N-Guanidyl and C-Tetrazole Leu-Enkephalin Derivatives: Efficient Mu and Delta Opioid Receptor Agonists with Improved Pharmacological Properties

Leu-enkephalin and d-Ala2-Leu-enkephalin were modified at their N- and C-termini with guanidyl and tetrazole groups. The resulting molecules were prepared in solution or by solid phase peptide synthesis. The affinity of the different analogues at mu (MOP) and delta opioid receptors (DOP) was then assessed by competitive binding in stably transfected DOP and MOP HEK293 cells. Inhibition of cAMP production and recruitment of β-arrestin were also investigated. Finally, lipophilicity (logD7.4) and plasma stability of each compound were measured. Compared to the native ligands, we found that the replacement of the terminal carboxylate by a tetrazole slightly decreased both the affinity at mu and delta opioid receptors as well as the half-life. By contrast, replacing the ammonium at the N-terminus with a guanidyl significantly improved the affinity, the potency, as well as the lipophilicity and the stability of the resulting peptides. Replacing the glycine residue with a d-alanine in position 2 consistently improved the potency as well as the stability of the analogues. The best peptidomimetic of the whole series, guanidyl-Tyr-d-Ala-Gly-Phe-Leu-tetrazole, displayed sub-nanomolar affinity and an increased lipophilicity. Moreover, it proved to be stable in plasma for up to 24 h, suggesting that the modifications are protecting the compound against protease degradation.