The translocator protein ligands as mitochondrial functional modulators for the potential anti-Alzheimer agents

Figures & data

Figure 1. Virtual screening process to identify the best hit compound.

Figure 2. Representative TSPO ligands used as a training set to generate the pharmacophore model.

Figure 3. The ligand-based pharmacophore model mapping with FGIN-1–27 (1). (C atom, black; N atom, blue; O atom, red; F atom, cyan). The pharmacophore features are drawn as colour-coded spheres: H-bond acceptor, green; hydrophobic feature, light blue; and ring aromatic feature, blue.

Table 1. In vitro cell-based assay results of 7 and 8.

Compound	Recovery of ΔΨm (%)^a	ATP Recovery (%)^a	MTT		ROS Inhibition (%)
			Recovery (%)^a	Viability (%)^b
7 (VH034)	78%	42%	28%	95%	97%
8 (VH050)	92%	45%	−184%	34%	−10%
Piracetam	60%	127%	29%	132%	129%
CsA	55%	−46%	61%	90%	ND

^aRecovery percent values were calculated by normalising each measured value against the untreated control; for example, 0% (Aβ-induced damaged condition) and 100% (normal condition in the absence of Aβ).

^bHT22 cell viability after 24 h of the treatment with 5 μM of each compound.

ND: Not determined.

Table 2. In vitro cell-based assay results of 9–20.

Compound	R1	R2	Recovery of ΔΨm (%)	ATP Recovery (%)	MTT		ROS Inhibition (%)
					Recovery (%)	Viability (%)
7 (VH034)			78%	42%	28%	95%	97%
9 (VH057)	OMe	Me	30%	ND	ND	ND	ND
10 (VH058)	OEt	Me	52%	49%	45%	87%	−177%
11 (VH059)	OEt	i-Bu	59%	ND	ND	ND	−234%
12 (VH060)	OEt	n-Bu	1%	ND	ND	ND	ND
13 (VH061)	OMe		66%	14%	ND	ND	ND
14 (VH062)	OMe		77%	122%	92%	120%	136%
15 (VH063)	OEt		86%	31%	59%	139%	−4%
16 (VH064)	OMe		74%	38%	32%	135%	112%
17 (VH065)			72%	−105%	ND	ND	ND
18 (VH066)			53%	−18%	ND	ND	ND
19 (VH067)			73%	−77%	ND	ND	ND
20 (VH068)			50%	−55%	ND	ND	ND
Piracetam			60%	127%	29%	132%	129%
CsA			55%	−46%	61%	90%	NDa

ND: Not determined.

Figure 4. Recovery of Y-maze spontaneous alternations in an acute AD model by the treatment of compounds 7 or 14 (30 mg/kg daily, 6 days, i.p.) Each group was evaluated by comparing with the piracetam-treated group (30 mg/kg daily, 6 days, i.p.). Data are expressed as a mean ± SEM (n = 7 per group): *p < 0.05.

Figure 5. Recovery of Y-maze spontaneous alternations in transgenic (Tg; APPswe/PSEN1dE9 2x TG) AD model mice by the treatment of 7. Each group was evaluated by comparing with the piracetam-treated group (30 mg/kg daily, 1 month, PO). Data are expressed as a mean ± SEM (n = 7 per group): *p < 0.05.

Figure 6. Contextual fear conditioning tests in wildtype and transgenic AD model mice. Each group was evaluated by comparing with the piracetam-treated group (30 mg/kg daily, 1 month, PO). Data are expressed as a mean ± SEM (n = 7 per group): *p < 0.05.

Scheme 1. Synthesis of 5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one derivatives 7, 14, and 27–57. Reagents and conditions: (a) ethyl 2-cyanoacetate, Sulphur, Et₃N, EtOH, reflux, 4 h, 96–100%, (b) 1,1'-thiocarbonylbis(pyridin-2(1H)-one), THF, reflux, 4 h, 80–86%, (c) substituted aniline, EtOH, reflux, 12 h, 84–93%, (d) (i) 10% NaOH/MeOH (1:3), reflux 3 h, (ii) 2 N HCl, 93–98%, (e) 2-bromo-N-substitued acetamide 26, Et₃N, MeCN, reflux, 5 h, 33–98%.

