A new serotonin 5-HT6 receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding

Abstract

Serotonin 5-HT₆ receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer's disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT₆ receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT₆ receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT₆ receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT_2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.

Figure 1. Benzimidazole-based compounds targeting the serotonin 5-HT₆R receptor.

(A) Pharmacophore elements for antagonists are a positive ionisable atom (PI, in red), an aromatic ring (AR, in yellow), a hydrogen bond acceptor group (HBA, in green), and a hydrophobic site (HYD, in blue). (B) Previously reported antagonist with benzimidazole ring as central AR core. (C,D) New series of compounds with the key structural elements anchored to the benzimidazole ring.

Figure 2. 5-HT₆R affinities of new compounds 2–19.

^aValues are the mean of two to four experiments performed in triplicate. ^b5-HT (K_i = 71 ± 6 nM) and SB-258585 (K_i = 4.5 ± 0.2 nM) were used as reference compounds.

Figure 3. Synthesis of target compounds 2–19.

Reagents and conditions: (a) pyridine, dichloromethane, rt, 15 h (2–6, 8–12, 14–19); (b) NaHCO₃, acetonitrile, rt, 22 h (7, 13).

Figure 4. Synthesis of aminobenzimidazoles 20–22.

Reagents and conditions: (a) i. Cl(CH₂)₂N(CH₃)₂·HCl, K₂CO₃, NaI, DMF, 60 °C, 15 h; ii. column chromatography (SiO₂, EtOAc/MeOH 98:2), 23: 33%, 24: 30%; (b) H₂, 10% Pd(C), MeOH, rt, 15 h, quantitative; (c) H₂N(CH₂)₂N(CH₃)₂, K₂CO₃, DMF, 90 °C, 12 h, 60–78%; (d) HCOOH, H₂O, reflux, 3 h, 92%; (e) 10% Pd(C), HCOOH, MeOH, reflux, 20 h, 69%; (f) 4 M H₂SO₄, THF, 50 °C, 1 h, quantitative.

Figure 5. Homology models and site-directed mutagenesis define the binding mode to the 5-HT₆R.

(A,C) Detailed view of the binding modes of compounds 7 (panel A) and 10 (panel C) in the energy-minimized computational models of the 5-HT₆R. The amino acids of the receptor (labeled according to Ballesteros GPCR nomenclature) are shown in green and compounds 7 and 10 in white sticks. The pharmacophore elements PI, AR, HBA, and HYD are labeled in panel C according to Fig. 1. Chlorine atoms (purple spheres) of compounds 7 and 10 halogen bond the carbonyl group at position 4.56 (i-4 relative to Pro 4.60, red dashed lines). (B) The antagonist effect (pIC₅₀ values are shown within the parenthesis) of compound 7 is represented as the percentage of inhibition of 5-HT-induced stimulation of cAMP taken as 100%, in WT and mutant receptors. SB-258585 (pIC₅₀ = 7.6 ± 0.1, dashed line) was assayed in WT as a reference compound. Values are represented as mean ± SEM of 3–5 independent experiments each performed in triplicate. (D) Main chain hydrogen bond network on the extracellular side of TM 4 in the computational model of the 5-HT₆R (left) and crystal structures of 5-HT_1BR (PDB ID 4IAR, middle) and 5-HT_2BR (PDB ID 4IB4, right). Exposed carbonyl oxygens (small spheres marked with an asterisk) in TM 4 are free to interact with halogen atoms. (E) Extension of our previously reported pharmacophore model for 5-HT₆R antagonists, and the predicted interacting amino acids in the TMs of the 5-HT₆R.

Figure 6. Procognitive activity tested in the novel object recognition task.

Effect of compound 7 (1 mg/kg, ip) and cognitive enhancer tacrine (0.5 mg/kg, po) on time-induced memory deficit tested in the NORT in rats. Data are means ± SEM of exploration times; *P < 0.05 and ***P < 0.001 (paired Student’s t test) indicate significant differences of time spent exploring the novel vs familiar objects. N = 8–10 animals/group.