Leurosine 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). 

The synthesis of Catharine (250), " to which catharinine was initially believed to be closely related, has in fact been achieved by a process which involves the fission of ring D of the velbanamine component of leurosine (249). This conversion was first reported as a result of the accidental over-oxidation that occurred in the preparation of leurosine from anhydrovinblastine by means of t-butyl hydroperoxide in the presence of trifluoroacetic acid. The by-product in this reaction was initially regarded as the 21-lactam related to leurosine, but it has now been recognised as Catharine, and can be prepared equally well by oxidation in the absence of acid (Scheme 41) a radical mechanism appears to be involved. In view of this facile conversion under oxidising conditions, the status of Catharine as a bona fide natural product is open to question. Indeed, the status of leurosine itself as an alkaloid has been questioned, in view of the ease with which anhydrovinblastine is oxidised to leurosine, even in the absence of specific oxidising agents. For example, anhydrovinblastine is oxidised to leurosine if not stored in an inert atmosphere, and the conversion is even more rapid in solution, particularly in the presence of adsorbents such as silica or alumina. A conversion of 40% has been observed after only 72 hours at room temperature. In view of these results it is perhaps not surprising that anhydrovinblastine has not been isolated from any Catharanthus species examined to date. 

Two deoxygenations of naturally occurring compounds have been reported. Anhydrovinblastine, an important intermediate for the anticancer drug navelbine, was prepared by Doris and coworkers from leurosine, an abundant alkaloid from the Madagascan periwinkle Catharantus roseus, in 70% yield by using Cp2TiCl (Scheme 5) [46]. 

The isolation of Catharine (10), C45H54N40,o, mp 271-275°C, an on-colytically inactive alkaloid, has been reported from several Catharan-thus species C. roseus (29-31), C. ovalis (32), and C. longifolius (33). The structure of Catharine (10) has been elucidated by X-ray crystallography (89-91) of its acetone solvate. Catharine (10) can be obtained by mild oxidation of either leurosine (11) (30) or anhydrovinblastine (8) (92-93). In view of the ease of this oxidation, Catharine (10) may be considered as an artifact of the isolation process. 

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. 

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 question of the possible artifactual nature of leurosine (11) was examined by Kutney and co-workers (229) using a cell-free preparation at pH 6.3. After a 3-hr incubation, a 22% yield of leurosine (11) was formed from anhydrovinblastine (8), and when anhydrovinblastine (8) was incubated with horseradish peroxidase in the presence of HjOj, leurosine (11) was formed in 65% yield. 

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. 

There are two especially relevant cases where Cp2TiCl-promoted epoxide deoxygenations have been demonstrated to conform to the requirements of selectivity, mildness, and wide functional group tolerance desirable in natural product synthesis the chemical correlation between cryptophycin-23 and cryptophycin-45 and the synthesis of anhydrovinblastine, the key intermediate in the synthesis of the anticancer drug Navelbine, from leurosine (Scheme 14) [91-93]. 

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. "°... 

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