CN107011227B - 基于微管蛋白的叠氮-β-内酰胺小分子探针及其制备方法和应用 - Google Patents
基于微管蛋白的叠氮-β-内酰胺小分子探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一类基于微管蛋白的叠氮‑β‑内酰胺小分子探针及其制备方法和应用，属于药物化学领域。本发明将β‑内酰胺母核与叠氮活性单元结合，简单高效。其具有如下结构通式：该类探针分子体外抗癌活性试验表明对多种肿瘤细胞MCF‑7、MGC‑803、A549均具有一定的抑制作用。特别是叠氮‑β‑内酰胺探针分子Ⅰ‑9对MGC‑803细胞的活性为85 nM。另一方面，本发明化合物能与微管蛋白结合，抑制微管蛋白聚合，可作为潜在的微管蛋白小分子探针。本发明解决了叠氮类微管蛋白探针种类少，合成繁琐，成本高昂的问题，有利于抑制微管蛋白聚合药物的开发。
Description
技术领域
本发明涉及药物化学领域，具体涉及一类基于微管蛋白的叠氮‐β‐内酰胺化合物、它们的制备方法及其作为一类新的抗肿瘤药物先导化合物及小分子探针的应用。
背景技术
光亲和标记技术主要是在照光后,配基与大分子的结合部位形成特异性、不可逆性结合。进一步分析此复合物的特点,以定性受体蛋白。目前，最理想的光亲和标记物是芳香叠氮类化合物(Nature,1969,224,511-512)。
目前已报道的含叠氮基的微管蛋白小分子探针结构为：
该化合物能与微管蛋白结合，抑制微管蛋白的聚合。合成方法为下式 ：
可以发现，该方法合成含叠氮基的微管蛋白小分子探针需要七步，路线繁琐。同时需要使用紫杉醇为原料，价格高昂，不利于大规模生产。
该类小分子探针，对研究微管蛋白的生物学功能具有重要的意义，目前尚未见文献报道。
发明内容
本发明目的在于提供系列新型的叠氮‐β‐内酰胺衍生物微管蛋白小分子探针，丰富相关领域的结构类型。
本发明的另一个目的在于提供一种简单高效，绿色环保的合成叠氮‐β‐内酰胺衍生物的方法。
本发明的再一个目的在于提供所述化合物在制备抗肿瘤药物和微管蛋白小分子探针中的应用。
为实现本发明目的，所述一类新型叠氮‐β‐内酰胺微管蛋白小分子探针结构式如下：
芳环为苯环或取代的苯环或者噻吩环。取代的苯环优选：甲氧基、卤素单取代苯环。
更优选如下化合物：
本发明所述新型叠氮‐β‐内酰胺微管蛋白小分子探针主要通过下列步骤制得：
H2SO4溶液中，加入3-氨基苄醇或4-氨基苄醇，亚硝酸钠，叠氮钠，0℃-5℃下搅拌反应，抽滤得化合物Ⅰ-A。取化合物Ⅰ-A，溶于二氯甲烷中，加入氯铬酸吡啶鎓盐，室温搅拌反应，反应结束后，萃取浓缩，重结晶得化合物Ⅰ-B。取化合物Ⅰ-B，加入3,4,5-三甲氧基苯胺，室温搅拌抽滤得化合物Ⅰ-C。无水二氯甲烷或二氯乙烷中，加入三乙胺，芳环乙酰氯和化合物Ⅰ-C，回流反应得叠氮-β- 内酰胺衍生物。
所述的芳环乙酰氯为苯乙酰氯、对甲氧基苯乙酰氯、间甲氧基苯乙酰氯、对氯苯乙酰氯、对氟苯乙酰氯、噻吩苯乙酰氯。
反应物摩尔比:化合物I‐C:芳环乙酰氯：三乙胺＝1：1：1。
本发明创新点及优点在于：以氨基取代的苄醇为原料，经四步反应制备得到叠氮‐β‐内酰胺衍生物，路线简短，成本低廉。另外，体外抗癌活性试验表明本发明化合物对多种肿瘤细胞MCF-7、MGC-803、A549均具有一定的抑制作用。特别是化合物I-9对MGC-803细胞的活性为85nM，可作为候选抗肿瘤药物；另一方面，本发明化合物能与微管蛋白结合，抑制微管蛋白聚合，可作为潜在的微管蛋白小分子探针。解决了叠氮类微管蛋白探针种类少，合成繁琐，成本高昂的问题，有利于抑制微管蛋白聚合药物的开发。
附图说明
图1为本发明化合物I-9的微管蛋白聚合实验图。
具体实施方式
为了对本发明进行更好的说明，特举实施例如下：
实施例化合物(I-9)的制备
100ml 2M H2SO4溶液中，加入39g的3-氨基苄醇或4-氨基苄醇，42g亚硝酸钠，42g的叠氮钠，0℃下搅拌反应，抽滤得化合物Ⅰ-A。取15g化合物Ⅰ-A，溶于150ml二氯甲烷中，加入22g氯铬酸吡啶鎓盐，室温搅拌3h，萃取浓缩， 10ml乙醇重结晶得化合物Ⅰ-B。取2.9g化合物Ⅰ-B，加入3.6g 3,4,5-三甲氧基苯胺，室温搅拌抽滤得化合物Ⅰ-C。20ml无水二氯甲烷中，加入0.03mol三乙胺，0.02mol芳环乙酰氯，0.02mol化合物Ⅰ-C，回流反应得叠氮-β-内酰胺衍生物I-1～I-9。
