New cyclopentaquinoline and 3,5-dichlorobenzoic acid hybrids with neuroprotection against oxidative stress for the treatment of Alzheimer’s disease

Figures & data

Figure 1. The idea of yeast three-hybrid (Y3H) system [31]. Components: I. Hybrid ligand composed of ligand 1 (L1), linker and ligand 2 (L2) II. First hybrid protein comprised of protein binding L1 and activating domain (AD) of yeast GAL4 transcription factor. III. Second hybrid protein comprised of protein binding L2 and DNA-binding domain (DBD) of yeast GAL4 transcription factor.

Table 1. Substances used to perform calibration curve.

Substance name	Experimental properties
	logP^53	pKa
Procainamide HCl	0.88	9.32^54
Tacrine	2.71	9.95^55
Thymol	3.30	–
Naphthalene	3.60	–
Promazine HCl	4.55	9.36^54
Promethazine HCl	4.81	9.10^54
Chlorpromazine HCl	5.41	9.30^53
Thioridazine HCl	5.90	9.50^56

Scheme 1. Synthesis of compounds 2a–2h and 3a–3h. Reagents: (a) 3,5-dichlorobenzoic acid, 2-Chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-methylmorpholine, THF; (b) HCl/ether.

Table 2. The activity of novel compounds 3a–3h against acetylcholinesterase from electric eel and equine butyrylcholinesterase.

Compound	AChE IC50 ± SD (µM)^a	BuChE IC50 ± SD (µM)^b	Selectivity for AChE^c	Selectivity for BuChE^d
3a	1.545 ± 0.121	0.225 ± 0.021	0.15	6.88
3b	1.179 ± 0.134	0.889 ± 0.027	0.56	1.78
3c	1.155 ± 0.130	0.126 ± 0.007	0.11	9.18
3d	0.211 ± 0.031	1.123 ± 0.180	5.33	0.19
3e	0.131 ± 0.016	0.116 ± 0.006	0.89	1.13
3f	0.253 ± 0.042	0.117 ± 0.027	0.46	2.16
3g	1.485 ± 0.133	0.330 ± 0.034	0.22	4.50
3h	0.145 ± 0.013	0.131 ± 0.011	0.91	1.10
Tacrine	0.118 ± 0.016	0.016 ± 0.002	0.14	7.38
Donepezil	0.123 ± 0.008	5.869 ± 0.379	47.72	0.02

^aInhibitor concentration (means ± SD of three experiments) for 50% inactivation of AChE. ^bInhibitor concentration (means ± SD of three experiments) for 50% inactivation of BuChE. ^cSelectivity for AChE is defined as IC₅₀(BuChE)/IC₅₀(AChE). ^dSelectivity for BuChE is defined as IC₅₀(AChE)/IC₅₀(BuChE).

Table 3. SH-SY5Y cell viability (%) after treatment with H₂O₂ (100 µM) or mixture of rotenone (30 µM) and oligomycin A (10 µM) at selected concentrations of 3e.

			Concentrations
	Cells without compounds	Compound	10 µM	1 µM	0.1 µM	0.01 µM
H2O2 100 µM	% cell viability
	84.27 ± 0.03	3e	41.11 ± 3.60	95.44 ± 3.09	96.75 ± 4.23	90.57 ± 7.65
		Trolox	99.22 ± 3.30	99.38 ± 9.81	98.62 ± 1.36	96.41 ± 1.51
			% neuroprotection
		3e		71	79.34	40.04
		Trolox	95.05	96.03	91.22	77.16
			0.1 µM	0.01 µM	0.001 µM	0.0001 µM
Rotenone/Oligomycin A 30/10 µM	% cell viability
	46.77 ± 2.17	Pre-incubation 3e	48.05 ± 1.73	50.84 ± 7.02	53.40 ± 7.03	56.15 ± 6.55
		Pre-incubation Trolox	44.26 ± 3.73	46.38 ± 2.06	47.91 ± 4.48	46.23 ± 3.65
	47.91 ± 4.02	Co-incubation 3e	46.76 ± 6.52	49.38 ± 4.59	52.43 ± 6.60	54.55 ± 6.72
		Co-incubation Trolox	48.09 ± 8.01	50.34 ± 8.11	50.83 ± 6.98	42.11 ± 6.28
			% neuroprotection
		Pre-incubation 3e	2.41	7.65	12.46	17.64
		Pre-incubation Trolox	–	–	2.15	–
		Co-incubation 3e	–	2.82	8.67	12.75
		Co-incubation Trolox	0.35	4.67	5.60	–

Cell viability was determined by the MTT test. The results were expressed as mean ± SD. The experiment was performed in quadruplicate and repeated three times.

Figure 2. Acute oral toxicity stages. ^a – Moribund or dead; ^b – GHS (Globally Harmonised Classification System mg/kg body weight); ^c – LD₅₀ cut-off mg/kg body weight.

Table 4. LD50 and toxicity class prediction of tacrine, bis-7-tacrine and new hybrids with 3,5-dichlorocarboxylic acid.

Compound	Predicted LD50 (mg/kg)	Predicted toxicity class
2a	2100	5
2b	200	3
2c	400	4
2d	400	4
2e	400	4
2f	400	4
2g	400	4
2h	400	4
Tacrine	40	2
Bis-7-tacrine	400	4

The prediction was performed with ProTox-II (http://tox.charite.de/protox_II), a webserver for the prediction of small molecules toxicity⁵³.

Table 5. Inhibition of β-amyloid (Aβ) aggregation by compound 3e at different concentrations. Thioflavin T assay (λ_exc = 446 nm; λ_em = 490 nm).

Concentration of 3e compound (µM)	Inhibition of Aβ(1-42) aggregation (%)
5	55.7 ± 4.7
10	62.7 ± 5.3
25	76.5 ± 3.7
50	79.8 ± 2.9
100	82.6 ± 1.7

Figure 3. Scatterplots of equation results including a regression line, marked as the continuous line. The dashed lines determine the area of the regression belt at a confidence level of 0.95. (A) Plot pH vs pH-log(Ax-Aa)/(Ab-Ax) 332/343 nm; (B) plot pH vs pH-log(Ax-Aa)/(Ab-Ax) 343/332 nm; (C) plot pH vs pH-log(Ax-Aa)/(Ab-Ax) 332/343 nm; (D) plot pH vs pH-log(Ax-Aa)/(Ab-Ax) 343/332 nm.

Table 6. Experimental and predicted pKa and logP values of 3e.

	Experimental	ChemAxon	ACD/Percepta
pKa1	8.40	8.89	9.17
pKa2	11.26	14.16	13.86
logP	5.438	5.80	6.04

Figure 4. Calibration curve for LogP assay. The regression line is marked as a continuous line. Dashed lines determine the area of the regression belt at a confidence level of 0.95.

Figure 5. Binding mode of compound 2e within acetylcholinesterase (A) and butyrylcholinesterase (B).

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