Catharanthin

After the stmcture and absolute stereochemistry of cleavamine (111), C22H24N2, was estabUshed, its synthesis was shortly completed and impetus to unravel the stmcture of the dimeric bases (22) was bolstered (77). Again, the fragment, now only slightly modified from that originally present in secologanin (102), is readily seen in catharanthine (107). 

Initial attempts to synthesize the compounds (22) were hampered by the failure to obtain the correct stereochemical configuration about the vindoline—catharanthine linkage, a most difficult problem eventually solved by insight and hard work (83). 

Imanishi, T., Shin, H., Yagi, N., Hanaoka, M. (1980) l,6-Dihydro-3(2H)-Pyridinones as Symmetric Intermediates. Formal Synthesis of ( )-Tabersonine and ( )-Catharanthine. Tetrahedron Letters, 21, 3285-3288. 

Two RCM reactions were employed in a new and efficient route to a key chiral intermediate, isoquinuclidine 150, in the synthesis of alkaloid (-F)-catharanthine <06AG(I)5334>. The first RCM makes use of chiral enone 151, derived from L-serine, to generate a chiral dihydropyridinone 152. Intramolecular alkene metathesis of dialkenyl piperidine 153 generates 150, which represents the first example of the use of RCM in the generation of an azabicyclo[2.2.2]alkene system. 

Finally, the last new alkaloid belonging to this subtype was found in T. mocquerysii, besides (205)-l,2-dehydropseudoaspidospermidine 169, already discussed in Volume 17 of this series and (20R)-pseudoaspidospermidine 174 (147). The new compound, (205)-pseudoaspidospermidine (175, cI9h26n2, mp 89°C, [a]D +60°), was identical in all respects, except optical rotation, with the pseudoaspidospermidine (304, [ot]D-60°), obtained by chemical elaboration of (+)-catharanthine (305) (109). 

Extensive biotransformation studies have been conducted with the As-pidosperma alkaloid vindoline, but much less work has been done with monomeric Iboga and dimeric alkaloids from this plant. The long-standing interest in this group of compounds stems from the clinical importance of the dimeric alkaloids vincristine and vinblastine, both of which have been used for more than 2 decades in the treatment of cancer. Few mammalian metabolites of dimeric Catharanthus alkaloids have been characterized. Thus the potential role of alkaloid metabolism in mechanism of action or dose-limiting toxicities remains unknown. The fact that little information existed about the metabolic fate of representative Aspidosperma and Iboga alkaloids and Vinca dimers prompted detailed microbial, mammalian enzymatic, and chemical studies with such compounds as vindoline, cleavamine, catharanthine, and their derivatives. Patterns of metabolism observed with the monomeric alkaloids would be expected to occur with the dimeric compounds. 

SCHEME 4. Catharanthine 3 and tabersonine 4, natural compounds containing a cyclohexene moiety. These may derive from in vivo Diels-Alder reactions53... 

Goodbody and co-workers (7/9) have examined the production of alkaloids in root and shoot cultures induced from seedlings of C. roseus. The pattern of alkaloids in the root cultures was similar to that of the roots from intact plants. Thus ajmalicine (39) and catharanthine (4) were produced, but no vindoline (3), a major leaf alkaloid, and no bisindole alkaloids. Similarly, the pattern of the alkaloid content of the shoot cultures was like that of the leaves of the intact plant, showing the presence of vindoline (3), catharanthine (4), and ajmalicine (39), with 3 predominating. A search for the bisindole alkaloids in the cultures indicated the presence of anhydrovinblastine (8) and leurosine (11) in the shoot cultures (2.6 and 0.3 xg/g fresh weight, respectively), but no vinblastine (1) or vincristine (2). 

The effect of light on alkaloid production in these cultures was also evaluated (7/9). More catharanthine was produced in the light than in the dark, and the same observation was made for ajmalicine (39). Knobloch et al. 120) examined the production of anthocyanins, ajmalicine (39), and serpentine (40) in cell suspension cultures and found that although serpentine levels increased 18-fold in the light, ajmalicine levels decreased 50%. In the work of Goodbody et al. 119) the biosynthesis of vindoline (3)... 

Miura and co-workers 121) have successfully induced multiple shoot cultures of C. roseus from seedlings in the presence of 1.0 mg/liter of the cytokinin benzyladenine. Vindoline (3) and catharanthine (4) were predominating alkaloids in the MSC-B-1 line, showing levels of 1.8 and 0.37 mg/g dry weight, respectively in the leaf tissue. In the case of catharanthine (4) this represented a 10-fold increase over the parent plant tissue, and such levels were sustained in the regenerated plants. When the benzyladenine was eliminated, overall growth was reduced, but vindoline (3) and catharanthine (4) concentrations increased to 3.2 and 1.1 mg/g dry weight, respectively. 

Continued work by the same group 123) has led to the first isolation of vinblastine (1) from a multiple shoot culture of C. roseus. The most productive line, MSC-B-1, consisted of two distinctly different tissues, multiple shoots and unorganized tissue, and was maintained growing and productive for 30 months. Vinblastine (1) was isolated by HPLC, and the content was estimated to be 15 jjig/g dry weight. Production of this alkaloid was greater than that in the callus culture but less than that observed for the parent plant, even though the levels of catharanthine (4) and vindoline (3) were about the same. 

