CN102432612A - 4,7-二氢四唑[1,5-a]嘧啶衍生物及其在制备抗肿瘤药物中的应用 - Google Patents
4,7-二氢四唑[1,5-a]嘧啶衍生物及其在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种4，7-二氢四唑[1，5-a]嘧啶衍生物，其特征在于，所述4，7-二氢四唑[1，5-a]嘧啶衍生物的化学结构式如以下通式所示：
式中，X为亚甲基CH2、亚氨基NH或0；R1为C1～C3的烷基、C6～C8的含有一个取代基的苯基、苯甲基、含有N的5元或6元芳香杂环基；R2为：含有1～3个取代基的苯基、含有N的5元或6元芳香杂环基、或苯并杂环基团；其中，所述取代基选自：氢、甲基、甲氧基、乙氧基、苯甲氧基、硝基。本发明首次发现了种4，7-二氢四唑[1，5-a]嘧啶衍生物或其可药用的盐具备ROCK抑制剂的功能，对肺癌细胞、乳腺癌细胞的增殖具有明显的抑制作用，可以用作制备抗肺癌、抗乳腺癌的药物。
Description
技术领域
本发明涉及一种4，7-二氢四唑[1，5-a]嘧啶衍生物及其在制备抗肿瘤药物中的应用。
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4，7-二氢四唑[1，5-a]嘧啶衍生物及其在制备抗肿瘤药物中的应用。
为达到上述发明目的，本发明采用的技术方案是：一种4，7-二氢四唑[1，5-a]嘧啶衍生物，其化学结构式如以下通式所示：
式中，X为亚甲基CH2、亚氨基NH或O；
R1为C1～C3的烷基、C6～C8的含有一个取代基的苯基、苯甲基、含有N的5元或6元芳香杂环基；
R2为：含有1～3个取代基的苯基、含有N的5元或6元芳香杂环基、或苯并杂环基团；
其中，所述取代基选自：氢、甲基、甲氧基、乙氧基、苯甲氧基、硝基。
优选的技术方案中，所述4，7-二氢四唑[1，5-a]嘧啶衍生物选自：7-(4-羟基-3-甲氧基苯基)-5-甲基-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶、7-(3，4-二乙氧基苯基)-6-[N-(2-甲氧基苯基)甲酰胺基]-5-甲基-4，7-二氢四唑(1，5-α)嘧啶、7-(4-苯甲氧基-3-甲氧基苯基)-6-(N-苯基甲酰胺基)-5-甲基-4，7-二氢四唑(1，5-α)嘧啶、7-(2-N-嘧啶)-5-氟-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶、7-苯基-6-(N-苯甲基)甲酰胺基-5-甲基-4，7-二氢四唑(1，5-α)嘧啶、7-(1，3-苯并间二氧杂环戊烯)-6-[N-(2-甲氧基苯基)甲酰胺基]-5-甲基-4，7-二氢四唑(1，5-α)嘧啶、7-(3-甲氧基-3′-硝基-4-羟基苯基)-6-[N-(2-甲氧基苯基)甲酰胺基]-5-甲基-4，7-二氢四唑(1，5-α)嘧啶、7-(4-苯甲氧基苯基)-6-[N-(2-吡啶)甲酰胺基]-4，7-二氢四唑(1，5-α)嘧啶、7-(4-甲氧基苯基)-6-羧酸异丙基酯-4，7-二氢四唑(1，5-α)嘧啶；其化学结构式依次如下所示：
本发明对上述4，7-二氢四唑[1，5-a]嘧啶衍生物进一步进行了生物学活性测定，发现它们对ROCK1都具有明显的抑制活性，在细胞水平对于乳腺癌和肺癌细胞都有不同程度的杀伤效果
因此，本发明同时要求保护上述4，7-二氢四唑[1，5-a]嘧啶衍生物或其可药用的盐在制备ROCK抑制剂的应用，所述可药用的盐包含盐酸盐、磷酸盐、硫酸盐、醋酸盐、马来酸盐、枸橼酸盐、苯磺酸盐、甲基苯磺酸盐、富马酸盐、酒石酸盐。
本发明同时要求保护上述4，7-二氢四唑[1，5-a]嘧啶衍生物或其可药用的盐在制备抗肺癌、乳腺癌药物中的应用。
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4，7-二氢四唑[1，5-a]嘧啶衍生物或其可药用的盐具备ROCK抑制剂的功能，对肺癌细胞、乳腺癌细胞的增殖具有明显的抑制作用，可以用作制备抗肺癌、抗乳腺癌的药物。
附图说明
图1实施例一中制备7-(4-羟基-3-甲氧基苯基)-5-甲基-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶的合成路线图；
图2为实施例二中抑制剂对ROCK1的抑制曲线；
图3.(a).抑制剂在ROCK1活性口袋中的结合构象(抑制剂采用棍状模型显示)；(b).抑制剂和ROCK1活性口袋残基之间的相互作用模式；
图4实施例二中抑制剂对HeLa细胞的杀伤效果；
图5实施例二中抑制剂对H460肺癌细胞的杀伤效果；
图6实施例二中抑制剂对MDA-231乳腺癌细胞的杀伤效果；
图7实施例二中小鼠体重随时间的变化图。
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：制备7-(4-羟基-3-甲氧基苯基)-5-甲基-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶。