Scheme 2. Synthesis of 5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidin-4(3H)-one derivatives 58–62. Reagents and conditions: (a) substituted amine, EtOH, reflux, 12 h, 57–99%, (b) (i) 10% NaOH/MeOH (1:3), reflux 3 h, (ii) 2 N HCl, 56–96%, (c) 2-bromo-N-(3,4-dimethoxyphenyl)acetamide 26, Et₃N, MeCN, reflux, 5 h, 50–90%.

Table 3. In vitro assay results of compounds 27–47.

Compound	R2	R3	Recovery of ΔΨm	ATP recovery	MTT
					Recovery	Viability
27	H	3,4-difluorophenyl	73%	−15%	−4%	92%
28	H	2-(Piperidin-1-yl)ethyl	78%	−10%	−22%	71%
29	H	3-(Piperidin-1-yl)propyl	69%	−22%	−10%	89%
30	H	1-Methylpiperidin-4-yl	68%	60%	6%	98%
31	H	Benzyl	81%	59%	18%	109%
32	Methyl	Benzyl	83%	7%	8%	88%
33	Ethyl	Benzyl	86%	7%	35%	104%
34	H	4-Chlorobenzyl	82%	40%	20%	97%
35	H	4-Chlorophenyl	82%	38%	18%	107%
36	H	4-Methylbenzyl	82%	35%	2%	96%
37	Methyl	Methyl	45%	ND	ND	ND
38	Ethyl	Ethyl	65%	12%	5%	77%
39	n-Propyl	n-Propyl	79%	15%	26%	99%
40	n-Butyl	n-Butyl	80%	33%	32%	107%
41	H	n-Propyl	51%	ND	ND	ND
42	H	Isopropyl	51%	ND	ND	ND
43	H	n-Butyl	73%	1%	−27%	79%
44	4-Phenylpiperazin-1-yl		83%	63%	9%	97%
45	4-Benzylpiperazin-1-yl		78%	54%	−31%	113%
46	4-Phenylpiperidin-1-yl		82%	80%	28%	86%
47	4-Benzylpiperidin-1-yl		81%	86%	31%	94%
7			73%	42%	28%	95%

ND: Not determined.

Table 4. In vitro assay results of compounds 48–57.

Compound	R1	R2	Recovery of ΔΨm	ATP Recovery	MTT
					Recovery	Viability
48	H	3,4-Dimethoxyphenyl	55%	62%	35%	106%
49	H	Benzyl	61%	36%	42%	99%
50	Methyl	Benzyl	78%	32%	33%	95%
51	Ethyl	Benzyl	74%	22%	39%	107%
52	H	4-Chlorobenzyl	67%	30%	19%	80%
53	H	4-Chlorophenyl	65%	10%	−21%	89%
54	H	4-Methylbenzyl	57%	1%	2%	107%
55	H	4-Methylphenyl	66%	56%	45%	127%
56	n-Propyl	n-Propyl	69%	50%	16%	108%
57	n-Butyl	n-Butyl	73%	50%	66%	110%
7			73%	42%	28%	95%

Table 5. In vitro assay results of compounds 58–62.

Compound	R	Recovery of ΔΨm	ATP Recovery	MTT
				Recovery	Viability
58	Ethyl	60%	23%	4%	99%
59	n-Propyl	79%	−1%	5%	112%
60	Isobutyl	66%	14%	1%	96%
61	n-Butyl	71%	−7%	0%	97%
62	Isopropyl	68%	15%	0%	94%
7		73%	42%	28%	95%

Table 6. CYP450, hERG liability, and human liver microsomal stability profiles of the selected compounds.

