key: cord-1020714-sdr4wixr authors: Sapi, J.; Massiot, G. title: Chapter 3 Noniridoid Bisindole Alkaloids date: 1995-12-31 journal: The Alkaloids: Chemistry and Pharmacology DOI: 10.1016/s0099-9598(08)60155-8 sha: 77f44da12ec24b461b58cd3b3384516595e8d1e6 doc_id: 1020714 cord_uid: sdr4wixr Publisher Summary This chapter discusses that indole is a reactive nucleus prone to dimerization when it is isolated or a part of tryptamine or tryptophan, which themselves are reactive toward many functionalities. For these reasons, bisindoles, the majority of which are of iridoid origin, are frequently isolated in nature. Besides these “dimers,” there exists a growing class of noniridoid bisindoles found in the marine environment, in microorganisms, and in plant species, many of which display interesting biological activities. The chapter tries to promote research on a series of compounds worthy of attention. Marine environments are a rich source of indole and bisindole alkaloids of great structural variety. In the absence of traditional use, the chemical constituents of marine organisms are separated following bioassay-guided fractionation, and, consequently, contrary to compounds from terrestrial sources, many of the structures are associated with significant biological activity. Indole is a reactive nucleus prone to dimerization when it is isolated or a part of tryptamine or tryptophan, which themselves are reactive toward many functionalities. For these reasons, bisindoles, the majority of which are of iridoid origin, are frequently isolated in nature; this field has been the subject of several reviews (I). Besides these "dimers," there exists a growing class of noniridoid bisindoles found in the marine environment, in microorganisms, and in plant species, many of which display interesting biological activities. It is the purpose of this chapter to review the field for compounds 2-6 is due to restricted rotation around the bond connecting the two indole halves (atropisomerism). The Okinawan red alga Laurencia brongniurtii contained a sulfur bromoindole dimer (7), whose symmetric nature was deduced from NMR data (nine signals in the 13C-NMR spectrum) and confirmed by X-ray analysis (3) . Antimicrobial and cytotoxic activities associated with extracts from the Fijian tunicate Polycitorellu mariae led to the isolation of citorellamine (4). The original structure-a monomer-was revised to 8 on the basis of total syntheses of the monomer and dimer and on comparison of the spectra (5). is often a recurring structural motif of marine natural products. 2,2-Bis(6'-bromo-3'-indolyl)ethylamine (lo), isolated from the tunicate Didemnum candidum, is a simple dimer derived from 9 and 6-bromindole (6) . Structurally more elaborated dimers have a spacer unit between the two indole nuclei. Such is the case for dragmacidin (11) (3, a cytotoxic metabolite of the deep-water marine sponge, Dragmacidon sp. Hallman 1017, which inhibits the growth of P388, A549, and HCT-8 cancer cell lines in in uitro assays. This dimer contains a 6-bromotryptamine and a dibromohydroxytryptamine unit as shown by IH-NMR and mass spectral fragments at d z 289, 291, and 293 for the dibromohydroxyindole (CsH5-NOBr,). The location of the substituents was facilitated by the analysis of the spectra of tridebromodragmacidin (12) . Coupling constant measurements and H-C COSY experiments suggested substitution at C-4' or C-7' atoms with a phenolic hydroxyl. The final confirmation was obtained on calculation of chemical shifts, which were in agreement with a 4-hydroxyindole nucleus. NOE experiments and coupling constants (& = 10.3 and 55-6 = 11.3 Hz) favored a chair conformation with the two bromoindole appendages in equatorial positions on the piperazine ring. Two other closely related dimers, dragmacidon A (13) and dragmacidon B (14), have been isolated as minor constituents of the deep-water sponge Hexadella sp., collected off the coast of British Columbia (8). The aromatic region of the 'H-NMR spectrum of 13 displayed signals for two 