were carried out by the customary Kohl- rausch method in a thermostat at 25. The water used was redistilled over barium hydroxide, rejecting all that gave a test for ammonia with Nessler's reagent, until it had a specific con- ductivity of approximately 2 X io~ 6 , and the conductivity vessel, pipettes, etc., were thoroughly steamed before using. The rise in conductivity was determined by preparing 20 cc. of exactly -$-% solution of the substance directly in the vessel and measuring its conductivity, at intervals, until it remained practically constant. The rise in specific conductivity of pure water, due to absorption of air, solution of soda from the walls of the vessel, etc., was also determined and found to be prac- tically zero (o . o 7 2 per hour) . In calculating results the amount of hydrolysis could not be determined as the amount of dissociation of the resulting ura- minoacrylic acids (?) was unknown. The specific and molec- ular conductivities were calculated and the latter plotted as ordinates with the time in hours as abscissas. As a reference line the rise in conductivity of water was also plotted, using 64,000 times the specific conductivity to agree with the molecular conductivity of the solutions (A 64 = 64,000 x). 68 Researches on Pyrimidins Molecular conductivity of the pyrimidins in ^ solution at 25, showing increase due to hydrolysis. Time. Thymin. 1 ,3-Dimethly- thymin. l-Methylthymin. 3-Methyl- thymin. 4-Methyl- uracil. hour 0.23 0.35 1 " 0.75 1 0.37 0.49 0.38 2 " ( + i) 0.41 (+i)1.04 0.90 3 " 0.42 1.17 4 0.44 1.36 5 " 0.45 1.54 6 " 0.46 7 0.47 1.98 8 " 17 " 0.68 2.50 18 " 0.69 2.53 19 " 0.72 2.67 20 " 0.74 22 ( + })0.75 4.51 23 " ( + i) 0.57 4.67 24 " 0.57 4.96 8.90 25 26 " 27 " 5.28 10.19 28 5.56 30 " 5.75 41 " 13.53 49 2.97 55i " 6.02 Treat B. Johnson and Samuel H. Clapp 69 Specific conductivity of pyrimidin solutions at 25, showing increase due to hydrolysis. Time. H 2 O. Thymin. 1,3-Di- methyl- thymm. l-Methyl- thymin. 3-Methyl- thymin. 4-Methyl- uracil. hour 0.0 5 21 0.0 5 36 0.0 6 55 I " 0.0 4 12 1 0.0 5 22 0.0 8 57 0.0 5 77 0.0 6 60 2 0.0 5 24 ( + $)0.0 5 65 ( + $)0.0 4 162 0.0 4 14 3 0.0 6 26 0.0 5 66 0.0 4 182 4 0.0 6 67 0.0 4 212 5 0.0 5 69 0.0 4 240 6 0.0 5 71 7 0.0 6 72 + ( + $)0.0 4 308 8 0.0 5 74 17 0.0 5 55 0.0 4 107 0.0 4 39 18 0.0 4 109 0.0 4 40 19$ " 0.0 4 42 20 0.0 4 113 22 0.0 4 115 0.0 4 704 23 ( + $)0.0 5 89 0.0 4 116 0.0 4 730 24 0.0 4 775 0.0 3 139 25 26 0.0 5 91 27 0.0 5 92 0.0 4 824 0.0 3 159 28 0.0 5 93 0.0 4 868 30 0.0 4 898 41 0.0 3 211 49f 0.0 4 47 55$ " 0.0 4 991 Researches on Pyrimidins /r Wafer X. RESEARCHES ON PYRIMIDINS: THE ACTION OF DIAZOBENZENE SULFONIC ACID ON THYMIN, URACIL AND CYTOSIN. (Thirty-fourth Paper.) BY TREAT B. JOHNSON AND SAMUEL H. CLAPP. (From the Sheffield Laboratory of Yale University.} (Received for publication, June 9, 1908.) We have, at the present time, practically no knowledge of the way in which the pyrimidins thymin, uracil and cytosin are linked in the nucleic acid molecule. The question whether they are actually as such contained in the nucleic acids, from which they are obtained, has not been settled. The recent work of Osborne and Heyl 1 on triticonucleic acid, and of Levene and Mandel 2 would seem to indicate that they do not result from the purin bases, 3 but that the pyrimidin nucleus is present in nucleic acids in the simple form. In order to obtain new data, which might prove of service in settling the question of the nature of the linking of pyrimidins in nucleic acids, we undertook this investigation. We shall describe the behavior of diazobenzene sulfonic acid on thymin, uracil, cytosin and some of their alkyl derivatives. 4 A sum- mary of the results of our experiments, and their significance, is given at the end of this paper. Burian 5 has investigated the action of diazobenzene sulfonic acid on several nucleic acids and purins. He examined the nucleic acids from sperma, thymus, yeast and spermatozoa of the 1 Amer. Journ. of Physiol., xxi, p. 157. 2 Biochem. Zeitschr., ix, p. 233. 3 Asher-Spiro: Ergeb. d. Physiol., v, p. 795, 1905; Burian: Zeitschr. f. physiol. Chew., li, p. 438, 1907. 4 Johnson and Clapp: This Journal, v, p. 49. 5 Ber. d. deutsch. diem. Gesellsch., xxxvii, p. 708; Zeitschr. f. physiol. Chem., li, p. 435. 163 164 Researches on Pyrimidins herring; and states that they do not react with this reagent. He showed, on the other hand, that the purins xanthin, hypoxan- thin, guanin, adenin and theophyllin in which the hydrogen in position 7 is unsubstituted, react with diazobenzene sulfonic acid, in presence of alkali, giving intensely colored compounds. He regards the compounds formed as diazoamino derivatives of the general formula I. Substitution in the pyrimidin (alloxan) 2C sc - N - N : NC 6 H 4 S0 3 H I I >C 8 3 N *C -N 9 I ring apparently had no influence on the reaction. On the other hand, he observed that purins substituted in position 7 theo- bromin, caffein and also uric acid, do not react with the diazo acid. Burian concludes from these results that the xanthin bases are linked in the nucleic acids at the nitrogen atom in posi- tion 7. So far as the writers are aware Evans 1 was the first investi- gator to observe that a diazobenzene derivative reacts with a pyrimidin giving a colored compound. He found, for example, that 2-oxy-4,6-dimethylpyrimidin, II, CO CH I II NH CCH 3 II combines with diazobenzene chloride, in presence of alkali, giv- ing a red compound. He says: "Es ist dies ein sehr kraftiger Farbstoff. " The compound was unstable and no formula was assigned it. Steudel 2 afterwards observed that natural thymin reacts with diazobenzene sulfonic acid, in alkaline solutions, giving an intense 1 Journ. f. prakt. Ghent., xlviii, p. 489. 2 Zeitschr. f. physiol. Chem., xlii, p. 170. Treat B. Johnson and Samuel H. Clapp 165 red color. Pauly also mentions this diazo reaction in a later publication 1 and states that it cannot be used to distinguish between the pyrimidin and imidazol rings. He observed, for example, that 4-methyluracil gives as intense a color with diazo- benzene sulfonic acid as histidin. We now find that not only thymin and 4-methyluracil, but also uracil, 4,5-dimethyluracil, cytosin, 5-methylcytosin and 5- bromuracil react under proper conditions with diazobenzene sul- fonic acid giving red colored solutions. The colors obtained with thymin, 4-methyluracil, 4,5-dimethyluracil and 5-methylcytosin are much more intense thant those obtained with uracil, cytosin and 5-bromuracil. Apparently, the character of the groups occupying the 4 and 5 positions has a decided influence on the intensity of the color. We have also made the interesting observation that the forma- tion of red colors is entirely inhibited by substitution in the 3 positions of the uracil, cytosin and thymin molecules. The assumption that the diazo compound might react at the 4 posi- tion of the pyrimidin ring 2 giving azo derivatives is excluded by Pauly's observation 3 and also by the fact that 4, 5-dimethyluracil reacts with the diazo acid giving a red color. Substitution in position i of thymin does not prevent the formation of a red color, i -Methyl thymin, 4 for example, gave as intense a color as thymin itself. On the other hand a red color was not obtained with the diazo acid when the hydrogens in positions i and 3 or 3 alone of thymin were substituted by methyl groups. Similar observations were also made in the cases of uracil and 5-bromuracil. While these pyrimidins gave red colors with the diazo acid, no colors were obtained when i,3-dimethyluracil 5 and 3 -methyl- 5-bromuracil were tested with the reagent. The corresponding 3-methyluracil is unknown. The formation of a color in the cases of cytosin, III, and 5-methylcytosin, VI, is not dependent upon the presence of the free amino radical. 2-Oxy- 1 Zeitschr. f. physiol. Chem., xlii, p. 512. 2 Burian: Zeitschr. f. physiol. Chem., xlii, p. 297; Mann's Chemistry of the Proteids, p. 431, 1906. 3 Loc. cit. 4 Johnson and Clapp : Loc. cit. 5 Ibid. 1 66 Researches on Pyrimidins 6-methylphenylaminopyrimidin V, 1 gave as intense a color as cytosin, III, while, on the other hand, no color was obtained with 3-methylcytosin, IV, 3, 5-dimethylcytosin, VII, and 2-0x7-3- methyl-6-methylphenylamino-pyrimidin, VIII. CN (CH 3 ) (C 6 H 5 ) CH N = = CNH 2 N = CNH 2 N CN (CH 3 ) (C 6 H 5 ) 1 I CO CCH 3 CO CCH 3 I II I II NH - CH CH 3 N CH VI VII VIII The remarkable tendency of certain pyrimidins to react with diazobenzene sulfonic acid, and the apparent inertness of the same ring in purins are of especial interest. According to Bu- rian 2 substitution in the pyrimidin nucleus of purins did not influ- ence the reaction with the diazo acid. While purins are closely related in structure to pyrimidins it is important to point out in this connection that positions i and 3 of the pyrimidin ring do not necessarily correspond to positions i and 3 in purins. While the pyrimidin nucleus is symmetrical, the purin, on the other hand, is unsymmetrical with respect to the 2 position. Two purins can theoretically be formed from a pyrimidin ring according as the glyoxalin ring is joined at the 4, 5 or 5,6 posi- tions. An inspection of the formulas below will show that this involves a change in the numbering of the atoms in the pyri- midin nucleus. c N . 2 C 5 C-N 7 C C I I >c- || SN C-N N C 4 9 Purin. Pyrimidin. 1 Johnson and Clapp : Loc. cit. 2 Loc. cit. Treat B. Johnson and Samuel H. Clapp 167 It is also of interest to note here that 6-oxypyrimidin 1 and 6-aminopyrimidin ? do not give a red color with diazobenzene sulfonic acid. Whether this reagent reacts to form colored com- pounds only with pyrimidins having a CO-NH grouping in the i, 2 or 2, 3 positions must be decided by further experi- ments. The compounds formed by the action of diazobenzene sul- fonic acid or pyrimidins appear to be more unstable than those formed in the case of purins. Two attempts to isolate the reac- tion-product in the case of thymin were unsuccessful. The diazo acid used in our work was prepared according to the directions of Pauly. 3 That the test shall not be found to be capricious it is absolutely necessary that the acid be pure and freshly prepared. METHODS OF APPLYING THE TEST. /. With jo per cent sodium hydroxide solution. Five to ten milligrams of the pyrimidin are dissolved in 0.5 cc. of 10 per cent sodium hydroxide solution and about 5.0 milli- grams of diazobenzene sulfonic acid then added to the solution. If no reaction takes place the solution usually assumes a yellow or orange color. When a red color is formed it usually develops quite rapidly. The red color is usually quite permanent, lasting in some cases (thymin) for several hours. 2. With ^ sodium hydroxide solution. Dissolve 5.0 to 10.0 milligrams of the pyrimidin in 2 cc. of *-Q sodium hydroxide solution and add 5.0 to 10.0 milligrams of the sulfonic acid. This method of testing is not as reliable as Method i. 3. Testing on a watch glass. Five to ten milligrams of the pyrimidin and an equal weight of the sulfonic acid are mixed together, with a glass rod, on a dry 1 Wheeler: This Journal, iii, p. 285, 1907. 2 Biittner : Ber. d. deutsch. chem. Gesellsch., xxxvi, p. 2232, 1903 : Wheeler: Loc. cit. 3 Zeitschr. f. physiol. Chem., xlii, p. 516. 1 68 Researches on Pyrimidins watch glass. A drop of 10 per cent sodium hydroxide solution is then allowed to flow into the mixture. The red color develops immediately under these conditions. This method of applying the diazo test is recommended on account of its delicacy and reliability. The results of our experiments are given, for comparison, in the following tables : TABLE I. Pyrimidins. 0.5 ce. 10 per cent NaOH solution. 2.0 cc. T ^ NaOH solution. Test on watch- glass. NH PO Intense red color, which disappears on diluting with water. Light red which fades rapidly. Intense red color. 1 1 CO CCH 3 1 1! NH PH (Thymin). PH N PO Intense red color, which disappears on diluting with water. No color.* Intense red color. 1 1 CO CCH 3 1 II NH CH (1-Methylthymin.) NH PO No color. No color. No color. 1 CO CCH 3 1 II PH N PH (3-Methylthymin.) PH N PO No color. No color. No color. CO CCH 3 1 II PH N PH (1, 3-Dimethylthy- min.) *Note The statement no color indicates that no red color was formed. Treat B. Johnson and Samuel H. Clapp 169 TABLE II. Pyrimidins. 0.5 cc. 10 per cent NaOH solution. 2.0 cc. N^NaOH solution. Test on watch- glass. NH CO 1 1 CO CH 1 II T\ITJ pTT Red color but not as intense as with thymin. Red color which developed slowly. Color permanent Red color. (Uracil.) PTT 1ST PO Red color. Red color. Red color. CO CH II ATTT pTT (1-Methyluracil). NH CO 1 ! CO CH 1 II PH N PTT (3-Methyluracil) CH 3 N CO i CO CH CR AT PTT No color. No color. No color. (1, 3-Dimethyluracil) 1 7 o Researches on Pyrimidins TABLE III. Pyrimidins. 0.5 cc. 10 per cant NaOH solution. 2.0 cc. ^NaOH solution, Test on watch- glass. NTT TO I I CO CBr 1 II Red color. Faint red color which develops Faint red color. ! 11 NTT PH slowly. (5-Bromuracil.) flTT AT pf) | 1 CO CBr 1 II Red color. NTT PTT (l-Methyl-5-brom- uracil.) NH CO I CO CBr II No color. No color. No color. CH 3 N CH (3-Methyl-5-brom- uracil). CH 3 N CO 1 1 CO CBr II No color. No color. No color. CH 3 N CH (1, 3-Dimethyl- ..': 5-bromuracil.) Treat B. Johnson and Samuel H. Clapp 171 TABLE IV. Pyrimidins. 0.5cc. 10 per cent NaOH solution. 2.0 cc.^ NaOH solution. Test on watch- glass. N = C.NH 2 1 1 1 1 CO CH.H 2 O II Red color which developed slowly. Faint red. Red color like uracil. NH CH (Cytosin.) N = CNH 2 CO CH No color. No color. No color. CH 3 N CH (3-Methyl-cytosin) . N C /CH 3 ji/ CO CH ii Red color. No color. Red color. II NH CH CO ( ^CA 3H t No color. No color. No color. 1 CH 8 N CH 172 Researches on Pyrimidins TABLE V. Pyrimidins. 0.5 cc. 10 per cent NaOH solution. 2.0cc. y N ff NaOH solution. Test on watch- glass. N = CNH 2 CO C ;CH 3 Intense red color. Faint red which Intense red. 1 faded. NH CH (5-Methyl-cytosin N = CNH 2 CO C II CH 3 No color. No color. No color. CH 3 N CH (3, 5-Dimethyl- cytosin.) SUMMARY. (1) Thymin, uracil and cytosin react with diazobenzene sul- fonic acid, in presence of alkali, giving red colored solutions. (2) The color is given by thymin with greater intensity than by uracil and cytosin. (3) Substitution in position 3 of the pyrimidin ring prevents the formation of a red color. (4) Accepting the statement of Burian, 1 that nucleic acids do not react with diazobenzene sulfonic acid, the foregoing ob- servations seem to indicate that thymin and probably uracil and cytosin as well are linked in nucleic acids at position 3 . N CO CO C (3) N- (5) Whether the pyrimidins are linked to phosphorus, a car- bohydrate complex or otherwise must be decided by further study. Ber. d. deutsch. chem. Gesellsch,, xxxvii, p. 708; Zeitschr. f. physiol. Chem,, li, p. 435. [Reprinted from the American Chemical Journal, Vol. XI,. No. 2. August, 1908.] [Contributions from the Sheffield Laboratory of Yale University.] CLVIL RESEARCHES ON PYRIMIDINES: THE AC- TION OF POTASSIUM THIOCYANATE UPON SOME IMIDECHLORIDES. [THIRTY-FIFTH PAPER.] By TREAT B. JOHNSON AND WALTER F. STOREY. Two papers entitled "The Action of Potassium Thiocyan- ate upon Imidechlorides" have previously been published from this laboratory. In the first paper 1 the authors showed that the three imide- chlorides, viz., 2-ethylmercapto-6-chlorpyrimidine, I., 2-ethyl- mercapto-5-methyl-6-chlorpyrimidine, II., and 2-ethylmer- ipto-5-brom-6-chlorpyrimidine, III., reacted with potassium thiocyanate, under certain conditions, giving isothiocyanates. icy made no attempts to obtain evidence of the interme- liate formation of thiocyanates. N=CC1 N = CC1 N CC1 II II II C 2 H 5 SC CH C,H 5 SC CCH 3 C 2 H 6 SC CBr. ' II II II II II II N CH N CH N CH I. II. III. 1 Wheeler and Bristol: THIS JOURNAL, 33, 448. 132 Johnson and Storey. In the second paper, 1 Johnson and McCollum described an unique case of the molecular rearrangement of a thiocyanate into an isothiocyanate. They observed that 2-ethylmercapto- 5-ethoxy-6-chlorpyrimidine, IV., reacted, in alcohol, with potassium thiocyanate, giving 2-ethylmercapto-5-ethoxy-6- thiocyanpyrimidine, V., which then underwent a metameric change into 2-ethylmercapto-5-ethoxy-6-isothiocyanpyrimi- dine, VI. N = CC1 N = CSCN N = CNCS I I II I I C,H 5 SC COC 2 H 5 C,H 5 SC COC 2 H 5 C 2 H 6 SC COC,H 5 . II I! II II II II N CH N CH N CH IV. V. VI. The work described in this paper was undertaken with the object of examining the behavior of potassium thiocyanate towards the three cyclic imidechlorides, viz., 2-paratoluidino- 6-chlorpyrimidine, VII., 2-orthotoluidmo-6-chlorpyrimidine, VIII., and 2-paratolyl-4-methyl-6-chlorpyrimidine, IX. In- cidentally we have also reinvestigated the action of potassium thiocyanate on the imidechlorides, I., II., and III., studied by Wheeler and Bristol. N = CC1 N = CC1 II II />-CH,.C 6 H,NHC CH o-CH 3 .C 6 H 4 NHC CH II II II II N CH N CH VII. VIII. N = CC1 I I 4 C CH. II II N CCH 3 IX. 2-Paratoluidino-6-chlorpyrimidine, VII., and 2-orthotolui- dino-6-chlorpyrimidine, VIII., were prepared from 2-ethyl- mercapto-6-oxypyrimidine. This mercaptopyrimidine reacted, in a smooth manner, with para- and orthotolmdine, at 100, giving quantitative yields of 2-paratoluidino-6-oxypyrimidine 1 THIS JOURNAL, 36, 136. Researches on Pyrimidines. 133 and 2-orthotoluidino-6-oxypyrimidine, respectively. The oxy- pyrimidines were then converted into the imidechlorides, VII. and VIII., by treatment with phosphorus oxychloride or phosphorus pentachloride. These two chlorides are more stable in alcoholic solutions than the corresponding 2-ethyl- mercapto-6-chlorpyrimidine. While the mercapto deriva- tive is decomposed by boiling alcohol the toluidinopyrimi- dines can be recrystallized repeatedly from this solvent with- out alteration. We now find that these two chlorides do not react with potassium thiocyanate under normal conditions. They were recovered unaltered after digesting, in alcohol or acetone, with potassium thiocyanate for several hours. On the other hand, 2-paratolyl-4-methyl-6-chlorpyrimidine, IX., which was obtained from 2-paratolyl-4-methyl-6-oxy- pyrimidine 1 by the action of phosphorus pentachloride, re- acted in a smooth manner with potassium thiocyanate, giv- ing a good yield of 2-paratolyl-4-methyl-6-thiocyanpyrimi- dine, X. This thiocyanate was stable at ordinary tempera- ture and reacted with thiobenzoic acid, giving a quantitative yield of 2-paratolyl-4-methyl-6-thiopyrimidine. When heated above its melting point, the thiocyanate was converted into a mixture of the isothiocyanate, XI., and apparently a poly- meric form of the isothiocyanate melting at 204. The iso- thiocyanate reacted with aqueous ammonia at ordinary tem- perature, giving 2-paratolyl-4-methyl-6-thioureapyrimidine, XII. N = CSCN N = CNCS II II />-CH 3 C 6 H 4 C CH />-CH 8 C fl H 4 C CH N CCH S x. N CCH 3 XI. N = CNHCSNH 2 I I />-CH 3 C 6 H 4 C CH. II II N CCH, XII. 1 Clock: Ber. d. chem. Ges., 21, 2658. 134 Johnson and Storey. Wheeler and Bristol, 1 in their investigation of the action of potassium thiocyanate on the imidechlorides, 2-ethylmer- capto-6-chlorpyrimidine, I., 2-ethylmercapto-5-methyl-6- chlorpyrimidine, II., and 2-ethylmercapto-5-brom-6-chlor- pyrimidine, III., worked with the object of converting these chlorides into isothiocyanates. They invariably digested the chlorides with potassium thiocyanate in toluene or alco- hol for 3 to 15 hours. Under these conditions the thiocyanates, if formed, would be converted completely into isothiocyanates. We now find that these imidechlorides give practically quan- titative yields of the thiocyanates XIII., XIV., and XV., when warmed in alcohol with potassium thiocyanate, if the time of digestion is limited to 20 to 60 minutes. The thio- cyanates are stable at ordinary temperature and can be re- crystallized from alcohol without alteration. They rearrange smoothly into isothiocyanates when heated above their melting points or when digested for long periods with alcohol. The isothiocyanates, when formed under the latter condition, re- act with the alcohol, giving the corresponding 6-thiourethane- py rimidines : N = CSCN N = CSCN N = CSCN II II II C 2 H 5 SC CH C 2 H 6 SC CCH 3 C 2 H 5 SC CBr. II II II II II II N CH N CH N CH XIII. XIV. XV. The melting points and boiling points of all the 6-thio- cyanpyrimidines, 6-isothiocyanpyrimidines, and corresponding 6-thioureapyrimidines, which have been prepared in this lab- oratory, are given in the following table : 1 Loc. cit. . - Iff lii? Researches on Pyrimidines. P P P B 0=0 o ^ O o=ri- w w q-w o 135 sfe O tn o O O ~ P W 00 P -. w - B- as o Jz; NX oo ^CO o z! 3 P W ^ S p 1 w 136 Johnson and Storey. P! i if H I B I W ?g==g gl 1^8=8-8 * s o c > " w o " s ^ i 3*hH W S M 0-= -3 BTo o o K& " Q^ ^2 O ^H flL '5 a v; 3 5 *&| M O O ^Cn ^ B* || ?! -| I w ^"W "W 5- P =L >s ^ a oCO ^ * /""N* W Ca H^ O < g' ^ .? P P! I Researches on Pyrimi dines. EXPERIMENTAL PART. 137 = CSCN 2-Ethylmercapto-6-thiocyanpyrimidine, C 2 H 5 S.C CH . N CH By DR. E. V. MCCOLLUM. Three and nine-tenths grams of potassium thiocyanate were dissolved in 40 cc. of 95 per cent alcohol and 7 grams of 2-ethylmercapto-6-chlorpyrimidine added to the solu- tion. There was an immediate reaction with separa- tion of potassium chloride. The mixture was warmed on the steam bath for twenty minutes and the insolu- ble potassium chloride separated by nitration. On cooling the alcohol solution, the thiocyanate deposited in prismatic crystals which were purified for analysis by recrystallization from alcohol. It separated in aggregates of rectangular prisms melting without effervescence at 82 to a clear oil. The com- pound was insoluble in alkali and did not react with ammonia or aniline at 100. Analysis (Kjeldahl) : Calculated for CT^NS^. I. N 21.31 20.89 Pound. II. 21.32 III. 2I.O9 When the alcohol filtrates were combined and evaporated to dryness a yellow, crystalline residue was obtained which partially dissolved in cold 10 per cent solution of sodium hydroxide. When this solution was acidified with acetic acid we obtained 0.3 gram of material which crystallized from alcohol in flat prisms or plates melting at92-93. The com- pound was identical with the 2-ethylmercapto-6-thionethylure- thanepyrimidine described by Wheeler and Bristol. 1 A quan- titative yield of the above thiocyanate can be obtained by boiling 2-ethylmercapto-6-chlorpyrimidine in acetone solu- tion with potassium thiocyanate. It was very soluble in ace- tone and separated in prismatic crystals melting at 8i-82. 1 Loc. cit. 138 Johnson and Storey. N=C.NCS 2-Ethylmercapto-6-isothiocyanpyrimidine, C 2 H 5 SC CH II II N CH BY DR. E. V. MCCOLLUM. 2-Ethylmercapto-6-thiocyanpyrimidine shows no tendency to rearrange to the isothiocyanate at ordinary temperature. On the other hand, a rearrangement took place when the thiocyanate was heated for 4 to 5 hours at 8o-9O. The isothiocyanate was obtained as an oil which reacted at once with aniline, giving 2-ethylmercapto-6-phenylthioureapyrimi- dine 1 melting at 205. The isothiocyanate was obtained as a yellow oil boiling at 200-205 (45-50 mm.) when the thiocyanate was distilled. It did not crystallize on standing but a yellow, crystalline compound slowly deposited. This was difficultly soluble in benzene and practically insoluble in alcohol. It melted at i75-i77 and did not react with ammonia. The compound is apparently identical with the product which Wheeler and Bristol obtained by treating 2-ethylmercapto-6-chlorpyrimi- dine with potassium thiocyanate. It is probably a polymeric form of the isothiocyanate, (CyHyNgS^. The isothiocyanate reacted at once with aqueous ammonia, with evolution of heat, giving 2-ethylmercapto-6-thiourea- pyrimidine 2 melting at 214. 2-Ethylmercapto-5-methyl-6-thiocyanpyrimidine, N^C.SCN I I C 2 H 5 SC CCH 3 . Five grams of 2-ethylmercapto-5-methyl- II II N CH 6-chlorpyrimidine and 3.5 grams of dry potassium thiocyanate were dissolved in 25 cc. of absolute alcohol and the solution boiled for i hour. The undissolved potassium chloride was then filtered off and the solution evaporated on the steam bath. We obtained a syrup which was triturated with cold water to remove any potassium chloride and then extracted 1 Wheeler and Bristol: Loc. cit. Researches on Pyrimidines. 139 with ether. When the ether was evaporated we obtained an oil which did not solidify after standing for several days. This oil partially dissolved in 10 per cent sodium hydroxide. The insoluble portion solidified on standing, and crystallized from 95 per cent alcohol in beautiful prisms melting without effer- vescence at 95 to a clear oil. It did not react with ammonia or aniline and a nitrogen determination agreed with the cal- culated value for a thiocyanate (Kjeldahl) : Calculated for C 8 H 9 N 8 Sj. Found. N 19-90 19.6 The thiocyanate dissolved at once in cold thioacetic acid without apparent evolution of heat. On standing, beautiful prismatic crystals deposited which melted at i79-i8o. This compound was not examined further but it probably was 2-ethylmercapto-5-methyl-6-thiopyrimidine. It dissolved in al- kalis and gave a test for sulphur. When the sodium hydroxide solution (above) was acidified with acetic acid we obtained an oil which soon solidified. It was insoluble in water but crystallized from 95 per cent alcohol in stout prisms melting at 90. It was identical with 2-ethyl- mercapto-j-methyl-d-thionurethanepyrimidine, N=C.NHCSOC 2 H 5 C 2 H 5 SC CCH 3 , which was described by Wheeler II II N CH and Bristol. 1 A mixture of the two compounds melted at 8 9 - 9 o . 2-Ethylmercapto-5-brom-6-thiocyanpyrimidine, N = CSCN .1 I C 2 H 5 S.C CBr. This thiocyanate was prepared in the II II N CH following manner: Five grams of 2-ethylmercapto-5-brom-6- chlorpyrimidine and 2.5 grams of potassium thiocyanate were dissolved in 50 cc. of 95 per cent alcohol and the solution boiled on the steam bath for 20 minutes. The insoluble potassium 1 Loc. cit. 140 Johnson and Storey. chloride was filtered off and the filtrate cooled, when the thiocyanate deposited in prisms melting at 8 1-82. They were very soluble in acetone, boiling ligroin, warm alcohol, and benzene. The compound did not dissolve in sodium hydroxide and could be warmed with ammonia without altera- tion. Analysis (Kjeldahl) : Found. Calculated for C 7 H 6 N 3 S2Br. I. II. N 15.21 15.32 15.28 Action of Thioacetic And Thiobenzoic Acids On 2-Ethyl- mercapto-^-brom-6-thiocyanpyrimidine: About 0.3 gram of the thiocyanate was dissolved in a small quantity of thioacetic acid. Reaction took place at once, on warming, without evolution of carbon bisulphide, giving large, well-developed prisms. The compound crystallized from benzene in rhombic prisms that melted at 198 with slight effervescence. It was identified as 2-ethylmercapto-5-brom-6-thiopyrimidine. 1 A mixture of the two products melted at the same temperature. The same 6-thiopyrimidine was obtained when the thiocyanate was warmed in a water bath with a molecular proportion of thiobenzoic acid. Rearrangement of 2-Ethylmercapto-^-brom-6-thiocyanpyrimi- dine into the Isothiocyanate: About 0.2 gram of the thiocyanate was heated for 2 hours at i5O-i6o. We obtained a red oil which solidified on cooling and melted at 75-8o. That a rearrangement had taken place was shown in the following manner: The compound (melting at 75-8o) reacted at once with ammonia, giving 2-ethylmercapto-5-brom-6-thiourea- pyrimidine 2 melting at 2i9-22O. A mixture of the two preparations melted at the same temperature. NH CO I I 2-Orthotoluidino-6-oxypyrimidine, o-CH 3 .C 6 H 4 NH.C CH. II II N CH Bighteen grams of 2-ethylmercapto-6-oxypyrimidine and one molecular proportion of orthotoluidine (14 grams) were heated together on the steam bath for 3 days. The evolution of 1 Wheeler and Bristol: Loc. cit. * Ibid. Researches on Pyrimidines. 141 mercaptan was then complete and a compound was obtained which was difficultly soluble in alcohol. It crystallized from hot alcohol or acetic acid in prismatic crystals melting at 2i9-22o. It was soluble in alkali. Analysis (Kjeldahl) : Found. Calculated for C U H U ON 3 . I. II. N 20.89 20.93 20 -74 2-Orthotoluidino-6-chlorpyrimidine, o-CH 3 .C 6 H 4 NH.C CH. II II N CH Ten grams of 2-orthotoluidino-6-oxypyrimidine were heated on the steam bath with 25 cc. of phosphorus oxychloride until the evolution of hydrochloric acid gas ceased. The excess of phosphorus oxychloride was then distilled under diminished pressure. We obtained a yellow oil, which assumed a crystal- line form when treated with ammonia. The compound crystallized from alcohol in corpuscular crystals melting at 78 . It was insoluble in alkali. Analysis (Kjeldahl) : Found. Calculated for C n H 10 N 3 Cl. I. II. N 19.1 18.8 18.92 Action of Potassium Thiocyanate on 2-Orthotoluidino-6- chlorpyrimidine: Four grams of the chlorpyrimidine and 3 grams of potassium thiocyanate were dissolved in 50 cc. of alcohol and the solution boiled for 2 hours. After filtering a small amount of insoluble material the excess of alcohol was evaporated on the steam bath. We obtained a crystalline residue which crystallized from alcohol and melted at 77-78. It responded to a test for chlorine and was identified as the unaltered chloride. Analysis (Kjeldahl): Calculated for CuH 10 N 3 Cl. Found. N 19.1 19.07 2-Orthotoluidino-6-aminopyrimidine t N^CNH, I I 0-CH 3 .C 6 H 4 NHC CH. 2-Orthotoluidino-6-chlorpyrimidine II II N CH 142 Johnson and Storey. does not react with ammonia at ordinary temperature. When heated with alcoholic ammonia for 2 hours at 140- 150 it gave a quantitative yield of the aminopyrimidine. This was very soluble in alcohol and crystallized from dilute alcohol in aggregates of small prisms melting at 124. Analysis (Kjeldahl) : Calculated for CnHi 2 N 4 . Found. N 28.00 27.7 2-Orthotoluidino-6-anilinopyrimidine, N = CNHC 6 H 5 o-CH 3 C 6 H 4 NHC CH. Three grams of 2-orthotolui- II II N CH dino-6-chlorpyrimidine and 4 grams of aniline were dissolved in 25 cc. of benzene and the solution boiled for 6 hours. It was then filtered and evaporated to dryness. We obtained a crystalline residue which was washed with water to remove aniline hydrochloride and then dissolved in warm dilute hydro- chloric acid. The hydrochloride of the anilinopyrimidine crystallized, on cooling, in slender needles melting at 126. Analysis (Kjeldahl): Calculated for C 17 H 16 N 4 .HC1. Found. N 17.92 17.73 When the hydrochloride was dissolved in water and ammonia added to the solution the free base separated. It crystallized from dilute alcohol in hexagonal plates melting at 128 to a clear oil. Analysis (Kjeldahl) : Calculated for C^H^N^ Found. N 20.28 20.29 NH CO I I s-Paratoluidino-d-oxypyrimidine, />-CH 3 .C 6 H 4 NHC CH. I! il N CH This compound is formed, in a smooth manner, by heating 2-ethylmercapto-6-oxyprimidine with a molecular proportion of paratoluidine at 100. It was difficultly soluble in warm alcohol, acetone, boiling water, and cold acetic acid. It crystal- Researches on Pyrimidines. 143 lized from acetic acid in clusters of needles melting at 270- 271. Analysis (Kjeldahl) : Calculated for CiiH u ON 3 . Found. N 20.89 21.0 2-Paratoluidino-6-chlorpyrimidine, />-CH 3 C 6 H 4 NH.C CH. II I! N CH Twenty-six grams of 2-paratoluidino-6-oxypyrimidine were digested with 65 cc. of phosphorus oxychloride until the evolu- tion of hydrochloric acid gas ceased. We obtained a dark colored solution which was heated at 100 under diminished pressure to remove the excess of phosphorus oxychloride. The residue was dissolved in ice water and the solution made alkaline with ammonia when the chlorpyrimidine deposited. It crystallized from alcohol in prisms melting at 112- 113 to an oil. Analysis (Kjeldahl) : Calculated for CiiH 10 N 3 Cl. Found. N 19.13 18.99 Action of Potassium Thiocyanate on 2-Paratoluidino-6- chlor pyrimidine: This pyrimidine was recovered unaltered, melting at ii2-ii3, after warming, in acetone solution, with potassium thiocyanate for 2.5 hours. 2-Paratoluidino-6-anilinopyrimidine, N = CNHC 6 H 5 -CH 3 C 6 H 4 NHC CH. The hydrochloride of this base II II N CH was obtained when 2-paratoluidino-6-chlorpyrimidine was warmed in benzene with aniline. The salt was very soluble in hot water and alcohol. It crystallized from alcohol in prisms which decomposed at 134. Analysis (Kjeldahl) : Calculated for Ci 7 Hi 6 N 4 .HCl. Found. N 17.92 17.96 When sodium hydroxide was added to an aqueous solution of this salt the pyrimidine base deposited in needles. It was 144 Johnson and Storey. insoluble in hot water, and crystallized from alcohol in needles melting at 135 to an oil. Analysis (Kjeldahl) : Calculated for Ci 7 H 16 N 4 . Found. N 20.28 20.15 NH CO I I 2-}-Naphthylamino-6-oxypyrimidine, /?-C 10 H 7 NH.C CH. II II N CH From /?-naphtylamine and 2-ethylmercapto-6-oxypyrimidine. It was insoluble in hot water and difficultly soluble in alcohol. It crystallized from alcohol in clusters of needles melting at 270. Analysis (Kjeldahl) : Calculated for Ci 4 H u ON 3 . Found. N 17.72 17.77 2-Paratolyi-4-methyl-6-chlorpyrimidine, N ==CC1 I I -CH 3 C 6 H 4 .C CH. This chloride was prepared by heating II II N CCH 3 on the steam bath 15 grams of 2-paratolyl-4-methyl-6-oxy- pyrimidine 1 with 17 grams of phosphorus pentachloride and about 20 cc. of phosphorus oxychloride for 7 hours. A dark colored solution was obtained which was slowly poured upon crushed ice to destroy the phosphorus halides. The chlorpy- rimidine separated at once, and crystallized from alcohol in beautiful prisms melting at 107 to a clear oil. Analysis (Kjeldahl) : Found. Calculated for C u H u NgCl. I. II. N 12. 81 12.75 12.83 2- Paratolyl-4-methyl-6-thiocyanp yrimidine , N = C.SCN I I />-CH 3 C 6 H 4 C CH. An alcoholic solution (30 cc.) of 4 II II N CCH 3 grams of 2-paratolyl-4-methyl-6-chlorpyrimidine and 3 grams 1 Loc. cU. Researches on Pyrimidines. 145 of potassium thiocyanate was boiled for 1.5 hours. The in- soluble potassium chloride was removed by filtration, and the solution cooled, when the thiocyanate separated in irregular prisms. It crystallized from alcohol in fern- shaped crystals melting at 123. When warmed with aniline or aqueous ammonia at 100 it was recovered unaltered. Analysis (Kjeldahl) : Calculated for Ci 8 H n N 3 S. Found. N 17.42 17-2 2-Paratolyl~4-methyl- 6-thiopyrimidine , NH CS I I />-CH 3 C 6 H 4 C CH. This compound was obtained when II II N CCH 3 2-paratolyl-4-methyl-6-thiocyanpyrimidine was gently warmed with one molecular proportion of thiobenzoic acid. It depos- ited from alcohol in slender prisms melting at 114. Analysis (Kjeldahl) : Calculated for Ci 2 Hi 2 N 2 S. Found. N 12.96 12. 81 Rearrangement of 2-Paratolyl-4-methyl-6-thiocyanpyrimidine into the Isothiocyanate: One and five- tenths grams of the thiocyanate were heated, in an oil bath, for 2.5 hours at 130- I 35- We obtained a thick oil which slowly crystallized after cooling. The compound was very soluble in warm alcohol and on cooling crystallized in radiating prisms melting at 2O7-2o8. It did not react with ammonia and a nitrogen determination agreed with the calculated value for the iso- thiocyanate. It probably is a polymeric form of the iso- thiocyanate, (C^nNgS),. Analysis (Kjeldahl) : Calculated for (CisHnNsS)*. Found. N 17.42 17-35 In a second experiment the thiocyanate was heated for 2 hours at 130- 135, cooled, and then suspended in aqueous ammonia for 2 days. We obtained a yellow amorphous pro- 146 Johnson and Storey. duct which crystallized from alcohol in irregular prisms melting at 1 45- 1 46. A nitrogen determination agreed with the calculated value for 2-Paratolyl-4-methyl-6-thioureapyrimidine, />-CH 3 C 6 H 4 C CH. II II N CCH 8 Calculated for C^H^^S. Found. N 21.70 21.45 2-Paratolyl-4-methyl-6-aminopyrimidine, N=C.NH, I I />-CH 3 C 6 H 4 C C.H. 2-Paratolyl-4-methyl-6-chlorpyrimidine II II N C.CH 3 was recovered unaltered after heating with alcoholic ammonia for 2 hours at 120 and again for 2 hours at i5O-i6o. When heated with ammonia at i8o-i9o for 2 hours a good yield of the aminopyrimidine was obtained. It deposited from alcohol in hexagonal tables melting at I78-I79. An- alysis (Kjeldahl) : Calculated for C 12 H 18 N 3 . Found. N 21.10 2 I. O6 2-Paratolyl-4-methyl-6-anilinopyrim 1 A complete list of our previous papers on pyrimidines up to the end of the year 1907 is given at the end of this article. 2 THIS JOURNAL, 37, 392 (1907). Ibid., 38, 594 (1907). * Ann. Chem. (Liebig), 297, 75 (1897). 234 Wheeler and Johns. condenses in alkaline solution with 2-ethylpseudothiourea, H 2 N C(SC 2 H 5 ) = NH, giving 2-ethylmercapto-5-carbethoxy-6- oxypyrimidine (Formula I., on page 237). The condensation is especially smooth. We have succeeded in greatly increasing the yield of pyrimidine over the amount obtained in our first experiments and this condensation now offers an excellent starting point for further syntheses. We have also pre- viously shown that 2-ethylmercapto-5-carbethoxy-6-oxypyrimi- dine is easily saponified by alkali and the yield of 2-ethyl- mercapto-6-oxypyrimidine-5-carboxylic acid, II., is very good. We now find that with proper precautions this mercapto acid can be practically quantitatively converted into the acid chloride of 2-ethylmercapto-6-chlorpyrimidine-5-carboxylic acid, III. This acid chloride is a very reactive substance and, as might be expected, the chlorine attached to the CO group is more reactive than that in the 6 position. When the dichloride is treated with cold aqueous ammonia one chlorine atom is replaced by the amino group and 2-ethyl- mercapto-6-chlorpyrimidine-5-carboxamide, IV., results. This is proved by the fact that on treating the chloramide with sodium ethylate an ethoxy derivative, V., is obtained which is isomeric and not identical with 2-ethylmercapto-5-carbeth- oxy-6-aminopyrimidine, X. When the dichlorpyrimidine is warmed with concentrated aqueous ammonia on the steam bath, both chlorine atoms are removed and it is quantitatively converted into 2-ethyl- mercapto-6-aminopyrimidine-5-carboxamide, VI. We have found that this acid amide combines with bro- mine with the evolution of heat and that the product has the properties and composition of a dibrom addition product, VII. When it was treated with alkali it regenerated unal- tered 2-ethylmercapto-6-aminopyrimidine-5-carboxamide. When it was heated under certain conditions, its loss in weight and percentage of nitrogen corresponded with that re- quired for the formation of a monobromamide. If a mono- bromamide was formed here it might be expected that, in common with such substances, on treatment with alkali the bromamide group, RCONHBr, would undergo a rearrange- Researches on Pyrimidines. 235 ment into the carbonamide form, RNCO. In this case the reactive grouping would probably unite with the adjacent amino group in the 6 position, forming a five membered ring, with the result that an 8-oxypurine would be obtained. When the product or mixture obtained by heating the di- brom addition product was treated with aqueous alkali, cyto- sine-5-carboxylic acid, VIII., sometimes 2-ethylmercapto-6- aminopyrimidine-5-carboxamide, VI., and a product free from sulphur was obtained. This latter substance and its salts gave results on analysis which closely agreed with the calculated for 2,8-dioxypurine, XI., the only one of the di- oxypurines that has not been described. The empirical formula of 2,8-dioxypurine, C 5 H 4 O 2 N 4 , differs fromcytosine-5-carboxamide, IX., C 5 H 6 O 2 N 4 , by only two hydrogen atoms. The percentage composition of these substances is therefore so closely similar that the analysis did not decide which one of the two we were dealing with. In our previous work we had reason to be- lieve that we had obtained cytosine-5-carboxamide, IX. With the object of preparing this substance, the behavior of the ethyl ester of cytosine-5-carboxylic acid, or 5-carbethoxy- cytosine, XII., with aqueous ammonia was examined at that time and it was found that this ether showed an exceptional inertness. On heating at 140 to 150 for two hours, about 25 per cent remained unaltered. On concentrating the mother liquor more soluble material crystallizing in needles was ob- tained. The analytical results in this case and the method of preparation were then taken to indicate that the expected cytosine-5-carboxamide had been obtained. Owing to the poor yield, nothing further was done with this substance. This material was not identical with that obtained in the present work. The facts then seemed to show that the ma- terial obtained from the brom addition product, VII., was 2,8-dioxypurine. The. question was finally settled in the nega- tive, as follows: The material obtained by heating the di- bromamide and then treating with alkali was carefully freed from 2-ethylmercapto-6-aminopyrimidine-5-carboxamide and cytosine-5-carboxylic acid. It was then boiled with strong hydrochloric acid and found to completely decompose into cytosine-5-carboxylic acid. 236 Wheeler and Johns. This shows that no Hofmann rearrangement had taken place and that the present substance is the true cytosine-5-carbox- amide. The inertness of 5-carbethoxycytosine, and also of 2,6- diamino-5-carbethoxypyrimidine, 1 on heating with ammonia, is comparable with that of the dialkylmalonic esters. 2 The ammonium salts of these acids break down with the evolu- tion of carbon dioxide, and since 5-carbethoxycytosine is a substituted malonic acid derivative this offers an explana- tion of our previous results. The material formerly viewed as cytosine-5-carboxamide was probably impure or partly hydrous cytosine. It is probable that no monobromamide was formed on heat- ing the dibrom addition product and that the dibrom addi- tion product has the bromine atoms attached to the double bond between the 4 and 5 positions. Similar additions of bromine to the double bond have been observed in other cases in this laboratory, e. g., thymine and 4,5-dimethyluracil. The compounds in general have little stability. An unusually stable dibrom addition product of this sort, prepared from 2-phenylhydrazine-4-methyl-6-oxypyrimidine, has been de- scribed by Pellizzari and Roncagliolo. 8 When 2-ethylmercapto-6-aminopy rimidine-5-c a r b o x amide, VI., was boiled with hydrochloric acid it was converted into cytosine-5-carboxylic acid. When given a less energetic treatment with acid, in addition to unaltered material, some cytosine-5-carboxamide along with cytosine-5-carboxylic acid was obtained. This amide was identical with that obtained from the brom product. The question whether or not the Hofmann rearrangement can be applied in the pyrimidine series still remains to be investigated. A 2-oxygen amide such as cytosine-5-carbox- amide, being soluble in alkali and free from sulphur, would be a more promising substance than the mercaptoamide to decide this question. The work will be undertaken as soon 1 THIS JOURNAL, 38, 598 (1907). 