Design and Synthesis of Dual-Targeting Inhibitors of sEH and HDAC6 for the Treatment of Neuropathic Pain and Lipopolysaccharide-Induced Mortality

Abstract

Epoxyeicosatrienoic acids with anti-inflammatory effects are inactivated by soluble epoxide hydrolase (sEH). Both sEH and histone deacetylase 6 (HDAC6) inhibitors are being developed as neuropathic pain relieving agents. Based on the structural similarity, we designed a new group of compounds with inhibition of both HDAC6 and sEH and obtained compound M9. M9 exhibits selective inhibition of HDAC6 over class I HDACs in cells. M9 shows good microsomal stability, moderate plasma protein binding rate, and oral bioavailability. M9 exhibited a strong analgesic effect in vivo, and its analgesic tolerance was better than gabapentin. M9 improved the survival time of mice treated with lipopolysaccharide (LPS) and reversed the levels of inflammatory factors induced by LPS in mouse plasma. M9 represents the first sEH/HDAC6 dual inhibitors with in vivo antineuropathic pain and anti-inflammation.

Figure 1.

Chemical structures of sEH and HDAC6 inhibitors in clinical trials.

Figure 2.

The effects of M8, M9, Belinostat on acetylated H3 and acetylated α-tubulin in THP-1 cells treated for 24 hours at 1 and 2 μM.

Figure 3.

Molecular dockings of B401, M9 and Ricolinostat: (A) binding mode of compound B401 in the sEH (PDB ID: 3WKE); (B) binding mode of compound M9 in the sEH (PDB ID: 3WKE); (C) binding mode of compound Ricolinostat in the HDAC6 (PDB ID: 5WGL); (D) binding mode of compound M9 in the HDAC6 (PDB ID: 5WGL); (E) binding mode of compound M9 in the HDAC1, HDAC1 was constructed by homology modeling using HDAC2 (PDB ID: 4LXZ); (F) binding mode of compound M9 in the HDAC3 (PDB ID: 4A69).

Figure 4.

MD simulations analyses of M9 in complex with 3WKE and 5WGL protein. (A) RMSD curve of M9/3WKE complexes; (B) RMSD curve of M9/5WGL complexes; (C) The RMSF maps of M9/3WKE complexes over the entire simulations; (D) The RMSF maps of M9/5WGL complexes over the entire simulations.

Figure 4.

MD simulations analyses of M9 in complex with 3WKE and 5WGL protein. (A) RMSD curve of M9/3WKE complexes; (B) RMSD curve of M9/5WGL complexes; (C) The RMSF maps of M9/3WKE complexes over the entire simulations; (D) The RMSF maps of M9/5WGL complexes over the entire simulations.

Figure 5.

Effect of compound M9 on body weight of mice. M9 was prepared in 0.5 % CMC-Na solution and was administered orally at the dose of 4.0 g/kg.

Figure 6.

The effects of co-administration of Ricolinostat and EC5026 against the SNI-induced pain of mouse. (A) Time-and dose-response curve of anti-nociceptive sensation induced by i.g. injection of Ricolinostat and EC5026; (B) The AUC of 0–7 hours calculated from these data were statistically analyzed and given in the text. n = 8, *p < 0.05 for between EC5026 and EC5026+Ricolinostat group; (C, D) The antinociception induced by i.g. injection of Ricolinostat and EC5026 were investigated at 3h and 5h after administration. n = 8, *p < 0.05 and ^**p < 0.01 compared with EC5026 group.

Figure 7.

M9 attenuates neuropathic pain in SNI model mice. (A) Analgesic efficacies of M9 and Gabapentin on the 1^st day. (B) Analgesic efficacy-area under the curve of M9 and Gabapentin treatment; (C) Analgesic efficaciues of M9 and Gabapentin on the 7^th day; (D) Analgesic efficacies of M9 and Gabapentin on the 7^th day-area under the curve, ^**p < 0.01 between M9 and SNI control group, ^&&p < 0.01 between Gabapentin and SNI control group, ^#p < 0.05 between M9 and Gabapentin group; (E) Analgesic efficacies test of M9 and Gabapentin for one week; (F) Analgesic efficacy and tolerance of Gabapentin for one week; (G) Analgesic efficacy and tolerance of M9 for one week. *p < 0.05 between 1^st day and 7^th day. Data are expressed as mean ± standard error (n=8); M9 and Gabapentin were given orally.

Figure 8.

M9 prevents death from LPS administration in mice. All mice except Blank group were give a lethal dose of LPS (30 mg/kg, ip). M9 (5 mg/kg, ip) and Dexamethasome (5 mg/kg, ip) were administered to mice 4 h and 12 h later. The mortality of the mice was monitored per hour. (A) the Kaplan-Meier survival curves were drawn (n=12–15 per group); (B) The plasma levels of IL-6, MCP-5 and TNF-α were measured at 12 h by ELISA. ^##P < 0.01 and ^#P < 0.05 compared with the Vehicle group; **P < 0.01 and *P < 0.05 compared with the blank group.

Scheme 1.