6-bromoindol-3-yl residues. The remaining aliphatic protons consisted of a singlet (N-CH3 at 6 2.09 ppm) and two separated three-spin systems, each corresponding to a methylene, adjacent to a methine, as found in the piperazine ring of 11. Dragmacidon B (14) had an additional methyl group dragmacidin d on the piperazine ring. Consequently, the molecule contained a twofold axis of symmetry similar to that of the simplest demethyl-dragmacidon A (W), isolated from the tunicate Didernnum candidurn (6) . Total synthesis of dragmacidon B (14) has been achieved (Scheme 1) by bromination of 1,Cdimethyl piperazine-2,5-dione (16) followed by coupling with 6bromoindole (9). Dragmacidon A (13) showed in uitro cytotoxicity in the L1210 assay. Dragmacidin d (17) has been isolated from a deep-water sponge of the genus Spongosorites (10) . It exhibited a broad spectrum of biological activities : growth inhibition of feline leukemia virus, of P388 murine, and of A549 human lung tumor cell lines. Based on detailed NMR experiments, the two indole rings were identified as a 6-bromoindol-3-yl unit and a 7hydroxy-3,4-dialkylated indole unit. Observation of carbon resonances at those of known bromo-and hydroxyindoles. The presence of an imidazole spacer was demonstrated by 'H-13C-NMR correlation techniques and confirmed by synthesis. After analysis of the structure for hidden symmetries, two short syntheses of topsentin A were produced (Scheme 2): thermolysis of the quaternary hydrazine derivative (21) gave 18 in a single step by selfcondensation of the presumed intermediate (22) whereas the same result was obtained by condensation of glyoxalylindole (23) with ammonia (23). 4,5-Dihydro-6"-deoxybromotopsentin (24) was isolated as a minor compound of a sponge tentatively identified as Spongosorites sp. (12) . In addition to the resonances attributed to unsubstituted and monosubstituted benzene rings, significant signals were observed for a deshielded ABX system, corresponding to an N-CH2-CH-N sequence. The COSY spectrum placed the methine group at the 3 position of the bromoindole in accordance with the proposed structure (24). All of the natural topsentins showed activity as antiviral and antitumor agents, and, consequently, several C-5"' analogues, named "neotopsentins" (25-27), were prepared to establish structure-activity relationships. Topsentin and bromotopsentin were active against the Herpes simplex virus (HSV-1) and corona virus A-59. Topsentin had in u i f o activity against P388 and human tumor (HCT-8, A549) cells and had in uiuo activity against P388 and B16 melanoma. As a general observation, the introduction of a hydroxyl group was found to enhance cytotoxicity, whereas a bromine atom decreased it (12) . Bromotopsentin (20) (14) and a new compound unfortunately also named topsentin C (28) were reported for the deep-water sponge Hexadella sp., collected off the coast of British Columbia (8). The 'H-NMR spectrum of 28 displayed two sets of resonances which could be assigned to 6-bromoindol-3yl residues by analogy with the characteristic shifts of 24. The methyl group (6 3.05 ppm) could be placed on the N-1 atom of the dihydroimidazole ring in accordance with the strong NOES observed with H-5 ( Grossularine-1 (32) is a marine metabolite of the tunicate Dendrodoa grossularia from New Caledonia (16). The presence of an indole nucleus substituted on C-3 by a carbonyl was deduced from the mass spectral fragmentations at d z 144 and 116 and by the NMR data (CO at 6 186.8 pprn). On the basis of I3C-NMR, a second 2,3-disubstituted indole ring was attached to an N,N-dimethyl-guanidine core and structure 33 was proposed. It was later revised after the isolation of grossularine-2 (34), a metabolite from the same source whose structure was solved by X-ray crystallography (I 7J8). These molecules were reported as the first natural products with an a-carboline moiety. Grossularine-1 displayed cytotoxicity toward L1210 leukemia cells at the pg/ml level. Eudistomin refers to a series of P-carbolines isolated from