2 Fischer and Dilthey: Ber. d. chem. Ges., 35, 844. a Gaz. chim. ital., 31, 1, 513. Researches on Pyrimidines. 237 as a more convenient method of preparing this amide, in quantities sufficient for synthetical purposes, is obtained. It appears from the above results that the unsaturated condition of the molecule, or the double bond in the 4,5 posi- tion, interferes with the Hofmann rearrangement, at least in the case of 2 -ethylmercapto-6-aminopyrimidine-5-carbox- amide. HN CO LSC CCOAH5 N CH I. N = C NH 2 HN CO CjHgSC CCOOH N==cq C 2 H 6 SC CCOC1 N CH II. N CH III. 0.1 *" **1 I I 2 H 5 SC icONH 2 <- C 2 H 5 SC CCONH 2 CCONH 2 N CH VI. N CH IV. *f N . Br N CHBr = C NH 2 | /CONH 2 N = C NH, OC CCONH 2 HN CH IX. N = C NH 2 OC CCOOH 1 II HN CH VIII. 1 1 / oc c- NH/ HN CH XI. C0 N CH v. N = C NH 2 C 2 H 6 SC CCOOC.H. II II N CH X. N=-C NH, OC CCOOCjH, I II HN CH XII. EXPERIMENTAL PART. Ethoxymethylenemalonic Ester, C 2 H 6 OCH = C(CO 2 C 2 H 5 ) 2 . Since this ester has proved to be of special importance for pyrimidine syntheses a detailed account is given of our methods of preparing and employing the substance. 238 Wheeler and Johns. The ester was prepared essentially according to Claisen's directions. 1 We have found that the mixture of malonic ester (100 grams), orthoformic ester (93 grams), acetic anhy- dride (128 grams), and zinc chloride (7 grams) reacts imme- diately on gently warming and enough heat is evolved to boil the solution. The mixture was kept boiling for an hour, then all that distilled up to 110 at ordinary pressure (the bulb of the thermometer being in the solution) was removed. The distillate usually weighed about 52 grams and represented the ethyl acetate formed in the reaction. A little anhydride was usually carried over. It was found that it is unnecessary to boil the above mix- ture for 1 1- 1 2 hours, as stated by Claisen. In fact, heating the flask attached to a return condenser in an oil bath at 120- 130, or so that the mixture boiled gently, for two more hours was sufficient. There were then obtained 14-15 grams of impure ethyl acetate or about 67 grams in all, which is 60 per cent of the calculated. The calculated yield of ethyl acetate is no grams. Boiling for a longer time gave very little more ethyl acetate. A portion of the ethyl acetate, however, proba- bly remains behind in the mixture. The mixture was then distilled at 15-20 mm. pressure and in almost every case 60-62 grams of ethoxymethylenemalonic ester were obtained. Sixty-two grams of ethoxymethylene- malonic ester is only 45.9 per cent of the calculated. How- ever, this yield is in perfect agreement with Claisen's state- ment that 500 grams of malonic ester, in five portions, gave 307 grams of distilled ethoxymethylenemalonic ester. The amount of ethyl acetate obtained indicated that at least 81 grams of ethoxymethylenemalonic ester was formed from 100 grams of malonic ester. We have now found that it is not necessary or even advisa- ble, on account of loss, to prepare pure distilled ethoxymethyl- enemalonic ester for the present condensation. In fact, a much better yield of condensation product is obtained if the oil is not distilled. 1 Ann. Chem. (Liebig), 297, 75 (1897). Researches on Pyrimidines. 239 Preparation of the Potassium Salt of 2-Ethylmercapto-^-carb- HN CO I I ethoxy-6-oxypyrimidine, C.jH 5 SC CCO 2 CjH 5 . In one experi- II II N CH ment, after heating the above mixture at ordinary pressure for three hours, distilling the ethyl acetate as stated, and filtering from zinc chloride, everything was then removed that dis- tilled below 117 at about 20 mm. pressure. The residue in this case weighed 105 grams. This was added to 100 grams of the addition product of ethyl bromide and thio- urea dissolved in 100 cc. of water. To this cold solution 60 grams of potassium hydroxide in 120 cc. of water were slowly added and the mixture was not allowed to warm. The solution soon became semisolid from the separation of the potassium salt. It was found that more separated on standing. The mixture was allowed to stand for two days at ordinary tem- perature. The salt which separated then, on filtering and drying, weighed 113 grams, or nearly twice as much crude salt as was obtained from 100 grams malonic ester in our previous work. In another experiment everything was removed that distilled below 120 at 25 mm. pressure. The remaining oil weighed 100 grams. When this was condensed with 2-ethylpseudo- thiourea as above, the weight of the potassium salt, after washing with a little alcohol, was 91 grams. The above yields, of course, do not represent the weights of pure substances, they merely serve to give a general idea. We conclude, however, that the above process yields almost 75 per cent of the calculated of ethoxymethylenemalonic ester; 105 grams is 77.7 per cent of the calculated. Preparation of 2-Ethylmercapto-6-oxypyrimidine-5-carboxylic HN CO Acid, C 2 H 5 SC CCOOH. The crude potassium salt of II II N CH 2-ethylmercapto-5-carbethoxy-6-oxypyrimidine was saponified 240 Wheeler and Johns. by warming with potassium hydroxide. For about 27-30 grams of this salt 15 grams of potassium hydroxide were used. If a smaller proportion of alkali was used the saponification was liable to be less smooth. The potassium salt was dissolved in alcohol, the alkali in water added, and the solution evaporated to dryness on the steam bath. The residue was taken up in water and evaporated again, when the saponification was usually found to be com- plete. The acid was then precipitated from the filtered aque- ous solution of this residue by means of hydrochloric acid. It separated in an almost pure condition as shown by its melting point (near 167). Care must be taken not to add too much hydrochloric acid since the mercapto acid is soluble in a certain excess. On the other hand, if the solution is not made dis- tinctly acid, the potassium salt of 2-ethylmercapto-6-oxy- pyrimidine-5-carboxylic acid is obtained. This melts with effervescence above 250. If the precipitate melts near 131 the saponification is not complete. Twenty-seven grams of the washed potassium salt gave 17 grams of 2-ethylmercapto-6-oxypyrimidine-5-carboxylic acid. The same quantity of salt in another experiment gave 1 8 grams of acid. This is 88.6 per cent of the calculated. The Acid Chloride of 2-Ethylmercapto-6-chlorpyrimidine-5- carboxylic Acid, C^HgSC CCOC1. The best yield of this I! II N CH compound was obtained when perfectly dry 2-ethylmercapto- oxypyrimidine-5-carboxylic acid and phosphorus oxychloride were mixed in the proportion of 5 cc. of the chloride to i gram of the acid and then heated, with a return condenser, in an oil bath. The temperature of the bath was kept at i3o-i5O or so that the solution boiled quietly and until hydrogen chloride was no longer evolved. This required from 2 to 3 hours, although solution took place soon after heating. Most of the phosphorus oxychloride was then removed under reduced pressure at 100 and the residue, a thick oil, was poured upon Researches on Pyrimidines. 241 cracked ice. It then changed to a granular solid. The yield of crude chloride was 85 per cent of the calculated. To prepare the amide of 2-ethylmercapto-6-aminopyrimidine-5-carboxylic acid, instead of pouring on ice, it is poured directly into con- centrated ammonia (see below). The yield of crude amide is then almost quantitative. In another experiment 18 grams of the acid and 45 cc. of phosphorus oxychloride were boiled on a sand bath. Under these conditions the material was completely decomposed. The yield of this chloride may vary decidedly under slight changes of conditions. This acid chloride was very soluble in cold benzene, ether, and ligroin. It was purified for analysis by dissolving in ligroin and evaporating at the temperature of the room. Radiating clusters of colorless prisms were formed, which melted to a clear oil at 38-4O. Analysis: Calculated for C7H 6 ON 2 C1 2 S. Found. N 11.81 11.76 The Amide of 2-Ethylmercapto-6-chlorpyrimidine-5-carboxylic I SC Acid, C 2 H 5 SC CCONH,. This is the first product obtained II II N CH by the action of aqueous ammonia on the above acid chloride. When the acid chloride was added to cold concentrated aqueous ammonia, an immediate reaction took place with the liberation of heat and a white powder was formed. This was washed with water to remove ammonium chloride. When dried it was found to be moderately soluble in hot alcohol, benzene, and toluene, and difficultly soluble in boiling ether. It crystallized from benzene in long, hairlike crystals which formed a fibrous mat. It then melted sharply to a colorless oil at 134 and the yield was almost quantitative. Analysis: Calculated for Found. C 7 H 8 ON 8 C1S. I. II. N 19.31 19.36 19-24 When this chloramido compound was first obtained it became a question which chlorine atom had been replaced in the above 242 Wheeler and Johns. acid chloride. In order to decide this point, two grams of the chloramido compound were dissolved in absolute alcohol and added to a solution of 0.25 gram of sodium, also in alcohol. An immediate precipitate resulted and heat was liberated. On cooling, a mass of well defined prisms separated. The sodium chloride was removed by washing with water. The residue was then found to be insoluble in hot water but it crystallized from alcohol, in which it was moderately soluble. The substance melted at 134, the same temperature as the chloramido compound. However, it was free from chlorine and when mixed with the chloramido compound the melting point of the mixture was lowered about 30. The yield and nitrogen determinations agreed with the calculated for an ethoxy derivative. Calculated for Found. C 9 Hi 3 2 N 8 S. I. II. N 18.50 18.54 18.45 This ethoxy derivative should have the structure represented by one of the following formulas: N = C NH 2 N COC 2 H 5 C CCO.OC 2 H 5 C 2 H 5 SC CCO.NH 2 . II II II II N CH N CH I. II. The first of these two compounds has already been described by us in our twenty-sixth paper, p. 597. It melts at 102 . To the ethoxy compound now obtained, melting at 134, is there- fore assigned the structure represented by formula II. It follows that the chloride from which it was prepared has the chlorine atom in the 6 position and is the amide of 2-ethyl- mercapto-6-chlorpyrimidine-5-carboxylic acid. The Amide of 2-Ethylmercapto-6-aminopyrimidine-5-carboxylic N = C NH 2 Acid, CaH 6 SC CCONH 2 . If the acid chloride of 2-ethyl- II II N CH mercapto-6-chlorpyrimidine-5-carboxylic acid is warmed with Researches on Pyrimidines. 243 concentrated ammonia both chlorine atoms are replaced by the amino group and the amide of 2-ethylmercapto-6-amino- pyrimidine-5-carboxylic acid is formed. We have prepared this compound by slowly adding the acid chloride (it reacts vigorously) to a considerable excess of aqueous ammonia in an evaporating dish and then warming on the steam bath. Not very much of the aminoamide thus formed dissolves in the hot ammonia nor is it soluble to any great extent in boiling water. It should be warmed until the insoluble material melts above 200. When purified by crystallizing from alcohol, in which it is moderately soluble, it separates in rec- tangular crystals, little stout prisms which separate slowly and melt to an oil at 2i8-2i9. It is soluble in glacial acetic acid but insoluble in alkali. The yield of crude product melting at 214 is almost quantitative. Thirteen grams of mercapto acid gave 12 grams of this amide. Analysis: Calculated for Found. C 7 H 10 ON 4 S. I. II. N . 28.28 27.99 2 7-9i Action of Hydrochloric Acid. When this amide is digested on the steam bath with concentrated hydrochloric acid ethyl mercaptan is evolved and the amido group attached to the carboxyl radical in position 5 is removed. If the resulting solution is allowed to cool slowly the hydrochloride of cytosine- 5-carboxylic acid separates in the form of large prisms which contain one molecule of water and melt sharply at 276 to a brown oil and with some effervescence. This salt was iden- tified by its properties previously described and by the fol- lowing analyses: Calculated for C 5 H 6 O3N 8 .HC1.H 2 O. Found. H 2 8.59 8.75 The water determination was made by heating the substance at I20-i3o. A nitrogen determination then gave the follow- ing results: Calculated for C 5 H 6 O 8 N3.HC1. Found. N 21.92 22.21 244 Wheeler and Johns. Prolonged digestion of the aminoamide with dilute hydro- chloric acid or potassium hydroxide also removed the amido group from the 5 position. For a description of the less energetic action of hydrochloric acid on this amide see under cytosine-5-carboxamide. It did not dissolve in a solution of potassium hypobromite (16 grams of bromine in 280 cc. of 10 per cent alkali). The Action of Dry Bromine: The Dibrom Addition Product of 2-Ethylmercapto-6-amino-5-carboxamide, N = C NH, I I C 2 H 5 SC CCONH 2 Br 2 . This compound is formed by adding II II N CH the amide directly to liquid bromine. They unite with evolu- tion of considerable heat. Bight grams of finely powdered and carefully dried amide were added in small portions, best through a sieve to prevent the formation of lumps, to about 15 cc. of dry bromine. The amide then dissolved at once in the bromine. When all of the amide had been added the excess of bromine was allowed to evaporate at the temperature of the room. This treatment left a yellow, crystalline crust that was powdered in a mortar in order to liberate the inclosed bromine. The yield at this point was quantitative if care was taken to exclude moisture ; otherwise some decomposition took place. Analyses of the air-dried material gave the following results: Calculated for Found. C 7 H 10 ON 4 Br,S. I. II. N 15.64 15.50 15.91 When the dibrom addition product was warmed with water unaltered amide was obtained. Action of Alkali. The unaltered amide was also obtained when this material was treated with alkali. One gram of the addi- tion product was added to 10 cc. of water containing i gram of potassium hydroxide. Only a small portion dissolved even after heating on the steam bath. On filtering and acidifying the filtrate with acetic acid a very small amount of material not soluble in ammonia was precipitated. This had all the Researches on Pyrimidines. 245 properties of cytosine-5-carboxamide. Its hydrochloric acid solution gave a precipitate with nitric acid. Behavior on Heating. When the dibrom addition product was heated at 120- 135 a point was finally reached where the evolution of hydrogen bromide seemed to stop or proceed more slowly. The brownish white material then agreed in weight with the loss of about one molecular proportion of hydrogen bromide. There was a loss from 13.5 grams of the addition product of 3.3 grams while the calculated loss is 3.1 grams. Nitrogen determinations gave the following results: Calculated for Found. C 7 H 9 ON 4 BrS. I. II. N 20. 2 i 20.47 20.76 This product had an odor similar to that of the sulphur chlorides. It dissolved only partially in hot water, alcohol, ethyl acetate, or amyl acetate, and it behaved like a mixture of more or less decomposed material. It dissolved partially in a solution of sulphur dioxide, and ammonia then precipitated unaltered mercaptoaminoamide. This might result from the hydrobromic acid salt of the amide possibly contained in the mixture, or from unaltered dibrom addition product. A solvent suitable to isolate a monobromamide, if formed here, was not found, and the results obtained on treating the material with alkali would suggest that in spite of the analyses such a compound was not formed. Sometimes the product would dissolve completely in alkali. When dark in color it usually left undissolved amorphous, dark colored decomposition prod- ucts mixed at times with unaltered mercaptoaminoamide. N = C NH, Cytosine-5-carboxamide, OC CCONH 2 . Ten grams of I II HN CH the brom product obtained by heating the above dibrom addition product of the amide of 2-ethylmercapto-6-amino- pyrimidine-5-carboxylic acid were dissolved in 50 cc. of water containing 10 grams of potassium hydroxide. In this particular experiment complete solution resulted. On acidifying with 246 Wheeler and Johns. acetic acid mercaptan was evolved and a granular precipitate was obtained. This was filtered and dissolved in hot water to which a little hydrochloric acid was added. The hot solution was then made strongly alkaline with ammonia which repre- cipitated the base. This treatment removed any cytosine-5- carboxylic acid that was present, the acid being soluble in ammonia. The best yield of aminoamide obtained at this point was about 75 per cent of the theory. It varied con- siderably. For analysis the aminoamide was purified by recrystallizing from dilute hydrochloric acid. The hydro- chloride was then dissolved in water, with the aid of a little hydrochloric acid, and the base reprecipitated with ammonia, washed thoroughly with water and alcohol, and finally dried over calcium chloride. It did not lose water when heated at I20-i30 (Analyses I. II. and III.). When we first obtained this material attempts were made to determine nitrogen by Kjeldahl's method and it was found that neither the free base nor any of its salts that we tried gave results agreeing with the calculated. The figures invariably came several per cent too low. It appears probable that the failure to obtain correct results by Kjeldahl's method was due to the fact that the weakly basic properties of the compound permitted the escape of material on heating the sulphuric acid solution. This might have escaped unnoticed if we had no theory to check the results. I. 0.0763 gram of substance gave 24.6 cc. of nitrogen at 22 and 770 mm. pressure. II. 0.2462 gram of substance gave 0.0877 gram of H 2 O and -3575 gram of CO 2 . III. 0.1440 gram of substance gave 0.0526 gram of H 2 O and 0.2093 gram of CO 2 . Calculated for Found. C 6 H 6 2 N4. I. II. III. IV. C 38.96 39.60 39-58 .../.. 3.89 4.00 3.90 N 36.36 37-oo 36.38 Cytosine-5-carboxamide was also obtained by the action of concentrated hydrochloric acid on 2-ethylmercapto-6-amino- Researches on Pyrimidines. 247 pyrimidine-5-carboxamide. One gram of the mercapto- aminoamide was evaporated to dryness with 15 cc. of con- centrated hydrochloric acid on the steam bath. The residue was made strongly alkaline with ammonia. This extracted cytosine-5-carboxylic acid and left a mixture which was boiled with alcohol. Unaltered mercaptoarninoamide dis- solved. The insoluble material was purified by dissolving in dilute hydrochloric acid and precipitating with an excess of ammonia while the solution was hot. This gave very small, microscopic, lenticular crystals that did not show signs of melting at 310. When a drop of nitric acid was added to the hydrochloric acid solution a precipitate of the nitrate was formed. (Analysis IV.). Cytosine-5-carboxamide is almost insoluble in water and the ordinary organic solvents. It dissolves readily in cold alkalies, but it is almost insoluble in ammonia. It does not dissolve in acetic acid. It dissolves in dilute mineral acids and yields beautiful crystalline salts which are dissociated by water. The aminoamide is a weak base. Its solubility in alkali is in accordance with the presence of NH CO groups. The free aminoamide prepared from the brom deriva- tive, when obtained by precipitating from alkaline solution by acetic acid or from the hydrochloric acid solution with ammonia, usually appears to be an amorphous powder. Be- fore purifying it has a more or less brownish color. Its method of separation along the lines of stirring indicates a crystalliza- tion but the crystals are too minute to be determined under the microscope. Action of Concentrated Hydrochloric Acid on Cy to sine- 5- carboxamide: Cytosine-5-carboxylic Acid. The amide obtained from the bromamide (0.6 gram) was boiled with 50 cc. of concentrated hydrochloric acid for about five minutes and the resulting solution evaporated to dryness on the steam bath. The residue dissolved completely in ammonia. On acidifying with acetic acid a precipitate of cytosine-5-carboxylic acid was obtained. This decomposed at 256. Analysis: Calculated for CjHsOsNa. Found. N 27.09 27.53 248 Wheeler and Johns. This acid was described in a previous paper. It should be added that, like the amide, it is almost insoluble in water and alcohol. It was obtained in an amorphous condition. When precipitated from its solution in ammonia by acetic acid it separates as a gelatinous precipitate which is difficult to filter. It is better to obtain the acid by evaporating the ammonia solution. It then separates as a finely divided powder which is readily filtered. It is also readily identified by its decomposing point, 256, and its crystalline hydro- chloride which contains i molecule of water of crystallization. Cytosine-5-carboxamide Hydrochloride, C 5 H 6 O 2 N 4 .HC1. The base dissolved in moderate quantities in hot dilute hydro- chloric acid. On cooling, the hydrochloride separated al- most completely in clusters of colorless, stout, pointed prisms which did not have a definite melting point. They did not lose weight on heating at 110, when dried over calcium chloride. When heated at I4o-i5o they lost their lustre, hydrochloric acid being slowly given off. The salt was dried at 110 for analysis: I. 0.0739 gram of substance gave 19.0 cc. of N at 22 and 768 mm. II. 0.0791 gram of substance gave 0.0607 gram of AgCl. Calculated for Found. C 6 H 6 2 N4.HC1. I. II. N 29.40 29 .42 Cl 18.64 l8 -97 Cytosine-5-carboxamide Nitrate, C 5 H 6 O 2 N 4 .HNO 3 . This is the most difficultly soluble and characteristic salt. It is formed not only by dissolving the amide in hot nitric acid but also on adding nitric acid to solutions of other salts of the base. Cytosine-5-carboxamide dissolved very slightly in hot nitric acid. On cooling, the nitrate separated in beautiful truncated octahedrons. When nitric acid was added to a solution of the base in dilute hydrochloric acid an immediate crystalline precipitate of the nitrate came down. It then formed tufts of short hairs, which did not decompose at 300, although they sublimed slowly below that temperature. They did not lose weight on heating at 115. Analysis: Researches on Pyrimidines. 249 Calculated lor C 5 H 6 O 2 N 4 .HNO3. Found. N 32.26 32.05 Cytosine-5-carboxamide Sulphate, (C 5 H 6 O 2 N 4 ) 2 H 2 SO 4 . A solu- tion of the amide was made in hot water containing about 10 per cent sulphuric acid. The sulphate separated, on cool- ing, in scales with a pearly lustre. They did not melt at 300 and no loss in weight took place on heating at 110. Analysis: 0-0653 gram of substance gave 0.0386 gram BaSO 4 . Calculated for (C 6 H 6 OaN4) 2 H t SO4. Found. S 7.88 8. II Cytosine-5-carboxamide Picrate, C 5 H 6 O 2 N 4 .C 6 H 2 (NO 2 ) 3 OH. The amide was dissolved in hot water to which a few drops of hydrochloric acid had been added. On adding an aqueous solution of picric acid an immediate precipitate was formed. This crystallized from an aqueous solution of picric acid. It dissociates in water into small, yellow prisms that began to decompose at about 26o-27o. The salt was found to be anhydrous. I. 0.0531 gram of substance gave n.6 cc. of N at 20 and 771 mm. II. 0.0967 gram of substance gave 21.2 cc. of N at 18 and 771 mm. Calculated for Found. CnH 9 O 9 N 7 . I. II. N 25.59 25.34 25.64 The papers on pyrimidines which have been published from this laboratory, under the direction of Dr. Treat B. Johnson and the writer, have appeared in two journals. Some of these papers have not been numbered and one has received an incorrect number, there being two twenty-first papers. For the sake of reference a complete list of these papers up to the end of the year 1907 is now given. The papers are ar- ranged and numbered in the order in which they appeared. The numbers correspond with those already given in the literature, with the exception of the paper which has been in- correctly numbered. By referring to this paper as the twenty- 250 Wheeler and Johns. fourth the order then remains otherwise undisturbed. The following is a complete list up to the end of the year 1907. 1. Researches on the Cycloamidines: Pyrimidine Derivatives. H. L. Wheeler: THIS JOURNAL, 20, 481 (1898). 2. On Some Condensation Products of the Pseudothioureas : Synthesis of Uratil, Thymine, and Similar Compounds. Henry L. Wheeler and Henry F. Merriam: THIS JOURNAL, 29, 478 (1903). 3. Syntheses of Aminooxypyrimidines having the Composition of Cytosine: 2-Amino-6-oxypyrimidine and 2-Oxy-6-aminopyrimidine. Henry L. Wheeler and Treat B. Johnson: THIS JOURNAL,, 29, 492 (1903). 4. On Cytosine or 2-Oxy-6-aminopyrimidine from Triticonucleic Acid. Henry L. Wheeler and Treat B. Johnson: THIS JOURNAL, 29, 505 (1903). 5. 5-Methylcytosine. Henry L. Wheeler and Treat B. Johnson: THIS JOURNAL, 31, 591 (1904). 6. Synthesis of 2-Amino-5-methyl-6-oxypyrimidine. Treat B. John- son and Samuel H. Clapp: THIS JOURNAL, 32, 130 (1904). 7. 2-Oxy-4,6-diaminopyrimidine. Henry L. Wheeler and George S. Jamieson: THIS JOURNAL, 32, 342 (1904). 8. The Structure of Some Substitution Products. Henry L. Wheeler and H. Stanley Bristol: THIS JOURNAL, 33, 437 (1905). 9. The Action of Potassium Thiocyanate upon Some Imidechlorides. Henry L. Wheeler and H. Stanley Bristol: THIS JOURNAL, 33, 448 (1905). 10. The Action of Aqueous and Alcoholic Ammonia and Aniline on Some Halogen- and Mercaptopyrimidines. Treat B. Johnson and Carl O. Johns: THIS JOURNAL, 34, 176 (1905). 11. 2-Ethylmercapto-5-amino-6-oxypyrimidine. Treat B. Johnson: THIS JOURNAL, 34, 191 (1905). 12. 2,5-Diamino-6-oxypyrimidine. Treat B. Johnson and Carl O. Johns: THIS JOURNAL, 34, 554 (1905). 13. Some lodopyrimidines: 5-Iodocytosine. Treat B. Johnson and Carl O. Johns: /. Biol. Chem., I, 305 (1906). 14. On Methods of Synthesizing Isobarbituric Acid and 5-Oxycytosine. Treat B. Johnson and Elmer V. McCollum: J. Biol. Chem., i, 437 (1906). 15. The Action of Potassium Thiocyanate upon Imidechlorides. Treat B. Johnson and Elmer V. McCollum: THIS JOURNAL, 36, 136 (1906). 1 6. The Formation of Purines from Ureapyrimidines. Treat B. John- son and Elmer V. McCollum: THIS JOURNAL, 36, 149 (1906). 17. 5-Nitrocytosine and its Reduction to 2-Oxy-5,6-diaminopyrimidine. Treat B. Johnson, Carl O. Johns, and Frederick W. Heyl: THIS JOURNAL, 36, 160 (1906). 1 8. 5-Ethylcytosine. Treat B. Johnson and George A. Menge: /. Biol. Chem., 2, 105 (1906). 19. Synthesis of Uracil-5-carboxylic Acid. Henry L. Wheeler, Treat B. Johnson, and Carl O. Johns: THIS JOURNAL, 37, 392 (1907). 20. Some Condensation Products of a Substituted Pseudothiourea: Researches on Pyrimidines. 251 Synthesis of i -Methyl uracil. Treat B. Johnson and F. W. Heyl: THIS JOURNAL, 37, 628 (1907). 21. On a Color Test for Uracil and Cytosine. Henry L. Wheeler and Treat B. Johnson: /. Biol. Chem., 3, 183 (1907). 22. On Some Salts of Cytosine, Isocytosine, 6-Aminopyrimidine, and 6-Oxypyrimidine. Henry L. Wheeler: /. Biol. Chem., 3, 285 (1907). 23. Uracil-4-carboxylic Acid. Henry L. Wheeler: THIS JOURNAL, 38, 358 (1907). 24. The Action of Methyl Iodide on 2-Anilino-6-oxypyrimidine and the Synthesis of 2-Anilinopyrimidine. Treat B. Johnson and Frederick W. Heyl: THIS JOURNAL, 38, 237 (1907). 25. Synthesis of Thymine-4-carboxylic Acid. Treat B. Johnson: /. Biol. Chem., 3, 299 (1907). 26. Synthesis of Cytosine-5-carboxylic Acid. Henry L. Wheeler and Carl O. Johns: THIS JOURNAL, 38, 594 (1907). 27 Synthesis of Thymine-5'-carboxylic Acid. (Uracil-5-acetic Acid). Treat B. Johnson and Carl Frank Speh: THIS JOURNAL, 38, 602 (1907). 28. Synthesis of 4-Methyluracil-5-acetic Acid. Treat B. Johnson and Frederick W^ Heyl: THIS JOURNAL, 38, 659 (1907). NEW HAVEN, CONN., March, 1908. [.Reprinted from the American Chemical Journal, Vol, XI,. No. 4. October, 1908.] CLX. RESEARCHES ON PYRIMIDINES: SYNTHESIS OF 4-METHYLCYTOSINE. [THIRTY-SEVENTH PAPER]. BY CARL O. JOHNS. There are two methyl derivatives of cytosine having the methyl group attached to carbon. Of these, 5-methylcy- tosine (Formula I.) has been described in the fifth paper of Researches on Pyrimidines. 349 this series, 1 while a description of the synthesis and properties of 4-methylcytosine (V.) will be given in the following pages. Acetoacetic ester condenses readily in an aqueous solution of sodium hydroxide with pseudoethylthiourea hydrobromide to give an excellent yield of 2-ethylmercapto-4-methyl-6-oxy- pyrimidine (II.)- This compound has been prepared by List 2 but his method requires several days, and, calculating from the weight of acetoacetic ester used, the yields are only about one-half as great as those obtained by the method here described. When 2-ethylmercapto-4-methyl-6-oxypyrimidine is boiled with phosphorus oxy chloride it yields 2-ethylmercapto- 4-methyl-6-chlorpyrimidine (III.)- When heated in a sealed tube at I4o-i5o with alcoholic ammonia the chlorine in this compound is replaced by an amino group and 2-ethyl- mercapto-4-methyl-6-aminopyrimidine (IV.) is obtained. This is readily converted into 4-methylcytosine (V.) by digest- ing with concentrated hydrochloric acid and precipitating the base with a slight excess of ammonium hydroxide. The chloride (III.) reacts more readily with alcoholic so- dium ethylate than it does with alcoholic ammonia. Action takes place at room temperature. Sodium chloride separates and 2-ethylmercapto-4-methyl-6-ethoxypyrimidine (VI.) is formed. 4-Methylcytosine can easily be distinguished from 5-methyl- cytosine. It does not melt at 310, while 5-methylcytosine melts with effervescence at 270. It is far less soluble in water than is 5-methylcytosine. Moreover, 4-methylcy- tosine crystallizes from water in long, anhydrous prisms, while 5-methylcytosine gives flat prisms containing one-half molecule of water. 4-Methylcytosine gives a very characteristic basic hydro- chloride containing 3 molecules of base to i molecule of hy- drogen chloride while 5-methylcytosine under similar con- ditions gives a hydrochloride containing 2 molecules of base to one of acid. 1 Wheeler and Johnson: THIS JOURNAL, 31, 591 (1904). 2 Ann. Chem. (Liebig), 236, 14 (1886). 350 Johns. When crystallized from concentrated solutions of the com- mon acids, 4-methylcytosine gives a series of normal salts. 4-Methylcytosine has been used for the preparation of some purine derivatives to be described in another paper. N = CNH 2 I I OC C.CH, I II HN CH I. N == CNH 2 I I OC CH I II HN C.CH 8 V. HN CO I I C 2 H 6 SC CH II II N C.CH 3 II. N^CCl I I C 2 H 6 SC CH N C.CH, III. / N = CNH a I I C 2 H 5 SC CH II II N C.CH 3 IV. EXPERIMENTAL PART. N = COC 3 H 5 C 2 H 6 SC CH II II N C.CH, VI. HN CO 2-Ethylmercapto-4-methyl-6-oxypyrimidine, C 2 H 5 SC CH . N C.CH, List 1 prepared this compound by heating 2-thio-4-methyl- 6-oxypyrimidine with ethyl iodide in the presence of sodium ethylate. It can be obtained more easily and in better yields by condensing pseudoethylthiourea hydrobromide with aceto- acetic ester in an aqueous solution of sodium hydroxide. This condensation is very smooth and the yields range from 80 to 90 per cent of the theoretical quantity. Ninety-three grams of pseudoethylthiourea hydrobromide are dissolved in 100 cc. of water and this solution is mixed with 65 grams of acetoacetic ester. To this mixture is slowly added a solution of 40 grams of sodium hydroxide in 80 cc. of water. Condensation takes place at once, but to insure complete reaction the mixture is allowed to stand at room temperature overnight. When the resulting clear solution i Ann. Chem. (Liebig), 236, 14 (1886). Researches on Pyrimidines. 351 is acidified with acetic acid about 75 grams of 2-ethylmer- capto-4-methyl-6-oxypyrimidine are obtained. When crys- tallized from dilute alcohol it melts at i44-i45 and pos- sesses all the properties of List's compound. Analyses (Kjeldahl) : Calculated for Found. C 7 H 10 ON 2 S. I. II. N 16.47 16.49 16.52 N = CC1 I | 2-Ethylmercapto-4-methyl-6-chlorpyrimidine, C 2 H 5 SC CH N CCH $ Twenty grams of 2-ethylmercapto-4-rnethyl-6-oxypyrimi- dine were heated with 60 cc. of phosphorus oxychloride in an oil bath kept at I3o-i35 until hydrogen chloride was no longer evolved. The excess of phosphorus oxychloride was removed under diminished pressure and the resulting thick oil was poured on cracked ice. The chloride was ex- tracted with ether and dried over calcium chloride. When the ether was evaporated a clear, red oil remained. This boiled at 142 at 15 mm. pressure. The yield of pure chlor- ide was about 75 per cent of the calculated quantity. This chloride is very stable towards water and alcohol. Analyses (Kjeldahl) : Calculated for Found. I. II. N 14-85 14-71 15-09 2-Ethylmercapto-4-methyl-6-ethoxypyrimidine, N = COC,H 5 C 2 H 5 SC CH . An ether solution of 2-ethylmercapto- ' II II N CCH 3 4-methyl-6-chlorpyrimidine was added to alcohol contain- ing an excess of sodium, calculating from the chloride. An immediate reaction took place with the liberation of heat. 352 Johns. The excess of alcohol was evaporated, and, after adding water, the oil was shaken out with ether and dried over cal- cium chloride. When the ether was evaporated an oil re- mained. This boiled at 154 at 20 mm. pressure. Analyses (Kjeldahl): Calculated for Found. C 9 H 14 ON2S. I. II. N 14-14 14.08 14-43 2-Ethylmercapto-4-methyl-6-aminopyrimidine, I I C,H 5 SC CH . Alcoholic ammonia does not react at II II N CCH, room temperature with 2-ethylmercapto-4-methyl-6-chlorpyr- imidine. Sixteen grams of the chloride were heated in a sealed tube for 4 hours at I4o-i5o with about 75 cc. of alcoholic ammonia saturated at o. The tube, wheen cooled, was lined with ammonium chloride and crystals of the new amino compound. The reaction was very smooth and the alcohol remained colorless. After evaporating to dryness, the resi- due was washed with cold water and recrystallized from di- lute alcohol. The crystals melted at ii5-n6 to a clear oil. They have a tetrahedral outline and show a great ten- dency to twin. They are easily soluble in cold alcohol and acetic acid, moderately soluble in cold benzene, and diffi- cultly soluble in boiling water. Analyses (Kjeldahl) : Calculated for Found. C 7 H U N 3 S. I. II. N 24.85 24.83 25.07 N = CNH 3 I I 4-Methylcytosine, OC CH . When 2-ethylmercapto- I II HN CCH S 4-methyl-6-aminopyrimidine is evaporated with concentrated hydrochloric acid ethyl mercaptan is evolved and an almost Researches on Pyrimidines. 353 theoretical yield of 4-methylcytosine monohydrochloride is obtained. The base is obtained from this salt by adding an excess of ammonium hydroxide. 4-Methylcytosine crys- tallizes from water in anhydrous, slender, brittle prisms. These crystals do not melt at 310, but decompose slowly without effervescence at higher temperatures. 4-Methylcytosine is moderately soluble in boiling water. At 24, 0.36 gram is dissolved by 100 cc. of water. It is soluble in cold aqueous ammonia and in cold acetic acid. Analyses (Kjeldahl): Calculated for Found. C 6 H 7 ON 3 . I. II. N 33-6o 33.52 33.39 4-Methylcytosine Monohydrochloride, C 5 H 7 ON 3 .HC1. This salt is obtained when 2-ethylmercapto-4-methyl-6-amino- pyrimidine is digested with concentrated hydrochloric acid. It is also formed by crystallizing 4-methylcytosine from 20 per cent hydrochloric acid. It is moderately soluble in hot hydrochloric acid and separates on cooling in blunt, flat prisms with a hexagonal appearance. When dried over potas- sium hydroxide it does not lose water at 110. In a capillary tube it begins to shrivel at about 280 and decomposes slowly above 300. Analyses : Calculated for Found. C 5 H 7 ON 3 .HC1. I. II. N 26.00 26.10 26.09 Cl 21.98 21.81 The anhydrous monohydrochloride of 4-methylcytosine is also obtained unaltered when its aqueous solution is saturated with hydrogen chloride and evaporated in a desiccator. The crystals thus obtained did not lose weight at 125. Analysis : Calculated for C 6 H 7 ON V HC1. Found. Cl 2 i . 98 2 i . 95 4-Methylcytosine Basic Hydrochloride, (C 5 H 7 ON 3 ) 3 .HC1. >uch a salt was formed when a hot aqueous solution of 0.25 354 J hns > gram of the base was mixed with an aqueous solution of 0.32 gram of the monohydrochloride. The basic salt separated in scales with a pearly lustre. These began to shrivel at about 270 and turned brown above 280 but did not melt at 310. These crystals lost weight when dried at 130- 140, but at- tempts to determine water of crystallization gave results that differed widely, but indicated that one molecule of water was probably present. The dried salt contained 3 molecules of the base and i molecule of hydrogen chloride. This same salt was obtained when equal weights of the base and mono- hydrochloride were recrystallized from water. Analyses of the dried salt: Calculated for Found. (CsH T ON 3 )3.HCI. I. II. III. N 30.61 ---- 30.26 ---- Cl 8.62 8.51 ..... 8.49 4-Methylcytosine Nitrate, C 5 H 7 ON 3 .HNO 3 . One-half gram of 4-methylcytosine was crystallized from 10 cc. of 25 per cent nitric acid. The salt separated in round clusters of blunt prisms. It did not contain water of crystallization. 0.0983 gram of substance gave 25.2 cc. of moist nitrogen at 18 and 770 mtn. pressure. Calculated for Found. N 29.79 29.94 4-Methylcytosine Siilphate, (C 5 H 7 ON 3 ) 2 .H 2 SO 4 .H 2 O. When 4-methylcytosine was crystallized from 25 per cent sulphuric acid, two molecules of the base united with one molecule of sulphuric acid to form a normal sulphate containing one mole- cule of water of crystallization. The crystals were large, flat prisms tjiat did not lose water at 110, but lost one mole- cule of water slowly at i6o-i7o. Analyses: Calculated for Found. (C 5 H 7 ON 3 ) 2 .H 3 S0 4 .H 2 O. I. II. HI. H 2 4.91 .... 4-83 N 22.95 22.79 22.82 S 8.74 .... 8-88 Researches on Pyrimidines. 355 4-Methylcytosine Picrate, C 5 H 7 ON 3 .C C H 2 (NO 2 ) 3 OH. When an aqueous solution of picric acid is added to a solution of 4-methylcytosine, an immediate precipitate is obtained. This picrate is difficultly soluble in hot water and crystal- lizes in beautiful, yellow needles that turn brown at about 265 and decompose slowly above that temperature. 0.0858 gram of substance gave 17.3 cc. of moist nitrogen at 18 and 772 mm. pressure. N NEW HAVEN, CONN., May, 1908. Calculated for C 5 H 7 ON 3 .C 6 H 2 (N0 2 ) a OH. 23-73 Found. 23.65 [Reprinted from the American Chemical Journal, Vol. XI,. No. 5. November, 1908.] [Contributions from the Sheffield Laboratory of Yale University.] CLXL RESEARCHES ON PYRIMIDINES: SYNTHESES OF SOME BENZYL DERIVATIVES OF URACIL AND THYMINE. [THIRTY-EIGHTH PAPER.] BY TREAT B. JOHNSON AND JOHN H. DERBY, JR. In order to determine the structure of some new nitrogen derivatives of uracil and thyrnine it was essential to have knowledge of some characteristic nitrogen-alkyl derivatives of Researches on Pyrimidines. 445 these pyrimidines of known constitution. The only nitrogen- alkyl derivatives of uracil and thymine mentioned in the litera- ture are 1,3-dimethylthymine 1 and the methyl and ethyl com- pounds, which have been described in previous papers from this laboratory. 2 These alkyl pyrimidines were not suitable for our work on account of their solubility, and difficulty of isolation when accompanied by impurities. Furthermore, 3-methyluracil has not been synthesized. With the anticipation that the nitrogen-benzyl derivatives of uracil and thymine might be found to be more insoluble, and suitable for our work, we undertook this investigation. We shall describe in this paper the methods of preparation and the properties of i-benzyluracil, I., 3-benzyluracil, II., i-benzyl- thymine, III., and 3-benzylthymine, IV. NH CO I CH II CH Johnson and Heyl 3 showed that methyl iodide reacts with 2-ethylmercapto-6-oxypyrimidine, 4 in presence of alkali, giving i-methyl-2-ethylmercapto-6-oxypyrimidine, V. They did not observe the formation of the isomeric 2-ethylmercapto-3- methyl-6-oxypyrimidine, VI. Wheeler and Liddle 5 observed, on the other hand, that ethyl chloracetate reacts with this mercaptopyrimidine, giving 2-ethylmercapto-3-ethylacetate-6- 1 Steudel: Z. physiol. Chem., 30, 539. * Johnson and Heyl: THIS JOURNAL, 87, 628; Johnson and Clapp: J. Biol. Chem. 5,49. 3 Loc. cit. 4 THIS JOURNAL, 29, 478. 492. 8 J. Am. Chem. Soc., 30, 1152. 446 Johnson and Derby, Jr. oxypyrimidine, VII., instead of the isomeric mercaptopyrimi- dine, VIII. CH 3 N CO N CO K SC CH C 2 H 5 SC CH i II CH 3 N CH VI. H VII. We now find that benzyl chloride reacts with 2-ethylmer- capto-6-oxypyrimidine in presence of alkali, giving a mixture of two isomeric mercaptopyrimidines melting at 77 and 139. They were converted quantitatively into the corresponding benzyluracils melting at 175 and 173, respectively, by hydrolysis with hydrochloric acid. The structures of the benzyluracils and incidentally of the mercaptopyrimidines were established in the following manner : The benzyluracil melting at 173 reacted with methyl iodide, giving i -methyl-3 -benzyluracil, XVI. This same pyrimidine was also obtained by the action of benzyl chloride on i-methyl- uracil, 1 XVIII. This benzyl derivative is therefore to be represented as 3 -benzyluracil, XIII., and the isomer, melting at 175, as i -benzyluracil, XII. The mercaptopyrimidines, melting at 77 and 139, are therefore to be assigned the struc- tural formulas IX. and X., viz., i-benzyl-2-ethylmercapto-6- oxypyrimidine and 2-ethylmercapto-3-benzyl-6-oxypyrimidine, since they are converted into i -benzyluracil, XII., and 3- benzyluracil, XIII., respectively, by hydrolysis with acids. 2-Ethylmercapto-5-brom-6-oxypyrimidine 2 reacted with ben- zyl chloride, giving an excellent yield of 2-ethylmercapto-3- benzyl-5-brom-6-oxypyrimidine, XI. This was converted 1 Johnson and Heyl: Loc. ctt. 2 THIS JOURNAL, 31, 603. Researches on Pyrimidines. 