CN103181927A - 一类三氮唑并嘧啶类表皮生长因子受体拮抗剂的抗肿瘤治疗用途 - Google Patents

一类三氮唑并嘧啶类表皮生长因子受体拮抗剂的抗肿瘤治疗用途 Download PDF

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CN103181927A
CN103181927A CN2012105444033A CN201210544403A CN103181927A CN 103181927 A CN103181927 A CN 103181927A CN 2012105444033 A CN2012105444033 A CN 2012105444033A CN 201210544403 A CN201210544403 A CN 201210544403A CN 103181927 A CN103181927 A CN 103181927A
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growth factor
epidermal growth
factor receptor
receptor antagonist
treatment application
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CN103181927B (zh
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严明
张陆勇
高鹏
王翔
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China Pharmaceutical University
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Abstract

本发明涉及式(I)的表皮生长因子受体拮抗剂或其药学上可接受的盐。此类化合物可以拮抗表皮生长因子受体活性，可以作为分子靶向抗肿瘤药物用途开发。

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一类三氮唑并嘧啶类表皮生长因子受体拮抗剂的抗肿瘤治疗用途
技术领域
本发明涉及一类三氮唑并嘧啶类表皮生长因子受体拮抗剂的抗肿瘤治疗用途。 
背景技术
表皮生长因子受体(epidermal growth factor receptor，EGFR)是一种广泛分布于人体各组织细胞膜上的多功能糖蛋白，是鸟类成红细胞白血病病毒致癌基因同源体，为HER/ErbB家族的四个成员之一，故又名HER1或ErbB-1。EGFR属于酪氨酸激酶I型受体家族，是原癌基因Cerb-1的表达产物。EGFR与配体表皮生长因子、转化生长因子结合后可激活受体酪氨酸激酶，导致受体本身及细胞内酪氨酸残基的磷酸化，从而引起细胞分裂增殖。EGFR在调节细胞生长和组织修复中起重要作用，其信号系统所引起的细胞效应包括细胞增殖、迁移、黏附等多个环节。相关研究也表明EGFR过度表达可以促进肿瘤细胞的增殖和肿瘤血管生成，促进细胞迁移，引起肿瘤转移。70％以上生长的肿瘤细胞存在该受体及其配基，其过度表达或调节障碍会改变胞内信号途径，以不同方式影响肿瘤细胞的存活及凋亡，因此该受体可作为肿瘤治疗的靶点，国外已有8种小分子EGFR抑制剂上市。本实验运用均相时间分辨荧光技术，对待测样品对激酶活性的抑制情况进行高通量筛选，选择复筛具有活性较好抑制活性的化合物进入抗肿瘤分子靶向药物深入开发程序。待测样品购置美国ChemDiv公司高通量筛选化合物库。 
吉非替尼是第一个靶向表皮生长因子受体的抗肿瘤药物，对包括基于铂类和多西紫杉醇在内的一种或多种化疗方案无疗效反应的非小细胞肺癌患者具有强大的抗肿瘤活性，美国食品和药品监督管理局(FDA)在2003年5月批准吉非替尼作为抗肿瘤治疗药物。另一个作用于该靶点的治疗药物埃罗替尼分别于2004年11月和2005年10月经过FDA和欧洲药品评审机构批准上市。研究发现，表皮生长应依赖性肿瘤细胞通过基因变异改变对药物的亲和力，并可以通过其他转化生长因子受体途径可以构成激活下游细胞内信号效应器，可以逃脱药物治疗导致的生长抑制产生耐药性，所以开发新一代的靶向表皮生长因子受体拮抗剂具有重要应用价值。 
发明内容
为了开发表皮生长因子受体拮抗剂从而为抗肿瘤的治疗寻找新的候选药物，本发明在采用表皮生长因子受体拮抗剂高通量筛选模型，通过功能性验证寻找先导化合物，发现了一类表皮生长因子受体拮抗剂，为新型的抗肿瘤分子靶向药物开发提供先导化合物。本发明可为 此类临床抗肿瘤治疗药物的开发提供先导化合物，为新型抗肿瘤分子靶向药物开发提供线索和实验依据。 
