Anhydrovinblastine from catharanthine

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). 

Peroxidase containing fractions of plant extracts were found to catalyze the formation of the bisindole dehydrovinblastine from catharanthine and vindoline.(120, 121) A peroxidase from C. roseus leaves has been demonstrated to convert vindoline and catharanthine to anhydrovinblastine in vitro (122, 123). Because the dimerization of these C. roseus alkaloids also can be catalyzed by peroxidase from horseradish in reasonable yields (124), it is interesting to speculate that anhydrovinblastine may be a by-product of isolation after lysis of the plant material, nonspecific peroxidases are released from the vacuole and may act on vindoline and catharanthine. 

The chemical coupling of catharanthine and vindoline to yield anhydrovinblastine led to the obvious hypothesis that this compound might also be the first product of dimerization in the plant, and the dimeric precursor of vinblastine and vincristine. For three years it was not possible to find anhydrovinblastine in the plant, until Scott et al. in 1978 [115], by modifying the established methods for extraction and purification of alkaloids, isolated anhydrovinblastine from C. roseus plants, with incorporation of radiolabelled catharanthine and vindoline, thus proving that anhydrovinblastine was actually a natural product. 

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). 

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... 

The conversion of anhydrovinblastine (8) to vinblastine (1) has been examined by several different groups, using intact plants, cell suspension systems, and cell-free preparations. From the studies discussed above it was clear that 3, 4 -anhydrovinblastine (8) was probably the initially formed intermediate in the condensation of vindoline (3) and catharanthine (4) prior to vinblastine (1). Kutney and co-workers have reported (225,226) on the biotransformation of 3, 4 -anhydrovinblastine (8) using cell suspension cultures of the 916 cell line from C. roseus a line which did not produce the normal spectrum of indole alkaloids. After 24 hr the major alkaloid products were leurosine (11) and Catharine (10) in 31 and 9% yields, respectively, with about 40% of the starting alkaloid consumed. 

The extremely low yield of vincristine (2) from intact plants has made pursuit of its biosynthesis a very challenging problem, which at this point in time remains unsolved. Kutney et al. have used both anhydrovinblastine (8) (227) and catharanthine N-oxide (107) (233) as precursors to vincristine (2) in a cell-free preparation, but incorporation levels were extremely low. Therefore, the question of whether vinblastine (1) is an in vivo, as well as an in vitro, precursor remains to be answered. Several possibilities exist for the overall oxidation of vinblastine (1) to vincristine (2), including a direct oxidation of the A-methyl group or oxidative loss of the N-methyl group followed by N-formylation. 

Coupling of other vindoline derivatives with ring D or E modified oxidation levels (92-96) to catharanthine N-oxide provided new binary products for biological evaluation 39, 95-97). The two diastereomeric C-16 -C-14 PARE anhydrovinblastines 42 and 97 were obtained in a 46 54 ratio (50% yield) from racemic catharanthine (98), and the corresponding 20 -desethyl compounds 98 and 99 were generated at - 20°C in a 1 1 ratio (16% yield each), and at -76°C in lower yields, together with the corre-... 

Endo, T., Goodbody, A., Vukovic, J. and Misawa, M. (1986) Enzymes from Catharanthus roseus cell suspension cultures that couple vindoUne and catharanthine to form 3, 4 -anhydrovinblastine. Phytochemistry, 27,2147-9. 

If the C-15, C-16 bond is oxidatively cleaved, the secodine skeleton results (the proposed progenitor of the Aspidosperma and the iboga systems) through alternative Diels-Alder type cyclizations to afford tabersonine and catharanthine. The bisindole alkaloids of Catharanthus roseus reflect the union of vindoline and catharanthine to afford anhydrovinblastine modification affords the clinically significant alkaloids, vinblastine (VLB) and vincristine (VCR Fig. 39). The alkaloids, particularly VCR, are important as anticancer agents and have led to the development of the semisynthetic derivatives vindesine and vinorelbine (Fig. 40). Synthetic approaches are available to join the monomeric precursors. The enzymatically controlled sequence of reactions from tabersonine to vindoline has been elucidated. 