447 quantitatively into 3-benzyl-5-bromuracil, XIV., by hydrolysis with hydrochloric acid. The same pyrimidine was also formed by the action of bromine on 3-benzyluracil, XIII. i-Methyl-3- benzyl-5-bromuracil, XVII., was formed by treatment of 3-benzyl-5-bromuracil, XIV., with methyl iodide, in presence of alkali, and also by the action of bromine on i-methyl-3- benzyluracil, XVI. 3-Benzyluracil, XIII., reacted with cold, fuming nitric acid, giving a dinitropyrimidine which we have provisionally repre- sented as 3-/>-nitrobenzyl-5-nitrouracil, XV. Johnson and Clapp 1 observed that 2-ethylmercapto-5- methyl-6-oxypyrimidine, XIX., reacted with methyl iodide, in presence of alkali, giving about equal proportions of 1,5- dimethyl-2-ethylmercapto-6-oxypyrimidine, XX., and 2-ethyl- mercapto-3,5-dimethyl-6-oxypyrimidine, XXI. We now find that benzyl chloride reacts with this mercaptopyrimidine, under the same conditions, giving i-benzyl-2-ethylmercapto-5-methyl- 6-oxypyrimidine, XXII., melting at 72, and 2-ethylmercapto- 3-benzyl-5-methyl-6-oxypyrimidine, XXIV., melting at 121- 122. These pyrimidines were converted quantitatively into i-benzylthymine, XXIII., melting at 205, and 3-benzyl- thymine, XXVI., melting at i59-i6o, by hydrolysis with con- centrated hydrochloric acid. The structures of these four pyrimidines was determined by the behavior of the two benzylthymines towards diazobenzene- sulphonic acid. Johnson and Clapp 2 have shown in a recent paper that this reagent can be used to distinguish between i- and 3-alkyl derivatives of uracil and thymine. They ob- served, for example, that i-alkyl derivatives of these pyrimi- dines reacted with this reagent, giving red colored solutions. The isomeric 3-alkylpyrimidines, on the other hand, did not react with the diazo acid with the formation of red colors. We now find that the benzylthymine, melting at 205, reacts with the sulphonic acid, giving a brilliant red solution, indicating that it is a i -benzyl thymine as represented by formula XXIII. The isomer gave no color with the sulphonic acid and therefore * Loc. tit. 2 J. Biol. Chem. f Vol. 5. 448 Johnson and Derby, Jr. p pq W 00=0 W o W W =0 __ O W W 00=0 p 00= g-8-i Researches on Pyrimidines. 449 a_| :;:;; 8-3=3, -M, B .. IB lias = ^~& 2 w w" w 1 * W h n rl o o PI r? " M " ' ;- : ' - 3 111 S 5 8-l-g. ?J=s d 3-ts, M S Q g_ =i R i-8-g H -8-i* 3 w w W ci q cJ 5 -: 5- 1 \ 1 HI I W* cThj C C 3-S=S ; ; ! 8- M M a x/ :z: o o W o V W -0=^0 o o o 8 i g J i i > ^, ^ = ^ ^,"" g, z K 1 { 3 nT o W o -^ -nitrobenzyloxynitrohydrothymine. The four pyrimidines, i-benzyluracil, 3-benzyluracil, i- benzylthymine, and 3-benzylthymine, are characterized by their insolubility in cold water. 3~Benzyluracil and 3-benzyl- thymine are less soluble in water than uracil and thymine. They all crystallize from hot water without water of crystal- lization. They are weak acids and can be heated above their melting points without decomposition. EXPERIMENTAL, PART. C 6 H 5 CH 2 N CO i-Benzyl-2-ethylmercapto-6-oxypyrimidine, C 2 H 5 SC CH. N CH Fifteen grams of 2-ethylmercapto-6-oxypyrimidine and 5.4 grams of finely pulverized potassium hydroxide were dissolved in about 75 cc. of boiling absolute alcohol. Twelve and two- tenths grams of benzyl chloride were then added and the solu- tion heated on the steam bath until it gave no alkaline reaction with moist turmeric paper (about 4 hours). After cooling, the undissolved potassium chloride was filtered off and the alcohol removed by evaporation on the steam bath. We ob- tained a crystalline product which was triturated with 50 cc. of a 5 per cent solution of sodium hydroxide to remove un- 1 Johnson: THIS JOURNAL, 40, 19; J. Biol. Chem., 4, 407. Researches on Pyrimidines. 451 altered pyrimidine and potassium chloride. When the alkaline solution was acidified with acetic acid we recovered 1.7 grams of 2-ethylmercapto-6-oxypyrimidine. The crystalline material, insoluble in sodium hydroxide, was then extracted thoroughly with an excess of ether and the insoluble material saved (see 3-benzyl derivative below). When the ether solution was allowed to evaporate we obtained some oil and about 3.0 grams of crystalline material which melted at 65-7o to a clear oil with no effervescence. The oil gradually assumed a crystalline form on standing. The pyrimidine separated from alcohol in well-developed prisms melting at 77. The weight of one crystal, which was selected for analysis, was 0.3+ gram. Analysis (Kjeldahl) : Calculated for CisHuONaS. Found. N 11.38 11.63 2-Ethylmercapto-3-benzyl-6-oxy pyrimidine, if "l C 2 H 5 SC CH. The weight of the crude 3-benzyl deriv- C C H 5 CH 2 .N - CH ative, insoluble in ether, in the above experiment, was 1 1 . i grams, corresponding to 5 1 per cent of the calculated. The pyrimidine is soluble in warm benzene, acetone, alcohol, very soluble in chloroform, and practically insoluble in cold ether and ligroin. It is difficultly soluble in hot water and crystallizes in long, slender prisms, and hexagonal prisms or flat tables melting at 139 to an oil. This melting point was not raised by recrys- tallization from benzene. Analysis (Kjeldahl) : Calculated for Found. I. II. N 11-38 11.58 ii. 61 2-Ethylmercapto- 3-benzyl- 5~brom-6-oxy pyrimidine, N --- CO II I C 2 H 5 S.C CBr. This compound was obtained when 5.0 i ii C,H 6 CH 2 N- 452 Johnson and Derby, Jr, grams of 2-ethylmercapto-5-brom-6-oxypyrimidine 1 were heated in absolute alcohol, for 4 to 5 hours, with the required propor- tions of potassium hydroxide and benzyl chloride. After filtration from the undissolved potassium chloride and evapo- ration of the alcohol we obtained a semisolid substance which immediately solidified when washed with ether. The yield was 4.2 grams, corresponding to 60.0 per cent of the calculated. The pyrimidine is difficultly soluble in water and separates from a hot, aqueous solution in needles melting at 129 to a clear oil. Analysis (Kjeldahl) : Calculated for Ci3Hi 8 ON 2 BrS. Found. N 8.62 9.0 C 8 H 5 CH 2 .N CO i-Benzyluracil, CO CH. A quantitative yield NH CH of this pyrimidine was obtained when 3.0 grams of the correspond- ing 2-mercaptopyrimidine were dissolved in 150 cc. of con- centrated hydrochloric acid and the solution evaporated to dryness. It crystallizes from hot water in prismatic crystals melting at 175 to a clear oil. It did not contain water of crystallization. When mixed with the isomeric 3-benzyluracil (see below) melting at 173, the fusion point was lowered to I40-i55. Thepyrimidine reacted with diazobenzenesulphonic acid, in presence of alkali, giving a red colored solution. Anal- ysis (Kjeldahl) : Calculated for CuHi O 2 N 2 . Found. N 13.86 14.1 NH CO I I 3-Benzyluracil, CO CH. This pyrimidine was I II C 6 H 5 CH 2 .N CH prepared by digesting 6.7 grams of 2-ethylmercapto-3-benzyl-6- oxypyrimidine with concentrated hydrochloric acid for 5 to 6 1 Wheeler and Johnson: THIS JOURNAL, 31, 603. Researches on Pyrimidines. 453 hours. The yield was 5.5 grams or 96 per cent of the theoretical. It crystallizes from alcohol in stout prisms melting at 173 to a clear oil with no effervescence. It is difficultly soluble in water and moderately soluble in cold acetone ; insoluble in cold benzene and ether; and difficultly soluble in chloroform. It was insoluble in cold 25 per cent hydrochloric and sulphuric acids and easily soluble in warm glacial acetic acid. Analysis (Kjeldahl) : Calculated for Pound. C u Hio0 2 N 2 . I. H. N 13-86 13.9 14.0 This pyrimidine does not react with diazobenzenesulphonic acid to give a red colored solution. The mixture assumed a yellow color, which was permanent for 30 minutes. ^Solubility of j-Benzyluracil in Water. One hundred parts of water dissolved at 25 i. n. 0.0998 gram. 0.1008 gram. NH CO I I 3-Benzyl-5-bromuracil, CO CBr. This pyrimi- I II C 6 H 5 CH 2 .N CH dine can be prepared by treating 3-benzyluracil in glacial acetic acid with the calculated quantity of bromine. It crystallizes from acetic acid in hexagonal prisms melting at 204 to a clear oil. The same brompyrimidine was also prepared by dissolving 0.5 gram of 3-benzyluracil in bromine water and allowing the solution to evaporate to dryness. The residue, about 0.8-0.9 gram, was then digested with absolute alcohol for 2 hours and the solution evaporated to dryness. The compound obtained by this treatment crystallized from alcohol in prisms melting at 204. A quantitative yield of the pyrimidine was also obtained by digesting 2-ethylmercapto-3-benzyl-5-brom- 6-oxypyrimidine with concentrated hydrochloric acid. Anal- ysis (Kjeldahl) : Calculated for Found. CiiH 9 OaN 2 Br. N 9.96 10.0 454 Johnson and Derby, Jr. NH - CO I I 3-p-Nitrobenzyl-5-nitrouracil, CO CNO 2 . NO 2 .C C H 4 CH 2 .N -- CH Five-tenths of a gram of 3-benzyluracil was dissolved, at ordinary temperature, in 10 cc. of fuming nitric acid of density 1.5. The acid solution was then allowed to stand at ordinary temperature when prismatic crystals separated. They were purified for analysis by recrystallization from hot water. The pyrimidine is difficultly soluble and separates in slender prisms, which decompose, according to the rate of heating, at 2^^-240 with effervescence . Analysis (Kjeldahl) : Calculated for Found. CnH 8 6 N 4 . CnH 9 4 N 8 . I. II. N 19.18 17.00 19-48 19-39 CH 3 N - CO I I i-M ethyl- 3-benzyluracil, CO CH. This pyrimi- C 8 H 5 .CH 2 N - CH dine is easily prepared by dissolving 3-benzyluracil and one molecular proportion of potassium hydroxide in alcohol and then warming with an excess of methyl iodide. The same pyrimidine was also obtained when i-methyluracil 1 was digested in alcohol with the calculated proportions of potassium hy- droxide and benzyl chloride. The pyrimidine is extremely soluble in alcohol and benzene. It crystallizes from dilute alcohol in needles melting at 75 to an oil without effervescence. Analysis (Kjeldahl): Calculated for Found. N 12.96 i-M ethyl- 3-benzyl-5-bromuracil, C 8 H 5 CH 2 N pyrimidine was prepared in two ways: 1 Johnson and Heyl: Loc. cit. Researches on Pyrimidines. 455 1. By dissolving i-methyl-3-benzyluracil in glacial acetic acid and then adding the calculated amount of bromine. They reacted with evolution of heat. The pyrimidine was very soluble in glacial acetic acid but was precipitated by dilu- ting with water. 2. By warming 0.8 gram of 3-benzyl-5-bromuracil in methyl alcohol with 1.9 grams of methyl iodide and o.i gram of me- tallic sodium. The alkylation was very smooth and the solu- tion was neutral in a few minutes. The pyrimidine crystallizes from alcohol in diamond-shaped prisms melting at 123 to a clear oil with no effervescence. Analysis (Kjeldahl) : Calculated for CiaHjiOjNaBr, Found. N 9.50 9.8 2-Ethylmercapto-i-benzyl-5-methyl-6-oxypyrimidine, C 8 H 5 CH 2 N CO C 2 H 5 SC CCH 3 . Fifteen grams of 2-ethylmercapto-5- II II N CH methyl-6-oxypyrimidine and 4.9 grams of finely pulverized potassium hydroxide were dissolved in 75 cc. of absolute alcohol. Eleven and two- tenths grams of benzyl chloride were then added and the solution heated on the steam bath for 6 hours. After filtering from undissolved potassium chloride and evaporation of the alcohol we obtained a crystal- line product which was washed with 50 cc. of a 5 per cent solution of sodium hydroxide. Four and four-tenths grams of unaltered pyrimidine deposited when the sodium hydroxide solution was acidified with acetic acid. The crystalline ma- terial, insoluble in sodium hydroxide, was then extracted in the usual manner with ether and the insoluble portion saved (see 3 -benzyl derivative below). From the ether washings we obtained about 3.0 grams of pyrimidine melting at 65-66. The compound is very soluble in acetone, benzene, and ether. It separates from dilute alcohol in stout prisms melting at 70 to a clear oil without effervescence. Analysis (Kjeldahl) : .'t Calculated for Ci 4 Hi 6 ON 2 S. Found. N 10.77 J o-9 456 Johnson and Derby, Jr. 2-Ethylmercapto-3-benzyl-$-methyl- 6 -oxy pyrimidine, N CO H 3 . The crystalline material, which was I II C 6 H 5 CH 2 N CH insoluble in ether, in the above experiment represented this pyrimidine and weighed 11.2 grams, corresponding to 68 per cent of the calculated. The pyrimidine is extremely soluble in alcohol and acetone. It was purified for analysis by re- crystallization from benzene. It separated in hexagonal plates melting at 121- 122. Analysis '(Kjeldahl) : Calculated for I. Ci 4 H 16 ON 2 S. Found. N 10.77 ii. i C 6 H 5 CH 2 N CO i-Benzyltkymine, CO CCH 3 . One and eight- NH CH tenths grams of i-benzyl-2-ethylmercapto-5-methyl-6-oxypyr- imidine were digested with concentrated hydrochloric acid until the evolution of ethyl mercaptan ceased. When this solution was evaporated to dryness the benzylthymine was obtained as a crystalline compound. The pyrimidine separated from hot alcohol in clusters of radiating prisms which melted at 204- 205 to a clear oil. The pyrimidine is difficultly soluble in water. Analysis (Kjeldahl) : Calculated for CuHi 2 O 2 N 2 . Found. . N 12.96 13.3 This compound reacted with diazobenzenesulphonic acid, in presence of sodium hydroxide, giving a beautiful, claret red solution. NH CO I I 3-Benzylthymine, CO CCH 3 . A quantitative I II CH 5 CH 2 N CH yield of this pyrimidine was obtained when the corresponding Researches on Pyrimidines. 457 2-mercaptopyrimidine was digested with concentrated hy- drochloric acid until the evolution of ethyl mercaptan ceased. The pyrimidine is easily soluble in alcohol, acetone, benzene, and difficultly soluble in ether. It is very soluble in glacial acetic acid and chloroform and difficultly soluble in cold 25 per cent hydrochloric and sulphuric acids. It separates from hot water in diamond-shaped prisms melting at 160 to a clear oil with no effervescence. Analysis (Kjeldahl) : Calculated for Found. N 12.96 13.2 This pyrimidine did not react with diazobenzenesulphonic acid to give a red color. The mixture assumed a yellow color which remained permanent for over one hour. Solubility of 3-Benzylthymine in Water. One hundred parts of water dissolved at 25 i. 0.1172 gram. i-Methyl-j-benzylthymine, CO CCH 3 . From 3- C 6 H 5 CH 2 N CH benzylthymine and methyl iodide. It crystallized from water in prismatic crystals melting at 101 to a clear oil. It did not react with diazobenzenesulphonic acid to give a red color. Analysis (Kjeldahl) : Calculated for C^HuOaNa. Found. N 12.17 12.07 3-p-Nitrobenzyloxynitrohydrothymine, NH CO I I CH 3 CO C< .Five-tenths of a gram of | I NO, NO 2 C e H 4 CH 2 N CHOH 3 -benzylthymine was dissolved in 10 cc. of cold fuming nitric acid of density 1.5. There was only slight evolution of heat 458 Johnson and Derby , Jr. and the pyrimidine dissolved to a clear, yellow solution. On standing exposed to the atmosphere well-developed prisms separated. They decomposed at 176 with violent efferves- cence. A nitrogen determination indicated that a nitro group had entered the benzene ring and that nitric acid had added to the double bond in the 4,5 positions of the pyrimidine ring. Analysis (Kjeldahl) : Calculated for Ci 2 H l2 7 N4. Ci 8 H 1 30 5 N3. C 12 Hn0 4 N3? Found. N 17.28 14.33 16.0 17.11 NBW HAVBN, CONN., June, 1908. -i [Reprinted from The American Chemical Journal, Vol. XI,. No. 6. December, 1908.] [Contributions from the Sheffield Laboratory of Yale University.] CLXIIL RESEARCHES ON PYRIMIDINES: SYNTHESES OF NEW DERIVATIVES OF 5-HYDROXY- URACIL (ISOBARBITURIC ACID). ( [THIRTY-NINTH PAPER.] BY TREAT B. JOHNSON AND D. BREESE JONES. Johnson andClapp 1 have shown that i- and 3 -alky 1 derivatives of 2,6-dioxytetrahydropyrimidines, I., can be distinguished by the difference in their behavior towards diazobenzene- sulphonic acid. The i-alkylpyrimidines, II., react with the sulphonic acid, in presence of alkali, giving red colored solutions, while the isomeric 3-alkylpyrimidines, III., do not give red colors under the same conditions. This interesting behavior of i-alkylpyrimidines enabled Johnson and Derby 2 to de- termine the structures of the isomeric i- and 3-benzyl deriva- tives of uracil and thymine. NH CO AlkN CO NH CO II II CO C CO C I II I II NH C AlkN C II. III. We have also found this diazo reagent to be of service for determining the structures of some new i- and 3-alkyl deriva- tives of isobarbituric acid. The name isobarbituric signifies a compound isomeric with barbituric acid, IV., and having the constitution of a hexahydropyrimidine, as represented by formula V. The reactions of this pyrimidine, however, do not indicate a hexahydro compound but rather a tetrahydro- pyrimidine, as represented by formula VI., viz., 5-hydroxyuracil. NH CO NH CO NH- II II CO CH 2 CO CO CO C.OH NH CO NH CH 2 iv. v. J. Biol. Chem., 5, 163 (1908). THIS JOURNAL, 40, 444 (1908). Researches on Pyrimidines. 539 Formula VI. is supported by the facts that the pyrimidine does not give an oxime 1 and also that the hydrogen of the 5-hydroxyl group is replaceable by the acetyl radical 2 when the pyrimidine is warmed with acetic anhydride. Further- more, the pyrimidine and its ethyl ether 3 react with diazo- benzenesulphonic acid, giving red colors as intense as that obtained with thymine. 4 We shall describe in this paper the preparation and prop- erties of i-benzyl-5-hydroxyuracil (i-benzylisobarbituric acid), VII., 3-benzyl-5-hydroxyuracil (3-benzylisobarbituric acid), VIII., and also i-benzylisodialuric acid, IX. C 6 H 5 CH,N- CO -CO COH NH- CO NH CH C 6 H 6 CH 2 VII. VIII. -CO COH -CH C 6 H 5 CH 3 N CO CO COH I II NH COH IX. Johnson and McCollum 5 have shown that pseudoethylthio- urea condenses smoothly with the sodium salt of ethyl a-ethoxy- /?-oxyacrylate, giving 2-ethylmercapto-5-ethoxy-6-oxypyrimi- dine, X. This mercaptopyrimidine reacted with benzyl chloride, in presence of alkali, giving two isomeric pyrimidines, wz., i-benzyl-2-ethylmercapto-5-ethoxy-6-oxypyrimidine, XII., melting at i4O-i4i, and 2-ethylmercapto-3-benzyl-5-ethoxy- 6-oxypyrimidine, XIII., melting at 85-86. We did not observe the formation of 2-ethylmercapto-5-ethoxy-6-benzoxy- pyrimidine, XI. These two mercaptobenzylpyrimidines, XII. and XIII., were converted smoothly by hydrolysis with hy- drochloric and hydrobromic acids into i-benzyl-5-hydroxy- uracil, XVII., melting at 230, and 3-benzyl-5-hyroxyuracil, XIX., melting at 2OO-2io, respectively. We also succeeded in isolating the two intermediate products of the hydrolysis, viz., i-benzyl-5-ethoxyuracil, XIV., melting at 150, and 3-benzyl-5-ethoxyuracil, XVI., melting at i63-i64. 1 Behrend and Roosen: Ann. Chem. (Liebig), 251, 240. 2 Behrend and Roosen: Loc. cit. 3 Johnson and McCollum: J. Biol. Chem., 1, 437 ; THIS JOURNAL, 36, 154. 4 Johnson and Clapp: Loc. cit. 6 Loc. cit. 540 Johnson and Jones. HfT 5 o o o o = =S j 8-8=g . 8-1 PI 6 1 M ffl o w ^ N ffl . & e* " 3 s ^ * O N a E d > I af . ffl HrT ffl " a ffi a o o a - c f|~ 0=C |M' 1 =S 8-8=S 8-2- 1 > X 1 M IP II i-g_ = a o a o -** 55 co 2> a nT ffi o cT M a y\ * v / \ h-p a 9 sXg, 8-^, 8-=S, X ffi K* w ^ ^ j _ t7 ^ 5 O ^J ^ ^; o - Jz; o ^ cf rf g w S- & o" ^ Researches on Pyrimidines. 541 The structures of these pyrimidines were established in the following manner: The i-benzyl-5-hydroxyuracil, XVII., melt- ing at 230, and the corresponding ethyl ether, XIV., reacted with diazobenzenesulphonic acid in presence of sodium hy- droxide, giving beautiful red colors. The isomeric benzyl hy- droxyuracil, XIX., melting at 2OO-2io, and its ethyl ether, XVI., did not react with the sulphonic acid, with formation of red colors, indicating that these pyrimidines are 3-benzyl derivatives, as represented by the formulas above. The benzyl-5-hydroxyuracils and their ethyl ethers are difficultly soluble in water. The hydroxyuracils are weak acids and stable in the presence of hydrochloric acid below 100. i-Benzyl-5-hydroxyuracil, XVII., was converted prac- tically quantitatively into 5-hydroxyuracil (isobarbituric acid), XV., when heated with concentrated hydrochloric acid at I5o-i6o. i-Benzyl-5-hydroxyuracil, XVII., was converted quantita- tively into i -benzylisodialuric acid, XVIII., when it was suspended in cold water and then dissolved by adding a molec- ular proportion of bromine. The isodialuric acid crystal- lized from hot water without decomposition and gave a violet colored salt with barium hydroxide. EXPERIMENTAL PART. i-Benzyl-2-ethylmercapto-5-ethoxy-6-oxypyrimidine, C 6 H 5 CH 2 N - CO C COC 2 H 5 . 2 -Ethylmercapto-5-ethoxy-6-oxy- N -- CH pyrimidine 1 (34 grams) and one molecular proportion of potassium hydroxide (9.0 grams) were dissolved in 250 cc. of boiling, absolute alcohol. Twenty-one and five-tenths grams of benzyl chloride were then added and the solution boiled until it gave no alkaline reaction when tested with moist turmeric (about 2 to 2.5 hours). The undissolved potassium chloride was then separated by filtration and the excess of 1 Johnson and McCollum: Loc. cit. 542 Johnson and Jones. alcohol removed by evaporation on the steam bath. We obtained a syrup which completely solidified after trituration with about 30 to 50 cc. of a 10 per cent solution of sodium hydroxide. When the alkaline solution was acidified with acetic acid we recovered 3.8 grams of the unaltered 2-ethyl- mercaptopyrimidine. The crude benzylpyrimidines were then extracted 3 times with boiling ether and the ether solutions saved (see 3-benzyl derivative below). The i-benzylpyrimi- dine was insoluble in ether and melted without further purifi- cation at i34-i36 to an oil. It was purified for analysis by recrystallization from acetone or hot water. It separated in slender prisms melting at 140 141 to an oil without effervescence. The compound is very soluble in alcohol, benzene, and acetone. It crystallizes from water in anhydrous condition. Analysis (Kjeldahl) : Calculated for Found. C 15 H 18 O2N 2 S. I. II. N 9.65 9.81 9.80 2-Ethylmercapto-3-benzyl-5-ethoxy-6-oxypyrimidine, N CO II I CaHgSC COC,H 6 . When the ether washings from the C 6 H 5 CH 2 N CH above experiment were heated on the steam bath to remove the ether we obtained a syrup which partially solidified after standing for 3 to 4 days. The crystalline material was sepa- rated from a small amount of oil by suction filtration and re- crystallized from 95 per cent alcohol. It deposited, on cooling, in heavy, prismatic crystals which melted at 85-86 to a clear oil with no effervescence. The pyrimidine is very soluble in acetone, alcohol, and ether, but insoluble in cold water. Anal- ysis (Kjeldahl): Calculated for C l6 Hi 8 O 2 N8S. Found. N 9 . 65 9 . 30 The total yields, and relative proportions of the crude iso- meric benzylpyrimidines obtained in 4 experiments by alkyla- Researches on Pyrimidines. 543 tion of the mercaptopyrimidine, are given in the following table: Its j ^ !1 if wo a P|O o-o a fill o **{ ^.2 2 ^ rt Percentage y of i- and 3-b derivatives. 36.0 24.7 2.5 93-4 or or 59-3% 40-7% 23-5 17-5 3-8 94.0 or or 57-3% 42.7% 16.0 4.0 3-0 92-5 or or 80.0% 20.0% -n . c >>a A e-s asg '^ i$o 47-5 34-0 18.0 4 5-0 4-5 C 6 H 6 CH 2 N CO i~Benzyl-5-ethoxyuracil, CO COC 2 H 5 . Thispyrim- NH CH idine was always obtained mixed with i-benzyl-5-hydroxy- uracil (see below) by hydrolysis of i-benzyl-2-ethylmercapto- 5-ethoxy-6-oxypyrimidine with concentrated hydrochloric acid. In one experiment, 5 grams of the mercaptopyrimidine were dissolved in 150 cc. of concentrated hydrochloric acid and the solution boiled for 12 hours. When the hydrochloric acid was removed, by heating on the steam bath, we obtained a mixture of the ethoxypyrimidine and benzylhydroxyuracil melting at i4O-i8o. They were separated from each other by frac- tional crystallization from 95 per cent alcohol. This pyrim- idine was more soluble in alcohol than the hydroxyuracil and crystallized in characteristic hexagonal prisms melting at 150 to an oil. This melting point was not raised by recrystal- lization from water. The pyrimidine did not contain water of crystallization and, did not give a test for sulphur. When 544 Johnson and Jones. it was dissolved in bromine water, and barium hydroxide added to the solution, a purple precipitate was obtained. The pyrimidine reacted with diazobenzenesulphonic acid, in presence of sodium hydroxide, giving a brilliant, red colored 1 solution. The color slowly faded on standing. Analysis: 0.1160 gram of substance gave n.8 cc. N at 19 and 759 mm. Calculated for C 13 H 14 O3N2. Found. N 11.38 ii. 6 i-Benzyl-5-hydroxyuracil (i-Benzylisobarbituric Acid), C 6 H 5 CH 2 .N CO O C.OH. 2-Bthylmercapto-i-benzyl-5-ethoxy-6- I II NH CH oxypyrirnidine is not converted quantitatively into this pyrimi- dine by boiling with hydrochloric acid. In order to obtain a complete hydrolysis within a few hours we found it necessary to proceed under the following conditions: 36 grams of the mercaptopyrimidine were dissolved in about 500 cc. of con- centrated hydrochloric acid and the solution evaporated to dryness. We obtained a crystalline mixture of the interme- diate ethoxypyrimidine (see above) and the benzylhydroxyura- cil, which was then dissolved in 40 to 50 cc. of hydrobromic acid. After 3 to 4 hours' digestion with this acid the hydrolysis was complete. Too long digestion with hydrobromic acid is not advisable because the benzylhydroxyuracil slowly undergoes decomposition with formation of benzyl bromide and 5-hy- droxyuracil. After removal of the hydrobromic acid the pyrim- idine was purified by several recrystallizations from alcohol and water. The yield was good. The pyrimidine is less soluble in alcohol than the corresponding ethoxy derivative (see above) . It crystallizes from alcohol in clusters of radiating prisms melting at 230 to an oil with effervescence. The pyrimidine separates from hot water in corpuscular crystals resembling in appearance the crystalline form of 5-hydroxyuracil (Analysis III.). A violet purple precipitate was obtained when the 1 Johnson and Clapp: LOG. cil. Researches on Pyrimidines. 545 pyrimidine was dissolved in bromine water and barium hy- droxide added to the solution. The pyrimidine reacts with diazobenzenesulphonic acid, in presence of sodium hydroxide, giving as intense a red color as i-benzyl-5-ethoxyuracil. Anal- yses: I. 0.0925 gram substance gave 10.2 cc. N at 19 and 756 mm. II. and III. Nitrogen by Kjeldahl method. Calculated for Found. CuH l0 3 N2. I. II. III. N 12.84 12. 60 12.95 ^Z- 5-Hydroxyuracil (Isobarbituric Acid) from i -Benzyl- 2-ethyl- mercapto-5~ethoxy-6-oxy pyrimidine. About 3.5 grams of the mercaptopyrimidine were dissolved in 50 cc. of concentrated hydrochloric acid and the solution heated for 3 hours at 150- 160. When the tube was opened there was no pressure, and oil floated on the surface of the solution. This was separated and identified as a mixture of ethyl mercaptan and benzyl- chloride. When the acid solution was evaporated to dryness, we obtained a colorless substance which was difficultly soluble in water. It crystallized from hot water in hard, corpuscular crystals, which decomposed without melting at about 300. It did not contain sulphur and chlorine. When the compound was dissolved in bromine water and barium hydroxide added to the solution, a purple precipitate was obtained. A nitrogen determination agreed with the calculated value for 5-hydroxy- uracil. 1 0.1321 gram substance gave 25.4 cc. N at 22 and 750 mm. Calculated for C 4 H 4 O3N 2 . Found. N 21.87 21.48 NH CO I I S-Benzyl-j-ethoxyuracil, CO COC^H,.. This pyrim- C 8 H 5 CH 2 .N CH idine was formed smoothly when 2-ethylmercapto- 3-benzyl-5-ethoxy-6-oxypyrimidine was digested with con- 1 Behrend and Roosen: Loc. cit. 546 Johnson and Jones. centrated hydrochloric acid. It appeared to be more stable, in presence of hydrochloric acid, than the isomeric i-benzyl- pyrimidine. The compound is difficultly soluble in cold water but separates from a hot solution in needlelike prisms. It is very soluble in alcohol and separates from this solvent in the form of plates. It melts at i63-i64 to a clear oil. When the pyrimidine is dissolved in bromine water and barium hydroxide is added to the solution a violet precipitate is ob- tained. The pyrimidine did not react with diazobenzene- sulphonic acid to give a red colored solution. The solution as- sumed a yellow color which was permanent for 20 minutes. Analysis (Kjeldahl): Calculated for Found. C 13 H 14 08N 2 . I. II. N 11.38 11.23 11.40 3-Benzyl-5-hydroxyuracil (j-Benzylisobarbituric Acid), NH CO CO COH. This compound was obtained when C 6 H B CH 2 .N CH 2-ethylmercapto-3-benzyl-5-ethoxy-6-oxypyrimidine was di- gested for several hours with hydrobromic acid. The pyrimi- dine crystallized from acetic acid in irregular crystals which melted from 200 to 210 according to the rate of heating. It was difficultly soluble in water and alcohol. Analysis (Kjeldahl) : Calculated for CuH l0 O3N2. Found. N 12.84 12.63 C 6 H 5 CH 2 N CO I'Benzylisodialuric Acid, CO COH. Five and I II NH COH eight-tenths grams of finely pulverized i-benzyl-5-hydroxyur- acil were suspended in 50 cc. of ice water, and 4.5 grams of bromine slowly added to the solution with constant stirring. The bromine immediately disappeared and the pyrimidine Researches on Pyrimidines. 547 completely dissolved. The excess of water was then removed by distillation under diminished pressure at a temperature of 50 to 70. The isodialuric acid finally separated from the warm, saturated solution in beautiful, prismatic crystals. The solution was then cooled and the crystals separated by filtration. The yield of crude material was 6.1 grams. The pyrimidine is very soluble in hot water and separates, on cool- ing, in lenticular crystals melting at 139 with effervescence. The pyrimidine dissolved in a solution of barium hydroxide with formation of a violet colored solution. When heated at 105 the pyrimidine slowly underwent decomposition. Analysis : 0.0979 gram substance gave 10 cc. of N.at 20 and 768 mm. Calculated for . Found. N 11.96 n. Si NEW HAVEN, CONN., June. 1908. [Reprinted from the American Chemical Journal, Vol, XL. No. 6. December, 1908.] [Contributions from the Sheffield Laboratory of Yale University.] CLXIV. RESEARCHES ON PYRIMIDINES: THE THIO DERIVATIVES OF URACIL AND THE PREP- ARATION OF URACIL IN QUANTITY. [FORTIETH PAPER.] BY HENRY L. WHEELER AND LEONARD M. LIDDLE. The impression is given in several text-books that the prep- aration of uracil, thymine, and cytosine by our synthetic methods 1 is no easier than the isolation of these substances from nucleic acids. Our syntheses are even given in Beil- stein 2 as a process of formation (Bildung) and not as the best method of preparation (Darstellung) . This view of our work is far from correct. In the past few years we have prepared a number of kilograms of these pyrimidines by our methods. The preparation of this amount of these substances from the nucleic acids would be a task for a factory. 1 Wheeler, Merriam, and Johnson: THIS JOURNAL, 29, 478 and 492 (1903). 2 Erganzungsbande, IV., 550, 551, 1162. 548 Wheeler and Liddle. For the preparation of uracil the sodium salt of formyl- acetic ester, NaOCH=CHCO 2 C 2 H 5 , 1 was condensed, in alkaline, aqueous solution, with a pseudothiourea, H 2 N C (SC 2 H 5 )NH. The condensation product, 2-ethylmercapto-6- oxypyrimidine, on boiling with hydrochloric acid, easily gave off mercaptan, and a quantitative yield of uracil was obtained. In this paper we describe a method of preparing uracil that is an improvement even over our previous synthesis. It has been our experience that thiourea gives uniformly larger yields of pyrimidine derivative than ethylpseudothiourea 2 when condensed with the sodium salt of formyl acetic ester. The condensation with thiourea takes place with the forma- tion of 2-thiouracil, I., 8 as follows: HNH C 2 H 5 OCO HN CO C 2 H 5 OH SC + CH = SC CH + I II I I! HNH NaOCH HN CH NaQp This condensation has the advantage over our previous method of avoiding the presence of mercaptan and since 2-thiouracil is more than ten times less soluble than 2-ethyl- mercapto-6-oxypyrimidine it separates more nearly com- pletely on acidifying. It remained then to discover a simple method of replacing the sulphur in this substance by oxygen. 2-Thiouracil, like other compounds of this class, is not desulphurized readily, if at all, by the ordinary methods. It forms more or less stable salts with the metallic oxides. List had a similar ex- 1 Wislicenus: Ber. d. chem. Ges., 20, 2931 (1887). 2 We have repeated our earlier attempts to condense the sodium salt of ethyl formylacetate with urea in aqueous solution, and thus obtain uracil directly, but with- out success. We have found that pyrimidine condensations which fail when water is used as the solvent may be brought about in alcoholic solution, in the presence of sodium ethylate. When the sodium salt of ethyl formylacetate, urea, and sodium ethylate were warmed for an hour on the steam bath and then tested for uracil with bromine water and barium hydroxide (J. Biol. Chem., 3, 183 (1907)) no color was obtained. No precipitate was formed on acidifying the concentrated solution and no evidence of the formation of uracil was observed. 3 Wheeler and Bristol: THIS JOURNAL, 33, 458 (1905). Researches on Pyrimidines. 549 perience with 2-thio-4-methyluracil. 1 To desulphurize this substance it was necessary to heat in a closed tube with hydro- chloric acid for a few hours at I5o-i6o. The thiohydan- toins evidently also belong to this class. 2 After a number of experiments we found that when 2-thio- uracil was simply boiled with an aqueous solution of chlor- acetic acid, in slight excess, sulphur was easily removed, and on evaporation uracil remained. The yield was practically quantitative. The reaction probably involves the inter- mediate formation of the unstable 6-oxypyrimidine-2-thio- gly collie acid, II., which, as we show below, on boiling with water gives uracil, III., and thioglycollic acid or its decom- position products, as follows: HN CO HN CO II II HOCOCH 2 SC CH 4- H 2 O = OC CH + HOCOCH 2 SH. II I II N CH HN CH ;-^- II. III. This reaction, which introduces nothing which can not be easily removed, is so smooth and satisfactory that we intend to investigate the use of chloracetic acid for desulphurization in other cases. At present, we have examined simply the new thio derivatives of uracil in this respect and have made the curious observation that while 2-thiouracil and 6-thiouracil, VII., are readily desulphurized, 2 ,6-dithiouracil, VIII., is not desulphurized on boiling with chloracetic acid but apparently forms the stable pyrimidine, 2, 6-dithiogly collie acid, IX. The preparation of the new thio derivatives of uracil was carried , out as follows: 2-Ethylmercapto-6-chlorpyrimidine, V., from 2-ethylmercapto-6-oxypyrimidine, 3 IV., was treated with potassium hydrosulphide, giving 2-ethylmercapto-6- thiopyrimidine, VI. When this compound was treated with concentrated hydro- chloric acid on the steam bath mercaptan was evolved and 1 Ann. Chem. (Liebig), 236, 1 (1886). 2 Bailey and Randolph: Ber. d. chem. Ges., 41, 2495 (1908). 3 Wheeler and Johnson: THIS JOURNAL, 29, 496 (1903). 550 Wheeler and Liddle. 6-thiouracil, VII., was formed. On heating with dry hydro- gen chloride, ethyl chloride was given off and 2,6-dithiouracil, VIII., was obtained. The latter was also prepared from 2,6-dichlorpyrimidine, X., and potassium hydrosulphide. HN CO N=CC1 HN CS II II II C 2 H 6 SC CH -> C^SC CH -> C 2 H 6 SC CH II II II II II II N CH N CH N CH IV. V. VI. / j N=CSCH 2 CO 2 H HN CS HN CS II II II HCO 2 CH 2 SC CH <- SC CH OC CH II II I II I II N CH HN CH HN CH IX. VIII. VII. t N=CC1 C1C CH II II N CH x. EXPERIMENTAL PART. HN CO 2-ThiouracU, SC CH. The sodium salt of formylacetic HN CH ester, prepared according to Wislicenus' 1 directions from 150 grams of ethyl formate, 150 grams of ethyl acetate, and 42 grams of sodium clippings in 500-600 cc. of dry ether, was dissolved in a cold, saturated, aqueous solution of 70 grams of thiourea. The solution was allowed to stand for an hour or so, then warmed on the steam bath, and finally cooled and acidified with acetic acid. Four such experiments gave about 280 grams of 2-thiouracil. 1 Ber. d. chem. Ges.. 20, 2933 (1887). Researches on Pyrimidines. 551 A portion of this material was crystallized from water until it was obtained in colorless bunches of prismatic plates which melted with decomposition at about 340. A solubility deter- mination was then made as follows : The substance was placed in cold water and a rapid stream of air was drawn by suction, first through a wash bottle containing distilled water, and then through the solution containing the suspended substance. After two hours the undissolved material was filtered off and a weighed portion of the solution was evaporated to dryness and the residue was heated to constant weight at 110. To check the results a parallel series of determinations were made in which the substance was dissolved in hot water and the saturated solution was allowed to cool while air was drawn through for two hours as before. Solubility determinations were also made in this manner in the case of 2-ethylmercapto- 6-oxypyrimidine and 2-methylmercapto-6-oxypyrimidine. It was found that 100 parts of water at 17 dissolved: Hot saturated Cold saturated solution. solution. 2-Thiouracil 0.0533 gram 0.0598 gram 2-Methylmercapto-6-oxypyrimidine 0.6170 " 0.6620 " 2-Ethylmercapto-6-oxypyrimidine 0.8000 " 0.7930 " The mercaptopyrimidines are therefore more than ten times as soluble in water as 2-thiouracil. 2-Thiouracil is al- most insoluble in alcohol while the mercapto derivatives are very soluble. In our pyrimidine condensations alcohol is al- ways present as a by-product, unless removed by evaporation; this reduces the yield in the case of the mercapto derivatives while in the case of thio derivatives just the opposite is true. In accordance with its thioamide character, 2-thiouracil is a much stronger acid than uracil. It readily dissolves in sodium or potassium hydroxides and it forms very soluble, crystalline, anhydrous salts with these bases. It can be very conveniently crystallized from aqueous ammonia in which it is far more soluble than in water. It forms an ammonium salt which loses ammonia on drying or on boiling with water. Mercuric oxide removes 2-thiouracil from solution, forming an amor- phous, anhydrous, insoluble salt. 552 Wheeler and Liddle. The Copper Salt, C 4 H 2 ON 2 SCu.H 2 O. Copper sulphate pro- duces in aqueous solutions of 2-thiouracil, with or without alkali, an amorphous, mustard- yellow, insoluble precipitate. This salt can be boiled with water without alteration. It was dried in a desiccator over calcium chloride for analysis. 0.2997 gram substance lost 0.0276 gram on heating two hours at io5-ii5. Calculated for C 4 H2ON2SCu.H 2 O. Found. H 2 O 8 . 67 9 . 20 0.1167 gram substance gave 0.045 gram CuO. Calculated for C^HsONzSCu.HaO. Found. Cu 30.60 30.76 When silver nitrate is added to an aqueous solution of 2-thiouracil an amorphous, gelatinous, white precipitate is formed which does not blacken on boiling. HN CO Preparation of Uracil, OC CH. Twenty grams of 2-thio- HN CH uracil were boiled with one and a half molecular proportions of chloracetic acid in a large amount of water for one hour, then evaporated to dry ness on the steam bath. The slightly colored residue was decolorized by means of animal charcoal, and on concentrating the solution 14.2 grams of pure white uracil, free from sulphur, were obtained. The yield in this case was 81 per cent of the calculated. More could have been obtained from the mother liquor. In another experiment 30 grams of 2-thiouracil were boiled with 33 grams of chloracetic acid in about 700 cc. of water. When the material had dissolved the solution was allowed to evaporate to dry ness on the steam bath. The residue was warmed with alcohol, filtered, and washed with alcohol. It was then free from sulphur and weighed 23.5 grams. This is 89.5 per cent of the calculated. Uracil from 6-Thiouracil. An aqueous solution of 2.1 grams of 6-thiouracil was mixed with 3 grams of chloracetic acid and Researches on Pyrimidines. 553 the solution was then evaporated to dryness on the steam bath. When the residue was crystallized from water 1.2 grams of pure uracil separated. This is 66 per cent of the calculated. Ethyl 6-Oxypyrimidine-2-thioglycollate } HN CO I I C 2 H 5 CO 2 CH 2 SC CH.|H 2 O. Five grams of 2-thiouracil were II II N CH mixed with a solution of 0.9 gram of sodium in 30 cc. of alcohol and then 4.8 grams of ethyl chloracetate were added. The mixture was heated on the steam bath under a return con- denser for one hour. The solution was then filtered and allowed to evaporate under reduced pressure in a desiccator. The crystalline residue after three recrystallizations from water yielded long, thin, glistening plates. These gave a strong test for sulphur and melted to a clear oil at i54-i55. The sub- stance was easily soluble in hot water and hot alcohol and nearly insoluble in cold water. 0.2852 gram substance lost 0.0138 gram when heated at ioo-ii5 for two hours. Calculated for Found. C 8 Hio0 3 N2S.iH 2 0. I. II. III. H 2 4.03 .... .... 4.83 N 12.55 12.44 12.57 6-Oxypyrimidine-2-thioglycollic Acid, HN CO I I HOCOCH 2 SC CH.H 2 O. This acid was obtained from the II II N CH above ester by warming its aqueous solution with an excess of potassium hydroxide for a few minutes on the steam bath. The solution was then cooled and acidified with hydrochloric acid. A heavy, white precipitate formed which crystallized from water in bunches of fine prisms. This proved to be the hydrous form which separates slowly and melts at 178. If the solution is stirred or the beaker scratched the anhydrous form generally results. The latter separates rapidly in loose 554 Wheeler and Liddle. bunches of fine needles. The substance can be crystallized from alcohol. When it is boiled with water, thioglycollic acid separates and uracil is obtained. 0.2676 gram substance lost 0.0238 gram when heated two hours at io5-no. Calculated for Found. C 6 H 6 3 N 2 S.H 2 0. I. II. III. H 2 O 8 . 82 8 . 89 .... N 13.72 13.90 13.65 Some experiments were made with the object of preparing alkyl derivatives of 2-thiouracil. If a smooth alkylation on the sulphur would take place it would be the best method for the preparation of 2-alkylmercaptopyrimidines. 2-Ethylmercapto-6-oxypyrimidine. 1 The action of sodium ,ethylate and ethyl iodide on 2-thiouracil in alcoholic solution gave 2-ethylmercapto-6-oxypyrimidine, melting at 152 . When 30 grams or more of 2-thiouracil were employed the yields were much below the calculated. Unaltered 2-thiouracil was recovered when the substances were allowed to act in molecular proportions. It required repeated recrystalliza- tions from alcohol to remove this impurity, so that the yields were unsatisfactory. If an excess of iodide and alkali were used the yield of 2-ethylmercapto-6-oxypyrimidine was still further reduced, owing to the formation of higher alkylated products. It was found that benzyl chloride acted more smoothly. HN CO 2-Benzylmercapto-6-oxypyrimidine, C 6 H 5 CH 2 SC CH. Ten N CH grams of 2-thiouracil and 4.4 grams of potassium hydroxide were dissolved in the smallest possible quantity of water on the steam bath and mixed with 9.9 grams of benzyl chloride. Alcohol was then added until the oil went into solution. The reaction took place immediately and gave long, slender, color- less prisms weighing 11.7 grams, or 68 per cent of the calculated. 