Multistep synthesis of novel indole–benzimidazole hydrazone hybrids as xanthine oxidase inhibitors: In-Vitro evaluation, molecular docking, MD simulations and DFT studies

This work reports the synthesis of a focused library of indole-benzimidazole hydrazone hybrids ( 17 - 35 ) via a multistep procedure involving thiolation, hydrazination, and Schiff base condensation. The final structures were confirmed using 1 H-NMR, 13 C-NMR, and FT-IR spectroscopy by observing diagnostic NH, azomethine, and carbonyl peaks consistent with the proposed frameworks. All of the synthesized compounds were evaluated for in vitro xanthine oxidase (XO) inhibition, where only eight compounds possessed significant activity (IC₅₀ = 26.8–91.5 µM). Amongst the active molecules, compound 18 emerged as the most potent inhibitor (IC₅₀ ≈ 26–27 µM), followed by 20 , 17 , and 33 , with several analogues outperforming allopurinol (IC₅₀ = 78.5 ± 1.34 µM), indicating promising lead potential. The structure-activity (SAR) analysis highlights that small, single electron-donating substituents at ortho/meta positions increased XO potency, whereas multiple, bulky, or strongly electron-withdrawing groups created steric clashes, hence reduced activity. Conceptual DFT results supported the biological trends, showing lower HOMO-LUMO gaps (3.75–4.37 eV) and reduced hardness relative to allopurinol, consistent with higher electronic softness and improved target interaction in active compounds. Molecular docking was performed against bovine XO (PDB: 3NRZ), which displayed strong binding of the active compounds ( 18 , 20 , 17 , and 33 ) via cooperative H-bonding (Ser876), π–π stacking with phenylalanine residues, and π-cation/π-sulfur interactions (Lys771, Met770), rationalizing their inhibitory mechanisms. Furthermore, MD simulations were applied to compound 18 , allopurinol as a standard, and APO, and the results reveal that after ligand binding, the protein was stabilized. The ligand-binding complex of compound 18 was found to be more stable than the allopurinol complex. The binding energy was calculated by MMPBSA; compound 18 has -28.839 ± 4.59 kcal/mol, and the allopurinol complex has -7.746 ± 2.53 kcal/mol. This interactive experimental-computational work identifies the indole-benzimidazole hydrazone scaffold, specifically compound 18 , as a viable platform for producing next-generation XO inhibitors for the management of hyperuricemia and gout.