Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (_?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against _?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H₂O₂), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a _?OH-trapping agent after UVB exposure (305?nm) to H₂O₂ for 1?min in the presence of rebamipide. The signal intensity of _?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10₁₀, 8.16?×?10₉ and 1.65?×?10₁₀?M_-1?s_-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the _?OH generated by UVB/H₂O₂. Specific formation of this product explained the molecular characteristics of rebamipide as a potential _?OH scavenger.