Polyphenols containing new azo-aroylhydrazone hybrids: theoretical insights and evaluation of antioxidant potential via CUPRAC and DPPH assays

The growing interest in multifunctional antioxidants has drawn attention to hybrid scaffolds that unify distinct redox-active pharmacophores. Azo groups, aroylhydrazones, and gallic acid derivatives are individually recognized for their notable antioxidant potential, yet molecular hybrids integrating all three remain scarce. To address this gap, a new series of compounds (5a-j) combining azo, aroylhydrazone, and polyhydroxyphenyl moieties was designed and synthesized to enhance antioxidant activity through structural synergy. The synthesized molecules were thoroughly characterized by FTIR, 1H, 13C NMR, 2D NOESY/COSY, UV-Vis, and mass spectrometry. Their electronic and structural properties were further evaluated using DFT calculations at the B3LYP/6-311++G(d,p) level. In silico ADMEt predictions and molecular docking studies revealed favorable pharmacokinetic profiles and strong binding affinities toward key antioxidant-related enzymes-glutathione reductase (PDB: 1XAN), myeloperoxidase (PDB: 3F9P), and NAD(P)H oxidase (PDB: 2CDU). In vitro antioxidant activity was assessed using CUPRAC and DPPH assays, with Trolox serving as the standard. Remarkably, this is the first study to report the combined use of DMF in the CUPRAC and DMSO in the DPPH assays, offering a novel solvent strategy that enhanced compound solubility and assay compatibility. Among the tested compounds, 5b and 5e showed the highest antioxidant activities, surpassing Trolox in both methods. These results suggest that the azo-aroylhydrazone-polyphenol hybrid structure holds strong promise as a platform for developing nextgeneration antioxidant agents.