乏氧选择性茚（1,2-b）喹喔啉-5,10-二氧-11-酮肟衍生物抗肿瘤前药的设计、合成与体外细胞毒性研究

发布时间：2018-03-05 17:01

本文选题：乏氧　切入点：喹喔啉　出处：《河北大学》2014年硕士论文　论文类型：学位论文

【摘要】：肿瘤的乏氧环境为肿瘤的治疗提供了靶点，，生物还原性药物能选择性地抑制乏氧肿瘤细胞生长，氮氧化物是其中具有代表性、有发展前景的一类化合物。喹喔啉类氮氧化物茚(1,2-b)喹喔啉-5,10-二氧-11-酮属于氮氧化物的一种，具有一定的乏氧选择活性，但其溶解性较差导致扩散能力弱，限制了其应用。针对茚(1,2-b)喹喔啉-5,10-二氧-11-酮脂溶性差的缺点，以邻苯二胺、水合茚三酮为起始原料，经缩合、肟化、醚化、氧化和胺化反应得到不同侧链长度及端位取代基的茚(1,2-b)喹喔啉-5,10-二氧-11-酮肟醚化合物。探讨溶剂、试剂等反应条件对收率的影响。采用IR、NMR、MS等手段对产物及中间体的结构进行表征；采用MTT法分别在常氧和乏氧条件下测定了茚(1,2-b)喹喔啉-5,10-二氧-11-酮肟醚对Hela、A549、MCF-7细胞系的体外细胞毒性，计算乏氧毒性比，探讨侧链长度及端位取代基的结构对乏氧毒性比的影响；采用PI单染法检测法及Annexin V/PI双染法凋亡检测II-a对Hela细胞周期的阻滞情况及凋亡作用；采用彗星实验在常氧及乏氧条件下检测IV-a对Hela细胞DNA的损伤情况，探究茚(1,2-b)喹喔啉-5,10-二氧-11-酮肟醚产生乏氧选择活性的机制。结果表明：肟醚链及端位取代基的引入，分子的脂溶性增大了3倍，水溶性增强了2倍。MTT结果显示茚(1,2-b)喹喔啉-5,10-二氧-11-酮肟醚具有较高的细胞毒性及一定的乏氧选择活性，化合物IV-a的乏氧条件下对于A549细胞IC50达到了2.34μmol/L，乏氧毒性比（HCR）为2.7。肟醚链及端位取代基引入后增大了化合物的细胞毒性与乏氧选择活性。当端位取代基相同时，侧链的长度越短化合物乏氧选择活性越强，当侧链长度相同时，端位取代基修饰的茚(1,2-b)喹喔啉-5,10-二氧-11-酮肟醚乏氧毒性比的顺序为哌啶吡咯二丁胺吗啉。细胞周期及细胞凋亡实验结果表明化合物II-a能将Hela细胞的细胞周期阻滞在G0/G1期，引起细胞凋亡，且随着化合物浓度的增大，细胞周期阻碍及细胞凋亡现象更加明显；当II-a浓度从5μmol/L升至10μmol/L后Hela细胞的G0/G1期比例从57.33%升至68.83%，早期凋亡比例从31.25%升至55.62%，晚期凋亡比例从0.13%升至2.20%。彗星实验结果表明化合物IV-a在乏氧条件下对Hela细胞DNA的损伤程度比在常氧条件下更加明显。在常氧条件下control组尾部DNA百分含量为0.01%，加药组为3.63%，乏氧条件下control组尾部DNA百分含量为0.01%，加药组为7.43%。茚(1,2-b)喹喔啉-5,10-二氧-11-酮肟醚在乏氧环境下所释放的羟基自由基是造成Hela细胞DNA损伤的可能原因，继而引起Hela细胞的细胞周期阻滞及凋亡。
[Abstract]:The hypoxic environment of tumor provides a target for the treatment of tumor. Bioreductive drugs can selectively inhibit the growth of hypoxic tumor cells. Quinoxaline indene 1 (2-b) quinoxaline -5o (10) -dioxo-11-one is a kind of nitrogen oxide, which has a certain hypoxic selective activity, but its poor solubility leads to weak diffusion ability, which limits its application. In view of the disadvantage of poor liposolubility of ninhydrin 1 / 2-b) quinoxaline-5 / 10-dioxo-11-one, starting from o-phenylenediamine and indenhydrin, it was synthesized by condensation, oximation and etherification. Different side chain lengths and end substituents were obtained by oxidation and amination to obtain quinoxaline 1 / 2-b) quinoxaline 10-dioxy-11-ketone oxime ether compounds. The effect of reaction conditions such as reagents on the yield was studied. The structure of the products and intermediates were characterized by IR NMRMS. The cytotoxicity of indene 12-b) quinoxaline -510-dioxo-11-ketoxime ether to HelaA549 MCF7 cell line was determined by MTT method under normoxic and hypoxic conditions, respectively. The effects of the length of side chain and the structure of the end substituent on the anoxia toxicity ratio were investigated. Pi single staining method and Annexin V / Pi double staining method were used to detect the cell cycle arrest and apoptosis of Hela cells induced by II-a, and comet assay was used to detect the DNA damage of Hela cells induced by IV-a under normoxic and hypoxic conditions. To explore the mechanism of anoxic selective activity of indene 1 2-b) quinoxaline-5-10-dioxo-11-ketone oxime ether. The results showed that the liposolubility of the molecule increased by 3 times and the water solubility increased by 2 times with the introduction of oxime ether chain and terminal substituents. The results showed that the indene quinoxaline-5o-10-dioxo-11-ketone oxime ether had high cytotoxicity and certain hypoxia selective activity. The anoxic toxicity of compound IV-a to A549 cells was 2.34 渭 mol / L and 2.7 渭 mol / L, respectively. When the oxime ether chain and the end substituents were introduced, the cytotoxicity and anoxic selective activity of the compounds were increased, and when the end substituents were the same, the cytotoxicity of the compounds was increased when the end substituents were the same. The shorter the length of the side chain, the stronger the anoxic selectivity of the compound, when the length of the side chain is the same, The order of anoxic toxicity ratio of quinoxaline to 10-dioxo-11-ketoxime ether was piperidine pyrrolidine dibutylamine morpholine. The results of cell cycle and apoptosis experiments showed that compound II-a could block the cell cycle of Hela cells in G _ 0 / G _ 1 phase. Apoptosis was induced, and with the increase of the concentration of compounds, cell cycle obstruction and apoptosis became more obvious. When the concentration of II-a increased from 5 渭 mol/L to 10 渭 mol/L, the G _ 0 / G _ 1 phase of Hela cells increased from 57.33% to 68.83, the proportion of early apoptosis increased from 31.25% to 55.62 and the proportion of late apoptosis increased from 0.13% to 2.20.The results of comet assay showed that the DNA of Hela cells was damaged by compound IV-a under hypoxic conditions. The degree of injury was more obvious than that under normoxic condition. Under normoxic condition, the content of DNA in tail of control group was 0. 01, that of drug added group was 3. 63, that of control group was 0. 01% and that of control group was 7. 4343%. Indanine 1 + 2-b) quinoxaline-510-dioxy-11-one oxime ether was added. The hydroxyl radical released in hypoxic environment is the possible cause of DNA damage in Hela cells. In turn, the cell cycle arrest and apoptosis of Hela cells were induced.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R914

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