Synthesis of Mixed Opioid Affinity Cyclic Endomorphin‑2 Analogues with Fluorinated Phenylalanines

As part of our continuing studies on the structure–activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c­[d-Lys-Phe-Phe-Asp]­NH₂ (where Dmt = 2′,6′-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF₃-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The <i>in vitro</i> potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c­[d-Lys-Phe-4-F-Phe-Asp]­NH₂ and Dmt-c­[d-Lys-Phe-2,4-F-Phe-Asp]­NH₂, were tested <i>in vivo</i> in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood–brain barrier

In Vitro Membrane Permeation Studies and in Vivo Antinociception of Glycosylated Dmt¹‑DALDA Analogues

In this study the μ opioid receptor (MOR) ligands DALDA (Tyr-d-Arg-Phe-Lys-NH₂) and Dmt¹-DALDA (Dmt-d-Arg-Phe-Lys-NH₂, Dmt = 2′,6′-dimethyltyrosine) were glycosylated at the N- or C-terminus. Subsequently, the modified peptides were subjected to in vitro and in vivo evaluation. In contrast to the N-terminally modified peptide (<b>3</b>), all peptide analogues derivatized at the C-terminus (<b>4</b>–<b>7</b>) proved to possess high affinity and agonist potency at both MOR and DOR (δ opioid receptor). Results of the Caco-2 monolayer permeation, as well as in vitro blood–brain barrier model experiments, showed that, in the case of compound <b>4</b>, the glycosylation only slightly diminished the lumen-to-blood and blood-to-lumen transport. Altogether, these experiments were indicative of transcellular transport but not active transport. In vivo assays demonstrated that the peptides were capable of (i) crossing the blood–brain barrier (BBB) and (ii) activating both the spinal ascending as well as the descending opioid pathways, as determined by the tail-flick and hot-plate assays, respectively. In contrast to the highly selective MOR agonist Dmt¹-DALDA <b>1</b>, compounds <b>4</b>–<b>7</b> are mixed MOR/DOR agonists, expected to produce reduced opioid-related side effects