Morpholine-modified thiosemicarbazones and thiazolidin-4-ones against Alzheimer’s key enzymes: From synthesis to inhibition

Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is a key therapeutic approach in the management of Alzheimer’s disease and other neurodegenerative disorders. A novel series of phenolic Mannich base-derived thiosemicarbazones and their cyclized thiazolidin-4-one analogs incorporating morpholine moieties were synthesized and characterized. Enzyme inhibition kinetics were evaluated against AChE and BChE, with cytotoxicity assessed on the BEAS-2B cell line. The most potent inhibitors were further examined via molecular docking, MM-GBSA binding free energy decomposition, and 250 ns molecular dynamics (MD) simulations to elucidate their binding mechanisms and stability. Compounds 12 (AChE, K_i = 32.83 ± 4.45 nM) and 6 (BChE, K_i = 30.13 ± 5.78 nM) exhibited the highest inhibitory activities without notable cytotoxicity at their effective concentrations. Kinetic analyses revealed competitive inhibition. Computational studies demonstrated that morpholine tertiary amine groups played a pivotal role in anchoring the ligands via persistent cation–π and hydrogen-bond interactions with key active site residues. In silico ADME analysis indicated that most of the synthesized compounds possess favorable pharmacokinetic properties. This combined in vitro and in silico study identifies compounds 6 and 12 as promising lead structures for cholinesterase inhibition, highlighting the critical contribution of tertiary amine moieties to binding affinity and selectivity.