Abstract

Subtle structural modifications at the 3-position of opioid peptides have been reported to modulate their receptor binding affinity and functional activity. Our previous work identified a novel cyclic hexapeptide, Tyr-c[D-Lys-Gly-Phe-Asp]-D-Pro-NH2, as a multitarget full agonist at μ-, κ-, and δ-opioid receptors (MOR, KOR, and DOR). In the present study, 16 novel analogs of this hexapeptide were synthesized by replacing the Gly3 residue with phenylalanine and its derivatives to investigate their structure–activity relationships. Functional characterization demonstrated that these modifications generated analogs exhibiting varying selectivity and efficacy profiles at opioid receptors. Notably, analog 9 containing p-F-Phe3 exhibited MOR and KOR agonism/DOR antagonism and produced potent antinociception following peripheral administration, with reduced antinociceptive tolerance, physical dependence, and respiratory depression. Surprisingly, analog 11 containing p-Br-Phe3 acted as a potent and selective MOR antagonist. Pharmacological blockade of the peripheral MOR by analog 11 significantly attenuated fentanyl-induced antinociceptive tolerance, highlighting its therapeutic potential.