New pyridazinone derivatives 3a-d, 4a, 4b, and 5a-f were synthesized and tested for in-vitro inhibition of human COX-1 and COX-2. Compounds 5a and 5f showed strong COX-2 inhibition (IC50 = 0.77 and 1.89 μM; SI = 16.70 and 13.38) compared to indomethacin (IC50 = 0.42 μM, SI = 0.50) and celecoxib (IC50 = 0.35 μM, SI = 37.03). In LPS-induced RAW264.7 macrophages, ELISA results showed that compound 5a reduced TNF-α and IL-6 levels by 87 % and 76 %, outperforming celecoxib (67 % and 81 %), while compound 5f reduced them by 35 % and 32 %. RT-PCR revealed that compound 5a suppressed TNF-α and IL-6 mRNA by 82 % and 62 % (vs celecoxib 68 % and 70 %), whereas compound 5f achieved 27 % and 47 % reductions. Both compounds inhibited LPS-mediated NO by 35.7 % for compound 5a and 20 % for compound 5f and ROS production (compound 5a: 42 %, compound 5f: 21.3 %). In vivo, rat paw edema inhibition showed that both had strong anti-inflammatory effects, comparable to indomethacin and celecoxib, with a lower ulcer number and index compared to the indomethacin group. Gastric mucosal protection was 99.77 % for compound 5a and 83.08 % for compound 5f. Histopathology revealed paw tissue from treated groups had healthy epidermal layers with reduced inflammation. Stomach tissue from compound 5a-treated rats showed moderate tunica mucosa improvement and epithelial layer degeneration; compound 5f showed mild fundic mucosa improvement with inflammatory infiltration and mucosal desquamation. In paw tissue, both compounds reduced iNOS protein expression and significantly suppressed NF-κB. Molecular modelling indicated strong COX-2 binding affinities. ADME profiling confirmed drug-likeness: compound 5a fully complied with Veber's rules; compound 5f met Lipinski and Egan criteria without violations. Overall, compounds 5a and 5f demonstrate potent COX-2 selectivity, anti-inflammatory activity, reduced gastric toxicity, and favorable pharmacokinetics, positioning them as promising leads for safe and effective anti-inflammatory drug development.
