Kinase experiment:

hCOX1 or hCOX2 is preincubated with COX2-IN-2 in 0.1 M Tris-HCl buffer containing 2 μM hematin and 5 mM L-tryptophan at 30℃ for 5 min, followed by a 5 min incubation with arachidonic acid. The enzyme reaction is stopped by the addition of 1 N HCl. The PGE2 formed is extracted with EtOAc and measured by RIA[1].

Animal experiment:

Rats: Ten male Sprague Dawley rats are used per group. A suspension of 0.5% brewer's yeast in 0.5% methyl cellulose is injected into the right hind paw. The pain threshold us determined 3h after yeast injection. COX-2-IN-2 is given orally 2 h after yeast injection. The pain threshold in the treated rats are compared with that in the control rats[1].

COX2-IN-1 is a selective and inducible COX2 inhibitor with an IC50 of 0.24 μM. COX2-IN-1 is an anti-inflammatory compound with anti-inflammatory and analgesic activities.

COX2-IN-1 shows no COX-1 inhibition even at 100μM[1].

COX2-IN-1 has oral ED50 values of 0.030 and 0.47mg/kg on adjuvant-induced arthritis and collagen-induced arthritis, respectively, and an ED30 value of 7.4mg/kg in the yeast-induced hyperalgesia (Randall-Selitto) assay. COX2-IN-1 shows good analgesic activity and no ulcerogenicity[1].

[1]. Tsuji K, et al. Studies on anti-inflammatory agents. IV. Synthesis and pharmacological properties of 1,5-diarylpyrazoles and related derivatives. Chem Pharm Bull (1997), 45(6), 987-995.