Synthesis And Anticancer Activity Of A Series Of Novel Celastrol Derivatives With Amino Acid Chain

Cancer has become the number one of disease killer in human life and health.According to statistics,there are 7,500 people died of cancer average daily in china in 2016.The incidence of malignant tumors is increasing dramatically.For human being,it is imminent to explore and develop new anticancer drugs.Celastrol,is a pentacyclic triterpenoid natural product,posses broad biological activities,including anticancer,antiinflammatory,antivirus,immunosuppressive,proteasome inhibitors and antiobesity.Among them,anticancer activity is the most significant activity of many pharmacokinetics of celastrol.A large number of domestic and foreign researches have shown that celastrol has significantly antiproliferation and direct killing effect on variety of cancer cell lines,which indicating great research value and development potential.Although celastrol has a significant inhibitory effect on a variety of cancer cell lines,its high toxicity,low oral bioavailability and poor water solubility restricts its application.Thus,it's necessary to study on structure modification of celastrol.Firstly,amino acid esters are intermediates of a variety of drugs and have certain biological activity,including special permeability,low toxicity and enzyme catalytic specificity.The introduction of the amino acid moiety at the structure of drugs would improve the dissolution,the absorption and the bioavailability of drugs inside the body.Secondly,the cancer cells due to their own vigorously metabolism,their demand for amino acids are far greater than that of the normal cells.In addition,numerous studies have been reported that many natural products with a certain biological activity and drugs are modified by amino acids,amino acid derivatives and peptides.Finally,some short peptides have selective anticancer activities.The main purpose of this thesis is to synthesize eight amino acid ester intermediates with five amino acids as raw materials,and then to realize the bonding of amino acids with celastrol.The purpose of the current work is to improve the solubility of celastrol and obtain novel celastrol derivatives which are less toxicity and higher bioactivities.These researches support further studies of targeted administration and action mechanism of celastrol in the future.The main contents of the current thesis include the following aspects:1.The synthesis of amino acid esters?including methyl esters,ethyl esters,isopropyl esters,etc.?intermediates were optimized by thionyl chloride method using five kinds of amino acids?alanine,glycine,serine,valine and phenylalanine?as raw materials.Then the eight title compounds were obtained by reacting the amino acid ester intermediates with celastrol by amide condensation reaction catalyzed by 1-ethyl-?3-dimethylaminopropyl?carbodiimide hydrochloride?EDCl?,1-hydroxybenzo triazole?HOBT?and triethylamine?TEA?in anhydrous CH2Cl2 conditions.2.The structures of all the target compounds were identified and analyzed by modern spectroscopy(IR,¹H-NMR,^13C-NMR and ESI-MS).3.The antiproliferation activity of celastrol and the target product was carried out by MTT assay with Hela,A549 and Hep G-2 cell lines,and the IC50 values of the compounds were calculated and compared with cisplatin as positive control.The most significant inhibitory activities of title compounds 2c?IC50 = 0.235 ?M?and 2d?IC50 = 0.109 ?M?were screened out.Further apoptosis assays were carried out by AO/EB and Annexin V-FITC / PI double staining method,and the results were detected by fluorescence microscopy and flow cytometry,respectively.4.Toxic effect of title compounds 2c,2d and celastrol on embryo development of zebrafish were studied;and the toxicity of Zebrafish embryos in vivo suggested that the derivative 2d(LC₅₀ = 2.0 ?M)has lower toxicity than 2c(LC₅₀ = 1.3 ?M)and the parent compound celastrol(LC₅₀ = 1.4 ?M)...