低氧激活O~6-苄基鸟嘌呤衍生物的合成与活性研究

发布时间：2018-04-28 10:48

本文选题：O6-烷基鸟嘌呤-DNA-烷基转移酶 + AGT抑制剂　；参考：《北京工业大学》2016年硕士论文

【摘要】：氯乙基亚硝基脲(CENUs)是一类重要的双官能团抗肿瘤烷化剂,广泛用于脑瘤、骨髓瘤、神经胶质瘤、恶性黑色素瘤等恶性肿瘤的治疗。目前临床上常用的CENUs主要包括尼莫司汀(Nimustine,ACNU),卡莫司汀(Carmustine,BCNU),洛莫司汀(Lomustine,CCNU),司莫司汀(Semustine,MeCCNU)等。CENUS可以导致DNA形成dG-dC股间交联,从而发挥其抗癌作用。但是,肿瘤细胞内的O6-烷基鸟嘌呤-DNA-烷基转移酶(O6-alkylguanine-DNA alkyltransferase,AGT)可以对CENUs导致的DNA损伤进行修复,使得肿瘤细胞对CENUs产生耐药性。O6-苄基鸟嘌呤(O6-benzylguanine,O6-BG)是一种有效的AGT抑制剂,它可以将O6位上的苄基转移到AGT的活性中心Cys145上,导致AGT失去活性,从而提高肿瘤细胞对CENUs的敏感性。然而,O6-BG由于缺少靶向性,在作用于肿瘤细胞的同时也会抑制正常细胞中AGT活性,从而增加正常细胞对CENUs的敏感性。本研究通过分子模拟的方法对AGT抑制剂的作用机理进行研究,合成了三种新型低氧激活AGT抑制剂,并通过细胞活性实验对三种AGT的活性进行评价。使用分子对接、分子动力学模拟以及量子化学计算的方法对O6-甲基鸟嘌呤(O6-MeG)、O6-苄基鸟嘌呤(O6-BG)和O6-溴噻吩基鸟嘌呤(4-BTG)对AGT的抑制作用机理进行了研究。构建了AGT-O6-MeG、AGT-O6-BG和AGT-4-BTG三种底物与AGT蛋白(1T38)形成的复合物分子结构;然后,截取复合物活性中心的关键氨基酸残基和底物分子作为计算模型,使用分层计算法(ONIOM)在M062X/6-31+G(d,p):AM1水平上对AGT活性中心第145位半胱氨酸(Cys145)转移O6-MeG、O6-BG和4-BTG上O6-烷基的作用机理进行了研究。结果表明,4-BTG中溴噻吩基转移的能垒显著低于O6-MeG中甲基和O6-BG中苄基转移的能垒,这与4-BTG比O6-MeG和O6-BG具有更高的AGT抑制活性相吻合。由于实体肿瘤生长迅速,使得肿瘤组织氧供减少,处于缺氧状态。利用肿瘤组织中存在低氧微环境的特点合成了三种低氧激活的O6-BG类似物(Hypoxia activated O6-BG derivatives,HA-BG)——N2-((((4-硝基苄基)氧)羰基)氨基)-N9-氯乙基-O6-苄基鸟嘌呤(HA-BG-1)、N2-((((4-硝基苄基)氧)羰基)氨基)-N9-溴乙基-O6-苄基鸟嘌呤(HA-BG-2)和N2-((((4-硝基苄基)氧)羰基)氨基)-N9-(2-胺基)乙基)-O6-苄基鸟嘌呤(HA-BG-3)。这三种化合物能够在低氧环境下发生分解,分别释放出N9-氯乙基-O6-苄基鸟嘌呤(HA-BG-1A)、N9-溴乙基-O6-苄基鸟嘌呤(HA-BG-2A)和N9-(2-胺基)乙基-O6-苄基鸟嘌呤(HA-BG-3A)。对三种AGT抑制剂的分子结构进行了1H NMR、IR、UV和ESI-MS鉴定。对HA-BG-1、HA-BG-2和HA-BG-3的AGT抑制效果和低氧激活作用进行研究。将3种HA-BG与ACNU联合处理神经胶质瘤细胞SF763,分别设置有氧条件和无氧条件下的ACNU组、ACNU与O6-BG联合用药组以及ACNU与3种HA-BG的联合用药组。使用CCK-8法测定各实验组的细胞存活率。结果表明,3种HA-BG与ACNU联合用药组在低氧条件下的细胞存活率显著低于有氧条件下的实验组(p0.01),且HA-BG-2的AGT抑制活性最强。由此可见,HA-BG-1、HA-BG-2和HA-BG-3能够选择性地在低氧条件下被激活,释放出O6-BG类似物,从而靶向性地发挥对肿瘤细胞的AGT抑制作用。分别将HA-BG-1、HA-BG-2和HA-BG-3以及它们被低氧激活后生成的HA-BG-1A、HA-BG-2A和HA-BG-3A对接到AGT的活性中心。结果表明,HA-BG-1、HA-BG-2和HA-BG-3并不能进入AGT的活性中心,HA-BG-1A、HA-BG-2A和HA-BG-3A可以进入AGT的活性中心,分子对接的实验结果与细胞活性试验结果一致。本课题阐明了O6-烷基鸟嘌呤对AGT的抑制剂作用机理,在此基础上设计合成了三种具有硝基苄氧羰基的低氧激活AGT抑制剂,并对其低氧反应活性和肿瘤细胞抑制效果进行了研究。结果表明,本研究所合成的3种N2-((((4-硝基苄基)氧)羰基)氨基)-O6-苄基鸟嘌呤能够特异性地抑制处于低氧环境中肿瘤细胞的AGT活性,因此可能成为一种新型靶向性AGT抑制剂应用于与亚硝基脲类烷化剂的联合用药。本研究为进一步设计合成同时具有抗耐药性和肿瘤组织靶向性的高效低毒烷化剂类抗癌药物提供了新的突破口。
[Abstract]:Chloroethyl nitroso (CENUs) is an important class of antitumor alkylating agents, which are widely used in the treatment of malignant tumors such as brain tumors, myeloma, glioma, and malignant melanoma. Currently, the commonly used clinical CENUs includes Nimmo Stine (Nimustine, ACNU), Carmustine, BCNU, and the division of Lomustine (CCNU). .CENUS, such as Semustine (MeCCNU), can cause DNA to form a dG-dC interstrand cross-linking and thus play its anticancer effect. However, the O6- alkyl guanine -DNA- alkyl transferase (O6-alkylguanine-DNA alkyltransferase, AGT) in the tumor cells can repair DNA damage caused by CENUs, making the tumor cells resistant to drug resistance. O6-benzylguanine (O6-BG) is an effective AGT inhibitor, which can transfer benzyl on O6 site to AGT's active center Cys145, causing AGT to lose activity and thus increase the sensitivity of cancer cells to CENUs. However, O6-BG, due to lack of targeting, also inhibits AG in normal cells while acting on tumor cells. T activity increases the sensitivity of normal cells to CENUs. In this study, the mechanism of the action of AGT inhibitors was studied by molecular simulation. Three new types of hypoxia activated AGT inhibitors were synthesized, and the activity of three kinds of AGT was evaluated by cell activity experiments. The use of sub docking, molecular dynamics simulation and quantum chemistry were used. The mechanism of the inhibitory action of O6- methyl