Belizatinib: Novel reactive intermediates and bioactivation pathways characterized by LC-MS/MS

Belizatinib (BZB; TSR-011) is a next-generation anaplastic lymphoma kinase inhibitor that also inhibits tropomyosin-related kinases A/B/C. In this in-vitro study, we examined the formation of reactive metabolites from BZB using rat liver microsomes or human liver microsomes in the presence of a trapping agent (potassium cyanide) to generate iminium reactive intermediates. Identification of the in vitro BZB metabolites indicated that the major in-vitro metabolic reaction involved hydroxylation of the piperidine moiety. We identified eight in-vitro phase I metabolites and three iminium reactive intermediates, suggesting two possible BZB-bioactivation pathways. We propose that the tertiary nitrogen in the piperidine ring activates the attached benzyl carbon in addition to the two α carbons inside the ring. To our knowledge, this is the first report on the structural identification of reactive metabolites derived from BZB.