key: cord-0724776-nmjkuh0m
authors: Głowacka, Iwona E.; Balzarini, Jan; Andrei, Graciela; Snoeck, Robert; Schols, Dominique; Piotrowska, Dorota G.
title: Design, synthesis, antiviral and cytostatic activity of ω-(1H-1,2,3-triazol-1-yl)(polyhydroxy)alkylphosphonates as acyclic nucleotide analogues
date: 2014-07-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2014.05.020
sha: 5e61ffdbc474a1e93643cb4b72c2abde03065167
doc_id: 724776
cord_uid: nmjkuh0m

The efficient synthesis of a new series of polyhydroxylated dibenzyl ω-(1H-1,2,3-triazol-1-yl)alkylphosphonates as acyclic nucleotide analogues is described starting from dibenzyl ω-azido(polyhydroxy)alkylphosphonates and selected alkynes under microwave irradiation. Selected O,O-dibenzylphosphonate acyclonucleotides were transformed into the respective phosphonic acids. All compounds were evaluated in vitro for activity against a broad variety of DNA and RNA viruses and for cytostatic activity against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells. Compound (1S,2S)-16b exhibited antiviral activity against Influenza A H3N2 subtype (EC(50) = 20 μM—visual CPE score; EC(50) = 18 μM—MTS method; MCC >100 μM, CC(50) >100 μM) in Madin Darby canine kidney cell cultures (MDCK), and (1S,2S)-16k was active against vesicular stomatitis virus and respiratory syncytial virus in HeLa cells (EC(50) = 9 and 12 μM, respectively). Moreover, compound (1R,2S)-16l showed activity against both herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC(50) = 2.9 and 4 μM, respectively) and feline herpes virus in CRFK cells (EC(50) = 4 μM) but at the same time it exhibited cytotoxicity toward uninfected cell (MCC ⩾ 4 μM). Several other compounds have been found to inhibit proliferation of L1210, CEM as well as HeLa cells with IC(50) in the 4–50 μM range. Among them compounds (1S,2S)- and (1R,2S)-16l were the most active (IC(50) in the 4–7 μM range).

Infectious diseases caused by different microorganisms such as bacteria, fungi and viruses, are still a problem of human civilization. Among all pathogenic microorganisms viruses are notorious, the most active and probably the most dangerous because they penetrate into cells, evolve rapidly and interfere with the genetic material of the host. Despite current achievements in the development of antiviral drugs, 1,2 there is still a need for new compounds with an unique mechanism of action and limited side-effects.

The successful search for acyclic nucleoside analogues started when acyclovir [9-(2-hydroxyethoxymethyl)guanine] was described as an antiherpesvirus agent. 3 Soon after, a few other acyclic nucleoside or acyclic nucleoside phosphonate analogues became available as antiviral compounds, namely, ganciclovir, cidofovir, tenofovir, adefovir, etc. 4, 5 Attempts to improve the solubility of compounds in aqueous media resulted in synthesis of hydroxylated analogues of nucleosides such as ganciclovir as well as other nucleoside mimetics shown on Figure 1 . 4, [6] [7] [8] [9] [10] [11] [12] [13] [14] The interest in investigation of hydroxylated nucleosides has also been stimulated by a previous discovery of extreme potency of the naturally accessible D-eritadenine 2, 7, 8 which acts as an inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH). This enzyme has earlier been shown to be an attractive target for poxviruses, (À)RNA viruses such as paramyxovirus and rhabdovirus, and (±)RNA viruses such as reovirus. 15, 16 Consequently, (2 0 S)-9-(2 0 ,3 0 -dihydroxypropyl)adenine 3 10 and other N(9)-substituted adenines and guanines possessing polyhydroxyalkyl chains have been obtained (Fig. 1) .

