Design And Synthesis Of (ph)Map-containing EM-1 Analogues And Their Pharmacological Characterization

Since the discovery of endomorphin-1 and endomorphin-2,they have been the most potent analgesia which bind to μ-opioid receptors(MORs),MORs mainly contribute to the control of pain transmission.Endogenous peptides EM-1 and EM-2 exhibited the highest affinity for MORs and a high selectivity for MOR over DOR(δ-opioid receptor).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 likely to induce respiratory depression and cardiovascular effects at effective antinociceptive doses.However,EMs are prone to be degraded by various enzymes,and their short duration of action,lack of oral activity,relatively inability to cross the blood-brain barrier(BBB)and access to the central nervous system(CNS),plus their low bioavailability in vivo,those drawbacks have seriously limited their use in pain alleviation.So we decided to explore the new EM-1 analogues that can be easy to cross the BBB and with high stability,along with enhanced bioavailability.Our group have synthesized a novel unnatural a-methylene-p-amino acids(Map)and introduced it to the third and fourth position of EM-1.Turns out that the(ph)Map4-EM-1 exhibited high affinity and selectivity toward MOR.So we employ this modification and based on that sequence,we use different unnatural amino acids such as Dmt1,D-Ala2,NMe-D-Ala2 to substitute Tyr1 and Pro2,replace Trp3 with Gly3,in order to obtain different analogues that synthesized by solution-phase method with segment-coupling peptide synthesis strategy.Further examinations were used to determine their pharmacological activities which including their binding to receptors and antinociceptive activity,metabolic stability and isolated tissue assay.We found that all these 6 analogues exhibited increased in vitro metabolic stability to EM-1.Among these compounds,H-Dmt1-NMe-Ala2-Trp3-(ph)Map4-NH2(analogue 8)exhibited a remarkable highest affinity to μ-receptors(Kiμ=0.0207nm),and its affinity to δ-receptors(Kiδ=27.1 nM)was also the highest,and with the increased stability of half-time period>300min,but this leads to relatively low selectivity toward MOR,we supposed that analogue8 could be used as a μ/δ-opioid agonist;H-Tyr1-Pro2-Trp3-(ph)Map4-NH2(analogue 3)displayed high affinity(Kiμ=0.535nM)and higher selectivity toward MORs,which was 4.9 fold and 45 fold compared with EM-1 relatively,with increased stability in mouse brain homogenate and better antinociceptive activity,this provide a binding mode with μ-opioid receptor;H-Tyr1-NMe-D-Ala2-Trp3-(ph)Map4-NH2(analogue 7)exhibited just a slightly higher binding affinity(Kiμ=1.45nM),and others had no significant impact on ligand binding and selecitivity,neither on antinociception and in vitro bioactivity.