CN105418526A - 一类具有单磺酸苯基四氮唑结构的化合物及其应用 - Google Patents

一类具有单磺酸苯基四氮唑结构的化合物及其应用 Download PDF

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CN105418526A
CN105418526A CN201510757078.2A CN201510757078A CN105418526A CN 105418526 A CN105418526 A CN 105418526A CN 201510757078 A CN201510757078 A CN 201510757078A CN 105418526 A CN105418526 A CN 105418526A
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sulphenyl
tetrazole
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阮奔放
阮健昵福
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HANGZHOU JENNIFER BIOTECH Co.,Ltd.
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Hangzhou Jiama Biological Technology Co Ltd
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Abstract

本发明涉及医药生物技术领域，公开了一类具有单磺酸苯基四氮唑结构的化合物，具有2-(R1苯基)-5(2-磺酸苯基)-2H-四氮唑的结构通式，本发明的一类含2-磺酸苯基四氮唑盐，具有低毒性、合成路径短、纯度质量易控制等优点。由于2-磺酸苯基四氮唑盐在450nm处几乎无光吸收，而其还原产物在450nm处具有较大光吸收，因此，采用分光光度法能够简单快速测定细胞内谷氨酸脱氢酶酶活力或NADH/NADPH含量。

Description

一类具有单磺酸苯基四氮唑结构的化合物及其应用
技术领域
本发明涉及医药生物技术领域，尤其涉及一类具有单磺酸苯基四氮唑结构的化合物及其应用。
背景技术
NADPH和NADH是生物体内重要的氧化还原反应介质。虽然它们的化学结构只差一个磷酸基团，但是生物功能具有很大差别。NADH参与分解反应在细胞呼吸营养物质代谢起重要作用。而NADPH参与和成反应，在反应中消耗能量和成大分子，也是细胞内抗氧化防御系统的重要组成成分在细胞防御性氧(ROS)损伤方面起重要作用。因此，准确定量NADPH和NADH的细胞浓度对生物活性检测和疾病的诊断具有重要意义。
NADPH和NADH可以通过四氮唑盐类化合物(Tetrazoliunsalts)的氧化还原颜色反应检测。1894年首次合成的TTC(2,3,5-三苯基四氮唑盐)是脂溶性光敏感复合物，用于检测种子的生存能力。MTT是另一个检测细胞存活和生长的方法，利用活细胞线粒体的琥珀酸脱氢酶使MTT还原成水不溶解的蓝紫色结晶甲瓒(Formazan)，经DMSO溶解后检测540nm或720nm波长吸收值。具有灵敏度高，经济性好等优点，但检测步骤繁多。1997年水溶性的WST(双磺酸苯基四氮唑盐)被合成，并被用于商业化活细胞活性测试中，但是，水溶性WST化合物不易被合成和纯化。化合物纯度和质量不易控制。
发明内容
本发明针对现有技术中现有的针对NADPH和NADH的检测步骤繁琐，专一性差等缺点，提供了一类针对NADPH或和NADH检测的高灵敏度结构新颖的一类具有单磺酸苯基四氮唑结构的化合物。
为了解决上述技术问题，本发明通过下述技术方案得以解决：
一类具有单磺酸苯基四氮唑结构的化合物，具有以下的化学分子结构通式，
式中：R1、R2、R3和R4每一个独立的取代基包含1～50个选自C、H、N、O、S、P、Si和卤素原子的原子。取代基为氢原子，或者取代基为由C、H、N、O、S、P、Si或卤素原子构成的基团。
作为优选，R1、R2、R3和R4独立地选自氢原子、烷基、烷氧基、硝基、氰基、羧基或卤素原子。
作为优选，R1、R2、R3和R4独立地选自烷基、烷氧基、羟基、羧基、磺酰胺基或氨基。
作为优选，R1、R2、R3和R4独立地选自取代烷基、取代烷氧基、取代羧基、取代磺酰胺基或取代胺基，取代基团选自烷基、芳基、芳烷基。
作为优选，R1、R2、R3和R4独立地选自包含1～50个选自C、H、N、O、S、P和卤素原子构成的杂环化合物或多环化合物。
作为优选，一类具有单磺酸苯基四氮唑结构的化合物，其选自
本发明提供的化合物，作为氢受体，被还原后在450nm具有最大光吸收，用于测定谷氨酸脱氢酶活性或谷胺酰胺酶活性或细胞中NADPH/NADH的含量。
本发明的苯基四氮唑盐克服了传统脂溶性四氮唑盐毒性高的不足，具有低毒性的优点，又克服了水溶性四氮唑盐合成困难，不易纯化的不足，具有合成路径短，纯度质量易控制的优点。由于四氮唑盐在450nm处几乎无光吸收，其还原产物甲瓒在450nm处具有较大光吸收，因此，采用分光光度法能够简单快速测定细胞内谷氨酸脱氢酶酶活力，或测定细胞中NADH的含量。
附图说明
图1是本发明实施例1的具有单磺酸苯基四氮唑结构的化合物和还原态苯基具有单磺酸苯基四氮唑结构的化合物的全波长扫描曲线。
图2是本发明实施例2的具有单磺酸苯基四氮唑结构的化合物与谷氨酸脱氢酶反应后，在450nm处光吸收情况。
图3是本发明实施例3的具有单磺酸苯基四氮唑结构的化合物与谷胺酰胺酶反应后，在450nm处光吸收情况。