化合物(I-1～I-9)结构表征如下：
I-1：Yellow soild,yield:46.2％,m.p:107～109℃.1HNMR(400MHz,CDCl3)δ7.39(d,J＝8.5Hz,2H),7.24(d,J＝8.6Hz,2H),7.07(d,J＝8.5Hz,2H),6.91(d,J＝8.7 Hz,2H),6.58(s,2H),4.84(d,J＝2.5Hz,1H),4.20(d,J＝2.4Hz,1H),3.81(s,3H), 3.78(s,3H),3.72(s,6H).13CNMR(100MHz,CDCl3)δ165.77,159.41,153.61, 140.58,134.67,134.21,133.55,128.61,127.47,126.48,119.93,114.52,94.87,64.62, 63.95,60.97,56.08,55.36.HRMS(ESI):calcd C25H25N4O5,[M+H]+m/z,461.1816； found,461.1825.
I-2：Yellow soild,yield:37.6％,m.p:66～68℃..1HNMR(400MHz,CDCl3)δ7.37(d,J＝24.2Hz,7H),7.09(s,2H),6.58(s,2H),4.90(s,1H),4.26(s,1H),3.75(d,J＝20.4Hz,9H).13CNMR(100MHz,CDCl3)δ165.30,153.63,140.67,134.73,134.46, 134.15,133.50,129.13,128.08,127.51,127.43,119.96,94.88,65.13,63.57,60.97, 56.09.HRMS(ESI):calcd C24H23N4O4,[M+H]+m/z,431.1715；found,431.1719.
I-3：Brown soild,yield:66.3％,m.p:64～66℃.1HNMR(400MHz,CDCl3)δ7.43(d,J＝8.4Hz,2H),7.32(dd,J＝8.7,7.8Hz,1H),7.10(d,J＝8.4Hz,2H),6.98–6.85 (m,3H),6.59(s,2H),4.92(d,J＝2.4Hz,1H),4.25(d,J＝2.4Hz,1H),3.83(s,3H), 3.80(s,3H),3.75(s,6H).13CNMR(100MHz,CDCl3)δ165.14,160.12,153.60, 140.66,135.87,134.68,134.10,133.48,130.20,127.52,119.95,119.62,113.37, 113.23,94.86,65.02,63.44,60.98,56.08,55.34.HRMS(ESI):calcd C25H25N4O5, [M+H]+m/z,483.1816；found,461.1825.
I-4：Brown soild,yield:35.5％,m.p:147～149℃.1HNMR(400MHz,CDCl3)δ7.46 –7.34(m,3H),7.28(s,2H),7.19(d,J＝7.0Hz,1H),7.08(d,J＝8.0Hz,1H),7.02(s, 1H),6.56(s,2H),4.84(s,1H),4.25(s,1H),3.79(s,3H),3.73(s,6H).13CNMR(100 MHz,CDCl3)δ164.63,153.66,141.47,139.45,134.91,134.09,133.28,132.79, 130.92,129.33,128.80,122.33,119.41,116.47,94.91,64.19,63.54,60.97, 56.11.HRMS(ESI):calcdC24H21ClN4NaO4,[M+Na]+m/z,487.1152；found, 487.1149.
