CHEMBL3888231

Inhibition Assay: V79 MZh11B1 and V79 MZh 11B2 cells (8˙10^5 cells per well) were grown to confluency on 24-well cell culture plates with 1.9 cm^2 culture area per well (Nunc, Roskilde, Denmark). Before the test, the DMEM culture medium present was removed, and 450 μl of fresh DMEM with inhibitor was added for at least three concentrations to each well to determine the IC50. After preincubation (60 min, 37 °C.), the reaction was started by adding 50 μl of DMEM with 2.5 μl of solution of the substrate 11-deoxycorticosterone (20 μM, containing 1.25 nCi of [4-14C]11-deoxycorticosterone in ethanol). Thereafter, the plate was stored at 37 °C. and 5% CO2 in a CO2 incubator. The V79 MZh 11B1 cells were incubated for 120 min, and the V79 MZh 11B2 cells were incubated for 40 min. Controls without inhibitor were treated in the same way. The enzyme reactions were quenched by extracting the supernatant with 500 μl of EtOAc. The samples were centrifuged (10,000 g, 2 min), the solvent was removed and evaporated. The residue was taken up in 10 μl of chloroform and analyzed by HPTLC (see below).