Design, synthesis and evaluation of quinolinone derivatives containing dithiocarbamate moiety as multifunctional AChE inhibitors for the treatment of Alzheimer’s disease

Figures & data

Figure 1. Rational design of quinolinone derivatives as multifunctional AChE inhibitors for the treatment of AD.

Scheme 1 Synthesis of compounds 4a-m and 9a-b. Reagents and conditions: (a) (i) cinnamoyl chloride, dry dichloromethane, 4 h, reflux; (ii) AlCl₃, chlorobenzene, 8 h, reflux; (b) α, ω-dibromoalkanes, K₂CO₃, acetone, reflux, 4 h; (c) appropriate secondary amines, CS₂, TEA, DMF, r.t., 12 h. (d) ethyl acetoacetate for 6a, ethyl 2-methylacetoacetate for 6 b, 150 °C, 48 h; (e) (i) NaNO₂, H₂SO₄, 0 °C; (ii) 10 M, H₂SO₄, reflux, 10 min.

Table 1. Inhibition of eeAChE and eqBuChE by compounds 4a-m and 9a-b.

					eeAChE^a	eqBuChE^b
Compd.	n	R	R1	R2	IC50 (μM)/Inhibition (%)	IC50 (μM)/Inhibition (%)
4a	2	A	H	H	45.3 ± 0.35%	16.69 ± 2.36%
4b	3	A	H	H	3.96 ± 0.71 μM	16.60 ± 4.01%
4c	4	A	H	H	0.22 ± 0.03 μM	45.37 ± 8.34%
4d	5	A	H	H	0.24 ± 0.02 μM	20.95 ± 2.61%
4e	6	A	H	H	0.35 ± 0.02 μM	17.49 ± 2.17%
4f	4	B	H	H	0.69 ± 0.22 μM	37.22 ± 1.22%
4g	4	C	H	H	9.61 ± 1.12 μM	31.43 ± 3.15%
4h	4	D	H	H	3.80 ± 0.35 μM	2.00 ± 0.23 μM
4i	4	E	H	H	20.17 ± 3.27%	15.81 ± 2.45%
4j	4	F	H	H	14.52 ± 2.44%	23.91 ± 2.03%
4k	4	G	H	H	23.14 ± 5.54%	28.65 ± 3.44%
4l	4	H	H	H	12.31 ± 1.76%	30.47 ± 4.11%
4m	4	I	H	H	31.43 ± 1.65%	7.63 ± 0.92%
9a	4	A	Me	H	0.30 ± 0.12 μM	18.31 ± 3.15%
9b	4	A	Me	Me	0.34 ± 0.01 μM	21.20 ± 1.60%
Donepezil	–	–	–	–	0.041 ± 0.002 μM	4.22 ± 0.20 μM
Galanthamine	–	–	–	–	1.65 ± 0.14 μM	11.68 ± 3.49 μM
Tacrine	–	–	–	–	0.21 ± 0.02 μM	0.057 ± 0.013 μM

^aThe 50% inhibitory concentration of eeAChE or percent inhibition with inhibitor at 10 μM (means ± SD of three experiments).

^bThe 50% inhibitory concentration of eqBuChE or percent inhibition with inhibitor at 10 μM (means ± SD of three experiments).

Figure 2. Lineal correlation between experimental and reported permeability of commercial drugs using the PAMPA-BBB assay. P_e (exp.) = 0.9082P_e(bibl.) – 0.3034 (R² = 0.9410).

Table 2. Permeability P_e(×10⁻⁶) in the PAMPA-BBB assay for 9 commercial drugs in the experiment validation.

Commercial drugs	Bibliography^a	Experiment^b
Testosterone	17.0	14.2 ± 3.6
Estradiol	12.0	10.2 ± 2.5
Progesterone	9.3	10.4 ± 1.9
Chlorpromazine	6.5	7.3 ± 2.3
Corticosterone	5.1	2.4 ± 0.8
Hydrocortisone	1.9	0.46 ± 0.12
Caffeine	1.3	1.2 ± 0.2
Atenolol	1.02	0.19 ± 0.23
Theophylline	0.1	0.16 ± 0.07

^aAdapted from Di et al.²⁶.

^bExperimental data are expressed as mean ± SD from three independent experiments, using PBS: EtOH (70:30) as solvent.

Table 3. Permeability P_e (×10⁶ cm/s) in the PAMPA-BBB assay for target compounds and their predicted penetration into CNS.

