We previously reported that various metabolites were detected in mice, monkeys, and humans after oral administration of radiolabeled DS-1971a. Among these metabolites, mono- (M6) and di-oxidized (M12 and M13) forms were recognized as being specific to monkeys. Here, we report the definitive structures and formation mechanisms of these metabolites to elucidate the monkey-specific metabolism of DS-1971a. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that mass fragments of standard compounds for M6, M12 and M13, which were biosynthesized by a microorganism, were identical to those of in vivo samples from monkeys, but their full structures could not be identified. Therefore, M6, M12, and M13 were additionally subjected to UV, nuclear magnetic resonance (NMR) and X-ray crystal structure analyses, revealing that these metabolites possess 4-hydroxy (M6), and 4-hydroxy and 5-oxo pyrazole ring moieties (M12 and M13). Notably, M12 and M13 were rearranged stereoisomers of each other. Incubation study with recombinant monkey cytochrome P450s (P450s) suggested that monkey CYP2C8 is responsible for producing not only M6 from DS-1971a but also M12 and M13, both of which are further oxidized metabolites of M6. These results demonstrate novel and unusual metabolism of the pyrazole ring moiety of DS-1971a via monkey CYP2C8-mediated oxidation and rearrangement.
