Introduction: Alzheimer's disease (AD) necessitates novel therapeutics. This study aimed to design and develop new heterocyclic cholinesterase inhibitors using a rational, integrated computational and experimental approach, with a focus on achieving potency and selectivity, particularly for butyrylcholinesterase (BuChE).

Methods: Novel piperidine-based analogues were designed via molecular docking simulations against acetylcholinesterase (AChE) and BuChE to predict binding modes. Seven designed compounds were synthesized, and their structures were confirmed using TLC, melting point, UV, IR, EI-MS, and ¹H-NMR. In vitro inhibitory activity was evaluated against both enzymes using the Ellman assay, with donepezil as a reference standard. Drug-likeness was assessed based on Lipinski’s Rule of Five and blood-brain barrier permeability predictions.

Results: Docking studies elucidated a binding mode involving anchoring in the catalytic site and π–π interactions in the peripheral site. In vitro testing identified two potent dual inhibitors, P8 (IC₅₀ = 11.5 µM for BuChE) and P14 (IC₅₀ = 15.3 µM for BuChE), which outperformed donepezil for BuChE inhibition. The series exhibited a selective profile favoring BuChE. Structure-activity relationship (SAR) confirmed terminal group planarity as critical for potency. All compounds exhibited favorable drug-like properties.

Conclusion: The study successfully identifies P8 and P14 as promising, drug-like lead compounds with a superior BuChE-inhibitory profile, making them viable scaffolds for developing next-generation AD therapeutics. The work validates the combined computational and experimental framework for rational inhibitor design