Structural properties, molecular modeling studies and antioxidant activities of novel benzoic acid-based azo molecules: A combined<i> in</i><i> vitro</i> and<i> in</i><i> silico</i> study

Two novel compounds, 2-((3-(tert-butyl)-2-hydroxy-5-methylphenyl)diazenyl)benzoic acid (o-HMB) and 4-((3(tert-butyl)-2-hydroxy-5-methylphenyl)diazenyl)benzoic acid (p-HMB), were synthesized and characterized using 1H- and 13C-NMR, HRESI-MS, FT-IR, and UV-vis spectroscopic methods. The optimized molecular geometries, MEP diagrams, and vibrational frequencies of the o-HMB and p-HMB were calculated with the 6-311G(d,p) basis set using the DFT method and B3LYP functional. Chemical shift values were calculated using the GIAO approach, while FMOs and electronic transitions were obtained using the TD-DFT method. Calculated global reactivity descriptors indicated that p-HMB is softer and more chemically reactive. The calculated spectroscopic data were in agreement with the experimental ones. ADMET predictions indicated good drug-likeness and low toxicity for both molecules. Molecular docking studies against the Keap1 Kelch domain (PDB ID: 5FNQ) revealed that p-HMB exhibited the best binding energy (-8.9 kcal/mol), and MD simulations confirmed the stability of the p-HMB5FNQ complex over 100 ns. The antioxidant activities of o-HMB and p-HMB were also evaluated in vitro using the CUPRAC method and p-HMB exhibited higher activity than o-HMB with a TEAC value of 0.76.