CN102389430B - 一种小分子化合物在制备抗肺癌药物中的应用 - Google Patents
一种小分子化合物在制备抗肺癌药物中的应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及一种小分子化合物在制备ROCK抑制剂以及抗肺癌药物中的的应用。
背景技术
癌症即恶性肿瘤是目前人类健康的头号杀手。2010年卫生部公布的统计数据显示,恶性肿瘤已成为中国人的首要死因。因此,癌症的治疗刻不容缓。癌症是一个多步骤的过程,一般涉及到多个基因的变异;当调控细胞生长的基因发生突变或损坏时,细胞的程序性死亡受到抑制,使得细胞失去控制,持续的生长及分裂而产生肿瘤,同时这些癌细胞的浸润和转移过程中能够加速肿瘤新生毛细血管的形成。最普遍的基因变异中,导致这些缺陷的是GTP酶Ras家族的异常激活。Ras蛋白的突变被发现发生在30%的人类肿瘤中,Rho家族蛋白质是小G蛋白的Ras超级家族的成员,具有GTP酶活性。
Rho激酶(ROCKs)属于AGC家族的丝氨酸/苏氨酸蛋白激酶,它是第一个被发现的Rho下游效应因子,通过对磷酸化轻链的影响调节了RhoA诱导的肌球蛋白细胞骨架的变化。研究表明,Rho家族蛋白与肿瘤发生发展的各个方面均有联系,包括肿瘤的生长和增殖、侵袭和转移、细胞凋亡、肿瘤新生血管的形成等。Rho蛋白质在人类肿瘤中持续的被激活,不受控制的增殖,侵袭机体和恶性转化。由于Rho/ROCK信号在肿瘤细胞增殖、分化和运动中的作用,使得通过阻断ROCK信号来治疗某些存在Rho信号过度激活的肿瘤成为人们关注的焦点。ROCK分为ROCK I和ROCK II两种。ROCK的过度表达或/和高度激活均可导致疾病的发生,与之相关的疾病主要包括心血管疾病、神经系统疾病、纤维化疾病以及肿瘤等。在抗肿瘤方面,人们在肝癌细胞、卵巢癌细胞、肝癌细胞、肺癌细胞株、乳腺癌细胞株、膀胱细胞株等上的研究中均发现ROCK抑制剂可抑制肿瘤细胞的侵袭和转移。
在抗肿瘤药物研究方面,国外许多医药公司和科研机构正在开发新的具有抗肿瘤效果的ROCK抑制剂。目前,虽然有若干ROCK抑制剂已经进入I期或II期临床研究阶段(如BA-210),但已经成功上市的ROCK抑制剂仅有一个,即法苏地尔(Fasudil),而且仅限于日本市场。因此,积极寻找和设计新的ROCK抑制剂,并探索其抗癌效果,具有重要的临床意义和广阔的应用前景。
发明内容
本发明的发明目的是提供一种小分子化合物在制备ROCK抑制剂的应用。
为达到上述发明目的,本发明采用的技术方案是:小分子化合物或其可药用的盐在制备ROCK抑制剂的应用,所述小分子化合物为6-{[4-(2-氟苯基)1-哌嗪基]甲基}-N-(萘基)-1,3,5-三嗪-2,4-二胺,其化学结构式如下所示:
上述技术方案中,哌嗪基和萘基之间的N上的氢一般不用画出来;该小分子化合物是从美国Chembridge公司购买得到的。
上述化合物6-{[4-(2-氟苯基)1-哌嗪基]甲基}-N-(萘基)-1,3,5-三嗪-2,4-二胺对ROCKI具有较强的抑制作用,对肺癌细胞的增殖具有明显的抑制效果,并且该化合物细胞毒性较小,因此,本发明同时要求保护上述小分子化合物或其可药用的盐在制备抗肺癌药物中的应用。
由于上述技术方案运用,本发明与现有技术相比具有下列优点:
1.本发明首次发现了小分子化合物为6-{[4-(2-氟苯基)1-哌嗪基]甲基}-N-(萘基)-1,3,5-三嗪-2,4-二胺具备ROCK抑制剂的功能,对肺癌细胞的增殖具有明显的抑制作用,可以用作制备抗肺癌的药物。
附图说明
图1实施例一中抑制剂对ROCK1的抑制曲线;