Among various structural modifications of nucleosides/nucleotides 1,2,3-triazole analogues have been of special interest. The applicability of a 1,2,3-triazole ring as a replacement of sugar [17] [18] [19] or nucleobase moieties 17, [19] [20] [21] as well as an additional linker between a phosphonoalkyl unit and a nucleobase has been widely explored [22] [23] [24] [25] [26] [27] [28] including our achievements. [29] [30] [31] [32] [33] [34] [35] Recently, several http://dx.doi.org/10.1016/j.bmc.2014.05.020 0968-0896/Ó 2014 Elsevier Ltd. All rights reserved. acyclic 1,2,3-triazolyl analogues of nucleosides/nucleotides with nucleobases attached via the methylene group at the C4 in the 1,2,3-triazole moiety have been obtained and some of them showed promising biological activity (Fig. 2) . While (1,2,3-triazol-1-yl)nucleosides 9-11 were found to be inactive against selected viruses, 22,23 the phosphonomethyl-12 (n = 1; B = Thy, Ade) and phosphonoethyl(1,2,3-triazoles) 12 (n = 2; R = Thy, Ura) showed moderate activity against hepatitis C virus (HCV). 25 Recently, we succeeded in the synthesis of 1-(3-phosphonopropyl)-1,2,3-triazole 13 substituted with benzoylbenzuracil via a methylene linker which exhibited activity against herpes simplex virus-1 (KOS), herpes simplex virus-2 (G) and feline herpes virus. 33 Moreover, the 1-(3-amino-3-phosphonopropyl)-1,2,3-triazole analogue (R)-14 having 3-acetylindole as a modified nucleobase showed moderate activity toward vesicular stomatitis virus. 35 In continuation of our research program towards 1,2,3-triazole nucleoside analogues, [14] [15] [16] [17] [18] [19] [20] [21] and taking into account the known biological activity of hydroxylated nucleoside analogues as well as the antiviral activity of 13 and 14 and the cytostatic properties of 15, 1,2,3-triazoles 16 and 17 possessing dibenzyloxyphosphono(polyhydroxy)alkyl residues have been designed (Fig. 3) . We assumed that incorporation of additional hydroxyl groups into an alkyl side-chain would assure better solubility and perhaps improve biological activity of 1,2,3-triazoles 16 and 17 in comparison with analogous compounds having unfunctionalised aliphatic moieties. 33 2. Results and discussion

Enantiomerically pure (1R,2S)-and (1S,2S)-azidophosphonates 18 were obtained from L-ascorbic acid, 36, 37 whereas for the synthesis of (1S,2R,3S)-and (1S,2R,3R)-azidophosphonates 19 38 tartaric acid and L-isoascorbic acid were used, respectively, as a source of chirality.

The 1,2,3-triazoles (1R,2S)-and (1S,2S)-16 were synthesised by the 1,3-dipolar cycloaddition of the corresponding (1R,2S)-and (1S,2S)-azidophosphonates 18 with N-propargyl nucleobases 20 (N 9 -propargyladenine 20a, 23 N 1 -propargylthymine 20b, 23 N 1 -propargyluracil 20c, 39 N 4 -acetyl-N 1 -propargylcytosine 20d 40 ) and several propargylated nucleobase mimetics 20 (N 1 -propargyl-6-azauracil 20e, 41 3-acetyl-N-propargylindole 20f, 42 N 1 -propargyltheobromine 20g, 43,44 N 7 -propargyltheophylline 20h, 45 8-chloro-N 7 -propargyltheophylline 20i, 46 N-propargyl-2-pyridone 20j, 47 N 3 -benzoyl-N 1 -propargyluracil 20k 33, 48 and N 3 -benzoyl-N 1 -propargylquinazolin-2,4dione 20l 33 ).

According to a standard protocol the regioselective formation of respective 1,4-disubstituted 1,2,3-triazoles was secured by the 1,3-dipolar cycloaddition of azides with terminal alkynes in the presence of a catalytic amount of Cu(I) at room temperature. 49, 50 However, under these conditions more than 3 days were required to complete the reaction of (1S,2S)-and (1R,2S)-azidophosphonates 18 with N-propargyl nucleobases 20. Structures of all new compounds were confirmed on the basis of 1 H, 13 C, 31 P NMR and IR spectra data as well as by elemental analysis. , dextran sulfate (molecular weight 5000, DS-5000), ribavirin, oseltamivir carboxylate, amantadine and rimantadine were used as the reference compounds. The antiviral activity was expressed as the EC 50 : the compound concentration required to reduce virus plaque formation (VZV) by 50% or to reduce virus-induced cytopathogenicity by 50% (other viruses).