guanine (O6-MeG), O6- benzyl guanine (O6-BG) and O6- bromide guanine (4-BTG) on AGT was studied. The molecular structure of the complex of AGT-O6-MeG, AGT-O6-BG and AGT-4-BTG three substrates with AGT protein (1T38) was constructed, and the key amino groups of the active center of the compound were intercepted. Acid residues and substrate molecules are used as calculation models to study the mechanism of the transfer of O6-MeG on AGT active center 145th - cysteine (Cys145) and the mechanism of O6- alkyl on O6-BG and 4-BTG on the level of M062X/6-31+G (D, P): AM1 at the level of AM1. The results show that the energy barrier of the transfer of bromothiophene in 4-BTG is significantly lower than that of the intermediate methyl and the AM1. The energy barrier of benzyl transfer in 6-BG is consistent with the higher AGT inhibitory activity of 4-BTG than O6-MeG and O6-BG. Due to the rapid growth of the solid tumor, the oxygen supply of the tumor tissue is reduced and the hypoxia state is in the condition of hypoxia. Three kinds of low oxygen activated O6-BG analogues (Hypoxia activated O6-BG d) are synthesized by the characteristics of the hypoxia microenvironment in the tumor tissues. Erivatives, HA-BG) - N2- ((((((((((((4- nitro benzyl) oxygen) amino) -N9- chloro ethyl -O6- benzyl guanine (HA-BG-1), N2- (((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((((of))) The decomposition of N9- chloro ethyl -O6- benzyl guanine (HA-BG-1A), N9- bromide ethyl -O6- benzyl guanine (HA-BG-2A) and N9- (2- amine) ethyl -O6- benzyl guanine (HA-BG-3A) were released in the low oxygen environment. 3 kinds of HA-BG and ACNU were combined to deal with SF763 of glioma cells, the oxygen condition and the ACNU group under oxygen free conditions, the combination of ACNU and O6-BG and the combination of the ACNU and the 3 kinds of HA-BG were set up respectively. The survival rate of each group was measured by CCK-8 method. The results showed that 3 HA-BG and A were used. The survival rate of the CNU group under the hypoxia condition was significantly lower than that under the aerobic condition (P0.01), and the AGT inhibitory activity of HA-BG-2 was the strongest. Thus, HA-BG-1, HA-BG-2 and HA-BG-3 could be selectively activated in the hypoxia condition and released the O6-BG analogues, thus targeting the AGT suppression of the tumor cells. The results show that HA-BG-1, HA-BG-2 and HA-BG-3 can not enter the active center of AGT, and that HA-BG-1, HA-BG-2 and HA-BG-3 do not enter the active center of AGT, and that HA-BG-1A, HA-BG-1, and HA-BG-3 can enter the active center of AGT, and the experimental results and finer results of molecular docking. The results of cell activity test were consistent. This subject clarified the mechanism of O6- alkyl guanine's inhibition of AGT. Based on this, three kinds of hypoxic activated AGT inhibitors with nitrobenzyl carbonyl were designed and synthesized. The activity of hypoxia and the inhibitory effect of tumor cells were studied. The results showed that 3 kinds of N2- (((4) synthesized in this study (((((4)) Nitrobenzyl) carbonyl) amino) -O6- benzyl guanine can specifically inhibit the AGT activity of tumor cells in the hypoxic environment. Therefore, it may become a novel targeting AGT inhibitor in combination with nitroso alkanating agents. This study is to further design and synthesize anti drug resistance and tumor tissue targets. A new breakthrough has been provided for the highly effective and low toxic alkylating agents.

【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R914.5

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