CHEMBL5738527

Cox-1 Assay: The effect of the compounds on COX-1 activity was determined by a continuous colorimetric assay that monitored the oxidation of N, N, N, N-tetramethyl-1,4-phenylenediamine (TMPD) when coupled to the COX catalyzed formation of PGH2 from PGG2 using arachidonic acid as substrate. In brief, 200 μL of reaction solution was composed of 100 mM Tris-HCl (pH 8.0), 2 μM Hemin (Sigma), 5% DMSO, a serial dilution of compounds, COX-1 enzyme (175 nM), 80 μM TMPD (Sigma), and 20 μM arachidonic acid (Sigma). Reagents were mixed and incubated at 25° C. for 5 min followed by adding a mixture of TMPD and arachidonic acid to initiate the reaction. A continuous colorimetric assay to measuring TMPD oxidation at 610 nm was conducted using a Synergy 2 plate reader at 25° C. for 5 min, and IC50 value of each compound was calculated (Adeniji, et al., 2012, J Med Chem 55:2311-2323).