Pseudoproline-Containing Analogues of Morphiceptin and Endomorphin-2: Evidence for a Cis Tyr−Pro Amide Bond in the Bioactive Conformation

Analogues of the opioid peptides [D-Phe3]morphiceptin (H-Tyr-Pro-D-Phe-Pro-NH2) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) containing the pseudoproline (ΨPro) (4R)-thiazolidine-4-carboxylic acid (Cys[ΨR1,R2pro]) or (4S)-oxazolidine-4-carboxylic acid (Ser[ΨR1,R2pro]) in place of Pro2 were synthesized. The pseudoproline ring in these compounds was either unsubstituted (R, R = H) or dimethylated (R1, R2 = CH3) at the 2-C position. 2-C-dimethylated pseudoprolines are known to be quantitative or nearly quantitative inducers of the cis conformation around the Xaai - 1−Xaai[ΨCH 3 ,CH 3 pro] imide bond. All dihydropseudoproline-containing analogues (R1, R2 = H) showed good μ opioid agonist potency in the guinea pig ileum (GPI) assay, high μ receptor binding affinity in the rat brain membrane binding assay, and, like their parent peptides, excellent μ receptor binding selectivity. 1H NMR spectroscopic analysis of the Cys[ΨH,Hpro]2- and Ser[ΨH,Hpro]2-containing analogues in DMSO-d 6 revealed that they existed in a conformational equilibrium around the Tyr−Xaa[ΨH,Hpro] peptide bond with cis/trans ratios of 40:60 and 45:55, respectively. The dimethylated thiazolidine- and oxazolidine-containing [D-Phe]morphiceptin- and endomorphin-2 analogues (R1, R2 = CH3) all retained full μ agonist potency in the GPI assay and displayed μ receptor binding affinities in the nanomolar range and high μ receptor selectivity. As expected, no conformers of the latter analogues with a trans conformation around the Tyr−Xaa[ΨCH 3 ,CH 3 pro] imide bond were detected by 1H NMR spectral analysis, indicating that in these compounds the cis conformation is highly predominant (>98%). These results represent the most direct evidence obtained so far to indicate that morphiceptin and endomorphin-2 have the cis conformation around the Tyr−Pro peptide bond in their bioactive conformations.