Prediction, in silico antioxidant activity, and targeted synthesis of sterically hindered phenol azomethine derivatives

Molecular design and synthesis of a new series of biologically active azomethines containing a sterically hindered phenolic fragment were carried out. Within the scope of the study, 8 compounds were synthesized, and their antioxidant activity was evaluated under in vitro conditions. To establish the mechanism of action, molecular docking was used to model the interaction of the synthesized ligands with the active site of glutathione peroxidase-4 (GPx-4). The conducted analysis revealed key structural features determining antioxidant efficacy and established a correlation between molecular structure and biological activity.

The aim. Synthesis, computer screening, and investigation of the antioxidant properties of new azomethines based on sterically hindered phenol, as well as establishing structure–activity relationships.

Materials and methods. A new series of 2,6-di-tert-butyl-4-[C-alkyl-(aryl)-(N-phenyl)-azomethine]phenols was synthesized by the condensation of corresponding ketones with aromatic amines in the presence of catalytic amounts of p-toluenesulfonic acid. The structure and purity of the obtained compounds were confirmed by a complex of physicochemical methods, including IR spectroscopy, H NMR spectroscopy, and elemental analysis. For the initial assessment of the biological potency of the synthesized compounds, computer prediction (in silico) of their antioxidant, antiradical, and cardiotonic properties was performed using the online platform PASS Online. Molecular modeling of potential inhibitory activity against human glutathione peroxidase-4 (GPx-4) was carried out using the Autodock 4.0 program. The conformational mobility of the ligands was taken into account, for which optimal torsion angles were previously determined and set. Experimental study of antioxidant activity (AOA) was conducted in two model systems: induction of lipid peroxidation (LPO) in a complex of corn oil fatty acids under UV irradiation; and the Fenton system (H₂O₂/Fe²⁺). To compare efficacy, ubiquinone and bottled hydroxytoluene (BHT, the active substance of the drug dibulin), representing the class of sterically hindered phenols, were used as reference standards.

Results. The spectrum of biological activity of the studied compounds was predicted in silico using the PASS Online service. As it was expected, all substances have cardiotonic, membrane-stimulating, and antioxidant potential. The presence of AOA and the ability to scavenge free radicals allows these molecules to be classified as antiradical agents. Experimental verification of AOA was carried out in two model systems: based on photooxidation (UV irradiation) of a complex of fatty acids from corn oil (system No. 1) and on the Fenton system (H₂O₂/Fe²⁺, system No. 2). In all the cases, the studied compounds demonstrated high efficacy, inhibiting lipid peroxidation LPO by 42–48%. This result significantly exceeds the activity of standard antioxidants — ubiquinone (11%) and BHT (39%) — in the same conditions.

Conclusion. The results of molecular docking indicate a high affinity of the new ligands to the GP-4 protein, with the calculated binding energy for the most promising structures being comparable to that of known standards—ubiquinone, dibulin (hydroxybutylated toluene), and mexidol. In vitro experimental data confirmed the pronounced antioxidant activity of the synthesized compounds. “Lead” structures were identified that surpass classical antioxidants—ubiquinone and dibulin — in efficacy.

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