Design, synthesis, in-vitro hydrolysis and biological evaluation of antioxidant conjugated mutual prodrugs of ibuprofen

Abstract A novel series of antioxidant-conjugated mutual prodrugs of ibuprofen (RJ-02-01 to RJ-02-10) was designed and synthesized to mitigate the gastrointestinal (GI) negative impact linked with the parent ibuprofen drug. In vitro hydrolysis results demonstrated the immense stability of the synthesized ibuprofen prodrugs in SGF (pH 1.2 simulated gastric fluid), with minimal reversion to parent compound, indicating reduced potential for gastric irritation. Conversely, at intestinal pH of SIF (simulated intestinal fluid, pH 7.4), a higher rate of reversion was observed, facilitating drug release and absorption in the intestine. During the docking study, compounds RJ-02-01, RJ-02-05, RJ-02-07, RJ-02-09 and RJ-02-10 exhibited the highest binding energies of -12.08, -12.49, -13.25, -12.87, and − 13.04 Kcal/mol, respectively, as compared to the standard ibuprofen. Among the selected derivatives based on molecular docking and hydrolysis profiles, RJ-02-07 exhibited superior pharmacological properties. RJ-02-07 demonstrated stronger COX-2 inhibition (IC₅₀ = 44.44 µM) compared to ibuprofen, and a comparable COX-1 inhibition profile (IC₅₀ = 53.22 µM), suggesting potent dual inhibitory activity. Treating with AGS cells, RJ-02-07 significantly reduced ROS levels relative to ibuprofen, indicating improved oxidative stress mitigation and led to a greater increase in SOD activity than the parent drug, suggesting an enhanced antioxidant defense mechanism. Western blot analysis confirmed downregulation of both COX-2 and COX-1 protein expression in AGS cells treated with RJ-02-07, and cytotoxicity assessment via MTT assay in HEK293 cells demonstrated that RJ-02-07 exhibited lower cytotoxicity than ibuprofen, with no significant reduction in cell viability, indicating a favorable safety profile. These findings support the concept of mutual prodrug as an encouraging strategy to enhance the therapeutic profile of ibuprofen through antioxidant conjugation.