For the first time, it was established that carboxylated chalcones containing hydroxy, methoxy, and ethoxy groups in ring A, as well as Δ3,9-homoisoflavonoids and flavone are submicromolar inhibitors of xanthine oxidase, more potent than carboxylated Δ2,3-homoisoflavonoids, dihydrochalcone, oxa-dihydrochalcones and oxa-homoisoflavonoid. Structure-activity relationship analysis suggests that the presence of a chalcone, ∆3,9-homoisoflavonoid or flavone scaffold and a carboxyl group are important for enzyme inhibition. Antioxidant properties of carboxylated chalcones and related flavonoids were determined by measuring the effect of the compounds on the degree of conversion of deoxyribose in the Fenton reaction. According to the obtained data, some chalcone-4-carboxylic acids, oxa dihydrochalcone-4-carboxylic acid, ∆2,3-homoisoflavonoid-4ꞌ-carboxylic acid, and 4′-carboxy-7-methoxyflavone showed the best activity. At the same time, the activity of all compounds was higher than that of trolox as a reference antioxidant. Kinetic studies conducted for both inhibitors indicate that their affinity for the binding sites on the surface of the free enzyme is much higher than that for the enzyme-substrate complex. Molecular docking was performed to predict the binding mode of inhibitors to the active site of xanthine oxidase. According to the obtained results, carboxyl groups of chalcone-4-carboxylic acid and Δ3,9 homoisoflavonoid-4′-carboxylic acid (E-isomers) form hydrogen bonds with amino acid residues Arg880 and Thr1010. The positions of the B-rings of the compounds are stabilized by π-π stacking interactions with the Phe914 residue. The oxygen atoms of the carbonyl groups of chalcone and Δ3,9-homoisoflavonoid have similar orientations, while the A-rings of chalcone and Δ3,9-homoisoflavonoid are located in the hydrophobic region formed by amino acid residues Leu648, Met770, Lys771, Leu873, Phe1013, and Leu1014, with differences in binding modes depending on the structure of the inhibitor. The results of the dissertation form new knowledges about the structure and inhibitory capacity of new inhibitors of xanthine oxidase, which can be the basis for the creation of effective and selective inhibitors of this enzyme, including carboxylated flavonoids and their structural analogues, as well as carboxylated derivatives of heterocyclic compounds. The new knowledge gained in this area of bioorganic chemistry demonstrates the potential of the approaches which can be used for further drug design.
