Of anhydrovinblastine

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 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... 

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). 

To examine whether a dihydropyridinium intermediate is involved in the conversion of anhydrovinblastine (8) to vinblastine (1), Scott et al. fed methyl- C]anhydrovinblastine (8) to a cell-free suspension... 

The relatively facile formation of anhydrovinblastine (42) by the modified Polonovski reaction, and the poor yields experienced on coupling of other catharanthine derivatives, made anhydrovinblastine an attractive precursor for the preparation of additional binary alkaloids, including vinblastine itself (50,57). Hydrogenation to 20 -deoxyleurosidine (61a), formation of its Af -oxide (78), and reaction with trifluoroacetic anhydride led to the enamine 59 (Scheme 23). Oxidation of this enamine with thal-... 

The stereochemistry of hydrogenation of anhydrovinblastine was initially misassigned. See also ref. 70-74. 

The product from the reaction of anhydrovinblastine Nfc-oxide 1 with trifluoroacetic anhydride in methylene chloride was treated, after evaporation of the excess of the reagent, with a mixture of water and THF. This gave a mixture of products, only one of which could be obtained pure (27%), namely 5 -noranhydrovinblastine 2. 

The critical dependence of the stereochemical and regiochemical course of the modified Polonovski reaction on the oxygen functionality in the catharanthine derivative has been well exemplified in recent synthetic studies. Indeed, in the reaction that ultimately provided the first synthesis of anhydrovinblastine, a minor product proved to be the result of an alternative fragmentation of the catharanthine Nb-oxide derivative in which the 5,6-bond was cleaved [->(266)] and subsequent coupling of vindoline occurred at position 6, with formation of the dimeric species (267).159 When an attempt was made to couple the N-oxide of the lactone (238) with vindoline under Polonovski conditions, this type of coupling occurred exclusively, and the products were the lactone (268) (major product)163-165, the... 

Further information is available on the biosynthesis of vinblastine-type alkaloids (cf. Vol. 11, p. 19 Vol. 10, p. 19). Anhydrovinblastine (61) was found to be metabolized to leurosine (63) and Catharine (64) in cultures of a C. roseus cell-line that do not normally produce these alkaloids.55 Ring-opening in the conversion of the skeleton of anhydrovinblastine (61) into that of Catharine (64) has been suggested to occur by Baeyer-Villiger-type oxidation of an imine las (62). The alternative 21 -imine could give catharinine.56... 

Oxo-15,205 -dihydrocatharanthine (275a), prepared as described above, has been employed in a new synthesis of anhydrovinblastine (323). Stereospecific reduction of (275a), followed by acetylation and formation of the A -oxide, gave an... 

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]. 

Scott AI, Gueritte F, Lee SL. Role of anhydrovinblastine in file biosynthesis of file antitumor dimeric indole alkaloids. J. Am. 

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. 

A. Purification and characterization of anhydrovinblastine syn- 140. thase (peroxidase like) from Catheranthus roseus. FEES Lett. 1998 428 299-303. 

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). ... 

Sundberg, R. J., Gadamasetti, K. G., Hunt, P. J. Mechanistic aspects of the formation of anhydrovinblastine by Potier-Polonovski oxidative coupling of catharanthine and vindoline. Spectroscopic observation and chemical reactions of intermediates. Tetrahedron 1992,48, 277-296. 

The Canadian group have also followed their synthesis of anhydrovinblastine (266) by studying the introduction of oxygen substituents to positions 15 and/or 20. Oxidation of anhydrovinblastine with t-butyl hydroperoxide afforded a moderate yield of leurosine following experience with other substrates in this reaction Kutney etal. formulate leurosine as the -epoxide, rather than the a-epoxide (258) favoured " by other workers. However, both formulations are at present tentative, and it will be intesting to note which one ultimately proves to be correct. 

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]. 

In 1979, Langlois and Potier [116] proposed that anhydrovinblastine could be the precursor of most, if not all, dimeric alkaloids of C. roseus, and feeding studies indicated the enzymatic incorporation of anhydrovinblastine into vinblastine and other dimeric alkaloids [117-120]. Incorporation studies were lurther confirmed by experiments with cell free homogenates of C. roseus cell suspension cultures [121, 122]. Several biosynthetic routes were proposed in which either anhydrovinblastine or its iminium were the pivotal intermediates of all dimeric alkaloids [114,... 

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]. 

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... 

The accessibility of anhydrovinblastine (255) affords an independent route to vinblastine derivatives, via functionalisation of the 15, 20 double-bond. Hydroboron ation-oxidation of (255), for example, gives a mixture of 15 j8-hydroxy-N. Langlois, F. Gueritte, Y. Langlois, and P. Potier, J. Amer. Chem. Soc., 1976, 98, 7017. 

Scheme 5 Total synthesis of vinblastine (117) by Boger and structure of anhydrovinblastine (126).

There have been several recent syntheses of anhydrovinblastine, stimulated in part by its role as a key intermediate in the synthesis of the anticancer drug, Navelbine (vinorelbine). Anhydrovinblastine has been prepared via an electrochem-ically mediated coupling of catharanthine (667) and vindoline (668). The oxidation of catharanthine on a platinum anode in MeCN-Et4NC104 at controlled potential (0.6 V vs. SCE) in the presence of vindoUne, gave, after in situ reduction with NaBH4, (16 5)- and (16. R)-anhydrovinblastine, in yields of 52 and 12%, respectively (Scheme 46) (459,460). 

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