Design, Molecular Docking and Neuroprotective Screening of Novel Pyrimidine-Based Acetylcholinesterase Inhibitors

Abstract

Alzheimer's disease (AD) remains a multifactorial neurodegenerative disorder and currently there are only a few symptomatic therapies available to treat the cholinergic system. The pyrimidine scaffold has become a privileged scaffold for the design of multi-target directed ligands (MTDLs) that are effective at targeting the cholinergic deficit and downstream neurotoxic cascades. A series of ten new pyrimidine derivatives, 3a-3j, were synthesized, characterized and evaluated for their biological activity as potential anti-AD agents. Molecular docking of compound 3e to Torpedo californica acetylcholinesterase (AChE, PDB: 4EY7) showed that compound 3e had superior binding affinity (–12.4 kcal/mol), which was achieved by dual binding mode with both catalytic anionic site (Trp84, Tyr121) and peripheral anionic site (Trp279, Tyr70). An in vitro enzymatic assay confirmed that 3e was the most potent AChE inhibitor (IC₅₀ = 38 ± 4 nM), similar in potency to donepezil (22 ± 2 nM) and demonstrated a good selectivity for AChE compared to butyrylcholinesterase (BChE) (5.5-fold). Structure-activity relationship (SAR) studies indicated that an unsubstituted pyrimidine ring for π–π stacking, hydrogen bond donors at position 4/5 and an extended lipophilic tail for PAS recognition are essential for potency. No cytotoxicity up to 50 μM was observed with 3e in differentiated SH-SY5Y neuroblastoma cells, where it also resulted in the highest level of neuroprotection against Aβ₁–₄₂-induced toxicity (81 ± 5% viability recovery, similar to donepezil at 77 ± 4%). These results prove that 3e is a promising multi-target lead that has a high degree of AChE inhibition activity and anti-amyloid neuroprotection activity with PAS, justifying further preclinical development as a disease-modifying drug in AD.