Novel bioactivation mechanism of reactive metabolite formation from phenyl methyl-isoxazoles

Recently, we described a series of phenyl methyl-isoxazole derivatives as novel, potent, and selective inhibitors of the voltage-gated sodium channel type 1.7 (Bioorg Med Chem Lett 21:3871-3876, 2011). The lead compound, 2-chloro-6-fluorobenzyl [3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbamate, showed unprecedented GSH and cysteine reactivity associated with NADPH-dependent metabolism in trapping studies using human liver microsomes. Additional trapping experiments with close analogs and mass spectra and NMR analyses suggested that the conjugates were attached directly to the 5'-methyl on the isoxazole moiety. We propose a mechanism of bioactivation via an initial oxidation of the 5'-methyl generating a stabilized enimine intermediate and a subsequent GSH attack on the 5'-methylene. Efforts to ameliorate reactive metabolite generation were undertaken to minimize the potential risk of toxicity. Formation of reactive metabolites could be significantly reduced or prevented by removing the 5'-methyl, by N-methylation of the carbamate; by replacing the nitrogen with a carbon or removing the nitrogen to obtain a carboxylate; or by inserting an isomeric 5'-methyl isoxazole. The effectiveness of these various chemical modifications in reducing GSH adduct formation is in line with the proposed mechanism. In conclusion, we have identified a novel mechanism of bioactivation of phenyl 5-methyl-isoxazol-4-yl-amines. The reactivity was attenuated by several modifications aimed to prevent the emergence of an enimine intermediate. Whether 5'-methyl isoxazoles should be considered a structural alert for potential formation of reactive metabolites is dependent on their context, i.e., 4'-nitrogen.