CN112876460B - 7-二乙胺基-3-乙酰基香豆素衍生物及其合成方法和应用 - Google Patents
7-二乙胺基-3-乙酰基香豆素衍生物及其合成方法和应用 Download PDFInfo
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Abstract
本发明提供了一种7‑二乙胺基‑3‑乙酰基香豆素衍生物及其合成方法和应用，所述衍生物为4‑(3‑(7‑(二乙氨基)‑2‑氧代‑2H‑铬‑3‑基)‑5‑(对甲苯基)‑4,5‑二氢‑1H‑吡唑‑1‑基)苯甲酸，英文名称为4‑(3‑(7‑(diethylamino)‑2‑oxo‑2H‑chromen‑3‑yl)‑5‑(p‑tolyl)‑4,5‑dihydro‑1H‑pyrazol‑1‑yl)benzoic acid，命名为Probe 1；或者为4‑(3‑(7‑(二乙氨基)‑4‑羟基‑2‑氧代‑2H‑铬‑3‑基)‑5‑(对甲苯基)‑4,5‑二氢‑1H‑吡唑‑1‑基)苯甲酸，英文名称为4‑(3‑(7‑(diethylamino)‑4‑hydroxy‑2‑oxo‑2H‑chromen‑3‑yl)‑5‑(p‑tolyl)‑4,5‑dihydro‑1H‑pyrazol‑1‑y l)benzoic acid，命名为Probe 2。在PBS(pH＝7.4)的缓冲溶液中，Probe 1与ClO‑不反应，Probe 2可以实现“turn‑off”型检测ClO‑。该检测过程简便、灵敏、快速，检测结果准确。
Description
技术领域
本发明涉及7-二乙胺基-3-乙酰基香豆素衍生物，具体属于7-二乙胺基-3-乙酰基香豆素衍生物及其合成方法，以及该衍生物作为检测试剂在检测ClO-中的应用。
背景技术
香豆素因其结构易于修饰和光物理性质多样等优点，在化学生物学探针领域取得了长足的发展。香豆素是一种具有苯并吡喃酮结构的染料，它本身没有荧光。在3、4位引入给电子基团或6、7位引入吸电子基团会产生强烈荧光。香豆素染料具有较大的斯托克斯位移、良好的水溶性和较高的量子产率。由于香豆素染料体积小，能够穿透细胞膜，近年来越来越多的研究者将其作为一种检测相关疾病标志物的小分子荧光探针进行研究。其中，7-二乙基氨基-3-乙酰香豆素，二乙胺基作为强的给电子基团，乙酰基作为强的吸电子基团，导致形成分子内电荷转移效应，使整个分子发出很强的荧光。目前报道的工作均在3位进行设计和优化。然而对7-二乙基氨基-3-乙酰香豆素的4位进行的研究相对较少。
针对以上存在的问题，对7-二乙基氨基-3-乙酰香豆素的4位进行研究并设计选择性好、灵敏度高、具有低细胞毒性的荧光探针用于疾病标记物的检测，已成为当前生物医学发展中具有挑战性的前沿课题之一。
在本发明中，合成了基于7-二乙胺基-3-乙酰基香豆素的化合物，通过探针与ClO-在反应前后荧光变化，实现对ClO-的检测。
发明内容
本发明的目的是提供7-二乙胺基-3-乙酰基香豆素衍生物及其合成方法，以及将该衍生物用于ClO-的检测，且检测方法简单，操作方便，选择性好，灵敏度高。
本发明提供的7-二乙胺基-3-乙酰基香豆素衍生物：(1)中文名称为4-(3-(7-(二乙氨基)-2-氧代-2H-色烯-3-基)-5-(对甲苯基)-4,5-二氢-1H-吡唑-1-基)苯甲酸，英文名称为4-(3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid，命名为Probe 1；(2)中文名称为4-(3-(7-(二乙氨基)-4-羟基-2-氧代-2H-色烯-3-基)-5-(对甲苯基)-4,5-二氢-1H-吡唑-1-基)苯甲酸，英文名称为4-(3-(7-(diethylamino)-4-hydroxy-2-oxo-2H-chromen-3-yl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzoic acid，命名为Probe 2；其结构式为：
本发明提供的7-二乙胺基-3-乙酰基香豆素衍生物Probe 1的合成方法，步骤为：
(1)按摩尔比为1：1将7-二乙胺基-3-乙酰基香豆素和4-甲基苯甲醛在无水乙醇中混合，搅拌下加入少量的哌啶，将混合物回流48小时。冷却至室温，过滤沉淀物并用乙醇洗涤，然后真空干燥以获得橙色固体化合物，即(E)-7-(二乙氨基)-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮；
