On resin click-chemistry-mediated synthesis of novel enkephalin analogues with potent anti-nociceptive activity

Abstract

Here, we report the chemical synthesis of two DPDPE analogues 7a (NOVA1) and 7b (NOVA2). This entailed the solid-phase synthesis of two enkephalin precursor chains followed by a Cu^I-catalyzed azide-alkyne cycloaddition, with the aim of improving in vivo analgesic efficacy versus DPDPE. NOVA2 showed good affinity and selectivity for the μ-opioid receptor (K_I of 59.2 nM, EC₅₀ of 12.9 nM, E_Max of 87.3%), and long lasting anti-nociceptive effects in mice when compared to DPDPE.

Figure 1

Main structural modifications applied to the cyclic opioid peptide DPDPE.

Figure 2

Reagents and conditions (a) Fmoc-Rink amide-linker, TBTU, HOBt anhydrous, DIPEA, DMF, at r.t., 24 h. (b) piperidine 20% in DMF at r.t. 30 min. (c) Fmoc-DAza-OH, HATU, DIPEA, DMF at r.t. or Fmoc-DPra-OH, HATU, DIPEA, DMF at r.t. (d) Fmoc-Phe-OH, TBTU, HOBt a., DIPEA, DMF at r.t. (e) Fmoc-Gly-OH, TBTU, HOBt a., DIPEA, DMF at r.t. (f) Fmoc-DAza-OH, HATU, DIPEA, DMF at r.t. or Fmoc-DPra-OH, HATU, DIPEA, DMF at r.t. (g) Fmoc-Tyr(OtBu)-OH, TBTU, HOBt a., DIPEA, DMF at r.t. (h) CuBr, sodium ascorbate, water, 2,6-Lutidine, DIPEA, DMSO anhydrous at r.t., N₂ atmosphere, 24 h. (i) TFA:TIS:DCM:water = 90%/2.5%/5%/2.5% at r.t. 2 h.

Figure 3

NOVA compounds preferentially bind to the MOP and DOP. (A) Competition binding curves for the NOVA compounds and naloxone (MOP, DOP) or U50,488 (KOP) positive controls shown, along with the scaffold/parent controls DPDPE, Leu-Enkephalin, and Met-Enkephalin. All compounds were competed against a fixed concentration of ³H-diprenorphine in the membranes of MOP, DOP, or KOP-expressing CHO cells. Summary curves shown with the mean ± SEM of each point from n = 3 independent experiments. (B) The K_I of each compound was calculated from the IC₅₀ of each curve using the previously established K_D of ³H-diprenorphine in each cell line. Each K_i value was calculated from each independent experiment and reported here as the mean ± SEM (n = 3 independent experiments).

Figure 4

NOVA compounds selectively activate the MOP in the ³⁵S-GTPγS coupling assay. (A) Summary agonist concentration curves for the NOVA compounds and a positive control agonist (DAMGO for MOP; SNC80 for DOP; U50,488 for KOP) in ³⁵S-GTPγS coupling shown. All compounds were normalized to the stimulation caused by the positive control (100%) and vehicle (0%) and reported as the mean ± SEM from n = 3 independent experiments. (B) The potencies (EC₅₀) and efficacies (E_Max) of each NOVA compound and positive control were calculated in each independent experiment (n = 3), then reported as the mean ± SEM. The E_Max was reported as a percent of stimulation caused by the positive control, which had a defined E_Max of 100%. For the DOP and KOP, E_Max values in parentheses report the maximum stimulation at 10 μM of compound, not the fully defined top of the agonist curve, as these curves are incomplete at 10 μM.

Figure 5

Effects induced by NOVA2 (N) and DPDPE (D) in the hot plate test in mice. In the left panel, the effects induced by i.c.v. administered NOVA2 and DPDPE at doses of 5 and 23 nmol are reported. In the right panel, the effects induced by i.v. administered NOVA2 and DPDPE at the dose of 23 μmol are reported. ** is for P < 0.01, *** is for P < 0.001 and **** is for P < 0.0001 comparing NOVA2 vs DPDPE at the same dose. N = 6–9.

Figure 6

Effects induced by s.c. administered NOVA2 and DPDPE in the formalin test in mice. DPDPE and NOVA2 were administered at the dose of 100 nmol. **** is for P < 0.0001 and ** is for P < 0.01 vs DPDPE. N = 8.

Figure 7

Best ranked docking poses of TIPP-NH₂ (A), DPDPE (B), NOVA1 (C) and NOVA2 (D) docked at the DOP (4RWD).

Figure 8

Best ranked docking poses of BU72 (A), NOVA1 (B), NOVA2 (C), docked at the ΜΟP (PDB:5C1M).