tunicates (19). Eudistomin U (39, isolated from the Caribbean ascidian Lissoclinum fragile (20) , is the first bisindole among these molecules (M' 283.1094 for CI9Hl3N3). Spectral data, especially 'H-and 13C-NMR data, were in good accordance with those reported for P-carboline and indole, thus supporting the proposed structure (35). Isoeudistomin U (36), from the same source, comprises indole and dihydro-a-carboline moieties. This structure was proposed on the basis of HMBC and NOE measurements (e.g., an NOE response of H-3 to irradiation at H-2') and by comparison of I3C-NMR chemical shifts : C-9a at 6 158.6 and at 6 159.2 pprn for 36 and 32, respectively. It is the personal opinion of the authors, however, that the structure of isoeudistomin U should be reconsidered based upon the following arguments: from a chemical standpoint, a dihydro a-carboline substituted as in 36 should not be stable and would readily be oxidized to the fully aromatic compound. The information extracted from the HMBC experiment may be explained by changing chemical shift assignments; for example, the carbon signal at 6 20.3 ppm could be reassigned to the C-6 of a 3,4-dihydro P-carboline unit. The most puzzling data concern the above-mentioned NOE between H-2' and H-3; the published value (12%) is much too high for a molecule of this size at 400 MHz! An artifactual observation must not be excluded. Last, but not least, both compounds display the same UV spectra and once again artifacts should be looked for. Whatever their structure, 35 and 36 possess strong antibacterial activity (Agrobucteriurn turnefuciens). Fascaplysin (37) is an antimicrobial, cytotoxic pigment, isolated from the Fijian sponge Fuscaplysinopsis sp. Bergquist (21) , whose unique structure (pyrido( 1,2-u:3,4,br)diindole) was determined by X-ray diffraction analysis. (26) . Extracts from the green alga C. rucemosu gave two new pigments (49 and 50), derivatives of 47 (27, 28) . Structural analogy among these compounds was established by simple hydrolysis and esterification reactions. Caulerpin is a plant growth regulator, and this is probably due to its 3-indolyl acrylic acid dimer structure (29) . A large survey of the distribution of caulerpin in algae from Bermuda, Florida, and Tasmania was conducted to reveal correlations with habitat, morphology, and taxonomy (30). Hyrtiosin B (51) is a symmetrical phenolic dimer, found in the Okinawan marine sponge Hyrfios erecfu (31). Its presumed precursor, 3-formyl- A. AMAUROMINE Amauromine (559, C32H36N402 (M' 508.283), isolated from a culture broth of Amauroascus, is a novel alkaloid with potent vasodilating activity (33) . Its 'H-and 13C-NMR spectra displayed signals for 18 protons and 16 carbons as a consequence of the symmetry of the molecule. These spectra showed the presence of a dimethylallyl group, as in roquefortine (56). The fused indoline moiety with amide functions (IR,v 1660 cm-'; 13C-NMR, 6 166.1 ppm) was hydrolytically degraded (6N HCl, llO°C, 4h) to L-tryptophan. Consequently, carbons C-15a and C-7a with their exchangeable protons (Na2C03,CH30D) were given the S absolute configuration. Irradiation of the methyl groups of the isoprenyl moiety induced NOES at the protons on the C-5a and C-13a positions, thus proving the B/C and E/F cis ring junctions. A fruns relative position between the proton at C-15a (C-7a) and that of C-5a (C-13a) was evidenced by the high-field absorption of the 15a proton (6 3.8 ppm), which experiences the anisotropic effect of an aromatic ring, A(G). Determination of the absolute configuration of the stereocenters was confirmed by comparison of the physical data (NMR, a, CD) of the natural product (and derivatives) with those of synthetic analogues of known stereochemistry (34). A diketopiperazine metabolite isolated at the same time from Penicillium nigricans was given the name nigrifortine (35). The published structure is identical to that of amauromine, and the spectroscopic data for the two compounds are similar. Therefore, the compounds are most probably one