Structure-activity Relationships Of The Novel Dimeric, Side Chain And C-terminal Modified Analogs Of Endomorphins

Two endogenous opioid peptides, endomorphin-1 (EM-1: Tyr-Pro-Trp-Phe-NH₂) and endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH₂) were discovered in 1997 which showed high affinity and selectivity toward μ-opioid receptor. They are putative μ-opioid receptor endogenous ligands with potent analgesic activity. Unfortunately, the exogenous application of these opioid peptides generally met with failure, owing to their biological instability and inability to be transmitted through the blood-brain barrier (BBB). Therefore we synthesized their analogues by rational design and hoped to improve some of these abilities of them. The other purpose is to study the structure-activity relationships between their interactions with the opioid receptors and present some evidence to the development of new analgesics based on endomorphins. The major methods used here are dimerization, side chain modification and C-terminal modification. I have made a review about the opiate and endogenous opioid peptides especially endomorphins in this thesis. The main results are as follows:1. Designed and synthesized endomorphins and 12 analogues of them with C-terminal modification. Designed and synthesized morphiceptin and 11 analogues of endomorphins with side chain modifications. Designed and synthesized 14 dimeric analogues of endomorphins. Conventional solution phase method was used. All the final products were purified by HPLC or other methods, their molecular weights were identified by mass spectrometry.2. Determined the affinity and selectivity of all the analogues to μ and δ opioid receptors in the rat brain P₂ membrane by radio ligand competition experiment. Their activity on GPI and MVD preparation were assayed. Antinociceptive activity on mouse hot-plate test and degradation properties on mouse brain homogenate of some analogues were tested.The main results obtained through these experiments were listed below:1. Changing the amide group of endomorphins in the C-terminal into a smaller, neutral or weak basic group would improve their affinity to the μ opioid receptor, bigger or acidic group would have reversal effects. The spatial direction of the aromatic group in the C-terminal could significantly affect the affinity and selectivity of endomorphins.2. Introducing Hfe into position 3 or Phg into position 4 of endomorphins or morphiceptin could mimic the role of Phe very well, while D-Phg could not. The roles of thearomatic groups in position 3 and 4 are different. The analogue of endomorphin with Hfe in position 3 could prolong its time course of antinociception significantly, the main in vivo degradative site of endomorphins was between the position 2 and 3.3. Dimeric dipeptide or tripeptide fragments of endomorphins could increase the affinity of their monomers. The selectivity of dimeric endomorphin-2 wavily increased along with the increase of the lengths of the spacers. When the length of spacer was 2 or 12 atoms, the relative analogs had good activity on MVD assay. So we could thoroughly change the selectivity of endomorphins just through the method of dimerization and get a kind of useful compounds. They were very important to the structure-activity relationship study and could serve as special pharmacological probes of opioid peptides research.