咔唑类小分子DNMT抑制剂的合成及抗肿瘤活性研究

发布时间：2018-03-01 12:44

本文关键词： DNMT1 DNMT抑制剂咔唑衍生物抗肿瘤药物　出处：《广州中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:肿瘤的发生是一个多步骤、多阶段的复杂过程,严重危害了人类生命健康,近年来肿瘤发病率逐渐上升,寻找治疗有效的抗肿瘤药物迫在眉睫。研究表明,肿瘤的产生与DNA甲基化异常紧密相关。以DNA甲基化转移酶(DNAmethyltransferases,DNMT)为靶点的抗肿瘤药物研究是当前的热门领域。课题组前期基于DNMT1复合物晶体,利用药物设计手段开展了 DNMT1抑制剂虚拟筛选,发现了两类含咔唑母核结构的小分子抑制剂,并测定了其抗肿瘤等生物活性,在此基础上,本文继续在咔唑骨架3位或者3,6位引入溴元素,同时改变母核侧链,以获得一系列咔唑类小分子DNMT抑制剂,同时筛选高活性抗肿瘤化合物。方法:1、咔唑类小分子化合物的设计及合成本文以咔唑为原料,经由溴化、N-烷基化等步骤,合成关键中间体3-溴-9-(环氧丙烷-2-甲基)-9H-咔唑和3,6-二溴-9-(环氧丙烷-2-甲基)-9H-咔唑,之后与一系列胺合成两个系列43个单分子咔唑和1个双分子咔唑衍生物。目标化合物结构经熔点、薄层色谱、LC-MS、1H-NMR、13C-NMR确证。2、咔唑类小分子化合物体外活性研究2.1采用酶联免疫吸附法(ELISA)测定了合成的44个化合物对DNMT1的抑制率。2.2通过MTT法检测目标化合物的抗肿瘤活性。该方法以HCT116、MNK-45和A549为测试细胞株,分别以吉西他滨、紫杉醇、顺铂为阳性药物,得到化合物对细胞增殖的半数抑制浓度,评价化合物的抗肿瘤活性。2.3采用HCT116细胞株,流式细胞术PI染色检测化合物2、12对肿瘤细胞周期的影响,Annexin V-FITC/PI双染检测化合物2、12、42是否造成肿瘤细胞的早期凋亡。结果:1、本课题共合成44个咔唑类小分子化合物。2、化合物 1、2、12、13、24、25、26、30、31、34、35、37、41、42 对 DNMT1催化活性有不同程度的抑制作用,并呈一定的剂量依赖,其中化合物2、12、25、26、41、42半数抑制率IC50分别为10、9.6、16、2.7、14、1.2 μM。MTT实验分析表明:1)绝大部分化合物能不同程度的抑制三种肿瘤细胞增殖,且对三种细胞增殖的影响与药物作用浓度呈线性相关,其中对HCT116、MNK-45细胞增殖的抑制作用呈时间依赖性,其中以化合物2抑制作用最好,该化合物与HCT116、MNK-45、A549作用24h的IC50分别为 4.12±0.22、5.53±0.20、2.68±0.08 μM;2)化合物 2、12能够诱导HCT116细胞周期的G1期阻滞,并呈剂量依赖性;3)化合物2、12、42能诱导HCT116细胞凋亡,并跟随浓度的增大,调亡细胞明显增加。结论:本文合成了 44个含咔唑母核结构的小分子化合物,并对它们进行了结构表征。通过酶活抑制实验和体外抗肿瘤活性筛选实验发现化合物2、12、42不仅对DNMT1有一定的抑制活性,而且对三种肿瘤细胞均有较强抑制作用,以上结果为进一步的抗肿瘤药物研究提供参考价值。
[Abstract]:Objective: tumorigenesis is a multi-step, multi-stage complex process, which seriously endangers human life and health. In recent years, the incidence of cancer has gradually increased, so it is urgent to find effective anti-tumor drugs. The production of tumor is closely related to the abnormal methylation of DNA. The research of anti-tumor drugs targeting DNA methyltransferase DNA-methyltransferasesof DNMTis is a hot field at present. Two kinds of small molecular inhibitors containing carbazole mother nuclear structure were found by virtual screening of DNMT1 inhibitors by drug design, and their antitumor activities were measured. In order to obtain a series of carbazole small molecule DNMT inhibitors, bromine elements were introduced into carbazole skeleton at 3 or 3 ~ 6 sites, and the side chain of parent nucleus was changed simultaneously. Methods the design and synthesis of small molecular compounds of carbazole with high activity were studied in this paper, carbazole was used as raw material and N-alkylation of carbazole was used as raw material. Synthesis of key Intermediates 3- Bromo-9-( Propylene oxide -2methyl-9H-Carbazole and 3OX-6-Dibromo-9-( Propylene oxide -2-Methyl Con -9H-Carbazole), Two series of 43 monomolecular carbazole derivatives and one bimolecular carbazole derivative were synthesized with a series of amines. LC-MS1H-NMR-13C-NMR confirmed in vitro activity of carbazole compounds 2.1 the inhibition rate of 44 compounds to DNMT1 was determined by enzyme linked immunosorbent assay (Elisa) .2.2 the inhibition rate of the synthesized compounds to DNMT1 was determined by MTT method. HCT116MNK-45 and A549 were used as test cells. Using gemcitabine, paclitaxel and cisplatin as positive drugs, the median inhibitory concentration of the compound on cell proliferation was obtained. The antitumor activity of the compound was evaluated by HCT116 cell line. Flow cytometry Pi staining to detect the effect of compound 2n12 on the cell cycle of tumor cells: Annexin V-FITC / Pi double staining to detect whether compound 2n12C42 causes early apoptosis of tumor cells. Results: 1, 44 carbazole small molecule compounds. 2 were synthesized in this study. The complex 1 ~ (2) C ~ (2) C ~ (12) C ~ (12) C ~ (2 +) ~ (24) C ~ (25) C ~ (26) C ~ (30) C ~ (31) C ~ (34) C ~ (3 +) ~ (3) 3 ~ (5) C ~ (3 +). In a dose-dependent manner, the half inhibition rate (IC50) of compound 212A12O25T26O2641O42 was 109.6U 162.7U 141.2 渭 M. MTT analysis showed that most of the compounds could inhibit the proliferation of three kinds of tumor cells in varying degrees, and the results showed that most of the compounds could inhibit the proliferation of three kinds of tumor cells in different degrees. The inhibitory effect on the proliferation of HCT116 MNK-45 cells was time-dependent, and compound 2 had the best inhibitory effect on the proliferation of HCT116MNK-45 cells. The IC50 of the compound treated with HCT116MNK-45A549 for 24 hours was 4.12 卤0.225.53 卤0.205.53 卤0.202.68 卤0.08 渭 MN ~ (2)) compound 2Y12 could induce the G1 phase arrest of HCT116 cell cycle, and in a dose-dependent manner, 2N _ (1242) could induce the apoptosis of HCT116 cells and follow the increase of the concentration. Conclusion: 44 small molecular compounds containing carbazole mother nuclear structure were synthesized. Through enzyme activity inhibition test and in vitro anti-tumor activity screening experiment, it was found that compound 2n12O42 not only had a certain inhibitory activity to DNMT1, but also had a strong inhibitory effect on three kinds of tumor cells. The above results provide a reference value for the further study of anti-tumor drugs.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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