Design principle of sEH/HDAC dual-targeting compound.

Scheme 2.

The synthetic route of compounds M1、M2 and M11. Reactions and conditions: (i) (S)- ethyl 3-piperidinecarboxylate, Et₃N, THF, 0 °C to rt., 8h; (ii) Fe, NH₄Cl, EtOH/H₂O, 70 °C, 2h; (iii) (1) BTC, Et₃N, DCM, −10 °C to reflux; (2) memantine, Et₃N, DCM, 0 °C to rt., 2h; (iv) (1) NaOH, H₂O, 50 °C, 4h; (2)1N HCl; (v) methyl 3-aminopropionate, EDCI, HOBt, Et₃N, DCM, rt., 2h; (vi) (1) SOCl₂, DMF, THF; (2) NH₄OH, THF, 2h; (vii) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N. HCl.

Scheme 3

The synthetic route of compounds M3-M5. Reactions and conditions: (i) SOCl₂, MeOH, 0 °C to rt., 12h; (ii) Fe, NH₄Cl, EtOH/H₂O, 70 °C, 2h; (iii) (1) BTC, Et₃N, DCM, −10 °C to rt.; (2) memantine, Et₃N, DCM, 0 °C to rt., 2h; (iv) (1) NaOH, H₂O, 50 °C, 4h; (2) 1N HCl; (v) corresponding amines, EDCI, HOBt, Et₃N, DCM, rt., 2h; (vi) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N HCl.

Scheme 4

The synthetic route of compounds M7-M9. Reactions and conditions: (i) (1) BTC, Et₃N, DCM, −10 °C to rt.; (2) memantine, Et₃N, DCM, 0 °C to rt., 2h; (ii) (1) NaOH, H₂O, 50 °C, 4h; (2) 1N HCl; (iii) corresponding amines, EDCI, HOBt, Et₃N, DCM, rt., 2h; (iv) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N HCl.

Scheme 5

The synthetic route of compounds M6, M10. Reactions and conditions: (i) (S)-ethyl 3-piperidine formate, EDCI, HOBt, Et₃N, DCM, rt., 2h; (ii) (1) NaOH, H₂O, 50 °C, 4h; (2) 1N HCl; (iii) corresponding amines, EDCI, HOBt, Et₃N, DCM, rt., 2h; (iv) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N HCl.

Scheme 6

The synthetic route of compounds M12-M17. Reactions and conditions: (i) (1) BTC, Et₃N, DCM, −80 °C to rt.; (2) 4-(Trifluoromethoxy)aniline or 3-fluoro-4-(trifluoromethoxy)aniline, Et₃N, DCM, −80 °C to rt., 2h; (ii) (1) NaOH, H₂O, 50 °C, 4h; (2) 1N HCl; (iii) corresponding amines, EDCI, HOBt, Et₃N, DCM, rt., 2h; (iv) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) Conc. HCl; (v) (1) NaOH, H₂O, 50 °C, 2h; (2) 1N HCl.

Scheme 7

The synthetic route of compound M18. Reactions and conditions: (i) 4-(tert-butoxycarbonylamino)piperidine, DIPEA, HATU, DCM, rt., 2h; (ii) TFA, DCM, rt., 5h; (iii) (1) BTC, Et₃N, DCM, −10 °C to rt.; (2) memantine, Et₃N, DCM, 0 °C to rt., 2h; (iv) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N HCl.

Scheme 8

The synthetic route of compound M19. Reactions and conditions: (i) (1) NaOH, H₂O, 50 °C, 4h; (2) 1N HCl; (ii) methyl 5-aminovalerate, DIPEA, HATU, DCM, rt., 2h; (iii) TFA, DCM, 0 °C to rt., 5h; (iv) BTC, Et₃N, DCM, −10 °C to rt.; (2) memantine, Et₃N, DCM, 0 °C to rt., 2h; (v) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N HCl.

Scheme 9

The synthetic route of compound M20. Reactions and conditions: (i) cis-N-Boc-4-amino-cyclohexanol, DIAD, PPh₃, THF, 0 °C to rt., 12h; (ii) (1) NaOH, H₂O, 50 °C, 4h; (2) 1N HCl; (iii) methyl 3-aminopropionate, DIPEA,HATU, DCM, rt., 2h; (iv) TFA, DCM, 0 °C to rt., 5h; (v) (1) BTC, Et₃N, DCM, −10 °C to rt.; (2) memantine, Et₃N, DCM, 0 °C to rt., 2h; vi) (1) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N HCl.

Scheme 10

The synthetic route of compounds M21-M24. Reactions and conditions: (i) corresponding amines, Et₃N, DCM, 0 °C, 8h; (ii) Fe, NH₄Cl, EtOH/H₂O, 70 °C, 2h; (iii) (1) BTC, Et₃N, DCM, −80 °C to rt.; (2) memantine, Et₃N, DCM, −80 °C to rt., 2h; (iv) 50 wt % NH₂OH solution, 1 M NaOH, MeOH, 0 °C to rt., 2 h; (2) 1N. HCl.