The inhibitory potential of 4,7-dihydroxycoumarin derivatives on ROS-producing enzymes and direct HOO ^• /o ₂ ^• – radical scavenging activity – a comprehensive kinetic DFT study

This study examined the antiradical activity of three synthesized coumarin derivatives: (<i>E</i>)-3-(1-((2-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (<b>A₁-OH</b>), (<i>E</i>)-3-(1-((3-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (<b>A₂-OH</b>), and (<i>E</i>)-3-(1-((4-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate (<b>A₃-OH</b>) against HOO•/O2•- radical species. The investigation included electron spin resonance (ESR) measurements and a DFT kinetic study. Thermodynamic and kinetic parameters of antiradical mechanisms-Formal Hydrogen Atom Transfer (<i>f</i>-HAT), Radical Adduct Formation (RAF), Sequential Proton Loss followed by Electron Transfer (SPLET), and Single-Electron Transfer followed by Proton Transfer (SET-PT)-were evaluated using the Quantum Mechanics-based test for Overall Free Radical Scavenging Activity (QM-ORSA) under physiological conditions. ESR results indicated antiradical activity decreased in the sequence <b>A₁-OH</b> (58.7%) > <b>A₂-OH</b> (57.5%) > <b>A₃-OH</b> (53.1%). Kinetic analysis revealed the <i>f</i>-HAT mechanism dominated HOO^• inactivation. A newly formulated Sequential Proton Loss followed by Radical Adduct Formation (SPL-RAF) mechanism described interactions with O₂^•-. The activity toward O₂^•- was <b>A₂-OH</b> (1.26 × 10⁶ M^-1s^-1) > <b>A₃-OH</b> (7.71 × 10⁵ M^-1s^-1) > <b>A₁-OH</b> (4.22 × 10⁵ M^-1s^-1). Molecular docking and dynamics studies tested inhibitory capability against enzymes producing reactive species: Lipoxygenase (LOX), Myeloperoxidase (MPO), NAD(P)H oxidase (NOX), and Xanthine Oxidase (XOD). Affinity to enzymes decreased in the order: XOD > LOX > NOX > MPO.