Analogues of the potent and moderately mu-opioid-receptor-selective cyclic beta-casomorphin-5 derivative H-Tyr-c[-D-Orn-Phe-D-Pro-Gly-] (2) were prepared by conventional solution synthesis. Replacement of the Phe3 residue by 2-naphthylalanine (2-Nal) led to a peptide (4) with high affinity for both mu and delta opioid receptors. This compound turned out to be an agonist in the mu-receptor-representative guinea pig ileum (GPI) assay but a moderately potent antagonist against various delta agonists in the delta-receptor-representative mouse vas deferens (MVD) assay. It thus represents the first known cyclic opioid peptide analogue with mixed mu agonist/delta antagonist properties. Interestingly, replacement of 2-Nal3 in compound 4 with 1-naphthylalanine (1-Nal) resulted in an analogue (5) showing high affinity for mu receptors and a full agonist effect in the MVD assay that was mediated by both mu and delta receptors. Substitution of Trp for Phe3 in 2 (compound 8) was well tolerated at both receptors and led to an analogue with agonist activity in both the GPI and MVD assays. Variation of the peptide ring size in 4 was achieved by substitution of D-Orn2 with D-Lys (compound 6) or D-2,4-diaminobutyric acid (compound 7). Analogue 6 was also a mixed mu agonist/delta antagonist with somewhat lower potency than 4, whereas compound 7 displayed mu agonist and partial delta agonist properties. Further reduction of the peptide ring size, as achieved by deletion of the Gly5 residue, produced a compound (9) which was a full agonist in both bioassays. Conformational analysis of analogues 2, 4, and 5 by 1H NMR spectroscopy and molecular mechanics studies suggested that the overall conformation of parent compound 2 and the 2-Nal-containing peptide 4 was similar, while the side-chain orientation of 1-Nal in peptide 5 was different. These results suggest that the delta antagonist properties of analogue 4 may not be due to a difference in its overall conformation as compared to the agonist 2 but may be a direct effect of the 2-naphthyl moiety per se preventing proper alignment of the peptide for receptor activation.
Attempts were undertaken to develop cyclic β‐casomorphin‐5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H‐Tyr‐Pro‐Phe‐Pro‐NH2). The tetrapeptide sequence Boc‐Tyr(tBu)‐D‐Xaa‐Phe‐Yaa‐OH (Xaa = Lys, Orn, A2bu; Yaa = Pro in L‐ or D‐configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the ω‐amino group of Xaa and the C‐terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and 1H‐NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L‐configuration could a cyclic monomer be detected, and the cyclodimer (7–9) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11‐membered ring of the cyclic [D‐A2bu2, D‐Pro4]‐morphiceptin analog 12, since only traces of the monomer were found. In the case of both the [D‐Lys2, D‐Pro4]‐analog 10 and the [D‐Orn2, D‐Pro4]‐ analog 11, the cyclomonomer/cyclodimer ratio was about 80: 20. The cyclic monomers 10 and 11 showed high opioid activity in the μ‐receptor‐representative guinea pig ileum assay (IC50= 2–5 nM) and in the δ‐receptor representative mouse vas deferens assay (IC50= 50–60 nM), whereas the potency of the cyclodimers was 2–3 orders of magnitude lower in both in vitro bioassays.
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