1 Wheeler and Merriam: THIS JOURNAL, 29, 478 (1903). Researches on Pyrimidines. 555 The same quantities of substances shaken repeatedly in a separatory funnel in aqueous solution without alcohol gave 12.4 grams, or 72 per cent of the calculated. The first method of preparation is smoother and is much easier, the product being more nearly pure. The substance is soluble in about 16 parts of hot alcohol and in 50 parts of cold. In water or ether it is practically insoluble. Recrystallization from alcohol gave slender, twinned prisms which melted to a clear oil at i92-i93. Calculated for C n H l0 ON2S. Found. N 12.88 12.88 When the substance was warmed with hydrochloric acid on the steam bath, benzyl mercaptan separated. On evaporating and extracting with alcohol, uracil was obtained. The Sodium Salt of 2-Benzylmercapto-6-oxypyrimidine t C 11 H 9 ON 2 SNa.3H 2 O. Themercapto derivative was dissolved in an aqueous solution of sodium hydroxide and allowed to crystal- lize slowly in a desiccator. Large talclike plates formed which were very soluble in water and fairly soluble in alcohol. 0.3069 gram of substance lost 0.056 gram when heated at ioo-iio for two hours. Calculated for Found. C u H 9 ON2SNa.3H 2 0. I. II. III. H 2 O 18.35 18.24 .... N 9.52 9.69 9.43 HN CS I I 2-Ethylmercapto-6-thiopyrimidine, C 2 H 5 SC CH. Eight N CH grams of 2-ethylmercapto-6-chlorpyrimidine 1 were treated with two and a half times the calculated quantity of potassium hydrosulphide in alcoholic solution. The solution was boiled for about 15 minutes and then evaporated to dryness on the steam bath and the residue taken up in a small quantity of * Wheeler and Johnson: THIS JOURNAL, 29, 496 (1903); 31, 596 (1904). 556 Wheeler and Liddle. water. The solution was shaken with ether, to remove any 6-ethoxy or unaltered chlor compound, and then made dis- tinctly acid in the cold with acetic acid. A white flocculent precipitate separated, weighing 6.4 grams, or 80 per cent of the calculated. The substance was recrystallized twice from water, when it gave brilliant needlelike prisms, having a pale yellow color, which melted to a yellow oil at 149. The mate- rial was easily soluble in alcohol and moderately in hot water. In cold water it was nearly insoluble. The properties of the compound were very similar to those of 2-ethylmercapto-6- oxypyrimidine, but a mixture of these substances melted at about 126. Calculated for CeH 8 N2S2. Found. N 16.27 16.05 HN - CS I I 6-Thwuracil, OC CH. Two grams of 2-ethylmercapto-6- I II HN - CH thiopyrimidine were evaporated to dryness on the steam bath with 10 cc. of concentrated hydrochloric acid. Mercaptan escaped. The residue was extracted with cold alcohol to re- move unaltered ethylmercaptothiopyrimidine and the in- soluble material was then found to weigh 0.8 gram, or 55 per cent of the calculated. In another experiment more prolonged action of acid gave a yield of 90.0 per cent; a small amount of uracil was also formed. Like uracil and 2-thiouracil, 6-thiouracil is practically in- soluble in strong alcohol but moderately soluble in hot water. It crystallized from hot water in small, light yellow needles. On rapidly heating it blackened above 270 and melted with effervescence at 328. The properties of this substance are closely similar to those of 2,6-dithiouracil but a mixture of these compounds melted at 295. Calculated for Found. N 21.87 21.66 Researches on Pyrimidines. 557 HN - CS I I 2,6-Dithiouracil, SC CH. A portion of 2 ,6-dichlorpyrimi- HN - CH dine 1 was warmed on the steam bath with an aqueous solu- tion containing somewhat more than four times the calculated quantity of potassium hydrosulphide. As soon as the oil disappeared, the hot solution was filtered from slight im- purities and acidified with hydrochloric acid. This gave a yellow flocculent precipitate which was recrystallized from water. It was found to be difficultly soluble and it gave a feltlike mass of fine, bright yellow needles, which turned dark and decomposed above 230 without showing a definite melt ing point. 0.3215 gram substance gave 1.0278 gram BaSO 4 . Calculated for Found. I. II. S 44-44 43-79 ---- N 19-44 I 9-46 2,6-Dithiouracil was also obtained when 2-ethylmercapto-6- thiopyrimidine was melted in an oil bath at 170 and treated with dry hydrogen chloride. The material quickly solidified in the hot bath. It was then removed and crystallized from water, whereupon the characteristic yellow needles of 2,6-di- thiouracil were obtained. It was also found that when 2-ethylmercapto-6-oxypyrimidine was treated in the same manner 2-thiouracil was formed. 2 Action of Chloracetic Acid on 2,6-Dithiouracil: Pyrimidine- N = CSCH 2 C0 2 H 2,6-dithioglycollic Acid, HCO 2 CH 2 SC CH .Two II II N - CH grams of 2,6-dithiouracil were dissolved in hot water and 5 grams of chloracetic acid were added. The solution was evaporated to dryness, the residue taken up in water and 1 J. Biol. Chem., 3, 287 (1907). 2 See also Wheeler and Liddle: J. Am. Chem. Soc., 30, 1157 (1908). 558 Wheeler and Liddle. evaporated again, the operation being repeated several times or until the yellow color disappeared. The residue was then moderately soluble in alcohol and very difficultly soluble in hot water. It was crystallized from 600 cc. of boiling water, and 2 grams of white powder separated. Boiling with water or even warming with concentrated hydrochloric acid did not desulphurize this substance. When heated it decomposed above 200. Calculated for C 8 H 8 04N2S2. Found. N 10.77 11.14 NEW HAVEN, CONN.. September, 1908. [Reprinted from The American Chemical Journal, Vol. XU. No. i. January, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXV. RESEARCHES ON PYRIMIDINES: ON THE FORMATION OF PURINE DERIVATIVES FROM 4-METHYLCYTOSINE. [FORTY-FIRST PAPER.] BY CARL O. JOHNS. It was found by Gabriel and Colman that 4-methyl-6- ammopyrimidine 1 and 2-chlor-4-methyl-6-aminopyrimidine 2 did not give the expected 5-nrtro compounds when nitrated, but instead yielded 6-nitramine derivatives. When 4-methylcytosine, I., 3 is nitrated in the presence of sulphuric acid, an almost quantitative yield of 4-methyl-5- nitrocytosine, II., is obtained. To determine whether the compound formed was a 6-nitramine or a 5-nitropyrimidine, the nitration product was heated with 30 per cent sulphuric acid in a sealed tube. This treatment gave Behrend's ni- tromethyluracil, 4 III., in which the nitro group occupies position 5. This result agrees with previous observations made when pyrimidines containing an amino group in position 6 have been heated with sulphuric acid. When cytosine 5 was heated with 20 per cent sulphuric acid it gave uracil, and when nitro- cytosine 6 was heated under similar conditions, nitrouracil was formed. The reduction of 4-methyl-5-nitrocytosine to 2-oxy-4- methyl-5,6-diaminopyrimidine, IV., presented considerable difficulty. This was not unexpected since it was only after many unsuccessful attempts that 2-oxy-5,6-diaminopyrimi- dine 7 was obtained by the reduction of 5-nitrocytosine. An attempt to reduce 4-methyl-5-nitrocytosine with stan- nous chloride did not give the desired diaminopyrimidine, and when ammonium sulphide was used, 4-methylcytosine 1 Ber. d. chem. Ges., 34, 1240 (1901). 2 Ibid. t 34, 1241 (1901). 8 THIS JOURNAL, 40, 348 (1908). 4 Ann. Chem. (Liebig), 240, 3 (1887). * Wheeler and Johnson: THIS JOURNAL, 29, 494 (1903). 8 Johnson, Johns, and Heyl: Ibid., 36, 116 (1906). ' Ibid., 36, 170 (1906). Researches on Pyrimidines. 59 was regenerated. The reduction was finally accomplished by the use of aluminium amalgam. Even then prolonged re- duction or a temperature above 45 liberated ammonia and regenerated 4-methylcytosine. If the solution which was obtained from the reduction was evaporated on a water bath at 100 a black residue was obtained. The evaporation was therefore carried out under diminished pressure. When the solution had been concentrated to a small volume, stout, flat crystals of the 2-oxy-4-methyl-5,6-diaminopyrimidine sepa- rated. These contained one and one-half molecules of water of crystallization. Ttiey could be recrystallized from water without undergoing decomposition and then separated in long prisms that contained but one molecule of water. It is probable that the decomposition observed when the solution obtained from the reduction was evaporated was due to the presence of some unstable by-product. Gabriel and Colman condensed urea 1 and thiourea 2 with 4-methyl-5,6-diaminopyrimidine and obtained purine deriva- tives. The writer has found that 2-oxy-4-methyl-5,6-diaminopyrim- idine also condenses with urea and thiourea to give 2,8- dioxy-6-methylpurine, V., and 2-oxy-6-methyl-8-thiopurine, VI. Moreover, formic acid acts on 2-oxy-4-methyl-5,6- diaminopyrimidine and produces a monoformyl derivative. This compound gives a sodium salt which loses water, when heated, and yields 2-oxy-6-methylpurine, VII. These reactions with urea and formic acid are such as might be expected of an orthodiamine but not of a hydrazine deriva- tive, and they confirm the assumption that 4-methylcytosine nitrates in position 5. Bmil Fischer 3 has called attention to the fact that the in- troduction of oxygen into purine decreases its solubility in water. In accordance with this, 2-oxy-6-methylpurine is easily soluble in hot water while the same purine with oxygen or sulphur in position 8 is almost insoluble. If 2-oxy-5,6-diaminopyrimidine were heated with urea it 1 Ber. d. chem. Ges., 34, 1247 (1901). * Ibid., 34, 1248 (1901). t * Untersuchungen in der Puringruppe, p. 74. 6o Johns. might be expected to yield 2,8-dioxypurine. This compound is the only one of the three possible dioxypurines which has not been prepared and it has the structure which was at first assigned to xan thine. Later, Emil Fischer showed that xan- thine was 2,6-dioxypurine. 1 6,8-Dioxypurine has been pre- pared by Fischer and Ach. 2 The writer has begun work on the synthesis of 2,8-dioxypurine. N=CNH 2 I I OC CH I II NH C.CH 8 I. N: :CNH 2 I I OC CNO, I II NH C.CH 3 II. N CNH a I I OC CNH 2 NH -- C.CH 3 IV. NH CO I OC CNO, NH C.CH, III. NH C.CH 3 OC N C NH C NH V. NH C.CH 3 I II OC C NH ^>co NH N=C NH NH C.CH 3 I II OC N: VI. EXPERIMENTAL PART. VII. C NH >CH C NH N = I I 4-M ethyl- 5-nitrocytosine, OC CNO 2 . Four grams of 4- I II NH C.CH 3 methylcytosine were dissolved in 1 1 cc. of concentrated sul- phuric acid. Heat was liberated. While the solution was still 1 Ber. d. chem. Ges., 30, 553 (1897). 2 Ibid., 30, 2218 (1897). Researches on Pyrimidines. 61 warm n cc. of nitric acid, density 1.5, were added gradually. The first portions of nitric acid produced considerable heat but no red fumes were evolved. The resulting solution was allowed to stand at room temperature for 10 minutes and was then poured into 100 cc. of cold water. When the acids were neutralized with ammonium hydroxide, 4.8 grams of a yellow precipitate, which corresponds to 90 per cent of the calculated yield, separated. This compound was difficultly soluble in boiling water and crystallized in small pointed prisms. Its slight solubility in water showed that it was not 4-methyl- cytosine. It began to turn brown at 260 and became black at 280 but did not melt. It was soluble in acids and ammonia. Analysis (Kjeldahl) : Calculated for C 6 H 6 O 8 N 4 . Found. N 32-94 33-17 The position of the nitro group was determined as follows: One gram of the nitro compound was dissolved in 20 cc. of 30 per cent sulphuric acid and heated in a sealed tube at 130- 140 for two hours. A deposit of large crystals was obtained when the tube cooled. These were filtered off and the filtrate was analyzed for nitrogen in the form of ammonia by adding sodium hydroxide and distilling into N/io hydrochloric acid. N calculated for one NH 2 group, 0.08 gram. Found, 0.09 gram. The crystals which were filtered off dissolved readily in boiling water and separated from the cold solution in stout prisms and toothed structures. They possessed all the properties of Behrend's nitromethyluracil, in which the nitro group occupies position 5. Calculated for C 5 H 5 O4N 3 . Found. N 24.56 24.99 N=CNH 2 I I 2-Oxy-4-methyl-5,6-diaminopyrimidine, OC CNH 2 . Two NH C.CH S and two-tenths grams of finely pulverized 4-methyl-5 -nitro- 62 Johns. cytosine were suspended in 100 cc. of water and reduced vigorously with i gram of aluminium amalgam for one-half hour. The temperature was kept below 45. The aluminium used was in the form of fine drillings. The aluminium hy- droxide formed was filtered off by suction and washed with hot water. The filtrate was concentrated under diminished pressure to about 15 cc. -Crystals separated during the evap- oration. These were redissolved by careful heating and a slight insoluble residue was filtered off. Stout, flat prisms separated on cooling. These became brown above 250 and decomposed slowly at about 28o-285 without melting. They contained one and one-half molecules of water of crystal- lization. The yield of pure hydrous crystals was from 50 to 60 per cent of the calculated. Water determinations on crystals obtained from different experiments gave the following results: I. 1.6340 gram lost 0.2637 gram of H 2 O. II. 0.5011 gram lost 0.0805 gram of H 2 O. III. 1.1859 gram lost 0.1907 gram of H 2 O. Calculated for Found. C 6 H 8 ON 4 .iiH 2 O. I. II. III. H 2 O 16.16 16.14 16.06 16.08 Nitrogen determinations resulted as follows : Calculated for C 5 H 8 ON4.iiH 8 O. Pound. N 33-53 33.6i Calculated for C 6 H 8 ON 4 . Found. N 40.00 39-78 When the flat crystals obtained above were recrystallized from water they separated in long prisms that contained but one molecule of water of crystallization. The following analyses were made on different lots of the recrystallized diaminopyrimidine. Calculated for Found. C 5 H 8 ON 4 .HjO. I. II. III. H 2 O 11.39 n-53 JI -49 11.42 Calculated for C 6 H 8 ON 4 . Found. N 40 . oo 40 . 02 Researches on Pyrimidines. 63 2-Oxy-4-methyl-5,6-diaminopyrimidine was easily soluble in hot water but it dissolved only slightly in cold water. It did not dissolve in alcohol. The dried crystals were very hygroscopic. Water solutions gave precipitates with mercuric chloride and picric acid. The picrate charred above 240 but did not melt. The best yield was obtained by using fine aluminium drillings and reducing rapidly. If the reduction is prolonged or the temperature raised above 45, ammonia is liberated and 4-methylcytosine is re-formed. NH - C.CH 3 I II 2,8-Dioxy-6-methylpurine, OC C NH. One gram of an- I t > co N=C NH hydrous 2-oxy-4-methyl-5,6-diaminopyrimidine was intimately mixed with i gram of urea by grinding in a mortar. This mixture was heated in an oil bath kept at I7o-i8o for one- half hour. The mass melted and ammonia was evolved. The fusion product was dissolved in boiling water containing a few drops of ammonium hydroxide and the solution was decolorized with animal charcoal. When the filtrate was boiled to drive off ammonia, minute crystals separated. These were almost insoluble in boiling water but dissolved readily in mineral acids and bases. They were insoluble in alcohol or acetic acid. They did not melt at 345. The yield was i.i grams, or 93 per cent of the calculated weight. Calculated for Found. C 6 H 6 2 N4. I. II. N 33-73 33-59 33-8i NH -- C.CH 3 I II 2-Oxy-6-methyl-8-thiopurine, OC C NH. One-half gram N=C NH of 2-oxy-4-methyl-5,6-diaminopyrimidine was ground in a mortar with one gram of thiourea. The mixture was heated in an oil bath kept at i8o-i9o for one hour. It melted and 64 Johns. turned black. Ammonia was evolved. The fusion mass was dissolved in water containing ammonium hydroxide. A clear solution was obtained by using animal charcoal. This solution gave an immediate precipitate when acidified with acetic acid. The compound thus obtained was purified by dissolving in very dilute ammonium hydroxide and reprecipi- tating with acetic acid. Extremely small crystals were ob- tained. These were not soluble in alcohol and were almost insoluble in boiling water and boiling acetic acid. They were freely soluble in alkalies and mineral acids. They did not melt at 345. The yield of the pure purine was 80 per cent of the calculated quantity. Calculated for CeH 6 ON 4 S. Found. N 30.77 30.90 S 17.59 17-81 The Monoformyl Derivative of 2-Oxy-4-methyl-5,6-diami- nopyrimidine, C 5 H 7 ON 4 .CHO. Four grams of the diamino- pyrimidine were dissolved in 4 cc. of formic acid, density 1.2, and the solution was heated on a water bath for one-half hour. The excess of formic acid was evaporated in an open dish and the residue was dissolved in water. When this solution was neutralized with ammonium hydroxide, the monoformyl derivative precipitated. It was moderately soluble in hot water and formed minute crystals when the solution was cooled. It dissolved readily in acetic acid and was slightly soluble in boiling alcohol. The crystals began to turn black at about 325 but did not melt at 345. Calculated for C 6 H 8 2 N 4 Found. N 33-33 33-29 Prolonged heating in an oil bath at 240 caused the formyl derivative to darken considerably but it did not lose water to form a purine. When recrystallized from water the heated product exhibited all the properties of the formyl derivative. It was dried at 130 for analysis. Calculated for C 6 H 8 O a N 4 . Found. N 33-33 33-46 Researches on Pyrimidines. 65 NH C.CH 3 I II 2-Oxy-6-methylpurine, OC C NH. Three grams of the - '; - ]=J-N monoformyl derivative of 2-oxy-4-methyl-5,6-diaminopyrimi- dine were dissolved in an excess of 10 per cent solution of sodium hydroxide. When alcohol was added the sodium salt of the formylpyrimidine separated as a white powder. This salt was dried and heated in an oil bath at 200. Water came off readily and considerable foaming ensued, leaving a porous crust. This dissolved freely in cold water and did not give a precipitate when the solution was neutralized with acetic acid. The solution was therefore evaporated to dryness and the residue was dissolved in a little hot water. Clusters of slender prisms were obtained when the solution cooled slowly. These turned brown at about 280 and became black above 300 but did not melt at 345. They were easily soluble in acetic acid and ammonium hydroxide and sparingly soluble in boiling alcohol. A water solution gave a precipitate with mercuric chloride but did not give precipitates with ferric chloride, copper sulphate, or chloroplatinic acid. Calculated for Found. C<,H 6 ON 4 . I. II. III. N 37-33 37-05 37.10 37.35 NEW HAVEN, CONN., November, 1908. [Reprinted from the Journal of The American Chemical Society, Vol. XXXI. No. 5. May, 1909 ] [CONTRIBUTIONS FROM THE SHEFFIELD LABORATORY OF YALE UNIVERSITY.] RESEARCHES ON PYRIMIDINES: SYNTHESIS OF i-METHYL-5- HYDROXYURACIL. [FORTY-SECOND PAPER.] BY TREAT B. JOHNSON AND I). BREESE JONES Received March 19, 1909. In a previous paper from this laboratory, Johnson and McCollum 1 described a new synthesis of 5-hydroxyuracil (isobarbituric acid). They showed that this pyrimidine is formed quantitatively by hydrolysis of 2-ethylmercapto-5-ethoxy-6-oxypyrimidine with hydrochloric acid. In a recent paper, Johnson and Jones 2 have shown that nitrogen-alkyl derivatives of this mercaptopyrimidine can be obtained easily and also undergo hydrolysis, giving nitrogen-alkyl derivatives of 5-hydroxyuracil. They prepared by this method i- and 3-benzyl-5-hydroxyuracils, I., and II. C e H 5 CH 2 N CO NH CO i I II CO COH CO COH I II I II NH CH C 8 H 5 CH 2 N CH I. II. The object of the work described in this paper was to prepare the nitrogen-methyl derivatives of 2-ethylmercapto-5-ethoxy-6-oxypyrimidine 3 and study their behavior on hydrolysis. 5-Hydroxyuracil and nitrogen-alkyl derivatives of this pyrimidine are prepared, according to BehrendV synthesis, by the reduction of 5-nitrouracil and its alkyl derivatives with tin and hydrochloric acid. The only nitrogen-alkyl derivatives of 5-hydroxyuracil that have been synthesized by this method, are methyl-5-hydroxyuracil (methyliso- barbituric acid) and ethyl-5-hydroxyuracil (ethylisobarbituric acid). These pyrimidines are incorrectly represented in Beilstein's Handbuch 5 as i-alkyl pyrimidines III, and IV, and were prepared by Lehmann 8 by reducing methyl- and ethylnitrouracils, to which he assigned, without proof, formulas V, and VI. 1 J. Biol. Chem., i, 437. 2 Am. Chem. J., 40, 538. 3 Loc. cit. 4 Ann., 249, 39; Ibid., 251, 239. 8 Vol. I, 1347, 1348- 8 Ann., 253, 77. SYNTHESIS OF I-METHYIv-5-HYDROXYURACIL. 591 C H S N CO C 2 H 5 N CO CH 3 N CO C,H 6 N CO COH CO CNO 3 CO CNO, NH CH NH CH NH CH IV. V. VI. The structures of these alkyl nitrouracils, and consequently the corre- sponding 5-hydroxyuracils of Lehmann's were correctly established by Behrend and Thurm 1 who showed that they are to be represented as 3-alkyl-pyrimidines ; for example, 3,4-dimethyluracil (a-dimethylur- acil), VII, and nitric acid reacted giving the same nitromethyluracil, VIII, as originally was obtained by Lehmann 2 by methylation of nitro- uracil. Furthermore, the oxidation of Lehmann's methyl-5-hydroxy- uracil with bromine water gave a methylisodialuric acid, IX, which condensed with urea, giving 3-methyluric acid (d-acid) X. 3 Lehmann's NH CO NH CO NH CO NH CO CO CH CO CNO 2 CO COH CO CNH> II II I II II CCH 3 CH 3 N CH CH 3 N COH CH 3 N ( VII. VIII. IX. X. CO CNH V I II >co [N CNH/ ethylnitrouracil reacted with methyl iodide, giving the same ethylmethyl- pyrimidine as was obtained from i-methylnitrouracil and ethyl iodide. 4 Johnson and Jones 5 observed that i-benzyl-2-ethylmercapto-5-ethoxy- 6-oxypyrimidine is the chief product of the reaction when benzyl chloride acts on 2-ethylmercapto-5-ethoxy-6-oxypyrimidine, XI, in presence of alkali. We now find that methyl iodide reacts with this mercaptopyrim- idine, giving a mixture of the corresponding i and 3-methylpyrimi- dines which contains about 70 per cent, of the theoretical yield of i- methyl-2-ethylmercapto-5-ethoxy-6-oxypyrimidine, XII. 2-Ethylmercapto-3-methyl-5-ethoxy-6-oxypyrimidine, XVI, possessed the unique property of combining with potassium iodide, giving a definite, double compound. We suspected this substance, at first, of being an addition product of methyl iodide and the potassium salt of i- or 3- methyl-2-ethylmercapto-5-ethoxy-6-oxypyrimidines. This assumption, however, proved to be incorrect, since the same compound was formed quantitatively by crystallizing 2-ethylmercapto-3-methyl-5-ethoxy-6-oxy- pyrimidine from an alcoholic solution of potassium iodide. The analytical determinations agreed with the calculated values for a double compound Ann., 323, 160. Loc. cit. E. Fischer and Ach, Ber., 32, 2721. Loeben, Ann., 298, 181. Behrend and Dietrich, Ann., 309, 260. Behrend and Thurm, Loc. cit. Loc. cit. 592 ORGANIC AND BIOLOGICAL. containing 3 molecules of the 3-methylpyrimidine and 2 molecules of potassium iodide, XIII. 2-Bthylmercapto-i-methyl-5-ethoxy-6-oxypyrimidine, XII, is converted smoothly into i-methyl-5-ethoxyuracil, XV, by digestion with hydro-) chloric acid. When this pyrimidine or the mercaptopyrimidine are heated with strong hydrochloric acid at 120-130, they are changed practically quantitatively into i-methyl-5-hydroxyuracil, XVII. Hydrolysis of 2-ethylmercapto-i-methyl-5-ethoxy-6-oxypyrimidine with boiling hydro- bromic acid gave a mixture of i-methyl-2-thio-5-hydroxyuracil, XIV, and i-methyl-5-ethoxyuracil, XV. The formation of 2-thiopyrimidines from the corresponding mercapto derivatives by hydrolysis with acids has previously been observed by Johnson and Clapp; 1 for example, 2-ethylmercapto-3,5-dimethyl-6-oxypyrimidine gave 2-thio-3,5-dimethyl- 6-oxypyrimidine when digested with hydrobromic acid. The sulphur in 2-thio-i-methyl-5-hydroxy-6-oxypyrimidine, XIV, is very firmly bound. It cannot be removed by digestion with chloracetic acid. 2 The pyrimidine combines with this acid, giving i -me thy 1-2- thioglycollic-acid-5-hydroxy-6-oxypyrimidine which can be digested with concentrated hydrochloric acid without decomposition. When the double compound of 2-ethylmercapto-3-methyl-5-ethoxy-6-oxypyrimidine and potassium iodide, XIII, was digested with hydrochloric acid, it was converted into 2-thio-3-methyl-5-ethoxy-6-oxypyrimidine, XVIII. CH 3 N CO NH CO ~ N CO COC 2 H 5 C^SC C 2 H 5 SC COC 2 H 5 II II CH 3 N CH XIII. . 2 KI _ 3 CH 3 N CO CO CO( N CO II I C 2 H 5 SC I II CH 3 N CH J XVL NH CO I I CS COC 2 H & I II CH 3 N CH XVIII. 1 Jour. Biolog. Chem., 5, 57. 2 Wheeler and Liddle, Am. Chem. /., 40, 547. SYNTHESIS OF I-METHYV5-HYDROXYURACII,. 593 Summary. 1. Alkylation of 5-nitrouracil with methyl iodide gives chiefly 3-methyl- 5-nitrouracil. Reduction of this nitropyrimidine gives 3-methyl-5- aminouracil and j-methyl-j-hydroxyuracil, NH CO CO COH. I I! CH 3 N CH 2. Alkylation of 2-ethylmercapto-5-ethoxy-6-oxypyrimidine with methyl iodide gives chiefly i-methyl-2-ethylmercapto-5-ethoxy-6-oxy- pyrimidine. Hydrolysis of this mercaptopyrimidine with hydrochloric acid at 120-130 gives quantitatively i -methyl- j-hydroxyuracti, CH 3 N CO CO COH. NH \ -CH Experimental Part. The 2-ethylmercapto-5-ethoxy-6-oxypyrimidine that was used in this work was prepared from ethyl formate and ethyl ethylglycollate according to the directions of Johnson and McCollum. 1 This pyrimidine can be obtained easily in good yield by this method. The yields of pyrimidine obtained in five different condensations are given in the following table: if i I f . I I tj Grams of pyrimidine obtained, *g H NH CO w9> < I I aw a aw c 2 H 6 sc coc 2 H 6 . % 2o 2 23 II II 53 O CO N CH 1 113 75 79-5 42-0 48.8 2 102 65 71.8 35.0 45.1 3 109 70 76.7 44.5 53.6 4 76 50 53-5 27.5 47.5 5 76 50 53-5 32.0 55.3 CH 3 N CO I I i- Methyl-2-ethylmercapto-5-ethoxy-6-oxy pyrimidine, C 2 H 6 S.C COC 2 H 6 . Fifteen I! II N CH grams of 2-ethylmercapto-5-ethoxy-6-oxypyrimidine 1 and 4.5 grams (i mol.) of pul- verized potassium hydroxide were dissolved in 150 cc. of boiling, absolute alcohol. Fourteen grams of methyl iodide were then added and the mixture warmed, in a water- bath, until it gave no alkaline reaction, when tested with moistened turmeric paper (45 minutes). After cooling, the insoluble potassium iodide (u grams) was filtered 1 Loc. cit. 594 ORGANIC AND BIOLOGICAL. off and the excess of alcohol expelled by evaporation under diminished pressure. This method of procedure was adopted after we had observed, in a preliminary experiment, that the methyl pyrimidine volatilized with alcohol vapors by distillation at ordinary pressure. We obtained a crystalline substance which was extracted several times with an excess of ether and the insoluble part saved (see below). After evaporation of the ether we obtained u.o grams of the i-methylpyrimidine melting at 45-50. This yield corresponds to about 69 per cent, of the theoretical. In a second experiment 10.0 grams of 2-ethylmercapto-5-ethoxy-6-oxypyrimidine and 2.85 grams of potassium hydroxide were dissolved in 100 cc. of boiling, absolute alcohol and the solution cooled to 10. Ten and six- tenths grams of methyl iodide (1.5 mols.) were then added and the solution allowed to stand at 20-5. Potassium iodide began to separate in one-half an hour and after 3 hours the reaction was com- plete. The weight of undissolved potassium iodide was 6.25 grams. After evapora- tion of the alcohol at 40-50 at 47 mm. pressure the residue was then extracted, as usual, with an excess of ether. The yield of i-methylpyrimidine corresponded to about 71 per cent, of the theoretical. The product insoluble in ether was saved (see below). The 2 -ethylmercapto- i-methylpyrimidine is extremely soluble in ethyl alcohol, ben- zene and acetone. It crystallized from ether and water in plates melting at 50 to a clear oil. Analysis (Kjeldahl): Calculated for C9H 14 O 2 N 2 S: N 13.08; found, 13.4, 13.20. The Material Insoluble in Ether. This substance was extremely soluble in water and difficultly soluble in cold alcohol. It crystallized from a saturated, aqueous solution in characteristic needles arranged in the form of rosettes and from 95 per cent, alcohol in well-developed prisms. It decomposed, when heated slowly, at 177-8 into an oil and a crystalline substance, which did not melt below 250. The compound con- tained sulphur and iodine and left an inorganic residue when fused on platinum foil. When some of the compound was dissolved in cold water, and silver nitrate solution added, a gelatinous, white silver salt separated which decomposed, when the solution was warmed, giving yellow silver iodide. The crystalline habit and characteristic properties indicated that we were dealing with a definite compound and not a mixture of a pyrimidine and potassium iodide. Our analytical determinations agreed with the calculated values in a double compound containing 3 molecules of 2-ethylmercapto-^- methyl-5-ethoxy-6-oxypyrimidine and 2 molecules of potassium iodide, (C 9 H 14 O 2 N 2 S) 3 .2KI. Calculated: N, 8.60 ; I, 25.00 Found: N. 8.26, 8.35, 8.40; I, 24.55 N CO II I 2-Ethylmercapto-3-methyl-5-ethoxy-6-oxypyrimidine t C 2 H 5 SC COC 2 H 5 . Some of the I II CH 3 N CH double compound (above) was heated, in an oil bath, at 175-90 for about 30 minutes. We obtained a crystalline substance, on cooling, which was thoroughly pulverized and digested for a long time with an excess of anhydrous ethyl acetate. After expelling the excess of ethyl acetate at 100, we obtained a crystalline substance which was extremely soluble in cold water and alcohol. It crystallized from ethyl acetate in clusters of small prisms melting at 149-51. They were free from iodine but gave a strong test for sulphur. The compound slowly sublimed when heated at 100. A mixture of the pyrimidine and 2-ethylmercapto-5-ethoxy-6-oxypyrimidine 1 (melting 1 Loc. cit. SYNTHESIS OF I-METHYIv-5-HYDROXYURACIIv. 595 at 169) melted at 118-25. Analysis (Kjeldahl): Calculated for C^O^S, N 13.0; found, 13.4. Crystallization t o/ 2-Ethylmercapto-3-methyl-5-ethoxy-6-oxypyrimidine from an Alco- holic Solution of Potassium Iodide. About 0.5 gram of the pyrimidine was dissolved in 25 cc. of hot, absolute alcohol, which had previously been saturated with potassium iodide. On cooling, the double compound, described above, separated in colorless prisms which decomposed at 178. Analysis (Kjeldahl): Calculated for (C 6 H 14 O 2 N 2 S) 3 . 2KI, N 8.60; found, 8.5. CH 3 .N - CO I I i-Methyl-2-thio-5-hydroxyuracil, CS COH. Three and five-tenths grams of I I! NH CH i-methyl-2-ethylmercapto-5-ethoxy-6-oxypyrimidine were digested with 20 cc. of hydrobromic acid for about 14 hours. The solution was then evaporated to dryness, when we obtained a crystalline substance which dissolved in boiling water. Upon cooling, about 2.0 grams of slender prisms separated which were extremely insoluble in cold water and boiling alcohol. The material had no definite melting point and gave a strong test for sulphur. It crystallized from hot water without water of crystallization. Analysis (Kjeldahl): Calculated for C 5 H 6 O 2 N 2 S, N 17.7; found, 17-39, 17-5. CH 3 N - CO I I i-Methyl-2,6-dioxy-5-ethoxypyrimidine, CO COC 2 H 5 . After separation of the I II NH CH 2-thiopyrimidine, in the preceding experiment, the aqueous filtrate was evaporated to dryness. We obtained a crystalline substance which crystallized from hot water in aggregates of short prisms. They turned brown, when heated above 220, and then melted to an oil at about 240 according to the rate of heating. The compound did not contain sulphur, and reacted with diazobenzene sulphonic acid giving a strong, red color. Analysis (Kjeldahl): Calculated for C 7 H 10 O 3 N 2 , N 16.47; found, 16.7. This same pyrimidine is obtained smoothly and practically free from i-methyl- 2-thio-5-hydroxyuracil by digestion of i-methyl-2-ethylmercapto-5-ethoxy-6-oxy- pyrimidine with concentrated hydrochloric acid for several hours. CH 3 N - CO I I i-Methyl-5-hydroxyuracil, CO COH. This pyrimidine was formed practically I II NH CH quantitatively when i -methyl- 2, 6-dioxy-5-ethoxy pyrimidine or i-methyl-2-ethyl- mercapto-5-ethoxy-6-oxypyrimidine was heated with concentrated hydrochloric acid at 120-30 for 2-3 hours. The pyrimidine is more soluble in water than the isomeric 3-methyl-5-hydroxyuracil. 1 It dissolves readily in boiling water and alcohol and separates from hot aqueous solutions in clusters of radiating prisms which melt at 247 to an oil with slight effervescence. The isomeric pyrimidine decomposes above 260 without melting, with violent effervescence. A mixture of the i- and 3-methyl-5-hydroxyuracils melted at 217-30 to an oil. Analysis: Calculated for C 6 H 6 O 3 N 2 , N 19.71; found, 19.9. CH 3 N-CO I I i~Methyl-2-thioglycollic-acid-5-hydroxy-6-oxypyrimidine, HOOCCHjS.C COH. II II N CH 1 Loc. cit. 596 ORGANIC AND BIOLOGICAL. Two grams of chloracetic acid and i gram of i -methyl- 2 -thio-5-hydroxyuracil were dissolved in boiling water and the solution evaporated to dryness. We obtained a crystalline compound, which separated from hot water in stout prisms, and decom- posed at 217 with effervescence. It gave a strong test for sulphur and crystallized from water without water of crystallization. Analysis' (Kjeldahl): Calculated for C 7 H 8 O 4 N 2 S, N 1 2. 96; found, 12.8. This pyrimidine was extremely stable in presence of hydrochloric acid. Some of the compound was dissolved in a large excess of concentrated acid and the solution evaporated to dryness. The pyrimidine was recovered unaltered and decomposed sharply at 217. Analysis (Kjeldahl): Calculated for C 7 H 8 O 4 N 2 S, N 12.96; found, 12.94. NH-CO I I 2-Thio-3-methyl-5-ethoxy-6-oxy pyrimidine, CS COC 2 H 6 . This pyrimidine was I I! CH 3 N CH obtained, in small amount, when the double compound of potassium iodide and 2- ethylmercapto-3-methyl-5-ethoxy-6-oxypyrimidine was digested with hydrochloric acid. After expelling the hydrochloric acid, we obtained a crystalline substance which was washed with^alcohol and then crystallized from hot water. It separated in long needles^ which melted at 2101 to an oil, without effervescence. It con- tained sulphur and gave a violet colored solution by treatment with bromine water and barium hydroxide. Analysis (Kjeldahl): Calculated for C 7 Hi O 2 N 2 S, N 15.05; found, 15-05- NEW HAVEN, CONN. [Reprinted from The American Chemical Journa Vol. XLJI. No. i. July, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXVIL RESEARCHES ON PYRIMIDINES: THE PREPARATION OF 3-METHYL- AND 3- BENZYLURACIL. [FORTY-THIRD PAPER.] BY HENRY L. WHEELER AND TREAT B. JOHNSON. From a theoretical standpoint a larger number of alkyl com- pounds may result by alkylating a uracil derivative than can be obtained by a similar treatment of a 2-mercapto-6-oxy- pyrimidine. With the mercaptopyrimidines only three mono- derivatives should result. With uracil, 4-methyluracil, thy- mine, or 5-methyluracil, in each case there are ten possible compounds, four mono- and six dialkyl derivatives. This includes oxygen derivatives, but not tautomeric forms. In the case of the 2-amino-6-oxypyrimidines, the guanidine derivatives, the alkylation may be still more complicated. The work done in this laboratory has therefore been con- fined almost entirely to the alkylation of mercaptopyrimidines. 2-Ethylmercapto-6-oxypyrimidine 1 and 2-ethylmercapto-5-eth- oxy-6-oxypyrimidine 2 gave with potassium hydroxide and methyl iodide the i -methyl derivatives in largest quantity, while 2-ethyl-mercapto-5-methyl-6-oxypyrimidine 3 gave a mix- ture of about equal amounts of the i- and 3-methylated products. 2-Ethylmercapto-5-ethoxy-6-oxypyrimidine 4 with benzyl chloride also gave a i -benzyl derivative as the chief product. On the other hand 2-ethylmercapto-6-oxypyrimidine 5 and 2-ethylmercapto-5-methyl-6-oxypyrimidine gave mostly 3- alkyl compounds with benzyl chloride. A similar result was obtained with the former pyrimidine and ethyl chloracetate. 6 Besides the N isomers there were probably formed, in most of these cases, some 6-oxyalkyl derivatives (oils), the amount 1 Johnson and Heyl: THIS JOURNAL, 37 628 (1907). 2 Johnson and Jones: Ibid., 40, 538 (1908). 3 Johnson and Clapp: J. Biol. Chem., 5, 51 (1908). 4 Johnson and Jones: Loc. cit, 6 Johnson and Derby: THIS JOURNAL, 40, 444 (1908). 6 Wheeler and Liddle: J. Am. Chem. Soc., 30, 1152 (1908). Researches on Pyrimidines. 31 of 6-oxy derivative being noteworthy in the methylation of 2-anilino-6-oxypyrimidine. 1 These examples show that no regularity is ^>bserved in the alkylation of 2-mercapto-6-oxypyrirriidines, that analogous salts can give different results with the same halide, and that with different halides the salts may be attacked in different positions. It will also be noticed from the above that the papers so far published have contained accounts only of the results obtained with 2-mercapto-6-oxypyrimidines. It was therefore of interest to study the alkylation of some isomeric 2-oxy-6-mercaptopyrimidines and to examine the effect pro- duced when the mercapto group and the oxygen atom were interchanged. We now find when 2-oxy-6-methyl-mercaptopyrimidine (V.) is treated with potassium hydroxide, in alcoholic solution, and either methyl iodide or benzyl chloride, that 3-alkyl deriva- tives result. The amount of isomeric products, if any, formed in this reaction is very small. The 6-mercapto derivatives, like the 2-mercapto com- pounds, on boiling with hydrochloric acid evolve mercaptan and pass smoothly into uracil derivatives. The new 3-methyl- uracil (VII.) thus obtained now completes the list of the theoretically possible monomethyluracils. It gives no color with diazobenzenesulphonic acid although the isomeric i- methyluracil gives a red coloration. 2 2-Oxy-6-methylmercaptopyrimidine (V.) was prepared by methylating 6-thiouracil (IV.), while the preparation of the latter substance has been described by Wheeler and Liddle. 3 The various steps in the synthesis of 3-methyluracil may be represented as follows: 1 Johnson and Heyl: THIS JOURNAL, 38, 238 (1907). 2 Johnson and Clapp: J, Biol. Chem., 5, 163 (1908) 3 THIS JOURNAL, 40, 547 (1908). Wheeler and Johnson. HN CO N= =CC1 HN [ 5 SC C 2 H 5 SC CH > C 2 H 5 SC CH C 2 H oi CH CH 3 N CH VI. \ HN CH 3 N VII. 3-Methyluracil dissolves in alkali and is precipitated unaltered by hydrochloric acid. Miiller 1 obtained a 3-methyluracil-4-carboxylic ester by condensing oxalacetic ethyl ester and methylurea. When the sodium salt of this substance was treated with hydrochloric acid he obtained /?-methyluramidoacrylic acid, H 2 N CO N(CH 3 )CH = CHCOOH. Neither acids nor al- kalies changed this substance into 3-methyluracil. Similar peculiar relations have been observed before in the pyrimidine series. Fischer and Roeder 2 in their synthesis of uracil obtained an acid which differed from uracil by one molecular proportion of water. It was left undecided whether this substance was uracilic acid (Uracilsaure) , H 2 N CO NHCH = CHCOOH, or oxyhydrouracil, HN CO NH CH 2 CHOHCO. They did not suc- ceed in converting the substance into uracil. Johnson and Clapp 3 obtained two a-methyl-/9-guanidine- acrylic acids. One could be dissolved in dilute alkali and be 1 J. prakt. Chem., 56, 498 (1897). 2 Ber. d. chem. Ges., 34, 3760 (1901). 3 THIS JOURNAL, 32, 130 (1904). Researches on Pyrimidines. 33 reprecipitated by acids unaltered, with the other this treatment closed the ring and gave 2-amino-5-methyl-6-oxy- pyrimidine. The explanation given for the existence of these two acids was that they represented the cis and trans modifica- tions. Such an explanation might also account for the fact that like Mtiller's pyrimidines certain uracil derivatives dissolve in alkali and on precipitating with acids give the corresponding uramidoacrylic acids, while others such as our 3-methyl- uracil are precipitated in the form having the closed, uracil ring. EXPERIMENTAL PART. The preparation of 2-ethylmercapto-6-thiopyrimidine from 2-ethylmercapto-6-chlorpyrimidine 1 has been described by Wheeler and Liddle. 2 In the present work instead of employ- ing two and a half times the calculated quantity of potassium hydrosulphide we have taken four times the calculated amount. By this method a yield of over 90 per cent of the calculated quantity of 2-ethylmercapto-6-thiopyrimidine was obtained. HN CS 6-Thiouracil, OC CH .When 2-ethylmercapto-6-thio- I II HN CH pyrimidine is treated with concentrated hydrochloric acid the pale yellow color of the compound changes to a bright yellow. If water is added the color disappears or becomes much lighter. If this solution is boiled too long the material is completely desulphurized and uracil is obtained. The following procedure, however, gave a yield of over 90 per cent of the calculated amount of 6-thiouracil: Nine and six- tenths grams of 2-ethylmercapto-6-thiopyrimidine were added to a mixture of 50 cc. of concentrated hydrochloric acid and 50 cc. of water. On warming, mercaptan was evolved and solution took place, then 6-thiouracil separated. The acid was evapo- rated on the steam bath and the residue was crystallized from water. 1 Wheeler and Johnson: THIS JOURNAL, 29,496 (1903) ; 31, 596 (1904). 2 Ibid., 40, 555 (1908). 34 Wheeler and Johnson. N==CSCH 3 I I 2-Oxy-6-methylmercaptopyrimidine, OC CH . One and HN -- CH one- tenth grams of sodium were dissolved in 25 cc. of methyl alcohol and 6.1 grams of 6-thiouracil were added. The mixture became semisolid owing to the separation of a colorless, diffi- cultly soluble sodium salt. Ten grams of methyl iodide and 25 cc. more methyl alcohol were added. On warming the mixture, solution soon took place and it then gave a neutral reaction. The alcohol was evaporated and the residue was treated with water. Crystalline material separated which, after washing with water and drying, weighed 5 grams, or 73.7 per cent of the calculated. This dissolved easily in hot water and on cooling, long, slender needles or prisms were obtained melting to an oil with no effervescence at 205. Calculated for C 5 H 6 ON 2 S. Found. N I9-7 1 I 9-5 2-Oxy-3-methyl-6-methylmercaptopyrimidine, OC CH CH 3 N - CH Four and four- tenths grams of sodium were dissolved in 100 cc. of absolute alcohol and then 10.5 grams of 6-thiouracil were added. On warming a clear solution resulted. The solution was then cooled and 28 grams of methyl iodide were added. A mass of colorless crystals consisting of the sodium salt of 2-oxy-6-methylmercaptopyrimidine separated. On add- ing 100 cc. more of alcohol and warming for a few hours a clear solution that had a neutral reaction was again obtained. The alcohol was then evaporated off, the final portions in a vacuum. The product was separated from sodium iodide by repeated extractions with chloroform. When about 9.5 grams of ma- terial had been extracted it was purified by crystallizing from benzene. It separated in needles which melted at 124 to a clear oil without effervescence. Researches on Pyrimidines. 