The 2′-Trifluoromethyl Analogue of Indomethacin Is a Potent and Selective COX-2 Inhibitor

[Image: see text] Indomethacin is a potent, time-dependent, nonselective inhibitor of the cyclooxygenase enzymes (COX-1 and COX-2). Deletion of the 2′-methyl group of indomethacin produces a weak, reversible COX inhibitor, leading us to explore functionality at that position. Here, we report that substitution of the 2′-methyl group of indomethacin with trifluoromethyl produces CF(3)–indomethacin, a tight-binding inhibitor with kinetic properties similar to those of indomethacin and unexpected COX-2 selectivity (IC(50) mCOX-2 = 267 nM; IC(50) oCOX-1 > 100 μM). Studies with site-directed mutants reveal that COX-2 selectivity results from insertion of the CF(3) group into a small hydrophobic pocket formed by Ala-527, Val-349, Ser-530, and Leu-531 and projection of the methoxy group toward a side pocket bordered by Val-523. CF(3)–indomethacin inhibited COX-2 activity in human head and neck squamous cell carcinoma cells and exhibited in vivo anti-inflammatory activity in the carrageenan-induced rat paw edema model with similar potency to that of indomethacin.