Scott and co-workers have also reported on the isolation of alkaloids from C. roseus cell suspension cultures 126). The cell line used, identified as CRW, afforded akuammicine (49), catharanthine (4), and strictosi-dine (33), and feeding experiments with labeled tryptophan led to incorporation into ajmalicine (39), akuammicine (49), catharanthine (4), and vindoline (3). The ability to produce alkaloids was carried through 8 successive generations. 

The 200GW line proved to be quite different, and of particular interest was the discovery that this line produced catharanthine (4) at levels about three times that of the intact plant (0.005%) (155,159,160). Curiously, the predominant alkaloid (60.48%) was strictosidine lactam (41), which is not normally seen in extracts of intact plants. Variation of the pH and added phytohormones did not significantly alter the pattern of alkaloids produced by this cell line (160). Further cell line studies (161) afforded one line (176G) which produced mainly ajmalicine (39) and lochnericine (73) and one (299Y) which apparently contained relatively inactive p-glucosi-dases, since the major alkaloids produced were strictosidine (33) (83%) and strictosidine lactam (41) (Table XIII). 

It is well established that the iridoids are derived from two units of mevalonic acid (97), which itself is derived from acetyl-CoA. Mevalonate is also known to be a metabolic product of leucine (172), and the latter is a precursor of the monoterpene linalool (173). Wigfield and Wen (174) pursued the incorporation of leucine into the monoterpene unit in both vindoline (3) and catharanthine (4), where levels of 0.07 and 0.02%, respectively, were found, irrespective of the amount of precursor fed. This was important because, although initial results were obtained with [2- C] leucine, the specificity of incorporation was determined with 2- C-la-beled precursor. Two carbons in vindoline (3), C-8 and C-24, were en-... 

In vivo feeding experiments with singly and doubly labeled strictosidine (33) in C. roseus shoots afforded labeled ajmalicine (39), serpentine (40), vindoline (3), and catharanthine (4). Vincoside (85, page 37) was not incorporated into the alkaloids, suggesting that it was biologically inert 188). Brown and co-workers 190) conducted somewhat parallel studies examining the precursor relationship of strictosidine in C. roseus. Incorporation into tetrahydroalstonine (75), ajmalicine (39), catharanthine (4), and vindoline (3) was observed. 

Almost nothing is known about the biosynthetic pathway between a Corynanthe intermediate and the first Aspidosperma alkaloid tabersonine (38) and, indeed, the first Iboga alkaloid catharanthine (4). Thus, the focus of further work has been the intermediates involved in the pathway... 

Dadonna and Hutchinson (2/7) investigated the incorporation of [3,6- H2,wct/ioj > - C]loganin into vinblastine (1), catharanthine (4), and vin-doline (3) in whole plants of C. roseus. The specific incorporation into vinblastine (1) was in accord with intact incorporation into both units. Interpretation of the experiment is so complex, however, that further conclusions regarding intermediates or the stereospecificity of the processes are extremely speculative. 

The enzyme-catalyzed formation of anhydrovinblastine (8) from catharanthine (4) and vindoline (3) was first examined by Kutney and co-workers (170,219) using a cell-free preparation. [ao f- H]Catharanthine (4) and [acety/- C]vindoline (3) were incubated for 3-8 hr, both separately and jointly with a preparation from C. roseus, which led to the isolation of labeled anhydrovinblastine (8) and leurosine (11) incorporations were of the order of 0.54 and 0.36%, respectively. On this basis, anhydrovinblastine (8) was proposed as the key biosynthetic intermediate en route to vinblastine (1) and vincristine (2). 

More recently, Kutney and co-workers (220) have investigated whether the same dihydropyridinium intermediate 109 is involved in the enzymatic conversion of catharanthine (4) and vindoline (3) to anhydrovinblastine (8) as is involved in the chemical conversion. Use of a cell-free preparation from a 5-day culture of the AC3 cell line gave 18% of the bisindole alkaloids leurosine (11), Catharine (10), vinamidine (25), and hydroxy-vinamidine (110), with 10 predominating. When the incubations were carried out for only 5-10 min, the dihydropyridinium intermediate was detected followed by conversion to the other bisindole alkaloids, with FAD and MnClj required as cofactors. Clearly a multienzyme complex is present in the supernatant, but further purification led to substantial loss of enzymatic activity. The chemical formation of anhydrovinblastine (3) is carried out with catharanthine A-oxide (107), but when this compound was used in the enzyme preparation described, no condensation with vindoline (3) occurred to give bisindole alkaloids. This has led Kutney and co-workers to suggest (220) that the A-oxide 108 is not an intermediate in the biosynthetic pathway, but rather that a 7-hydroperoxyindolenine... 

FMN. Similar conversion yields for each of the isozymes were in the range 34-50%. Paralleling these data was the observation that horseradish peroxidase was also capable of converting vindoline (3) and catharanthine (4) to anhydrovinblastine (8) with the correct C-18 stereochemistry (222). 

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