参见(Yao，C.Heterocyl.Chem.2008，45，1609；Gein，V.L.Russ.J.Org.Chem.2010，46，699；Zeng，L.Y.J.Comb.Chem.2010，12，35)，按照图1的合成路线：以香草醛(5)、5-氨基四唑(2)和乙酰乙酸苄酯(6)这三个商品化的试剂为原料，通过三组分一锅煮的方法就可以一步得到化合物7。该反应可以在无溶剂的条件下直接加热到130-170℃时进行；在加入氨基磺酸为催化剂后，无溶剂或乙醇为溶剂的条件下反应温度降到85℃就可以很好进行；用10mol％的碘作为催化剂，异丙醇为溶剂，加热回流的条件下也能得到很好的产率。重结晶后，化合物的纯度可以到95％以上。对产物进行分析，分析结果显示所得产物为7-(4-羟基-3-甲氧基苯基)-5-甲基-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶，其化学结构式为：
实施例二：
(1)对实施例一所得化合物进行ROCK 1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技术，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2.5μLATP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。
实验结果：不同浓度的7-(4-羟基-3-甲氧基苯基)-5-甲基-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶进行ROCK 1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13.7μM/L(图2)。
(2)抑制剂和ROCK1之间相互作用模式的评价
实验原理：基于分子对接和分子动力学模拟，从原子尺度上预测抑制剂和ROCK1之间的相互作用模式。
实验步骤：基于分子对接预测得到的结果，对抑制剂/ROCK1进行5ns的分子动力学模拟，模拟采用了AMBER9.0。
实验结果：通过分子对接预测和分子动力学模拟得到的抑制剂和ROCK1之间的相互作用如图3所示。预测结构表明，抑制剂和ROCK1之间的分子识别主要通过范德华和氢键相互作用。抑制剂上的羟基和氨基会和Gly88以及Asn203形成两个稳定的氢键；两个苯环会和周围的多个疏水性残基产生强的范德华相互作用，包括Leu 107、Ile82、Val90以及Leu205；此外Lys的正离子会和苯环形成π阳离子相互作用。
(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，利用酶标仪测定在490nm处的吸光度，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PBS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。通过酶联免疫检测仪在490nm处测量吸光值。而ROCK 1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4)，但化合物对MDA-231乳腺癌尤其是A549肺癌细胞有很好的抗癌细胞活性(图5和6)，A549肺癌细胞的半数致死浓度为ED50＝7.5μM/L。所以化合物7-(4-羟基-3-甲氧基苯基)-5-甲基-6-羧酸苯甲酯-4，7-二氢四唑(1，5-α)嘧啶不仅仅具有好的ROCK 1抑制活性，还具有良好的肺癌细胞及明显的乳腺癌细胞杀伤效果，有可能成为治疗肿瘤的一类新药。
(4)小鼠急性毒性实验
实验原理：急性毒性实验主要研究一定时间内单次或多次给予化学物质后动物所产生的毒性反应及死亡情况。该实验可以得到半数致死剂量(LD50)，即能引起实验动物50％死亡的受试物剂量。按照1998年OECD化学物质毒性分级标准，以5、50、300、2000、5000mg/kg为分界点，将物质毒性分为5级。当LD50＞2000mg/kg时，此种化合物可以被认为基本无毒。
实验步骤：实验采用balb/c小鼠10只，体重20g左右。采用口服给药的方式，给药剂量为2000mg/kg，给药后观察14天。
实验结果：实验结果表明，小鼠并无任何异常状况，无死亡。如图7所示，在给药后，小鼠的体重稳中有升，说明该化合物是非常安全的，基本可以认定无毒，可以作为一种有潜力的药物进行后续的研究和开发。
Claims (3)
1.一种4，7-二氢四唑[1，5-a]嘧啶衍生物，其特征在于，所述4，7-二氢四唑[1，5-a]嘧啶衍生物的化学结构式如以下通式所示：
式中，X为亚甲基CH2、亚氨基NH或O；
R1为C1～C3的烷基、C6～C8的含有一个取代基的苯基、苯甲基、含有N的5元或6元芳香杂环基；
R2为：含有1～3个取代基的苯基、含有N的5元或6元芳香杂环基、或苯并杂环基团；
其中，所述取代基选自：氢、甲基、甲氧基、乙氧基、苯甲氧基、硝基。
2.权利要求1所述4，7-二氢四唑[1，5-a]嘧啶衍生物或其可药用的盐在制备ROCK抑制剂的应用。
3.权利要求1所述4，7-二氢四唑[1，5-a]嘧啶衍生物或其可药用的盐在制备抗肺癌、乳腺癌药物中的应用。
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