Dimethoxybenzohomoadamantane-based soluble epoxide hydrolase inhibitors: in vivo efficacy in a murine model of chemotherapy-induced neuropathic pain

Abstract

The soluble epoxide hydrolase (sEH) has recently emerged as a promising target for the treatment of several pain-related conditions. Herein, we report the design and synthesis of a peripherally restricted sEH inhibitor with high potency and good Drug Metabolism and Pharmacokinetics (DMPK) properties. Molecular dynamics and X-ray crystallography helped reveal the binding of these inhibitors to sEH. The selected compound showed a robust analgesic effect in a dose-dependent manner in a murine model of chemotherapy-induced neuropathic pain (CINP). Moreover, the compound also prevented the development of paclitaxel-induced neuropathic pain. Overall, these results suggest that peripheral inhibition of sEH might constitute a novel therapy to prevent and treat CINP.

Keywords: Benzohomoadamantane; chemotherapy-induced neuropathic pain; epoxyeicosatrienoic acids; soluble epoxide hydrolase; urea.

Graphical abstract

Figure 1.

Structures and IC₅₀ values in the hsEH of clinical candidate EC5026, 1, and compounds 2–5. All compounds were evaluated under the same conditions. The blue circle shows the RHS of 2.

Figure 2.

Benzohomoadamantane amines 6a–g used in this work^,.

Scheme 1.

Synthesis of amine 6g. Reagents and conditions: (i) Ph₃PCH₃ I, NaH, anh. DMSO, 90 °C, overnight, 24% yield. (ii) ClCH₂CN, conc. H₂SO₄, AcOH, 0 °C to room temperature, overnight, 39%. (iii) Diethylaminosulfur trifluoride, DCM, −30 °C to room temperature, overnight, 81% yield. (iv) (1) Thiourea, glacial AcOH, abs. Ethanol, reflux, overnight; (2) HCl/Et₂O, 55% overall yield. See the ‘Experimental’ section and Supplementary Information for further details.

Scheme 2.

Synthesis of the new sEHI. Reagents and conditions: (i) Triphosgene, NaHCO₃, DCM, 30 min; (ii) DCM, overnight; (iii) H₂ 1 atm, PtO₂, ethanol, room temperature, 8 days. See the ‘Experimental’ section and Supplementary Information for further details.

Figure 3.

(A) hsEH active site regions and volume. (B) BenzoH-RHS-1 binding pose. Overlay of most populated conformations of 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. The benzohomoadamantane moiety occupies the RHS pocket, while the piperidine group is placed in the LHS pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. Water molecules are depicted as spheres. Relative binding affinity calculated with respect of BenzoH-RHS-1. (C) BenzoH-LHS-1 binding pose. Overlay of most populated conformations of 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. The benzohomoadamantane moiety occupies the LHS pocket while the piperidine group is placed in the RHS pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. Water molecules are depicted as spheres. (D) BenzoH-RHS-2 binding pose. Overlay of most populated conformations of compound 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. The benzohomoadamantane moiety occupies the RHS pocket, while the piperidine group is placed in the LHS pocket. The central urea unit establishes hydrogen bonds with Asp335, Tyr466, and Tyr383. (E) Rotation and orientation of the benzohomoadamantane moiety represented by the dihedral angle: C (urea moiety), N (urea moiety), C (benzohomoadamantane), C (benzohomoadamantane). Histogram plot of the dihedral angle that describes the rotation of the benzohomoadamantane moiety along the MD simulations of BenzoH-RHS-1 (blue), BenzoH-LHS-1 (grey), and BenzoH-RHS-2 (purple). (F) Water occupation in the sEH active site. Representation of the normalised kernel density plot of the water distribution in the vicinity of methoxy groups (<4 Å) along the MD simulations of BenzoH-RHS-1 (blue), BenzoH-LHS-1 (grey), and BenzoH-RHS-2 (purple). PDB 6I5E was used as starting point for molecular modelling.

Figure 4.

X-ray crystal structures of 20 in complex with hsEH CTD (9F1A). (A) Crystallographic symmetry. The crystallographic homodimeric structure (comprising chain A and chain B) is shown as cartoon with the catalytic pocket being presented as surface (cyan). The 20 ligand is shown as sticks (orange). (B–D) Interactions of 20 with hsEH CTD in conformation 1 (B), 2 (C), and 3 (D) on chain A. Residues involved in hydrogen bonding in all conformations are shown in cyan, while residues participating in hydrophobic or van der Waals contacts in all conformations are coloured in purple. Residues stabilising specific conformations (1, 2, or 3) are represented with green sticks. Hydrogen bonds are displayed as yellow dotted lines. (E,F) Superpositions of different conformations of ligand 20 in chain A (E) and chain B (F). The two epoxide positioners Y383 and Y466 and the catalytic triad residue D335 are in white. Ligand conformations 1, 2, and 3 are coloured in pink, magenta, and brown, respectively.

Figure 5.

Effect of the subcutaneous (s.c.) compound 21 in paclitaxel (PTX)-induced neuropathic pain. Threshold force for paw withdrawal (g) was tested as a measure of PTX-induced sensory hypersensitivity. Baseline sensitivity to mechanical stimulation was evaluated (PRE), and then animals were treated once daily for 5 days with PTX (2 mg/kg, intraperitoneal) or its vehicle (Vh). (A) Time-course of the effect of a single s.c. administration of 21 (2.5 and 5 mg/kg) or its solvent (5% DMSO), 10 days after the first PTX administration (n = 8 animals in all groups except in the group treated with PTX and 5% DMSO, which had n = 10). (B) Effect of the administration of 21 (5 mg/kg, s.c.) or gabapentin (GBP) associated with MSPPOH (20 mg/kg, s.c.) or their solvent (5% DMSO) at day 10 after PTX (n = 8, 10, 8, 8, 10, 8, 8 from left to right in the bars represented in the figure). (C) Time-course of the effect of PTX associated with 21 (5 mg/kg, s.c.) or its solvent 30 min before each PTX administration (n = 8 animals in each group). (A–C) Each point or bar and vertical line represents the mean ± SEM of the values obtained in the mice tested. Statistically significant differences between values from animals treated with PTX vs Vh: *p < 0.05; and 21 vs 5% DMSO: ^#p < 0.05. (B) 21 associated with MS-PPOH or its solvent: ^†p < 0.05. (A,C) Two-way repeated measures ANOVA. (B) One-way ANOVA. Tukey post hoc test was used in all cases.