CYP1A1 and CYP1B1-Mediated Biotransformation of the Antitrypanosomal Methamidoxime Prodrug DB844 Forms Novel Metabolites Through Intramolecular Rearrangement

Abstract

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 if 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 release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of an oxygen into the amidine C=N to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of nitric oxide. 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.

The Revised Human Liver Cytochrome P450 “Pie”: Absolute Protein Quantification of CYP4F and CYP3A Enzymes Using Targeted Quantitative Proteomics

“…Notably, CYP4F11 metabolizes erythromycin and ethylmorphine (Kalsotra et al, 2004), and CYP4F12 metabolizes ebastine (Hashizume et al, 2002). CYP4F2 and/or CYP4F3B are the major hepatic enzymes that catalyze the v-hydroxylation of fingolimod (FTY720), an oral drug for relapsing multiple sclerosis (Jin et al, 2011), and O-demethylation of the antitrypanosomal methamidoxime prodrugs pafuramidine (DB289), DB844, and DB868 (Wang et al, 2006;Generaux et al, 2013;Ju et al, 2014). In addition, a CYP4F2 genetic polymorphism has been shown to significantly affect the required maintenance dose of warfarin (Caldwell et al, 2008).…”

Reactive Metabolites from Thiazole-Containing Drugs: Quantum Chemical Insights into Biotransformation and Toxicity

“…The formation of an oxaziridine metabolite is a rarely suggested pathway in drug metabolism. The oxaziridine reactive metabolite may be involved in apoprotein modification or covalent modification of DNA. − In the case of a 3-substituted indazole compound, the formation of oxaziridine was reported, mediated by several rat CYPs enzymes such as CYP3A1 and CYP3A2. − In contrast, the oxaziridine formation on TZ has not been reported. The barrier for the formation of an oxaziridine ring is 14.56 kcal/mol via a transition state TS-A (Figures , ).…”

DMAKO-20 as a New Multitarget Anticancer Prodrug Activated by the Tumor Specific CYP1B1 Enzyme

“…When the prodrug underwent trafficking to the cytosolic region within cancer cells, the protecting group was cleaved by CYP1 enzymes to produce active multitarget drugs that would kill the cancer cells. The oxime moiety had already been well-established as a nitric oxide donor in previous studies 6 and a few oxime-contained structures underwent CYP1B1-catalyzed bioactivation to release nitric oxide (NO). Thus, the protection of an active ketone group or a quinone moiety of a drug might produce effective CYP1B1activated anticancer prodrugs.…”