Compound	CYP450 (% remaining Activity at 10 μM)					hERG (IC50, μM)	Human Liver Microsomal Stability (% remaining after 30 min)
	CYP1A2	CYP2D6	CYP2C9	CYP3A4	CYP2C19
30	119.7	118.5	93.0	31.0	107.6	12.30 ± 1.25	99.1
31	143.1	88.1	51.1	18.3	70.0	8.80 ± 2.36	32.7
32	117.3	91.7	30.9	49.3	23.3	6.53 ± 2.46	8.1
33	150.5	82.0	32.8	56.6	35.7	2.77 ± 1.18	9.6
34	160.6	92.6	32.9	15.0	56.8	4.13 ± 1.39	0.0
35	120.0	113.8	64.0	70.7	72.0	30.00 ± 10.2	0.1
36	123.6	98.9	50.4	17.3	92.7	22.00 ± 12.7	0.0
39	143.1	95.1	61.0	51.8	64.0	5.87 ± 1.18	0.0
40	103.0	79.8	33.6	38.9	57.4	2.43 ± 0.48	1.5
44	92.0	117.3	84.4	72.2	28.3	8.90 ± 0.22	15.3
45	110.5	107.0	14.9	89.1	23.2	ND	4.3
46	84.4	72.3	6.7	81.2	16.5	ND	6.6
47	81.5	64.0	15.6	66.2	15.0	ND	2.2
48	35.8	57.8	33.7	20.7	20.3	ND	63.4
49	95.3	43.1	99.6	28.1	90.0	15.00 ± 4.24	87.9
50	97.3	30.0	66.4	36.5	47.9	10.60 ± 0.97	73.1
51	98.7	61.4	105.8	44.1	52.5	10.00 ± 3.60	82.0
52	117.2	36.6	86.9	42.6	16.1	8.60 ± 0.83	67.6
55	69.2	59.2	43.0	23.0	31.2	5.63 ± 1.75	49.1
56	71.9	89.2	44.5	30.9	30.5	12.90 ± 3.03	1.3

ND: not determined (due to the low solubility).

Figure 7. Recovery of Y-maze spontaneous alternations in acute AD model mice by treating each compound. Each group was evaluated by comparing with the piracetam-treated group (30 mg/kg daily, 6 days, i.p.). Data are expressed as a mean ± SEM (n = 7 per group): *p < 0.05.

Table 7. Pharmacokinetic parameters (mean ± SD) of compounds 31 and 44 after intravenous (n = 3) and oral (n = 3) administration (10 mg/kg) to SD male rats.^a

	31		44
	Intravenous	Oral	Intravenous	Oral
AUC0–∞ (mg min/mL)	330.66 ± 93.61	26.59 ± 22.32	1070.23 ± 114.39	614.11 ± 120.15
AUClast (mg min/mL)	273.60 ± 64.45	11.67 ± 5.99	1058.16 ± 109.82	487.49 ± 87.29
Terminal half-life (min)	173.83 ± 32.38	448.10 ± 27.60	80.17 ± 9.34	199.34 ± 134.70
Cmax (mg/mL)	–	0.04 ± 0.02	–	2.11 ± 0.91
Tmax (min)	–	48 (30 ∼ 60)	–	72 (60 ∼ 120)
CL (mL/mim/kg)	32.53 ± 10.08	–	9.43 ± 1.05	–
Vss (mL/kg)	7736.8 ± 1114.3	–	785.49 ± 39.64	–
F (%)	8.0		78.4

^aAUC_0–∞, total area under the plasma concentration–time curve from time zero to time infinity; AUC_last, total area under the plasma concentration–time curve from time zero to last measured time; C_max, peak plasma concentration; T_max, time to reach C_max; CL, time-averaged total body clearance; V_ss, apparent volume of distribution at steady state; F, bioavailability.

Table 8. TSPO binding affinity.^a

Compound	KD
7	0.63 ± 0.33 μM
14	1.69 ± 0.40 μM
31	1.25 ± 0.40 μM
44	2.74 ± 0.66 μM
PK11195	1.1 ± 0.45 nM

^aThe K_D values are expressed as the mean ± SD from three independent measurements.

Figure 8. Interactions of 7 and compound 44 with the human TSPO homology model. (A) Surface depiction of the TSPO binding site with the binding orientation overlapped with 7 (red stick) and compound 44 (blue stick). Detailed view of the interactions between 7 (B) and compound 44 (C) with the human TSPO. TSPO is represented with cyan helices and the interacting amino acids are depicted with grey sticks. Corresponding 2D interactions of 7 (D) and compound 44 (E).