Eugenol-derived carbamates as sub-nanomolar dual cholinesterase inhibitors with neuroprotective activity against Alzheimer's disease: Structure-activity relationships and molecular dynamics insights.

Carbamate-based cholinesterase inhibitors represent a clinically validated therapeutic strategy for Alzheimer's disease (AD); however, sub-nanomolar AChE inhibitory activity within natural phenol-derived scaffolds has not been previously reported. To address this gap, a series of eleven eugenol-based carbamate derivatives (E1-E11) was rationally designed, synthesized, and evaluated as multifunctional anti-AD agents. Systematic variation of the carbamate N-substituent enabled fine modulation of inhibitory potency and isoform selectivity, revealing well-defined structure-activity relationships. Among the synthesized compounds, E11 emerged as the most potent AChE inhibitor (IC50 = 0.62 nM), surpassing rivastigmine (0.80 nM), the only clinically approved carbamate-based inhibitor, while displaying a markedly AChE-selective profile (SI = 24.0). To our knowledge, this represents the most potent AChE inhibitory activity reported for a carbamate within a natural phenol-derived framework. E10 established a genuine dual inhibitory profile (AChE IC50 = 4.25 nM; BChE IC50 = 3.20 nM), relevant to multitarget therapy across AD disease stages. Enzyme kinetic studies confirmed mixed-type inhibition for both lead compounds, with nanomolar Ki values indicating simultaneous engagement of the catalytic and peripheral anionic sites. These findings were corroborated by 200 ns molecular dynamics simulations and MM-PBSA binding free energy analyses, which revealed stable dual-site binding modes consistent with the kinetic data. Although DPPH radical scavenging was attenuated by carbamate masking of the phenolic hydroxyl group, CUPRAC analysis revealed significant electron transfer-based antioxidant capacity for E10 and E11. Importantly, both lead compounds demonstrated significant neuroprotective effects in H2O2-challenged HT-22 mouse hippocampal neuronal cells, a well-established oxidative stress model relevant to AD neurodegeneration, with acceptable cytotoxicity profiles. In silico ADME profiling predicted favorable gastrointestinal absorption, blood-brain barrier permeability, and absence of P-glycoprotein efflux liability for all compounds. Collectively, these findings establish eugenol-based carbamates as a pharmacologically competitive and structurally accessible multifunctional scaffold for AD, with E10 and E11 identified as priority candidates for further preclinical investigation; however, the present study is limited to in vitro and in silico evaluation, and in vivo pharmacological assessment and detailed metabolic profiling, particularly regarding the predicted mild CYP450 liability of the aryl-substituted derivatives, are warranted in future studies.