(2)按摩尔比为1：3将(E)-7-(二乙氨基)-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮和4-肼基苯甲酸在无水乙醇中混合，并加入少量的冰乙酸，混合物搅拌回流4h。冷却至室温后，过滤沉淀物并用无水乙醇洗涤，然后真空干燥以获得红色固体，即4-(3-(7-(二乙氨基)-2-氧代-2H-色烯-3-基)-5-(对甲苯基)-4,5-二氢-1H-吡唑-1-基)苯甲酸(Probe 1)。
本发明提供的7-二乙胺基-3-乙酰基香豆素衍生物Probe 2的合成方法，步骤为：
(1)按摩尔比为1：1将7-二乙胺基-4-羟基-3-乙酰基香豆素和4-甲基苯甲醛在无水乙醇中混合，搅拌下加入少量的哌啶，将混合物回流48小时。冷却至室温，过滤沉淀物并用乙醇洗涤，然后真空干燥以获得橙色固体化合物，即(E)-7-(二乙氨基)-4-羟基-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮；
(2)按摩尔比为1：3将(E)-7-(二乙氨基)-4-羟基-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮和4-肼基苯甲酸在无水乙醇中混合，并加入少量的冰乙酸，混合物搅拌回流4h。冷却至室温后，过滤沉淀物并用无水乙醇洗涤，然后真空干燥以获得红色固体，即4-(3-(7-(二乙氨基)-4-羟基-2-氧代-2H-色烯-3-基)-5-(对甲苯基)-4,5-二氢-1H-吡唑-1-基)苯甲酸(Probe 2)。
本发明合成的Probe 2可用于ClO-的检测。
本发明提供的一种非疾病诊断或治疗的检测ClO-的方法，步骤为：
(1)、用二甲基亚砜(DMSO)配制2mM Probe 2的荧光探针储备液；
(2)、将2mL的PBS(pH＝7.4)缓冲溶液和10μL荧光探针储备液加到一个荧光比色皿中，在荧光分光光度仪上检测，随着ClO-的加入，520nm的荧光强度逐渐减弱，荧光呈“turn-off”型变化；
(3)、以ClO-浓度为横坐标，以荧光强度F520nm为纵坐标绘制图，得到ClO-的工作曲线；线性回归方程为：y＝-303.7x+3578.6，x的单位为10-6mol/L。
与现有技术相比，本发明具有如下优点和效果：
1、本发明7-二乙胺基-3-乙酰基香豆素衍生物合成简单，成本低廉；
2、本发明7-二乙胺基-3-乙酰基香豆素衍生物Probe 2能实现对ClO-的检测，检测结果灵敏度高，选择性好；同时还可用于细胞中ClO-检测，也可用于动物活体如斑马鱼ClO-检测；
3、本发明检测手段简单，只需要借助荧光光谱仪即可实现；
4、本发明采用“turn-off”荧光检测，检测信号明显。
附图说明
图1实施例1制备的荧光探针Probe 1的核磁氢谱图
图2实施例1制备的荧光探针Probe 1的核磁碳谱图
图3实施例1制备的荧光探针Probe 1的质谱图
图4实施例1制备的荧光探针Probe 2的核磁氢谱图
图5实施例1制备的荧光探针Probe 2的核磁碳谱图
图6实施例1制备的荧光探针Probe 2的质谱图
图7荧光探针Probe 2与ClO-作用的荧光发射图
图8荧光探针Probe 2与各种分析物的荧光柱状图
图9荧光探针Probe 2测定ClO-的工作曲线
图10荧光探针Probe 2测定ClO-的细胞成像图
图11荧光探针Probe 2测定ClO-的斑马鱼成像图
具体实施方式
下面结合实施例和附图对本发明做进一步说明，但本发明不受下述实施例的限制。
实施例1
Probe 1的制备和表征
将7-二乙胺基-3-乙酰基香豆素(1.55g,6mmol)和4-甲基苯甲醛(0.72g,6mmol)在30mL无水乙醇中混合，搅拌下加入200μL哌啶，将混合物回流48小时。冷却至室温，过滤沉淀物并用乙醇洗涤，然后真空干燥以获得橙色固体化合物(1.25g,产率:57.6％)即(E)-7-(二乙氨基)-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮。1H NMR(600MHz,Chloroform-d)δ8.57(s,1H),8.13(d,J＝15.7Hz,1H),7.84(d,J＝15.7Hz,1H),7.61(d,J＝7.9Hz,2H),7.45(d,J＝8.9Hz,1H),7.22(d,J＝7.8Hz,2H),6.65(d,J＝8.9Hz,1H),6.52(s,1H),3.49(q,J＝7.1Hz,4H),2.40(s,3H),1.27(t,J＝7.0Hz,6H).13C NMR(150MHz,Chloroform-d)δ186.62,160.89,158.61,152.72,148.67,143.58,140.74,132.63,131.81,129.55,128.83,123.86,117.03,110.04,108.87,96.91,45.36,45.29,21.59,12.46,12.44.
