Synthesis, In Vitro Profiling, and In Vivo Evaluation of Benzohomoadamantane-Based Ureas for Visceral Pain: A New Indication for Soluble Epoxide Hydrolase Inhibitors

Abstract

The soluble epoxide hydrolase (sEH) has been suggested as a pharmacological target for the treatment of several diseases, including pain-related disorders. Herein, we report further medicinal chemistry around new benzohomoadamantane-based sEH inhibitors (sEHI) in order to improve the drug metabolism and pharmacokinetics properties of a previous hit. After an extensive in vitro screening cascade, molecular modeling, and in vivo pharmacokinetics studies, two candidates were evaluated in vivo in a murine model of capsaicin-induced allodynia. The two compounds showed an anti-allodynic effect in a dose-dependent manner. Moreover, the most potent compound presented robust analgesic efficacy in the cyclophosphamide-induced murine model of cystitis, a well-established model of visceral pain. Overall, these results suggest painful bladder syndrome as a new possible indication for sEHI, opening a new range of applications for them in the visceral pain field.

Figure 1

Simplified AA cascade.

Figure 2

Structures and IC₅₀ values in the human sEH of AR9281, 1, t-AUCB, 2, EC5026, 3, t-TUCB, 4, 5, 6, and 7 and general structure, I, of the new derivatives reported on this work.

Figure 3

Benzohomoadamantane amines 8a–g used in this work.

Scheme 1. Synthesis of the New sEHI

Reagents and conditions: (a) triphosgene, NaHCO₃, DCM, 30 min; (b) DCM, overnight; and (c) n-BuLi, anhyd THF, anhyd DCM, overnight. See the Experimental Section and Supporting Information for further details.

Figure 4

(a) Representative structure of compound 21 bound in the active site of sEH obtained from the most visited conformations along MD simulations. PDB ID 5AM3 has been used as the starting point for 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. (b) Most relevant molecular interactions in the rhs. Average distances (in Å) obtained from three replicas of 500 ns of MD simulations are represented. Hydrogen bonds between the oxygens of the tetrahydropyran group of 21 and the hydrogen of the OH group of Ser415 is shown. The hydrophobic interaction average distances are computed between the terminal heavy atom of amino acid side chains and the centroid of each ring. Hydrogen bond distances between the carboxylic group of the catalytic Asp335 and the amide groups of the inhibitor and the distance between the carbonyl group of the urea inhibitor and the OH group of Tyr383 and Tyr466 residues. (c) Most relevant molecular interactions in the lhs. Average distances (in Å) obtained from the three replicas of 500 ns of MD simulations are represented. The CH−π interaction is calculated between the hydrogens of the benzohomoadamantane unit and the centroid of the benzoid ring of Trp336. The NH−π interaction is monitored between the amide hydrogen of Gln384 and the center of the aromatic ring of the benzohomoadamantane scaffold.

Scheme 2. Synthesis of the Probe 28

See the Experimental Section for details.

Figure 5

Target engagement and off-target profile of 28 in HEK293T cell lysates. (a) Fluorescence scan showing probe 28 labeling pattern in lysates, purified EPHX2- and EPHX2-spiked lysates, revealing that EPHX2 is the only target visibly outcompeted by the parent compound 15 and hence, the only target with high occupancy (Coomassie-stained gel in Figure S11). (b) Western blot analysis of selected proteins further confirms EPHX2 target engagement by 28 and proves that neither EPHX1, MAPK38, nor VEGFR are targeted by this compound. Compound 28 used at 10 μM and compound 15 used at 100 μM.

Figure 6

Reduction of capsaicin-induced secondary mechanical hypersensitivity in mice by the systemic administration of AS2586114, and compounds 15 and 21, is due to sEH inhibition. The data shown represent the effect of the sc administration of AS2586114, 15, and 21 administered alone or associated with the CYP450 oxidase inhibitor MS-PPOH (sc) on paw withdrawal latency in mice-treated intra-plantarly (i.pl.) with capsaicin. Each bar and vertical line represent the mean ± SEM of the values obtained in 8–10 animals. Statistically significant differences: **p < 0.01 between nonsensitized mice (open bar) and the other experimental groups; #p < 0.05 and ##p < 0.01 between capsaicin-treated mice injected with the sEHI or their solvent (black bar); ++p < 0.01 sEHI-treated mice associated or not with MS-PPOH (one-way ANOVA followed by Student–Newman–Keuls test).

Figure 7

Effects of compound 21 on pain-related behaviors and referred mechanical hyperalgesia induced by CTX. (a) Behavioral score was recorded at 30 min intervals over the 150–240 min observation period after the intraperitoneal (ip) injection of (CTX, 300 mg/kg) or its vehicle. (b) 50% mechanical threshold was evaluated by stimulation of the abdomen with von Frey filaments at 240 min after the administration CTX or its vehicle and was used as an index of referred hyperalgesia. Each bar and vertical line represents the mean ± SEM of values obtained in at least six animals per group. Statistically significant differences: **p < 0.01, between nonsensitized mice (open bar) and the other experimental groups; #p < 0.05, ##p < 0.01 between CTX-treated mice injected with the sEHI or their solvent (black bar); ++p < 0.01 mice injected with compound 21 associated or not with MS-PPOH (one-way ANOVA followed by Student–Newman–Keuls test).