35 It was very soluble in cold water, alcohol, chloroform, and boiling ethyl acetate. Calculated for C 6 H 8 ON2S. Found. 17.95 I7-76 When the above mercapto- CH 3 N -- CH pyrimidine was boiled with strong hydrochloric acid mercaptan was readily given off and on evaporating the acid under di- minished pressure colorless prisms were obtained. The crude product melted at 22^-226 while i-methyl uracil melts at i74-i75. 1 The material was very soluble in water and boiling alcohol. It crystallized from absolute alcohol in rectangular prisms and from hot saturated, aqueous solutions in needles which melted at 232 to an oil without effervescence. It gave no red color with diazobenzenesulphonic acid in presence of sodium hydroxide. With bromine water and barium hy- droxide it gave a purple precipitate. 3-Methyluracil is less soluble in cold water than i-methyluracil. Calculated for Found. N 22.22 21.90 Some of the above material was dissolved in cold dilute sodium hydroxide and the solution carefully neutralized with dilute hydrochloric acid. The pyrimidine was recovered unaltered and melted at 232. 2-Oxy-3-benzyl-6-methylmercaptopyrimidine, N=CSCH 3 I I OC CH . Two grams of 2-oxy-6-methylmercapto- C 6 H 5 CH ? N -- CH pyrimidine and 1.8 grams of benzyl chloride were dissolved in about 30 cc. of alcohol containing one molecular proportion of potassium hydroxide (0.8 gram). There was an immediate 1 Johnson and Heyl: THIS JOURNAL; 37, 633 (1907). 36 Wheeler and Johnson. reaction when the mixture was heated and potassium chloride separated. The solution was warmed on the steam bath until it gave no alkaline reaction. The potassium chloride was then filtered off and the solution was cooled, whereupon the benzyl derivative separated in prisms melting at 148- 149 to an oil without effervescence. This melting point was not raised by recrystallizing from alcohol. The alcohol filtrates were evaporated to dryness on the steam bath and the residue was treated with dilute sodium hydroxide to remove any un- altered material. The insoluble portion melted without further purification at i46-i48 and was practically pure 3-benzyl derivative. The yield of this pyrimidine was 2.9 grams, or 90 per cent of the calculated. No evidence of the formation of an isomeric benzyl derivative was obtained. Ca Iculated for C 12 H 12 ON,S. Found. N 12. 06 12.19 When this mercaptobenzylpyrimidine was boiled with hydrochloric acid it gave a quantitative yield of 3-benzyl- uracil 1 melting at i72-i73. A mixture of this compound and a sample of 3-benzyluracil melted at i72-i73, while a mixture of the pyrimidine in question and i-benzyluracil (m. p. 175) melted at i4O-i45. Action of Methyl Iodide on 2-Ethylmercapto-6-oxy pyrimidine. We have reexamined the action of methyl iodide on the potassium salt of 2-ethylmercaptp-6-oxypyrimidine 2 and we now find that both i- and 3-methyl derivatives are formed. In the previous work only the i -methyl derivative was iso- lated. A mixture of 2-ethylmercapto-6-oxypyrimidine (15.2 grams), potassium hydroxide (5.6 grams), and methyl iodide (18 grams), in 75 cc. of alcohol was warmed on the steam bath until the solution gave a neutral reaction. It was then evap- orated to a syrup and a little water was added. This caused the separation of 3.6 grams of i-methyl-2-ethylmercapto-6- oxypyrimidine which was filtered off. The filtrate was boiled with hydrochloric acid and evaporated to dryness and the 1 Johnson and Derby: THIS JOURNAL, 40, 444 (1908). 2 Johnson and Heyl: Loc. cit. Researches on Pyrimidines. 37 residue was repeatedly extracted with boiling alcohol. The part insoluble in alcohol proved to be uracil. The alcoholic solution was decolorized with animal charcoal and some sulphur dioxide. The alcohol was evaporated and the residue was fractionally crystallized from water. We then obtained the 3-methyluracil melting at 232 and the i-methyl deriva- tive melting at 174. Owing to their similar solubility in alcohol, water, and other solvents it is impossible to make even an approximately quantitative separation by this treat- ment. It seems to us, however, that the i -methyl derivative is formed in largest quantity. Further work may show that these proportions vary with change of conditions. NEW HAVEN, CONN., April, 1909. [Reprinted from The American Chemical Journal Vol. XLII. No. 2. August, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXVIIL RESEARCHES ON PYRIMIDINES: THE PREPARATION OF i,4-DIMETHYLURACIL AND OF THE MONOBENZYL DERIVATIVES OF 4-METHYLUR ACIL. * [FORTY-FOURTH PAPER.] BY HENRY L. WHEELER AND DAVID F. MCFARLAND. Behrend 2 has shown, in his work on the methylation of 4-methyluracil with alkali and methyl iodide, that the three products i ,4-dimethyluracil, 3,4-dimethyluracil, and i,3,4-tri- methyluracil, are all formed and have to be dealt with in addi- tion to unaltered material. List 3 found that 2-thio-4-methyluracil (I.) is readily methyl- ated and that the alkyl group unites with the sulphur, producing 2-methylmercapto-4-methyl-6-oxypyrimidine (II.). List did not determine what was formed on further alkyla- tion. 1 Part of a Thesis presented by David F. McFarland for the Degree of Ph.D., Yale, 1909. 2 Behrend and Dietrich: Ann. Chem. (Liebig), 309, 265 (1899). Behrend and Meyer: Ber. d. chem. Ges., 33, 624 (1900). Behrend andThurn: Ann. Chem. (Liebig), 323, 160 (1902). 3 Ann. Chem. (Liebig), 236, 12 (1886). IO2 Wheeler and McFarland. In previous papers from this laboratory it has been shown that 2-mercapto-6-oxypyrimidines usually give a mixture of the i- and 3-alkyl derivatives on further alkylation. 1 We will now show that the potassium salt of List's 2-methyl- mercapto-4-methyl-6-oxypyrimidine, in alcoholic solution, al- kylates almost entirely in the i -position with methyl iodide and that i,4-dimethyl-2-methylmercapto-6-oxypyrimidine (III.) is formed. There are indications also, in this reaction, of the formation of 2-methylmercapto-4-methyl-6-methoxypyr- imidine in small amount. Since the alkylmercaptopyrimidines easily evolve mercaptan and form the corresponding uracil derivatives on boiling with hydrochloric acid, this method is the best (Darstellung) for the preparation of i ,4-dimethyluracil (IV.). By condensing acetoacetic ethyl ester with thiourea in alcoholic solution and in the presence of two molecular pro- portions of sodium ethylate, a quantitative yield of List's 2-thio-4-methyluracil (I.) may be obtained. The condensa- tion also takes place in alkaline aqueous solution but the yield is not as good. Even this process is far superior to the original methods. 2 The steps in the synthesis of i,4-dimethyluracil may be represented as follows : HN CO I I SC CH > I II HN CCH 3 CH 3 N CO I I OC CH Acetoacetic ethyl ester condenses with ethylpseudothiourea, 1 For a list of these cases see our forty-third paper on pyrimidines, THIS JOURNAL, 42, (1909). 2 Nencki and Sieber: J. prakt. Chem., [2] 25, 72 (1882). List: Loc. tit. Researches on Pyrimidines. 103 H 2 N C(SC 2 H 5 )NH, in alkaline aqueous solution more smoothly and gives a larger yield (over 80 per cent) of mer- captopyrimidine than in the case of the corresponding con- densations of the sodium salts of either ethyl formylacetate or ethyl formylpropionate. When 2-ethylmercapto-4-methyl-6-oxypyrimidine (V.), pre- pared in this manner, was treated with sodium hydroxide and benzyl chloride, a mixture of i-benzyl-2-ethylmercapto- 4-methyl-6-oxypyrimidine (VI.) and 2-ethylmercapto-4-methyl- 6-benzyloxypyrimidine (VII.) was obtained. This mixture, which was obtained as an oil insoluble in alkali, could not be separated by distillation under reduced pressure. When it was boiled with hydrochloric acid mercaptan escaped and a new benzyl-4-methyluracil, melting at 194, along with 4-methyluracil, was obtained. C C H 5 CH 2 N CO HN CO N=COCH 2 C 6 H 6 * C 2 H 5 SC CH -3 FX1IN V^V^Xlj CH 3 N VIII. IX. Hagen 1 described a benzyl-4-methyluracil melting at 233 which he obtained in very small amount the quantity being only sufficient for analysis by heating the dry potassium salt of 4-methyluracil at a high temperature with benzyl chloride. This substance is given in Beilstein's Handbuch? as i -benzyl-4-methyluracil (VIII.). We now find that this is incorrect and that the compound is 3-benzyl- 4-methyluracil (XIII.). The proof of the struc- ture of these compounds was obtained as follows: 1,4- Dime thyl- 1 Ann. Chem. (Liebig), 244, 9 (1888). 2 [3] 2, 529. IO4 Wheeler and McFarland. uracil (XI.) was treated with potassium hydroxide and benzyl chloride in alcoholic solution. This gave i ,4-dimethyl-3-benzyl- uracil (XII.), melting at 85-86. Hagen's benzylmethyl- uracil, which we find is easily obtained, along with the isomer melting at 194, when 4-methyluracil, potassium hydroxide and benzyl chloride are warmed in alcoholic solution, was then methylated; the same dimethylbenzyluracil melting at 85-86 was thereby obtained. CH,N CO CH 3 N CO HN CO II II OC CH < OC CH I II I II C 6 H 5 CH 2 N CCH 3 C 6 H 5 CH 2 N CCH 3 XII. XIII. When the new i-benzyl-4-methyluracil (VIII.) was methyl- ated it gave the isomeric i-benzyl-3,4-dimethyluracil (IX.), melting at 164. The possibility of an alkylation taking place in the 5 -position in the case of pyrimidines derived from acetoacetic ester had to be considered, at this point, before the above results could be taken as proof of the structures assigned to the benzyl compounds in question. Barbituric acid or 2,4,6-trioxy- pyrimidine alkylates under the same conditions in the 5-posi- tion. 1 From the analogy which exists between acetoacetic ester and malonic ester it might be expected that 4-methyluracil or 2-ethylmercapto-4-methyl-6-oxypyrimidine might react in the tautomeric forms represented by formulas (XIV.) and (XV.) and yield 5 -benzyl derivatives. N CO N CO II I II I HOC CH 2 C 2 H 5 SC CH 2 II II N^=<:CH 3 N=CCH 3 XIV. XV. In order to prepare 4-methyl-5-benzyluracil (XVIII.) it was necessary to condense benzylacetoacetic ester with a urea 1 Conrad and Gutzeit: Ber. d. chem. Ges., 14, 1643 (1881) ; 15, 2846 (1882). Fischer and Dilthey: Ann. Chem. (Liebig), 333, 338 (1904). Researches on Pyrimidines. 105 derivative. This ester proved to be one of those /?-ketone esters which failed to condense satisfactorily by the general method with the pseudothioureas in aqueous solution. We find, however, that it condenses in alcoholic solution with thiourea in the presence of sodium ethylate, giving the sodium salt of 2-thio-4-methyl-5-benzyluracil (XVI.). In order to desulphurize this it was found best to convert it into 2-ethyl mercapto-4-methyl-5-benzyl-6-oxypyrimidine (XVII.) which, en boiling with hydrochloric acid, easily gave the desired 4-methyl-5-benzyluracil (XVIII.) melting at 249-25o. HN CO HN CO HN II -> I I * I SC CCH 2 C 6 H 5 C 2 H 5 SC CCH 2 C 6 H 5 OC HN CCH 3 N CCH 3 HN XVI. XVII. XVIII. Since this compound, melting at 249-25O, is different from either of the above benzylmethyluracils it follows that in the alkylation of the pyrimidines derived from acetoacetic ester, under the conditions stated, the benzyl group does not enter the 5-position, and that the various benzyl compounds are correctly represented by the formulas given above. The isomeric benzyloxypyrimidines would not be stable when boiled with hydrochloric acid and are therefore excluded. This otherwise completes the list and settles the structure of the monobenzyl derivatives of 4-methyluracil. EXPERIMENTAL PART. i,4-Dimethyl-2-methyimercapto-6-oocypyrimidine, CH 3 N CO CH 3 SC CH. An almost quantitative yield of 2-thio-4- N CCH 3 methyluracil was usually obtained when 30 grams of aceto- acetic ester and two molecular quantities of sodium ethylate, made by dissolving 10.6 grams of sodium in 200 cc. of absolute alcohol, were warmed for half an hour with 17.5 grams of thio- io6 Wheeler and McFarland. urea. On evaporating the alcohol on the steam bath, which ap- pears to render the condensation more nearly complete, the sodium salt of 2-thio-4-methyluracil remained. This salt easily dissolved in water. On addition of acetic acid to the solu- tion, 2-thio-4-methyluracil was precipitated. The precipi- tate is apt to carry down with it some of the sodium salt, and it was found advisable to warm it, after filtering, with water acidified with acetic acid. Then on washing with water it is obtained pure and free from sodium. When 2-thio-4-methyluracil is treated with two molecular proportions of potassium hydroxide and methyl iodide a considerable quantity of 2-methylmercapto-4-methyl-6-oxy- pyrimidine is obtained. In order to produce a satisfactory alkylation on the nitrogen it is necessary to use a greater excess of alkali and methyl iodide. Potassium hydroxide gives better results in nitrogen alkylation than sodium hydr- oxide. Thirty grams of 2-thio-4-methyl-6-oxypyrimidine were dissolved with 35 grams of potassium hydroxide in 150 cc. of absolute alcohol. The potassium salt formed was much more soluble than the free pyrimidine. After cooling the solution, 94 grams of methyl iodide, an excess of 4 grams over that calculated for 3 molecular quantities, were gradually added and the mixture was allowed to stand two days. At the end of that time, about 6 hours' gentle heating on the steam bath was required to complete the reaction so that the solu- tion gave no alkaline test with moist turmeric paper. The alcohol was then evaporated, under diminished pressure, and the solid residue was treated with dilute sodium hydrox- ide. The undissolved portion was filtered and washed with a little water. Twenty-five and one- tenth grams of i,4-di- methyl-2-methylmercapto-6-oxypyrimidine melting at 8 5-9O were obtained. An additional 2.3 grams were recovered by shaking the alkaline mother liquor with ether, making the total yield of the compound 27.4 grams, or 76.4 per cent, of the calculated yield. From the alkaline solution 4.5 grams of 2-methylmercapto-4-methyl-6-oxypyrimidine, formed by incomplete methylation, were obtained on acidifying. Al- Researches on Pyrimidines. 107 lowing for this, the yield of i,4-dimethyl-2-methylmercapto- 6-oxypyrimidine becomes 91.2 per cent of the calculated. In another experiment where only two molecular propor- tions of alkali and of methyl iodide were used with 27 grams of 2-thio-4-methyluracil, the amount of intermediate 2-methyl- mercapto-4-methyl-6-oxypyrimidine was largely increased, 8 grams of it being formed, with a corresponding decrease of dimethylmercapto compound. This shows that a large excess of alkali and of methyl iodide is necessary in order to completely methylate 2-thiouracil. A small quantity of oily substance, with a pungent odor resembling that of parsnips, coated the material insoluble in alkali. It is probable that this oil is a 6-methoxy derivative but not enough was obtained to permit of its identification. When the material insoluble in alkali was crystallized from water it gave needles which melted sharply at 94 and the results on analysis agreed with those calculated for i,4-dimethyl- 2 - me thy Imercap to-6-oxy py rimidine . Calculated for C 7 H 10 ON2S. Found. N 16.22 16.47 The substance is readily soluble in alcohol and boiling water, crystallizing from aqueous solutions in silky needles. It is extremely soluble in ether, chloroform and benzene, and rather difficultly soluble in petroleum ether. It sublimes readily at the temperature of the steam bath, condensing again in fine silky needles. It is easily volatile with steam, and solutions containing it cannot be evaporated on the steam bath without loss. Action of Hydrochloric Acid: i,4-Dimethyluracil. The above i, 4-dimethyl-2-me thy Imercap topyrimidine was boiled for one hour with strong hydrochloric acid under a reflux condenser to prevent loss of the volatile compound. By this treatment it was quantitatively converted into Behrend's /?-dimethyl- uracil ( 1,4- dime thyluracil). One recrystallization from hot water gave material melting sharply to a clear oil at 260 - 261. io8 Wheeler and McFarland. No trace of Behrend's isomeric -dimethyluracil (3,4-di- methyluracil) melting at 220 was found in any of the mother liquors from the above. It is reasonably certain, therefore, that the corresponding 2-methylmercapto-3,4-dimethyl-6-oxy- pyrimidine was not formed in appreciable quantities. i-Benzyl-2-ethylmercapto-4-methyl-6-oxypyrimidine, C C H 5 CH 2 N CO C 2 H 5 SC CH. 2-Ethylmercapto-4-methyl-6-oxypyrimi- N CCH 3 dine was prepared by condensing acetoacetic ethyl ester with ethyl pseudothiourea in aqueous, alkaline solution; from 54.5 grams of acetoacetic ester a yield of 58.5 grams of crude mer- capto compound was obtained, or 81.5 per cent of the calcu- lated. Twenty grams of this compound were added to a solution of 2.95 grams of sodium in 60 cc. of alcohol. After complete solution had been effected 16.2 grams of benzyl chloride were added and the mixture was heated on the water bath until no further alkaline reaction could be obtained with moist turmeric paper. This required about four hours. The alcohol was then evaporated under diminished pressure and the residue treated with 20 cc. of water and 10 cc. of dilute sodium hydroxide. A heavy oil remained undissolved. This was shaken out with ether; the ether solution was washed with water to remove alcohol, dried over solid potassium hydroxide, and filtered into a weighed flask. After evaporating the ether, 20.7 grams of oil remained, or 68 per cent of the calcu- lated monobenzyl derivative. From the above alkaline solution 9 grams of unaltered substances and from the oil 6 grams of low-boiling material were obtained. Allowing for these weights, the yield of monobenzyl derivative is 87.5 per cent. The oil was distilled at 22 mm. pressure and after about six grams of low-boiling oil (benzyl chloride?) had distilled over, the temperature rose to 224 and a little over 10 grams of material distilled between this and 236. The greater part boiled near 227. The temperature rose slowly, toward Researches on Pyrimidines. 109 the last to 260, where the distillation was stopped. The fractions obtained were only slightly colored. None of them gave any solid even when cooled to 15. Analyses were made of two separate fractions, one (A) boiling at 227-228, and the other (B) at 228-26o. Both gave excellent re- sults for a monobenzyl derivative of 2-ethylmercapto-4-methyl- 6-oxypyrimidine. Calculated for Found. CuHi 6 ON 2 S. A. B. N 10.77 10.63 10.62 Action of Hydrochloric Acid: i- Benzyl- 4-methyluracil. Two grams of the above fraction B were boiled for two hours with 15 cc. of concentrated hydrochloric acid, during which time mercaptan was evolved. The solution was evaporated to dry ness, a solid residue weighing 1.3 grams, which nearly all dissolved in boiling water remaining. After filtering off the small amount of gummy residue which did not dissolve, a crystalline mass separated on cooling. It dissolved in boiling alcohol, and, on cooling gave colorless, rounded, lozenge- shaped crystals melting to a clear oil at 194. Analysis showed this to be a monobenzyl-4-methyluracil. Calculated for C 12 H ]2 O 2 No. Found. N 12.96 12.93 The compound dissolves easily in cold chloroform, moder- ately in hot alcohol, and rather difficultly in hot water. The crystals from alcohol are characteristic. By evaporation of the aqueous mother liquor from the above compound, material was obtained which crystallized in clus- ters of fine needles. It had no sharp melting point but decom- posed at 270-30O. Analyses of this material gave results which agreed with those calculated for 4-methyluracil. When it was mixed with pure 4-methyluracil, the decomposition point was not lowered. From the two grams of oil hydrolyzed there were obtained about 0.3 gram of i-benzyl-4-methyl- uracil and 0.6 gram of 4-methyluracil. This result indicates that the fraction of oil boiling at 228-26o consisted about no Wheeler and McFarland. one- third of i-benzyl-2-ethylmercapto-4-methyl-6-oxypyrim- idine and two-thirds of the isomeric 2-ethylmercapto-4-methyl- 6-benzyloxypyrimidine. A similar hydrolysis of fraction A of the oil yielded approx- imately equal proportions of 4-methyluracil and i-benzyl- 4-methyluracil, so that this fraction consisted of a corre- sponding mixture of the mercaptobenzyl compounds. Re- peated fractional crystallization of all the residues from this experiment failed to give any other substance besides the two described. No trace of the monobenzyl-4-methyluracil described by Hagen 1 was found, although a painstaking search was made for it. Action of Benzyl Chloride upon 4-Methyluracil: j-Benzyl- 4-methyluracil. The 4-methyluracil for this experiment was made by boiling 2-ethylmercapto-4-methyl-6-oxypyrimidine with strong hydrochloric acid until mercaptan ceased to be evolved. It was purified by crystallization from water. Five and a half grams of 4-methyluracil were heated with 2.44 grams of potassium hydroxide in 50 cc. of absolute alcohol. Complete solution did not take place until a further addition of 50 cc. of alcohol and 28 cc. of water had been made. Six grams of benzyl chloride, or one-half gram excess over the calculated, was then added and the mixture heated for three hours on the steam bath or until no alkaline reaction was ob- tained. On evaporating the alcohol, a partially solidified mass remained. Part of this dissolved when warmed with dilute sodium hydroxide. The insoluble part formed a resinous semisolid on cooling; this gum could not be induced to crys- tallize. From the alkaline solution acetic acid precipitated a mix- ture. This was boiled with about 50 cc. of alcohol. The portion which did not dissolve proved to be chiefly 4-methyl- uracil. On cooling the alcoholic solution, pearly, irridescent,, diamond-shaped plates separated which melted about 220 235, at least 30 higher than i-benzyl-4-methyluracil. Three crystallizations from alcohol raised the melting point to 233- 1 Loc. cil. Researches on Pyrimidines. 1 1 1 235. It therefore agreed with that given by Hagen for his benzyl-4-methyluracil. The compound is very difficultly soluble in hot alcohol and still more difficultly in boiling water. It is almost insoluble in cold chloroform. An analysis of the substance melting at 233-235 gave results agreeing with those calculated for monobenzyl-4-methyluracil. Calculated for Found. N 12.96 13.18 The alcoholic mother liquors from this were evaporated to dryness. The remaining material was then shaken with chloroform. The insoluble part proved to be almost pure 3-benzyl-4-methyluracil (Hagen's compound). The chloro- form solution was evaporated to dryness and the residue was crystallized from alcohol, whereupon the isomeric i -benzyl- 4-methyluracil was obtained. A second benzylation of 4-methyluracil, with 7 grams of the pyrimidine and two molecular proportions of potassium hydroxide (6.22 grams) and of benzyl chloride (14.5 grams) in 115 cc. of alcohol and 35 cc. of water, gave 3.6 grams of gum, insoluble in alkali, 2 grams of unaltered 4-methyluracil, and about 2 grams each of the isomeric nitrogen benzyl- 4-methyluracils. CH 3 N - CO I I i ,4-Dimethyl-3-benzyluracil, OC CH . Hagen's C 6 H 5 CH 2 N -- CCH 3 benzyl-4-methyluracil melting at 233 (1.6 grams) was dis- solved in 90 cc. of methyl alcohol with three molecular propor- tions of potassium hydroxide and warmed for three hours and a half with methyl iodide (3.25 grams). The alcohol was then evaporated and the residue was stirred with dilute sodium hydroxide. This left 1.8 grams of oil which on cooling solidified. It was dissolved in ether and the clear, filtered solution was allowed to evaporate slowly in a tall test tube. Beautiful, clear, transparent, prismatic tables were then obtained which melted sharply at 85-86. When these H2 Wheeler and McFarland. were mixed with i ,4-dimethyl-3-benzyluracil prepared by benzylating i,4-dimethyluracil the melting point was not lowered. The two preparations agreed in all respects and are identical. The preparation of this compound from i ,4-dimethyluracil was as follows: Two and eighty-five hundred ths grams of i,4-dimethyluracil and one molecular quantity of potassium hydroxide were dissolved in 120 cc. of 95 per cent alcohol. One molecular portion of benzyl chloride was added and the mixture was heated on the steam bath until a neutral reac- tion was obtained. A considerable amount of potassium chloride which separated was filtered off and the alcohol was evaporated, a partially solid mass remaining. A portion of this dissolved when treated with sodium hydroxide, leaving 2.1 grams of oil, which finally solidified. When crystallized from ether as described above it melted sharply at 85-86 and the analytical results were as follows: Calculated for Found. C 18 H 14 2 N 2 . I. II. N 12.17 12.41 12.22 This substance was found to be extremely soluble in alcohol and benzene, much less so in ether, and only slightly in petro- leum ether. C 6 H 5 CH 2 N CO i-Benzyl-3,4-dimethyluracil, OC CH This com- I II CH 3 N CCH 3 pound, isorneric with the above, was produced when 1.45 grams of i-benzyl-4-methyluracil melting at 194 was warmed for about four hours with molecular proportions of potassium hydroxide and methyl iodide in 50 cc. of 95 per cent alcohol. The potassium iodide which separated during the reaction was filtered off and the alcohol evaporated to dryness. The residue was treated with dilute sodium hydroxide, washed with water, and dried. It weighed 1.2 grams, or 76 per cent of the calculated. When crystallized from alcohol, beautiful, Researches on Pyrimidines. 113 long, colorless prisms melting at 164 to a clear oil, were obtained. An analysis gave results agreeing with the calculated for i-benzyl-3,4-dimethyl uracil. Calculated for Found. C 13 H U 2 N 2 . I. II. N 12.17 12.43 12.32 The alkaline solution above gave i,4-dimethyluracil when acidified with acetic acid, the alkylation not being complete. HN CO U, SC 2-Thio-4-melhyl- j-benzyluracil, SC CCH 2 C 6 H 5 . Benzvl- I II HN - CCH 3 acetoacetic ethyl ester was prepared by the action of benzyl chlo- ride upon the sodium salt of acetoacetic ester, according to the method of Bhrlich. 1 The portion boiling at i94-2O4 at 40 mm. pressure was used in the following work, part of which was done by Mr. McKay S. Howard : Ten grams of benzy lace toace tic ester and 4 grams of thiourea were added to a solution of sodium ethylate made by dissolving three grams of sodium in 75 cc. of absolute alcohol. The mix- ture was heated on the steam bath for four hours. The alco- hol was then evaporated and the residue was dissolved in water. On the addition of dilute acetic acid 2-thio-4-methyl- 5-benzyl-6-oxypyrimidine was precipitated. The yield was 7.4 grams, or 70 per cent of the calculated. The substance was very difficultly soluble in water but more easily in alcohol, from which it crystallized in colorless, leafy plates or scales. After two recrystallizations it melted to a clear oil at 257- 258. Calculated for Found. I. II. N 12.07 12.12 12.13 In another experiment, 20 grams of benzy lace toace tic ester gave 13.1 grams of the condensation product, or 62.4 per cent of the calculated. 1 Ann. Chem. (Liebig), 187, 12 (1875). H4 Wheeler and McFarland. 2-Ethylmercapto-4-methyl- ^-benzyl- 6-oxypyrimidine, HN CO I I C 2 H 5 SC CCH 2 C 6 H 5 . The above 2-thiouracil derivative was I! II N CCH 3 dissolved in alcohol containing one molecular proportion of sodium ethylate and then allowed to digest with a slight excess of ethyl bromide until the solution was neutral. The product, which was almost insoluble in water, and soluble only with difficulty in cold alcohol, formed fine, prismatic needles melt- ing sharply at 166. Calculated for Found. C W H 16 ON 2 S. I. II. N 10.77 ii. oo 10.56 HN CO 4-M ethyl- 5-benzyluracil, OC CCH 2 C 8 H 5 . Two grams of HN CCH 3 the above mercapto compound were allowed to digest for 24 hours on the steam bath with 50 cc. of concentrated hydro- chloric acid. Mercaptan escaped, and, on cooling, 1.6 grams of 4-methyl-5-benzyluracil, or 96 per cent of the calculated, separated. The substance was recrystallized from water and alcohol. It gave diamond- shaped plates melting to a clear oil at 249-25o. Calculated for Ci 2 H 12 O 2 N 2 . Found. N 12.96 13.06 2 - Benzylmercapto-4-methy I- 5 - benzyl- 6-oxypyrimidine. This was prepared by benzylating 2-thio-4-methyl-5-benzyluracil with molecular quantities of benzyl chloride and sodium ethylate in alcoholic solution. It is nearly insoluble in water. From alcohol it separates in matted masses of long, colorless needles and it melts to a clear oil at 194. Calculated for Found. C w Hi 8 ON 2 S. I. II. N 8.69 8.58 8.75 Researches on Pyrimidines. 115 This benzylmercapto compound proved to be very stable when boiled with hydrochloric acid. It was not desulphurized when given the same treatment as that in the case of the corre- sponding ethylmercapto derivative. When boiled with hydro- bromic acid it was finally desulphurized and 4-methyl-5-benzyl- uracil was obtained. In view of this behavior it is therefore not a matter of indifference what mercapto derivative is chosen for these desulphurizations. NEW HAVEN, CONN., May, 1909. [Reprinted from The American Chemical Journal, Vol. XLII. No. 3. September, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXIX. RESEARCHES ON PYRIMIDINES: SULPHUR DERIVATIVES OF 5-HYDROXYURACIL: PREPARATION OF 5-BENZYLMERCAPTOURACIL AND 5-BENZYLMERCAPTOCYTOSINE. [FORTY-FIFTH PAPER.] BY TREAT B. JOHNSON AND HERBERT H. GUEST. Several investigators have shown that the methylene hydro- gens of cyclic compounds which contain the S CH 2 CO 272 Johnson and Guest. grouping rhodanic acids 1 (I) and pseudothiohydantoins 2 (II) and (III) react with aldehydes, in presence of alkali, giving unsaturated condensation products (IV). It has also CH 2 S CH 2 S CH 2 S CO CS CO CO(NHR) CO CNR 2 \/ V V NR NR N I. II. III. RCH:C S HOOCCOCH S I I CO C:NC 8 H 5 NH V. been shown that the methylene group of arylpseudothiohydan- toins is capable of condensing with diethyl oxalate in presence of sodium ethylate. Wheeler and Jamison, 3 for example, prepared in this manner phenylpseudothiohydantoinglyoxylic acid (V), from stable phenylpseudothiohydantoin. Loven 4 examined the behavior of the methylene groups in thiodigly- collic acid towards benzaldehyde and obtained the sulphide of a-mercaptocinnamic acid, (C 6 H 5 CH : CCOOH) 2 S, but no attempts, so far as the writer is aware, 5 have been made to condense ethyl formate or diethyl oxalate with any acyclic compound containing the grouping S CH 2 CO . A search of the literature 6 reveals the fact that no a- or 1 Nencki: Ber. d. chem. Ges., 17,2278 (1884). Giesberg and Bondzynski: Ibid.. 19, 113 (1886). Bondzynski: Monats. Chem.. 8, 349 (1887). Zipser: Ibid., 23, 958; Centralb., 1903, I, 283. Andreasch and Zipser: Monats. Chem., 24, 499; 25, 159; 26, 1191; Chem. Ztg., 26, 54, 623. Stuchetz: Monats. Chem., 26, 1209. Andreasch: Ibid., 27, 1211. Wagner: Ibid., 27, 1223. Bargellini: Atti. Accad. Lincei, [5] 15, I, 35, 181; Centralb., 1906, I, 1436, 1438; Gazz. Chim. Ital., 36, II, 129. 2 Andreasch: Monats. Chem., 8, 407; 10, 73, 75; Ber. d. chem. Ges.. 31, 138; Centralb., 1899, II, 804. Wheeler and Jamieson: J. Am. Chem. Soc.. 25, 366 (1903). 3 Loc. cit. 4 Ber. d. chem. Ges., 18, 3243 (1885). 5 The writer has signified in a previous paper (Tins JOURNAL, 31, 290) his intention of condensing diethyl oxalate with diethyl thiodigly collate. 8 Beilstein's Handbuch, Vol. I. Researches on Pyrimidines. 273 ^-mercapto derivatives of acrylic acid (VI), corresponding to a-hydroxy- (VII), /?-hydroxy- (VIII), and a,/?-dihydroxy- acrylic acids (IX), have been described. CH 2 :CHCOOH. VI. ft. a. f. a. CH 2 : C(OH)COOH CH 2 : C(SR)COOH. VII. HOCH : CHCOOH RSCH : CHCOOH. VIII. HOCH : C(OH)COOH RSCH : C(SR)COOH. RSCH : C(OH)COOH HOCH : C(SR)COOH. X. It was, therefore, of especial interest to determine whether ethyl formate would condense with a thioether of ethyl thio- glycollate, HSCH 2 COOC 2 H 5 , giving an a-mercapto derivative of /?-hydroxy aery lie acid (X). We prepared, for our experiments, ethyl benzylthiogly- collate 1 and now find that ethyl formate condenses smoothly with this ester, in presence of metallic sodium, giving ethyl a-benzylmercapto-/?-hydroxy aery late (XIII). We have also condensed ethyl formate with ethyl benzoylthiogly collate, 2 C 6 H 5 COSCH 2 COOC 2 H 5 , under the same conditions, but have reserved the description of this work for publication in another paper. The sodium salt of ethyl a-benyzlmercapto-/?-hydroxy- acrylate (XI) condensed smoothly with pseudoethylthio- urea, in an aqueous solution, giving 2-ethylmercapto-5-benzyl- mercapto-6-oxypyrimidine (XII). This new condensation is represented by the following equation: 1 Gabriel: Ber. d. chem. Ges., 12, 1641. 2 Wheeler and Johnson: THIS JOURNAL, 26, 198. 274 Johnson and Guest. NH 2 COOC 2 H 5 C 2 H 5 S.C + CSCH 2 C 6 H 5 = II II NH NaOCH XI. NH C 2 H S SC II CSCH 2 C 8 H 5 + NaOH + C 2 H 5 OH. II II N- II CH XII. The study of the properties of this pyrimidine was of par- ticular interest because it is the first mercaptopyrimidine to be described which has the mercapto group linked to the 5-position of the pyrimidine ring. Our previous work in this laboratory has been confined to the study of 2- and 4- or 6-mercapto derivatives. The 5-mercapto group in 2- ethylmercapto-5-benzylmercapto-6-oxypyrimidine (XII) is very firmly bound and is not removed by hydrolysis with acids. When the pyrimidine was digested with concentrated hydro- chloric acid, it was converted quantitatively into 5-benzyl- mercaptouracil (XV). On the other hand, when heated above its melting point in a stream of dry hydrochloric acid gas, it was converted smoothly into 2-thio-5-benzylmercapto- 6-oxypyrimidine (XIV). 2-Ethylmercapto-5-benzylmercapto-6-oxypyrimidine reacted in a smooth manner with phosphorus oxychloride, giving 2-ethylmercapto-5-benzylmercapto-6-chlorpyrimidine (XVI) . When this chlorpyrimidine was heated with ammonia, a quantitative yield of 2-ethylmercapto-5-benzylmercapto-6- aminopyrimidine (XVIII) was obtained. This compound was then converted quantitatively into 5-behzylmercapto- cytosine (XVII) by hydrolysis with hydrochloric acid. These different transformations are represented by the following formulas : Researches on Pyrimidines. K to 275 co ro o a f "U-i : s en f a I Q I P ffi ffi O o * :=o ^ o M ' r r o \ ' 3Q2 co o * W ? I ffi " a . There are 7 possible thiopyrimidines that can be derived from 5-hydroxyuracil by replacement of the oxygen atoms with sulphur, viz., the three monothio derivatives, 2-thio-5- hydroxy-6-oxy-, 2,6-dioxy-5-mercapto-, and 2-oxy-5-hydroxy- 6-oxypyrimidines (XIX, XX, XXI), the three dithio deriva- tives, 2,6-dithio-5-hydroxy-, 2-thio-5-mercapto-6-oxy-, and 2- oxy-5-mercapto-6- thiopyrimidines (XXII, XXIII, XXIV) , and finally 2,6-dithio-5-mercaptopyrimidine (XXV). 2 7 6 NH Johnson and Guest. NH CO CSH XXII. XXIV. NH CN XXV. 5-Benzylmercaptouracil (XV) and 2-thio-5-benzylmercapto-6- oxypyrimidine (XIV) are the benzyl ethers of 2,6-dioxy-5- mercaptopyrimidine (XX) and 2-thio-5-mercapto-6-oxypy- rimidine (XXIII), respectively, and also the first thio ethers of this series of thiopyrimidines to be described. We have prepared the first member of the series, 2-thio- 5-hydroxy-6-oxypyrimidine (XIX), by heating 2-methyl- mercapto-5-ethoxy-6-oxypyrimidine 1 (XXVI) in a stream of dry hydrochloric acid gas. 2 An attempt was also made to synthesize 2-oxy-5-hydroxy-6-thiopyrimidine (XXI) from 2- methylmercapto-5-ethoxy-6-oxypyrimidine. The latter com- pound reacted smoothly with phosphorus oxychloride, giv- ing 2-methylmercapto-5-ethoxy-6-chlorpyrimidine (XXVIII), which was then converted into 2-methylmercapto-5-ethoxy-6- thiopyrimidine (XXIX) by the action of potassium hydro- sulphide. All attempts to hydrolyze this pyrimidine to 2- oxy-5-ethoxy-6-thiopyrimidine and 2-oxy-5-hydroxy-6-thio- pyrimidine (XXI) were unsuccessful. The 6-sulphur atom is not firmly bound and the pyrimidine underwent hydrolysis with formation of methyl mercaptan and hydrogen sulphide 1 Johnson and McCollum: J. Biol. Chem., 1, 447. 2 Wheeler and Liddle: J. Am. Chem. Soc., 30, 1157. Researches on Pyrimidines. 277 and was converted into 5-ethoxyuracil (XXX). 2,6-Dichlor- 5-ethoxypyrimidine (XXXII) was prepared by the action of phosphorus oxychloride on 5-ethoxyuracil (XXX). When this dichlorpyrimidine was warmed with potassium hydro- sulphide it was converted quantitatively into the ethyl ether of 2,6-dithio-5-hydroxypyrimidine (XXIII) or 2,6-dithio-5- ethoxypyrimidine (XXXI). These various transformations are represented by the following formulas : O H O=O - O n g Q Ci a H w 8- I =- O 1=00 I O Q o p M : dS|| B f ^0=0-0 XXVI. \ S a O !z! 278 Johnson and Guest. EXPERIMENTAL, PART. Ethyl Benzylthioglycollate, C 6 H 5 CH 2 SCH 2 COOC 2 H 5< This es- ter has been described by Gabriel, 1 who prepared it by esteri- fication of benzylthioglycollic acid with ethyl alcohol. We prepared it by the action of sodium benzylmercaptide on ethyl chloracetate. The benzyl mercaptan was dissolved in alcohol containing a molecular proportion of sodium ethylate and the required amount of ethyl chloracetate added. The solution was then heated on the steam bath until it 5 failed to give an alkaline reaction, cooled, and the undissolved sodium chloride filtered off. The excess of alcohol was then removed by heating the mixture to 120 and the crude ester washed with water, dissolved in the ether, and dried over calcium chloride. It was purified by one distillation under diminished pressure and the fraction boiling within 10 degrees at a constant pressure saved for our experiments. The boiling points of four different preparations were i9O-2OO at 30-33 mm., i85-20o at 33 mm., i98-2io at 50 mm., and i79-i89 at 23 mm. Thirty- two, 35 and 72 grams of the distilled thio- glycollic ester were obtained from 34, 38 and 75 grams, re- spectively, of benzyl mercaptan. Ethyl a-Benzylmercapto-p-hydroxyacrylate, HOCH:C(SCH 2 C 6 H 5 )COOC 2 H 5 . The sodium salt of this ester was prepared by adding a mixture of 32 grams of ethyl benzyl- thioglycollate and 20 grams of ethyl formate, in small portions, to dry ether in which was suspended 4 grams of finely divided sodium. The salt began to deposit at once as a yellow powder, and within 24 hours the sodium had completely disappeared and the condensation was complete. The yield of crude salt was 45 grams. When the salt was dissolved in water and the solution acidified with hydrochloric acid the acrylic ester separated as an oil which solidified when cooled at o. After drying in a vacuum over potassium hydroxide and sulphuric acid it melted at 57-58 to a clear oil. The ester was very soluble in the common organic solvents and reacted with ferric chloride, giving a bright red color. An attempt to dis- 1 Ber. d. chem. Ges.. 12, 1641. Researches on Pyrimidines. 279 til it under diminished pressure was unsuccessful. Sulphur determination (Carius) : 0.2527 gram substance gave 0.2654 gram of BaSO 4 . Calculated for Ci2H 14 O 8 S. Found. S 13.44 i4-4 2-Ethylmercapto-5-benzylmercapto-6-oxy pyrimidine, NH CO I I C 2 H 5 SC CSCH 2 C C H 5 . Molecular proportions of pseudo- N CH ethylthiourea hydrobromide (28 grams) and potassium hy- droxide (8.5 grams) were dissolved in water and the solutions added successively to a cold, aqueous solution of 45 grams of the sodium salt of ethyl a-benzylmercapto-/?-hydroxy- acrylate. The mixture was then allowed to stand at ordinary temperature for about 12-14 hours and heated one hour on the steam bath to complete the reaction. The alkaline solu- tion was then cooled, filtered, and acidified with hydrochloric acid. A heavy oil separated and finally crystallized in prisms, melting at i5O-i53 to a clear oil. The weight of this crude pyrimidine was 10 grams, and 2 grams more were obtained by extracting the acid solution with ether. The pyrimidine is insoluble in water and difficultly soluble in ether. It crystallizes from 95 per cent, alcohol in four- and six-sided, tabular crystals, which melt at i55-i56 to a clear oil without effervescence. The pyrimidine dissolves in dilute sodium hydroxide solution and is reprecipitated unaltered by addition of acids. Analysis (Kjeldahl) : Calculated for CiaH^ONaSa. Found. N 10.07 10.10 NH CO I I 5-Benzylmercaptouracil, CO CSCH 2 C C H 5 . A quantita- NH CH tive yield of this pyrimidine was obtained by digesting 2- 280 Johnson and Guest. ethylmercapto-5-benzylmercapto-6-oxypyrimidine (3 grams) with strong hydrochloric acid for 4 hours. It is difficultly soluble in hot water and cold alcohol. It crystallizes from boiling 95 per cent, alcohol in rhombic plates or tables, which melt at 290 to a clear oil with decomposition. They gave a strong test for uracil when treated with bromine water and barium hydroxide. Analysis (Kjeldahl) : Calculated for CnHioOsNjS. Found. N 11.96 n-74 Five-tenths gram of this pyrimidine was heated with 10 cc. of concentrated hydrochloric acid at i3O-i4o for 2 hours. It was recovered unaltered and melting sharply at 290. The pyrimidine was then heated again at i6o-i7o for 3 hours, when it had been partly decomposed. However, even after this energetic treatment, a small amount of the pyrimi- dine was recovered unaltered and melting at 290. This pyrimidine also dissolves in boiling aniline without change and separates on cooling in plates melting at 290. 2-Ethylmercapto- i )-benzylmercapto-6-chlor pyrimidine, N== CC1 I I C 2 H 5 SC CSCH 2 C tt H 5 . 2-Bthylmercapto-5-benzylmercapto- II II N CH 6-oxypyrimidine reacts smoothly with phosphorus oxychloride, giving this chlorpyrimidine. Ten grams of the mercapto- pyrimidine and 40 cc. of phosphorus oxychloride were heated at 120 for 45 minutes, when the evolution of hydrochloric acid had practically ceased. The excess of phosphorus halide was then expelled by heating at 100 under diminished pres- sure, when a viscous oil was obtained. This was poured into water, warmed gently to decompose any double compound of phosphorus oxychloride, and then extracted with ether. After drying over calcium chloride and evaporating the ether, we obtained the chlorpyrimidine in the form of a dark oil. This solidified on cooling and melted at 4O-45. The pyrimi- dine crystallizes from ligroin in prisms melting at 47-48 Researches on Pyrimidines. 