Microbial transformations of the antimelanoma agent betulinic acid

Betulinic acid (1), a pentacyclic lupane-type triterpene, is a natural product that has been reported to be a melanoma-specific cytotoxic agent based on both in vivo and in vitro studies. Due to its high antitumor activity and lack of toxicity, the bioactive triterpene 1 is currently undergoing preclinical development for the treatment of malignant melanoma.; An essential part of preclinical development of a drug is to elucidate its mammalian biotransformation and evaluate the biological activity and/or toxicity of its mammalian metabolite(s). It is the main objective of this project to utilize microbial cultures as in vitro model systems to predict and prepare potential mammalian metabolites of betulinic acid. Preparative-scale biotransformation with Bacillus megaterium ATCC 13368, Bacillus megaterium ATCC 14581, Cunninghamella elegans ATCC 9244, Cunninghamella species NRRL 5695, and Mucor mucedo UI-4605 have produced several metabolites of betulinic acid (1), which have been characterized as 3-oxo-lup-20(29)-en-28-oic acid (2), 3-oxo-11α-hydroxy-lup-20(29)-en-28-oic acid ( 3), 1β-hydroxy-3-oxo-lup-20(29)-en-28-oic acid (4), 3β,7β,15α-trihydroxy-lup-20(29)-en-28-oic acid (5), 3β,7β-dihydroxy-lup-20(29)-en-28-oic acid (6), 3β,6α,7β-trihydroxy-lup-20(29)-en-28-oic acid (7), 1β,3β,7β-trihydroxy-lup-20(29)-en-28-oic acid (8), and 28-O-β-D-glucopyranosyl 3β-hydroxy-lup-20(29)-en-28-oate (9) based on 1D- and 2D-NMR experiments and high resolution mass spectral analyses. The isolated metabolites 2–9 were evaluated for antimelanoma activity against the cultured human melanoma cell lines, Mel-1 and Mel-2.; CYP102, a barbiturate-inducible P450 enzyme from Bacillus megaterium ATCC 14581, exhibits a 51.7% overall structural similarity with the human enzyme CYP2C9. Both CYP102 and CYP2C9 show selectivity for compounds containing a free carboxylic acid moiety in their structures. The biotransformation of 1 by B. megaterium ATCC 14581 suggested that 1 may act as a substrate for human CYP2C9. In an effort to predict the sites of metabolism mediated by CYP2C9 in 1, a molecular modelling study was carried out. Based on our study, CYP2C9 is expected to produce oxygenated metabolites of 1 at the 23-methyl group and 6α positions. These predicted sites of metabolism are consistent with the distance ranges encountered for other substrates of human CYP2C9.