Design, Synthesis, In Silico Docking, Multitarget Bioevaluation and Molecular Dynamic Simulation of Novel Pyrazolo[3,4-d]Pyrimidinone Derivatives as Potential In Vitro and In Vivo Anti-Inflammatory Agents

<b>Background:</b> A novel series of pyrazolo[3,4-<i>d</i>]pyrimidinone derivatives were synthesized, characterized, and examined for their anti-inflammatory effects. <b>Results:</b> The findings indicated that compounds <b>5d</b>, <b>5j</b>, <b>5k</b>, and <b>5m</b> 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 <b>5k</b> 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 <b>5j</b> and <b>5k</b> 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 <b>5k</b> 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 <b>5k</b>. <b>Conclusions:</b> Compound <b>5k</b> 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.