The cytotoxicity of the tested compounds toward the uninfected host cells was defined as the minimum cytotoxic concentration (MCC) that causes a microscopically detectable alteration of normal cell morphology. The 50% cytotoxic concentration (CC 50 ), causing a 50% decrease in cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay system.

It was established that compound (1S,2S)-16b containing a 1,2,3-triazole moiety substituted at C4 0 with thymine exhibited antiviral activity against Influenza A H3N2 subtype (EC 50 = 20 lM by visual CPE score; EC 50 = 18 lM by MTS score; MCC >100 lM, 

The cytostatic activity of the tested compounds was defined as the 50% cytostatic inhibitory concentration (IC 50 ), causing a 50% decrease in cell proliferation and was determined against murine (Table 1 ).

As far as cytostatic properties are considered, within a series of hydroxylated (1,2,3-triazol-1-yl)nucleotide analogues 16 and 17, compounds 16 containing three-carbon phosphonoalkyl chain are more cytostatic toward the tested tumor cell lines when compared with four-carbon phosphonates 17 having the same nucleobases (16a vs 17a, 16c vs 17c, 16d vs 17d, 16g vs 17g, 16i vs 17i). Moreover, the configurations at stereogenic centres have slight or negligible impact on the cytostatic properties of (1,2,3-triazol-1-yl)nucleo- Analogous structure-antiviral activity relationship studies on a series of (1,2,3-triazol-1-yl)nucleotide analogues 16 and 17 were performed.

Thus, 1-hydroxy-2-benzyloxypropylphosphonate (1R,2S)-16l, which contains a N 3 -benzoylbenzyuracil residue, showed promising antiviral activity against herpes simplex viruses (HSV-1, HSV-2) in HEL cell cultures (EC 50 = 2.9 and 4 lM, respectively) and feline herpes virus in CRFK cells (EC 50 = 4 lM), whereas its N 3 -benzoyluracil and uracil analogues (1R,2S)-16k and (1R,2S)-16c, respectively, appeared inactive. This trend well correlates with our previous observations on structurally analogous (1,2,3-triazol-1-yl)nucleotides having an unsubstituted phosphonopropyl chain, since compound 13 was the most active against HSV-1, HSV-2 (EC 50 = 17 lM) and against feline herpes virus (EC 50 = 24 lM). 33 Furthermore, the stereochemistry of the aliphatic chain substituted with hydroxyl groups is essential for activity since analogue (1S,2S)-16l showed no activity. On the other hand, the significant activity toward vesicular stomatitis virus and respiratory syncytial virus in HeLa cell cultures was observed for compound (1S,2S)-16k containing an N 3 -benzoyluracil unit (EC 50 = 9 and 12 lM, respectively) since analogous compounds (1S,2S)-16l (B = N 3 -benzoylbenzuracil) and (1S,2S)-16c (B = uracil) were inactive against these viruses. Again, stereochemistry of an aliphatic fragment appeared important and resulted in lack of activity for (1R,2S)-16k. 

1 H NMR were taken in CDCl 3 or CD 3 OD on the following spectrometers: Varian Mercury-300 and Bruker Avance III (600 MHz) with TMS as an internal standard; chemical shifts d in ppm with respect to TMS; coupling constants J in Hz. 13 C NMR spectra were recorded for CDCl 3 ,CD 3 OD or DMSO-d 6 solutions on a Varian Mercury-300 and Bruker Avance III (600 MHz) spectrometer at 75.5 and 150.5 MHz, respectively. 31 P NMR spectra were taken in CDCl 3 or CD 3 OD on Varian Mercury-300 and Bruker Avance III at 121.5 and 242 MHz.

IR spectral data were measured on an Infinity MI-60 FT-IR spectrometer. Melting points were determined on a Boetius apparatus and are uncorrected. Elemental analyses were performed by the Microanalytical Laboratory of this Faculty on a Perkin Elmer PE 2400 CHNS analyzer. Polarimetric measurements were conducted on an Optical Activity PolAAr 3001 apparatus.

The following adsorbents were used: column chromatography, Merck silica gel 60 (70-230 mesh); analytical TLC, Merck TLC plastic sheets silica gel 60 F 254 . TLC plates were developed in chloroformmethanol solvent systems. Visualization of the spots was effected with iodine vapours. All solvents were purified by methods described in the literature.

