Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain

Abstract

Soluble epoxide hydrolase (sEH) is related to arachidonic acid cascade and is over-expressed in a variety of diseases, making sEH an attractive target for the treatment of pain as well as inflammatory-related diseases. A new series of memantyl urea derivatives as potent sEH inhibitors was obtained using our previous reported compound 4 as lead compound. A preferential modification of piperidinyl to 3-carbamoyl piperidinyl was identified for this series via structure-based rational drug design. Compound A20 exhibited moderate percentage plasma protein binding (88.6%) and better metabolic stability in vitro. After oral administration, the bioavailability of A20 was 28.6%. Acute toxicity test showed that A20 was well tolerated and there was no adverse event encountered at dose of 6.0 g/kg. Inhibitor A20 also displayed robust analgesic effect in vivo and dose-dependently attenuated neuropathic pain in rat model induced by spared nerve injury, which was better than gabapentin and sEH inhibitor (±)-EC-5026. In one word, the oral administration of A20 significantly alleviated pain and improved the health status of the rats, demonstrating that A20 was a promising candidate to be further evaluated for the treatment of neuropathic pain.

Keywords: Analgesia; Inhibitor; Neuropathic pain; Soluble epoxide hydrolase; Synthesis.

Graphical abstract

Figure 1

Chemical structures of sEH inhibitors in clinic trials.

Figure 2

Docked pose of compound 4 in blue bound to sEH (PDB ID: 3WKE).

Scheme 1

Design strategy based on lead compound 4.

Scheme 2

Synthetic route to compounds A1−A7. Reactions and conditions: i) piperidine-4-carboxylic acid, K₂CO₃, THF/H₂O; (ii) H₂ (g), 5% Pd-C, EtOH, 60 °C, 12 h; (iii) K₂CO₃, PhOCOCl, THF, 0 °C→ rt; iv) memantine, Et₃N, THF, 75 °C, 8 h; v) SOCl₂, EtOH, 0 °C→reflux, 2 h; vi) (1) SOCl₂, DMF, THF; (2) NH₃ (g), THF; vii) amines, EDCI, HOBt, Et₃N, DCM, rt.

Scheme 3

Synthetic route to compounds A8−A14. Reactions and conditions: i) piperidine-3-carboxylic acid, K₂CO_3, THF/H₂O; (ii) H₂ (g), 5% Pd-C, EtOH, 60 °C, 12 h; (iii) K₂CO₃, PhOCOCl, THF, 0 °C→rt; iv) memantine, Et₃N, THF, 75 °C, 8 h; v) SOCl₂, EtOH, 0 °C→reflux, 2 h; vi) (1) SOCl₂, DMF, THF; (2) NH₃ (g), THF; vii) amines, EDCI, HOBt, Et₃N, DCM, rt.

Scheme 4

Synthetic route to compounds A15−A17. Reactions and conditions: i) (1) SOCl₂, DMF, THF, 0 °C→rt; (2) ethyl piperidine-3-carboxylate, Et₃N,THF, 0 °C→rt; ii) Fe, NH₄Cl, EtOH/H₂O, 70 °C; iii) (1) BTC, Et₃N, DCM, 0 °C→reflux; (2) memantine, Et₃N, DCM, 0 °C→rt; iv) (1) NaOH, H₂O, EtOH; (2) Conc. HCl; v) (1) SOCl₂, DMF, THF, 0 °C→rt; (2) NH₃ (g), THF, 0→rt; vi) SOCl₂, MeOH, 0 °C→reflux, 2 h; vii) 50% (w/w) NH₂OH solution, 1 mol/L NaOH, MeOH, 0 °C.

Scheme 5

Synthetic route to compounds A18 and A19. Reactions and conditions: i) R- or S-ethyl piperidine-3-carboxylate, Et₃N, THF, 0 °C→rt; ii) H₂ (g), 5% Pd-C, EtOH, 60 °C, 12 h; (iii) K₂CO₃, PhOCOCl, THF, 0 °C→rt; iv) memantine, Et₃N, THF, 75 °C, 8 h; v) (1) NaOH, H₂O, EtOH; (2) Conc. HCl; vi) (1) SOCl₂, DMF, THF; (2) NH₃ (g), THF.