281 to a clear oil. It is extremely soluble in benzene and alcohol, but insoluble in water. Analysis (Kjeldahl) : Calculated for C 13 Hi 3 N 2 ClS 8 . Found. 9.8 N 9.44 2-Ethylmercapto-5-benzylmercapto-6-aminopyrimidine, C 2 H 5 SC CSCH 3 C C H 5 . A quantitative yield of this pyrimi- II II N CH dine was obtained when 5 grams of the above chlorpyrimidine were heated with an excess of an alcoholic solution of ammonia, at I20-i4o, for 2 hours. After evaporating the excess of alcohol at 100, the pyrimidine was then separated from ammonium chloride by dissolving it in ether. It is very soluble in ether, ligroin, alcohol and benzene, but insoluble in water. It crystallizes from ligroin in sheaves of needles melting at 68-69 to a clear oil. Analysis (Kjeldahl): N Calculated for C 13 H 16 N 8 S 2 . I5.I6 Found 14.88 5-Benzylmercaptocytosine, CO CSCH 2 C 6 H 5 . Five grams NH CH of 2-ethylmercapto-5-benzylmercapto-6-aminopyrimidine were dissolved in 50 cc. of strong hydrochloric acid and the solution boiled for 1.5 hours, when the evolution of ethyl mercaptan had practically ceased. The solution was then evaporated to dryness, the hydrochloride dissolved in water, and the pyrimidine base precipitated by addition of ammonia. It is soluble in hot alcohol and insoluble in water and ether. It crystallizes from 95 per cent, alcohol in plates melting at 240-24i. Analysis (Kjeldahl) : N Calculated for CuHnONsS. I8.0I Found. iS.OO 282 Johnson and Guest. 2-Thio-5-benzylmercapto-6-oxypyrimidine, NH CO CS CSCH 2 C 6 H 5 . Seven-tenths gram of 2-ethylmercapto-5- NH CH benzylmercapto-6-oxypyrimidine was heated in a current of dry hydrochloric acid gas, at i6o-i7o, until the evolution of ethyl chloride ceased. We obtained a brown substance, which was washed with ether to remove traces of oil and then crystallized from 95 per cent, alcohol. The pyrimidine sepa- rated, on cooling, in blocks melting at i95-i96 to an oil without effervescence. It gave a strong test for sulphur. Analysis (Kjeldahl) : Calculated for C n H 10 ON 2 S. C 4 H 4 ONjSj. Found. N 11.20 17.50 11.57 Attempts to prepare this pyrimidine by condensation of the sodium salt of ethyl a-benzylmercapto-/?-hydroxyacrylate with thiourea in aqueous solution, and in alcohol in presence of sodium ethylate, were unsuccessful. For example: one gram of sodium and 3 grams of thiourea were dissolved in alcohol, ten grams of the sodium salt suspended in the solution, and the mixture then digested on the steam bath for 6 hours. There was no indication of any reaction and 8.0 grams of the unaltered sodium salt separated on cooling. The alcohol filtrate was then evaporated to dryness and the residue left behind dis- solved in a little cold water and the solution carefully acidified with hydrochloric acid. The solution became turbid, but on standing no pyrimidine separated, showing that no condensa- tion had taken place. 2-Methylmercapto- 5- ethoxy-6-chlor pyrimidine, N==CC1 I I CH 3 SC COC 2 H 5 . Twenty-five grams of 2-methylmercapto- N CH S-ethoxy-G-oxypyrimidine 1 were suspended in 40 cc. of phos- 1 Johnson and McCollum: Loc. cit. Researches on Pyrimidines. 283 phorus oxychloride. There was an immediate reaction and the pyrimidine dissolved completely. The solution was heated in an oil bath at 120- 130 for a few hours to complete the reaction, and the excess of phosphorus oxychloride re- moved by heating at 100 under diminished pressure. An oil was obtained which immediately solidified when poured into cold water. This was a double compound of the pyrimi- dine and phosphorus oxychloride, and in order to decompose it and destroy the phosphorus halide, it was necessary to triturate it with hot water. The pyrimidine was then dis- solved in ether, washed with dilute sodium hydroxide solution and dried over calcium chloride. When the ether was removed we obtained the pyrimidine as a crystalline solid melting at 70. The yield was 21 grams, or 95 per cent, of the calculated. The pyrimidine is insoluble in water and very soluble in ben- zene and ether. It crystallizes from 95 per cent, alcohol in slender prisms melting at 75 to an oil. Analysis (Kjel- dahl) : Calculated for C 7 HoON 2 ClS. Found. N 13.69 13.63 2-Methylmercapto-5-ethoxy-6-thiopyrimidine, NH CS I I CH 3 SC COC 2 H 5 . The potassium salt of this pyrimidine II II N CH was obtained when 5 grams of 2-methylmercapto-5-ethoxy- 6-chlorpyrimidine were dissolved in an alcoholic solution of potassium hydrosulphide and the mixture digested on the steam bath for several hours. The excess of alcohol was then removed by evaporation at 100 and the potassium salt of the pyrimidine and potassium chloride dissolved in water. When this solution was acidified with acetic acid, the 6-thiopyrimidine separated in yellow crystals. It is difficultly soluble in hot water but crystallizes from alcohol in light yellow prisms melting at 190 to a clear oil. The yield was 3.5 grams. Analysis (Kjeldahl) : 284 Johnson and Guest. Calculated for C 7 HioON 2 S 2 . Found. N 13.86 13.78 An attempt to prepare 2-oxy-5-ethoxy-6-thiopyrimidine from this compound, by hydrolysis with hydrochloric acid, was unsuccessful. About 7 grams of the mercaptopyrimidine were digested with a large excess of concentrated hydrochloric acid on the steam bath for several hours. Methyl mercaptan and hydrogen sulphide were evolved and the pyrimidine finally dissolved. The acid solution was then evaporated to dryness and the crystalline substance obtained purified by repeated crystallizations from hot water. It separated in irregular prisms which melted at about 275 to a dark oil. This melting point varies according to the rate of heating. The compound gave no test for sulphur, and a nitrogen de- termination (Kjeldahl) agreed with the calculated value for 5-ethoxyuracil. 1 The small amount of material which accompanied this 5-ethoxypyrimidine and rendered the puri- fication difficult contained sulphur, but we did not succeed in isolating a sufficient quantity of the substance in pure state for analysis. Its melting point, i9O-23O, indicated a mix- ture. Calculated for C 6 H 8 O 8 N 2 . Found. N 17-94 17.80 2, 6-Dichlor- 5-ethoxypyrimidine, Cl.C COC 2 H 5 . Thispyr- II II N - CH imidine was prepared by the action of phosphorus oxychloride on 5-ethoxyuracil. 1 Twelve grams of the ethoxyuracil and 70 cc. of phosphorus oxychloride were digested at 120- 130 for nearly 3 hours before the evolution of hydrochloric acid ceased. The dark liquid was then filtered from a small amount of insoluble, amorphous substance and the excess of phosphorus halide removed, in the usual manner, by heating at 100 under diminished pressure. We obtained a thick syrup 1 Johnson and McCollum: Loc. cit. Researches on Pyrimidines. 285 which solidified when poured into cold water. In order to destroy all phosphorus halide, this substance was then melted under water by heating the liquid to 6o-7o, and after thor- ough mixing the pyrimidine was dissolved in ether. The ether solution was then dried over calcium chloride and the excess of ether removed by spontaneous evaporation, when the pyrimidine separated in well-developed prisms. It is soluble in cold alcohol, benzene and petroleum ether and crystallizes from the latter in clusters of radiating prisms and needles which melt at 4i-42 to a clear oil. It crystal- lizes from water in prisms melting at the same temperature. The yield was about 75 per cent, of the calculated. Analysis (Kjeldahl) : Calculated for C 6 H 6 ON 2 C1 2 . Found. N 14-50 14-35 2, 6-Diihio-5-eihoxy pyrimidine (5-Ethoxydithiouracil) , NH CS I I CS COC 2 H 5 . This compound was prepared by the action NH CH of potassium hydrosulphide on 2,6-dichlor-5-ethoxypyrimidine in alcoholic solution. It is soluble in hot alcohol and crystal- lizes, on cooling, in needles which slowly decompose when heated above 255, and effervesce violently at about 267 268. The pyrimidine is insoluble in water. Analysis (Kjel- dahl) : Calculated for C 6 H 8 ON 2 S2. Found. N 14.7 14.9 Sodium Salt of Ethyl a-Phenoxy-fi-hydroxyacrylate, NaOCH:C(OC 6 H 5 )COOC 2 H 5 . Johnson and Heyl 1 prepared this salt by condensation of ethyl formate with ethyl phenoxy- acetate in presence of sodium ethylate. The use of sodium ethylate is unnecessary and a practically quantitative yield of the salt can be obtained if the esters are condensed in ether in the presence of metallic sodium. Thirty-one grams of 1 THIS JOURNAL, 37, 636. 2 86 Johnson and Guest. crude, dry salt were obtained from 25 grams of ethyl phenoxy- acetate. NH CO 2-Thio-$-phenoxy-6-oxy pyrimidine, CS COC 6 H 5 . John- NH CH son and Heyl 1 have shown that the above sodium salt con- denses with pseudoethylthiourea, in aqueous solution, giving 2-ethylmercapto-5-phenoxy-6-oxypyrimidine. Fifteen grams of thiourea and 4.5 grams of sodium were dissolved in alcohol and 31 grams of the sodium salt dissolved in the solution by warming on the steam bath. After digesting for 10 hours, the excess of alcohol was removed by evaporation at 100 and the residue remaining dissolved in cold water, filtered, and the solution acidified with acetic acid. Sixteen grams of the crude pyrimidine separated as a colorless, granular powder. It is insoluble in hot water, moderately soluble in alcohol, insoluble in benzene and soluble in acetic acid. It crystallizes from acetic acid in clusters of prisms showing a twinning habit, and melts at 253 -254 to a brown oil. Nitro- gen (Kjeldahl) and sulphur (Carius) determinations gave the following results: Calculated for Found. Ci H 8 OjN 2 S. I. II. N 12.73 12.82 3 J4-56 14-30 H-72 NH CO 5-Phenoxyuracil, CO COC 8 H 5 . This pyrimidine was pre- NH CH pared by heating 2-thio-5-phenoxy-6-oxypyrimidine (2 grams) with concentrated hydrochloric acid (35 cc.) at i4O-i6o, and also by digestion with hydrobromic acid for several hours. It is practically insoluble in cold water and alcohol and difficultly soluble in hot water. It crystallizes from boiling acetic acid i Loc. cit. Researches on Pyrimidines. 287 in distorted needles melting at 290 with effervescence. Analy- sis (Kjeldahl) : Calculated for C 10 H 8 OjN 3 . Found. N 13-72 13-62 NEW HAVEN, CONN., June, 1909. [Reprinted from The American Chemical Journal, Vol. XVI. No. 4. October, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXXL RESEARCHES ON PYRIMIDINES: DIMETHYL DERIVATIVES OF 2-AMINOPYRIMIDINE. PREPARATION OF 2-METHYLAMINO-5-METHYL- PYRIMIDINE. [FORTY-SIXTH PAPER.] BY TREAT B. JOHNSON AND KENNETH G. MACKENZIE. 1 This paper is a preliminary contribution to the study of aminomethylpyrimidines having the empirical formula C 6 H 9 N 3 . Our interest in aminopyrimidines of this compo- sition was incited by a paper entitled "Ueber die chemische Zusammensetzung der japanischen Soja-Souce oder Schoyu," 2 in which the authors have described two decomposition products of Schdyu to which they have assigned the empirical formulas C 6 H 9 N 3 and C 4 H 12 N 2 . They state that the substance, C 6 H 9 N 3 , is probably an isorner of aminodimethylpyrimidine, but is not identical with any one of three pyrimidines of this constitution which have been described in the literature, 8 mz. y 2,6-dimethyl-4-aminopyrimidine, 4 2-amino-4,5-dimethyl- pyrimidine, 5 and 2-amino-4,6-dimethylpyrimidine. 6 1 A part of this paper was presented as a thesis by Mr. Kenneth Gerard Mackenzie to the Faculty of the Graduate School of Yale University for the Degree of Master of Science (June, 1909). 2 Suzuki, Aso, and Mitarai: Bull. Coll. Agriculture (Tokio Univ.), 7, 477 (1907). 3 The Japanese investigators failed to recognize that a fourth isomer, 4,5-dimethyl- 6-aminopyrimidine, has been prepared by Schlenker (Ber. d. chem. Ges., 34, 2823). No salts of this pyrimidine have been described. T. B. J. 4 Schwarze: J. prakt. Chem., 42, 1 (1889). Schmidt: Ber. d. chem. Ges., 35 1577 (1902). 6 Schlenker: Ber. d. chem. Ges., 34, 2819 (1901). 6 Angerstein: Ber. d. chem. Ges., 34, 3962. 354 Johnson and Mackenzie. If we disregard the tautomeric form of 2-aminopyrimidine 1 (II), there are only five possible dimethyl derivatives of this pyrimidine (1), viz., the two isomers described in the litera- ture, 4,5-dimethyl 2 - and 4,6-dimethyl-2-aminopyrimidine 3 (III and IV), 2-methylamino-5-methylpyrimidine (V), 2-methylamino-4-methylpyrimidine (VI), and 2-dimethyl- aminopyrimidine (VII). H 2 N.C CH HN= C CH II II II II N CH HN CH I. II. N=CH N=CCH 3 H 2 NC CH II II N CCH 3 IV. N=< CH 3 HNC CCH 3 CH 3 HNC CH (CH 3 ) 2 NC CH . II II II II N CH N CCH 3 V. vi. We shall describe, in this paper, the preparation and prop- erties of 2-methylamino-5-methylpyrimidine (V), and also some derivatives of the isomeric 2-methylamino-4-methyl- pyrimidine (VI). A description of a new method of pre- paring thymine has also been appended to this paper. It has been shown in several papers from this laboratory* that aryl substituted aminopyrimidines can be obtained, in practically quantitative yields, by heating 2-mercaptopyr- imidines with aromatic bases. The action of aliphatic amines on mercaptopyrimidines has not been investigated. Since 1 Biittner: Ber. d. chem. Ges., 36, 2229. 2 Schlenker: Loc. cit. 3 Angerstein: Loc. cit. 4 Wheeler and Bristol: THIS JOURNAL, 33, 438, 448. Johnson and Johns: Ibid., 34, 175. Johnson and Heyl: Ibid., 38, 238. Johnson and Storey: Ibid., 40, 131. Researches on Pyrimidines. 355 it was necessary for us to devise a practical method of pre- paring alphylaminopyrimidines easily and in sufficient quan- tities for our work, we therefore investigated the action of methylamine on some 2-mercaptopyrimidines. We now find that 2-methylmercapto-5-methyl-6-oxypyr- imidine 1 (VIII), and 2-methylmercapto-4-methyl-6-oxypyr- imidine 2 (IX), react with methylamine at i4o-i5O, giving quantitative yields of 2-methylamino-5-methyl-6-oxypyrimi- dine (X) and 2-methylamino-4-methyl-6-oxypyrimidine (XI), respectively. The latter pyrimidine was identical with the 2-methylaminopyrimidine obtained by Jager 3 by alkylation of 2-amino-4-methyl-6-oxypyrimidine 4 with methyl iodide. The same pyrimidine has also recently been prepared by Majima 5 by condensing methylguanidine with ethyl aceto- acetate. The yield by this method, however, corresponds to only about 50 per cent, of the theoretical, because part of the methylguanidine condenses, giving the isomeric 2-amino- 3,4-dimethyl-6-oxypyrimidine. These two 2-methylaminopyrimidines, (X) and (XI), reacted smoothly with phosphorus oxychloride, giving prac- tically quantitative yields of 2-methylamino-5-methyl-6-chlor- pyrimidine (XII) and 2-methylamino-4-inethyl-6-chlorpyr- imidine (XIII), respectively. Both of these pyrimidines underwent hydrolysis to the original 6-oxypyrimidines (X) and (XI), when an attempt was made to reduce them with hydriodic acid. On the other hand, they were converted into 2-methylamino-5-methylpyrimidine (V) and 2-methyl- amino-4-methylpyrimidine (VI), respectively, by reduction with zinc dust. We have not isolated the free 2-methyl- amino-4-methylpyrimidine, 6 but have procured evidence of its formation, since we have obtained a picrate of the base and a characteristic zinc chloride double compound of definite 1 Wheeler and Merriam: THIS JOURNAL, 29, 487. 2 List: Ann. Chem. (Liebig), 236, 12. Ann. Chem. (Liebig), 262, 365. 4 Jager: Loc. cit. Kohler: Ber. d. chem. Ges., 19, 220. 6 Ber. d. chem. Ges., 41, 176. 6 We were obliged to discontinue experiments on the reduction of 2-methylamino- 4-methyl-6-chlorpyrimidine, because of the unexpected departure of one of us from the laboratory and on account of pressure of other work. T. B. J. 356 Johnson and Mackenzie. constitution. The experimental evidence indicates that this methylaminopyrimidine is not identical with the base found in Schdyu. NH CO I I CH 3 SC II N- CCH< II CH VIII. NH- CH 3 SC II N CO IX. CCH 3 NH CO I I CH 3 HNC CCH 8 N- X. CH i N == CC1 CH 3 HNC II N CCH, II CH XII. NH CO I 1 CH 3 HNC CH II II N CCH, XI. CH 3 HNC N == CC1 I I CH N CCH 3 XIII. CH 3 HNC CCH, CH,HNC CH N -- CH N -- CCH 3 V. VI. 2-Methylamino-5-methylpyrimidine (V) is not identical with the base C 6 H 9 N 3 which Suzuki and his coworkers isolated from Schdyu. They state that their base reacted with diazo- benzenesulphonic acid, in the presence of alkali, giving an intense red color. Our pyrimidine and the diazo reagent gave at first no color, but on long standing a brilliant red color finally developed. A characteristic feature of their base was its tendency to form acid salts, viz., the picrate Researches on Pyrimidines. 357 C 6 H 9 N 3 (C 6 H 3 O 7 N 3 ) 2 , decomposing at 230, and the hydrochloride C 6 H 9 N 3 .2HC1, melting at 232-233. Our pyrimidines, on the other hand, are characterized by their tendency to form basic salts. The isomeric picrates, (C 6 H 9 N 3 ) 2 .C 6 H 3 O 7 N 3 , of 2-methylamino-5-methylpyrimidine and 2-methylamino-4- methylpyrimidine both decomposed when heated above 150. 2-Methylamino-5-methylpyrimidine formed a hydrous hydrochloride, (C 6 H 9 N 3 ) 2 HC1.H 2 O, melting at i62-i63. It is an interesting fact that 2-amino-5,6-dimethylpyrimidine 1 is the only dimethyl derivative of 2-aminopyrimidine, so far examined, that forms acid salts, viz., the picrate and hy- drochloride. A New Method of Preparing Thymine. In a previous paper from this laboratory one of the writers 2 showed that pseudomethylthiourea condenses smoothly, in aqueous solution, with the sodium salt of diethyl oxalpro- pionate, giving 2-methylmercapto-4-carboxyl-5-methyl-6-oxy- pyrimidine. When this acid, in small quantities, is heated above its melting point it undergoes a quantitative decom- position, with evolution of carbon dioxide, giving 2-methyl- mercapto-5-methyl-6-oxypyrimidine. 3 A quantitative yield of thy mine is then obtained by hydrolysis of tMs mercapto- pyrimidine. The writer did not, at that time, lay stress on this method of synthesizing thymine because of the progress of other work in this laboratory on different methods of pre- paring this pyrimidine. We have now investigated this method of preparation and find that the sodium salt of diethyl oxalpropionate condenses as smoothly, in aqueous solution, with pseudoethylthiourea as with pseudomethylthiourea, giving the corresponding 2-ethylmercapto-4-carboxyl-5-methyl-6-oxypyrimidine (XIV) . This pyrimidine melts lower than the corresponding 2-methyl- mercapto compound 4 and undergoes decomposition at its melting point, giving 2-ethylmercapto-5-methyl-6-oxypyrimi- 1 Loc. cit. 2 Johnson: J. Biol. Chem., 3, 299 (1907). 3 Wheeler and Merriam: Loc. cit. 4 Johnson: Loc. cit. 358 -Johnson and Mackenzie. dine 1 (XV), which can be converted into thymine (XVI) by hydrolysis with hydrochloric acid. The success of this method of preparation was evidently dependent upon the fact whether large amounts of the mercapto acid (XIV) could be changed smoothly and quantitatively into 2-ethyl- mercapto-5-methyl-6-oxypyrimidine. 2 Working with small quantities, less than 15-16 grams of the acid, the decompo- sition into 2-ethylmercapto-5-methyl-6-oxypyrimidine was practically quantitative and the final yield of thymine corre- sponded to nearly 85-90 per cent, of the theoretical. On the other hand, when large amounts of 2-ethylmercapto-4- carboxyl-5-methyl-6-oxypyrimidine are decomposed under the same conditions the method is not practicable because it is then necessary to heat a long time, at a high temperature, in order to complete the reaction. Under such conditions secondary reactions set in with formation of products which contaminate the 2-ethylmercapto-5-methyl-6-oxypyrimidine and render its purification difficult. It is probable that these mercaptopyrimidines, when heated at high temperatures and in presence of impurities, slowly undergo dissociation into ethylene hydrocarbons and 2-thiopyrimidines. The ethyl ester of 2-ethylmercapto-4-carboxyl-5-methyl-6-oxypyrimidine (XVII) was obtained in one experiment as the chief product of the condensation. It was also formed quantitatively by the action of ethyl iodide on the silver salt of the mercapto acid (XIV). When the mercapto acid (XIV) was heated with methylamine, a quantitative yield of 2-methylamino- 4-carboxyl-5-methyl-6-oxypyrimidine (XVIII) was obtained. These various transformations are represented by the follow- ing formulas : 1 Wheeler and Johnson: THIS JOURNAL, 31, 595. . cit, t Researches on Pyrimidines. 359 PART. 2-Methylamino-5-methyl-6-oxypyrimidme, NH - CO I I CH 3 HNC CCH 3 . Ten grams of 2-methylmercapto-5- II II N -- CH methyl-6-oxypyrimidine 1 were heated with 18 grams of 33 per cent, methylamine solution (6 grams CH 3 NH 2 ) at 140- 150 for two hours. The clear solution was then transferred to a dis- 1 Loc. cit. 360 Johnson and Mackenzie. tillation flask and the excess of methylamine expelled by passing a current of air through the boiling solution. On cooling, 8 grams of the aminopyrimidine separated, corre- sponding to 91 per cent, of the theoretical yield. In two other experiments, when 6 grams of the 2-methylmercaptopyr- imidine were heated with 7.2 grams of 33 per cent, methyl- amine solution, under the same conditions, we obtained 5.0 and 5.3 grams of the crude pyrimidine. This 2-methylaminopyrimidine is difficultly soluble in cold, but very soluble in hot water; soluble in boiling alcohol and benzene. It crystallizes from water in colorless, hairy crys- tals, which melt at 213 to a clear oil. The pyrimidine is characterized by its tendency to form supersaturated, aqueous solutions. It reacts with diazobenzenesulphonic acid in presence of sodium hydroxide, giving a strong, claret-red color. It contained one molecule of water of crystallization, which was determined by heating at ioo-uo for one hour. 0.5821 gram substance lost 0.0584 gram H 2 O. Calculated for C 6 H g ON 3 .H s O. Found. H 2 O 11.4 10.03 Nitrogen determination in anhydrous substance (Kjeldahl) : Calculated for C 6 H 9 ON 3 . Found. N 30.22 30.23 Sulphate, C 6 H 9 ON 3 .H 2 SO 4 . Five- tenths gram of the pyr- imidine base and 0.7 gram of sulphuric acid were dissolved in water and the solution allowed to concentrate in the air. The salt finally crystallized in large, transparent, tabular crystals, which melted at 202 to a brown oil. The salt is insoluble in alcohol but very soluble in water. It was dried at 100 for analysis (Kjeldahl): Calculated for Found. C 6 H 9 ON 8 .HS04. I. II. N 17.72 17.51 17.83 Picrate, (C 6 H 9 ON 3 ) 2 .C 6 H 3 O 7 N 3 .H 2 O. This salt was pre- pared by adding a solution of picric acid to an aqueous solu- tion of the base. The picrate deposited in distorted prisms Researches on Pyrimidines* 361 which melted with decomposition at 240. Analysis (Kjel- dahl) : Calculated for C 6 H 9 ON 3 .C C H 3 O 7 N 3 = 22 .82 per cent N. Calculated for C 6 H 8 ON 3 .(C 6 H 3 O 7 N 3 ) 2 = 21 . 10 per cent N. Calculated for (C 6 H 9 ON 3 ) 2 .C 6 H 3 O 7 N 8 .H 2 O = 24 . oo per cent N. I. II. III. IV. Nitrogen found : 23.89 24.12 24.20 24.25 Platinum Chloride Salt, (C 6 H 9 ON 3 .HCl) 2 .PtCl 4 , was pre- pared by addition of hydrochloroplatinic acid to an aqueous solution of the pyrimidine base. It did not contain water of crystallization. Platinum determinations: I. 0.0889 gram of salt gave 0.0248 gram of Pt. II. 0.0989 gram of salt gave 0.0277 gram of Pt. Calculated for Pound. (C6H 9 ON.HCl)2.PtCl4. I. II. Pt 28.3 27.90 28.06 2-Methylamino-5-methyl-6-chlor pyrimidine, I I CH S HNC CCH 3 . Six and five-tenths grams of 2-methyl- II II N -- CH amino-5-methyl-6-oxypyrimidine were dissolved, by warm- ing, in 30 cc. of phosphorus oxychloride and the solution boiled for 30 minutes. The excess of phosphorus oxychloride was then removed by heating at 100 under diminished pressure, when a viscous liquid was obtained. This was dissolved in cold water and the solution made alkaline with sodium hydroxide, when the chlorpyrimidine separated. After dry- ing in a vacuum over concentrated sulphuric acid it weighed 6.9 grams, corresponding to 95 per cent, of the theoretical. In a second experiment, when we used 10.6 grams of the 2-methylmercaptopyrimidine and 53 cc. of phosphorus oxy- chloride we obtained a yield of 11.5 grams of the chlorpyr- imidine. The pyrimidine is insoluble in water but soluble in alcohol and benzene. It sublimes at 100 and melts at 131 to a 362 Johnson and Mackenzie. clear oil with no effervescence. It crystallizes from 95 per cent, alcohol in prismatic crystals. Analysis (Kjeldahl): Calculated for C 6 H 8 N 3 C1. Found. N 26.67 26.83 2-Methylamino-5-methylpyrimidine, CH 3 HNC CCH 3 . -- N -- CH Five grams of 2-methylamino-5-methyl-6-chlorpyrimidine and 15 grams of zinc dust were suspended in 200 cc. of water and the mixture digested for 8 hours. The excess of zinc was then removed by nitration and the filtrate concentrated on the steam bath. We obtained a thick syrup which was dissolved in 25 cc. of water and an excess of a saturated, aqueous solu- tion of potassium hydroxide added. A light colored pre- cipitate was formed which was filtered by suction, and dried in a desiccator over sulphuric acid. An attempt to purify this substance by sublimation was unsuccessful. The crude, dry material was finally extracted several times with boiling ether. When the excess of ether was evaporated, about 2 grams of a colorless, crystalline sub- stance which melted at 97-ioo to a clear oil was obtained. In order to purify it for analysis it was dissolved in dry ligroin and the solution allowed to concentrate by slow evapora- tion. The pyrimidine separated under these conditions in beautiful, transparent prisms, which melted sharply at 102 to a clear oil. The pyrimidine is extremely soluble in cold water, ether, and benzene. It showed no alkaline reaction when tested with turmeric and was precipitated from an aqueous solution by mercuric chloride. It gave at first no color when tested with diazobenzenesulphonic acid, but on allowing the mixture to stand a strong red color finally de- veloped. The pyrimidine sublimes when heated above 100. Analysis : Calculated for C 6 H 9 N 3 . Found. N 34.14 34.20 Researches on Pyrimidines. 363 An attempt was made to reduce the 6-chlorpyrimidine with hydriodic acid, but it underwent hydrolysis, giving 2-methylamino-5-methyl-6-oxypyrimidine. Picrate, (C 6 H 9 N 3 ) 2 .C H 3 O 7 N 3 . This salt separated imme- diately when an aqueous solution of picric acid was added to a solution of the pyrimidine base. It had no definite melting point, but began to shrivel at about 100 and de- composed when heated above 150. Analysis: Calculated for (C 6 H 9 N 3 )2.C 6 H 3 7 N 8 . Found. N 26.53 26.36 Hydrochloride, (C 6 H 9 N 3 ) 2 .HC1.H 2 O. This salt was prepared by dissolving the base in warm hydrochloric acid and con- centrating the solution on the steam bath. It was extremely soluble in water, but crystallized from methyl alcohol in well- developed prismatic crystals. The salt melts at i62-i63 to a clear oil and does not effervesce below 250. Analysis: Calculated for Found. ^CeHpNg.HCl. N 26-33 2i .64 . (C 8 HgN 3 ) 2 .HCl.H 2 O. 27-95 I. II. 27.7 27.88 NH CO 2-Methylamino-4-methyl-6-oxy pyrimidine, CH 3 HNC CH II II N - CCH 3 We prepared this pyrimidine 1 by heating 2-methylmer- capto-4-methyl-6-oxypyrimidine 1 with an excess of methyl- amine at I4o-i5o. The yield was quantitative and the pyrimidine melted at 2Oi-2O2 after one recrystallization from hot water. Analysis (Kjeldahl) : Calculated for CH 9 ON 8 . Found. N 30.22 30.00 2-Methylamino-4-methyl-6-chlorpyrimidine t CH.HNC CH . Fourteen and five-tenths grams of II II N -- CCH, > Loc. cit f 364 Johnson and Mackenzie. 2-methylamino-4-methyl-6-oxypyrimidine and 72 cc. of phos- phorus oxychloride were heated in an oil bath at 120- 130 for 30 minutes, when the evolution of hydrochloric acid gas had practically ceased. We obtained a clear solution which was then heated at 100 under diminished pressure to remove the excess of phosphorus oxychloride. A brown oil remained, which was dissolved in cold water, and the solution made alkaline with sodium hydroxide. The pyrimidine separated in colorless crystals and was dried in a desiccator over sul- phuric acid. It is very insoluble in water but easily soluble in alcohol. It separates from an alcohol solution in long needles which melt at 135 to a yellow oil without efferves- cence. It slowly sublimes when heated above 100. Analysis (Kjeldahl) : Calculated for C 9 H 8 N 3 C1. Found. N 26.67 26.70 Reduction of 2-Methylamino-4-methyl-6-chlorpyrimidine with Zinc. This chlorpyrimidine is very slowly reduced by di- gestion with zinc dust because of the insolubility of the pyrimi- dine in water. Unaltered chlorpyrimidine was recovered after 12 hours' digestion of 12 grams of the pyrimidine and 50 grams of zinc dust in 1000 cc. of water. One hundred cc. of alcohol were then added and the mixture boiled again for 10 hours, cooled and filtered. By extraction of the zinc residue with alcohol, we recovered 6 grams of unaltered 6-chlor- pyrimidine. The aqueous nitrate was concentrated to a volume of 15-20 cc. and allowed to stand at ordinary tem- perature, when well-developed, red crystals separated. They melted sharply at I7o-i72 to an oil without effervescence and contained zinc and chlorine. The substance was extremely soluble in water and did not lose weight when heated at ioo- 110. A nitrogen determination (Kjeldahl) agreed with the calculated value for a double compound containing 3 mole- cules of 2-methylamino-4-methylpyrimidine and one mole- cule of zinc chloride, (C 6 H 9 N 3 ) 2 .ZnCl 2 : Calculated for (CH 9 N,),ZnCl2. Found. N 22.97 22.80 Researches on Pyrimidines. 365 The filtrate obtained after separation of the zinc chloride compound was concentrated on the steam bath to a viscous liquid and then triturated with about 25 cc. of a strong, aqueous solution of potassium hydroxide. An amorphous substance was precipitated, which was separated by suction filtration, washed with a few drops of cold water, and dried in a desic- cator over sulphuric acid. This substance was then extracted in a Soxhlet apparatus with ether for three days. When the ether was evaporated we obtained an oil which dissolved at once in water, alcohol and benzene. In order to estab- lish the presence of 2-methylamino-4-methylpyrimidine, the oil was dissolved in water and a solution of picric acid added to the aqueous solution. A picrate separated at once in the form of irregular prisms which melted at I5o-i55 to an oil with no appreciable effervescence. This salt did not give a test for chlorine. Analysis (Kjeldahl) : Calculated for (C 6 H 9 N 3 )2.C 6 H 8 07N3. C 6 H 9 N3.CH 3 07Na. Found. N 26.5 23.8 25.98 2-Methylamino-4-methyl-6-chlorpyrimidine was converted into 2-methylamino-4-methyl-6-oxypyrimidine when heated with hydriodic acid. 2-Ethylmercapto-4-carboxyl-5-methyl-6-oxypyrimidine, NH CO I I C 2 H 5 S.C CCH S . An excellent yield of this pyrimidine II II N CCOOH is obtained by condensation of pseudoethylthiourea with the sodium salt of diethyl methyloxalacetate in aqueous solution. In our preliminary experiments this sodium salt was pre- pared by condensation of diethyloxalate with ethyl propionate in presence of sodium ethylate. 1 We found, however, that the use of dry sodium ethylate is unnecessary and that as good a yield of the salt can be obtained by condensation of the esters in anhydrous ether in presence of metallic sodium. This fact is shown by comparison of the yields of pyrimidine given in the table. Our method of preparing the pyrimidine i Arnold: Ann. Chem. (I^iebig), 246, 329. 366 Johnson and Mackenzie. was as follows: A mixture of 50 grams of ethyl propionate and 7 1 grams of diethyl oxalate was added slowly to dry ether, in which was suspended 11.5 grams of metallic sodium. After the esters had been added the mixture was allowed to stand about 2 days to insure a complete reaction. Cold water (about 500-600 cc.) was then cautiously added to dissolve the sodium salt, and the ether layer separated. To this aqueous solution was then added a concentrated, aqueous solution of 52 grams of pseudoethylthiourea hydrobromide and finally two molecular proportions of potassium hydroxide (32 grams). After allowing to stand for 2 days at 4o-5o the solution was concentrated to about one-half its original volume, cooled, and acidified with an excess of strong hydro- chloric acid. The pyrimidine usually separated in the form of a light pink or nearly colorless, granular powder melting at 2io-2i5 with evolution of carbon dioxide. The yield of mercaptopyrimidine is decreased by using, for the con- densation, more than two molecular proportions of potassium hydroxide because the pyrimidine is slowly converted into thymine-4-carboxylic acid, 1 with evolution of ethyl mercaptan, when warmed with strong alkaline solutions (Experiment 5 in table). The proportion of pseudoethylthiourea hydrobromide to be used for a condensation was calculated upon the basis that the yield of sodium salt of diethyl oxalpropionate is only about 50 per cent, of the theoretical (Arnold 2 says 45 percent.). Under these conditions, fifty grams of ethyl propionate require for condensation 45 grams of the pseudo- thiourea salt, which we increased to 5254 grams or about 0.6 molecular proportion. The yield of pyrimidine was not increased by using more than this proportion (see Experiment 2 in table). 1 Johnson: J. Biol. Chem.. 3, 299. 2 Loc. cit. Researches on Pyrimidines. 367 Table I. i o H I 1 ^ I n W o 5 C CQ o o^ fc JM M Grams. Grams. Grams. Grams. Grams. I 50.0 71.0 "5 52.0 16.0 2 50.0 71.0 ii. 5 72.O 22. O 3 50.0 71.0 ii .5 54-o 32-0 4 50-0 71.0 1 1 . 5 in form 54-0 32.0 NaOC2H 5 5 IOO.O 144.0 22.5 IOO.O 108.0 Ifil Grams. 36.0 36.5 34-o 37-0 60.0 and 15.0 (2 . 5 mols.) of thymine- 4-carboxylic 11.5 in form acid 1 6 50.0 71.0 NaOC 2 H 5 45.0 30.0 22.0 2- Ethylmercapto-4-carboxyl - 5 - methyl-6-oxypyrimidine is soluble in boiling water but separates on cooling in clusters of slender needles and prismatic crystals. It is more soluble in alcohol than in water. It decomposes, after crystalliza- tion from water, from 2i5-2i8 with effervescence, giving 2-ethylmercapto-5-methyl-6-oxypyrimidine 2 (see below). If the acid is dissolved in dilute sodium hydroxide solution and reprecipitated by addition of hydrochloric acid it sepa- rates in colorless crystals melting sharply at 220. The acid is not precipitated from aqueous solutions of its sodium salt by addition of acetic acid. Analysis (Kjeldahl) : Calculated for Found. C 8 HioO 8 NjtS. I. II. N 13.08 13.3 13.2 Stability of the Sodium Salt of Diethyl Oxalpropionate, C 2 H 5 OOC.C(ONa) : C(CH 3 )COOC 2 H 5 . Seventy-one grams of diethyl oxalate were condensed with 50 grams of ethyl pro- pionate, in presence of sodium ethylate, 3 on June 10, 1908. This salt was then allowed to stand exposed to the air, in an 1 Johnson: Loc. cit. 2 Loc. cit. 3 Arnold: Loc. cit. 368 Johnson and Mackenzie. open flask, until October 15, 1908. It was then condensed with pseudoethylthiourea in the usual manner, when we obtained 22 grams of pyrimidine melting at 2O9-2i4 (Ex- periment 6 in table). Ethyl Ester of 2-Ethylmercapto-4-carboxyl-5-methyl-6-oxypyr- NH CO imidine, C 2 H 5 SC CCH 3 .The sodium salt of diethyl II II N CCOOC 2 H 5 oxalpropionate was prepared in the usual manner by con- densing 71 grams of diethyl oxalate with 50 grams of ethyl propionate in presence of metallic sodium. This salt was then dissolved in 400-500 cc. of water and mixed with an aqueous solution containing 0.6 molecular proportion of pseudoethylthiourea. The mixture was then allowed to stand, at ordinary temperature, for several days, when 17.0 grams of the ethyl ester had separated. After concentrating the aqueous filtrate and then acidifying with hydrochloric acid, we obtained the corresponding acid. This ester is difficultly soluble in water but very soluble in boiling alcohol. It crystallizes from 95 per cent, alcohol in prisms which melt at i72-i73 to a clear oil. It dissolves in sodium hydroxide solution without decomposition but on warming it easily undergoes saponification. The ester is also formed quanti- tatively by the action of ethyl iodide on the silver salt of 2-ethyl- mercap to -4- carboxyl-5-methyl - 6 - oxy pyrimidine. Analysis (Kjeldahl) : Calculated for CioHi4O 8 N 2 S. Found. N 11.56 ii. 8 Behavior of 2-Ethylmercapto-4-carboxyl-5-methyl- 6- oxy pyr- imidine on Heating. A few grams of this mercaptopyrimidine (3-4 grams) were heated in an oil bath at 22o-23o until effervescence ceased. A dark oil was obtained which solidified on cooling. This substance was purified by crystallization from hot water and separated in prisms melting at 159. It was identified as 2-ethylmercapto-5-methyl-6-oxypyrimi- dine. 1 Analysis (Kjeldahl) : 1 Wheeler and Johnson: Loc. cit. Researches on Pyrimidines. 369 Calculated for C 7 HioONjS. Found. N 16.47 16.6 Conversion of 2-Eihylmercapto-4-carboxyl-5-methyl-6-oxypyr- imidine into Thymine. Sixteen grams of this mercapto- pyrimidine, melting at 2i8-22O, were heated in an oil bath at 220-230 until the evolution of carbon dioxide ceased (about 1.5 hours). We obtained 13.0 grams of crude 2-ethyl- mercapto-5-methyl-6-oxypyrimidine (theoretical yield is 12.7 grams), which was then digested for several hours with con- centrated hydrochloric acid and converted into thymine. The acid solution was evaporated to dryness and the crude thymine decolorized in hot, aqueous solution with animal charcoal and then crystallized from water. It separated in colorless crystals, decomposing at about 325-335. The yield was 8.0 grams, or about 85 per cent, of the theoretical. Since 36-37 grams of 2-ethylmercapto-4-carboxyl-5-methyl- 6-oxypyrimidine can be obtained from 50 grams of ethyl propionate (see table) this amount of thymine (8 grams) therefore corresponds to 21.6 grams of the ester. A quantitative yield of thymine from 21.6 grams of ethyl propionate would be 26.5 grams. Analysis (Kjeldahl) : Calculated for C 5 H 6 OjN 2 . Found. N 22.22 22.OO 2-Methylamino-4-carboxyl-5-methyl-6-oxypyrimidine, NH CO I I CH 3 HNC CCH 3 .Practically a quantitative yield of N CCOOH this pyrimidine was obtained when 7 grams of 2-ethylmercapto- 4-carboxyl-5-methyl-6-oxypyrimidine and 9 grams of a 33 per cent, solution of methylamine were heated at i4O-i5o for 4 hours. It was precipitated from cold, aqueous solutions of its sodium, ammonium and methylamine salts by addition of acetic acid. It was very insoluble in cold water and crys- tallized from hot water in long, hairy crystals which melted 370 Johnson and Mackenzie. at 27O-28o, according to the rate of heating, with effervescence. Analysis (Kjeldahl) : Calculated for C 7 H 9 O 8 N 8 . Found. N 22.9 22.4 Methylamine Salt, C 7 H 9 O 3 N 3 .CH 3 NH 2 . This salt crystal- lized from hot water in granular crystals, which decomposed with violent effervescence at 274. Two of the crystals that were used for analysis weighed 0.0393 an d 0.03170 gram, respectively. Analysis (Kjeldahl) : Calculated for C 7 H908N 8 .CH 8 NH 2 . Found. N 26.1 25.91 Hydrochloride, C 7 H 9 O 3 N 3 .HC1. This salt was prepared by dissolving the pyrimidine base in hot, dilute hydrochloric acid and allowing the solution to cool. The salt separated in prismatic crystals, which decomposed at 276-283 with effervescence. Analysis (Kjeldahl) : Calculated for CjHsOaNa.HCl. Found. N I9-I4 I 9-3 2 NEW HAVEN, CONN., June 1, 1909. [Reprinted from The American Chemical Journal, Vol. XI.II. No. 5. November, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXXIL RESBARCHKvS ON PYRIMIDINES: THE ACTION OF METHYL IODIDE AND OF BENZYL CHLORIDE UPON 2-OXY-4-METHYL-6-METHYL- MERCAPTOPYRIMIDINE. 2 [FORTY-SEVENTH PAPER. ] BY HENRY L. WHEELER AND DAVID F. MCFARLAND. The papers so far published on the alkylation of mercapto- pyrimidines, with but one exception, have been confined to a description of the results obtained from a study of 2-mer- 2 Part of a thesis presented by David F. McFarland for the degree of Ph.D., Yale, 1909. 432 Wheeler and McFarland. capto-6-oxypyrimidines. In most cases 2-mercapto-6-oxy- pyrimidines were found to alkylate both in the i- and, more or less, in the 3-position. 1 In the exception mentioned, Wheeler and Johnson 2 showed that when a compound having the opposite configuration, the me reap to group and the oxygen atom being interchanged, 2-oxy-6-methylmercaptopyrimidine for example, was treated with potassium hydroxide and either methyl iodide or benzyl chloride, 3-alkyl derivatives were obtained. No i -derivative was observed in this particular alkylation. Since the 3-alkyl- 6-mercapto derivatives pass quantitatively into the 3-alkyl compounds of uracil when boiled with hydrochloric acid, this procedure is therefore the best for the preparation of these compounds. With this result at hand, it then became a question whether or not 2-oxy-6-mercaptopyrimidines in other cases or in general would be the best to select for the preparation of 3-alkyl derivatives. Cytosine, or 2-oxy-6-aminopyrimidine, which has a constitution analogous to these 2-oxy-6-mercapto- pyrimidines, also gives the 3-alkyl derivative on methylation. 3 It seems to us that any new facts in regard to methods which lead to the formation of 3-alkyl derivatives or 3-attach- ments are of interest since results have been obtained by Johnson and Clapp 4 which can be explained by the assump- tion that uracil, thymine, and cytosine are substituted or attached to other groups in these positions in the nucleic acids. In our preceding paper we have shown that 2-methylmer- capto- and 2-ethylmercapto-4-methyl-6-oxypyrimidine give i-alkyl derivatives, the formation of the isomeric 3-deriva- tives in these cases not being observed. It was there- fore of especial interest, in the above connection, to study, in this case, the alkylation of the compound having the opposite configuration, namely, 2-oxy-4-methyl-6-methylmercapto- 1 For a list of these papers see THIS JOURNAL, 42, 30 (1909). 2 Ibid. 3 Johnson and Clapp: J. Biol. Chem., 5, 163 (1908). 4 Loc. cit. Researches on Pyrimidines. 