All microwave irradiation experiments were carried out in a microwave reactor Plazmartonika RM 800. The reaction carried out in 50 mL-glass vials. 

Yield 

04 (dd, J = 11.4 Hz, J = 8.1 Hz, 4H, 2 Â POCH 2 Ph), 4.98 (AB, J = 14.4 Hz, 1H, CH a H b -Ura), 4.88 (AB, J = 14.4 Hz, 1H, CH a H b -Ura), 4.79 (dd, J = 14.4 Hz, J = 3.0 Hz, 1H, H-3a), 4.61 (dd, J = 14.4 Hz, J = 6.9 Hz, 1H, H-3b), 4.41 (d, J = 11.1 Hz, 1H, OCH a H b Ph), 4.33 (d, J = 11.1 Hz, 1H, OCH a H b Ph), 4.41 (dddd

acetylamino-2-oxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-2-benzyloxy-1-hydroxypropylphosphonate (1R,2S)-16d Yield: 75%; white powder

41 (d, J = 14.4 Hz, 1H, CH a H b -Cyt), 5.08-4.80 (m, 5H, 2 Â POCH 2 Ph, H-3a), 4.89 (d, J = 14.4 Hz

5-dioxo-1,2,4-triazin-2-yl)methyl]-1H-1,2,3-triazol-1-yl}-1-hydroxypropylphosphonate (1R,2S)-16e Yield: 91%; colorless oil

CDCl 3 ): d = 11.41 (br s, 1H, NH), 7.68 (s, 1H, HC5'), 7.31 (s, 1H, HC@N), 7.29-7.21 (m, 10H, H aromat ), 7.20-7.16 (m, 3H, H aromat ), 7.09-7.03 (m, 2H, H aromat ), 5.13 (AB, J = 14.7 Hz, 1H, CH a H b ), 5.10 (AB, J = 14.7 Hz, 1H, CH a H b ), 5.06-4.98 (m, 4H, 2 Â POCH 2 Ph), 4.71 (dd, J = 14.4 Hz, J = 3.3 Hz, 1H, H-3a), 4.60 (dd, J = 14.4 Hz, J = 6.3 Hz, 1H, H-3b), 4.39 (d, J = 11.1 Hz, 1H, OCH a H b Ph), 4.27 (d, J = 11.1 Hz, 1H, OCH a H b Ph

40 ppm. Anal. Calcd for C 30 H 31 N 6 O 7 P: C, 58

1R,2S)-Dibenzyl 3-{4-[(3-acetyl-1H-indol-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-2-benzyloxy-1-hydroxypropylphosphonate (1R,2S)-16f Yield: 91%; white powder

39 (s, 2H, CH 2 ), 5.30-4.96 (m, 4H, 2 Â POCH 2 Ph), 4.66 (dd, J = 14.5 Hz, J = 3.4 Hz, 1H, H-3a), 4.53 (dd, J = 14.5 Hz

CDCl 3 ): d = 23.68 ppm. Anal. Calcd for C 37 H 37 N 4 O 6 P: C, 66

7-dimethyl-2,6-dioxopurin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1-hydroxypropylphosphonate (1R,2S)-16g Yield: 80%; white powder

29 (s, 2H, CH 2 ), 5.02 (dd, J = 13.2 Hz, J = 8.4 Hz, 4H, 2 Â POCH 2 Ph), 4.67 (dd, J = 14.7 Hz, J = 3.6 Hz, 1H, H-3a), 4.58 (dd, J = 14.7 Hz, J = 6.3 Hz, 1H, H-3b), 4.40 (d, J = 11.1 Hz, 1H, OCH a H b Ph), 4.29 (d, J = 11.1 Hz, 1H, OCH a H b Ph), 4.16 (dddd, J = 9.6 Hz, J = 6.6 Hz, J = 6.3 Hz, J = 3.6 Hz, 1H, H-2), 3.95 (dd, J = 9.6 Hz, J = 6.0 Hz, 1H, H-1), 3.94 (s, 3H, CH 3 ), 3.52 (s, 3H, CH 3 ), 1.93 (br s, 1H, OH)

3-dimethyl-2,6-dioxopurin-7-yl)methyl]-1H-1,2,3-triazol-1-yl}-1-hydroxypropylphosphonate (1R,2S)-16h Yield: 91%; colorless oil

