Opioid Receptor Activity and Analgesic Potency of DPDPE Peptide Analogues Containing a Xylene Bridge

Abstract

d-Pen²,d-Pen⁵ enkephalin (DPDPE) is one of the most selective synthetic peptide agonists targeting the δ-opioid receptor. Three cyclic analogues of DPDPE containing a xylene bridge in place of disulfide bond have been synthesized and fully characterized as opioid receptors agonists. The in vitro activity was investigated showing a good affinity of 7a-c for μ- and δ-receptors. In vivo biological assays revealed that 7b is the most potent analogue with the ability to maintain high level of analgesia from 15 to 60 min following intracerebroventricular (i.c.v.) administration, whereas DPDPE was slightly active until 45 min. Compound 7b induced long lasting analgesia also after subcutaneous administration, whereas DPDPE was inactive.

Keywords: DPDPE; Opioids; antinociception; peptides; xylene bridge.

Figure 1

DPDPE cyclization strategy from Leu/Met-enkephalins.

Scheme 1. Synthesis of Final Compounds 7a–c

Reagents and conditions: (a) 1.1 equiv of BocPhe-OH, 1.1 equiv of EDC·HCl, 1.1 equiv of HOBt anhydrous, 3.3 equiv of DIPEA, DMF under N₂ atmosphere, r.t., overnight; (b) TFA/DCM = 1:1 under N₂ atmosphere, r.t., 1 h; (c) 1.1 equiv of BocGly-OH, 1.1 equiv of EDC·HCl, 1.1 equiv of HOBt anhydrous, 3.3 equiv of DIPEA, DMF under N₂ atmosphere, r.t., overnight; (d) 1.1 equiv of Boc(d)Pen-OH, 1.1 equiv of EDC·HCl, 1.1 equiv of HOBt anhydrous, 3.3 equiv of DIPEA, DMF under N₂ atmosphere, r.t., overnight; (e) 2.1 equiv of o-dibromo-xylene, 6 days for 5a, 1.3 equiv of m-dibromo-xylene, 4 days for 5b, 2.1 equiv of p-dibromo-xylene, 6 days for 5c, 2.6 equiv of DIPEA in DMF under N₂ atmosphere, r.t.; (f) 1.1 equiv of BocTyr-OH, 1.1 equiv of EDC·HCl, 1.1 equiv of HOBt anhydrous, 3.3 equiv of DIPEA, DMF under N₂ atmosphere, r.t., overnight; (g) 4 equiv of 1 M NaOH in THF, 5 h for 7a, 2 equiv of 1 M NaOH in THF, 2 h for 7b, 3.5 equiv of 1 M NaOH in THF, 3 h for 7c, r.t.

Figure 2

Tail flick, hot plate and formalin test assays on cyclic peptides 7a–c. V is for vehicle; D is for DPDPE. In the HP and TF test, drugs were injected i.c.v. at the dose of 23 nmol/mouse. In the formalin test, drugs were administered s.c. at the dose of 150 nmol/mouse, 15 min before formalin. ****P < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 vs D. N = 8–10.

Figure 3

Best docking poses of DPDPE (A), 7a (B), 7b (C), and 7c (D) at the DOR (only the involved residues are depicted).