Novel Pyrazole-3-Cyano-2-Pyridinone Hybrids as Multitarget Anti-Inflammatory Agents: Synthesis, Computational Modeling, and Biological Evaluation

Introduction: The development of multi-target anti-inflammatory agents represents a promising strategy to improve therapeutic efficacy while minimizing adverse effects associated with single-target drugs. In this study, a rational hybridization approach was employed to design pyrazole/3-cyano-2-pyridinone hybrids aimed at modulating key inflammatory mediators. Methods: A novel series of pyrazole/3-cyano-2-pyridinone hybrids was synthesized and evaluated for anti-inflammatory activity. Nitric oxide (NO) production and iNOS activity were assessed in LPS-stimulated RAW 264. 7 macrophages. COX-1/COX-2, LOX (5-LOX and 15-LOX), PGE 2, and TNF-α inhibition assays were performed. Cytotoxicity was determined using MTT assays. Molecular docking and 100-ns molecular dynamics (MD) simulations were conducted to investigate binding modes and stability, while in silico ADME profiling was used to predict pharmacokinetic properties. Results: Compounds 5f, 5g, 5k, and 5m significantly inhibited NO production and iNOS activity, with compound 5m showing the strongest effect (IC₅₀ = 203. 9 μM). COX inhibition assays revealed selective COX-2 activity, with compound 5k exhibiting the highest potency (IC₅₀ = 0. 92 μM) and a selectivity index of 19. 6. Compound 5g most effectively suppressed PGE 2 production (IC₅₀ = 152. 7 pg/mL), while 5m markedly reduced TNF-α levels, comparable to ibuprofen. In LOX assays, compound 5f showed potent inhibition of both 5-LOX (IC₅₀ = 0. 34 μM) and 15-LOX (IC₅₀ = 0. 21 μM), outperforming zileuton. All tested compounds exhibited low cytotoxicity (IC₅₀ > 85 μM). Docking and MD simulations confirmed stable and favorable binding interactions of 5k, 5f, and 5m with COX-2, 5-LOX, and iNOS, respectively. In silico pharmacokinetic analysis predicted good oral bioavailability and drug-like properties. Conclusion: The synthesized pyrazole/3-cyano-2-pyridinone hybrids demonstrated promising multi-target anti-inflammatory activity with favorable safety and pharmacokinetic profiles, highlighting their potential as lead candidates for further development.