Catharanthus alkaloids processes

Catharanthus alkaloids, particularly vinblastine (Al) and vincristine (A2), are well known anticancer drugs, which are used clinically to treat Hodgkin s lymphoma and acute childhood lymphoblastic leukemia, respectively. These alkaloids interact with tubulin, a protein necessary for cell division, and are inhibitors of mitosis (the process of cell division). 

Another cultured cell line of Catharanthus roseus (EU4A), which does not produce detectable amounts of vinblastine and other bisindole alkaloids, was also examined for its ability to transform 78 (183). Cell-free extracts of the culture line were prepared, and the 35,000 X g supernatant solution was used. Incubations with 2r-tritioanhydiovinblastine yielded a mixture from which radioactive vinblastine (52) was isolated. The labeled vinblastine was reisolated after unlabeled carrier was added and rigorously purified by successive thin-layer chromatography, reversed-phase HPLC, and crystallization to constant specific activity. Boiled extracts could not produce labeled 52, thus supporting the involvement of enzymes in the conversion process. 

The observation that fractions of the rosey periwinkle, Catharanthus rosea, produced severe leukopenia, resulted in the isolation and development of two major anticancer drugs, vincristine and vinblastine. These two complex, dimeric indole-indoline alkaloids are important in the treatment of acute childhood leukemia (vincristine), Hodgkin s disease (vinblastine), and metastatic testicular tumors (vinblastine), and continue to be manufactured today by mass cultivation and processing of their natural source. 

Plants possess an incredibly diverse biosynthetic capacity leading to the production of a myriad of compounds that, although not having an apparent function for fundamental life processes (growth, development and reproduction), seem to have vital roles as mediators of ecological interactions, being very important for the survival of plants. This chemical wealth is the basis of the use of plants in medicine, and is still largely unexplored. One example of application of the so called plant secondary metabolites are the terpenoid indole alkaloids of Catharanthus rose us, used in cancer therapy, and known as the Vinca alkaloids. 

Induction and production media are used in a two-stage process in the first stage biomass is produced, and the second stage allows alkaloid production. Morris, however, selected conditions for a Catharanthus roseus culture in which high alkaloid accumulation could be combined with high biomass accumulation (37). 

The release of sanguinarine from the cells into the medium after elicitation was used as the basis for the design of an industrial process for production of the alkaloid 72,99,487,492,493). As the cells of P. somniferum remain viable after a 72-hr exposure to a fungal elicitor, it is possible to recycle the cells after induction of alkaloid biosynthesis. Because about 40-60% of the alkaloid is released to the medium, a simple change of medium is sufficient to collect the alkaloid and to recover viable cells. By a repeating sequence of elicitation and medium replenishment, a semicontinuous production process was obtained. Such a process was also developed for alkaloid production in Catharanthus roseus, Ruta grave-olens, and P. somniferum cell cultures using different elicitors (99). 

Catharanthus roseus is probably the most extensively studied plant for secondary metabolite production in cell cultures. As such it is an excellent model system however, the levels of alkaloids produced are still far below amounts necessary for a commercially feasible process. The technology for the large-scale culture of C. roseus cells is available. Future studies should thus focus on the regulation of alkaloid production, with the aim of eventually applying genetic engineering to increase alkaloid levels. 

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. "°... 

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. 

Zhao J, Verpoorte R (2007) Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors from biochemical processing to metabolic engineering. Phytochem Rev 6 435 57. doi 10.1007/sl 1101-006-9050-0... 

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