Design, Synthesis, In Vitro Hydrolysis and Biological Evaluation of Antioxidant-Conjugated Mutual Prodrugs of Naproxen

Objective: A novel series of antioxidant-conjugated mutual prodrugs of naproxen (RJ-01-01–RJ-01-10) was designed and synthesized to mitigate the gastrointestinal (GI) adverse effects associated with the parent drug, naproxen. Methods: The study included molecular docking, synthesis and characterization, in vitro hydrolysis, and biological evaluation. Results and Discussion: The synthesized prodrugs demonstrated high stability in simulated gastric fluid (SGF, pH 1.2), with minimal hydrolysis to the parent naproxen (18.62–24.14%), indicating reduced potential for gastric irritation. Conversely, in simulated intestinal fluid (SIF, pH 7.4), a significantly higher hydrolysis rate (75.07–84.01%) was observed, facilitating drug release and intestinal absorption. In docking studies, compounds RJ-01-04, RJ-01-05, RJ-01-06, RJ-01-08, and RJ-01-09 exhibited the highest binding energies (–13.68, –12.23, –13.74, –12.01, and –13.06 kcal/mol, respectively) compared to naproxen. During in vitro evaluation, RJ-01-04 showed significantly more potent COX-2 inhibition (IC50 = 44.18 ± 1.7 µM) than naproxen (IC50 = 61.48 ± 0.8 µM) and comparable COX-1 inhibition (IC50 = 55.34 ± 0.6 vs. 59.43 ± 0.5 µM for naproxen), indicating dual inhibitory activity. In AGS cells, RJ-01-04 reduced reactive oxygen species (ROS) levels and increased superoxide dismutase (SOD) activity more effectively than naproxen, demonstrating enhanced antioxidant effects. Western blot analysis confirmed downregulation of COX-2 and COX-1 protein expression in AGS cells treated with RJ-01-04. Cytotoxicity assessment (MTT assay) in HEK293 cells revealed that RJ-01-04 exhibited significantly lower cytotoxicity than naproxen. Conclusions: These findings support the mutual prodrug strategy as a promising approach to improve the therapeutic profile of naproxen through antioxidant conjugation.