Elucidating the Mechanism of Cytochrome P450–Mediated Pyrimidine Ring Conversion to Pyrazole Metabolites with the BACE1 Inhibitor GNE-892 in Rats

An uncommon biotransformation of pyrimidine during the metabolism of GNE-892, a β-secretase 1 inhibitor, was investigated. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the 14C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and NMR. Though these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. Following hydrolysis of the formamide, a carbon is lost as formic acid together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.