Design, synthesis, and evaluation of chalcone-Vitamin E-donepezil hybrids as multi-target-directed ligands for the treatment of Alzheimer’s disease

Figures & data

Figure 1. Design strategy for chalcone-Vitamin E-donepezil hybrids.

Scheme 1. Synthesis of target chalcone-Vitamin E-donepezil hybrids 8, 9a–f, and 10. Reaction conditions: (i) Br(CH₂)_nBr, K₂CO₃, CH₃CN, 65 °C, 6–10 h; (ii) NHR₁R₂ (3a–c), K₂CO₃, CH₃CN, refluxed, 6–8 h; (iii) 1, 4a–b, 5a–c, and 6a–b, 50% KOH, r.t., 3–4 days.

Scheme 2. Synthesis of target chalcone-Vitamin E-donepezil hybrids 16 and 17a–f. Reagents and conditions: (i) chloromethyl methyl ether, (i-Pr)₂EtN, acetone, 50 °C, 6–8 h; (ii) 4a–b, 5a–b, and 6a–b, 50% KOH, r.t., 3–4 days; (iii) 1, 50% KOH, r.t., 3–4 days; (iv) 10% HCl, room temperature, overnight.

Table 1. AChE and BuChE inhibitory potency and oxygen radical absorbance capacity (ORAC, Trolox equivalent) by chalcone-Vitamin E-donepezil hybrids, Vitamin E, and donepezil.

Compds.	IC50 (μM) ± SD^a			SI^e	ORAC^f
	Rat AChE^b	EeAChE^c	RatBuChE^d
8	>500	>500	>500	–	5.4 ± 0.01
16	>500	>500	>500	–	7.8 ± 0.01
9a	3.12 ± 0.02	1.46 ± 0.02	>500	>160.3	1.01 ± 0.01
9b	0.67 ± 0.01	1.62 ± 0.01	>500	>746.3	0.93 ± 0.01
9c	1.77 ± 0.01	1.50 ± 0.02	>500	>282.5	0.97 ± 0.01
9d	0.32 ± 0.02	1.20 ± 0.01	451 ± 1.68	1409.4	1.01 ± 0.2
9e	1.68 ± 0.01	1.00 ± 0.03	>500	>297.6	0.99 ± 0.02
9f	0.61 ± 0.01	1.78 ± 0.02	>500	>819.7	0.96 ± 0.01
10	4.94 ± 0.02	2.72 ± 0.02	>500	>101.2	1.02 ± 0.01
17a	3.45 ± 0.03	2.30 ± 0.01	326 ± 10.7	94.5	3.1 ± 0.02
17b	0.73 ± 0.01	3.16 ± 0.02	155 ± 5.8	212.3	2.9 ± 0.02
17c	1.63 ± 0.02	3.74 ± 0.03	>500	>306.7	2.8 ± 0.01
17d	0.58 ± 0.01	3.68 ± 0.02	406 ± 15.2	700	3.1 ± 0.01
17e	1.40 ± 0.02	2.16 ± 0.01	>500	>357.1	3.4 ± 0.02
17f	0.41 ± 0.02	1.88 ± 0.01	>500	>1219.5	3.3 ± 0.01
Donepezil	0.015 ± 0.002	0.12 ± 0.01	20.7 ± 1.36	1380	n.t.^g
Vitamin E	–	–	–	–	2.6 ± 0.06

^aIC₅₀ values were expressed as ±SD μM by three independent experiments.

^bratAChE was from cortex homogenate of rat.

^ceeAChE was from electric eel AChE.

^dratBuChE was from serum of rat.

^eSI = Selectivity index = IC₅₀ (BuChE)/IC₅₀ (AChE).

^fThe ORAC values are expressed as μM of Trolox equivalent/μM of compounds.

^gn.t. = no test.

Figure 2. Steady-state inhibition by 17f of AChE hydrolysis of acetylthiocholine (ATCh).

Figure 3. Compound 17f (green stick) interacted with AChE (PDB code: 1eve) (A) Interactions in the active site. (B) 3D docking model. (C) 2D docking model.