and the same product. The key intermediate, diketopiperazine (57), was prepared from N-benzyloxycarbonyl tryptophan by classical reactions. The methylthio function was introduced by phosphorous pentasulfide thionation and methylation. Coupling of the active ester, derived from 58 by DCC-HOSu with amine (59), afforded dipeptide (60) in 52% yield. The two 1,l-dimethyl-2prenyl groups were introduced by the thio-Claisen rearrangement through the corresponding sulfonium salt. Converting 61 into amauromine (55) (38) while preserving the inverted prenyl group was realized by the combined use of TiC14 and LiAlH4. Two diketopiperazine containing dimers (62 and 63) were isolated from extracts of the sclerotia of Aspergillus ochruceus Wilhelm (39). According to the spectroscopic data, their gross structure was related to that of amauromine, but they showed different sets of NMR signals for each half of the molecule. A cis relationship among H-2, H-11, and the isoprenyl group was established by a nuclear Overhauser effect. Another set of diketopiperazines was isolated from Aspergillus sp., SC319 (ATCC74177) (40). The main compounds, named WIN 64821 (64) and WIN 64745 (65), are derived from tryptophan and from phenylalanine and valine, respectively. Compound 64 is a potent antagonist of substance P, and it was shown that the configurations of C-2 and C-3 were of paramount importance for biological activity. Staurosporine (66) was first isolated from a culture of Streptomyces staurosporeus Awaya (41) and subsequently from other Actinomyceres (46-49). Its structure, determined by X-ray crystallography (50,51), is a hexacyclic ring system and an aminohexose unit, attached by two glycosidic linkages to the indole nitrogen atoms. Complete and unambiguous 'H-and I3C-NMR assignments were obtained by one-and two-dimensional techniques, including HMQC and HMBC (52) . In accordance with the X-ray solution, the six-membered pyran ring was found to be in a chair conformation with the N-methyl group in axial position (53). Its unusual shielding (6 1.54 ppm) is probably due to the anisotropic effect of the adjacent aromatic ring system. Significant changes in the NMR spectrum upon protonation revealed a dominant (95%) boat conformation with an equatorial NH+-CH3 group (6 2.79 ppm) (54). The absolute configuration of staurosporine (66) was not directly proved until 1994, when an X-ray analysis, performed on 4'-N-methylstaurosporine methiodide and using anomalous diffusion, determined the stereocenters to be 2'4 3'R, 4'R, and 6'R (55). As a consequence, the commonly used stereostructural presentation of 66 ought to be revised. The absolute configurations of staurosporine deri atives ll-hydroxystaurosporine (67) (56), 7- The origin of marine natural products is not always known with certainty: marine organism or symbiotic microorganisms? For this reason and because of the structural relationship with the above-mentioned compounds, derivatives containing the indolo[2,3-a]carbazole skeleton, isolated from the marine environment, are reviewed in this chapter. A brown tunicate, Eudistoma sp., collected in Micronesia is the source of two highly cytotoxic and powerful protein kinase C inhibitors: 11hydroxystaurosporine (92) and 3,11-dihydroxystaurosporine (93) (56). Comparison of the 'Hand 13C-NMR spectra of these molecules with those of staurosporine allowed the determination of the position of the phenol hydroxyl groups at C-11 (6 144.1 ppm) and at C-3 and C-11 in 92 and 93, respectively. The blue-green alga Nostoc sphaericum produces simpler indolo[2,3a]carbazoles (94-96), with weak activity against the Herpes simplex-2 virus and human cancer cell lines (80) . Another blue-green alga, Tolypothrix (43,77) . Arcyriaverdin C (116) from A. denudata is a formal condensation product of maleimide and 6-hydroxyisatin (43) . In arcyriacyanin A (117) and in its dihydro derivative 118 from A. nutans (43,44) , the two indole nuclei are attached by a C-2 to C-4' bond. In a similar fashion, an oxygen atom bridges the C-2 and