CN104817487A - 一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物及其应用 - Google Patents

一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物及其应用 Download PDF

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CN104817487A
CN104817487A CN201510227785.0A CN201510227785A CN104817487A CN 104817487 A CN104817487 A CN 104817487A CN 201510227785 A CN201510227785 A CN 201510227785A CN 104817487 A CN104817487 A CN 104817487A
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CN104817487B (zh
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林森森
顾铭
袁胜涛
孙立
严明
张陆勇
吴亮
高鹏
练进
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China Pharmaceutical University
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    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/34—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

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Abstract

本发明属于医药技术领域，公开了一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物及其应用。该化合物为一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物，其结构如通式(I)所示，其中R1代表C4-C7的环烷基、芳基或取代芳基，R2代表C1-C10的烷基、C4-C7的环烷基、芳基或取代芳基。该化合物可用于制备表皮生长因子受体拮抗剂，为制备靶向抗肿瘤药提供了更多选择。

Description

一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物及其应用
技术领域
本发明属于医药技术领域，涉及一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物及其应用。
背景技术
表皮生长因子受体(Epidermal growth factor receptor，EGFR)是一类生长因子家族的细胞膜受体，包括4个主要成员：EGFR(ErbB1)、HER2(ErbB2)、HER3(ErbB3)和HER4(ErbB4)。EGFR家族是一种具有酪氨酸激酶活性的跨膜受体，结构上可分为胞外区、跨膜区和细胞内区3个部分。EGF等配体与EGFR家族受体的胞外区结合，通过配体自身介导EGFR二聚化，形成同源或异源的二聚体，进而发生受体的磷酸化，激活酪氨酸激酶受体，引发下游一系列的信号转导通路活化，如:Ras/Raf/MEK/ERK-MAPK、PI3K/Akt/mTOR等，最终作用于转录因子，导致肿瘤的增殖、浸润、转移并抑制肿瘤细胞凋亡、促进肿瘤组织的血管生成。研究表明，70％的人类肿瘤与EGFR家族有关，EGFR已成为肺癌、胃癌等高发肿瘤的“驱动基因”。作用于EGFR的酪氨酸激酶抑制剂(EGFR-TKI)已成为当今肿瘤药物研发的重要方向。FDA于2003年、2004年首先批准了第一代分子靶向药物，吉非替尼和厄洛替尼，用于常规化疗手段无效的难治性肺癌的治疗。迄今为止，已有8种小分子EGFR抑制剂上市，处于临床前和临床阶段的候选药物更多。临床实践中，尽管第一代EGFR抑制剂对肿瘤的治疗效果良好，但肿瘤细胞往往在一段时间的治疗后产生耐药突变，如T790M的突变等。现今的EGFR抑制剂基本都是基于氨基喹唑啉类的母核结构，寻找新型有效的EGFR抑制剂已成为创新药物开发的关键。
发明内容
本发明的目的是针对上述技术问题提供了一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物。
本发明另一个目的是提供上述化合物在制药中的应用。
本发明通过建立EGFR高通量抑制剂筛选模型，在体外酶学水平上寻找先导化合物(化合物样品均购自ChemDiv公司筛选化合物库)，发现了一类结构新颖的EGFR抑制剂，都是基于哌啶基乙酰胺类的母核结构。随后，以细胞学实验证实了该类化合物可抑制EGF诱导的细胞增殖。
本发明的目的是通过下列技术方案实现的：
一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物，其结构如通式(I)所示：
其中：
R1代表C4-C7的环烷基、芳基或取代芳基，所述取代基为烷基、链烯基、烷氧基、亚甲二氧基、卤代烷基、卤代烷氧基、卤素、硝基、羟基、羟烷基、巯基、氰基、羧基、甲酰基、氨基、烷氨基或二烷基氨基；
R2代表C1-C10的烷基、C4-C7的环烷基、芳基或取代芳基，所述取代基为烷基、链烯基、烷氧基、亚甲二氧基、卤代烷基、卤代烷氧基、卤素、硝基、羟基、羟烷基、巯基、氰基、羧基、甲酰基、氨基、烷氨基或二烷基氨基。
所述的化合物在制备靶向抗肿瘤药中的应用，所述的靶向抗肿瘤药为表皮生长因子受体拮抗剂。
