Bioactivation of a Dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide) Scaffold to a Putative p-Chlorophenyl Isocyanate in Rat Liver Microsomes and In Vivo in Rats

Compound I (4,5-dihydropyrazole-1,5-dicarboxylic acid-1-[(4-chlorophenyl)-amide] 5-[(2-oxo-2H-[1,3′]bipyridinyl-6′-yl)-amide] was found to undergo metabolic activation in rat liver microsomes in the presence of NADPH. A reactive intermediate, postulated to be p-chlorophenyl isocyanate (CPIC), was trapped by GSH in vitro and characterized by liquid chromatography tandem mass spectrometry (LC/MS/MS). Subsequently, the structure of the GSH conjugate was confirmed by a comparison with a synthetic standard. The GSH conjugate was also found in the bile of rats that received an oral dose (10 mg/kg) of compound I. Further analyses of rat bile and urine using online electrochemical derivatization coupled to LC/MS demonstrated the presence of p-chlorophenyl aniline (CPA), a hydrolytic product of the intermediate isocyanate. This provided further evidence for the potential existence of CPIC. Approximately 7% of the dose was accounted by the products of CPIC, which included the GSH conjugate and CPA excreted in bile ...