本文關鍵詞： DNMT1 DNMT抑制劑 咔唑衍生物 抗腫瘤藥物　出處：《廣州中醫(yī)藥大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:腫瘤的發(fā)生是一個多步驟、多階段的復雜過程,嚴重危害了人類生命健康,近年來腫瘤發(fā)病率逐漸上升,尋找治療有效的抗腫瘤藥物迫在眉睫。研究表明,腫瘤的產生與DNA甲基化異常緊密相關。以DNA甲基化轉移酶(DNAmethyltransferases,DNMT)為靶點的抗腫瘤藥物研究是當前的熱門領域。課題組前期基于DNMT1復合物晶體,利用藥物設計手段開展了 DNMT1抑制劑虛擬篩選,發(fā)現(xiàn)了兩類含咔唑母核結構的小分子抑制劑,并測定了其抗腫瘤等生物活性,在此基礎上,本文繼續(xù)在咔唑骨架3位或者3,6位引入溴元素,同時改變母核側鏈,以獲得一系列咔唑類小分子DNMT抑制劑,同時篩選高活性抗腫瘤化合物。方法:1、咔唑類小分子化合物的設計及合成本文以咔唑為原料,經由溴化、N-烷基化等步驟,合成關鍵中間體3-溴-9-(環(huán)氧丙烷-2-甲基)-9H-咔唑和3,6-二溴-9-(環(huán)氧丙烷-2-甲基)-9H-咔唑,之后與一系列胺合成兩個系列43個單分子咔唑和1個雙分子咔唑衍生物。目標化合物結構經熔點、薄層色譜、LC-MS、1H-NMR、13C-NMR確證。2、咔唑類小分子化合物體外活性研究2.1采用酶聯(lián)免疫吸附法(ELISA)測定了合成的44個化合物對DNMT1的抑制率。2.2通過MTT法檢測目標化合物的抗腫瘤活性。該方法以HCT116、MNK-45和A549為測試細胞株,分別以吉西他濱、紫杉醇、順鉑為陽性藥物,得到化合物對細胞增殖的半數(shù)抑制濃度,評價化合物的抗腫瘤活性。2.3采用HCT116細胞株,流式細胞術PI染色檢測化合物2、12對腫瘤細胞周期的影響,Annexin V-FITC/PI雙染檢測化合物2、12、42是否造成腫瘤細胞的早期凋亡。結果:1、本課題共合成44個咔唑類小分子化合物。2、化合物 1、2、12、13、24、25、26、30、31、34、35、37、41、42 對 DNMT1催化活性有不同程度的抑制作用,并呈一定的劑量依賴,其中化合物2、12、25、26、41、42半數(shù)抑制率IC50分別為10、9.6、16、2.7、14、1.2 μM。MTT實驗分析表明:1)絕大部分化合物能不同程度的抑制三種腫瘤細胞增殖,且對三種細胞增殖的影響與藥物作用濃度呈線性相關,其中對HCT116、MNK-45細胞增殖的抑制作用呈時間依賴性,其中以化合物2抑制作用最好,該化合物與HCT116、MNK-45、A549作用24h的IC50分別為 4.12±0.22、5.53±0.20、2.68±0.08 μM;2)化合物 2、12能夠誘導HCT116細胞周期的G1期阻滯,并呈劑量依賴性;3)化合物2、12、42能誘導HCT116細胞凋亡,并跟隨濃度的增大,調亡細胞明顯增加。結論:本文合成了 44個含咔唑母核結構的小分子化合物,并對它們進行了結構表征。通過酶活抑制實驗和體外抗腫瘤活性篩選實驗發(fā)現(xiàn)化合物2、12、42不僅對DNMT1有一定的抑制活性,而且對三種腫瘤細胞均有較強抑制作用,以上結果為進一步的抗腫瘤藥物研究提供參考價值。
[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 鹵0.225.53 鹵0.205.53 鹵0.202.68 鹵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.
【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R914;R96

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本文編號：1552011