图4是本发明实施例3的具有单磺酸苯基四氮唑结构的化合物与活细胞反应后，在450nm处光吸收情况。
具体实施方式
下面结合实施例对本发明作进一步详细描述。
实施例1
制备如下化合物
2-(4-硝基苯基)-3-(2-甲氧基-4-硝基苯基)-5(2-磺酸苯基)-2H-四氮唑单钠盐
代表性合成步骤
步骤A：向30mL甲醇中加入对硝基苯肼6mmol，溶解后，加入苯甲醛-2-磺酸钠6mmol，升温至60℃搅拌反应1～24hour，过滤，干燥，得到橙红色固体产物苯甲醛-2-磺酸钠对硝基苯腙，产率87.1％。
步骤B：将2mmol的2-甲氧基-4-硝基苯胺加入至烧瓶，冰浴条件下加入1ml水，再加入0.6ml浓盐酸，然后加入已溶解在1ml水中的2.2mmolNaNO2，继续搅拌反应20min～5hour，得到2-甲氧基-4-硝基苯胺的重氮盐。
步骤C：将2mmol苯甲醛-2-磺酸钠对硝基苯腙溶解于甲醇中，降温到0℃，加入步骤B现制的2-甲氧基-4-硝基苯胺的重氮盐2mmol，再加入NaOH溶液，搅拌反应1～24hour。过滤干燥，柱层析分离得甲瓒，产率为87.2％。
步骤D：取1mmol步骤C制备的甲瓒，溶解于甲醇中，冰浴条件下加入浓盐酸18mmol，在加入18mmol双氧水，搅拌反应5小时。过滤干燥，柱层析分离得暗棕黄色固体，四氮唑盐，产率为33％。
1HNMR(500MHz,DMSO):δ8.54(d,J＝8.9Hz,2H),8.37(d,J＝8.8Hz,1H),8.24(dd,J＝8.8,2.0Hz1H),8.05(d,J＝9.4Hz,3H),7.81-7.70(m,3H),7.44-7.35(m,1H),3.71(s,3H).
MP:105℃
FAB-MS：m/z＝499[M+H]+
权利要求书中的一类具有单磺酸苯基四氮唑结构的化合物，都能够根据上述步骤选择合适底物合成。
实施例2
由类似的合成程序，步骤B中，底物为1-萘胺，产物为1-萘胺的重氮盐，制备如下化合物。
2-(4-硝基苯基)-3-(1-萘基)-5(2-二磺酸苯基)-2H-四氮唑单钠盐黑色固体，产率47.4％
1HNMR(500MHz,DMSO):δ8.46(d,J＝8.4Hz,1H),8.29(d,J＝7.4Hz,1H),8.21(dd,J＝9.3Hz1H),8.06(d,J＝7.9Hz,2H),7.88(d,J＝9.5Hz,2H),7.62(d,J＝7.8Hz,2H),7.43(t,J＝7.6Hz,2H),7.36(t,J＝7.4Hz,2H),7.25(d,J＝6.6Hz2H).
MP:102℃
FAB-MS：m/z＝429[M+H]+
实施例3
由类似的合成程序，步骤B中，底物为8-氨基喹啉，产物为8-氨基喹啉的重氮盐，制备如下化合物。
2-(4-硝基苯基)-3-(8-喹啉基)-5(2-磺酸苯基)-2H-四氮唑单钠盐黑红色固体，产率67.17％
1HNMR(500MHz,DMSO):δ8.81(d,J＝4.3Hz,1H),8.72(d,J＝7.8Hz,1H),8.66-8.55(m,2H),8.35(d,J＝9.1Hz,1H),7.81(t,J＝7.6Hz,2H),7.72-7.68(m,2H),7.58-7.50(m,2H),7.42(d,J＝6.4Hz,1H),7.36(t,J＝7.1Hz,1H),7.25(d,J＝7.4Hz,1H).
MP:102℃
FAB-MS：m/z＝475[M+H]+
实施例4
苯基四氮唑盐及还原态苯基甲瓒的全波长扫描情况如图1所示。
其中的苯基四氮唑盐为实施例1中合成的2-(4-硝基苯基)-3-(2-甲氧基-4-硝基苯基)-5(2-磺酸苯基)-2H-四氮唑单钠盐。
苯基四氮唑盐(10mM；在450nm几乎无吸收；下方曲线)在1-甲氧基PMS和50mMTrispH8存在的条件下，可以被NADH还原成橘黄色的甲瓒(最大吸收波长为450nm；上方曲线)。因此可作为NADH类还原剂的检测方法。
实施例5
谷氨酸脱氢酶活性检测是通过催化谷氨酸脱氢生成NADPH/NADH，NADH将苯基四氮唑还原成橘黄色的甲瓒，如图2。
其中的苯基四氮唑盐为实施例2中合成的2-(4-硝基苯基)-3-(1-萘基)-5(2-二磺酸苯基)-2H-四氮唑单钠盐。
苯基四氮唑盐在450nm几乎无吸收；苯基四氮唑盐与1-甲氧基-5-甲基吩嗪硫酸甲酯盐，NADP，谷氨酸，谷胺酸脱氢酶作用后，在450nm波长处存在光吸收。且吸收光的强度和谷胺酸脱氢酶的量成正比。
苯基四氮唑盐可以用于测定谷胺酸脱氢酶活性和药物筛选。
实施例6
谷胺酰胺酶催化反应生成谷氨酸，谷氨酸可以用实施例5中的谷氨酸脱氢酶反应来定量，在450nm处测量光吸收情况，如图3。
苯基四氮唑盐在450nm几乎无吸收；测定0.1-25mMol/L的苯基四氮唑盐在1-甲基PMS，NADP，谷氨酰胺，谷胺酰胺酶，谷胺酸脱氢酶的缓冲体系中反应2h，测定不同浓度的苯基四氮唑底物在450nm波长处光吸收。
吸收光的强度和谷胺酰胺酶的量成正比。
苯基四氮唑盐可以用于测定谷胺酰胺酶活性和药物筛选。
实施例7
苯基四氮唑盐与活细胞反应后，在450nm处测量光吸收情况，如图4。
其中的苯基四氮唑盐为实施例3中合成的2-(4-硝基苯基)-3-(8-喹啉基)-5(2-磺酸苯基)-2H-四氮唑单钠盐。
苯基四氮唑盐在450nm几乎无吸收；苯基四氮唑盐与细胞中的NADH/NADPH作用后，在450nm波长处存在光吸收。并且吸收光的强度和细胞数量呈相关性。
因此，苯基四氮唑盐可以用于测定活细胞的量。
总之，以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所作的均等变化与修饰，皆应属本发明专利的涵盖范围。