C-4' carbons of the two indole halves in arcyro- (122), and arcyroxepin B (123) (43,44,77 ). Cordell proposed that two tryptophan units with intact carbon side chains were the biogenetic precursor of staurosporine aglycone (77) (83) . Participation of acetate in the biosynthesis was excluded on the basis of similar arguments. The following steps in the biosynthesis and the origin of the aminosugar moiety need further study. In order to determine the biogenetic origin of rebeccamycin (84), S. uerocolonigenes cultures were fed labeled D-glucose, L-methionine, and Ltryptophan (84) . The observed incorporations confirmed the involvement of these metabolites in biosynthesis, but the origin of the phthalimide nitrogen atom remains unknown. Gill Sarstedt and Winterfeldt's synthesis of staurosporine aglycone (77) used a photochemical cyclization as the key step (Scheme 8) (85) . Amide (124), prepared from tryptamine and P-indolyl-acetylchloride, was transformed into diketo-amide (125) with DDQ. Selective borohydride reduction, followed by acetylation, led directly to the pentaacetyl derivative (l26), which was then transformed into 127 under TiC1,-mediated reduction conditions and after deacylation. Irradiation of 127 smoothly afforded the target molecule (77). Magnus and Sear (86) made use of the indole-2,3-quinodimethane methodology to synthesize staurosporine aglycone (77) (Scheme 9). Na,Nb-bis-2-formyltryptamine (128) was condensed with 2-aminoStyrene, and, under standard conditions, the product (129) afforded. the pentacyclic carbamate with NBS opened a route to the preparation of unsymmetrical osazones and, hence, to unsymmetrical indolo[2,3-a]carbazole alkaloids (88) . The requirement of a selective bis-N-glycosylation method to transform 77 into staurosporine justified the introduction of a mobile N-protecting group on the lactam moiety. Use of N-benzylmaleimide as a dienophile seemed to provide a solution to the problem, but the sequence failed as it proved impossible to remove the N-benzyl protecting group (89) . Versatility and adaptability to large-scale operations were offered by a route involving ammonolysis of the terphenyl-anhydride (136), prepared by a Diels-Alder reaction between bis-(2-nitrophenyl)butadiene (135) and methyl acetylenedicarboxylate (Scheme 11). Because of low yields, the Clemmensen reduction of the imide into the corresponding lactam (137) was replaced by a two-step-procedure involving the intermediacy of a hydroxylactam. The lactam function was protected by a tetrahydropyranyl group (138) prior to triphenylphosphine-mediated deoxygenation and double-nitrene insertion (139) in order to avoid the formation of an extremely stable complex between the generated triphenylphosphine-oxide and the staurosporine aglycone. Arcyriaflavin-B (88) was obtained as a product of a Diels-Alder addition between the E,E-diene (140, R-OCH,) and maleimide, followed by triphenylphosphine-assisted deoxygenataion to the nitrene and demethylation (90) . Moody and Rahimtoola's route to the unsubstituted indolocarbazole system is based on an intramolecular Diels-Alder reaction of a pyrano [4,3- was treated with indolylmagnesium bromide to give diindolylmaleimide (145) . Oxidative cyclization to the hexacyclic ring system (146) was effected in high yield with 4-toluenesulfonic acid and DDQ in refluxing benzene. Partial reduction of the imide to the corresponding lactam could be achieved only under Clemmensen conditions (89,90) . The absolute configuration of rebeccamycin (84), which remained unknown after the first stages of spectroscopic investigation (74) , was determined by total synthesis (95) . The first approach to the heteroaromatic system was based on the well-known Grignard method (77,78), followed by photocyclization or silver-oxide-mediated oxidative thermal cyclization (Scheme 14) . The second route made use of the Diels-Alder reaction of maleimide and the 2,2'-bisindole (148), prepared from 7,7' -dichloroindigo (147) by Wolff-Kishner reduction. The sugar moiety was introduced