Figure 4. (A) RMSD analysis of compound 17f (green stick) in AChE (PDB code: 1eve). (B) The docking model for 17f into the protein crystal structure of AChE (PDB code: 1eve).

Table 2. The results of propidium iodide displacement assay and inhibition of huAChE-induced Aβ aggregation towards compound 17f and donepezil.

Compound	Propidium iodide displacement from AChE PAS (% inhibition)^a		% Inhibition of huAChE- induced Aβ aggregation^b
	10 μM	50 μM
17f	23.9 ± 1.6	34.2 ± 2.3	53.9 ± 3.7
Donepezil	20.4 ± 1.3	32.7 ± 2.6	24.3 ± 2.1

^aPropidium iodide displacement assay was performed on AChE to test the ability of compounds to displace propidium with reference to the donepezil at 10 and 50 μM. Data are presented as the mean ± SEM of three independent experiments.

^bInhibition of human AChE-induced Aβ_1–40 aggregation was tested using ThT assay, the concentration of tested compounds and Aβ_1–40 was 100 and 230 μM, respectively, and the Aβ_1–40/HuAChE ratio was equal to 100/1. Data are presented as the mean ± SEM of three independent experiments.

Table 3. Inhibition and disaggregation potency of Aβ_1–42 aggregation by donepezil, curcumin, and chalcone-Vitamin E-donepezil hybrids.

Compds.	Inhibition of Aβ1–42 aggregation (%)^a		Disaggregation of Aβ1–42 aggregation (%)^a
	Self-induced^b	Cu^2+-induced^c	Self-induced^d	Cu^2+-induced^e
8	23.1 ± 0.01	33.2 ± 0.01	n.t.^f	n.t.^f
16	27.7 ± 0.02	38.1 ± 0.01	n.t.^f	n.t.^f
9a	57.4 ± 0.01	66.2 ± 0.02	26.8 ± 0.02	54.2 ± 0.02
9b	63.4 ± 0.03	71.6 ± 0.01	22.2 ± 0.03	55.1 ± 0.22
9c	65.0 ± 0.01	74.1 ± 0.02	45.7 ± 0.12	66.6 ± 0.36
9d	72.7 ± 0.02	72.4 ± 0.01	33.0 ± 0.08	63.2 ± 0.63
9e	65.3 ± 0.02	76.2 ± 0.04	58.2 ± 0.01	77.0 ± 0.57
9f	69.0 ± 0.01	75.0 ± 0.02	62.8 ± 0.03	81.4 ± 0.68
10	37.7 ± 0.02	49.4 ± 0.02	25.6 ± 0.03	26.2 ± 0.28
17a	67.4 ± 0.04	91.1 ± 0.01	29.9 ± 0.03	84.6 ± 0.03
17b	82.1 ± 0.01	90.2 ± 0.02	60.2 ± 0.03	82.8 ± 0.03
17c	68.4 ± 0.02	92.5 ± 0.03	75.9 ± 0.03	79.1 ± 0.15
17d	73.8 ± 0.01	91.7 ± 0.02	45.7 ± 0.16	79.8 ± 0.03
17e	65.7 ± 0.03	96.2 ± 0.02	74.8 ± 0.31	83.7 ± 0.03
17f	78.0 ± 0.02	93.5 ± 0.01	72.3 ± 0.06	84.5 ± 0.35
Donepezil	n.a.^g	n.t.^f	n.t.^f	n.t.^f
Curcumin^h	47.3 ± 0.01	76.5 ± 0.02	n.t.^f	56.5 ± 0.21

^aInhibition and disaggregation experiments of Aβ_1–42 aggregation using ThT assay. The experiments were performed three times and the results were expressed as ±SD.

^bInhibition potency of self-mediated Aβ_1–42 aggregation at 25 μM.

^cInhibition potency of Cu²⁺-mediated Aβ_1–42 aggregation at 25 μM.

^dDisaggregation potency of self-mediated Aβ_1–42 aggregation at 25 μM.

