Metabolic Stability and CYP-Mediated Metabolism

The metabolic stability was evaluated using mouse and human liver microsomes (Xenotech LLC, USA) as previously described.^{38 (link),39} For the cytochrome P450 (CYP)-mediated metabolism, the reaction was carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/mL glucose 6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions, in triplicate, were initiated by addition of the liver microsomes to the incubation mixture (compound final concentration was 1 μM; 0.2 mg/mL microsomes). Controls in the absence of cofactors were carried out to determine the specific cofactor-free degradation. After 60 min of incubation, aliquots of the mixture were removed, and the reaction was quenched by addition of three times the volume of ice-cold acetonitrile spiked with the internal standard. Compound disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC–MS/MS) method. Additionally, metabolite identification (compound 1) was performed by full scan analysis on Thermo Q Exactive Orbitrap mass spectrometer, and phase-I metabolites were monitored.