Design, synthesis, and biological evaluation of caffeic acid-based novel multifunctional molecules for the management of Alzheimer's disease

Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder characterized by low levels of acetylcholine, oxidative stress, amyloid-beta (Aβ) aggregation, and metal dysregulation. This study aimed to address the limitations associated with natural products for Alzheimer's disease (AD) management by designing and synthesizing in vivo efficacious caffeic acid derivatives as multifunctional cholinesterase inhibitors. A comprehensive structure-activity relationship (SAR) analysis was carried out in a calibrated manner to improve upon the cholinesterase inhibitory potential of caffeic acid along with its multifunctional properties. Among the synthesized compounds, 12d emerged as a lead molecule, exhibiting selective AChE inhibition (IC₅₀=3.72±0.34 μM) over BChE (IC₅₀=9.65±0.08 μM), with significant improvement over parent caffeic acid (AChE and BChE 35.39 ± 0.66 % and 18.94 ± 0.91 %, respectively). Enzyme kinetic studies revealed 12d as a mixed inhibition mechanism against human AChE (hAChE) and equine BChE (eqBChE). Molecular docking and molecular dynamics simulations demonstrated that 12d interacts with key active-site residues of AChE (TRP86, HIS447, SER293, and ARG296), and BChE (TRP82, LEU286, HIS438, PHE329, G197, and TRP231). Additionally, 12d displayed potent antioxidant activity in the DPPH assay, exhibiting a strong free radical scavenging effect with an IC₅₀ value of 6.32 ± 0.15 μM. 12d also demonstrated metal-chelating properties, suggesting its potential to prevent metal-induced neurotoxicity. Furthermore, blood-brain barrier (BBB) permeability predictions (Pe = 4.12 ± 0.40) indicated that 12d can cross the BBB. 12d’s ability to modulate Aβ_1-42 self-aggregation was confirmed through Thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM) analyses. The neuroprotective potential of 12d against oxidative stress was further evaluated in SH-SY5Y neuronal cells. Pretreatment followed by the co-treatment with 12d at varying concentrations effectively protected the cells from H₂O₂-induced neurotoxicity. Also, 12d significantly reduced intracellular reactive oxygen species (ROS), as MitoSOX and H₂DCFDA assays demonstrated. Finally, in an in vivo scopolamine-induced AD mouse model, 12d improved spatial memory in the Y-maze test. Moreover, it effectively restored AChE and BChE levels while displaying potent ex vivo antioxidant activity. These findings collectively highlight 12d as a promising multitarget-directed ligand (MTDL) with therapeutic potential for AD management.