Design, synthesis and evaluation of novel cinnamic acid derivatives bearing N-benzyl pyridinium moiety as multifunctional cholinesterase inhibitors for Alzheimer's disease

Abstract

A novel family of cinnamic acid derivatives has been developed to be multifunctional cholinesterase inhibitors against AD by fusing N-benzyl pyridinium moiety and different substituted cinnamic acids. In vitro studies showed that most compounds were endowed with a noteworthy ability to inhibit cholinesterase, self-induced Aβ (1-42) aggregation, and to chelate metal ions. Especially, compound 5l showed potent cholinesterase inhibitory activity (IC₅₀, 12.1 nM for eeAChE, 8.6 nM for hAChE, 2.6 μM for eqBuChE and 4.4 μM for hBuChE) and the highest selectivity toward AChE over BuChE. It also showed good inhibition of Aβ (1-42) aggregation (64.7% at 20 μM) and good neuroprotection on PC12 cells against amyloid-induced cell toxicity. Finally, compound 5l could penetrate the BBB, as forecasted by the PAMPA-BBB assay and proved in OF1 mice by ex vivo experiments. Overall, compound 5l seems to be a promising lead compound for the treatment of Alzheimer's diseases.

Keywords: Alzheimer’s disease; benzylpyridinium; cinnamic acid; metal chelator; neuroprotection; β-amyloid aggregation.

Figure 1.

Drug design strategy for multi-target-directed ligands.

Scheme 1.

Synthesis of cinnamic acid derivatives 5a–n. Reagents and conditions: (i) DMAP/EDCI, CH₂Cl₂, rt., 12 h; (ii) CH3CN, reflux, 1–3 h.

Figure 2.

Kinetic study on the mechanism of EeAChE inhibition by compound 5l. Overlaid Lineweaver–Burk reciprocal plots of AChE initial velocity at increasing substrate concentration (0.05–0.50 mM) in the absence of inhibitor and in the presence of compound 5l are shown. Lines were derived from a weighted least-squares analysis of the data points.

Figure 3.

(A) 3D docking model of compound 5l with TcAChE. Atom colors: yellow – carbon atoms of 5l, gray – carbon atoms of residues of TcAChE, dark blue – nitrogen atoms, red – oxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (B) 2D schematic diagram of docking model of compound 5l with TcAChE. The figure was prepared using the ligand interactions application in MOE.

Figure 4.

(A) UV absorbance spectrum of 5l (50 μM) alone (red) or in the presence of 100 μM CuSO₄ (green) and 100 μM FeSO₄ (yellow) in MeOH. (B) Determination of the stoichiometry of complex 5l−Cu²⁺ by molar ratio method.

Figure 5.

Inhibition of Cu²⁺-induced Aβ (1–42) aggregation by compound 5l comparing with that of clioquinol (CQ) ([Aβ = 25 μM, [5l] = 50 μM, [CQ] = 50 μM, [Cu²⁺] = 25 μM, 37 °C, 24 h). Values are reported as the mean ± SD of three independent experiments. #p < .05, **p < .01.

Figure 6.

Docking study of compound 5l with Aβ (1–42) generated with MOE. Atom colors: yellow – carbon atoms of 5l, gray – carbon atoms of residues of Aβ (1–42), dark blue – nitrogen atoms, red – oxygen atoms. The dashed lines represent the interactions between the protein and the ligand.

Figure 7.

(A) Effects of compound 5l on cell viability in PC12 cells. The cell viability was determined by the MTT assay after 24 h of incubation with various concentrations. The results were expressed as a percentage of control cells. Values are reported as the mean ± SD of three independent experiments. (B) Neuroprotection against Aβ (1–42) toxicity. Compound 5l was tested for neuroprotective activity against Aβ (1–42) toxicity in PC12 cells. Data represent the mean SD of three observations. *p < .05 and **p < .01 compared to the Aβ (1–42)-treated control group.