Design, synthesis, structure-activity relationship and in-silico studies of novel pyrazolo[1,5-a]pyrimidine derivatives as selective COX-2 inhibitors with promising anti-inflammatory activity and safety profile.

Selective COX-2 inhibitors offer effective anti-inflammatory therapy with minimized gastrointestinal side effects, yet developing novel scaffolds with superior safety profiles remains critical. In this study, a novel series of pyrazolo[1,5-a]pyrimidine derivatives (7a-b, 8a-j, 9a-b, and 12a-e) was designed using ligand-based pharmacophore modeling, and their structures were confirmed by 1H/13C NMR and LC-MS. In vitro enzyme assays identified compound 9a as highly potent (IC50 = 0.050 μM), comparable to celecoxib (IC50 = 0.046 μM), while 8a (IC50 = 0.060 μM) and 8i (IC50 = 0.059 μM) exhibited the highest selectivity indices at 201 and 153.05, respectively. Nine representative compounds spanning both high- and low-selectivity chemotypes were evaluated in a formalin-induced paw edema model, chosen over carrageenan for its chronic, biphasic inflammatory profile: 8a displayed 1.5-fold higher potency than indomethacin and 1.45-fold over celecoxib (p < 0.0001), while 8i achieved 1.25-1.30-fold and 1.6-fold relative potency (p < 0.01). Histopathological examination confirmed that 8a and 8i caused the least inflammatory infiltration and tissue damage, prompting their selection for ELISA-based mechanistic evaluation, which revealed substantial reductions in NF-κB, COX-2, and IL-6 levels (2.9-3.6-fold for 8a; 2.2-2.6-fold for 8i). Molecular docking, 100 ns MD simulations, and MM-GBSA binding free energy calculations further confirmed stable active-site binding (ΔGbind = -54.32 and - 56.14 kcal/mol for 8a and 8i, respectively). Collectively, these integrated results establish 8a and 8i as potent, selective, and structurally stable lead candidates for preclinical COX-2 inhibitor development.