Novel tacrine–benzofuran hybrids as potential multi-target drug candidates for the treatment of Alzheimer’s Disease

Figures & data

Figure 1. General structure of the tacrine-benzofuran (TAC-BF) hybrids under study.

Figure 2. Molecular modelling of four representative compounds inside the binding site of AChE, superimposed with the original ligand (N-4′-quinolyl-N′-9″-(1″,2″,3″,4″-tetrahydroacridinyl)-1,8-diaminooctane), green Coloured. These compounds have the same TAC substituent (R = Cl), but different BF substituent (R₁) and spacer length: 15 (turquoise Coloured; n = 1; R₁ = H), 19 (dark blue Coloured; n = 1; R₁ = -OCH₃), 23 (yellow Coloured; n = 1; R₁= -OH), 24 (orange Coloured; n = 2; R₁ = H).

Scheme 1. Synthesis of TAC-BF hybrids. Reagents and conditions: (a) cyclohexanone, POCl₃, 180 °C, 3 h; (b) DMF, K₂CO₃ (1.2 eq), ethylbromoacetate (1.05 eq), 135-140 °C, 5–6 h; (c) diaminoalkane (3.0 eq), dry MeOH, overnight; (d) phenol, KI, 165–170 °C 35–60 min; (e) BCl₃, TBAI, DCM, under N₂ (−78 °C), 2 h.

Figure 3. Species distribution curves for compound 21 along with molar extinction coefficients at the maximum absorption wavelengths (C_L = 4.0 × 1 0 ⁻⁵M).

Table 1. Stepwise protonation constants for the ligand 21 and global formation constants^a for the corresponding Fe(III) and Cu(II)) complexes (T = 25.0 ± 0.1 °C, I = 0.1 M KCl, 25% w/w DMSO/water) as well as pM^b values.

Compound	MmHhLl	log Ki	log ${\beta }_{{\text{Fe}}_{\mathrm{m}}{\mathrm{H}}_{\mathrm{h}}{\mathrm{L}}_{\mathrm{l}}}$	log ${\beta }_{{\text{Cu}}_{\mathrm{m}}{\mathrm{H}}_{\mathrm{h}}{\mathrm{L}}_{\mathrm{l}}}$
	(011)	9.01(1)
	(021)	7.99(3)
	(101)		–	10.87(6)
	(111)		22.90(6)	19.02(2)
	(102)		–	20.68(8)
	(112)		38.37(6)	–
	(103)		36.95(7)	–
	(1–11)		–	2.25(3)
	(1–22)		12.21(8)	-
	pM		22.2	11.9
	(011)	9.02(3)
	(021)	8.18(5)
	(031)	3.32(6)
	(111)		–	17.27(5)
	(122)			34.38(6)
	(102)			20.14(8)
	pM			11.1

^a ${\beta }_{{\mathrm{M}}_{\mathrm{m}}{\mathrm{H}}_{\mathrm{h}}{\mathrm{L}}_{\mathrm{l}}}$= [M_mH_hL_l]/[M]^m[H]^h[L]^l.

^b pM = −log[M] at pH 7.4 (C_L/C_M = 10, C_M = 1 0 ⁻⁶M).

^c In 50/50 w/w DMSO/water³¹.

Figure 4. Species distribution curves for the systems (a) Fe(III)/21 1:3 and (b) Cu(II)/21 1:2 (C_L = 4.0 × 1 0 ⁻⁵M).

Figure 5. Graphical summary of the effect of different structural parameters on the inhibition of AChE (AChEi, IC₅₀) and on the inhibition of Aβ aggregation (%): (A) size of alkylchain linker in AChEi: n = 1 (propylic chain, n = 2 (butylic chain); (B) substituents at C6 of TAC (R = H, Cl) in AChEi; (C) substituents at C7 of BF (R₁ = H, -OCH₃, -OH) in AChEi; (D) inhibition of Aβ aggregation (self- and Cu-induced).

Table 2. Summary of results for the biological assays of TAC-BF hybrids

	n	R	R1	AChE inhibition^a IC50 (µM)	Aβ aggregation inhibition^b,c (%)
					Self-Aβ aggr	Cu-ind Aβ aggr
13	1	H	H	0.58	60.6	–
14	2	H	H	0.72	51.5	–
15	1	Cl	H	0.12	51.0	–
16	2	Cl	H	0.34	43.0	–
17	1	H	OCH3	0.36	57.5	–
18	2	H	OCH3	0.76	51.9	–
19	1	Cl	OCH3	0.13	57.8	–
20	2	Cl	OCH3	0.67	64.4	–
21	1	H	OH	0.81	44.0	50.1
22	2	H	OH	0.98	30.4	54.0
23	1	Cl	OH	0.13	31.5	52.5
24	2	Cl	OH	0.61	43.4	53.4
TAC	–	–	–	0.35	20	–

^a The values are mean of five independent experiments ± SD; AChE from Electric eel.

