Vinblastine from anhydrovinblastine

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). 

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...

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 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. 

When 3, 4 -[ao - H]anhydrovinblastine (8) was incubated with a cell-free preparation at pH 6.3 for 50 hr, leurosine (11) and Catharine (10) were labeled to the extent of 8.15 and 15.15%, respectively, and vinblastine (1) was labeled to 1.84% (776,227). Approximately the same level of incorporation was obtained by Scott s group, using 3, 4 -[21 - H]anhydrovinblas-tine (8) and isolating vinblastine (1) from cell-free extracts of C. roseus (228). Scott s failure (87) to observe incorporation of the same precursor into vinblastine (1) in whole plants was explained by the established (82) instability of anhydrovinblastine (8). 

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. 

The chemical reactivity of N-6 (or N ), directed entirely by the basicity of this atom, is controlled by the nature and stereochemistry of the substituents at C-4 (vide supra). Oxidation of N-6 occurs under mild conditions in several naturally occurring bisindole alkaloids. Thus, treatment of a dichloromethane solution of leurosine (4) with m-chloroperben-zoic acid at -20°C for 4 hr gives the N -oxide (15) in greater than 90% yield after preparative reversed-phase chromatography (46). Leurosine A/ -oxide has also been isolated from Catharanthus roseus and should therefore be considered a naturally occurring bisindole (50). The analogous conversion of vinblastine (1) to its A/ -oxide (16) proceeds under similar conditions but requires longer exposure to the peraeid (24 hr) (5/) 3, 4 -anhydrovinblastine is converted to its N -oxide (17) in 10 min at 0°C... 

Further investigations have been carried out into the direct preparation of 5 -nor-alkaloids from parent alkaloids of the vinblastine group.141 The 7 -chloro-indolenines (328) and (329) derived from anhydrovinblastine and leurosine fragment in the presence of silver fluoroborate to give, after re-cyclization, the 5 -nor-bases (330) and (331). In the formation of (328) and (329), some chlorination also occurs at position 12, and this can, under appropriate conditions, form the major reaction. Products similar to (330) and (331) were also obtained from the analogous reactions with anhydrovincristine and leuroformine. 

Baxster RI, Dorschel CA, Lee SL, Scott AI. Biosynthesis of the antitumor cafiiaranfiius alkaloids. Conversion of anhydrovinblastine into vinblastine. J. Chem. Soc. Chem. Comm. 1979 257-259. Gueritte F, Bac NV, Langlois Y, Potier P. Biosynthesis of antitumour alkaloids from Cafiiaranfiius roseus. Conversion of 20 deoxyleurosidine into vinblastine. J. Chem. Soc. Chem. Comm. 1980 452-453. 

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. 

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 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. 

Whether leurosine, Catharine, and their congeners are true alkaloids, or artefacts derived from anhydrovinblastine, the fact remains that the aerial oxidation of anhydrovinblastine is a facile process which does not need to be enzyme-mediated, and a further examination of this reaction has revealed that all the alkaloids of the vinblastine group are produced. The oxidations were performed in acetonitrile solution, and in one experiment, conducted at 26 for 48 hours, the composition of the alkaloid mixture obtained was roughly similar to the relative abundances of the dimeric alkaloids isolated from Catharanthus species. In the oxidation the lone electrons on Nb are presumably involved, since anhydrovinblastine Nb-oxide is inert towards oxidation by air, and while the presence of moisture promotes the reaction, oxygen from the water is not incorporated into the oxidized alkaloids. On the basis of the available evidence, a mechanism, shown in truncated form in Scheme 37, was proposed for the oxidative transformation of anhydrovinblastine into the various alkaloids iso-lated. "°... 

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... 

Recently however, a basic peroxidase, anhydrovinblastine synthase, which couples eatharanthine and vindoline to yield anhydrovinblastine, the putative precursor to vinblastine and vincristine, has been purified and characterized from C. roseus leaves 421,422). The enzyme showed a specifie anhydrovinblastine synthase activity of l.Snkatmg and a molecular weight of 45.40kDa. It was shown to be a high-spin ferrie heme protein belonging to the plant peroxidases superfamily (class HI peroxidases), and eytochemical studies showed that the enzyme is localized in the mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. On the basis of the ability of the monomeric alkaloid substrates to reduce the C. roseus basic... 

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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