Molecularly diverse lonazolac-inspired pyrazole-cyanopyridin-2-one conjugates: design, synthesis, in vitro, in vivo and in silico studies as multi-target anti-inflammatory agents.

New pyrazole/3-cyanopyridin-2-one conjugates 4a-f and 5a-h were designed and synthesized; their anti-inflammatory activities were evaluated in vitro and in vivo. In the LPS-induced RAW 264.7 cell line, nitric oxide (NO) inhibitory activity revealed that 4b, 4e, and 5c were the most powerful NO inhibitors, with % inhibition of 64.97, 71.45 and 73.61, respectively, compared with indomethacin (60.97). Hybrids 4f (IC50 = 2.64 µM) and 5e (IC50 = 0.34 µM) were the most potent COX-2 inhibitors with selectivity indexes of 8.1 and 12.6, respectively, compared with Celecoxib (IC50 = 1.33 µM, SI = 4.8), while 4e and 5c were the most potent 5-LOX inhibitors, with IC50 values of 2.75 and 0.71 µM, respectively, compared to the zileuton (IC50 = 0.45 µM). Treatment of carrageenan-induced paw inflammation rats, with hybrids 4b, 4e, 4f, 5c, and 5e resulted in outstanding anti-inflammatory activity with significant inhibition of paw inflammation, reduction in PGE2, and proinflammatory cytokines (TNF-α, and IL-1β levels). Moreover, the in vivo ulcerogenic study in rats suggested the safety of the tested hybrids when administered at high concentrations on gastric mucosa. Molecular docking using GNINA v1.3, validated by redocking against the native 3LN1-celecoxib and 6N2W-30Z complexes, together with molecular dynamics simulations, produced binding poses and interaction networks that are consistent with the recorded potency and selectivity, particularly for 5e in COX-2 and 5c in 5-LOX. In parallel, ligand-based 3D-QSAR (CoMFA/CoMSIA) and quantum-chemical descriptors established a coherent structure-activity relationship across the series and delineated the steric and electronic features governing anti-inflammatory activity. It could be preliminarily suggested that conjugates 4e and 5c could be considered as NO/5-LOX inhibitor, while conjugate 4f is a selective COX-2/5-LOX inhibitor, and 5e is a selective COX-2 inhibitor. These results indicate that 4e, 5c, and 4f are promising multitarget anti-inflammatory candidates that require further optimization.