Synthesis And Pharmacological Activity Of Novel Endomorphin-1 Analogues With Combined Chemical Modification

Two potent endogenous opioid peptides, endomorphin-1 (EM-1, Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂), which showed high affinity and selectivity towardμ-opioid receptor, were isolated from bovine in 1997. They are found to elicit equipotent analgesia to morphine, but without some of the undesirable side effects of morphine and other opioid drugs selective for theμ-opioid receptor. Herein, EM-1, instead of EM-2, has been chosen as the parent compound because many studies have demonstrated that EM-1 has a more favorable therapeutic profile than EM-2, thus could provide better basis for novel and safer analgesics. However, with relatively rapid degradation and limited delivery to the central nervous system (CNS), it is unlikely that EM-1 could be used for the clinical treatment of pain. The scope of the present study was to optimize the physicochemical properties of EM-1 by modifying its molecular structure, and to produce novel analogues with enhancing metabolic stability and ability to cross the blood-brain barrier (BBB) into the CNS, thus being able to induce analgesia after systemic administration. In addition, by determining the potential contribution of these alterations to the biological activity, we can further our knowledge of the structural requirements necessary for pharmacological effects.In the present study, we have synthesized a series of novel EM-1 analogues by combined chemical modifications including cationization of EM-1 by guanidino-addition on the Tyr¹ , chloro-halogenation at the para position of Phe⁴, replacement of L-Pro in position 2 with dipeptide fragment D-Pro-Gly and O-methylation of para hydroxyl group on the aromatic nucleus of Tyr¹. EM-1 and its four analogues were synthesized by solution-phase method, and their binding and bioassay activity, lipophilicity, stability, protein binding affinity as well as antinociceptive activity after i.c.v. and s.c. administrations were determined. All of the analogues had low binding to theμ-/δ-opioid receptor in rat brain membrane and showed low GPI and MVD potencies. Cationization of EM-1 by guanidino-addition on Tyr¹ led to a significant increase in metabolic stability in both brain and serum. The introduction of D-Pro-Gly in place of L-Pro not only drastically enhanced their stability, but also resulted in a slight increase in lipophilicity. All the analogues with C-terminal chloro-halogenation had increased lipophilicity and protein binding affinity when compared with their corresponding non-halogenated forms. O-methylation increased the overall lipophilicity and showed no significant influence on stability in both brain and serum, while at the same time decreasingμ-opioid receptor affinity and functional bioactivity. Antinociception studies showed that i.c.v. administration of the four analogues elicited less potent but longer-lasting analgesia. Compared with mother peptide EM-1, the selected analogue 3 was found to display potent and prolonged antinociceptive activity upon s.c. administration through a central mechanism. Overall, due to its enzyme resistance and antinociceptive activity after peripheral administration, analogue 3 can be regarded as a promising modified endomorphin model with little side effects and may help identify the best possible drug candidate for clinical nociceptive pain management.