Development of novel diaryl ether-phenolic Mannich Base derivatives with potent cholinesterase inhibition and low neuronal toxicity.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cholinergic dysfunction, making acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) attractive therapeutic targets. In the present study, twelve novel phenolic Mannich base-derived hydrazones incorporating a diaryl ether scaffold were rationally designed, synthesized, and evaluated as cholinesterase inhibitors. All synthesized compounds exhibited potent nanomolar inhibition against both cholinesterases, with Ki values ranging from 24.27 to 98.56 nM for AChE and 28.68-148.41 nM for BChE. Compound 5 was identified as the most potent AChE inhibitor (Ki = 24.27 nM), whereas compound 6 showed the highest BChE inhibitory activity (Ki = 28.68 nM). Both compounds were considerably more potent than the reference inhibitors tacrine and donepezil. Enzyme kinetic studies revealed that all compounds acted as competitive inhibitors. Cytotoxicity evaluation against SH-SY5Y neuronal cells demonstrated low toxicity, with IC50 values ranging from 33.41 to 46.79 μM, indicating that effective cholinesterase inhibition occurred at concentrations far below those affecting neuronal cell viability. Molecular docking and MD simulations demonstrated stable binding of the most active compounds within the catalytic gorge of both enzymes through persistent hydrogen bonds, cation-π, and π-π interactions with key active-site residues. MM-GBSA and energy decomposition analyses further supported their favorable binding affinities. In addition, the synthesized compounds exhibited acceptable predicted pharmacokinetic properties, including favorable parameters related to blood-brain barrier permeability and oral absorption. These results identify phenolic Mannich base-derived hydrazones as promising lead compounds for the development of new cholinesterase inhibitors for the treatment of Alzheimer's disease.