将(E)-7-(二乙氨基)-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮(0.36g,1mmol)和4-肼基苯甲酸(0.46g,3mmol)在无水乙醇(25mL)中混合，并加入1mL冰乙酸，混合物搅拌回流4h。冷却至室温后，过滤沉淀物并用无水乙醇洗涤，然后真空干燥以获得红色固体Probe1(0.20g,产率:40.4％)。1H NMR(600MHz,DMSO-d6)δ8.41(s,1H),7.72(d,J＝9.0Hz,2H),7.60(d,J＝9.0Hz,1H),7.14(q,J＝8.3Hz,4H),7.02(d,J＝8.7Hz,2H),6.77(dd,J＝9.0,2.3Hz,1H),6.57(d,J＝2.1Hz,1H),5.53(dd,J＝12.1,4.9Hz,1H),3.96(dd,J＝18.0,12.1Hz,1H),3.47(q,J＝6.9Hz,4H),3.22(dd,J＝18.0,4.9Hz,1H),2.25(s,3H),1.14(t,J＝7.0Hz,6H).(图1)13C NMR(150MHz,DMSO-d6)δ167.68,159.56,156.77,151.57,147.50,147.24,141.23,139.32,137.17,131.23,130.77,130.08,126.00,120.09,112.36,111.43,110.08,108.53,96.58,62.24,45.56,44.66,21.12,12.85.(图2)ESI-MS m/z:[M+H]+calcdfor 496.2236；Found496.2224；[M+Na]+calcd for 518.2056；Found 518.2046.(图3)
Probe 2的制备和表征
将7-二乙胺基-4-羟基-3-乙酰基香豆素(1.65g,6mmol)和4-甲基苯甲醛(0.72g,6mmol)在30mL无水乙醇中混合，搅拌下加入200μL哌啶，将混合物回流48小时。冷却至室温，过滤沉淀物并用乙醇洗涤，然后真空干燥以获得橙色固体化合物(1.40g,产率:61.9％)即(E)-7-(二乙氨基)-4-羟基-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮。1H NMR(600MHz,DMSO-d6)δ8.27(d,J＝15.8Hz,1H),7.91(d,J＝15.8Hz,1H),7.75(d,J＝9.1Hz,1H),7.64(d,J＝7.3Hz,2H),7.31(d,J＝7.4Hz,2H),6.79(d,J＝9.1Hz,1H),6.51(s,1H),3.49(dd,J＝10.6,7.1Hz,4H),2.36(s,3H),1.14(t,J＝6.7Hz,6H).13C NMR(150MHz,DMSO-d6)δ190.89,179.62,160.72,157.42,154.42,145.29,141.94,132.29,130.33,129.35,127.35,122.30,110.14,103.04,98.10,96.36,44.89,21.64,12.80.
将(E)-7-(二乙氨基)-4-羟基-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮(0.37g,1mmol)和4-肼基苯甲酸(0.46g,3mmol)在无水乙醇(25mL)中混合，并加入1mL冰乙酸，混合物搅拌回流4h。冷却至室温后，过滤沉淀物并用无水乙醇洗涤，然后真空干燥以获得红色固体Probe 2(0.25g,产率:48.9％)。1H NMR(600MHz,DMSO-d6)δ13.48(s,1H),7.78–7.74(m,3H),7.18–7.15(m,4H),6.88(d,J＝8.6Hz,2H),6.81(d,J＝9.1Hz,1H),6.53(s,1H),5.49(dd,J＝12.0,5.2Hz,1H),4.13(dd,J＝18.7,12.1Hz,1H),3.48–3.46(m,4H),3.45(s,1H),2.26(s,3H),1.14(t,J＝7.0Hz,6H).(图4)13C NMR(150MHz,DMSO-d6)δ167.56,166.72,160.95,155.82,153.10,152.42,146.38,138.94,137.34,131.50,130.13,126.07,125.55,120.64,112.06,109.92,102.50,96.52,91.88,60.76,46.79,44.65,21.13,12.79.(图5)ESI-MS m/z:[M+H]+calcd for 512.2185；Found 512.2175.(图6)