Catharanthine (LIV) and vindoline (Lin) are regarded as the monomeric precursors of the dimeric alkaloids vinblastine and vincristine, via a-3 ,4 -anhydrovinblastine. C. roseus peroxidase catalyzes the coupling reaction of catharanthine and vindoline (Scheme XXVI) to lead to a-3 ,4 -anhydrovinblastine (XLVH) or, more properly, to an iminium intermediate (LVI) from which a-3 ,4 -anhydrovinblastine is directly derivated [52,74,166]. a-3 ,4 -Anhydrovinblastine is then converted to vinblastine (XLIX, R = CH3) and vincristine (XLIX, R = CHO) in C. roseus plants [167-169], a-3 ,4 -Anhydrovinblastine (XLVn), or the unstable iminium intermediate (LVI) formed during the coupling reaction, is then assumed to be the precursor of all dimeric alkaloids in C. roseus. 

Although the precise mechanism of the coupling reaction is not thoroughly established, one can visualize the formation of (71) as arising from initial fragmentation of the C(16)—C(21) bond of (69), followed by condensation of vindoline with the more accessible a face of the iminium ion (73). The impact of the Polonovski approach in this area is emphasized by the fact that all other attempts to couple vindoline with 16,21-seco derivatives of catharanthine lead invariably to formation of the unnatural dimer. A Polonovski reaction was also a key step in the subsequent elaboration of anhydrovinblastine (71) to (68). ... 

Fig. (2). Biosynthesis of vinblastine from the monomeric precursors catharanthine and vindoline. Anhydrovinblastine is the direct product of the dimerization reaction and the precursor of the anticancer drugs. Shaded areas indicate the structural differences between the precursor catharanthine and the deavamine part of...

In 1975, Potier and collaborators proposed that, in planta, the dimeric vinblastine type alkaloids resulted from the coupling of catharanthine and vindoline and, in light of this hypothesis, they reported for the first time the chemical synthesis of a dimer with the natural configuration through a modified Polonovski reaction [18, 19]. This reaction resulted in the formation of an iminium dimer which, after reduction with NaBH4, yielded a-3 ,4 -anhydrovinblastine, Fig. (2), later proved to be the first dimeric biosynthetic precursor of vinblastine in the plant. The group of Potier investigated possible modifications of anhydrovinblastine and produced vinorelbine, Fig. (1), which was the first active derivative with an altered cleavamine (catharanthine) moiety [20, 21]. 

The search of the enzyme responsible for the dimerization reaction, i.e. for the biosynthesis of anhydrovinblastine, resulted in the finding that peroxidase-like activities extracted from cell suspension cultures were capable of performing the coupling of catharanthine and vindoline into anhydrovinblastine [125-127]. Horseradish peroxidase, a commercial plant peroxidase, was also capable of performing the coupling reaction [128]. 

At this point, anhydrovinblastine had been proved to actually be a major alkaloid present in C. roseus leaves [106, 107] representing together with catharanthine and vindoline the three major alkaloids of the plant. This indicated the presence of high in vivo anhydrovinblastine synthase activity in leaves and that this was the appropriate biological material to search for the enzyme. Work with leaves started at the laboratory of Prof. Frank DiCosmo from the University of Toronto, Canada, and has mostly been developed in our labs, at the University of Murcia and the University of Porto. 

It would thus appear that the presence of an a-acetoxy-group at C-15 severely inhibits the fission of the 16,21-bond in the coupling reaction, since the isovinblastine O-acetate (258) was obtained in yields of only 6 and 4%, respectively, from (257) and (260). The effect of a /3 -acetoxy-group is less well defined Honma and Ban " report the formation of anhydrovinblastine (255), but only as the minor product of the reaction, whereas Kutney and Worth report the formation of (253) and (254), but in unspecified yield. For the synthesis of vinblastine derivatives the absence of a C-15 substituent, as in catharanthine and dihydro-catharanthine, seems preferable for example, catharanthine N-oxide was... 

When LEE is used, it is advisable to do either very quickly or from only a moderately alkaline aqueous solution, which was basified by sodium carbonate or ammonia. The Catharanthus alkaloids was prepared by LEE from the 75 % ethanol extract at pH 3.5, then adjusted the basic to pH 12 and finally extracted by chloroform [7]. Three major alkaloids, vinblastine and its monomeric precursors (vindoline and catharanthine), were monitored in transformed root cultures of Catharanthus roseus, after rapid sample preparation by LEE in our previous work [8]. The extraction method was the same as above. Other Catharanthus alkaloids, such as vindoline, catharanthine, and anhydrovinblastine, were prepared by LEE from the methanol extraction [9]. 

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