*4.6. Microsomal Stability Assay*

In the microsomal stability assay, an analytical high-performance liquid chromatography (HPLC) system was used with the following condition: column, Merck Lichrospher 100 RP18 125 mm 4.6 mm (5 μm); mobile phase A = 0.1% trifluoroacetic acid in water, B = 0.1% trifluoroacetic acid in acetonitrile, isocratic; flow rate, 1 mL/min; detection, UV at 400 nm injection, 20 μL; column temperature, ambient. The metabolic stability assays were carried out using the cosolvent method, appropriate for assessing the metabolic stability of compounds poorly soluble in aqueous medium [40]. For the cosolvent method, a 0.5 mM DMSO stock solution of Oxa12 was prepared. Then, a diluted solution of the compound was prepared by adding 50 μL of the previous 0.05 mM solution with 200 μL of acetonitrile, to make a 0.1 mM solution of Oxa12 in 20% DMSO/80% acetonitrile. Cosolvent assay conditions were: substrate concentration, 1 μM; microsomal protein, 0.5 mg/mL; organic solvents, 0.2% DMSO, 0.8% acetonitrile; incubation time, 30 min; number of assays, duplicates for T0 and T30 min. Time 0 and time 30 batches, after quenching with acetonitrile, were centrifuged at 11,000× *g* for 5 min and the supernatants were analyzed by HPLC, in order to quantify compound Oxa12.
