Design, synthesis, and biological evaluation of novel multifunctional caffeic acid-piperazine derivatives for the management of Alzheimer's disease.

Alzheimer's disease (AD), the most prevalent form of dementia, is marked by progressive memory impairment, cognitive decline, reduced acetylcholine level, oxidative stress, amyloid-β (Aβ) aggregation, and disturbances in metal homeostasis. Our earlier work on caffeic acid-based multifunctional inhibitors prompted us to explore structure-activity relationships (SARs) to improve BChE cholinesterase inhibition while maintaining or enhancing the multifunctional activity of the previously identified lead molecule 12d. For this purpose, the glycinamide linker was rationally replaced with benzylpiperazine and glycinamide-piperazine, yielding six new series of derivatives. Comprehensive SAR analysis identified 7b as the most promising molecule, displaying markedly enhanced potency compared to caffeic acid and our earlier analog EJMC-12d. 7b exhibited approximate 3.6-fold improvement in BChE inhibition compared to EJMC-12d. While AChE inhibition of 7b was found to be comparable to EJMC-12d. The enzyme kinetic studies showed a mixed-type inhibitory mechanism for 7b at both AChE and BChE. Molecular docking studies also confirmed that 7b binds to the CAS and PAS sites of AChE. The DPPH assay on synthesized derivatives revealed strong antioxidant properties of 7b (IC₅₀ = 4.89 ± 0.72 μM) compared to EJMC-12d (IC₅₀ = 6.32 ± 0.15 μM).7b also showed efficient metal-chelating properties. 7b exhibited potent inhibitory activity against Aβ1-42 aggregation, as confirmed by the ThT fluorescence assay and validated by fluorescence microscopy. The PAMPA-BBB permeability assessment showed that 7b (Pe = 3.96 ± 0.38) can cross the blood-brain barrier. Cell viability studies with 7b in SH-SY5Y cells demonstrated cytocompatibility at higher doses. The neuroprotection studies against H₂O₂ and ROS generation confirmed that 7b exhibits strong antioxidant activity and a neuroprotective effect against oxidative stress. 7b was found to be safe in the acute toxicity studies at a higher dose of 500 mg/kg. In the in vivo Aβ-induced stereotaxic mouse model, 7b significantly restored spatial working memory. The overall findings highlighted 7b as a promising, efficacious, multifunctional candidate with significant therapeutic potential for AD. Despite this, there is scope for structural modifications in 7b to further improve the multifunctional properties. Overall, this scaffold demonstrates strong potential and can be further explored as a promising therapeutic candidate for AD management.