Design, Synthesis, In Silico and In Vitro Analysis of Newly Synthesized 2-Mercaptobenzazole Derivatives as Potential Acetylcholinesterase Inhibitors.

Abstract

Introduction/background: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cholinergic dysfunction and excessive oxidative stress. Targeting acetylcholinesterase (AChE) alongside antioxidant mechanisms represents a rational multitarget therapeutic strategy. The present study aimed to design, synthesize, and evaluate a new series of 2-mercaptobenzimidazole derivatives as potential dual-acting anti-Alzheimer agents.

Methods: A series of eight novel 2-mercaptobenzimidazole derivatives (2a-2h) was synthesized through a two-step synthetic route involving acylation of substituted amines with chloroacetyl chloride, followed by coupling with 2-mercaptobenzimidazole. Structural elucidation was performed using standard spectroscopic techniques. in silico ADMET profiling and molecular docking were conducted against human AChE (PDB ID: 4EY7). The compounds were further evaluated for in vitro AChE inhibitory activity, enzyme kinetics, and antioxidant potential using the DPPH radical scavenging assay.

Results: ADMET analysis predicted favorable drug-like characteristics, including acceptable physicochemical properties, good intestinal absorption, and low hepatotoxicity risk. Molecular docking studies revealed enhanced binding affinity for derivatives bearing electron-withdrawing substituents, with compound 2d demonstrating the strongest interaction (-10.6 kcal/mol) through π-π stacking and hydrogen bonding within the active site. in vitro AChE inhibition assays supported the computational findings, where compounds 2c (IC50 = 12.9 ± 0.7 μM) and 2h (IC50 = 15.1 ± 0.9 μM) exhibited promising activity relative to Donepezil. Kinetic analysis confirmed mixed- type inhibition by compound 2c, yielding a Km of 51.6 ± 1.5 μM, a Vmax of 0.61 ± 0.03 μmol/min/mg, and a Ki value of 6.8 ± 0.4 μM. Antioxidant evaluation indicated notable DPPH radical scavenging activity, with compound 2h showing 79.69% inhibition at 50 μg/mL.

Discussion: The consistency between molecular docking, enzyme inhibition, and kinetic findings suggests that substituent-driven interactions play an important role in AChE inhibition. Additionally, the observed antioxidant activity highlights the therapeutic potential of these derivatives as multitarget agents capable of addressing both cholinergic dysfunction and oxidative stress associated with AD.

Conclusion: Collectively, the synthesized 2-mercaptobenzimidazole derivatives demonstrated promising acetylcholinesterase inhibitory and antioxidant properties. These findings support their potential as lead scaffolds for the development of novel multitarget therapeutic candidates for Alzheimer's disease.