CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl} -nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine Cï£N to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:337-349, 2014 © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.