New Endomorphin-2 Analogues Incorporating Constrained Homologated Phenylalanine Residues

Endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2, EM-2) is a potent and selective endogenous mu-opioid receptor (MOR) agonist neuropeptide, playing a relevant role in the regulation of pain perception and in analgesia. Since the X-ray structure of MOR is not available yet, structure–activity studies performed on modified analogues are the only way to supply insight into the structural properties responsible for activity and selectivity. To determine the bioactive conformation of EM-2 is also a difficult task, because of its high flexibility, and divergent opinions have been reported with respect to this aspect. A common strategy adopted to study the structure-activity relationship of native peptides towards the search of new therapeutics, is the incorporation of unnatural amino acids and/or conformational constraints. For instance, the repertoire of conformations can be expanded by structures incorporating homologated amino acids. Advantages may also come from heterogeneous (mixed alfa- and beta-) backbone peptidomimetics. Since a definitive model is not available yet for the bioactive conformation of EM-2, we planned the synthesis of a small library of analogues, characterized by various substitutions of Phe residues at position 3 and 4, with the aim of better establishing the relevant role on bioactivity of the proper spatial orientation of the benzyl side chains. We identified some constrained beta-Phe-beta-Phe dipeptide mimics, which were synthesised and evaluated by the conformational point of view. Starting from constrained scaffold and from linear beta2-Phe-beta3-Phe and beta3-Phe-beta2-Phe dipeptides, EM-2 tetrapeptide mimics have been synthesised and subjected to conformational investigation and biological evaluation. From preliminary results, we observed the maintenance of opioid affinity in some compounds, even if the C-terminal Phe-Phe dipeptide is simultaneously substituted by a peptidomimetic structure. A type of modification seems able to completely abate the high mu-selectivity of EM-2 providing compounds endowed with a little preference for delta-opioid receptors.