Further exploration of the structure-activity relationship of dual soluble epoxide hydrolase/fatty acid amide hydrolase inhibitors

Abstract

Fatty acid amide hydrolase (FAAH) is a membrane protein that hydrolyzes endocannabinoids, and its inhibition produces analgesic and anti-inflammatory effects. The soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids. EETs have anti-inflammatory and inflammation resolving properties, thus inhibition of sEH consequently reduces inflammation. Concurrent inhibition of both enzymes may represent a novel approach in the treatment of chronic pain. Drugs with multiple targets can provide a superior therapeutic effect and a decrease in side effects compared to ligands with single targets. Previously, microwave-assisted methodologies were employed to synthesize libraries of benzothiazole analogs from which high affinity dual inhibitors (e.g. 3, sEH IC₅₀ = 9.6 nM; FAAH IC₅₀ = 7 nM) were identified. Here, our structure-activity relationship studies revealed that the 4-phenylthiazole moiety is well tolerated by both enzymes, producing excellent inhibition potencies in the low nanomolar range (e.g. 6o, sEH IC₅₀ = 2.5 nM; FAAH IC₅₀ = 9.8 nM). Docking experiments show that the new class of dual inhibitors bind within the catalytic sites of both enzymes. Prediction of several pharmacokinetic/pharmacodynamic properties suggest that these new dual inhibitors are good candidates for further in vivo evaluation. Finally, dual inhibitor 3 was tested in the Formalin Test, a rat model of acute inflammatory pain. The data indicate that 3 produces antinociception against the inflammatory phase of the Formalin Test in vivo and is metabolically stable following intraperitoneal administration in male rats. Further, antinociception produced by 3 is comparable to that of ketoprofen, a traditional nonsteroidal anti-inflammatory drug. The results presented here will help toward the long-term goal of developing novel non-opioid therapeutics for pain management.

Keywords: 4-Phenylthiazole moiety; ADMET predictions; Docking experiments; Enzyme inhibition; Formalin test; Microwave-assisted synthesis; Polypharmacology; Structure-Activity Relationship study.

Figure 1.

Metabolic pathways of enzymes fatty acid amide hydrolase and soluble epoxide hydrolase and interactions with arachidonic acid.

Figure 2.

Design strategy used to optimize new dual FAAH/sEH inhibitors. Key pharmacophoric features required to interact with both targets are merged in one united pharmacophore (shown in red box). The site of interest where SAR is performed in this study is shown in blue box.

Figure 3A.

Binding of 6o in human FAAH active site (2D representation): green shading represents hydrophobic regions; gray parabolas represent accessible surfaces for large areas; gray dotted lines represent hydrogen bonds; broken thick line around 6o shape indicates accessible surfaces; size of residue ellipse represents the strength of the contact.

Figure 3B.

Binding of 6o in human FAAH active site (3D representation): Important amino acid residues in the proximity of 6o are shown and labeled. Hydrogen bond with G485 is shown in green with the distance in Å.

Figure 4A.

Binding of 6o in human sEH active site (2D representation): green shading represents hydrophobic regions; gray parabolas represent accessible surfaces for large areas; gray dotted lines represent hydrogen bonds; broken thick line around 6o shape indicates accessible surfaces; size of residue ellipse represents the strength of the contact.

Figure 4B.

Binding of 6o in human sEH active site (3D representation): Important amino acid residues in the proximity of 6o are shown and labeled.

Figure 5.

Proposed pharmacophore for dual inhibitors based on the binding of 6o in both FAAH and sEH enzymes. Potential hydrogen bond donors are represented with blue cone, hydrogen bond acceptors with red cones, the lipophilic part of the molecule are shown as yellow spheres and aromatic parts are shown as grey barrels. The distances between major pharmacophoric parts are represented with dotted lines.

Figure 6.. Antinociceptive effects of 3 against formalin-induced inflammatory pain.

A) Pain-related behaviors (licking and guarding of the injected hindpaw) in the first phase of the Formalin Test. B) Pain-related behaviors in the second phase of the Formalin Test. n = 6/group. * indicates p < 0.05 from vehicle-treated rats.

Scheme 1.

Reagents and conditions: (a) DIPEA, DCM, 20 min, 80 °C, microwave irradiation, 74%; (b) LiOH/H2O, 16 h, rt, TFA, DCM, rt, 24 h, 91%; (c) 7l-p (see the inner box) or R-aniline (see Table 1 for R), EDC, DMAP, DCM, 20 min, 80 °C, microwave irradiation, 24–91%; (d) iPrOH, 2.5 h, 60 °C, 57–95%.