Design And Synthesis Of Endothelin Receptor Antagonists

Endothelin(ET) is a family of peptides containing 21 amino acids with potent vasoconstrictor properties that was originally identified in conditioned medium from cultured porcine endothelial cells. It has since been found in different animal and human cells including endothelial cells, smooth muscle, macrophages, epithelial cells, hepatocytes, glial cells, mesangial cells and astrocytes. The endothelin family comprises of four related peptides: ET-1, ET-2, ET-3 and ET-4, whose biological functions are mediated in mammals. Most scholars divided ET receptor into three subtypes: ETA, ETB and ETC. In the vasculature ETA receptors were first identified on smooth muscle and were recognized to mediate the ET-1-induced vasoconstrictor actions. ETB has the close affinity to the ET isomerism peptides, is also called the non-specificity ET acceptor, mainly distributes in the heart, the lung and the adrenal gland. ETB was divided into two hypotypes: ETB1 and ETB2. ETB1 acceptor leads the dilivery of endothelium-derived factor (EDRF), nitrogen monoxide (NO) and prostaglandin-II (PGI2), caused the diastole of blood vessel. ETB2 leads the vein blood vessel's contraction located at blood vessel smooth muscle as well as ETA. ETC is the ET-3 specificity union acceptor, mainly distributes in the nerve and outside the marrow tubulus renalis epithelial cell. Endothelin has been implicated in the pathophysiology of a large number of diseases. Significantly raised levels of ET-1 have been detected in the plasma, and other biological fluids, of patients suffering from congestive heart failure, stable and unstable angina, acute myocardial infarction, primary pulmonary hypertension, acute renal insufficiency/failure, shock (septic and cardiogenic), cerebral schaemia/stroke (haemorrhagic and nonhaemorrhagic), asthma, and metastatic prostate cancer.To overcome the unwanted effects of an abnormally regulated ET system, ET receptor antagonists are used in the treatment of these diseases, and a number of different peptidyl and nonpeptidyl ligands, both selective and nonselective, have been developed. Early small peptide inhibitors included BQ-123, BQ-485 and FR-139317, all of them are potent ETA selective antagonists. Additional several non-peptide non-selective antagonists have appeared in the literatures including Tezosentan, Bosentan, Enrasentan and SB 209670, as well as the more ETA selective antagonists Sitaxsetan,Ambrisentan , BMS 182874, PD156707, A-127722 and TBC-3711.Referring to plenty of informations on ET receptor antagonists and previous studies, we selected ETA antagonists BQ-485 as leading compound to develop new derivatives. By changing the ureido-structure of N-terminal, and introducing some unnatural amino acids as well as modifying the structure amino acid sequence, we could change peptide characters such as molecular conformation definition, hydrophilic property and so on. Subsequently the new structure type endothelin acceptor antagonists could be obtained. Through the ET receptor antagonistic activities were screened, we do our best to find one kind of tripeptide ET acceptor antagonists maintaining the high activity, increasing aqueous solubility, further lightening the skin irritation brought by the intramuscular injection, reducing the liver poisonous side effect, and antagonizing enzymolysis.In this thesis, the aimed tripeptide sequences as follows:Profiting from the functional group structural research achievement of the ETA acceptor antagonist BQ485 and Atrasentan(ABT-627), we used dibutylamine (DBA) as the RN groups and synthesized the ammonia acetazolamide and the ammonia armor carbonyl structure for changing the ureido-structure at the N-terminus. The N-terminal AA1 was selected from those hydrophobic amino acids such as Leu,modified Leu. The second amino acid AA2 was D-Trp and modified D(L)-Pro alternatively; The C-terminal amino acid AA3 was preferred to the aromatic D- amino acids such as D-Trp, D-(4-Cl)-Phe.Concerning the modification of amino acids structure, two aspects were conducted: (1) First of all, N-alkylation, acetyl modification on the Leu could decrease the degradation by aminopeptidase, inhibit protease hydrolysis prospectively. On the other hand, aromatic groups attached to the 4 position of proline introduced as the second amino acid AA2 in the tripeptides. At the same time, this strategy of linking the residues of C-terminal aromatic amino acids could be expected to improve the chemical physics nature of the compounds. Simulation the nature of D-Trp residues, we choosed the group of (1H-indol-3-yl)acetyl, which might have some impacts on antagonist activities. Further more, as pepper methyl included the epoxy ether linkage introduce at the appropriate position such AA2, it would increase the conformation restriction and hydrophilic. (2) Based on preliminary biological active evaluation of the designed and synthesized peptides which contained the non-ureido-structures at N-terminus and AA2 was replaced by modified proline. In this study, retaining the group of (1H-indol-3-yl)acetyl in the modified proline and introducing non-ureido-structures to tripeptide sequences which could expected to increase compound activity, aqueous solubility, the capacity of anti-enzyme and reduce irritation.In this thesis, the designed endothelin receptor antagonists were all small peptides and the N-terminus also were introduced by small molecules. In order to enhance the efficiency of the peptide synthesis, 8 starting material amino acids have been synthesized by the strategy of liquid. The structure of these amino acids has been identified by the mass spectrum, the nuclear magnetic resonance and the polariscopy. Subsequently, 20 new peptidyl compounds have been synthesized and purified by the middle pressure liquid chromatography (MPLC). The purity analyzed by the HPLC. The structure of compounds has been determined by the mass spectrum, the nuclear magnetic resonance.Comparing to the reported ETA antagonists BQ-485 and ETP-508, the results of preliminary screening tests in vitro showed that 6 compounds had similar activities. The primary structure-activity relationships of these compounds were deduced roughly according to the current data: 1) the ureido-structure at the N-terminus could be not an indispensable group; 2) the residue of D-Trp might be a necessary group; 3) containing indole ring non-amino acid unit such as (1H-indol-3-yl) acetyl could simulate the feature of D-Trp residue. Simultaneously the preliminary relationship between the partial irritation and structure of the compound were discussed that changing the ureido-structure at the N-terminus would probably reduce or eliminate partial the skin irritation.