^eDisaggregation potency of Cu²⁺-mediated Aβ_1–42 aggregation at 25 μM.

^fn.t. = not tested.

^gn.a. = no active, meaning inhibition rate was <5.0% at 25 μM.

^hThe concentration of Curcumin was 25 μM.

Figure 5. TEM images analysis of self-medicated Aβ_1–42 aggregation by curcumin and compound 17f. (A) Inhibition experiments. (B) Disaggregation experiments.

Figure 6. The UV spectrum of compounds 9d and 17f alone or in the presence of CuCl₂, AlCl₃, ZnCl₂, and FeSO₄. The final concentration was 37.5 μM.

Figure 7. Determination of the stoichiometry of complex compound-Cu²⁺ by using the molar ratio method. The final concentration of compounds 9d and 17f was 37.5 μM, with ascending amounts of CuCl₂.

Figure 8. TEM images analysis of Cu²⁺-mediated Aβ_1–42 aggregation by curcumin and compound 17f. (A) Inhibition experiments. (B) Disaggregation experiments.

Figure 9. Docking studies of 17f with Aβ_1–42 (PDB ID: 1BA4). (A) Cartoon model. (B) Interactions in the C-terminus of the active site. (C) 2D docking model.

Table 4. Inhibition potency of huMAO-A and huMAO-B) and selectivity index (SI) values of clorgyline, rasagiline, iproniazid, and chalcone-Vitamin E-donepezil hybrids.

Compds.	IC50 (μM) ± SD^a		SI^b
	MAO-B	MAO-A
8	n.t.^c	22.9%^d	–
16	n.t.^c	15.4 ± 0.16	–
9a	3.8 ± 0.02	13.9%^d	–
9b	8.1 ± 0.03	19.8 ± 0.22	2.4
9c	23.6 ± 0.12	35.7 ± 0.36	1.5
9d	12.3 ± 0.08	38.2 ± 0.63	3.1
9e	2.5 ± 0.01	37.8 ± 0.57	15.1
9f	7.2 ± 0.03	43.6 ± 0.68	6.1
10	14.6 ± 0.03	27.9 ± 0.28	1.9
17a	4.4%^d	18.2%^d	–
17b	2.1%^d	14.6%^d	–
17c	11.0%^d	18.3 ± 0.15	–
17d	15.1 ± 0.16	16.7%^d	–
17e	12.8 ± 0.31	17.9%^d	–
17f	8.8 ± 0.06	37.5 ± 0.35	4.3
Clorgyline	20.8 ± 0.27	0.0027 ± 0.0001	0.0001
Rasagiline	0.0281 ± 0.0068	0.587 ± 0.038	20.9
Iproniazid	1.35 ± 0.02	5.48 ± 0.03	4.1

^aThe experiments were performed three times and the IC₅₀ values were expressed as the mean ± SEM.

^bSI = selectivity index = IC₅₀ (hMAO-A)/IC₅₀ (hMAO-B).

^cn.t. = not tested.

^dPercent inhibition rate at 10 μM.

Figure 10. The interactions between compound 17f (green stick) and the residues of the active site in huMAO-B (PDB code: 2V60).

Figure 11. (A) Cytotoxicity of 17f in PC12 cells. (B) Attenuation of H₂O₂-induced PC12 cell injury by compound 17f was tested using MTT assay. (C) The LDH activity of compound 17f on H₂O₂-induced PC12 cell injury was evaluated using LDH assay. Three independent experiments were carried out. Data were expressed as mean ± SD and percentage of control value. ^##p < 0.01 vs. control; **p < 0.01, *p < 0.05 vs. H₂O₂ group. VE: Vitamin E.

Table 5. The predictive penetration of 17f by PAMPA-BBB assay.

Compounds	Pe (×10^−6 cm/s)	Prediction
17f	4.23 ± 0.37	CNS+
Verapamil	17.93 ± 1.26	CNS+
Diazepam	13.12 ± 0.79	CNS+
Enoxacine	0.53 ± 0.02	CNS−