^b Inhibition of self-mediated Aβ₄₂ aggregation (%) with or without copper (40 µM). The thioflavin-T fluorescence method was used, and the measurements were carried out in the presence of an inhibitor (80 µM).

^c The values are the mean of two independent measurements in duplicate (SEM < 10%).

Figure 6. TEM images of Aβ aggregation inhibition performed with samples incubated (37 °C) for 24 h. Experimental conditions: [Aβ_1–42] = [CuCl₂] = 25 μM; [21] = 50 μM; pH = 6.6.

Table 3. Summary of calculated pharmacokinetic molecular descriptors for the new hybrids by QikProp v.2.540³⁹

Comp. Ref.	(n,R,R1)	MW^a	clog P^b	log BB^c	Caco-2 Permeability^d (nm/sec)	Violations of Lipinski's rule of 5^e	CNS activity^f
13	(1,H,H)	399.491	5.143	−0.690	1764	1	+/−
14	(2,H,H)	413.518	5.106	−0.488	2173	1	+/−
15	(1,Cl,H)	433.936	5.685	−0.396	2149	1	+/−
16	(2,Cl,H)	447.963	6.039	−0.559	1893	1	+/−
17	(1,H,OCH3)	429.518	5.327	−0.573	2423	1	+/−
18	(2,H,OCH3)	443.544	5.745	−0.802	1867	1	–
19	(1,Cl,OCH3)	463.963	5.852	−0.431	2324	1	+/−
20	(2,Cl,OCH3)	477.989	5.907	−0.597	1890	1	+/−
21	(1,H,OH)	415.491	4.567	−1.522	460	0	–
22	(2,Cl,OH)	429.518	4.755	−1.466	520	0	–
23	(1,Cl,OH)	449.936	4.764	−1.261	469	0	–
24	(2,Cl,OH)	463.963	5.208	−1.324	493	1	–

^a MW < 500.

^b −2 < clog P < −6.5.

^c −3.0 < log BB < 1.2.

^d <25 poor and >500 great.

^e Maximum is 4.

^f (–) inactive toxicologically – (++) very active toxicologically³⁹.

Figure 7. Dose-response screening to select non-toxic concentrations of TAC-BF hybrids. Cells were treated with varying concentration of TAC-BF conjugates (from 10 μM to 40 μM) for 25 h and cell viability was determined using the MTT reduction assay. Results are expressed relatively to SH-SY5Y untreated cells, with the mean ± SEM derived from three different experiments. *p < 0.05; **p < 0.01, significantly different when compared with SH-SY5Y untreated cells.

Figure 8. Neuroprotective effect of TAC-BF hybrids from Aβ₄₂-induced toxicity on SH-SY5Y cells. Cells were treated with Aβ₄₂ peptide (2.5 μM) for 24 h in the absence or in the presence of the compounds (1 h pre-incubation + 24 h co-incubation). Evaluation of cell viability was performed using the MTT reduction assay. Results are expressed relatively to SH-SY5Y untreated cells, with the mean ± SEM derived from 4 different experiments. *p < 0.05, significantly different when compared with SH-SY5Y untreated cells; #p < 0.05, significantly different when compared with Aβ₄₂ treated SH-SY5Y cells. (compounds: 18, 19, 20 and 23 – 20 µM; 21, 24 – 35 µM).

Figure 9. Neuroprotective effect of TAC-BF conjugates against L-Ascorbic Acid (AscH(-))/Ferrous Sulphate (Fe) toxicity on SH-SY5Y cells. Cells were treated with Asc/Fe (5 mM/500 μM, respectively) for 24 h, after treatment for 1 h in the absence or in the presence of the compounds. Evaluation of cell viability was performed by using MTT reduction assay. Results are expressed relatively to SH-SY5Y untreated cells, with the mean ± SEM derived from 4 different experiments. **p < 0.01, significantly different when compared with SH-SY5Y untreated cells. #p < 0.05, significantly different when compared with Asc/Fe treated SH-SY5Y cells. (compounds: 18, 19, 20 and 23 – 20 µM; 21, 24 – 35 µM).

Chaves S, Hiremathad A, Tomás D, el al. Exploring the chelating capacity of 2-hydroxyphenylbenzimidazole based hybrids with multi-target ability as anti-Alzheimer’s agents. New J Chem 2018;42:16503–15.

 Web of Science ®Google Scholar

QikProp, version 2.5, Schrödinger. New York: LLC; 2005.

 Google Scholar