本发明的技术方案为：采用均相时间分辨荧光方法建立体外表皮生长因子受体拮抗剂高通量筛选模型，初筛，复筛发现一类具有抗肿瘤活性的候选药物。具体步骤如下： 
步骤一：建立表皮生长因子受体拮抗剂高通量筛选模型。 
步骤二：阳性药测试和模型稳定性验证。 
步骤三：建立高通量筛选实验方案并开展相关待测样品活性筛选。 
附图说明：
图1：EGFR激酶浓度梯度优化实验.n＝3，
Figure DEST_PATH_GSB00001071089900011
图2：EGFR激酶反应时间优化实验.n＝3，
图3：EGFR激酶底物浓度优化实验.n＝3，
Figure DEST_PATH_GSB00001071089900013
图4：EGFR激酶ATP浓度优化实验.n＝3，
Figure DEST_PATH_GSB00001071089900014
图5：阳性药吉非替尼量效曲线.n＝3，
图6：先导化合物活性列表 
具体实施方式
以下结合附图说明本发明的具体实施方式： 
1检测方法 
本实验采用均相时间分辨荧光方法对反应体系进行检测，该方法分为两个步骤：一是酶促反应，即当加入ATP后启动反应，激酶使底物发生磷酸化反应，将磷酸根连接在有生物素标记的底物上；二是终止及检测过程，在这个过程中EDTA终止了反应的进行，有铕元素标记抗磷酸化酪氨酸抗体靠近底物的磷酸根上，标记XL665的异藻蓝蛋白结合在底物的生物素标记上，两个荧光基团在互相靠近的过程中发生能量共振转移并在665nm处产生荧光，游离的TK antibody(标记铕离子)在620nm处发生荧光，此信号可作为背景信号，而游离的SA-XL665只产生短暂的荧光，通过延后检测时间(加入终止剂后1小时再进行检测)可将其忽略。表皮生长因子高通量筛选模型优化实验中，酶浓度优化实验结果见图1，酶反应时间优化实验结果见图2，底物浓度优化时间结果见图3，ATP浓度优化实验结果见图4，阳性化合物进行质控实验结果见图5。 
2模型建立及化合物筛选 
以384低体积白色板为反应容器，反应体系中每孔加入0.8μL酶溶液，0.8μL底物溶液， 1.6μL缓冲液(或1.6μL化合物溶液)，0.8μLATP溶液，共4μL反应体系，在室温条件下反应指定时间。终止反应时再加入2μL抗体溶液，2μL异藻蓝蛋白溶液，在室温下孵育60min。之后用Envision进行检测。其中实验反应中的试剂溶液使用Biomek NXP自动化加样仪器和Multidrop自动分液器进行加样。模型建好后对待测小分子化合物进行了初筛，对初筛结果活性较好的进行复筛，采用Prism5.0(Graphpad Software，USA)统计分析软件计算活性化合物IC50，先导化合物活性结果见图6。 
表皮生长因子受体拮抗剂活性筛选实验结果 
筛选得到的表皮生长因子受体拮抗剂其结构通式与IC50如下： 
Figure BSA00000824041000031
I：[1，2，4]-三氮唑并[1，5-a]嘧啶 
Figure BSA00000824041000032
Figure BSA00000824041000041
Figure BSA00000824041000051

Claims (2)

1.权利要求1要求保护的式(I)的化合物或其药学上可以接受的盐在表皮生长因子受体拮抗剂的用途。
Figure FSA00000824040900011
其中：
R1代表氢原子或甲基；
R2代表芳基、烷基或芳烷基取代的仲胺或叔胺
R3、R4各自独立的代表甲基或与其相连的碳原子组成的-CH2CH2CH2-和-CH2CH2CH2CH2-。
2.按照权利要求1的用途，作为表皮生长因子受体拮抗剂，用于开发分子靶向抗肿瘤药物的用途。
CN201210544403.3A 2012-12-11 2012-12-11 一类三氮唑并嘧啶类表皮生长因子受体拮抗剂的抗肿瘤治疗用途 Expired - Fee Related CN103181927B (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114805367A (zh) * 2022-05-24 2022-07-29 郑州大学 一种三氮唑并嘧啶类衍生物及其制备方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114805367A (zh) * 2022-05-24 2022-07-29 郑州大学 一种三氮唑并嘧啶类衍生物及其制备方法和应用
CN114805367B (zh) * 2022-05-24 2023-04-07 郑州大学 一种三氮唑并嘧啶类衍生物及其制备方法和应用

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