Design, Synthesis, And Pharmacological Characterization Of Novel Endomorphin Analogues Containing Unnatural Amino Acids Map

The opioid system is one of the most studied pain relieving systems, and it consists of three subtype receptors: the ? opioid receptor (MOR), the ? opioid receptor (DOR),and the ? opioid receptor (KOR). Opioid peptides serve as endogenous neurotransmitters and exert their pharmacological functions through these receptors.Opioid peptides have been studied extensively since their discovery, and many efforts have been dedicated to the determination of their intrinsic nature. In 1997, the MOR endogenous tetrapeptides, endomorphin-1 (EM-1, H-Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH2), were isolated from the bovine brain and the human cortex. These peptides exhibited the highest affinity for the MOR and a high MOR over DOR selectivity. Furthermore, EMs are thought to inhibit pain without some of the undesirable side effects of morphine. Particularly, the rewarding effect of EMs can be separated from analgesia, and they are less prone to induce respiratory depression and cardiovascular effects at effective antinociceptive doses.For these reasons, EMs have attracted much attention from the peptide chemist/pharmacologist teams and have been considered as useful pharmacological tools with a tremendous potential in pain alleviation. However, EMs still suffer from serious limitations including lack of oral activity, short duration of action, poor metabolic stability, and relative inability to cross the blood-brain barrier (BBB) and access the central nervous system (CNS). Therefore, it is essential to enhance their bioavailability in order to facilitate therapeutic use.A new class of endomorphin-1 (EM-1) analogues were synthesized by introduction of novel unnatural a-methylene-?-amino acids (Map) at position 2, 3 or/and position 4.Their binding and functional activity, metabolic stability, and antinociceptive activity were determined and compared. Most of these analogues showed high affinities for the ?-opioid receptor (MOR) and an increased stability in mouse brain homogenates compared with EM-1. Examination of cAMP accumulation in HEK293 cells confirmed the agonist properties of these analogues. Among these new analogues,H-Tyr-Pro-Trp-(2-furyl)Map-NH2 (analogue 18) exhibited the highest binding potency (Kl? = 0.221 nM) and efficacy (EC50 = 0.0334 nM, Emax = 97.14%). This analogue also displayed enhanced antinociceptive activity in vivo in comparison to EM-1. Molecular modeling approaches were then carried out to demonstrate the interaction pattern of these analogues with the opioid receptors. We found that,compared to EM-1, the incorporation of our synthesized Map at position 4 would bring the analogue to a closer binding mode with the ?-opioid receptor.Then we synthesized new EM-1 analogues containing Dmt1, (R/S)-?Pro2, and(ph)Map4/(2-furyl)Map4. All of the analogues showed a high affinity for the MOR and increased stability in mouse brain homogenates. Of the new compounds,Dmt-(R)-?Pro-Trp-(2-furyl)Map-NH2 (analogue 32) displayed the highest affinity toward MOR, in the picomolar range (Kl? = 3.72 pM). Forskolin-induced cAMP accumulation assays indicated that this analogue displayed an extremely high agonistic potency,in the subpicomolar range (EC50=0.0421 pM,Emax=99.5%). This compound also displayed stronger in vivo antinociceptive activity after iv administration when compared to morphine in the tail-flick test, which indicates that this analogue was able to cross the blood-brain barrier.