52 (s, 2H, CH 2 ), 5.09-4.97 (m, 4H, 2 Â POCH 2 Ph), 4.70 (dd, J = 14.1 Hz, J = 3.3 Hz, 1H, H-3a), 4.61 (dd, J = 14.1 Hz, J = 6.6 Hz, 1H, H-3b), 4.44 (d, J = 11.4 Hz, 1H, OCH a H b Ph), 4.25 (d, J = 11.4 Hz, 1H, OCH a-H b Ph), 4.14 (dddd, J = 9.3 Hz, J = 6.6 Hz, J = 5.7 Hz, J = 3.3 Hz, 1H, H-2), 3.97 (dd

-chloro-1,3-dimethyl-2,6-dioxopurin-7-yl)methyl]-1H-1,2,3-triazol-1-yl}-1-hydroxypropylphosphonate (1R,2S)-16i Yield: 85%; white powder

58 (s, 2H, CH 2 ), 5.09-4.96 (m, 4H, 2 Â POCH 2 Ph), 4.70 (dd, J = 14.4 Hz, J = 3.3 Hz, 1H, H-3a), 4.59 (dd, J = 14.4 Hz, J = 6.6 Hz, 1H, H-3b), 4.40 (d

oxopyridin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}propylphosphonate (1R,2S)-16j Yield: 81%; brown oil

m, 1H, H aromat ), 6.14 (dt, J = 6.9 Hz, J = 1.2 Hz, 1H, H aromat ), 5.17 (br s, 1H, OH), 5.12 (s, 2H, CH 2

-benzoyl-2,4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1-hydroxypropylphosphonate (1R,2S)-16k Yield: 81%; colorless oil

CDCl 3 ): d = 7.90-7.86 (m, 2H), 7.68-7.56 (m, 3H), 7.47-7.42 (m, 2H, H aromat ), 7.33-7.26 (m, 11H, H aromat ), 7.24-7.18 (m, 2H, H aromat ), 7.08-7.05 (m, 2H, H aromat ), 5.78 (d, J = 7.7 Hz, 1H, HC@CH), 5.08-5.00 (m, 4H, 2 Â POCH 2 Ph

1R,2S)-Dibenzyl 2-benzyloxy-3-{4-[(3-benzoyl-2,4-dioxoquinazolin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1-hydroxypropylphosphonate (1R,2S)-16l Yield: 88%; white solid

IR (KBr): m = 3418

Hz, 1H), 7.95-7.91 (m, 2H, 2 Â o-CH), 7.86 (br d, J = 8.5 Hz, 1H), 7.71 (ddd, J = 8.8 Hz, J = 7.3 Hz, J = 1.6 Hz, 1H), 7.61 (s, 1H, HC5 0 ), 7.64-7.38 (m, 1H, p-CH), 7.47-7.41 (m, 2H, 2 Â m-CH)

85 (br s, 1H, OH); 13 C NMR (151 MHz

135.8 (d, J = 5.4 Hz, C ipso )

CDCl 3 ): d = 23.37 ppm. Anal. Calcd for C 42 H 38 N 5 O 8 P: C, 65

-amino-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3S)-17a Yield: 83%; white powder

55 (s, 2H, HC@CCH 2 ), 5.185.08 (m, 4H, 2 Â POCH 2 Ph), 4.74 (dd, J = 14.5 Hz, J = 2.9 Hz, 1H, H-4b), 4.64 (dd, J = 14.5 Hz, J = 6.8 Hz, 1H, H-4b), 4.50 (dt, J = 6.8 Hz, J = 2.9 Hz

-methyl-2,4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3S)-17b Yield: 83%; white powder

IR (KBr): m = 3238

H-1), 3.40 (dd, J = 12.6 Hz, J = 5.3 Hz, 1H), 1.95 (s, 3H, CH 3 ), 1.38 (s, 3H, CH 3 ), 1.22 (s, 3H, CH 3 )

(2,4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3S)-17c Yield: 85%; yellow pale powder

3 (c 1.07 in CH 3 OH)