实施例2
用二甲基亚砜(DMSO)配制2mM Probe 2的荧光探针储备液；将2mL的PBS(pH＝7.4)缓冲溶液和10μL荧光探针储备液加到一个荧光比色皿中，取ClO-溶液，逐渐用微量进样器加到此比色皿中，加样的同时在荧光分光光度仪上检测。随着ClO-的加入，520nm的荧光强度逐渐减弱呈“turn-off”型变化。(图7)
实施例3
用二甲基亚砜(DMSO)配制2mM Probe 2的荧光探针储备液；在荧光比色皿中，加入2mL的PBS(pH＝7.4)溶液和10μL荧光探针储备液，再分别加入10倍当量的其它分析物和ClO-(1-Blank；2-Na+；3-K+；4-Mg2+；5-Cu2+；6-Zn2+；7-Fe2+；8-Fe3+；9-Al3+；10-Ca2+；11-NO2 -；12-NO3 -；13-ClO2 -；14-ClO3 -；15-ClO4 -；16-MnO4 -；17-Cl-；18-H2O2；19-ClO-)，在荧光分光光度仪上检测(图8)。ClO-使得检测体系在520nm处荧光强度明显降低，其它的分析物基本没有引起检测体系荧光强度的变化。
实施例4
以ClO-浓度为横坐标，以荧光强度F520nm为纵坐标绘制图，得到ClO-的工作曲线(图9)；线性回归方程为：y＝-303.7x+3578.6，x的单位为10-6mol/L。
实施例5
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2mM Probe 2的DMSO溶液；把10μLProbe 2的DMSO溶液加入到2mL的PBS中；将探针溶液加入HeLa细胞培养液中，使得其浓度为10μM，与HeLa细胞在37℃下，反应10min，体系在绿色通道有明显的荧光出现；然后再加入外源的ClO-，在37℃下，反应15min，体系在荧光成像仪下可以观察到绿色通道荧光消失。当HeLa细胞先孵育LPS，再加入探针溶液，在绿色通道荧光有所下降。见图10。
实施例6
配制pH＝7.4、浓度为10mM的PBS缓冲溶液，配制2mM Probe 2的DMSO溶液；把10μLNSSN的DMSO溶液加入到2mL的PBS中；将探针溶液与斑马鱼一起培养，使得其浓度为10μM，在37℃下，反应10min，体系在绿色通道有明显的荧光出现；然后再加入外源的次氯酸，在37℃下，反应15min，体系在荧光成像仪下可以观察到绿色通道荧光消失，见图11。
Claims (6)
1.一种7-二乙胺基-3-乙酰基香豆素衍生物，其特征在于，结构式为Probe 2所示：
2.如权利要求1所述的7-二乙胺基-3-乙酰基香豆素衍生物Probe 2的合成方法，其特征在于，步骤如下：
(1)按摩尔比为1：1将7-二乙胺基-4-羟基-3-乙酰基香豆素和4-甲基苯甲醛在无水乙醇中混合，搅拌下加入少量的哌啶，将混合物回流48小时；冷却至室温，过滤沉淀物并用乙醇洗涤，然后真空干燥以获得橙色固体化合物，即(E)-7-(二乙氨基)-4-羟基-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮；
(2)按摩尔比为1：3将(E)-7-(二乙氨基)-4-羟基-3-(3-(对甲苯基)丙烯酰)-2H-色烯-2-酮和4-肼基苯甲酸在无水乙醇中混合，并加入少量的冰乙酸，混合物搅拌回流4h；冷却至室温后，过滤沉淀物并用无水乙醇洗涤，然后真空干燥以获得黄色固体，即4-(3-(7-(二乙氨基)-4-羟基-2-氧代-2H-色烯-3-基)-5-(对甲苯基)-4,5-二氢-1H-吡唑-1-基)苯甲酸。
3.如权利要求1所述的7-二乙胺基-3-乙酰基香豆素衍生物Probe 2在非疾病诊断或治疗的ClO-检测中的应用。
4.一种非疾病诊断或治疗的检测ClO-的方法，其特征在于，包括如下步骤：
(1)、用二甲基亚砜(DMSO)配制2mM权利要求1所述的Probe 2的荧光探针储备液；
(2)、将2mL的pH＝7.4的PBS缓冲溶液和10μL荧光探针储备液加到一个荧光比色皿中，在荧光分光光度仪上检测，随着ClO-的加入，520nm的荧光强度逐渐减弱，荧光呈“turn-off”型变化；
(3)、以ClO-浓度为横坐标，以荧光强度F520nm为纵坐标绘制图，得到ClO-的工作曲线；线性回归方程为：y＝-303.7x+3578.6，x的单位为10-6mol/L。
5.如权利要求1所述7-二乙胺基-3-乙酰基香豆素衍生物Probe 2在制备细胞中ClO-检测试剂中的应用。
6.如权利要求1所述7-二乙胺基-3-乙酰基香豆素衍生物Probe 2在制备动物活体ClO-检测试剂中的应用。
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