I-5：White soild,yield:32.9％,m.p:164～166℃.1HNMR(400MHz,CDCl3)δ7.34(t, J＝7.8Hz,1H),7.23(dd,J＝8.9,7.7Hz,1H),7.12(d,J＝7.8Hz,1H),7.03–6.94 (m,2H),6.89–6.77(m,3H),6.50(s,2H),4.81(d,J＝2.5Hz,1H),4.18(d,J＝2.5 Hz,1H),3.73(s,3H),3.71(s,3H),3.66(s,6H).13CNMR(100MHz,CDCl3)δ165.06, 160.12,153.62,141.35,139.72,135.75,134.73,133.43,130.86,130.22,122.43, 119.62,119.30,116.52,113.43,113.23,94.86,64.84,63.48,60.98,56.09,55.34. HRMS(ESI):calcdC25H25N4O5[M+H]+m/z,461.1828；found,461.1825.
I-6：Yellow soild,yield:35.6％,m.p:112～114℃.1HNMR(400MHz,CDCl3)δ7.35(t,J＝7.8Hz,1H),7.28–7.20(m,2H),7.12(d,J＝7.8Hz,1H),7.05–6.97(m,3H), 6.95(d,J＝1.8Hz,1H),6.50(s,2H),4.77(d,J＝2.5Hz,1H),4.19(d,J＝2.4Hz, 1H),3.71(s,3H),3.66(s,6H).13CNMR(100MHz,CDCl3)δ163.94,162.71,160.25, 152.63,140.41,138.52,133.85,132.31,129.88,129.13,128.15,121.32,118.34, 115.20,93.89,63.12,62.72,59.95,55.08.HRMS(ESI):calcd C24H21FN4NaO4,[M+ Na]+m/z,471.1443；found,471.1445.
I-7：White soild,yield:48.9％,m.p:128～129℃.1HNMR(400MHz,CDCl3)δ7.47 –7.30(m,6H),7.20(d,J＝7.7Hz,1H),7.11–7.01(m,2H),6.58(s,2H),4.89(d,J ＝2.5Hz,1H),4.28(d,J＝2.5Hz,1H),3.79(s,3H),3.73(s,6H).13CNMR(100MHz, CDCl3)δ165.18,153.65,141.38,139.79,134.82,134.36,133.46,130.84,129.15, 128.13,127.44,122.40,119.28,116.54,94.90,64.96,63.61,60.98,56.11.HRMS (ESI):calcd C24H23N4O4,[M+H]+m/z,431.1716；found,431.1719.
I-8：Brown soild,yield:10.3％,m.p:122～124℃.1H NMR(400MHz,CDCl3)δ7.45(d,J＝8.2Hz,2H),7.36–7.27(m,1H),7.07(dd,J＝15.1,10.0Hz,4H),6.59(d,J＝6.4Hz,2H),4.97(d,J＝3.9Hz,1H),4.49(d,J＝4.1Hz,1H),3.78(dd,J＝20.8,6.4 Hz,9H).13CNMR(100MHz,CDCl3)δ164.11,153.64,140.80,135.81,135.80, 134.82,133.56,133.44,127.46,125.91,125.53,119.99,94.96,64.30,60.97,60.24, 56.10.HRMS(ESI):calcdC22H20N4NaO4S,[M+Na]+m/z,459.1104；found, 459.1103.
I-9：Yellow soild,yield:26.8％,m.p:106～108℃.1HNMR(400MHz,CDCl3)δ7.32(t,J＝7.8Hz,1H),7.19(dd,J＝5.1,0.9Hz,1H),7.11(d,J＝7.7Hz,1H),6.98(dd,J ＝8.9,5.7Hz,3H),6.92(dd,J＝5.0,3.6Hz,1H),6.48(s,2H),4.85(d,J＝2.5Hz, 1H),4.39(d,J＝2.3Hz,1H),3.69(s,3H),3.63(s,6H).13CNMR(100MHz,CDCl3) δ163.98,153.64,141.36,139.19,135.68,134.85,133.38,130.90,127.45,125.97, 125.59,122.35,119.42,116.47,94.95,64.30,60.96,60.07,56.09.HRMS(ESI):calcd C22H20N4NaO4S,[M+Na]+m/z,459.1107；found,459.1103.
