Synthesis, Biological Evaluation, Molecular Docking, Molecular Dynamics Simulation, Enzyme Kinetic Studies, and In silico Pharmacokinetic Evaluation of Novel Benzimidazole–Pyrazoline Derivatives as Dual Cholinesterase Inhibitors

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder in which impaired cholinergic neurotransmission substantially contributes to cognitive decline. In this study, a series of novel benzimidazole–pyrazoline derivatives was designed and synthesized as potential dual inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Their inhibitory activities were evaluated, and enzyme kinetics, molecular docking, molecular dynamics (MD), and in silico pharmacokinetic analyses were conducted. Among the synthesized derivatives, compound 22 exhibited the highest dual inhibitory activity, with IC50 values of 52.5 μM against AChE and 45.46 μM against BuChE, whereas the remaining compounds showed moderate-to-weak inhibitory activity. Compared with galantamine, compound 22 showed approximately 3.8-fold lower inhibitory activity against AChE and relatively comparable activity against BuChE (approximately 1.3-fold lower). Enzyme kinetic analysis using Lineweaver–Burk plots indicated that compound 22 exhibited an inhibition pattern consistent with noncompetitive inhibition against both enzymes. Molecular docking and 300-ns MD simulations using AChE (PDB: 7E3H) and BuChE (PDB: 4BDS) provided insights into the binding interactions and conformational stability of the compound 22–enzyme complexes, including persistent hydrogen-bonding and hydrophobic interactions. In addition, in silico pharmacokinetic assessment suggested that several derivatives possess predicted physicochemical and drug-likeness profiles compatible with further investigation; however, these computational predictions require experimental validation. Overall, the experimental and computational findings identify the benzimidazole–pyrazoline scaffold as a preliminary framework for further optimization of dual cholinesterase inhibitors, while compound 22 provides a starting point for subsequent structure-based medicinal chemistry studies.