吖丙啶环喹唑啉类非可逆EGFR抑制剂的设计合成、生物活性筛选及类药性化合物库的构建

发布时间：2018-03-18 13:30

本文选题：EGFR酪氨酸激酶　切入点：耐药性　出处：《浙江大学》2014年博士论文　论文类型：学位论文

【摘要】：恶性肿瘤是一类严重威胁人类健康的常见病和多发病,目前因恶性肿瘤引起的死亡占据人类所有疾病死亡率的第二位。而肿瘤细胞在用药过程中产生的耐药问题是肿瘤难以根治的重要原因之一。EGFR(表皮细胞生长因子受体)的信号传导关乎细胞的凋亡、增殖、分化、迁移和细胞周期循环,它在肿瘤细胞中往往过度表达,并且与肿瘤的形成和恶化息息相关,成为近年来备受关注的热门靶点。通过对EGFR结构的分析,选择特定部位作为靶点,干扰其信号传导,已经成为开发抗癌和抗肿瘤药物的有效手段。然而,目前临床上针对该靶点的第一代EGFR抑制剂如Gefitini、Erlotinib等也同样存在着用药过程中产生的耐药性问题,导致后期肿瘤细胞对药物的脱敏。其中,T790M突变是产生该获得性耐药的重要原因。以Afatinib为代表的第二代EGFR抑制剂通过药物分子结构中所含有的亲电反应官能团可以实现与靶蛋白之间的共价结合,提高抑制剂与靶蛋白的亲和力,从而增强抗癌效果,这为解决第一代EGFR抑制剂的耐药性问题提供了新的思路。本论文第一、二部分对临床应用的小分子EGFR抑制剂的构效关系以及文献中所报道的配体-受体蛋白复合物信息的进行了综述,在此基础上设计合成了4类4-芳胺基喹唑啉类衍生物。该部分工作主要的创新点是在传统的喹唑啉母核的6-位侧链引入三元吖丙啶环片段。吖丙啶环是一类较为活泼的半胱氨酸捕获基团,以吖丙啶环替代第二代非可逆EGFR抑制剂中的丙烯酰胺片段,有望开发出一类结构新颖并拥有自主知识产权的药物分子。所有合成的目标化合物均经过1HNMR、 HPLC-HRMS和熔点表征。在体外肿瘤细胞筛选中,所设计的化合物均对EGFR高表达的人表皮癌细胞株A431WT, overexpression以及Gefitinib耐药的人非小细胞肺癌细胞株H1975L858R/T790M有较好的增殖抑制活性。在激酶水平上,化合物在EGFRWT、EGFRT790M以及EGFRL858R同样展示了较好的抑制活性。部分化合物在细胞和激酶水平上的抑制活性与阳性Afatinib/Canertinib相当。此外,所选取的苗头化合物在EGFR低表达的细胞株SW620和SKBr上增殖抑制效果较差。体外Cysteine捕获实验和Western Blotting洗脱实验也验证了该类化合物可与Cysteine发生共价结合,但是其共价结合能力稍弱于阳性Canertinib。这样的结果预示着该类化合物可能具备较好的选择性和安全性。所合成的上述化合物的后续成药性评价也正在进行之中。化合物库的高通量筛选是新药开发的源头之一,是整个新药开发的关键部分。构建结构多样性的化合物库对先导化合物的发现、先导化合物结构的优化具有重要的现实意义。论文的第三部分,利用新化学方法学结合液态组合化学构建了6类共计800余个全新结构的化合物。其中,对于吗啡类衍生化合物库,在合成方法学研究的同时,对该类化合物进行了较为详细的体内外镇痛活性研究,发现了3个先导化合物；其余的化合物库分子也进行了初步的体外抗肿瘤活性筛选,在本论文中选取其中三个化合物库进行了介绍。6个化合物库针对其他不同生物靶点的活性评价也正在进行之中。
[Abstract]:Malignant tumor is a serious threat to human health common disease, the death caused by malignant tumor accounted for second of all human diseases mortality. Drug resistance and tumor cells in the treatment process is one of the important causes of cancer to cure.EGFR (epidermal growth factor receptor) signaling related to cell the apoptosis, proliferation, differentiation, migration and cell cycle in tumor cells, it is often over expression and tumor formation and progression are closely related, has become a popular target of concern in recent years. Through the analysis of the structure of EGFR, select the specific site as a target, the interference signal transduction, has become effective means the development of anticancer and antitumor drugs. However, the current clinical according to the target of the first generation of EGFR inhibitors such as Gefitini, Erlotinib etc. also exists in the course of medication The problem of drug resistance in the late stage of tumor cells to cause desensitization of drugs. Among them, T790M mutation is an important reason of the acquired resistance. To achieve a combination of target protein covalent electrophilic reaction between functional groups can be the second generation of EGFR inhibitors to Afatinib as the representative of the drug molecules contained, improve inhibitor and target protein affinity, thus enhance the anti-cancer effect, which provides a new way to solve the problem of resistance to the first generation of EGFR inhibitors.
In the first, second part, small molecule EGFR inhibitor to the clinical application of the structure-activity relationship and ligand - reported the receptor protein complexes were reviewed, based on the design and synthesis of 4 kinds of 4- aromatic amino quinazoline derivatives. The innovation of the main part of the work is the introduction of three yuan a C in the traditional pyridine quinazoline nucleus 6- side chain ring fragment. Aziridine ring is a kind of active cysteine capture groups, instead of the second generation of non reversible EGFR inhibitor of acrylamide fragment to aziridine ring, is expected to develop a novel structure and has independent intellectual property rights of the drug. All the target compounds were synthesized by 1HNMR, HPLC-HRMS and melting point characterization.
In the screening of tumor cells in vitro, compounds are designed for high EGFR expression of human epidermal carcinoma cell line A431WT, overexpression and Gefitinib resistant human non-small cell lung cancer cell line H1975L858R/T790M has good inhibitory activity. The proliferation level of EGFRWT kinase, EGFRT790M and EGFRL858R in the compound, also showed good inhibitory activity. Some of the compounds in the cell and inhibit the activity of kinase level and positive Afatinib/Canertinib. In addition, the signs of compounds in the low expression of EGFR and SKBr on SW620 cells proliferation inhibition effect. In vitro Cysteine capture experiment and Western Blotting elution experiments demonstrated that the compounds can be covalently combined with Cysteine, but its covalent binding ability weaker than positive Canertinib. this result indicates that the compounds may have a better choice The evaluation of the subsequent drug resistance of the synthesized compounds is also under way.
High throughput screening of compound libraries is one source of new drug development, is the key part of the development of new drugs. To construct the diversity of compound libraries found on the lead compound, has important practical significance to optimize the structure of the lead compound. The third part of the thesis, using the new chemical methodology 6 categories totaling more than 800 new structure the liquid compounds combining the construction of combinatorial chemistry. Among them, the morphine derivative compound libraries, research in the synthesis method, the compounds were studied in vitro and in vivo analgesic activity in detail, 3 compounds were found; the rest of the molecular compound library also conducted a preliminary screening of their antitumor activity in vitro and in this paper selected three compounds were introduced.6 compounds library for activity evaluation of different biological targets is underway.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R914.5;R91

【共引文献】