as 4methoxy-triacetobromoglucose, prepared from D-glucose. Removal of the protecting groups by hydrogenolysis and ammonolysis gave synthetic rebeccamycin, in all respects identical to the natural product. The above example shows that 2,2'-biindolyls are attractive starting materials for the preparation of potent PKC inhibitors via the Diels-Alder reaction. This approach relies on 2,2'-bisindoles, whose preparation has been reviewed (96,97). Bergman's method involves the conversion of indoles into 1,l'-carbonylindoles (149), followed by Pd(OAc)2-assisted 2,2'coupling (98). A later approach is based on a triethyl-phosphite-induced nitrene insertion as the key step in producing symmetrical or unsymmetrical 2,2'-biindolyls (Scheme 15) (150) (99). Arcyriarubin-A (112) is a pigment produced by slime molds of the Myxomycetes family; its synthesis was achieved by Steglich et al. (77) , using indolyl-Grignard chemistry in the crucial step (Scheme 16). The condensation of indolylmagnesium bromide and dibromomaleimide (151) gave monoindolyl (152) and diindolyl (153) compounds, whose ratio depended on the reaction solvent (200). In toluene, the bisindolyl derivative (153) was obtained, whereas in THF the monosubstituted compound (152) was the major product. The transformation of 153 into arcyriarubin-A (112) was performed in two steps: alkaline hydrolysis led to anhydride (154) which was also directly obtained from indolyl-3-glyoxylyl-chloride and indole acetic acid (202) or by iodine-promoted coupling of the trianion of indole acetic acid (102) . Treatment of 154 with ammonium acetate yielded arcyriarubin-A. Maleic anhydrides are also converted into maleimides under mild conditions, using a mixture of methanol and hexamethyldisilazane (103) . The monosubstituted product (152) was used in the synthesis of unsymmetrically substituted arcyriarubins (113) and arcyriaflavins (87-90) (77,94,95,202) . N-boc-protected monoindolyl derivative (156) was reacted with the Grignard derivative of 6-tetrahydropyranyloxyindole to afford the desired coupling product (157). Thermal deprotection, followed by the usual conversion of the methylimido group into an imide, led to arcyriarubin-B rearrangement, discovered at Schering-Plough, gave aldehyde (159) which was transformed into 160 in a five-step sequence (Scheme 18). This compound proved to be as good an inhibitor of PCK as staurosporine (IC5,, lOnM) (206). A solution to the problem of the formation of N-glycosidic linkages was proposed by Danishefsky and co-workers, who found that sodium salts of indoles opened a-1,2-anhydrosugar epoxides with an inversion of configuration at the anomeric carbon to afford the desired indole-N-glycosides (Scheme 19) (207) . Application of the method to the'total synthesis of rebeccamycin (84) called for the preparation of the a-1,2-anhydrosugar (161), resulting from deprotection, protection, and 2,2-dimethyldioxiranemediated epoxidation of the secoaglycone (162). This latter compound was obtained in 60% overall yield using Kaneko et al.'s'route (95). Coupling between 161 and 163 was achieved in 48% yield of the desired p-N-glycopyranoside (164) when 3 eq. of the anhydrosugar was used. Fluoride-induced desilylation, followed by photocyclization, led to benzyloxyrebeccamycin, whose deprotection required special precautions to avoid reductive cleavage of the chlorine atoms. In the total synthesis of the staurosporine analogue ( proved to be of paramount importance for the completion of the synthesis (109) . A monosubstituted derivative of the aglycone of staurosporine (172) was prepared by Winterfeldt and co-workers in order to circumvent the difficulties inherent in the selective glycosylation of the two indole halves (110) . Base-catalyzed and photo-induced cyclizations were applied to the construction of the planar ring system (Scheme 22). Naturally occurring indolo[2,3-a]carbazole, bisindolylmaleimide alkaloids, and their synthetic analogues display diverse biological activities which have been the subject of a review (45). Protein kinase inhibition is the most significant biological activity of staurosporine (66) ( I I I ) , of its aglycone (77) (212) , and a wide range of synthetic analogues (113) . Staurosporine is an inhibitor of low specificity (114, 115) , but the aglycone and numerous synthetic analogues (173-175) show specific inhibitions of particular protein kinases (A, G, etc.) (116, 117) . As an example, amine derivatives (176) have better PKC inhibitory activity than K-252a (75) (128) . The 7-0x0-derivative (177) or aromatic ether analogues were also prepared (119) . Surprisingly, N,N'-dialkyl-substituted aglycones (178 and 179) and bisindolyl maleimide analogues (180-184) exhibited strong, and sometimes extremely selective (180) and (184), protein kinase activities (120, 121) . with an IC,, < 0.01 p M (122, 123) , and was even found to be active against vincristine-resistant cell lines (124). Rebeccamycin (84) displays significant activity against P-388 and L-1210 leukemia and inhibits the growth of human lung adenocarcinoma cells (75). Numerous compounds, especially tertiary or quaternary amine derivatives activities (46,47,132) , are worth mentioning. Studies of mycotoxin production by fungi belonging to the genus Chaetomium revealed the production of several secondary metabolites, possessing remarkable antimicrobial and cytotoxic activities (Table I) (133) . This is the case with chaetocin (185), which was isolated from Chaetomium minuturn (134). Its symmetrical structure has been elucidated by chemical transformations and spectroscopy. The absolute configuration (S for C-3 and C-lla of the disulfide bridge) was determined by X-ray diffraction analysis, including sulfur anomalous diffusion, and was found to be opposite the configurations observed in gliotoxin (186) and sporidesmin (187); this was also proved by circular dichroism Chetracin-A (193) is a metabolite from Chaetomium abuense Lodha and C. retardatum Carter & Khan exhibiting strong cytotoxic activity (140). Its absolute configuration and the conformation of the tetrasulfide bridge were established by X-ray crystallography. Despite the symmetrical nature of the molecule, it formed a triacetate (194) instead of a tetraacetate on treatment with AczO and pyridine, which can be explained by steric hindrance of the C-ll'a-hydroxyl group and by hydrogen bonding between ll'a-OH and the carbonyl group located at the C-1' carbon. This effect might also be responsible for the incomplete acetylations of melinacidin IV (190) and the verticillins under identical conditions. Ditryptophenaline (195) is a metabolite from Aspergillus flauus (strains MIT-M25, -26, and -27) and did not exhibit any significant biological activities (141). Its symmetrical structure was established from spectroscopic data and X-ray diffraction studies. Formally, 195 is derived from the coupling of tryptophan, phenylalanine, and a methyl unit, followed by oxidative dimerization. Its absolute configuration was not determined. 1% were isolated from the culture of Chaetomium gfobosum Kinze ex. FR and were shown to possess antimicrobial activity against E. cofi W 3110 and Staphylococcus aureus 2097 (148). Their 'H-NMR spectra were similar, except for the presence of four additional thiomethyl singlets between 6 2.02 and 6 2.32 ppm in the spectrum of 197. All ambiguities in these structures were erased by X-ray crystallographic analysis, chemical correlations, and the reductive methylation of 1% to 197. By far, bisindoles in plants are of iridoid origin; some families of plants, however, produce bisindolic metabolites with a different biosynthesis. This is exemplified by a series of alkaloids from the Simaroubaceae and by the pigments of the isatin type. A. THE Picrasma DIMERS Bis-P-carboline-type dimers have been isolated from the roots of Picrasma quassioides Bennet (Simaroubaceae), a plant used in Japan as a bitter stomachic. The P-carboline nuclei were recognized by typical UV absorptions as found in the simple monomeric substances isolated from the plant. The 'H-NMR