上述化合物的抗肿瘤活性测定：
1.实验方法：
筛选所用化合物均是由本发明合成、纯化而得；样品储备液：称取2～3mg 样品置于2mL EP管中，然后用DMSO配制成浓度是128×103μg/mL的溶液，4℃保存放置，实验时根据所需浓度利用培养基稀释。
2.初筛
取对数生长期的细胞，消化计数后，用培养基调整细胞密度，接种至96孔板中，培养24h后，弃去培养基，加入用培养基稀释好的药物(50μg/mL、100 μg/mL)，每个浓度设6个复孔，另设空白对照组及阴性对照组。药物作用72h 后，每孔加入20μL MTT，继续培养4h后，吸去液体，加入150mL的DMSO，振荡均匀，酶标仪490nm处检测吸光度值，计算抑制率，计算公式如下：
抑制率(％)＝(1-给药组吸光度值/空白组吸光度值)×100％
3、细筛
50μg/mL时抑制率大于50％的样品，重新设置浓度进行细筛。即将待测样品以0.5μg/mL、1μg/mL、2μg/mL、4μg/mL、8μg/mL、16μg/mL、32μg/mL 浓度加入96孔板中，培养48h后，检测。试验结果采用SPSS软件计算IC50值和相关系数。
4、实验结果：
表1上述化合物对三种肿瘤细胞的抗肿瘤活性评价数据
a人类胃癌细胞b人类乳腺癌细胞c人类肺癌细胞d临床对照药
上述化合物的微管蛋白结合实验验证：
提取的微管蛋白重悬于冰冷的G-PEM缓冲液(80mM PIPES pH 5.9,5mM MgCl2,1mMEGTA,1mM ATP,5％(v/v)glycerol)，取100ul加至包含100ul本发明化合物I-9的96孔板内，微管蛋白终浓度为5.6g/L，药物浓度设置0uM，1uM， 2uM，4uM四个梯度，样品充分混匀，分光光度计检测微管蛋白的聚合，间隔 5min，总计60min，IC50值在30分钟使用GraphPad软件计算得到。本发明化合物I-9的微管蛋白聚合实验如附图：说明本发明化合物I-9确实能够结合微管蛋白，抑制其聚合。
Claims (5)
1.一类基于微管蛋白的叠氮-β-内酰胺小分子探针，其特征在于，具有如下所示结构：
芳环为苯环或取代的苯环或者噻吩环；所述取代的苯环为：甲氧基或卤素单取代苯环。
2.如权利要求1所述的一类基于微管蛋白的叠氮-β-内酰胺小分子探针，其特征在于，优选如下化合物之一：
。
3.如权利要求1或2所述的一类基于微管蛋白的叠氮-β-内酰胺小分子探针的应用，其特征在于，将其做为活性成分用于制备抗肿瘤药物或作为检测微管蛋白的小分子探针。
4.制备如权利要求1所述的一类基于微管蛋白的叠氮-β-内酰胺小分子探针，其特征在于，通过如下步骤实现：
H2SO4溶液中，加入化合物1，亚硝酸钠，叠氮钠，0℃-5℃下搅拌反应，抽滤得化合物Ⅰ-A；取化合物Ⅰ-A，溶于二氯甲烷中，加入氯铬酸吡啶鎓盐，室温搅拌反应，反应结束后，萃取浓缩，重结晶得化合物Ⅰ-B；取化合物Ⅰ-B，加入3,4,5-三甲氧基苯胺，室温搅拌抽滤得化合物Ⅰ-C；无水二氯甲烷或二氯乙烷中，加入三乙胺，芳环乙酰氯和化合物Ⅰ-C，回流反应得叠氮-β-内酰胺衍生物；
所述的芳环乙酰氯为苯乙酰氯、对甲氧基苯乙酰氯、间甲氧基苯乙酰氯、对氯苯乙酰氯、对氟苯乙酰氯、噻吩苯乙酰氯。
5.如权利要求4所述的一类基于微管蛋白的叠氮-β-内酰胺小分子探针的制备方法，其特征在于，反应物摩尔比:化合物I-C:芳环乙酰氯：三乙胺＝1：1：1。
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