Design, Synthesis, and Enantioselective Cholinesterase Inhibition of Novel Chiral Anthranilic Diamide Derivatives: In Vitro and In Silico Studies.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes are responsible for the hydrolysis of acetylcholine and play a vital role in Alzheimer's disease (AD) pathology. The observation of decreased AChE activity and a significant increase in BChE activity in the advanced stages of AD makes the identification of novel inhibitors targeting both AChE and BChE enzymes crucial. In this study, novel chiral anthranilic acid-based diamide derivatives (5a-5d, 7a, and 7b) with pure enantiomer structures were synthesized, and their inhibitory potential on AChE and BChE was comprehensively investigated. The structures of the synthesized compounds were confirmed by 1H NMR, 13C NMR, IR, and LC-MS analyses. In vitro cholinesterase inhibition studies showed that all compounds exhibited significant inhibitory activity against both enzymes. Specifically, compounds 7a and 7b, containing 2,6-pyridine dicarbonyl, exhibited higher AChE and BChE inhibition compared with the reference drug tacrine, displaying IC50 values at the nanomolar level. Enantioselective analyses showed that S-enantiomers were significantly more effective in AChE inhibition, while enzyme-specific inverse stereoselective preferences emerged in BChE inhibition. Experimental findings were supported by induced coherent molecular docking (IFD), MM-GBSA binding free energy calculations, and 250-ns molecular dynamics simulations. In silico analyses confirmed that 7b exhibited high stability and strong interactions in the AChE active site and 7a in the BChE active pocket. Furthermore, ADME analyses revealed that the compounds possessed favorable pharmacokinetic and drug-like properties. In conclusion, this study demonstrates that chiral anthranilic diamide derivatives are promising novel AChE/BChE inhibitor candidates that act as potent and selective cholinesterase inhibitors.