Design, synthesis, and biological evaluation of Chromene-Phenylpiperazine-Chalcone hybrids as multi-target-directed ligands for the treatment of Alzheimer's disease.

A novel series of chromene-phenylpiperazine-chalcone hybrids was rationally designed, synthesized and evaluated as multi-target-directed ligands (MTDLs) for Alzheimer's disease (AD). Among them, compound 9 s exhibited selective equine serum butyrylcholinesterase (eqBuChE, IC50 = 0.13 μM) and concurrent monoamine oxidase B (MAO-B, IC50 = 1.63 μM) inhibitory activities. Kinetic and molecular docking studies indicated that 9 s acts as a mixed-type dual-site inhibitor. In silico modeling suggests that the binding pose is stabilized by a predicted pseudo-seven-membered ring. Furthermore, 9 s facilitated the disassembly of self-aggregated and Cu2+-induced Aβ1-42 fibrils. In BV-2 microglial cells, it demonstrated a high safety margin (> 380-fold effective concentration), accelerated intracellular Aβ clearance, and subsequently attenuated LPS-induced NO production. In vivo evaluations revealed low acute toxicity (LD50 > 1000 mg/kg). Oral administration of 9 s successfully reversed scopolamine-induced spatial working memory deficits in mice. These findings validate the drug-like 9 s as an orally efficacious MTDL candidate that provides symptomatic cognitive relief, while possessing promising in vitro disease-modifying potential for AD therapy.