Claims (7)

1.一类具有单磺酸苯基四氮唑结构的化合物，其特征在于，具有以下的化学分子结构通式：
式中：R1、R2、R3和R4每一个独立的取代基包含1～50个选自C、H、N、O、S、P、Si和卤素原子的原子。
2.根据权利要求1所述的一类具有单磺酸苯基四氮唑结构的化合物，其特征在于：R1、R2、R3和R4独立地选自氢原子、烷基、烷氧基、硝基、氰基、羧基或卤素原子。
3.根据权利要求1所述的一类具有单磺酸苯基四氮唑结构的化合物，其特征在于：R1、R2、R3和R4独立地选自羟基、磺酰胺基或氨基。
4.根据权利要求1所述的一类具有单磺酸苯基四氮唑结构的化合物，其特征在于：R1、R2、R3和R4独立地选自取代烷基、取代烷氧基、取代羧基、取代磺酰胺基或取代胺基，取代基团选自烷基、芳基、芳烷基。
5.根据权利要求1所述的一类具有单磺酸苯基四氮唑结构的化合物，其特征在于：R1、R2、R3和R4独立地选自包含1～50个选自C、H、N、O、S、P和卤素原子构成的杂环化合物或多环化合物。
6.根据权利要求1所述的一类具有单磺酸苯基四氮唑结构的化合物，其选自：
7.应用权利要求1所述的化合物测定谷氨酸脱氢酶活性或谷胺酰胺酶活性或NADPH/NADH在细胞中的含量。
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