696 cm À1 ; 1 H NMR (CD 3 OD, 600 MHz): d = 7.95 (s 1H, HC5 0 ), 7.70 (d, J = 7.9 Hz, 1H, HC@CH), 7.427.32 (m, 10H, 2 Â C 6 H 5 ), 5.68 (d, J = 7.6 Hz, 1H, HC@CH), 5.175.09 (m, 4H, 2 Â POCH 2 Ph), 5.06 (AB, J AB = 15.3 Hz, 1H, CH a H b ), 5.03 (AB, J AB = 15.3 Hz, 1H, CH a H b ), 4.76 (dd, J = 14.5 Hz, J = 2.9 Hz, 1H, H-4b

4 (s, N-CH@CH) 142.2, 136.3 (d, J = 5.6 Hz, C ipso ), 136.2 (d, J = 5.6 Hz, C ipso ), 128.2, 128.2, 128.2, 127.9

acetylamino-2-oxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-O-isopropylidenebutylphosphonate (1S,2R,3S)-17d Yield: 89%; white powder

IR (KBr): m = 3395

HC@CCH 2 ), 4.86 (dd, J = 14.1 Hz, J = 1,9 Hz, 1H, H-4b), 4.60 (dd, J = 14,1 Hz, J = 8.8 Hz, 1H, H-4a), 4.52 (dt, J = 8.8 Hz

242 MHz): d = 23.14 ppm. Anal. Calcd for C 30 H 35 N 6 O 8 P: C, 56

7-dimethyl-2,6-dioxopurin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3S)-17g Yield: 88%; white powder

IR (KBr): m = 3275

58 (s, 3H, NCH 3 ), 3.29 (dd, J = 12.9 Hz, J = 5.5 Hz, 1H), 1.36 (s, 3H, CH 3 ), 1.24 (s, 3H, CH 3 ), 13 C NMR (151 MHz

242 MHz): d = 21.64 ppm. Anal. Calcd for C 31

-chloro-1,3-dimethyl-2,6-dioxopurin-7-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-O-isopropylidenebutylphosphonate (1S,2R,3S)-17i Yield: 92%; white powder

IR (KBr): m = 3229

56 (s, 3H, NCH 3 ), 3.41 (s, 3H, NCH 3 ), 3.14 (dd, J = 13.0 Hz, J = 5.2 Hz, 1H), 1.36 (s, 3H, CH 3 ), 1.21 (s, 3H, CH 3 ); 13 C NMR (151 MHz

242 MHz): d = 21.35 ppm. Anal. Calcd for C 31 H 35 ClN 7 O 8 P: C, 53

-amino-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3R)-17a Yield: 81%; white powder

+21.8 (c 1.43 in DMSO)

IR (KBr): m = 3324

H-1), 3.33 (br s, 1H, OH), 1.41 (s, 3H, CH 3 ), 1.27 (s, 3H, CH 3 ); 13 C NMR (151 MHz

Found: C, 56.15

-methyl-2,4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-O-isopropylidenebutylphosphonate (1S,2R,3R)-17b Yield: 89%; white powder

H-4a), 4.24 (br s, 1H, OH), 4, 13 (dt, J = 8.7 Hz, J = 5.5 Hz, 1H, H-1), 1.63 (s, 3H, CH 3 ), 1.42 (s, 3H, CH 3 ), 1.25 (s, 3H, CH 3 ); 13 C NMR (151 MHz

4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3R)-17c Yield: 93%; white powder

CH a H b ), 4.90 (AB, J AB = 15.0 Hz, 1H, CH a H b ), 4.98 (dd, J = 14.2 Hz, J = 2.5 Hz, 1H, H-4b

acetylamino-2-oxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-O-isopropylidenebutylphosphonate (1S,2R,3R)-17d Yield: 95%; white powder

H-2), 4.20 (dt, J = 10.3 Hz, J = 3.6 Hz, 1H, H-1), 2.26 (s, 3H, C(O)CH 3 ), 1.49 (s, 3H, CH 3 ), 1.38 (s, 3H, CH 3 ); 13 C NMR (151 MHz

CDCl 3 ): d = 25.31 ppm. Anal. Calcd for C 30

7-dimethyl-2,6-dioxopurin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3R)-17g Yield: 96%; white powder