具体地说，本发明首先建立EGFR抑制剂筛选模型；通过初筛、复筛，发现一类可抑制EGFR酶活的化合物；最后以细胞学实验验证该类化合物对EGF诱导的细胞增殖的作用。步骤如下：
步骤一：建立EGFR抑制剂高通量筛选模型；
步骤二：相关化合物样品的筛选；
步骤三：细胞学实验验证。
本发明有益效果：
本发明运用均相时间分辨荧光技术建立筛选模型，对化合物样品进行高通量筛选，以评价化合物对EGFR激酶活性的作用；随后，以MTT法分析活性样品对EGF诱导的肿瘤细胞增殖的作用。经初筛、复筛，选择4个对EGFR激酶活性抑制率较高的化合物样品进行抗肿瘤细胞增殖作用的实验，通过核对ChemDiv公司筛选化合物库，这4个样品分别为41107(1号化合物)、41119(2号化合物)、41111(3号化合物)和41126(4号化合物)，均为哌啶基乙酰胺类化合物。实验证明本发明筛选出的哌啶基乙酰胺类化合物均具有抑制EGF诱导的细胞增殖的作用，可用于制备EGFR抑制剂，作为靶向抗肿瘤药的一种，本发明的新型有效的EGFR抑制剂为制备抗肿瘤药提供更多选择。
附图说明
图1-1：1号化合物抑制EGFR激酶活性的量效曲线(n＝3)；
图1-2：2号化合物抑制EGFR激酶活性的量效曲线(n＝3)；
图1-3：3号化合物抑制EGFR激酶活性的量效曲线(n＝3)；
图1-4：4号化合物抑制EGFR激酶活性的量效曲线(n＝3)；
图2-1：1号化合物抑制EGF诱导的细胞增殖(n＝3)；
图2-2：2号化合物抑制EGF诱导的细胞增殖(n＝3)；
图2-3：3号化合物抑制EGF诱导的细胞增殖(n＝3)；
图2-4：4号化合物抑制EGF诱导的细胞增殖(n＝3)；
图3：EGFR高通量筛选模型中，EGFR激酶浓度的优化(n＝3)；
图4：EGFR高通量筛选模型中，EGFR酶反应时间的优化(n＝3)；
图5：EGFR高通量筛选模型中，EGFR酶底物浓度的优化(n＝3)；
图6：EGFR高通量筛选模型中，ATP浓度的优化(n＝3)；
图7：EGFR高通量筛选模型中，阳性药吉非替尼的抑制曲线(n＝3)。
具体实施方式：
以下通过实施例对本发明作进一步的阐述。
实施例1：
一、检测方法
均相时间分辨荧光(HTRF)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TKantibody(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。最终仪器给出的信号值由下列公式进行计算：Ratio＝(665nm/620nm)×104
1.模型建立与筛选
(1)以细胞培养基配制化合物溶液。在384孔板中每孔加入EGFR激酶溶液2μl，底物溶液2μl，缓冲液或待筛化合物4μl，ATP 2μl(n＝2)。室温反应1小时。(试剂盒KinEASETM，Cat.no 62TK0PEJ，厂家Cisbio Bioassays)
(2)每孔加入TK-Ab 5μl，SA-XL6655μl，室温孵育1小时。
(3)利用Beckman Coulter检测平台HTRF模块进行检测。
(4)筛选模型的优化见图3～图6。
(5)阳性药选择EGFR抑制剂吉非替尼(图7)。
2.MTT检测样品对EGF诱导的细胞增殖的作用
(1)取处于指数生长期状态良好的人肺癌NCI-H1299细胞一瓶，消化后计数，取180μl细胞悬液接种于96孔板上，2-4×103个/孔，置恒温CO2培养箱中培养24小时。
(2)第二天，弃去培养液，换入新鲜的无血清培养液180μl，加入待筛化合物以及EGF(终浓度100ng/ml)至总体积200μl，置于37℃恒温CO2培养箱中继续培养24小时。
(3)24小时后，将5mg/ml的MTT溶液加入96孔板中，20μl/孔，置于37℃恒温CO2培养箱中反应4小时。
(4)吸去上清液并拍干，加入DMSO，150μl/孔，平板摇床上室温振摇5分钟。用酶联免疫检测仪在波长为570nm处测定每孔的吸光值，并计算细胞抑制率。
(5)阴性对照为无菌PBS溶液，阳性对照为10μM的紫杉醇。
二、实验结果
筛选得到的该类化合物结构通式与IC50值如下(见图1-1～图1-4)：
以100ng/ml的EGF作用于人肺癌NCI-H1299细胞(无血清培养基)，可见EGF显著促进H1299细胞的增殖。活细胞计数显示，EGF上调细胞数目约1.9倍。该哌啶基乙酰胺类化合物均可一定程度上抑制EGF促进H1299细胞增殖的作用，且呈剂量依赖性(见图2-1～图2-4)。2号化合物作用最为显著；由于其对EGFR的酶活抑制效率最高，这提示，该哌啶基乙酰胺类化合物对H1299细胞增殖的作用与其对EGFR酶活的抑制密切相关。

Claims (3)

1.一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物，其结构如通式(I)所示：
其中：
R1代表C4-C7的环烷基、芳基或取代芳基，所述取代基为烷基、链烯基、烷氧基、亚甲二氧基、卤代烷基、卤代烷氧基、卤素、硝基、羟基、羟烷基、巯基、氰基、羧基、甲酰基、氨基、烷氨基或二烷基氨基；
R2代表C1-C10的烷基、C4-C7的环烷基、芳基或取代芳基，所述取代基为烷基、链烯基、烷氧基、亚甲二氧基、卤代烷基、卤代烷氧基、卤素、硝基、羟基、羟烷基、巯基、氰基、羧基、甲酰基、氨基、烷氨基或二烷基氨基。
2.权利要求1所述的化合物在制备靶向抗肿瘤药中的应用。
3.根据权利要求2所述的应用，其特征在于所述的靶向抗肿瘤药为表皮生长因子受体拮抗剂。
CN201510227785.0A 2015-05-06 2015-05-06 一类可抑制表皮生长因子受体的哌啶基乙酰胺类化合物及其应用 Expired - Fee Related CN104817487B (zh)

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