Phase i metabolic profiling and unexpected reactive metabolites in human liver microsome incubations of X-376 using LC-MS/MS: Bioactivation pathway elucidation and: In silico toxicity studies of its metabolites

Abstract

X-376 is a novel new generation anaplastic lymphoma kinase (ALK) inhibitor that can cross the blood brain barrier, so it can be used in patients with both ALK-positive NSCLC with CNS metastases. In this study, X-376 was incubated with human liver microsomes, and in vitro phase-I metabolic reactions were performed to generate X-376 reactive intermediates, which cannot be detected directly as they are unstable. Utilizing LC-MS/MS, we characterized X-376 metabolites and checked for the reactive electrophile generation using nucleophilic trapping agents, namely, potassium cyanide and GSH, which form stable adducts for characterization by mass spectrometry. Four X-376 phase-I metabolites and three reactive intermediates (one quinone methide and two iminium ions) were characterized and the bioactivation pathways were proposed. X-376 bioactivation occurred through unexpected novel pathways. Although the pyridazine ring was found to be bioactivated, no bioactivation was detected in the expected N-methyl piperazine ring that is often found for similar structures. The dichloro-phenyl group was bioactivated by a novel mechanism that was verified by LC-MS/MS. We propose that the X-376 reported side effects may be due to the formation of reactive metabolites. The in silico toxicity assessment of X-376 metabolites was carried out using DEREK software and structural modification were proposed to reduce their side effects and to validate the bioactivation pathway theory using StarDrop DEREK module. Further drug discovery studies can be done according to this concept, thus allowing the development of new drugs with more safety profile that was confirmed by using StarDrop software. To our knowledge, this is the first study of in vitro metabolic profiling or structural characterization and toxicological properties of the generated metabolites for X-376.