93 (s, 3H, NACH 3 ), 3.52 (s, 3H, NACH 3 ), 1.39 (s, 3H, CH 3 ), 1.26 (s, 3H, CH 3 ); 13 C NMR (151 MHz

3-dimethyl-2,6-dioxopurin-7-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-Oisopropylidenebutylphosphonate (1S,2R,3R)-17h Yield: 94%; colorless oil

644 cm À1 ; 1 H NMR (600 MHz

H-3), 4.45 (dt, J = 8.6 Hz, J = 6.1 Hz, 1H, H-2), 4.38 (dd, J = 14.2 Hz, J = 9.7 Hz, 1H, H-4a), 4.11 (dt, J = 8.7 Hz, J = 5.6 Hz, 1H, H-1), 3.66 (br s, 1H, OH), 3.59 (s, 3H, NACH 3 ), 3.43 (s, 3H, NACH 3 ), 1.45 (s, 3H, CH 3 ), 1.30 (s, 3H, CH 3 ); 13 C NMR (151 MHz

-chloro-1,3-dimethyl-2,6-dioxopurin-7-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2,3-trihydroxy-2,3-O-isopropylidenebutylphosphonate (1S,2R,3R)-17i Yield: 94%; white powder

997 cm À1 ; 1 H NMR (600 MHz

54 (s, 3H, NACH 3 ), 3.39 (s, 3H, NACH 3 ), 1.41 (s, 3H, CH 3 ), 1.28 (s, 3H, CH 3 ); 13 C NMR (151 MHz

Synthesis of phosphonic acid 21b and 21k (general procedure) The dibenzyl phosphonates (1S,2S)-16b and

mL) and 10% Pd-C (10 mg) was added. The suspension was stirred under hydrogen atmosphere at room temperature for 48 h. The catalyst was filtered through a layer of Celite and the aqueous solution was concentrated in vacuo to give pure phosphonic acids 21b and 21k

-Methyl-2,4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2-dihydroxypropylphosphonic acid (1S,2S)-21b Yield: 87%

02 (s, 2H, CH 2 ), 4.67 (dd, J = 14.4 Hz, J = 3.8 Hz, 1H, H-3a), 4.52 (dd, J = 14.4 Hz, J = 9

-benzoyl-2,4-dioxopyrimidin-1-yl)methyl]-1H-1,2,3-triazol-1-yl}-1,2-dihydroxypropylphosphonic acid (1S,2S)-21k Yield: 89%

689 cm À1 ; 1 H NMR (300 MHz, CD 3 OD): d = 8.03 (s, 1H, HC5 0 ), 7.97-7.95 (m, 2H, H aromat ), 7.85 (d, J = 8.0 Hz, 1H, HC@CH), 7.75-7.68 (m, 1H, H aromat

151 MHz, CD 3 OD): d = 170.0 (s, C@O), 162.7 (s, C@O)

Modified Nucleosides in Biochemistry

Antiviral Nucleosides: Chiral Synthesis and Chemotherapy

Immunosupressive effects of 8-substituted xanthine derivatives

Xanthine derivatives, the preparation thereof and use as pharmaceutical compositions

The authors wish to express their gratitude to Mrs. Małgorzata 

The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK À ) HSV-1 KOS strain resistant to ACV (ACV r ), herpes simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK À VZV strain 07À1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, Parainfluenza 3, Influenza virus A (subtypes H1N1, H3N2), influenza virus B, Reovirus-1, Sindbis, Reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain III B and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on inhibition of virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK) . Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID 50 of virus (1 CCID 50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (VZV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virusinfected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC 50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%.

Inhibition of HIV-1(III B )-and HIV-2(ROD)-induced cytopathicity in CEM cell cultures was measured in microtiter 96-well plates containing $3 Â 10 5 CEM cells/mL infected with 100 CCID50 of HIV per milliliter and containing appropriate dilutions of the test compounds. After 4-5 days of incubation at 37°C in a CO 2 -controlled humidified atmosphere, CEM giant (syncytium) cell formation was examined microscopically. The EC 50 (50% effective concentration) was defined as the compound concentration required to inhibit HIV-induced giant cell formation by 50%.

All assays were performed in 96-well microtiter plates. To each well were added (5-7.5) Â 10 4 tumor cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37°C in a humidified CO 2 -controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC 50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.