P154 Bioactivation of 6-fluoro-1H-indazole to quinone methide intermediate: Oxidative defluorination via an arene oxide formation

Steve Bowlin , Takeda, SAN DIEGO, CA
Kirk Kozminski , Takeda, SAN DIEGO, CA
Shane Alexis Apostol , Takeda, SAN DIEGO, CA
Amin Kamel , Takeda, San Diego, CA

Compound 1 was identified as a lead molecule during lead optimization efforts within the 1H-indazole series.  Compound 1 has a fluorine atom at the 6 position to mitigate against direct oxygen insertion.  In vivo and in vitro studies suggested that compound 1 undergoes phase II conjugative pathways as well as phase I oxidative pathways including oxidative defluorination to form the major 6-hydroxy metabolite (M1).  The formation of M1 is likely to proceed via an intermediate. Therefore, in vitro metabolite identification and glutathione trapping studies were conducted to provide more insights on the bioactivation potential of compound 1.  Bioactivation studies were carried out using rat induced liver S9 fortified with NADPH and glutathione (GSH) as a trapping agent, as per protocols reported in the literature.  Structure elucidation of metabolites was carried out on an AB Sciex TripleTOF 5600 LC/MS/MS system using positive ESI mode and full scan TOF-MS with Mass Defect Filter triggered MS/MS acquisition.

The results revealed the formation of M1 and 3 isomeric GSH adducts M2, M3 and M4.  In addition to M1, the identification of the GSH adducts further support the formation of a quinone methide intermediate, a precursor for the formation of such adducts.   Furthermore, incubations with rat live microsomes, rat liver microsomes pre-incubated with 1-aminobenzoriazole (ABT), a nonselective inhibitor of CYP-450, and recombinant human FMO (rFMO) enzymes (rFMO1, 3, and 5), revealed that the formation of M1 is CYP-450 mediated and none of the three rFMO isoforms tested is involved in its formation.  CYP-450 phenotyping reactions using major isoforms CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP1A2 are under investigation to determine which enzyme is responsible for the formation of M1.