Structure-Directed Discovery of Potent Soluble Epoxide Hydrolase Inhibitors for the Treatment of Inflammatory Diseases

Abstract

Soluble epoxide hydrolase (sEH) has been identified as an attractive target for anti-inflammatory drug design in recent years. Picomolar level compound G1 against sEH was obtained by introducing the hydrophilic group homopiperazine and hydrophobic fragment propionyl onto the structure of lead compound A. G1 showed good microsomal stability, a moderate plasma protein binding rate, and good oral bioavailability and was well tolerated in rats. G1 has significant analgesic effects on CFA-induced AIA mice, ameliorated the pancreatic injury in acute pancreatitis induced by l-arginine, reversed pancreatic injury, edema, and neutrophil infiltration, and increased the survival time of C57BL/6 mice in a lipopolysaccharide (LPS)-induced sepsis model. Moreover the expression levels of sEH, COX-2, NOS-2, vascular cell adhesion molecule (VCAM), IL-6, MCP-5, and tumor necrosis factor α (TNF-α) were measured by Western blot or enzyme-linked immunosorbent assay (ELISA), with varying degrees of decrease. These results suggested that G1 is a drug candidate worthy of further evaluation for the treatment of inflammation-induced diseases such as arthritis, acute pancreatitis, and sepsis.

Figure 1.

Chemical structures of sEH inhibitors that moved into clinical trials.

Figure 2.

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

Figure 3.

Molecular simulation of compounds G1 bound to sEH (PDB ID: 3WKE)

Figure 4.

Effect of compound G1 on body weight of mice. G1 was prepared in 0.5 % CMC-Na solution and administered orally at a dose of 200 mg / kg for seven consecutive days.

Figure 5.

Evaluation of compound G1 for the treatment of (CFA)-induced arthritis in a mouse model (AIA); (A) Body weight changes of mice in CFA group, CFA + G1 group and CFA + Celecoxib group; (B) Changes in pain thresholds of mice in CFA group, CFA + G1 group and CFA + Celecoxib group; (C) Curves of pain threshold improvement in mice in CFA + G1 group and CFA + Celecoxib group；(D) Curves of paw thickness swelling rate of mice in CFA group, CFA + G1 group and CFA + Celecoxib group; (E) Curves of paw width swelling rate of mice in CFA group, CFA + G1 group and CFA + Celecoxib group; G1 (10 mg / kg, ip) and Celecoxib (10 mg / kg, ip) were administered to mice 24 h and 44 h after induction of CFA by AAI (25 μL / 25g). (n = 8 per group); Significance: ^##P < 0.01 and ^#P < 0.05 compared with the Mod group; **P < 0.01 and *P < 0.05 compared with the Con group.

Figure 6.

Results of the histologic analysis of pancreas from mice treated with control, Model, Celecoxib and compound G1. (A) Representative H&E-stained sections of the pancreas from the in vivo efficacy study. Arrows indicate inflammatory cells and edema. Bold arrows indicate intracellular vacuoles (the red arrows on the image represent edema and inflammatory cells). (B-E) Histologic scoring of pancreatic tissues. (B), edema. (C), inflammatory cells (mononuclear and polymorphonuclear). (D), parenchymal atrophy. (E), total scoring (edema, mononuclear and polymorphonuclear and parenchymal atrophy). (n = 4 per group); Significance: *p < 0.05, and **p < 0.01vs control. ^#p < 0.05 and ^##p < 0.01vs Mod.

Figure 7.

Prophylactic and therapeutic treatment of G1 prevents death from LPS administration in mice. G1 (5 mg / kg, ip) and Dexamethasome (5 mg / kg, ip) were administered to mice 4 h and 12 h after induction of ALI by a lethal dose of LPS (30 mg / kg, ip). The mortality of the mice was monitored per hour, (A) the percent survival rate was expressed as a Kaplan-Meier survival curve (n = 12–14 per group); (B) The expression of inflammatory factors of COX-2, sEH, NOS-2 and VCAM in mouse plasma was detected by Western blot; (C) Measurement of plasma IL-6, MCP-5 and TNF-α inflammatory factors in mice by ELISA. Significance: ^##P < 0.01 and ^#P < 0.05 compared with the LPS group; **P < 0.01 and *P < 0.05 compared with the blank group.