Scheme 6

Synthetic route to compound A20 and A21. Reactions and conditions: i) (1) SOCl₂, DMF, THF, 0 °C→60 °C; (2) (S)-ethyl piperidine-3-carboxylate or (R)-ethyl piperidine-3-carboxylate, Et₃N, THF, 0 °C→rt; ii) Fe, NH₄Cl, EtOH/H₂O, 70 °C; iii) (1) BTC, Et₃N, DCM, 0 °C→reflux; (2) memantine, Et₃N, DCM, 0 °C→rt; iv) (1) NaOH, H₂O, EtOH; (2) Conc. HCl; v) (1) SOCl₂, DMF, THF, 0 °C→rt; (2) NH₃ (g), THF, 0 °C→rt.

Scheme 7

Synthetic route to compounds B1–B8. Reactions and conditions: (i) tert-butyl piperidin-4-yl-carbamate, Et₃N, THF, 0 °C→rt, 6 h; (ii) H₂ (g), 5% Pd-C, EtOH, 60 °C, 12 h; (iii) K₂CO₃, PhOCOCl, THF, 0 °C→rt, 7 h; (iv) memantine, Et₃N, THF, 75 °C, 8 h; v) TFA, DCM, 0 °C→rt, 2 h; vi) (a) corresponding acyl chlorides, Et₃N, DCM; or (b) corresponding acids, EDCI, HOBt, Et₃N, DCM, rt.

Scheme 8

Synthetic route to compound B9. Reactions and conditions: i) (1) SOCl₂, DMF, THF, 0 → 60 °C; (2) tert-butyl (S)-piperidin-3-yl-carbamate, Et₃N, THF, 0 °C→rt; ii) Fe, NH₄Cl, EtOH/H₂O, 70 °C; iii) (1) BTC, Et₃N, DCM, 0 °C→reflux; (2) memantine, Et₃N, DCM, 0 °C→rt; iv) TFA, DCM, 0 °C→rt; vi) MsCl, Et₃N, DCM, 0 °C→rt.

Scheme 9

Synthetic route to compounds C1–C8. Reactions and conditions: (i) tert-butyl piperazine-1-carboxylate, Et₃N, THF, 0 °C→rt, 6 h; (ii) H₂ (g), 5% Pd-C, EtOH, 60 °C, 12 h; (iii) K₂CO₃, PhOCOCl, THF, 0 °C→rt, 7 h; (iv) memantine, Et₃N, THF, 75 °C, 8 h; v) TFA, DCM, 0 °C→rt, 2 h; vi) (a) corresponding acyl chlorides, Et₃N, DCM; or (b) corresponding acids, EDCI, HOBt, Et₃N, DCM, rt.

Figure 3

Molecular simulation of compounds A20 and B9 bound to sEH (PDB ID: 3WKE). (A) showed docked poses of compounds A20 (purple), B9 (blue) and t-AUCB (green) in sEH binding pocket. (B) indicated sEH bound to compound A20 in purple. (C) displayed sEH bound to compound B9 in blue. Hydrogen bond was showed as dash in yellow.

Figure 4

The effect of the compound A20 on the body weight of ICR mice. A20 formulated in 0.5% CMC-Na solution was administered orally at the dose of 6.0 g/kg.

Figure 5

sEH inhibitors attenuate neuropathic pain in SD rat model induced by spared nerve injury. Data are expressed as mean ± standard error of mean (n = 6); ∗P < 0.05, ∗∗P < 0.01, vs. Treatment/Model; ^#P < 0.05, vs. Treatment/Model; ^$P < 0.05, ^$$P < 0.01, vs. Treatment/Model; ^&P < 0.05, vs. Treatment/Model; ^@P < 0.05, vs. Treatment/Model; ^▲P < 0.05, vs. Treatment/Model. A20 formulated in 0.5% CMC-Na solution was administered orally at the indicated doses.

Figure 6

sEH inhibitors attenuate neuropathic pain in rat model induced by spared nerve injury. Data are expressed as mean ± standard error of mean (n = 6).