Compound	Pe (×10^6 cm/s)^a	Prediction^b
4a	8.57 ± 1.23	CNS+
4b	8.78 ± 1.11	CNS+
4c	8.91 ± 2.03	CNS+
4d	8.82 ± 1.34	CNS+
4e	9.85 ± 0.98	CNS+
4f	6.30 ± 0.86	CNS+
4g	7.14 ± 1.97	CNS+
4h	6.42 ± 1.02	CNS+
4i	2.83 ± 0.65	CNS±
4j	3.25 ± 0.82	CNS±
4k	4.54 ± 0.84	CNS+
4l	7.35 ± 1.05	CNS+
4m	10.00 ± 2.63	CNS+
9a	8.79 ± 2.10	CNS+
9b	8.67 ± 1.76	CNS+

^aPermeability P_e (×10⁶ cm/s) values are expressed as mean ± SD from three independent experiments, using PBS: EtOH (70:30) as solvent.

^bCNS + is predicted as high BBB permeation with P_e (×10⁶ cm/s) > 3.33, CNS ± is uncertain for BBB permeation with 1.51 < P_e (×10⁶ cm/s) < 3.33.

Table 4. Inhibition of hAChE, and AChE- and self-induced Aβ_1-42 aggregation by selected compounds.

	hAChE	Inhibition of Aβ1–42 aggregation (%)
Compd.	IC50 (μM)^a	AChE-induced^b	Self-induced^c
4c	0.16 ± 0.02	29.02 ± 2.06	30.67 ± 6.54
4d	0.57 ± 0.01	24.11 ± 3.04	26.01 ± 3.01
4e	0.38 ± 0.02	20.28 ± 2.21	25.82 ± 1.43
4f	1.21 ± 0.33	12.06 ± 3.18	20.35 ± 3.22
9a	0.76 ± 0.10	14.19 ± 2.85	33.36 ± 4.85
9b	0.39 ± 0.01	24.37 ± 3.03	35.29 ± 2.17
Donepezil	0.021 ± 0.001	26.16 ± 2.55	–
Tacrine	0.47 ± 0.03	–	–
Curcumin	–	–	37.27 ± 2.76

^aThe 50% inhibitory concentration of hAChE (means ± SD of three experiments).

^bInhibition of AChE-induced Aβ_1–42 aggregation. The thioflavin-T fluorescence method was used, and the concentration of the tested inhibitor was 100 μM (means ± SD of three experiments).

^cInhibition of self-induced Aβ_1-42 aggregation. The thioflavin-T fluorescence method was used, and the measurements were carried out in the presence of 25 μM inhibitor (means ± SD of three experiments).

Figure 3. Kinetic study on the inhibition mechanism of hAChE by compound 4c. (a) Overlaid Lineweaver-Burk reciprocal plots of hAChE initial velocity at increasing substrate concentrations (0.05–0.50 mM) in the absence of inhibitor and in the presence of different concentrations (0.4, 0.2 and 0.1 μM) of 4c are shown. (b) The plot of the slopes of the Lineweaver-Burk plots versus inhibitor concentration.

Figure 4. (a) 3 D docking model of compound 4c with hAChE. Atom colours: green-carbon atoms of 4c, gray-carbon atoms of residues of hAChE, dark blue-nitrogen atoms, red-oxygen atoms, yellow-sulfur atoms. (b) 2 D schematic diagram of docking model of compound 4c with hAChE. The figure was prepared using the ligand interactions application in MOE.

Figure 5. (a) 3 D docking model of compound 9b with Aβ_1-42. Atom colours: green-carbon atoms of 9b, gray-carbon atoms of residues of Aβ, dark blue-nitrogen atoms, red-oxygen atoms, yellow-sulfur atoms. (b) 2 D schematic diagram of docking model of compound 9b with Aβ. The figure was prepared using the ligand interactions application in MOE.

Figure 6. Cytotoxicity of compound 4c on human neuroblastoma cells SH-SY5Y. SH-SY5Y cells were incubated with different concentrations of compound 4c (12.5–100 µM) for 24 h. The results are shown as the percentage of viable cells after treatment with compound 4c vs untreated control cells. Date are expressed as mean ± SD from three independent experiments.

Di L, Kerns EH, Fan K, et al. High throughput artificial membrane permeability assay for blood-brain barrier. Eur J Med Chem 2003;38:223–32.

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