Scheme 1

Design strategy of target compounds based on lead compound A

Scheme 2

Synthetic route to compounds A1-A5. Reactions and conditions: (i) SOCl₂, MeOH, rt; (ii) H₂ (g), 5 % Pd-C, EtOH, 60 °C, 12 h; (iii) (1) memantine, triphosgene, Et₃N, DCM, −78 °C; (2) Et₃N, DCM, 0 °C, 2 h; (vi) NaOH, THF/H₂O, 60 °C, 1 h; (v)methyl piperidine-4-carboxylate, HATU, DIPEA, DCM, rt 2 h; (vi) NaOH, THF / H₂O, 60 °C, 1 h; (vii) amines, HATU, DIPEA, DCM, rt.

Scheme 3

Synthetic route to compounds B1-B4. Reactions and conditions: (i) ethyl piperidine-3-carboxylate, HATU, DIPEA, DCM, rt; (ii) NaOH, THF / H₂O, 60 °C, 1 h; (iii) amines, HATU, DIPEA, DCM, rt.

Scheme 4

Synthetic route to compounds C1-C8. Reactions and conditions: (i) methyl 4-aminocyclohexane-1-carboxylate, HATU, DIPEA, DCM, rt, (ii) NaOH, THF / H₂O, 60 °C, 1 h; (iii) amines, HATU, DIPEA, DCM, rt, 2h.

Scheme 5

Synthetic route to compounds D1-D10. Reactions and conditions: (i) Borane-tetrahydrofuran complex, THF, 0 °C, 2h; (ii) phosphorus tribromide, DCM, rt, 8 h; (iii) ethyl piperidine-3-carboxylate, K₂CO₃, ACN, 50 °C, 4h; (iv) H₂ (g), 5 % Pd-C, EtOH, 60 °C, 12 h; (v) (1) memantine, triphosgene, Et₃N, DCM, −78 °C; (2) Et₃N, DCM, 0 °C, 2 h; (vi) NaOH, THF / H₂O, 60 °C, 1 h; (vii) amines, HATU, DIPEA, DCM, rt, 2h.

Scheme 6

Synthetic route to compounds E1-E5. Reactions and conditions: (i) ethyl piperidine-3-carboxylate, K₂CO₃, ACN, 50 °C, 4h; (ii) H₂ (g), 5 % Pd-C, EtOH, 60 °C, 12 h; (iii) (1) memantine, triphosgene, Et₃N, DCM, −78 °C; (2) Et₃N, DCM, 0 °C, 2 h; (iv) NaOH, THF / H₂O, 60 °C, 1 h; (v) amines, HATU, DIPEA, DCM, rt, 2h.

Scheme 7

Synthetic route to compounds F1-F3. Reactions and conditions: (i) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DCM, rt, 2h. (ii) TFA, DCM, rt, 2 h; (iii) acids, HATU, DIPEA, DCM, rt, 2h.

Scheme 8

Synthetic route to compounds F1-F3. Reactions and conditions: (i) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DCM, rt, 2h. (ii) TFA, DCM, rt, 2 h; (iii) acids, HATU, DIPEA, DCM, rt, 2h.

Scheme 9

Synthetic route to compounds H1-H4. Reactions and conditions: (i) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DCM, rt, 2h; (ii) H₂ (g), 5 % Pd-C, EtOH, 60 °C, 12 h; (iii) (1) memantine, triphosgene, Et₃N, DCM, −78 °C, 2h; (iv) TFA, DCM, rt, 2 h; (v) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DCM, rt, 2h; (vi) TFA, DCM, rt, 2 h; (vii) acids, HATU, DIPEA, DCM, rt, 2h; (viii) 2-chloro-N,N-dimethylpropan-1-amine, K₂CO₃, ACN, 50 °C, 4h.

Scheme 10

Synthetic route to compounds I1-I8. Reactions and conditions: (i) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DCM, rt, 2h; (ii) H₂ (g), 5 % Pd-C, EtOH, 60 °C, 12 h; (iii) (1) memantine, triphosgene, Et₃N, DCM, −78 °C, 2h; (iv) TFA, DCM, rt, 2 h; (v) tert-butyl 1,4-diazepane-1-carboxylate, HATU, DIPEA, DCM, rt, 2h; (vi) TFA, DCM, rt, 2 h; (vii) acids, HATU, DIPEA, DCM, rt, 2h; (viii) chlorides, K₂CO₃, ACN, 50 °C, 4h.