433 pyrimidine. This has now been done and we find that this pyrimidine, with methyl iodide and benzyl chloride, not only gives 3-alkyl derivatives but also the isomeric i -compounds. In the case of the action of methyl iodide, the i- and 3-iso- mers are formed in about equal proportions. Benzyl chloride gave more of the 3- than of the i -benzyl compound. The presence of a mercapto group or the absence of oxygen in the 6-position therefore appears to favor the formation of 3-alkyl derivatives. There is, however, no strict regularity in these alkylations, so that in new cases it is impossible to predict whether a i- or 3-derivative, or a mixture of both, will result. The starting point for the present work was the prepara- tion of List's 1 2-thio-4-methyluracil (I) from acetoacetic ester and thiourea. This gave 2-methylmercapto-4-methyl- 6-oxypyrimidine (II) by List's method of alkylation. The new 2-methylmercapto-4-methyl-6-chlorpyrimidine (III) was then prepared by warming the material with phos- phorus pentachloride and some oxychloride. When this compound was warmed with an excess of alcoholic potassium sulphhydrate, 2-methylmercapto-4-methyl-6-thiopyrimidine (IV) was obtained, and when the latter was boiled with hydrochloric acid 4-methyl-6-thiouracil (V) resulted. The calculated quantity of sodium ethylate and methyl iodide then gave 2-oxy-4-methyl-6-methylmercaptopyrimi- dine (VI). An excess of potassium hydroxide and methyl iodide, in alcoholic solution, gave 2-oxy-3,4-dimethyl-6- methylmercaptopyrirnidine (VIII) and 2-oxy-i,4-dimethyl- 6-methylmercaptopyrimidine(?) (VII). The latter was not obtained in a state of purity but its presence in the oily fraction was indicated by the behavior of this fraction toward hydrochloric acid. When boiled with hydrochloric acid the compounds VII and VIII gave the correspond- ing dimethyluracils described by Behrend, 2 namely, i,4-di- methyluracil (IX) and 3,4-dimethyluracil (X). 1 Ann. Chem. (Liebig), 236, 12 (1886). 2 Behrend and Dietrich: Ann. Chem. (Liebig), 309, 265 (1899). Behrend and Thurn: Ibid., 323, 160 (1902). 434 Wheeler and McFarland. The formation of i-alkyl derivatives in this reaction could be explained otherwise, without the assumption of the forma- tion of the compound represented by formula VII, if 2-0x7-4- methyl-6-methylmercaptopyrimidine lost mercaptan during the alkylation. 4-Methyluracil being formed, i-alkyl de- rivatives would result. However, we have treated 2-oxy-4- methyl-6-methylmercaptopyrimidine with alkali, under the same conditions as those in the alkylations, and we have found that no 4-methyluracil was formed. The mercapto compound was recovered unaltered. The various steps in the synthesis of these pyrimidines may, therefore, be repre- sented as follows : N==< N CCH 3 IV. CH 3 N CO :H oc a CH 3 N CCH 3 N=CCH 3 HN CCH 3 VIII. VII. IX. Researches on Pyrimidines. 435 PART. 2-Methylmercapto-4-methyl-6-chlorpyrimid'ine, N===CC1 [ 3 SC CH . Fi CH 3 SC CH . Fifty grams of 2 - methylmercapto - 4 - II II N -- CCH 3 methyl-6-oxypyrimidine were moistened with phosphorus oxychloride in a distilling bulb and then one molecular pro- portion (66.8 grams) of phosphorus pentachloride was added. No action was observed in the cold, and even when the mix- ture was warmed the reaction began only very slowly. The mass slowly liquefied with a regular, quiet evolution of hydro- gen chloride. After two hours warming, the liquefaction was complete and the evolution of gas was greatly diminished. The phosphorus oxychloride was removed by distilling under reduced pressure, and the oil which remained was poured on cracked ice. It solidified to a yellow solid, which was allowed to remain in the ice water for some time and then extracted with ether. The solution was dried with calcium chloride and, on evaporation, the oil obtained was distilled at 32-35 mm. pressure. The greater part (42 . 8 grams) boiled steadily at 147. This is 77 per cent, of the calculated. On cooling, this fraction solidified and then melted at 39-4O. The substance is very soluble in all of the common solvents. When dissolved in the smallest possible quantity of petroleum ether it separated in beautiful, long needles. These were pressed on filter paper and dried in a desiccator for analysis: Calculated for Found. N 16.02 16.02 2-Methylmercapto-4-methyl-6-thiopyrimidine, HN - CS [ 3 SC CH 3 SC CH . Ten grams of the above chlorpyrimidine II II N CCH 3 were mixed with an alcoholic solution of potassium hydro- sulphide, made by saturating a solution of 19.2 grams of potas- 436 Wheeler and McFarland. slum hydroxide in 60 cc. of alcohol with hydrogen sulphide. The chlor compound dissolved and very soon crystals of the potassium salt of 2-methylmercapto-4-methyl-6-thiopy- rimidine began to separate. The mixture was warmed for half an hour on the steam bath and the alcohol was then evaporated. The residue dissolved almost completely in water and the solution was filtered from a slight reddish sedi- ment. On acidifying with dilute acetic acid, the free thio compound was precipitated. The quantity obtained (9.85 grams) agreed almost exactly with the theoretical yield. This substance is very difficultly soluble in boiling water, from which it separates in light yellow, minute needles. It dissolves easily in ammonium hydroxide and separates again when the ammonia is boiled off. It is unstable in aqueous alkali and the solution takes on a dark red color. Dilute alcohol or water should therefore not be used in its prepara- tion. Alcohol dissolves it with some difficulty, giving needle- like prisms. On standing, the alcoholic mother liquors take on a deep red color. Calculated for C 6 H 8 N2S 2 . Found. N 16.27 16.12 2-Methylmercapto-4-methyl-6-thiopyrimidine exhibited a characteristic behavior when heated, the product obtained at high temperatures being 2,6-dithio-4-methyluracil. This substance has been described by Gabriel and Colman. 1 When slowly heated, such as is commonly done in taking melting points, it partially melted, like a mixture, at 212 to a milky mass, which effervesced on further heating and then solidified. If the tube was placed in a bath already heated to 200 and the temperature rapidly raised, it then melted completely to a clear yellow oil at 214. A slight elevation of tempera- ture caused the oil to effervesce and finally solidify to a crys- talline solid. On further heating this solid grew red, the color deepening until the material melted or decomposed to a red-brown oil at 28o-29O. One gram of the mercapto-6-thiopyrimidine was heated for 1 Ber. d. chem. Ges., 32, 2921 (1899). Researches on Pyrimidines. 437 a short time at 2i5-223. The product was treated with hot alcohol and the portion which did not dissolve was found to be also almost insoluble in boiling water. On heating, decomposition set in above 260, but the material did not melt at 310. A nitrogen determination gave 17.53 P er cent. while the calculated for 2,6-dithio-4-methyluracil is 17.72 per cent. HN -- CS 6-Thio-4-methyluracil, OC CH . This compound is I II HN - CCH 3 formed quantitatively by boiling 2-methylmercapto-4-methyl- 6-thiopyrimidine with strong hydrochloric acid for several hours and then evaporating to dryness. Long heating is re- quired to completely expel the mercaptan from the com- pound. The product is almost insoluble in alcohol and diffi- cultly soluble in boiling water. It crystallizes in irregular, prismatic forms, has a sulphur-yellow color, but no definite melting point. When heated it decomposes above 250. Calculated for Found. N 19-71 !9-4 2 The sulphur in this compound is more firmly bound than that in 6-thiouracil or 6-thiothymine. It was not removed on long heating on the steam bath with hydrochloric acid. 2-Oxy-4-methyl-6-methylmercaptopyrimidine, OC CH . Seven and six-tenths grams of 6-thio-4- I II HN - CCH 5 methyluracil were dissolved by warming with 2.2 grams of sodium hydroxide in 50 cc. of water and 30 cc. of methyl alcohol, and 8.5 grams of methyl iodide were added. The mixture was warmed until no further alkaline reaction was obtained. It was then evaporated to dryness. The product dissolved easily in water, leaving only a little oil, which was removed by filtration. On standing, pale yellow, flat, needle- 43 8 Wheeler and McFarland. like crystals formed. The yield in one case was 83 per cent. of the calculated. When recrystallized from water the ma- terial melted at i74-i75 to a clear oil. The substance was very soluble in water and alcohol, and it had a tendency to form supersaturated solutions. On this account it crys- tallized from water only after standing for some time. It is not appreciably volatile with steam. Calculated for Found. N 17.94 17-67 Action of Benzyl Chloride. Four grams of 2-oxy-4-methyl- 6-methylmercaptopyrimidine were dissolved in alcohol with 1.45 grams of potassium hydroxide. To the cooled solution, 3 . 3 grams of freshly distilled benzyl chloride were added and the mixture was allowed to stand for several days. The re- action then still being incomplete, the mixture was warmed for two hours on the steam bath. Some potassium chloride which separated was filtered off and the solution was allowed to evaporate spontaneously. A small amount of solid re- mained, mixed with a larger quantity of red oil, which had a very pungent odor. When the mixture was spread on a porous plate the oil was absorbed, leaving i . 6 grams of the sticky solid. This was very soluble in alcohol and benzene and very difficultly soluble in water. From none of these solutions could a pure crystalline substance be obtained, so the crude solid was treated directly with hydrochloric acid. One and a half grams of the solid were digested on the steam bath with a large excess of concentrated hydrochloric acid and then evaporated to dryness. This operation was twice repeated before mercaptan was completely eliminated. The residue weighed i . i grams. On crystallizing from alcohol it was found to consist principally of 4-methyluracil, decom- posing at 265-3oo, and, by extracting the product with cold chloroform a very small amount of i -benzyl- 4-methyl- uracil was obtained. The red oil, which was absorbed in a porous plate, was extracted with boiling alcohol and treated with concentrated hydrochloric acid as above. When mer- Researches on Pyrimidines. 439 captan ceased being evolved, the dry residue was stirred with alcohol. This left i . i grams of substance undissolved, while the red color of the residue was removed. When the ma- terial was crystallized once from alcohol, Hagen's j-benzyl-4- methyluracil, melting at 2^i-2^2, was obtained. The mother liquor appeared to contain a mixture of this compound and of the i -benzyl derivative. We conclude from the above that of the two isomers the 3-benzyl derivative is formed in larger amount. Action of Methyl Iodide', 2-Oxy-3,4-dimethyl-6-methylmer- captopyrimidine. Four and three- tenths grams of potassium hydroxide were dissolved in 50 cc. of methyl alcohol and 4 grams of 2-oxy-4-methyl-6-methylmercaptopyrimidine were added. This dissolved at once without warming. Three molecular proportions (n grams) of methyl iodide were then added and the mixture was allowed to stand two days. At the end of this time considerable potassium iodide had sepa- rated and the solution no longer gave an alkaline reaction. The alcohol was then evaporated under reduced pressure and the dry residue was extracted with three portions of boil- ing chloroform. On evaporation of the chloroform at ordi- nary temperatures, slightly yellow, large, prismatic crystals mixed with oil separated. The oil did not solidify on stand- ing for some time. It was removed by absorption in filter paper and saved for later treatment (see below). The crystals, which weighed 2 . 3 grams or 53 per cent, of the calculated for dimethyl-6-methylmercaptopyrimidine, were twice recrystallized from benzene. Some brownish color was removed by boiling the solution with animal charcoal, where- upon, on filtering, splendid prisms, somewhat irregular in outline, were obtained. They melted to a clear oil at 170- 171, and were found to be very soluble in water and in alco- hol, but only moderately so in benzene. Calculated for CyHioONjjS. Found. N 16.47 16.53 This material was boiled with hydrochloric acid until it gave no test for sulphur. It was then crystallized twice from alcohol, whereupon it melted at 220 and agreed in proper- 440 Wheeler and McFarland. ties with Behrend's 3,4-dimethyluracil. A nitrogen deter- mination gave 20.06 per cent, nitrogen while the calculated for this compound is 20 . oo per cent. 1,4- Dimethyl - 2 - oxy-6-mefhylmercaptopyrimidine (?). This compound was not isolated in a pure state but its presence in the oil obtained above was indicated by the hydrolysis of the oil with strong hydrochloric acid. After digesting on the steam bath until mercaptan was no longer evolved, the solu- tion was evaporated to dryness and the residue was crystal- lized from water. It separated in small prisms melting sharply, to a clear brown oil, at 260 and when mixed with a sample of /?-dimethyluracil or i,4-dimethyluracil the melting point was not lowered. Calculated for C6H 8 O 2 N 2 . Found. N 20 . 00 20 . 02 2 - Oxy -4- methyl - 6 - orthonitrobenzylmercaptopyrimidine. Molecular proportions of 6-thio-4-methyluracil, potassium hydroxide, and o-nitrobenzyl chloride were mixed in dilute alcoholic solution. The mixture was then warmed until it gave a neutral reaction. When the alcohol was evaporated and the residue was treated with water the yield of crude product agreed with the calculated amount. This material dissolved fairly easily in hot alcohol and, on cooling, it gave clusters of yellow, lancet-shaped crystals melting to a clear oil at 205. Calculated for Found. N 15.16 15.24 2 - Oxy - 4 - methyl - 6 - metadinitrophenylmercaptopyrimi- dine, prepared in a similar manner to the above, from 6-thio- 4-methyluracil, potassium hydroxide, and i-brom-2,4-dinitro- benzene, formed very small bunches of fine, mustard-yellow needles when crystallized from alcohol. It melted at 208. Calculated for CiiH 8 06N 4 S. Found. N 18.18 18.05 NEW HAVEN, CONN., May, 1909. [Reprinted from The American Chemical Journal, Vol. XLII. No. 6. December, 1909.] [Contributions from the Sheffield Laboratory of Yale University.] CLXXV. RESEARCHES ON PYRIMIDINES: SYNTHESIS OF 5-CYANURACIL. [FORTY-EIGHTH PAPER.] BY TREAT B. JOHNSON. The present work was undertaken with the object of pre- paring some pyrimidines to be used for the syntheses of new 506 Johnson. derivatives of thymine. I shall describe the preparation and properties of the nitrile of uracil-5-carboxylic acid (I), viz., 5-cyanuracil (II), and some of its derivatives. NH CO NH CO I I II CO CCOOH CO CCN I II I II NH CH NH CH (I) (II) It has been shown in a previous paper from this laboratory 1 that diethyl ethoxymethylenemalonate, 2 C 2 H 5 OCH : C(COOC 2 H 5 ) 2 , condenses smoothly with pseudothioureas, H 2 NC(SR) : NH, in aqueous solution, in the presence of alkali, giving 2-alkyl- mercapto-5-carboxyl-6-oxypyrimidines. Uracil - 5 - carboxylic acid (I) is then obtained easily by hydrolysis of these mer- captopyrimidines with acids. It seemed very probable to the writer that 5-cyanuracil (II) might be synthesized in an analogous manner from ethyl ethoxymethylenecyanacetate, C 2 H 5 OCH : C(CN)COOC 2 H 5 . DeBollemont 3 has described several esters of this character, which he has prepared by condensation of orthoformic esters, CH(OR) 3 , with esters of cyanacetic acid in presence of acetic anhydride. He has also examined the behavior of several of them towards organic bases and shown that they react smoothly with formation of the corresponding amino derivatives, RHNCH : C(CN)COOR. The action of ureas, thioureas, pseudoureas, and pseudo- thioureas on these esters, however, has not been investigated. Ethyl ethoxymethylenecyanacetate was prepared for my experiments according to the directions of deBollemont. 4 I now find that pseudoethylthiourea condenses in alcoholic 1 THIS JOURNAL, 37, 392 (1907). ( 2 Claisen: Ann. Chem. (Liebig), 297, 75 (1897). 3 Compt. Rend., 128, 1338; 129, 5; Bull. Soc. Chim., [3] 25, 18, 28, 39. 4 Loc. cit. Researches on Pyrimidines. 507 solution with this ester, in the presence of alkali, in two ways, giving a mixture of two mercaptopyrimidines. The main reaction is a condensation with the ester, similar to that with diethyl ethoxymethylenemalonate, 1 giving 2-ethyl- mercapto-5-cyan-6-oxypyrimidine (III). This condensation is represented by the following equation: NH 2 COOC 2 H 5 NH CO II II C 2 H 5 SC + CCN = 2C 2 H 5 OH + C 2 H 5 SC CCN II II II II NH CHOC 2 H 5 N CH (III) A part of the acrylic ester, however, reacts with the pseudo- thiourea, giving 2-ethylmercapto-5-carbethoxy-6-amino- pyrimidine (IV), which has previously been described in a paper from this laboratory. 2 NH 2 CN N = CNH 2 II II C 2 H 5 SC + CCOOC 2 H 5 = C 2 H 5 OH + C 2 H 5 SC = CCOOC 2 H 5 II II || II NH CHOC 2 H 5 N CH (IV) This interesting observation suggests that other cyanides containing the grouping C 2 H 5 OCH : C CN will be found to I condense with pseudothioureas, giving aminopyrimidines. It is interesting to note here that ethyl acetylcyanacetate, CH 3 COCH(CN)COOC 2 H 5 , does not condense with pseudoethylthiourea to give a pyrimidine. 3 On the other hand, it has been shown that the pseudoureas, 4 H 2 NC(OR) : NH, condense with ethyl cyan- acetate, in presence of alkali, giving the corresponding 2-alkyl- oxy-4-amino-6-oxypyrimidines. 5 1 Loc. cit. 2 Wheeler and Johns: THIS JOURNAL, 38, 594. 3 Wheeler: THIS JOURNAL, 28, 358. 4 Stieglitz and McKee: Ber. d. chem. Ges., 32, 1494; 33, 807, 1517; Stieglitz and Noble: Ibid., 38, 2243. "Central., 1904,11, 631. 508 Johnson. I also investigated the action of pseudoethylthiourea on ethyl ethoxymethylenecyanacetate (V) in alcohol containing no free alkali. When they were allowed to react under these conditions in molecular proportions, there was no evidence of the forma- tion of 2-ethylmercapto-5-cyan-6-oxypyrimidine (III), and 2-ethylmercapto-5-carbethoxy-6-aminopyrimidine (IV) and ethyl a-cyan-/?-pseudoethylthiourea-acrylate (VIII) were the only products of the reaction. If more than one molecular proportion of pseudoethylthiourea is used for the condensation the ethyl a-cyan-/?-pseudoethylthiourea-acrylate that is formed then reacts with the excess of pseudothiourea, giving a-cyan-/?- pseudoethylthiourea-acrylpseudoethylthiourea (VI) . Ethyl a-cyan-/?-pseudoethylthiourea-acrylate (VIII) and a-cyan-/?-pseudoethylthiourea-acrylpseudoethylthiourea (VI) are both converted quantitatively into 2-ethylmercapto-5- cyan-6-oxypyrimidine (III) when warmed with alkali. A quantitative yield of 5-cyanuracil (XI) is then obtained by hydrolysis of this mercaptopyrimidine, in alcohol, with sul- phuric acid. Attempts to hydrolyze this mercaptopyrimidine (III) smoothly to 5-cyanuracil, in aqueous solution, were unsuccessful. The nitrile group slowly undergoes hydrolysis under these conditions, giving a mixture of 5-cyanuracil (XI) and uracil-5-carboxamide (X). These are both converted quantitatively into uracil-5-carboxylic acid (IX) and finally into uracil (VII) by long digestion with concentrated hydrochloric acid. These various transformations are represented by the following formulas : Researches on Pyrimidines. 509 O 3 o tti O O o H L 4~* H L Q _ 8 O w 503 P JU 2=0 szj W 0=00 o o W O o o o 1 0= w 1 8^ % r < * < O O I / i ^ o \ ^ o * W W M I M i ~ p w 0=00 Wgo W 0=00 W o o ffi o P W o a; W 5 io Johnson. EXPERIMENTAL PART. Ethyl Ethoxymethylenecyanacetate, C 2 H 5 OCH : C(CN)COOC 2 H 5 . This ester was prepared ac- cording to the directions given by de Bollemont. 1 It melts at 5 2 ~53 anc * is insoluble in water but very soluble in alcohol, ether, and benzene. Condensation of Ethyl Ethoxymethylenecyanacetate with One Molecular Proportion of Pseudoethylthiourea in Alcohol. Ethyl a-Cyan-fl-pseudoethylthiourea-acrylate, NH 2 COOC 2 H 5 C 2 H 5 SC CCN . Molecular proportions of potassium N- CH hydroxide (0.45 gram) and the hydrobromide of pseudoethyl- thiourea (1.4 grams) were dissolved separately in 7 cc. of 95 per cent, alcohol, the solutions cooled to o, and combined. The undissolved potassium bromide was then separated quickly by filtration and 1.2 grams of ethyl ethoxymethylene- cyanacetate added to the cold alcohol solution of pseudo- thiourea. The ester dissolved at once and within 1-2 minutes a yellow, crystalline substance separated. After standing a few minutes this was filtered off, washed thoroughly with ether, and the ether washings saved (see below). The sub- stance obtained here, insoluble in ether, was the ethyl acrylate represented above and melted without further purification at I2o-i25. The ester is soluble in alcohol, insoluble in cold water and ether, and crystallizes from benzene in tufts of yel- low distorted needles melting at 130 with slight effervescence. It separates from a hot aqueous solution as an oil, which finally crystallizes in colorless needles melting at the same temperature. Nitrogen (Kjeldahl) : Calculated for Found. N 18.50 18.7 Loc. cit. Researches on Pyrimidines. 511 2-Ethylmercapto-5-carbethoxy-6-aminopyrimidine, N = CNH 2 I I C 2 H 5 SC CCOOC 2 H to . This pyrimidine was obtained when II II N CH the ether solution described above was allowed to evaporate. It was insoluble in cold, dilute sodium hydroxide solution but soluble in hydrochloric acid, and crystallized from 95 per cent, alcohol in rectangular plates melting at iO2-iO3 to a clear oil with no effervescence. This same pyrimidine has previously been described by Wheeler and Johns. 1 A mixture of my derivative and their pyrimidine melted at 102. Ni- trogen (Kjeldahl) : Calculated for CH,30 2 N3S. Found. N 18.50 18.6 The comparative yields of the above acrylic ester and this 6-aminopyrimidine were irregular, but the ester was always the chief product of the reaction. They were apparently the only compounds formed under the conditions employed. Action of Alkali on Ethyl a-Cyan-ft-pseudoethylthiourea- acrylate. This ester is not decomposed by cold, dilute sodium hydroxide solution. On the other hand, when warmed with alkali it immediately dissolved, giving the sodium salt of 2-ethylmercapto-5-cyan-6-oxypyrimidine. When the alkaline solution was acidified with hydrochloric acid, 2-Ethylmercapto-^-cyan- 6 -oxy pyrimidine, NH CO I I C 2 H 5 SC CCN, separated in colorless crystals melting at II II N CH 2i8-2o to an oil. It crystallizes from hot water in prisms, which melt at 222 to a clear oil without effervescence. The pyrimidine gives a strong test for uracil when treated with bromine water and barium hydroxide solution. 2 Nitrogen determinations (Kjeldahl) : 1 Loc. cit. 2 Wheeler and Johnson: J. Biol. Chcm., 3, 183 (1907). 512 Johnson. Calculated for Found. C 7 H 7 ON 8 S. I. II. N 23.22 23.0 23.15 This pyrimidine and 2-ethylmercapto-5-carbethoxy-6-amino- pyrimidine are the only products of the reaction when pseudo- ethylthiourea is condensed with ethyl ethoxymethylenecyan- acetate in alcohol in the presence of an excess of alkali. Condensation of Ethyl Ethoxymethylenecyanacetate with More than One Molecular Proportion of Pseudoethylikiourea in Alcohol. An alcoholic solution of pseudoethylthiourea was prepared by dissolving 20 grams of the hydrobromide of pseudoethyl- thiourea (1.7 mols.) in 70 cc. of absolute alcohol and then adding, at o, a solution of 6.1 grams of potassium hydroxide (1.7 mols.) in 30 cc. of 95 per cent, alcohol. After filtering from potassium bromide, 10.7 grams of ethyl ethoxymethylene- cyanacetate (i mol.) were added to the pseudourea solution at once when there was an immediate reaction and a thick mush of crystalline material was obtained. A portion of this material was filtered off and washed with ether. It then melted at i28-i3O. This insoluble substance was identified as ethyl a-cyan-/?-pseudoethylthiourea-acrylate, which was ob- tained in my previous condensation (see above). On evapora- ting the ether solution I obtained 2-ethylmercapto-5-carb- ethoxy-6-aminopyrimidine 1 which was identified by its melt- ing point, IO2-I03. The main portion of the reaction mixture, however, was allowed to stand, at ordinary temperature, for about 2-3 hours, when, to my surprise, practically all of the acrylic ester, with the exception of about 1-2 grams, had dissolved. The solution was then filtered, concentrated on the steam bath, and cooled whenl obtained about 12-13 grams of crystalline material. This was washed with ether to remove any 2-ethylmercapto-5- carbethoxy-6-aminopyrimidine, and then triturated with cold, dilute sodium hydroxide solution. I obtained 10.5 grams of a crystalline substance insoluble in the alkaline solution. It 1 Wheeler and Johns: Z,oc. cit. Researches on Pyrimidines. 513 was soluble in hot water and alcohol and crystallized from hot water in well-developed prisms which melted at 164- 165 with effervescence. A nitrogen determination agreed with the calculated value for a-cyan-ft-pseudoethylthiourea-acrylpseudo- ethylthiourea, NH 2 C(SC 2 H 5 ) :NCH :C(CN)CO.N :C(SC 2 H 5 )NH 2 . Calculated for Found. N 24.56 24.43 When the sodium hydroxide solution (above) was carefully acidified with hydrochloric acid, 2-ethylmercapto-5-cyan-6- oxypyrimidine separated. It crystallized in prisms melting at 222 to an oil. Analysis (Kjeldahl) : Calculated for C 7 H 7 ON3S. Found. N 23.22 23.0 Action of Alkali on a-Cyan-p-pseudoethylthiourea-acrylpseudo- ethylthiourea. About 1.5 grams of this compound were warmed with 10 cc. of a 10 per cent, aqueous solution of sodium hydroxide for a few minutes. Ethyl mercaptan was evolved and the compound dissolved, giving a clear solution. When this was acidified with hydrochloric acid 2-ethylmercapto-5-cyan-6- oxypyrimidine separated in prisms melting at 22O-222. Analysis (Kjeldahl): Calculated for C 7 H 7 ON3S. Found. N 23.22 23.16 Behavior with Hydrochloric Acid. One gram of a-cyan-/?- pseudoethylthiourea-acrylpseudoethylthiourea was dissolved in about 40-50 cc. of concentrated hydrochloric acid and the solution evaporated to dryness. I obtained practically a quantitative yield of 5-cyanuracil (see below) which melted without purification at 29i-293. After one crystallization from water it melted at 293-295 and a nitrogen determina- tion (Kjeldahl) gave: 4 * Calculated for C 5 H 3 O 2 N3. Found. N 30.6 30.2 514 Johnson. 2,6-Dioxy-5-cyanpyrimidine (5-Cyanuracil) , NH CO C.CN. CO C.CN. A quantitative yield of this pyrimidine was also NH CH obtained by hydrolysis of 2-ethylmercapto-5-cyan-6-oxypyrimi- dine with sulphuric acid as follows: 0.7 gram of the mercapto- pyrimidine was dissolved in a mixture of 30 cc. of alcohol and 3 cc. of concentrated sulphuric acid and the solution boiled until the evolution of ethyl mercaptan ceased. The 5-cyan- pyrimidine separated in hard, granular crystals melting at 29i-292. It is difficultly soluble in cold alcohol and crystal- lizes from hot water in prisms which melt at 295 with de- composition. Analysis (Kjeldahl) : Calculated for Found. I. II. N 30.6 30.1 30-55 When this pyrimidine was digested with concentrated hydrochloric acid for several hours it was converted quan- titatively into uracil. Analysis (Kjeldahl) : Calculated for C4H 4 O2N2. Found. N 25.00 24.6 NH CO Uracil- j-carboxamide, CO CCONH 2 . One gram of 2-ethyl- I II NH CH mercapto-5-cyan-6-oxypyrimidine was dissolved in 50 cc. of concentrated hydrochloric acid and the solution evaporated to dryness on the steam bath. The crystalline material which was obtained was then dissolved in hot water and the solution cooled slowly, when this pyrimidine separated in small prisms. It is difficultly soluble in cold water and does not melt below 300. It gives a strong test for uracil when treated with bromine water and barium hydroxide. After heating for 5 hours at ioo-iio a nitrogen determination (Kjeldahl) gave: Calculated for CsHsOaNa. Found. N 27.00 26.80 Researches on Pyrimidines. 515 This amide was converted quantitatively into uracil when digested with concentrated hydrochloric acid. The aqueous mother liquor (above), after being filtered from uracil- 5 -carboxamide, was concentrated to a small volume and cooled, when the characteristic prisms of 5-cyan- uracil separated. After two crystallizations from hot water it melted at 293-295. Analysis (Kjeldahl) : Calculated for CsHaOzNs. Found. N 30.6 30.1 NEW HAVEN, CONN., July, 1909. [Reprinted from The American Chemical Journal, Vol. XI,III. No. i. January, 1910.] [Contributions from the Sheffield Laboratory of Yale University.] CLXXVIL RESEARCHES ON PYRIMIDINES: THE THIO DERIVATIVES OF THYMINE AND THE PREPARATION OF THYMINE. 1 [FORTY-NINTH PAPER.] BY HENRY L. WHEELER AND DAVID F. MCFARLAND. In preparing uracil from ethylpseudothiourea hydrobromide and the sodium salt of ethyl formylacetate, 2 for some unknown reason it has sometimes happened that the yield obtained was very poor. A more satisfactory result was invariably obtained when sodiumformylacetate and thiourea were used. 3 The employment of thiourea instead of the pseudothiourea had the additional advantage of being a more direct process. It rendered unnecessary the preparation of the pseudothio- urea and was therefore less expensive, and, perhaps better than all, the presence of mercaptan was avoided. These results suggested that the preparation of thymine might be improved in a similar manner. When the sodium salt of ethyl formylpropionate was condensed with thiourea in aqueous solution (four experiments) we were surprised to find that the yield of 2-thiothymine (I) was invariably about one-half what would be expected from the pseudothiourea condensation under similar conditions. Obviously this was no improvement over our original method. We have recently found, in other cases, that certain /?-ketone esters condense with thiourea in alcoholic solution when they 1 Part of a thesis presented by David F. McFarland for the degree of Ph.D., Yale, 1909. 2 Wheeler and Merriam: THIS JOURNAL, 29, 478 (1903). 3 Wheeler and Liddle: Ibid., 40, 547 (1908). 2o Wheeler and McFarland. failed to do so in water. The condensation of ethyl sodium- formylpropionate with thiourea was therefore carried out in alcoholic solution (eight experiments). This brought about the desired improvement. The yields were over twice those obtained in the aqueous condensation. For comparison with our original method ethyl sodium- formylpropionate was condensed with ethylpseudothiourea in aqueous (four experiments) and alcoholic (one experiment) solutions. It was then found that the average yield of 2-ethyl- mercapto-5-methyl-6-oxypyrimidine, in these experiments, when expressed in the equivalent weight of thymine, was nearly identical (20.9 per cent, of the calculated) with that calculated from the average yield of 2-thiothymine obtained in the alcoholic condensations (22.6 per cent, of the calcula- ted). In other words, almost the same amount of thymine is ob- tained in these condensations whether we use thiourea in alco- hol or pseudothiourea in water or alcohol. The use of thio- urea, however, gives a little better yield and it is also to be recommended for the reasons stated above. The condensa- tion takes place as follows : HNH C 2 H 5 OCO HN CO C 2 H 5 OH I III SC + CCH 3 = SC CCH 3 + I II I II HNH NaOCH HN CH NaOH It is peculiar that a similar condensation failed to take place in the case of urea and ethyl sodiumformylpropionate or ethyl sodiumformylacetate 1 in either absolute alcohol or aqueous solutions. 2-Thiothymine (I), like 2-thiouracil, is desulphurized and quantitatively converted into thymine (V) by simply dis- solving in water and evaporating to dryness with a slight ex- cess of chloracetic acid. The thymine prepared in this manner 1 Wheeler and Liddle: Loc. cit. Researches on Pyrimidines. 21 melts higher and appears to be more readily purified than when prepared by means of a pseudothiourea. We have prepared 6-thiothymine (II) and 2,6-dithiothy- mine (IV) in a manner similar to that used in the case of the corresponding uracil 1 and 4-methyluracil derivatives. 2 The preparation of the 6-thio derivatives in the thy mine series is far less satisfactory than in the case of those previously de- scribed. The 2-ethylmercapto-5-methyl-6-thiopyrimidine (III), on boiling with hydrochloric acid, evolves mercaptan less easily than in the previous cases and the sulphur in the 6-position is also more or less removed at the same time, so that the yield is poor, or nothing but thymine is obtained. A similar behavior was observed in the preparation of 6-thiouracil but only on long warming with acid. On the other hand, 6-thio-4-methyluracil evolved mercaptan smoothly without being desulphurized in the 6-position. 2, 6-Dithio thymine (IV) was prepared from 2-ethylmer- capto-5-methyl-6-thiopyrimidine (III) by heating with dry hydrogen chloride. We have found that the methylation of 6-thiothymine first gives 2-oxy-5-methyl-6-methylmercaptopyrimidine (VI), as would be expected, and on further methylation this com- pound yields the 3-methyl derivative, 2-oxy-3,5-dimethyl-6- methylmercaptopyrimidine (VII). This result is in accord- ance with the general tendency, previously observed by us, of a 6-mercapto-2-oxypyrimidine to alkylate in the 3-posi- tion. 2-Oxy-3,5-dimethyl-6-methylmercaptopyrimidine (VII), on boiling with hydrochloric acid, gives a quantitative yield of 3-methylthymine (VIII). These compounds and their trans- formations may be represented as follows: 1 Wheeler and Liddle: Loc. cit. 2 Wheeler and McFarland: THIS JOURNAL, 42, 431 (1909). 22 Wheeler and McFarland. HN CS HN CS HN CS CCH, J.JLJ.TI V^WJ J.JLJ.-* ^>y-f JLJL-l.1l OC CCH 3 < C 2 H 5 SC CCH 3 > SC \ N=CSCH 3 N=CSCH 3 H 3 > OC CCH 3 A 1 -*^*-x' OC CCJ HN CH CH 3 N CH VI. VII. CH 3 N CH VIII. EXPERIMENTAL PART. Ethyl Sodiumformylpropionate, NaOCH = C(CH 3 )CO 2 C 2 H 5 . This salt was prepared, as Wislicenus directs, 1 in a manner similar to that used in the case of ethyl sodiumformylacetate. We have obtained the best results in the preparation of these salts when one atomic proportion of sodium was covered with dry ether in a flask attached to a return condenser and the mixture of esters slowly added. An excess of ethyl formate was employed and the amount of ether was sufficient to pre- vent the mixture from becoming thick. Three or four times the volume of the mixed esters was usually sufficient. If evaporation of the ether took place, owing to the esters being added too rapidly, or if not enough was present, the product frequently was sticky and difficult or impossible to filter. It therefore could not be washed with ether and dried satisfac- torily. When 100 to 150 grams of each ester were used about a day and a half or two days was taken to add the mixture to the sodium. The sodium disappeared and the reaction was complete in about four days altogether. In one experiment from 150 grams of ethyl propionate and Ber. d. chem. Ges., 20, 2934 (1887). Researches on Pyrimidines. 23 900 cc. of ether with one atomic proportion of sodium and 1 50 grams of ethyl formate, 145 grams of dry sodium salt were ob- tained. In the ether filtrate 9.6 grams of 2-thiothymine were found, or an equivalent of 32 . 3 grams more of sodium salt (see experiment XIV below). The total yield of crude sodium salt was therefore 177.3 grams for 150 grams of ethyl propionate, or 118.2 grams for 100 grams of ester. In another experiment which was carried out in the same manner as the above, 50 grams of ethyl propionate gave 43 . 2 grams of dry sodium salt and an amount of 2-thiothymine (4.3 grams) in the ether filtrate (experiment XV) equivalent to 1 6. 8 grams more of salt, making a total of 60 grams or 120 grams for 100 grams of ethyl propionate. This is 79 per cent, of the calculated. (See also experiment XIII below.) In the condensations in which we have used this crude salt, the yields of pyrimidine derivatives have in no case amounted to much over 30 per cent, of the calculated. The salt is obviously not pure, but whether or not this fact alone accounts for the low yield, or whether stereochemically different salts exist here, one form condens- ing and the other not, will have to be determined later. Preparation of 2-Ethylmercapto-5-methyl-6-oxy pyrimidine* HN CO I I C 2 H 5 SC CCH 3 . I. One hundred grams of propionic ester II II N CH and 80 grams of formic ester were added to 22.5 grams of sodium covered with ether. The ethyl sodiumformylpropion- ate was not filtered off but was condensed with 100 grams of pseudoethylthiourea hydrobromide by means of 65 grams of potassium hydroxide in about 750 cc. of water. On acidify- ing with acetic acid, after warming the mixture, 33 grams of 2-ethylmercapto-5-methyl-6-oxypyrimidine melting at 150 were obtained. II. In another experiment 150 grams of propionic ester, 120 grams of formic ester and 33.7 grams of sodium were used. The product was mixed with a solution of 150 grams of ethylpseudothiourea hydrobromide and 97 grams of potas- 1 See Wheeler and Johnson: THIS JOURNAL, 31, 595 (1904). 24 Wheeler and McFarland. suim hydroxide in about a liter of water. The yield of mer- captopyrimidine was 57.5 grams. III. This experiment was carried out by Dr. Victor C. Myers. One hundred and fifteen grams of propionic ester, TOO grams formic ester and 26 grams of sodium in 250 cc. of ether were taken. The sodium salt was not filtered off but was condensed with 97 grams of ethylpseudothiourea hydro- bromide and 30 grams of potassium hydroxide. The solu- tion was allowed to stand for 24 hours and then concentrated to one-half volume (300 cc.?) before precipitating with dilute acetic acid. The precipitate weighed 61.5 grams. This probably contained some potassium and sodium salts since on recrystallizing from water 40 grams of material melting at i58-i59 was obtained. IV. The above experiment was repeated with the same quantities and under the same conditions. The first portion of mercapto compounds separated weighed 53 . 3 grams. When recrystallized from water 38 . 3 grams of pure material were ob- tained. The mother liquor contained sodium and potas- sium salts. V. In this experiment the condensation was tried in alco- hol. Thirteen and a half grams of dry ethyl sodiumformyl- propionate were mixed with 16.4 grams of ethylpseudothio- urea hydrobromide in 300 cc. of absolute alcohol in which 2.0 grams of sodium had been dissolved. Complete solution did not take place. The mixture was shaken and let stand for 20 hours, then warmed on the steam bath, evaporated to dry- ness and dissolved in 60 cc. of water and precipitated with acetic acid. The precipitate weighed 3.85 grams. The following table gives a further comparison of the above results : Calculated weight of Calculated Weight of ethyl Weight of ethyl so- diumforrayl- Weight of mercapto derivative mercapto derivative per 100 Per cent, yield of mercapto weight of thymine from 100 propionate. propionate. obtained. grams ester. derivative. grams ester. I 100 I20 1 33-o 33-o 19-75 24.4 II I 5 ISO 1 57-5 38.3 22.9 28.4 HI II 5 I38 1 61.5 53-3 31-9 39-5 IV 115 I38 1 53-2 46-3 27-7 34-3 V ... 13-5 3-85 33-7 20.2 24.9 1 Calculated from average yield of salt. Researches on Pyrimidines. 25 If we exclude experiments III and IV, in which the results are a little too high, as already stated, the average yield of thymine is 25.9 grams from 100 grams of ethyl propionate (or including these results it is 30.3 grams). Twenty-five and nine- tenths grams of thymine is 20.9 per cent, of the theoretical amount calculated directly from the ethyl pro- pionate. HN CO I I Preparation of 2-Thiothymine, SC CCH 3 , in Alcoholic I II HN CH Solution. VI. Nineteen grams of ethyl sodiumformylpro- pionate, pressed on paper and dried until all odor of ether had disappeared, were added to a solution of 9.5 grams (one molecule) of thiourea and 2 . 9 grams of sodium in 350 cc. of absolute alcohol. The mixture was warmed for 3 hours, evaporated to dryness, dissolved in 80 cc. of water and pre- cipitated with dilute acetic acid. The crystalline precipitate weighed 6 . 8 grams. VII. Twenty-eight grams of the sodium salt were warmed for five hours with 14 grams (i molecule) of thiourea in 400 cc. of absolute alcohol, then evaporated to dryness, dissolved in water and precipitated with acetic acid. The yield in this case was 8 . o grams. VIII. Fourteen and one-tenth grams of the dry sodium salt were treated with the same relative proportions of the thiourea, sodium ethylate, and alcohol and under the same conditions as in experiment VI. The yield of 2-thiothymine was 3 . 4 grams. IX. Fifteen grams of the sodium salt, 4 grams of thiourea (about one-half molecule) and 2 . 2 grams of sodium dissolved in 150 cc. of absolute alcohol were mixed and heated for 3 hours. Then 25 cc. of water were added and the whole evap- orated to dryness. The residue was taken up in 60 cc. of water and precipitated with acetic acid. The precipitate weighed 4 . o grams. On recrystallizing from 17.5 cc. of alco- hol 3 . 4 grams separated. 26 Wheeler and McFarland. X. Twenty-seven grams of the sodium salt were warmed with 13.5 grams (one molecule) of thiourea and 4.3 grams of sodium in 350 cc. of 95 per cent, alcohol for three and a half hours. There remained considerable material undissolved. The alcohol was evaporated and the residue was dissolved in 140 cc. of water. The addition of acetic acid to this solution gave 8 . o grams of 2-thiothymine. XI. Thirteen and two- tenths grams of sodium salt were warmed with 3.5 grams of thiourea (a little over one-half molecule) and 2 grams of sodium in 150 cc. of 95 per cent, alcohol for 3 hours. Twenty-five cc. of water were added and the solution was warmed for a half hour, then evaporated to dryness. The residue was taken up in 60 cc. of water and precipitated, whereupon 3 . 3 grams of 2-thiothymine were obtained. XII. Fifteen grams of dry sodium salt were warmed 3 hours with 4 grams of thiourea (one-half molecule + 0.5 gram) and 2.3 grams of sodium dissolved in 100 cc. of 85 per cent, alcohol. The sodium salt was first dissolved in 15 cc. of water and then the sodium ethylate and thiourea were added. Some solid separated after warming, 10 cc. more water were added and the solution heated for a half hour longer. On evaporating to dryness, dissolving in 60 cc. of water and precipitating, 3.8 grams of 2-thiothymine were obtained. On recrystallizing from 175 cc. of water 3.2 grams separated. XIII. Fifty grams of ethyl propionate, 43 . 5 grams of ethyl formate and 11.3 grams of sodium gave 47 . 5 grams of dry ethyl sodiumformylpropionate. The ether filtrate was evap- orated to dryness and the residue was combined with the above. Calculating on the basis of 65 grams of sodium salt in the entire lot, twenty grams of thiourea (over half mole- cule) in 500 cc. of 95 per cent, alcohol were added. The mix- ture was heated three and a half hours, the alcohol was then evaporated and the residue dissolved in 175 cc. of water. When precipitated as in the previous cases, 15 grams of 2-thio- thymine were obtained. XIV. The ether filtrate from a condensation in which 150 grams of ethyl propionate were converted into sodium salt Researches on Pyrimidines. 