| Sumario: | This work reports the design, synthesis, and multilevel evaluation of 20 new S‐alkyl derivatives of 6‐(5‐mercapto‐4‐ethyl‐4H‐1,2,4‐triazol‐3‐yl)pyrimidine‐2,4(1H,3H)‐dione as potential selective cyclooxygenase‐2 (COX‐2) inhibitors with antioxidant properties. The aim was to establish structure–property relationships between the S‐alkyl fragment, antioxidant mechanisms, and COX‐2 inhibition and to identify lead structures for further optimization. Compounds were obtained by stepwise construction of the 1,2,4‐triazole fragment from orotic acid, followed by selective S‐alkylation of the mercaptotriazole, and their structures were confirmed by 1H/13C NMR, LC‐MS, and elemental analysis. Antiradical activity in vitro was assessed by the DPPH assay with determination of percent inhibition and IC50, whereas COX‐1/COX‐2 inhibition was evaluated using a fluorescent enzyme assay to obtain IC50 and selectivity index (SI). Density functional theory (DFT) calculations were employed to compare the thermodynamic feasibility of HAT, SET–PT, and SPLET pathways, while molecular docking and molecular dynamics were used to study the affinity and stability of binding to COX‐1 and COX‐2. A clear increase in antiradical activity was observed in the series 17 < ascorbic acid < 14 < 20 < 13 < 19, reaching 72.34% DPPH inhibition for Compound 19 with IC50(DPPH) = 12.15 ± 3.98 μM, considerably surpassing ascorbic acid. Compound 19 also exhibited the lowest IC50(COX − 2) = 24.5 ± 0.9 μM with SI ≈ 8.2; Compounds 13 and 20 showed moderate COX‐2 selectivity (SI ≈ 1.2–1.3), whereas 14 and 17 had a COX‐1‐shifted profile reminiscent of classical NSAIDs. DFT analysis indicated predominance of the SPLET mechanism for most systems, with the lowest ΔG values for 19 and 20, consistent with their high antioxidant activity. The combined experimental and in silico data identify Compound 19 as a primary lead candidate that combines potent antioxidant effects with pronounced, selective COX‐2 inhibition and highlight 13 and 20 as promising backup structures for further preclinical studies.
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