2.3. Liver microsome stability assay

The assay was performed with liver microsomes from male CD-1 mouse (Xenotech) and pooled human (Bioreclamation). Compounds HTB-03, HTB-04 and HTB-05 were tested at 1 μM with a final concentration of microsomal protein of 1 mg/mL. The reaction was initiated by the addition of NADPH (1 mM), and samples were incubated for up to 60 min at 37 °C in a shaking incubator. The reaction was terminated at 0, 5, 15 and 30 min by the addition of ice-cold ACN/MeOH (50:50) spiked with internal standard. Samples were centrifuged at 4000 rpm at 4 °C for 15 min and the supernatants analyzed by LC-MS/MS. The assay evaluated the metabolic stability of compounds by measuring the amount of parent remaining to test compounds with or without NADPH cofactor. LC-MS/MS: AB Sciex 4500 Qtrap; Kinetex 2.6 μm C18 100 Å column (3.0 mm × 30 mm); mobile phase A, 0.1% formic acid in H₂O; mobile phase B, 0.1% formic acid in ACN; flow rate, 0.8 mL/min; room temperature.