Chemical reactivity of methoxy 4-o-aryl quinolines: identification of glutathione displacement products in vitro and in vivo.

AMG 458 {1-(2-hydroxy-2-methylpropyl)-N-[5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl]-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide} is a potent, selective inhibitor of c-Met, a receptor tyrosine kinase that is often deregulated in cancer. AMG 458 was observed to bind covalently to liver microsomal proteins from rats and humans in the absence of NADPH. When [(14)C]AMG 458 was incubated with liver microsomes in the presence of glutathione and N-acetyl cysteine, thioether adducts were detected by radiochromatography and LC/MS/MS analysis. These adducts were also formed upon incubation of AMG 458 with glutathione and N-acetyl cysteine in buffers at pH 7.4. In vivo, the thioether adducts were detected in bile and urine of bile duct-cannulated rats dosed with [(14)C]AMG 458. The two adducts were isolated, and their structures were determined by MS/MS and NMR analysis. The identified structures resulted from a thiol displacement reaction to yield a quinoline thioether structure and the corresponding hydroxyaryl moiety. The insights gained from elucidating the mechanism of adduct formation led to the design of AMG 458 analogues that exhibited eliminated or reduced glutathione adduct formation in vitro and in vivo.