Background: A novel series of pyrazolo[3,4-d]pyrimidinone derivatives were synthesized, characterized, and examined for their anti-inflammatory effects. Results: The findings indicated that compounds 5d, 5j, 5k, and 5m demonstrated significant anti-inflammatory effects through the selective inhibition of the COX-2 isozyme, with IC₅₀ values ranging from 0.27 to 2.34 μM, compared to celecoxib (IC₅₀ = 0.29 μM). Compound 5k emerged as the most potent, exhibiting a selectivity index (SI) of 95.8 for COX-2 relative to COX-1. In vivo tests additionally validated that compounds 5j and 5k demonstrated significant anti-inflammatory efficacy, exhibiting greater suppression percentages of generated paw edema than indomethacin, comparable to celecoxib, while preserving excellent safety profiles with intact gastric tissue. Mechanistic studies demonstrated that the anti-inflammatory efficacy of the target compounds was associated with a substantial decrease in serum levels of TNF-α and IL-6. Moreover, molecular modeling investigations corroborated the in vitro findings. Compound 5k displayed a binding free energy ΔG of −10.57 kcal/mol, comparable to that of celecoxib, which showed a ΔG of −10.19 kcal/mol. The intensified binding contacts in the COX-2 isozyme indicated the augmented inhibitory efficacy of 5k. Conclusions: Compound 5k exhibited dual activity by inhibiting the COX-2 isozyme and suppressing the pro-inflammatory cytokines TNF-α and IL-6, therefore providing a remarkable anti-inflammatory effect with increased therapeutic potential.

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), PGE₂, 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 PGE₂ 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.