本文選題：EGFR抑制劑 + 二苯乙烯基嘧啶類　； 參考：《大連醫(yī)科大學》2017年碩士論文

【摘要】：目的:設計合成二苯乙烯基嘧啶類新型表皮生長因子受體(epidermal growth factor receptor,EGFR)抑制劑,通過初步評價其對非小細胞肺癌(non-small-cell carcinoma,NSCLC)的抑制活性及毒性,以期篩選出活性更好、毒性更低、抗耐藥性更加優(yōu)良的化合物。方法:以第三代EGFR抑制劑Rociletinib為先導物,基于分子雜合策略,設計、合成一系列新型二苯乙烯基嘧啶衍生物;借助1H NMR、13C NMR、HRMS、MS對新化合物進行分子結(jié)構(gòu)鑒定;采用體外熒光激酶檢測方法,測定所得到新化合物分別對野生型EGFR(wild-type EGFR,EGFRWT)和EGFRT790M/L858R(EGFR 790位Thr→Met,EGFR 858位Leu→Arg)激酶的抑制作用活性;以MTT比色法測定不同濃度新化合物作用于選定細胞株72小時以后對于細胞存活率的影響;采用DAPI染色及MTT法測定優(yōu)選化合物與上市藥物Gefitinib在相同條件下抗腫瘤活性;以流式細胞術(shù)來考察優(yōu)選化合物對肺癌細胞H1975凋亡的影響;利用薄膜分散法將優(yōu)選化合物SAR-007制備成藥物脂質(zhì)體;利用MTT法檢測由化合物SAR-007制備的藥物脂質(zhì)體、空白脂質(zhì)體及化合物SAR-007對非小細胞肺癌細胞H1975存活率影響。結(jié)果:合成純化獲得12個新型二苯乙烯基嘧啶類EGFR抑制劑,通過核磁共振及高分辨質(zhì)譜技術(shù)確定目標化合物結(jié)構(gòu)正確;激酶試驗中新化合物普遍對EGFRT790M/L858R顯示出良好的抑制作用,IC50值(half maximal inhibitory concentration,半抑制濃度)在6.8-29.4 nMol/L范圍內(nèi),較參照藥Gefitinib(IC50:1202 nMol/L)活性提高40-176倍;化合物SAR-007對EGFRT790M/L858R激酶(IC50:11.0 nMol/L)表現(xiàn)出強的抑制作用,和較高的選擇性(SI=49,SI=EGFRWT:EGFRT790M/L858R);MTT實驗結(jié)果顯示,絕大部分新化合物對A431鱗癌細胞和A549肺腺癌細胞有強的抗增殖活性,較Gefitinib提高2-35倍,對肺上皮正常細胞HBE的毒性性較上市藥物有所降低。尤其是化合物SAR-006(IC50:2.318μMol/L)和SAR-007(1C50:2.91μMol/L)對耐藥型H1975肺癌細胞也表現(xiàn)出較為良好的抑制活性,且對HBE細胞的半抑制濃度大于20μMol/L,遠大于其有效抑制H1975細胞增殖的濃度;DAPI熒光染色結(jié)果直觀顯示,化合物SAR-007在相同條件下對于H1975肺癌細胞的抑制活性較上市藥物效果更為顯著;藥物濃度-存活率曲線實驗結(jié)果表明,SAR-007對于H1975的殺傷作用強于Gefitinib;細胞凋亡試驗結(jié)果表明備選化合物SAR-007對H1975細胞的抑制作用以濃度依賴性方式明顯增加,凋亡率范圍為40.4-79.8%;通過薄膜分散法制備的SAR-007脂質(zhì)體粒徑分布均勻(Z-Average:197.7nm,PdI:0.354),外觀良好,通過MTT法所測得結(jié)果顯示藥物制備成脂質(zhì)體后對于H1975細胞的抑制活性有小幅度提高。結(jié)論:本研究得到12個新型二苯乙烯基嘧啶類EGFR抑制劑,該系列化合物具有良好的抑制野生型的EGFR和突變型EGFRT790M活性;發(fā)現(xiàn)化合物SAR-007對突變型EGFRT790M表現(xiàn)更加優(yōu)良的抑制活性、選擇性和細胞毒性;化合物SAR-007通過作用于EGFRT790M激酶,進而有效抑制肺癌細胞產(chǎn)生的耐藥性;SAR-007脂質(zhì)體實驗為此類分子的活性改善提供了借鑒;本論文的結(jié)果為二苯乙烯基嘧啶類新型靶向EGFR抑制劑的深入開發(fā)提供了依據(jù)。
[Abstract]:Objective: to design and synthesize a new type of epidermal growth factor receptor (EGFR) inhibitor for two styrene pyrimidine, and to evaluate its inhibitory activity and toxicity on non small cell lung cancer (non-small-cell carcinoma, NSCLC) by preliminary evaluation, in order to screen the compounds with better activity, lower toxicity and better resistance to resistance. Methods: a series of new two styrene pyrimidine derivatives were designed and synthesized based on the molecular heterozygosity strategy based on the third generation of EGFR inhibitor Rociletinib. The molecular structure of the new compounds was identified by 1H NMR, 13C NMR, HRMS, MS, and the new compounds were detected by in vitro fluorescence kinase assay, and the new compounds were determined to be in the wild type EGFR (wild) respectively. The inhibitory activity of -type EGFR, EGFRWT) and EGFRT790M/L858R (EGFR 790 Thr to Met, EGFR 858 Leu to Arg), and the effect of different concentrations of new compounds on cell viability after 72 hours of selected cell lines by MTT colorimetric method; Antitumor activity under the same condition; the effect of optimal compounds on H1975 apoptosis in lung cancer cells was investigated by flow cytometry; liposomes were