27 was evaporated to dryness and treated with 4 grams of sodium in 400 cc. of absolute alcohol and 20 grams of thiourea. The mixture was heated one hour on the steam bath and then evaporated to dryness. Two hundred and fifty cc. of water were added and the solution, on precipitating with acetic acid, gave 9.6 grams of 2-thiothymine. From the average yield of four condensations with the filtered salt it is calculated that the ether solution in this case contained 32 . 3 grams of ethyl sodiumformylpropionate. XV. The ether filtrate from the preparation of ethyl so- diumformylpropionate in which 50 grams of ethyl propionate were used was treated as above with 3 grams of sodium in loo cc. of absolute alcohol and 5 grams of thiourea. The mix- ture was warmed for 3 hours, then 35 cc. of water were added and the solution was warmed for a half hour longer. On evaporating to dryness, taking up in 50 cc. of water, and add- ing acetic acid 4.3 grams of 2-thiothymine were obtained. The following table gives the results of the condensation of ethyl sodiumformylpropionate with thiourea in alcoholic solu- tions : Calculated Number of Weight weight Calculated Weight of cc. of alcohol of of 2-thio- Per cent. weight of ethyl so- diumformyl- for i gram of sodium 2-thio- thymine thymine from 100 yield of 2-thio- thymine from 100 propionate. salt. obtained grams ester. thymine. grams ester. VI IQ.O 18.4 6.8 42.3 30-4 37-6 VII 28.0 H-3 8.0 33-8 24.2 30.0 VIII I4.I 18.4 3-4 28.6 20.5 25-3 IX 15.0 10. 4.0 32.0 23-0 28.4 X 27-0 13.0 8.0 35-o 25-1 31.0 XI 13.2 n-3 3-3 30.0 21.6 26.6 XII 15.0 5-7 3-8 30-4 21.8 27.0 XIII 6O.O? 8-3 15-0 30.0 20. 1 24.8 Experiments VI, VII, VIII and IX were carried out in ab- solute alcohol, X, XI and XIII in 95 per cent, alcohol, and XII in 85 per cent, alcohol. Experiment VIII is a duplicate of VI. Sodium ethylate and the calculated quantity of thio- urea were present. Number VII was similar except that no sodium ethylate was used. In IX sodium ethylate was pres- ent but only one-half the calculated quantity of thiourea 28 Wheeler and McFarland. was added. In the remaining experiments sodium ethylate and about one-half the calculated quantity of thiourea was employed, except in X, where one molecular proportion of thiourea was used. If we exclude experiment VI, which for some unknown rea- son gave a much higher yield of 2-thiothymine than the others, and also experiment XIII, in which the amount of salt taken was determined by calculation, we find that the average yield of thy mine by this method is 28.0 grams from 100 grams of ethyl propionate. This is 22.6 per cent, of the calculated. Preparation of 2-Thiothymine in Aqueous Solution. XVI. Seventeen grams of ethyl sodiumformylpropionate were dis- solved in a solution of 8.5 grams (one molecule) of thiourea in 65 cc. of water. The solution was allowed to stand for 10 hours, then precipitated with acetic acid. The yield of 2-thio- thymine was 2 . 5 grams. XVII. (By WAITER F. STOREY.) One hundred grams of propionic ester were condensed with an excess of ethyl formate and a little over (2 grams) one atomic proportion of sodium. When all the sodium had disappeared the calculated quantity (one molecule) of thiourea was added in ice water and the mixture thoroughly shaken. After standing overnight the ether was removed and, after warming, concentrating and cooling, acetic acid was added. The yield was 14.5 grams of the thiopyrimidine. XVIII. One hundred and fifty grams of ethyl propionate, 115 grams of ethyl formate and 36 grams instead of 33.8 of sodium were used in this experiment. When the sodium had disappeared a solution of 55 grams of thiourea in 400 cc. of water was prepared (38 grams is one- half the calculated amount). Lumps of ice were added to the solution of the salt and the ether solution. The whole was shaken and allowed to stand overnight. The deep red solution was concentrated on the steam bath and finally cooled and acetic acid added. The precipitate weighed 27 grams, and from the filtrate 3 grams more were obtained on standing. XIX. One hundred grams of each ester were condensed with 2 grams more than the calculated amount of sodium. Researches on Pyrimidines. 29 The whole was mixed with a saturated solution of 70 grams of thiourea in ice water. After standing a few minutes it was warmed and acetic acid added. Fifteen grams of 2-thio- thymine were obtained. The following table gives the results of the condensation of ethyl sodiumformylpropionate with thiourea in aqueous solu- tion. Calculated weight of Calculated Weight of Weight 2-thio- weight of ethyl of thymine Per cent. thymine Weight of sodium- 2-thio- from 100 yield of from propionic ester. formyl- propionate. thymine grams obtained, ester. a-thio- thymine 100 grams ester. i? 1 2-5 17-4 12.5 15-4 100 I20 1 14-5 H-5 10-4 12.8 150 iSo 1 30.0 20. o 14.4 17.7 IOO I20 1 15.0 15.0 10.7 13-3 XVI XVII XVIII XIX The average yield of thymine by this method is 14.8 grams from 100 grams of ethyl propionate. This is only 11.9 per cent, of the calculated. HN CO I I The Preparation of Thymine, OC CCH 3 . It was shown by I II HN CH Wheeler and Merriam that 2-methylmercapto-5-methyl-6- oxypyrimidine 2 gave 99 per cent, of the calculated amount of thymine when boiled with hydrochloric acid. The ethyl- mercapto derivative gives a similar result. 2-Thiothymine, like 2-thiouracil, is easily desulphurized by simply evaporating its aqueous solution with chloracetic acid. 3 For example, 3 grams of 2-thiothymine were dissolved in 250 cc. of boiling water and 3 grams of chloracetic acid were added. The solution was then evaporated to dry ness on the steam bath and the residue was washed with 15-20 cc. of alcohol. It was free from sulphur and weighed 2.55 grams or 95 . 8 per cent, of the calculated. On evaporating the alcoholic washings more was obtained. The washed ma- 1 Calculated from average yield of salt. 2 THIS JOURNAL, 29, 487 (1903). 3 Ibid., 40, 552 (1908). 30 Wheeler and McFarland. terial consisted of snow-white crystals or scales and it melted at 340. This is a higher melting point than that observed for thymine from other sources. Fischer and Roeder 1 give 321, Wheeler and Merriam 2 326. A nitrogen determina- tion by W. F. Storey gave: Calculated for C6H 6 O 2 N 2 . Found. N 22.22 22.21 In desulphurizing larger quantities it is unnecessary to dis- solve all the material at first and relatively less chloracetic acid may be used. Properties of 2-Thiothymine. The properties of 2-thio- thymine and its salts have been investigated by W. F. Storey. When the substance is first obtained by acidifying the alka- line solutions with acetic acid it separates slowly, usually not all at once, as a crystalline brownish- white precipitate. It has about the same solubility in water as in alcohol and it crystallizes from these solvents in fairly stout prisms, which have a tendency to retain the color of the solutions. One hun- dred parts of water at 20 dissolved 0.133 part of 2-thio- thymine. This is the average of two closely agreeing deter- minations. Thymine is more than twice as soluble in water. Mr. Storey found that 100 parts of water at 23 dissolved 0.303 part thymine. Calculated for C 6 H 6 ON 2 S. Found. N 19-71 19-67 2-Thiothymine has more pronounced acid properties than thymine. When crystallized from aqueous ammonia it re- tains ammonia. On drying, the amount of ammonia re- tained (21.86 per cent, nitrogen) was less than that calcula- ted for a i : i salt (26.41 per cent, nitrogen). The salts of the alkali metals are very soluble in water. A solution of the sodium salt gave with silver nitrate an amorphous white precipitate, which did not blacken on boiling. The boiling 1 Ber. d. chem. Ges., 34, 3751 (1901). 2 Loc. cit. Researches on Pyrimidines. 31 aqueous solution gives a white precipitate with mercuric chloride. It did not blacken on boiling alone or in the pres- ence of alkali. The sodium salt of 2-thiothymine, C 5 H 5 ON 2 SNa.i.5H 2 O, ap- pears to be less soluble than the potassium salt. It was formed by dissolving the pyrimidine in water containing the calcula- ted quantity of sodium hydroxide, concentrating and then crystallizing from aqueous alcohol. It formed radiating prisms which did not melt at 300. Calculated for Found. C 5 H B ON 2 SNa.i.5H 2 0. N 14.73 14.73 14.58 H 2 O 14.21 14.65 The potassium salt, C 5 H 5 ON 2 SK.H 2 O, is soluble in less than three parts of water. From dilute alcohol it forms radiating, colorless prisms, similar to the sodium salt. Calculated for C 6 H 6 ON 2 SK.H2O. Found. N 14.14 '13-83 The Copper Salt, C 5 H 4 ON 2 SCu.H 2 O. This salt was prepared by adding copper sulphate in aqueous solution to a hot satura- ted aqueous solution of 2-thiothymine. It formed a green, amorphous precipitate, which was washed with water and dried over sulphuric acid. Calculated for oO. Found. N 12.58 12.52 H 2 O 8.08 7.79 It may be mentioned that the corresponding copper salt of 2-thiouracil, which also contains one molecule of water, 1 has a mustard-yellow color. These salts do not blacken on boiling. List 2 found that 2-thio-4-methyluracil in hot, satura- ted, aqueous solution gave a yellow precipitate with copper sulphate (this salt was anhydrous), while if the potassium salt was used a green copper salt was obtained. 1 Wheeler and Liddle: Loc. cit. 2 Ann. Chem. (Liebig), 236, 9. 11 (1886). 32 Wheeler and McFarland. 2-Benzylmercapto-5-methyl-6-oxypyrimidine, HN CO I I C 6 H 5 CH 2 SC CCH 3 (By W. F. STOREY) .This was prepared II II N CH from the above potassium salt by warming in alcoholic solu- tion with benzyl chloride. From dilute alcohol it formed colorless needles, which melted to a clear oil at 2O4-2O5. Calculated for Ci,H lt ON 2 S. Found. N 12.07 12.25 2- Ethylmer capto-j- methyl- 6 - thiopyrimidine , HN CS I I C 2 H 5 SC CCH 3 . An alcoholic solution of potassium hydro- II II N CH sulphide was prepared by saturating a solution of 57 grams of potassium hydroxide in 250 cc. of alcohol with hydrogen sulphide. To this solution 24 grams of 2-ethylmercapto-5- methyl-6-chlorpyrimidine 1 were added. The reaction began immediately and after warming on the steam bath for nearly an hour the solution was evaporated to dryness. The resi- due dissolved completely in water and on acidifying with acetic acid a bulky precipitate separated. After washing with water and drying it was found that the yield was practically quantitative. The crude material melted at about 175 to a clear oil and on further heating it solidified at i8o-i9O and remained solid until 280 was reached, when it melted with some effervescence. The substance dissolved easily in boiling alcohol. When the solution was quickly cooled the material crystallized in plates, but when allowed to cool slowly, pris- matic needles separated. It then melted at 181 to a clear oil ; the pure material, on further heating, did not solidify. Calculated for C7Hi N s S a . Found. N I5-5 I 4-9 I 1 Wheeler and Johnson: THIS JOURNAL, 81, 595 (1904). Researches on Pyrimidines. 33 HN CS I I 2,6-Dithiothymine, SC CCH 3 . Two grams of 2-ethyl- I II HN CH mercapto-5-methyl-6-thiopyrimidine were heated in an oil bath at 215. The substance melted and a small portion sublimed. On passing perfectly dry hydrogen chloride into the molten mass the material almost immediately solidified. The passage of the gas was continued for about five minutes. After cooling, the solid was treated with ammonium hydrox- ide, which dissolved the greater part, leaving a small amount of a pungent smelling oil. This was separated from the am- monia solution and the latter was acidified with acetic acid. A bulky yellow precipitate resulted, which was only slightly soluble in water but dissolved fairly easily in alcohol, and then, on cooling, gave bunches of small, bright yellow needles. They melted at 281 with decomposition and effervescence. Calculated for C 6 H 6 N 2 S2. Found. N 17.94 18.14 HN CS I I 6-Thiothymine, OC CCH 3 . When 2-ethylmercapto-5- HN- CH methyl-6-thiopyrimidine is heated with strong hydrochloric acid it does not smoothly go into 6-thiothymine as might be expected from analogy with the corresponding 4-methyl compound, but instead it shows a strong tendency to lose sulphur from the 6-position and pass directly into thymine. In fact, by boiling ten grams of the 2-ethylmercapto-6-thio- pyrimidine for one and a half hours with strong hydrochloric acid and then twice evaporating to dryness with additional acid, very pure thymine was obtained which, after one recrys- tallization, gave no test for sulphur and melted at 340. A nitrogen determination gave 22.22 per cent, nitrogen. This is the calculated value for thymine. By a less vigorous treatment with acid, the decomposition 34 Wheeler and McFarland. was not carried so far and a small yield of 6-thiothymine was obtained. Several experiments were required to show the con- ditions best adapted to obtaining this result. It was found advisable to stop the hydrolysis while there still remained some unchanged mercapto derivative. This was then ex- tracted by means of alcohol, leaving a mixture of 6-thiothy- mine with a small amount of thymine. These were separa- ted by crystallizing from water. Nine grams of 2-ethylmercapto-5-methyl-6-thiopyrimidine were twice evaporated to dryness on the steam bath with 45 cc. of concentrated hydrochloric acid and an equal quantity of water. The dry residue was boiled for a few minutes with loo cc. of alcohol and the portion which remained undissolved was filtered off. This weighed 4.2 grams. It was dissolved in boiling water and on cooling gave bright yellow, matted, silky needles, which melted with some effervescence at 330. Calculated for C 5 H 6 ON,S. Found. N 19-72 J 9-98 2-Oxy-5-methyl-6-methylmercaptopyrimidine, N=CSCH 3 I I OC CCH 3 . Four grams of 6-thiothymine were dissolved HN- CH with 1.58 grams of potassium hydroxide in 125 cc. of alcohol and 20 cc. of water. The change from the yellow pyrimidine to the potassium salt was marked by an immediate change to a white color. Four grams of methyl iodide were added to the solution and the mixture was warmed for 15 minutes on the water bath and then allowed to stand overnight. The re- action was complete at the end of this time and the solution became neutral. The alcohol was evaporated and the resi- due was found to be easily soluble in hot water. On crystal- lizing from this solvent bunches of light yellow, prismatic needles were obtained which, after two recrystallizations from alco- hol, melted at 205 -2 1 1 . Calculated for Found. C 6 H 8 ON S S. I. ^ II. N 17.94 18.08 18.14 Researches on Pyrimidines. 35 2-Oxy-3,5-dimethyl-6-methylmercaptopyrimidine, N=CSCH 3 I I OC CCH 3 . Four and seven- tenths grams of potas- I II CH 3 N CH slum hydroxide and 4.4 grams of 2-oxy-5-methyl-6-methyl- mercaptopyrimidine were dissolved in 150 cc. of 95 per cent, alcohol. Twelve grams of methyl iodide, or three molecular quantities, were added and the mixture was warmed for an hour on the steam bath under a return condenser. The solu- tion then became neutral. After evaporating off the alcohol, the solid residue was extracted three times with boiling chloro- form. An oily residue separated from the chloroform solu- tion which solidified to a white solid on being stirred. It weighed 4.5 grams, or 94 per cent, of the theoretical amount of dime thylme thy Imercaptopyrimidine. This material was extremely soluble in alcohol. It also dissolved easily in boil- ing water and benzene. From benzene solutions it crystaj- lized in clusters of crystals resembling grains of wheat. From the water solutions, which had a marked tendency to remain supersaturated even after standing several hours, the sub- stance crystallized in white, prismatic needles, which melted to a colorless oil at 83 . Calculated for C 7 Hi ON 2 S. Found. N 16.4 16.6 Treatment with Hydrochloric Acid: j-Methylthymine. In order to determine the structure of the above compoud two grams of it were boiled for 3 hours with 25 cc. of concentrated hydrochloric acid and an equal quantity of water, under a return condenser. On evaporating off the acid i . 4 grams of a white solid was left behind, which melted at 28i-283. One crystallization of this from water gave one gram of material melting at 284 and exhibiting all the properties of 3-methyl- thymine, as described by Johnson and Clapp. 1 Especially noticeable were the two forms of crystals (needles and prisms) 1 J. Biol. Chem., d(^49 (1908). 36 Wheeler and McFarland. described. When this sample was mixed with a sample of their material the melting point was unchanged. Calculated for Found. N 2O. O 20.1 The mother liquor from this compound was carefully ex- amined for the isomeric i-methylthymine, but this substance was not found. In order to determine whether or not any of the isomeric 2-oxy-i,5-dimethyl-6-methylmercaptopyrimidine had been formed with the 3,5-dimethyl derivative, the mother liquors from the crystallization of 2-oxy-3,5-dimethyl-6-methyl- mercaptopyrimidine were boiled for 3 hours with hydrochloric acid, under a return condenser. After evaporating the acid the solid residue weighed i . i grams. From this, by crystal- lizing from water, were recovered 0.8 gram of nearly pure 3-methylthymine, melting at 275-28i and showing the characteristic properties. There was also obtained a very small quantity (0.15 gram) of another substance which sub- limed in white needles. This substance melted at 153- 155 and was extremely soluble in water and alcohol. Not enough was obtained for analysis, but it is probable that it was 1,3- dimethylthymine, described by Johnson and Clapp. They give the melting point as 153. When our material was mixed with theirs the melting point was not lowered. The formation of i,3-dimethylthymine may be explained by sup- posing a small amount of thymine to have been left in the 6-thiothymine. The difficulty of obtaining 6-thiothymine free from thymine has been noted above. Thymine, on methyl- ation, would give i,3-dimethylthymine. No evidence could be found here of the presence of i-methyl- thymine. It seems fair to conclude, therefore, that under the conditions employed in the methylation of this 6-methyl- mercaptopyrimidine only a 3-derivative was formed. NEW HAVKN, CONN.; September, 1909. VII. ON SOME PICROLONATES: GUANIDINS. BY HENRY L. WHEELER AND GEORGE S. JAMIESON. (From the Sheffield Laboratory of Yale University,) (Received for publication, December 13, 1907.) In a paper recently published by one of us 1 on some salts of cytosin, isocytosin, 6-aminopyrimidin and 6-oxypyrimidin the melting or effervescing points of the picrolonates were given. When the these salts were first prepared it was found that Kjel- dahl's method of analysis gave low results. The method was as follows: About 0.110.12 gram of substance was added to 20 cc. of sulphuric acid, then 2 or more grams of zinc dust and finally 15 grams of potassium sulphate ; the digestion then being carried on as usual. On the other hand this method proved successful in the case of the corresponding picrates. The picrolonic acid which we used was obtained from Kahl- baum. It was shown to be a pure sample by an absolute nitro- gen determination and by a carbon and hydrogen determina- tion, but the analysis of this material by Kjeldahl's method also gave results that were several per cent less than the calcu- lated. The difficulty was finally found to be due to the insolubility of picrolonic acid in concentrated sulphuric acid since Kjeldahl's procedure gave results agreeing with the calculated when the reduction was carried out in dilute sulphuric acid with the addi- tion of some alcohol. Although numerous picrolonates have recently been described it appears that nitrogen in these salts has invariably been determined by the absolute method. 2 Before correct results had been obtained in the analysis of our sample of picrolonic acid and in order to identify the material 1 This Journal, iii, p. 285, 1907. 2 For a list of papers on picrolonates up to the year 1907, see Matthes and Rammstedt: Archiv d. Pharmazie, ccxlv, p. 112, 1907. Since then, Levene: Biochem. Zeitschr., iv, p. 320, 1907, and Warren and Weiss: This Journal, iii, p. 327, 1907. ii2 On Some Picrolonates : Guanidins the guanidin salt was prepared. This salt agreed in properties with the description of guanidin picrolonate given by Schenck. 1 The action of aqueous solutions of amines, primary or second- ary, or ammonia on the alkylhalide addition products of thio- ureas affords a very convenient method for the preparation of certain substituted guanidins, especially those that have been found in meat extract 2 and urine. 3 The action takes place at ordinary temperature, mercaptan is liberated, and on evaporating, a guanidin salt is obtained. For example, when the methyl iodide addition product of thiourea, I (2-methylpseudothioureahydriodide), was dissolved in an excess of a 33 per cent solution of dimethylamine bubbles of methyl - mercaptan escaped. After evaporating off the excess of amine by warming, then adding a hot aqueous solution of picrolonic acid, unsymmetrical dimethyl guanidin (2, 2 -dimethyl guanidin) picrolonate separated. This salt agreed with the description of dimethyl guanidin picrolonate given by Kutscher. 4 NH 2 NH 2 NHCH 3 NHCH 3 II II CH 3 SCI - (CH 3 ) 2 NCI C 2 H 5 SCBr -> CH 3 NHCBr II II NH 2 NH 2 NH 2 NH 2 I II III IV The hydrobromide of the isomeric symmetrical dimethyl guanidin, IV.; (i, 2-dimethyl guanidin), was obtained by treat- ing the ethylbromide addition product of methyl thiourea, III, ( i -methyl- 2-ethylpseudothiourea) with methylamine. The melting point of the picrolonate prepared from this substance was identical with that assigned to symmetrical di- methyl guanidin picrolonate by Schenck. 5 Monomethyl guanidin hydrobromide was prepared in two ways, by dissolving the ethylbromide addition product of thiourea in an aqueous solution of methylamine and by dissolving the corre- sponding addition product of methylthiourea in concentrated 1 Zeitschr. f. physiol. Chem., xliv, p. 427, 1905. 2 Kutscher and Lohmann: Zeitschr. f. physiol Chem., xlviii, p. i, 1906. 3 Achelis: Ibid., 1, p. 10, 1906; Kutscher: Ibid, li, p. 457, 1907. 4 Loc. cit. 5 Zeitschr. f. physiol. Chem., xlviii, p. 423, 1906. Henry L. Wheeler and George S. Jamieson 113 ammonia. As would be expected from the non-existence of tautomeric forms in the case of the amidines 1 the picrolonates of the material prepared by these two methods were identical. The reactions and the substances may be represented as follows : (i)NH 2 (i)NH 2 I I CH 3 NH 2 + (2) C 2 H 5 S - C - Br = (2) CH 3 NH - C - Br + C 2 H 5 SH. I I (3)NH 2 ( 3 )NH 2 CH 3 NH CH 3 NH I I NH 3 + C 2 H 5 S - C - Br = H 2 N - C - Br + C 2 H 5 SH. I I NH 2 NH 2 The nomenclature hitherto employed for pseudothioureas and guanidins (symmetrical and unsymmetrical) is not sufficient to define substituted derivatives in general. The writer therefore proposes to use that system which has been found so convenient in the purin 2 and pyrimidin series, namely, of assigning numbers (as shown above) to indicate the position of substituents in the case of the pseudourea, pseudothiourea and guanidin derivatives. Dieckmann 3 has used this system for parabanic acid compounds. This then brings about a systematic nomenclature for all these related substances. EXPERIMENTAL PART. Cytosin Picrolonate, C 4 H 5 ON 3 .C 10 H 8 O 5 N 4 . A half a gram of cytosin in a 100 cc. of water was added to one gram of picrolonic acid in about 350 cc. of hot water. On rapidly cooling the solu- tion an amorphous, gelatinous precipitate separated. On slowly cooling or on crystallizing from water little balls of fine bright yellow needles or slender prisms separated. This salt is very difficultly soluble in alcohol from which it is obtained in flat prisms or plates. It melts with effervescence about 27o-273. The nitrogen determinations in the case of this and the other picrolonates were determined by Kjeldahl's method as follows: 1 Wheeler and Johnson: Amer. Chem. Journ., xxxi, p. 577, 1904. 2 Ber. d. deutsch. chem. Gesellsch., xxx, p. 549, 1897. 3 Ibid., xl, p. 3737, 1907. H4 On Some Picrolonates : Guanidins About o.i gram of substance was dissolved in a mixture of 35 cc. of alcohol and 20 cc. of dilute sulphuric acid. Two grams of zinc dust were used for the reduction and the solution was warmed until colorless. Then 10 cc. of concentrated sulphuric acid and 12 grams of potassium sulphate were added, the water and alcohol was evaporated and the residue was digested for two or three hours. The analysis was then finished in the usual manner. Calculated for Ci4H 13 O 6 N 7 : Found: N ........................ 26.13 26.15 Since this salt was prepared by us it has been obtained by Levene. 1 He gives the formula C 4 H 6 N 3 .C 10 H 8 N 4 O 5 , which is evidently a typographical error. 2-Amino-6-oxypyrimidin (Isocytosin) Picrolonate , C 4 H 5 ON 3 , C 10 H 8 O 5 N 4 . A half a gram of isocytosin and 1.2 gram of picro- lonic acid, on mixing the hot aqueous solutions (about 400 cc.), gave an immediate, bulky precipitate consisting of very fine, yellow hair-like needles. This salt is practically insoluble in alcohol and its melting or effervescing point varies with the rate of heating. It usuallv decomposes about 273- 275. Calculated for Found: N ...... . ................. 26.13 26.05 ' 6-Aminopyrimidin Picrolonate, C 4 H 5 N 3 .C 10 H 8 O 5 N 4 . A solution of o.i gram of 6-aminopyrimidin in 4 cc. of water was added to 0.3 gram picrolonic acid in 100 cc. of hot water. This gave an immediate precipitate and on cooling, the salt separated in the form of interlaced, yellow, hairlike needles. The yield was 0.35 gram, calculated, 0.4 gram. It melted with effervescence at 261 ; on rapidly heating it melted a few degrees higher. Calculated for Ci 4 H, 3 5 N 7 : Found: N ..... ................... 27.29 27.10 6-Oxypyrimidin Picrolonate, C 4 H 4 ON 2 .C 10 H 8 O 5 N 4 . A solution of 0.2 gram of 6-oxypyrimidin in 6 cc. of water was added to a hot solution of 0.55 gram picrolonic acid in 125 cc. of water. 1 Biochem. Zeitschr., iv, p. 320, 1907. Henry L. Wheeler and George S. Jamieson 115 This gave an immediate precipitate and on cooling, a bulky mass of slender, yellow needles separated. The yield was 0.55 gram. When this was heated it showed signs of change at 1 80 and then melted with vigorous effervescence at 222. The salt wasrecrys- tallized from water and from alcohol. It then melted suddenly, in both cases, at i9i-i93 It is difficultly soluble in alcohol and it separates from this solvent in thin scales or plates. Calculated for Found: N ......... ............... 23.33 23.33 MethylguanidinPicrolonate, C 2 H 7 N 3 .C 10 H 8 O 5 N 4 . 2-Ethyl-pseu- dothiourea hydrobromide was dissolved in an excess of a 33 per cent solution of methylamine and allowed to stand over night. The excess of amine and mercaptan was then evaporated and the solution, after filtering from a slight turbidity, was divided into two parts. One part was precipitated with picric acid; the picrate formed needles which melted sharply at 200. Fischer 1 gives the melting point of methyl guanidin picrate at 200. The remaining solution was precipitated with an aqueous solu- tion of picrolonic acid. The salt formed a compact, not bulky, yellow precipitate which when recrystallized from water formed minute, diamond shaped tables or blocks. It decomposed with effervescence at 291. Calculated for Ci2Hi 5 O 5 N 7 : Found: N ........................ 28.24 28.23 i-Methylguanidin was also prepared by dissolving 1.5 gram of i-methyl-2-ethylpseudothiourea hydrobromide in concentrated ammonia. On evaporating the solution and adding an aque- ous solution of picrolonic acid the precipitate obtained was iden- tical with the above. 2 ,2-Dimethylguanidin Picrate, C 3 H 9 N 3 .C 6 H 3 O 7 N 3 . The guani- din was prepared by dissolving 2-ethylpseudothiourea hydro- bromide in a strong, aqueous solution of dimethylamine. The precipitate produced by adding an aqueous solution of picric acid was crystallized from water. It formed small, pointed, 1 Ber. d. deutsch. chem. Gesellsch. xxx, p. 2414, 1897. n6 On Some Picrolonates : Guanidins yellow prisms or branch-like growths. It melted to an oil with- out effervescence at 224. Calculated for C 9 H 12 7 N 6 : Found: N 26.58 26.53 2,2-Dimethylguanidin Picrolonate , C 3 H 9 N 3 .C 10 H 8 O 5 N 4 . The guanidin was prepared as above and also by the action of dimethyl - amine on 2-methylpseudothiourea hydriodide. The solutions- gave an immediate precipitate with aqueous picrolonic acid. The salt was crystallized from water, it then formed small, flat, four-sided, yellow prisms which decomposed with effervescence quite sharply at 278. Kutscher and Lohmann give the melting point of their dimethylguanidin picrolonate obtained from urine at 275-278. 1 This salt is very difficultly soluble in alcohol from which it forms needle-like prisms. Calculated for CisHiyOsN?: Found: N 27.13 27.13 i ,2-Dimeihylguanidin Picrate, C 3 H 9 N 3 .C 6 H 3 O 7 N 3 . One gram of i-methyl-2-ethylpseudothiourea hydrobromide was treated with an excess of strong aqueous methylamine solution. The picrate formed yellow prisms with uneven striated faces. It melted to a clear oil at 178. Calculated'for C 9 Hi 2 O 7 N 6 : Found: N 26.58 26.58 A portion of the guanidin solution was precipitated with picro- lonic acid. This precipitate crystallized from water in thin, yellow plates which melted at 262. It agreed with the descrip- tion given by Schenck 2 who gives the melting point 26o-262. This also confirms the conclusion of Kutscher and Lohmann that the dimethyl guanidin from urine has the isomeric structure. In preparing guanidins by the above method an excess of amine must be used and the solutions should be strong, in order to bring about a complete reaction. Otherwise unaltered pseudo- thiourea will be precipitated as picrate or picrolonate. 1 Zeitschr. f. physiol. Chem., xlviii, p. 423, 1906. Henry L. Wheeler and George S. 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Jour. 36, 160. Centr. B. 1906, II, 1066 . T. B. Johnson and G. A. Menge, 5-Ethylcytosin. Jour. Biolog. Chem. 2, 105. Centr. B. 1906, II, 1507. _ H. Steudel, Ueber die Oxydation der Nucleinsaure. Zeit physiol. Chem. 48, 425. M. A. Whitely, 1, 3-Diphenylbarbituric Acid and Some Colored Derivatives of the Same. Synthesis of 1, 3-Diphenyl- uric Acid. Proceedings Chem. Soc. (London), 22, 200. Centr. B. 1906, II, 1404. J. K. Wood, The Acidic Constants of Some Ureides and Uric Acid Derivatives. Jour. Chem. Soc. (London), 89, 1831. Centr. B. 1907, I, 539. P. A. Levene und J. A. Mandel, Darstellung und Analyse einiger Nucleinsauren. Nucleinsaure der Spermatozoen des Maifisches. Zeit. physiol. Chem. 50, 1. Centr. B. 1907, I, 354. BIBLIOGRAPHY 19 J. A. Mandel and P. A. Levene, On the Pyrimidine Bases of the Nucleic Acid Obtained from Fish Eggs. Jour. Biolog. Chem. 1, 425. Centr. B. 1906, 1, 1790. 1907. H. Burrows and C. A. Keane, The Condensation of Diethylmalonamide with Aldehydes. Jour. Chem. Soc. (Lon- don), 91, 269. Centr. B. 1907, I, 1270. H. L. Wheeler, T. B. Johnson and C. O. Johns, Synthesis of Uracil-5-carboxylic Acid. Amer. Chem. Jour. 37, 392. Centr. B. 1907, I, 1632. E. F. J. Atkinson, H. Ingham and J. F. Thorpe, The Formation and Reactions of Iminocomponds. The Formation of 1, 3-Naphthylenediamine and Its Derivatives ' from o- Tolunitrile. Jour. Chem. Soc. (London), 91, 583. Centr. B. 1907, II, 69. R. Burian, Weitere Beitrage zur Kenntnis der Diazoam- inoverbindungen der Purinbasen. Zeit. physiol. Chem. 51, 425. Ibid. Pyrimidinderivate aus Purinbasen. Ziet. physiol. Chem. 51, 438. Centr. B. 1907, II, 141. R. Behrend und O. Hoebel, Ueber Alkylderivate des Methyluracils. Ann. 353, 242. Centr. B. 1907, II, 304. R. Behrend und G. Offe, Ueber die Oxydation von Uracil- derivaten. Ann. 353, 267. Centr. B. 1907, II, 305. T. B. Johnson and F. W. Heyl, Some Condensation Pro- ducts of a Substituted Pseudothiourea : Synthesis of 1-Methyl- uracil. Amer. Chem. Jour. 37, 628. Centr. B. 1907, II, 449. S. J. M. Auld, Mercury Derivatives of Pseudo Acids Con- taining the Group CONH. Jour. Chem. Soc. (London), 91, 1045. Centr. B. 1907, II, 831. P. A. Levene, Notiz iiber die Pikrolonate einiger Nuclein- basen. Biochem. Zeit. 4, 320. Centr. B. 1907, II, 587. Ibid. Ueber die diuretische Wirkung des Thymins. Biochem. Zeit. 4, 316. Centr. B. 1907, II, 619; also Jour. Exper. Medicine, 9, 229. W. Gossling, Dialkylbarbitursauren. Chem. Ztg. 31, 711. Centr. B. 1907, II, 689. M. A. Whiteley, Studies in the Barbituric Acid Series. 1, 3-Diphenylbarbituric Acid and Some Coloured Derivatives. Jour. Chem. Soc. (London), 91, 1330. Centr. B. 1907, II, 1065. H. L. Wheeler and T. B. Johnson, On a Color Test for Uracil and Cytosin. Jour. Biolog. Chem. 3, 183. Centr. 1907, II, 1087. A. P. N. Franchimont and H. Friedmann, L'action de 1'acide azotique reel sur la trimethylene-ureine et sur 1'hy- drouracil. Rec. trav. chim. 26, 218. Centr. B. 1907, II, 1248. 20 BIBLIOGRAPHY J. Tafel und P. A. Houseman, Zur Kenntniss des Isopurons. Ber. 40, 3743. Centr. B. 1907, II, 1401. H. L. Wheeler, On Some Salts of Cytosin, Isocytosin, 6- Aminopyrimidin and 6-Oxypyrimidin. Jour. Biolog. Chem. 3, 285. H. L. Wheeler, Uracil-4-Carboxylic Acid. Amer. Chem. Jour. 38, 358. Centr. B. 1907, II, 1634. T. B. Johnson and F. W. Heyl, The Action of Methyl Iodide on 2-Anilino-6-oxypyrimidine, and the Synthesis of 2- Anilinoyprimidine. Amer. Chem. Jour. 38, 237. Centr. B. 1907, II, 1248. T. B. Johnson, Synthesis of Thymin 4-Carboxylic Acid. Jour. Biolog. Chem. 3, 299. Centr. B. 1907, II, 1531. H. L. Wheeler and C. O. Johns, Synthesis of Cytosine-5- Carboxylic Acid. Amer. Chem. Jour. 38, 594. Centr. B. 1908, I, 289. T. B. Johnson and C. F. Speh, Synthesis of Thymine-5- Carboxylic Acid. Amer. Chem. Jour. 38, 602. Centr. B. 1908, I, 390. T. B. Johnson and F. W. Heyl, Synthesis of 4-Methyl- uracil-5-Acetic Acid. Amer. Chem. Jour. 38, 659. Centr. B. 1908, I, 391. M. Conrad, Zur Kenntnis der Hydurilsaure. Ann. 356, 24. Centr. B. 1907, II, 1609. U. Suzuki, K. Aso und H. Mitarai, Ueber die chemische Zusammensetzung der japanischen Sojasauce oder Schoyu. Bull, college Agric. Tokio, 7, 477. Centr. B. 1907, II, 1649. J. Tafel und H. B. Thompson, Elektrolytische Reduktion der Aethylbarbitursauren. Ber. 40, 4489. Centr. B. 1908, I, 122. H. Steudel, Ueber die Bildung von Pyrimidinderivate aus Purinkorpern. Zeit. physiol. Chem. 53, 508. Centr. B. 1908, I, 123. A. Einhorn und H. v. Diesbach, Ueber die Reduktion der Diathylthiobarbitursaure. Ber. 40, 4902. Centr. B. 1908, I, 455. H. L. Wheeler and G. S. Jamieson, On Some Picrolonates. Jour. Biolog. Chem. 3, 111. 1908. F. Sachs und E. Appenzeller, Uber den Tetramethyl-2, 4-diaminobenzaldehyde. Ber. 41, 91. Centr. B. 1908, 1,^519. R. Majima, Uber die Kondensation der Alkylguanidine mit Acetessigester usw. Ber. 41, 176. Centr. B. 1908, I, 1044. F. Baum, Zur Kenntniss der Traubeschen Pyrimidin-syn- thesen. Ber. 41, 532. Centr. B. 1908, I, 1167. BIBLIOGRAPHY 21 S. Gabriel, Uber einige synthetisch verwerthbase Derivate der Aminosauren. Ber. 41, 242. Centr. B. 1908, I, 729. A. Einhorn, Ueber neue Arzneimittel. Ann. 359, 145. Centr. B. 1908, I, 1535. P. A. Levene und J. A. Mandel, Zur Herkunst des Cytos- ins beider Hydrolyse der tierischen Nucleinsauren. Biochem. Zeit. 9, 233. Centr. B. 1908, I, 1710. T. B. Osborne and F. W. Heyl, The Pyrimidine Deriva- tives in Nucleic Acid. Amer. Jour, of Physiol. 21, 157. T. de Haan, La condensation 5-dicetones avec Turee. Rec. trav. chim. 27, 162. Centr. B. 1908, II, 35. O. Kuhling, Uber Phenacyldialursaure, Tartronursaure und Isohydantoinsaure. Ber. 41, 1658. Centr. B. 1908, II, 53. F. Pohl, Zur Kenntniss des Dicyandiamids. Jour. pr. Chem (2) 77, 533. Centr. B. 1908, II, 151. P. A. Levene und J. Mandel, Uber die Konstitution der Thymonucleinsaure. Ber. 41, 1905. Centr. B. 1908, II, 424. R. Behrend und K. Beer, Uber Trioxydihydromethyl- uracil. Ann. 362, 115. Centr. B. 1908, II, 886. T. B. Johnson, A Method of Separating Thymin from Uracil. Jour. Bilog. Chem. 4, 407. Centr. B. 1908, II, 1043. T. B. Johnson, The Action of Nitric Acid on 2, 6-Dioxy- pyrimidines. Oxynitrohydrothymine. Amer. Chem. Jour. 40, 19. Centr. B. 1908, II, 802. L. Hugouneng et A. Morel, Contribution a 1'etude de la constitution des nucleoproteides. Recherches sur les consti- tuants de la pepsine. Compt. rend. 147, 212. Centr. B. 1908, II, 805. H. L. Wheeler and L. M. Liddle, Synthesis of Uracil-3- acetic Acid. Jour. Amer. Chem. Soc. 30, 1152. Centr. B. 1908, II, 1045. Ibid: Synthesis of Uracil-4-acetic Acid. Jour. Amer. Chem. Soc. 30, 1156. Centr. B. 1908, II, 1045. T. B. Johnson and S. H. Clapp, Syntheses of Some Nitro- gen-Alkyl Derivatives of Cytosine, Thymine and Uracil. Jour. Biolog. Chem. 5, 49. Centr. B. 1908, II, 1264. T. B. Johnson and S. H. Clapp, The Action of Diazo- Benzene Sulphonic Acid on Thymin, Uracil and Cytosin. Jour. Biolog. Chem. 5, 163. Centr. B. 1908, II, 1872. J. Mandel, Jacobs and P. Levene, On Nucleic Acids. Proceedings Soc. Exp. Biolog. Med. 5, 92. T. B. Johnson and W. F. Storey, The Action of Potassium Thiocyanate Upon Some Imide-Chlorides. Amer. Chem. Jour. 40, 131. Centr. B. 1908, II, 1105. 22 BIBLIOGRAPHY E. L. Pinner, Uber 2-Phenyl-4,6-dioxypyrimidine, Malonylbenzamidine. Ber. 41, 3517. Centr. B. 1908, II, 1692. H. L. Wheeler and C. O. Johns, Synthesis of Cytosine- 5-Carboxamide. Amer. Chem. Jour. 40, 234. Centr. B. 1908, II, 1781. C. O. Johns, Synthesis of 4-Methylcytosine. Amer. Chem. Jour. 40, 348. Centr. B. 1908, II, 1933. T. B. Johnson and J. H. Derby, Jr., Syntheses of Some Benzyl Derivatives of Uracil and Thymine. Amer. Chem. j our . 40, 444. Centr. B. 1909, I, 85. F. Sachs und G. Meyerheim, Uber Azinpurine. Ber. 41, 3957. Centr. B. 1909, I, 31. T. B. Johnson and D. B. Jones, Synthesis of new Deriva- tives of 5-Hydroxyuracil (Isobarbituric Acid). Amer. Chem. Jour. 40, 538. Centr. B. 1909, I, 193. H. L. Wheeler and L. M. Liddle, Thio Derivatives of Uracil and the Preparation of Uracil in Quantity. Amer. Chem. Jour. 40, 547. Centr. B. 1909, I, 447. 1909. M. Engelmann, Uber eine Synthese des 1-Methylxanthins. Ber. 42, 177. Centr. B. 1909, I, 524, 806. A. Guyot et E. Michel, Condensation des ethers mes- oxaliques avec les amines aromatique tertiaires. Compt. rend. 148, 229. C. O. Johns, On the Formation of Purine Derivatives from 4-Methyl Cytosine. Amer. Chem. Jour. 41, 58., Centr. B. 1909, I, 925. M. Conrad und A. Schulze, Uber Malonylamidderivate. Ber. 42, 729. Centr. B. 1909, I, 1087. O. Stark, Zur Konstitution des Acetylacetonharnstoffs (4, 6-Dimethyl-2-ketopyrimidin). Kondensation mit aroma- tischen Aldehyden. Ber. 42, 699. Centr. B. 1909, I, 1243. ^ Ibid: Zur Konstitution des Acetylacetonharnstoffs. Ein- wirkung von Brom auf Acetylacetonharnstoff und seine Kon- densationsprodukte mit Aldehyden. Ber. 42, 708. Centr. B. 1909, I, 1245. E. Fourneau, Sur les acides oxyamines. Derives amines de 1'acide oxyisobutyrique. Bull. soc. chim. (4) 5, 229. Centr. B. 1909, I, 1318 (Hydrothymin). A. Hantzsch, Uber Pantochromie und Chromoisomerie von Violuraten und verwandten Oximinoketonsalzen. Ber. 42, 966. Centr. B. 1909, I, 1390. A. Hantzsch und B. Issaias, Uber polychrome und Ester der Violursauregruppe. Ber. 42, 986. Centr. B. 1909, I, 1393. BIBLIOGRAPHY 23 H. Hantzsch und B. Issaias, Uber polychrome und chromotrope Violurate. Ber. 42, 1000. Centr. B. 1909, I, 1395. R. Behrend und R. Schultz, Uber die Oxydation des Harn- saure in alkalischer Losung. Ann. 365, 21. Centr. B. 1909, I, 1378. O. Kiihling und B. Schneider, Uber Kondensationspro- dukte des Alloxans. Ber. 42, 1285. Centr. B. 1909, I, 1548, 1952. F. G. Dunan and W. Schneider, The Colour of Aqueous Solutions of Violuric Acid. Jour. Chem. Soc. (London), 95, 956. Centr. B. 1909, II, 348. J. K. Wood and E. A. Anderson, The Constitution of the Salts of Barbituric Acid. Jour. Chem. Soc. (London), 95, 979. Centr. B. 1909, II, 426. T. B. Johnson and D. B. Jones, Synthesis of 1-Methyl- 5-oxyuracil. Jour. Amer. Chem. Soc. 31, 590. Centr. B. 1909, II, 545. J. Posner und K. Rohde, Beitrage zur Kenntniss der ungesattigten Verbindungen. Uber die Addition von Hy- droxylamin auf ungesattigte Sauren mit Konjugierten Doppel- bindungen. Ber. 42, 2785. Centr. B. 1909, II, 705. (Cin- namenyldihydrouracil) . H. L. Wheeler and T. B. Johnson, The Preparation of 3-Methyl and 3-Benzyluracil. Amer. Chem. Jour. 42, 30. Centr. B. 1909, II, 1047. H. L. Wheeler and D. F. McFarland. The Preparation of 1, 4-Dimethyluracil and the Monobenzyl Derivatives of 4- Methyluracil. Amer. Chem. Jour. 42, 101. Centr. B. 1909, II, 1048. T. B. Johnson and H. H. Guest, Sulphur Derivatives of 5-Oxyuracil. Pheparation of 5-Benzylmercaptouracil and 5- Benzylmercaptocytosine. Amer. Chem. Jour. 42, 271. Centr. B. 1909, II, 1637. T. B. Johnson and K. G. Mackenzie, Dimethyl Derivatives of 2-Aminopyrimidine. Preparation of 2-Methylamino-5- Methylpyrimidine. Amer. Chem. Jour. 42, 353. Centr. B. 1910, I, 284. H. L. Wheeler and D. F. McFarland, The Action of Methyl Iodide and of Benzyl Chloride Upon 2-Oxy-4-Methyl- 6-Methylmercaptopyrimidine. Amer. Chem. Jour. 42, 431. Centr. B. 1910, I, 1030. T. B. Johnson, Synthesis of 5-Cyanuracil. Amer. Chem. Jour. 42, 505. H. L. Wheeler and D. F. McFarland, The Thio Deriva- 24 BIBLIOGRAPHY tives of Thymine and the Preparation of Thymine. Amer. Chem. Jour. 43, 19. C. Billow und K. Haas, Synthetische Versuche zur Dar- stellung von Derivaten des heterokondensierten heterocyclis- chen 1, 3-Triazo-7, 0-pyrimidins. Ber. 42, 4638. Centr. B. 1910, I, 286. J. Zerewitinow, Uber die organischen Salze der Violur- saure. Ber. 42, 4802. Centr. B. 1910, I, 342. PATENTS 1900. Centr. B. 1. 113. 1901. Centr. B. I. 547, 1219. 1902. Centr. B. II. 1165, 1229. 1903. Centr. B. II. 778, 813, 1483. 1904. Centr. B. I. 68, 69, 619. II. 1631. 1905. Centr. B. 1. 54, 58, 636, 784, 841. II. 182, 728, 798, 1141, 1756. 1906. Centr. B. 1. 300, 370, 514, 618, 620. 881, 1199, 1200, 1383, 1809, 1810. Centr. B. II. 386, 574, 725, 835, 984, 1093, 1371. 1465, 1696, 1792. 1907. Centr. B. I. 198, 774, 1295, 1648. II. 198, 276, 655, 956, 2001. 1908. Centr. B. I. 919. 1909. Centr. B. I. 233, 1283. II. 1182. THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $1.OO ON THE SEVENTH D-' OVERDUE. /Fit 13 1934 SEP 16 1337 LD21- 259663 m A r ' , -\ . , A. V V