data for 198 (149) displayed signals for two methylene groups as triplets (6 3.60 and 3.92 ppm), suggesting the presence of a 1,2disubstituted ethane function as a spacer. The location of methoxyl groups at C-4 (6 149.9 pprn) and C-8 (6 146.0 ppm) was deduced from a comparison of the 'Hand 13C-NMR data for 198 with those of P-carbolines (199 and 200). Further support of the structure (198) was obtained from the mass Compound 201 is a second dimer of the same origin (150), which differed from 198 in the non-conjugated part. The 'H-NMR spectrum showed a doublet of doublets at 6 5.55 ppm, assigned to a methine, a supplementary methoxyl group (6 3.51 ppm), and signals for two contiguous methylenes. The near superimposition of the aromatic parts of the I3C-NMR spectra of 201 and 198 led to the conclusion that the structural differences between the compounds belonged to the spacer chain. Structure 201 was deduced to account for these arguments and the observed mass spectrum fragmentations. Indirubin (214) was isolated from the fruits of the cannonball tree, Couroupifa quianensis Aubl. (158) , and shown to inhibit Lewis lung carcinoma and Walker carcinosarcoma. The structurally related dimer, candidine, is a violet indolic constituent from the culture of Candida lipolytica (159) . Mass spectral fragmentations led to the proposal of two structures, with the correct one (214) being identified by synthesis from tryptanthrin and N-acetylindoxyl. Candidine (215) proved to be identical to compounds isolated from Baphicacanthus cusia and Isatis finctoria and was given the name quingdainone (160,161). The Alkaloids Studies in Natural Products Chemistry Studies in Natural Products Chemistry 236750~ (1987)l; (b) Japanese Kokai Tokkyo Koho JP 62 PCT International Application WO 89 185264~ (1991)l; (g) PCT International Application WO 93 PCT International Application WO 93 The Biosynthesis of Mycotoxins In continuation of the work on P. quassioides, picrasidine H (202) and R (203) were isolated from the root bark (151) . They displayed a Pcarboline-like UV spectrum with an end absorption at 360-370 nm, attributed to a conjugated carbonyl function (1660 cm-'). The 'H-NMR spectrum of 202 showed an exchangeable hydroxyl proton at 6 4.83 ppm. Spindecoupling experiments allowed the determination of the structure of the hydroxyl-containing four-carbon subunit, which linked the two 0-carboline moieties. Irradiation of the methoxyl groups at Each displayed typical P-carbolinium-type UV spectra, whose maxima were shifted by the addition of alkali following transformation into P-carboline anhydro bases. The structure of picrasidine-F (204) was confirmed by Xray crystal structure analysis (152) . Picrasidines-G (205), -S (206), and -T (207) had almost identical 'H-NMR spectra and differed in their aromatic substituents, being proton, methoxyl, and hydroxyl, respectively (153) . Picrasidines-M (208), -N (209), and -U (210) are composed of P-carboline and canthin-5,6-dione moieties. The presence of the latter common structural feature in 208 was suggested by two carbonyl functions with resonances at 6 156.3 and 169.9 pprn and by the observation of a prominent mass spectral fragment at m/z 236. The location of the methoxy substituents was determined by treatment of 208 with acetic anhydride which afforded 5acetoxycanthin-6-one (211) and 4,8-dimethoxy-l-vinyl-~-carboline (213) (155) (Scheme 23) .The close resemblance of spectroscopic data for 208 and 210 suggested similar structures (156). The main difference between the NMR spectra of the aromatic regions of 210 and those of 208 concerned the C-4 position of the canthin-5,6-dione moiety in 210, in which the hydrogen was replaced by another methoxyl group (6 3.89 ppm). The cleavage reaction with acetic anhydride also proved the assignment. The 'H-NMR spectrum of picrasidine-N (209) showed that the methoxyl group at 6 64.7 ppm was located on the indole nitrogen. The compound was degraded into 4,9dimethoxy-1-vinyl-P-carboline (213), thus supporting structure 209 (157).