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