Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder involving cholinergic dysfunction, amyloid-β (Aβ) and Tau aggregation, oxidative stress, and neuroinflammation. To address the limited efficacy of single-target therapies, a series of chromone-2-carboxamide derivatives (E1-E29) were rationally designed, synthesized, and evaluated as potential multi-target-directed ligands (MTDLs). The target compounds exhibited moderate to potent inhibitory activity against acetylcholinesterase (AChE), with several derivatives (E3, E5, E12) showing submicromolar IC50 values comparable to donepezil and pronounced selectivity over butyrylcholinesterase. Selected compounds also demonstrated preferential inhibition of monoamine oxidase-B (MAO-B), with E7 and E8 approaching the activity of selegiline. Mechanistic studies revealed that E3 and E12 acted as reversible noncompetitive AChE inhibitors. Both compounds effectively inhibited Aβ40/42 and Tau fibrillization, promoted Aβ fibril disaggregation, and suppressed intracellular amyloid accumulation, accompanied by significant antioxidant and neuroprotective effects in SH-SY5Y cells. In vivo, E12 markedly improved scopolamine-induced memory deficits in mice without observable systemic toxicity. Molecular docking and dynamics simulations supported stable binding of E3 and E12 to AChE, Aβ, and Tau targets via π-π and H-π interactions, accompanied by disruption of hydrophobic networks. Collectively, these findings identify chromone-2-carboxamide hybrids, particularly E3 and E12, as promising scaffolds for the development of multi-target anti-AD drug candidates.