prepared by the method of thin film dispersion, and liposomes prepared by compound SAR-007 were detected by MTT method, and the blank liposomes and compound SAR-007 were fine for non-small cell lung cancer. The effect of H1975 survival rate. Results: 12 new two styrene pyrimidine EGFR inhibitors were synthesized and purified. The structure of the target compounds was correctly determined by NMR and high resolution mass spectrometry; the new compounds in the kinase test showed a good inhibitory effect on EGFRT790M/L858R, and the IC50 value (half maximal inhibitory concentrati). On, semi inhibitory concentration) in the range of 6.8-29.4 nMol/L, the activity of Gefitinib (IC50:1202 nMol/L) was 40-176 times higher than that of the reference drug Gefitinib (IC50:1202 nMol/L); the compound SAR-007 showed strong inhibition to EGFRT790M/L858R kinase (IC50:11.0 nMol/L), and higher selectivity (SI=49, SI=EGFRWT:). 431 squamous cell carcinoma cells and A549 lung adenocarcinoma cells have strong anti proliferative activity, 2-35 times higher than that of Gefitinib, and the toxicity of HBE in normal lung epithelial cells is lower than that of the listed drugs. Especially, compound SAR-006 (IC50:2.318 mu Mol/L) and SAR-007 (1C50:2.91 u Mol/L) also exhibit better inhibitory activity to drug-resistant H1975 lung cancer cells. The semi inhibitory concentration of HBE cells was greater than 20 mu Mol/L, which was far greater than the inhibitory concentration of H1975 cells. The results of DAPI fluorescence staining showed that the inhibitory activity of compound SAR-007 on H1975 lung cancer cells was more significant than that of the listed drug; the experimental results of drug concentration survival curve showed that SAR-007 was a H for H. The killing effect of 1975 was stronger than that of Gefitinib; the results of apoptosis test showed that the inhibitory effect of SAR-007 on H1975 cells increased significantly in a concentration dependent manner, and the range of apoptosis rate was 40.4-79.8%; the SAR-007 liposomes prepared by the membrane dispersion method were evenly distributed (Z-Average:197.7nm, PdI:0.354), and the appearance was good, through MTT. The results measured by the method showed that the inhibitory activity of H1975 cells was slightly improved after the preparation of the liposomes. Conclusion: 12 new two styrene pyrimidine EGFR inhibitors have been obtained. This series of compounds have a good inhibition of the wild type EGFR and the mutant EGFRT790M activity, and the compound SAR-007 to the mutant EGFRT790M is found. Better inhibition of activity, selectivity and cytotoxicity, compound SAR-007 effectively inhibits the drug resistance of lung cancer cells by acting on EGFRT790M kinase, and the SAR-007 liposome experiment provides a reference for the improvement of the activity of such molecules; the results of this paper are the depth of the new target EGFR inhibitor of two styrene pyrimidine. It provides a basis for development.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2

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