Synthesis, <i>In Vitro</i> Cholinesterase Inhibition, Molecular Docking, Density Functional Theory Analysis of Benzimidazole Based Hydrazone Schiff Bases

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the main therapeutic targets for the treatment of neurodegenerative diseases, predominantly Alzheimer's disease. This work reports the synthesis, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of synthesized hydrazone Schiff base derivatives of benzimidazole. The compounds have been structurally deduced by means of HR-ESI-MS, ¹H-NMR and ¹³C-NMR techniques and finally assessed for their in vitro AChE and BuChE inhibitory activities. All the compounds attributed notable inhibitory potential with IC₅₀ values ranging from 34.7 ± 0.02 to 185.2 ± 2.47 μM for AChE and 40.8 ± 0.32 to 188.8 ± 2.47 μM for BuChE enzymes. The molecular docking and TD-DFT studies attributed that the compound with methoxy groups, specifically compound (7) displays increased electronic properties and strong dual binding to AChE and BuChE enzymes. Molecular dynamics (MD) simulations for the most active compound (7) were performed, which showed that compound (7) exploits the integral flexibility of AChE and BuChE to encourage conformational variations that lock both enzymes into a two-site inhibitory state. These compounds presented orbitals and favorable electrostatic profiles that improve the inhibitory potential. The results authenticate the SAR trends and highlight the significance of methoxy groups in planning potent cholinesterase inhibitors.