二苯乙烯基嘧啶类新型靶向EGFR抑制剂的合成、抗NSCLC药理活性初步评价及相关药物脂质体研究

发布时间：2018-07-01 15:15

本文选题：EGFR抑制剂 + 二苯乙烯基嘧啶类　；参考：《大连医科大学》2017年硕士论文

【摘要】：目的:设计合成二苯乙烯基嘧啶类新型表皮生长因子受体(epidermal growth factor receptor,EGFR)抑制剂,通过初步评价其对非小细胞肺癌(non-small-cell carcinoma,NSCLC)的抑制活性及毒性,以期筛选出活性更好、毒性更低、抗耐药性更加优良的化合物。方法:以第三代EGFR抑制剂Rociletinib为先导物,基于分子杂合策略,设计、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对新化合物进行分子结构鉴定;采用体外荧光激酶检测方法,测定所得到新化合物分别对野生型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比色法测定不同浓度新化合物作用于选定细胞株72小时以后对于细胞存活率的影响;采用DAPI染色及MTT法测定优选化合物与上市药物Gefitinib在相同条件下抗肿瘤活性;以流式细胞术来考察优选化合物对肺癌细胞H1975凋亡的影响;利用薄膜分散法将优选化合物SAR-007制备成药物脂质体;利用MTT法检测由化合物SAR-007制备的药物脂质体、空白脂质体及化合物SAR-007对非小细胞肺癌细胞H1975存活率影响。结果:合成纯化获得12个新型二苯乙烯基嘧啶类EGFR抑制剂,通过核磁共振及高分辨质谱技术确定目标化合物结构正确;激酶试验中新化合物普遍对EGFRT790M/L858R显示出良好的抑制作用,IC50值(half maximal inhibitory concentration,半抑制浓度)在6.8-29.4 nMol/L范围内,较参照药Gefitinib(IC50:1202 nMol/L)活性提高40-176倍;化合物SAR-007对EGFRT790M/L858R激酶(IC50:11.0 nMol/L)表现出强的抑制作用,和较高的选择性(SI=49,SI=EGFRWT:EGFRT790M/L858R);MTT实验结果显示,绝大部分新化合物对A431鳞癌细胞和A549肺腺癌细胞有强的抗增殖活性,较Gefitinib提高2-35倍,对肺上皮正常细胞HBE的毒性性较上市药物有所降低。尤其是化合物SAR-006(IC50:2.318μMol/L)和SAR-007(1C50:2.91μMol/L)对耐药型H1975肺癌细胞也表现出较为良好的抑制活性,且对HBE细胞的半抑制浓度大于20μMol/L,远大于其有效抑制H1975细胞增殖的浓度;DAPI荧光染色结果直观显示,化合物SAR-007在相同条件下对于H1975肺癌细胞的抑制活性较上市药物效果更为显著;药物浓度-存活率曲线实验结果表明,SAR-007对于H1975的杀伤作用强于Gefitinib;细胞凋亡试验结果表明备选化合物SAR-007对H1975细胞的抑制作用以浓度依赖性方式明显增加,凋亡率范围为40.4-79.8%;通过薄膜分散法制备的SAR-007脂质体粒径分布均匀(Z-Average:197.7nm,PdI:0.354),外观良好,通过MTT法所测得结果显示药物制备成脂质体后对于H1975细胞的抑制活性有小幅度提高。结论:本研究得到12个新型二苯乙烯基嘧啶类EGFR抑制剂,该系列化合物具有良好的抑制野生型的EGFR和突变型EGFRT790M活性;发现化合物SAR-007对突变型EGFRT790M表现更加优良的抑制活性、选择性和细胞毒性;化合物SAR-007通过作用于EGFRT790M激酶,进而有效抑制肺癌细胞产生的耐药性;SAR-007脂质体实验为此类分子的活性改善提供了借鉴;本论文的结果为二苯乙烯基嘧啶类新型靶向EGFR抑制剂的深入开发提供了依据。
[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.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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