Design, synthesis, and biological evaluation of novel chalcone derivatives: integrating molecular docking with in vivo anti-inflammatory and antinociceptive studies

Chronic inflammatory disorders are often accompanied by persistent pain, yet current therapies remain limited by modest efficacy and adverse effects. To address this challenge, a series of eleven chalcone-based derivatives was synthesized via Claisen-Schmidt condensation and structurally characterized by FTIR, ¹H-NMR, ¹³C-NMR, and EIMS analyses. Cytotoxicity assessment on 3T3 fibroblasts using the MTT assay revealed no toxicity compared to doxorubicin, while brine shrimp lethality and hemolytic assays further confirmed their safety profile. In vitro COX-1/2 assays demonstrated preferential inhibition of COX-2, with AS1-6 showing a superior selectivity index relative to nimesulide. In vivo screening corroborated these results: AS1-2, AS1-6, and AS1-8 produced significant anti-inflammatory effects in carrageenan-induced paw edema, while AS1-2, AS1-6, and AS1-10 suppressed nociceptive behaviours in the formalin test, indicating both peripheral and central antinociceptive activity. Ex vivo ELISA further verified down-regulation of COX-2-associated inflammatory mediators compared to negative group. Molecular docking revealed strong and selective interactions of AS1-2 and AS1-6 within the COX-2 active site, while sparing COX-1. Molecular dynamics simulations confirmed the stability of AS1-6 in the active pocket throughout a 100 ns trajectory, consistent with the pharmacological outcomes. Collectively, the concordance of in vitro COX-1/2 inhibition, in vivo efficacy, and in silico analyses underscores the potential of these chalcone analogues as selective COX-2 inhibitors. These findings highlight the value of structure-guided design in developing dual-function Chalcones with anti-inflammatory and antinociceptive activity, warranting further optimization and preclinical evaluation.