Novel pyrazole carboxylate derivatives as lonazolac bioisosteres with selective COX-2 inhibition: design, synthesis and anti-inflammatory activity.

Two novel series of di-aryl/tri-aryl substituted pyrazole ester derivatives 15a-h and 19a-d were designed, synthesized as novel non-acidic lonazolac analogs and tested for its COX-2, 5-LOX, 15-LOX, iNOS, pro-inflammatory cytokines TNF-α and PGE2 inhibitory activities. All the tested compounds showed excellent COX-2 inhibitory activity (IC(50) = 0.059-3.89 μM), compared to that of celecoxib (IC(50) = 0.22 μM), where derivatives 15c, 15d, 15 h and 19d were found to be the most potent showing COX-2 selectivity index in range of (S.I. = 28.56-98.71) compared to celecoxib (S.I. = 13.65). Moreover, the most potent four derivatives 15c, 15d, 15 h and 19d showed outstanding 5-LOX and 15-LOX inhibitory activities (IC(50) = 0.24-0.81, 0.20-2.2 respectively, compared to zileuton IC(50) = 1.52 and 0.54, respectively). Further investigation of the anti-inflammatory mechanistic study of derivatives 15c, 15d, 15 h and 19d revealed that these four compounds exhibited comparable TNF-α and PGE2 (LPS-induced pro-inflammatory cytokines) inhibitory activities (IC(50) = 0.77-1.20 μM and 0.28-0.52 μM respectively) when compared to celecoxib (IC(50) = 0.87 μM and 0.38 μM respectively) as reference drug using lipopolysaccharide-activated RAW 264.7 macrophages. Based on the advanced inhibitory activity of compounds 15c, 15d, 15 h and 19d against LPS-induced pro-inflammatory mediators (TNF-α and PGE2), inducible nitric oxide synthase (iNOS) inhibition assay was carried out. Remarkably, compounds 15c, 15d, 15 h and 19d showed higher potency with lower IC(50) (0.41-0.61 µM) when compared to the reference drug celecoxib (0.48 µM). Prior to in vivo anti-inflammatory activity screening, cytotoxicity testing was performed to ascertain safe and non-toxic concentrations of each compound. Safe doses of compounds were determined using lipopolysaccharide-activated RAW 264.7 macrophages, moreover results showed that compounds 15c, 15d, 15 h and 19d were more safer (less cytotoxic) with higher IC(50) (178.95-301.40 µM) when compared to the reference drug celecoxib (148.90 µM). In vivo anti-inflammatory activity of the target compounds 15c, 15d, 15 h and 19d reinforced the results of in vitro screening as the derivatives 15c, 15d, 15 h and 19d showed (ED(50) = 8.22-31.22 mg/kg, respectively) and were more potent than celecoxib (ED(50) = 40.39 mg/kg). All screened derivatives 15c, 15d, 15 h and 19d were less ulcerogenic (ulcer indexes = 1.22-3.93) than lonazolac (ulcer index = 20.30) and comparable to celecoxib (ulcer index = 3.02). In silico docking and ADME studies were carried out in order to clarify the interactions of the most active derivatives 15c, 15d, 15 h and 19d with the target enzymes and their pharmacokinetic parameters.