Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer's Disease

Abstract

Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer's disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I₂ receptors (I₂-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I₂-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I₂-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I₂-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson's disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I₂-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration.

Keywords: 2-(benzo[b]thiophen-2-yl)-1H-imidazole; 3D-QSAR; 5XFAD; Alzheimer’s disease; imidazoline I2 receptor ligand; imidazoline-linked heterocycle; neuroprotection.

Figure 1

(A) Structure of clinical candidates CR4056 and [¹¹C]BU99008. (B) Structure of standard I₂-IR ligands BU224, idazoxan, and 2-BFI (C) Structure of compounds LSL42, LSL39, LSL60101, and LSL33 (D) Structure of compounds LSL35, LSL34, LSL29, and LSL33 The aromatization, isomerism, and bioisoster relationships are indicated.

Scheme 1

Condition reactions and yields for the synthesis of LSL42, LSL39, and LSL23 (i) Et₂O/HCl 2 M (0.25 mmol/mL), MeOH, 4 °C, 48 h, 2a, 77%; 2b, 94%; 2c, 85% yield, respectively. (ii) 2,2-Dimethoxyethylamine (1.1 equiv.), MeOH, 60 °C, 16 h, quantitative for 3a, 3b, and 3c. (iii) 2 M HCl (0.1 mmol/mL), 60 °C, 16 h, LSL42, 58%; LSL39, 64%; LSL33, 81% yield, respectively.

Scheme 2

Condition reactions and yields for the synthesis of LSL35, LSL34, and LSL29 (i) 2-Bromoquinoline (0.5 mmol), imidazole (0.25 mmol), HOBt (0.025 mmol), CuI (0.013 mmol), KOtBu (0.38 mmol), DMSO, reflux, 24 h, LSL35, 48% yield. (ii) 2-Bromobenzofurane (1 mmol), imidazole (1.5 mmol), CuI (0.2 mmol), Cs₂CO₃ (2 mmol), acetonitrile, reflux, 24 h, LSL34, 55% yield. (iii) Indole (0.5 mmol), imidazole (4 mmol), HCO₂NH₄, I₂ (1.25 mmol), dioxane, rt, 24 h, LSL29, 32% yield.

Figure 2

Favourable Var285: DRY-N1 and favourable Var408: O-N1 of compound LSL33 ((A), 3D-QSAR (I₂-IR) model); favourable Var280: DRY-N1 of LSL33 ((B), 3D-QSAR (α₂-AR) model); the hydrophobic regions (DRY) are labelled in yellow, H-bond donor regions in red, and H-bond acceptor regions in blue.

Figure 3

Analysis of the cytotoxicity of I₂-IR ligands in vitro. HT-22 cells, derived from the murine hippocampus, and SH-SY5Y cells, representing human dopaminergic neurons, were exposed to escalating concentrations of the specified compounds for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) test was employed to assess the cytotoxic potential of the compounds. The reported values represent the mean ± SD obtained from triplicate determinations repeated a minimum of three times.

Figure 4

In vitro screening of I₂-IR ligands: Assessment of neuroprotective and anti-inflammatory effects HT-22 cells exposed to 10mM glutamate for at least 16 h Similarly, SH-SY5Y was damaged with 35 μM 6-OHDA (35 μM). Some cultures were pretreated with the I₂-IR ligands. Cell viability was evaluated using the MTT assay, and nitrite production in the cell supernatant was quantified using the Griess reaction. The reported values represent the mean ± SD obtained from triplicate determinations repeated at least three times. Statistical analysis was performed: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (versus glutamate- or 6-OHDA-treated cultures); ^# p ≤ 0.05, ^### p ≤ 0.001 (versus idazoxan-treated cultures); ^& p ≤ 0.1, ^&&& p ≤ 0.001 (versus 2-BFI-treated cultures).

Figure 5

Role of I₂-IR ligands in an in vitro model of neuroinflammation. Nitrite production in the supernatant of glial cells was assessed using the Griess reaction after a 24 h exposure to lipopolysaccharide (LPS, 10 μg/mL) in the presence of the indicated I₂-IR ligands at a concentration of 1 μM. The values presented represent the mean ± SD from triplicate determinations, repeated at least three times. Statistical analysis revealed significant differences: * p ≤ 0.05 ** p ≤ 0.01, *** p ≤ 0.001 compared to LPS-treated cultures; ^# p ≤ 0.05, ^### p ≤ 0.001 compared to idazoxan-treated cultures; ^&& p ≤ 0.01, ^&&& p ≤ 0.001 compared to 2-BFI-treated cultures.

Figure 6

Results from cognition assays (A,B) Discrimination index of NORT of short- and long-term memories obtained in 7-month-old wild-type (WT) and 5XFAD mice, non-treated and treated for 4 weeks with LSL33 at 2 mg/Kg, respectively. (C) OLT obtained in 7-month-old wild-type (WT) and 5XFAD mice, non-treated and treated for 4 weeks with LSL33 at 2 mg/Kg, respectively. The data correspond to the mean ± SEM (n = 10–12 mice per group). Two-way ANOVA, Bonferroni’s post hoc test * p < 0.05, ** p < 0.01. ns: no significant.

Figure 7

Results from an in vivo study of synaptic plasticity markers. (A) Representative protein levels and quantification by WB for PSD95. (B,C) Representative gene expression for Ngf and Bdnf Gene expression levels were determined by real-time PCR. The data correspond to the mean ± SEM (n = 3–5 mice per group). Two-way ANOVA and Bonferroni’s post hoc test; * p < 0.05.

Figure 8

Results from an in vivo study Neuroinflammatory markers. Representative gene expression for Tnf-a and Il-6 Gene expression levels were determined by real-time PCR. The data correspond to the mean ± SEM (n = 3–5 mice per group). Two-way ANOVA, Bonferroni’s post hoc test. * p < 0.05, ** p < 0.001.