图2实施例一中抑制剂对HeLa细胞的杀伤效果;
图3实施例一中抑制剂对H460肺癌细胞的杀伤效果;
图4实施例一中抑制剂对A549肺癌细胞的杀伤效果。
具体实施方式
下面结合附图及实施例对本发明作进一步描述:
实施例一:ROCK1的酶抑制活性实验
实验原理:化合物ROCK1抑制活性的测量采用了Invitrogen公司的Z′-LYTE术,该技术基于荧光共振能量转移(FRET)原理,以磷酸化和非磷酸化多肽对蛋白水解切割的敏感性差异为基础。
实验方法:
(1)试剂的准备。激酶缓冲液:将2ml的5X激酶缓冲液用水稀释到10ml;化合物:将所要测试的化合物用水稀释到一个浓度梯度;ROCK激酶/底物的混合溶液:配制2250μL ROCK/底物的混合溶液;酶的浓度为10ng/ml,底物的浓度为4μM,溶剂为激酶缓冲液;磷酸化多肽溶液:将2μL的丝氨酸/苏氨酸磷酸化7肽加到498μL的激酶缓冲液中,充分混匀;ATP溶液:配制1110μL的ATP溶液。浓度为50μM,溶剂为激酶缓冲液;显色剂:按1∶32768的比例稀释显色剂A。
(2)实验步骤:1.将2.5μL配制好的化合物溶液加到黑色384孔板;2.加入5μLROCK激酶/底物的混合溶液;3.加入2.5μL的ATP溶液;4.将384孔板在室温下震荡并培养1小时;5.将5μL显色剂加入到孔板中,继续反应1小时;6.将孔板置于酶标仪中读数。
结果发现:用不同浓度的6-{[4-(2-氟苯基)1-哌嗪基]甲基}-N-(萘基)-1,3,5-三嗪-2,4-二胺进行ROCK1的酶抑制活性实验,发现该化合物有很好的抑制活性,其半抑制浓度IC50为12.8μM/L(图1)。
(3)细胞敏感性实验
实验原理:MTT分析法以活细胞代谢物还原剂MTT噻唑蓝为基础,利用酶标仪测定490nm处的光密度OD值,以反映出活细胞数目,从而测定化合物对肿瘤细胞的杀伤效果。
实验步骤:1.收集对数期细胞,调整细胞悬液浓度,每孔加入100μL,铺板使待测细胞密度为1000-10000个/孔(边缘孔用无菌PB S填充);2.将96孔板放在5%CO2,37℃培养箱中孵育,至细胞单层铺满孔底,加入浓度梯度的药物。原则上,细胞贴壁后即可加药,或两小时,或半天时间,但我们常在前一天晚上铺板,次日上午加药。一般5-7个梯度,每孔5μL,设3-5个平行孔;3.5%CO2,37℃孵育16-48小时,倒置显微镜下观察;4.每孔加入20μL MTT溶液(5mg/ml,即0.5%MTT),继续培养4h。若药物与MTT能够反应,可先离心后弃去培养液,小心用PB S冲2-3遍后,再加入含MTT的培养液;5.终止培养,小心吸去孔内培养液;6.每孔加入150μL二甲基亚砜,置摇床上低速振荡10min,使结晶物充分溶解。酶联免疫检测仪在490nm处测量各孔的吸光值。而ROCK1在多种癌细胞中过高表达,所以我们用多种癌细胞来尝试这种化合物的抗癌细胞活性,使用的癌细胞有H460、HepG-2、A549、7721、K562、KB、MCF-7、MDA-231、Lp1和OPM-2。通过HeLa细胞实验来确定此种化合物的毒性大小。
实验结果:实验结果表明,化合物的HeLa细胞毒性较低(图2),但化合物对H460尤其是A549肺癌细胞的增值具有明显的抑制效果(图3和4),H460肺癌细胞的半数有效浓度为ED50=8.1μM/L。所以化合物6-{[4-(2-氟苯基)1-哌嗪基]甲基}-N-(萘基)-1,3,5-三嗪-2,4-二胺是很好的ROCK1抑制剂,其具有有效的肺癌细胞杀伤效果。
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