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: ( E )-3-(1-((2-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate ( A 1 -OH ), ( E )-3-(1-((3-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate ( A 2 -OH ), and ( E )-3-(1-((4-hydroxyphenyl)amino)ethylidene)-2,4-dioxochroman-7-yl acetate ( A 3 -OH ) 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 ( f -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 A 1 -OH (58.7%) > A 2 -OH (57.5%) > A 3 -OH (53.1%). Kinetic analysis revealed the f -HAT mechanism dominated HOO • inactivation. A newly formulated Sequential Proton Loss followed by Radical Adduct Formation (SPL-RAF) mechanism described interactions with O 2 •- . The activity toward O 2 •- was A 2 -OH (1.26 × 10 6 M -1 s -1 ) > A 3 -OH (7.71 × 10 5 M -1 s -1 ) > A 1 -OH (4.22 × 10 5 M -1 s -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.