2-(4-Fluorophenyl)-1 H-benzo[ d]imidazole as a Promising Template for the Development of Metabolically Robust, α1β2γ2GABA-A Receptor-Positive Allosteric Modulators

Abstract

Modulation of α1β2γ2GABA-A receptor subpopulation expressed in the basal ganglia region is a conceptually novel mode of pharmacological strategy that offers prospects to tackle a variety of neurological dysfunction. Although clinical findings provided compelling evidence for the validity of this strategy, the current chemical space of molecules able to modulate the α1/γ2 interface of the GABA-A receptor is limited to imidazo[1,2-a]pyridine derivatives that undergo rapid biotransformation. In response to a deficiency in the chemical repertoire of GABA-A receptors, we identified a series of 2-(4-fluorophenyl)-1H-benzo[d]imidazoles as positive allosteric modulators (PAMs) with improved metabolic stability and reduced potential for hepatotoxicity, where lead molecules 9 and 23 displayed interesting features in a preliminary investigation. We further disclose that the identified scaffold shows a preference for interaction with the α1/γ2 interface of the GABA-A receptor, delivering several PAMs of the GABA-A receptor. The present work provides useful chemical templates to further explore the therapeutic potential of GABA-A receptor ligands and enriches the chemical space of molecules suitable for the interaction with the α1/γ2 interface.

Keywords: 1H-benzo[d]imidazole; GABA-A receptor; benzodiazepine site; positive allosteric modulator.

Figure 1

Basal ganglia region and a ligand (zolpidem) bound to the GABA-A receptor at the α1/γ2 interface.⁻

Figure 2

Reported positive allosteric modulators (PAMs) of the GABA-A receptor based on the imidazo[1,2-a]pyridine scaffold and their main metabolic sites, conferring high metabolic degradation. GSH—glutathione.

Figure 3

Design concept of a series of 1H-benzo[d]imidazoles toward improved metabolic stability. R = CH₃ or H.

Scheme 1

Reagents and conditions: (a) Na₂S₂O₅, EtOH, H₂O, 70 °C, and 24 h; (b) K₂CO₃, acetone, rt, and 12 h; (c) NaOH, EtOH, H₂O, 70 °C, and 12 h; (d) TBTU, DIPEA, DCM, rt, 5 min then proper amine, 30 °C, 5 min, and microwaves; and (e) CDI, THF, 10 °C, 2 h then proper amine, 40 °C, and 12 h. R₁, R₂ = H, CH₃, R₃ = aliphatic amine, cyclic amine (see Table 1).

Figure 4

Zolpidem (yellow stick) docked to the benzodiazepine binding site of hGABA_A. α1-subunit marked by cyan and the γ2-subunit by green color with interacting amino acids represented by sticks. Red dots present hydrogen bonds and orange presents the aromatic interaction, both CH−π and π–π.

Figure 5

Compounds 10 (left panel) and 9 (right panel) docked to the benzodiazepine binding site of hGABA-A. Ligands represented by yellow sticks. α1-Subunit marked by cyan and γ2 by green colors with interacting amino acids represented by sticks. Red dots present hydrogen bonds and orange presents aromatic interaction, both CH−π and π–π.

Figure 6

Compound 16 docked to the benzodiazepine binding site of hGABA-A. Ligands represented by yellow sticks. α1-Subunit marked by cyan and γ2 by green colors with interacting amino acids represented by sticks. Red dots present hydrogen bonds and orange presents the aromatic interaction, both CH−π and π–π.

Figure 7

MetaSite 6.0.1. Software prediction of the most probable sites of 23 and 9 metabolism.

Figure 8

Hepatotoxicity assays for selected lead molecules. Cell viability (A) was measured using the PrestoBlue reagent, cell membrane damage (B) was assessed using the ToxiLight bioassay, and mitochondrial membrane potential (C) was assessed using a JC-1 probe. Assays performed after 24 h treatment with tested compounds and a reference alpidem. Results are presented as mean % of untreated controls ± S.D. (n = 3). Differences among groups were evaluated by one-way ANOVA followed by post-hoc analysis (Dunnett’s multiple comparison tests) and were considered statistically significant if p < 0.05 (****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05).