CN115583920B - 一种四嗪类化合物及其制备方法和应用 - Google Patents
一种四嗪类化合物及其制备方法和应用 Download PDFInfo
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- CN115583920B CN115583920B CN202211056081.8A CN202211056081A CN115583920B CN 115583920 B CN115583920 B CN 115583920B CN 202211056081 A CN202211056081 A CN 202211056081A CN 115583920 B CN115583920 B CN 115583920B
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- tetrazine
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- -1 Tetrazine compound Chemical class 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 claims abstract description 32
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 3
- 150000001875 compounds Chemical class 0.000 claims description 28
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 150000004905 tetrazines Chemical class 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 9
- 150000003839 salts Chemical class 0.000 claims description 9
- 230000001590 oxidative effect Effects 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 230000003647 oxidation Effects 0.000 claims description 4
- 238000007254 oxidation reaction Methods 0.000 claims description 4
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 claims description 3
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 claims description 3
- 239000007800 oxidant agent Substances 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 2
- 239000001301 oxygen Substances 0.000 claims description 2
- 229910052760 oxygen Inorganic materials 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 14
- DPOPAJRDYZGTIR-UHFFFAOYSA-N Tetrazine Chemical compound C1=CN=NN=N1 DPOPAJRDYZGTIR-UHFFFAOYSA-N 0.000 abstract description 11
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 29
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 20
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 15
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- WZZBNLYBHUDSHF-DHLKQENFSA-N 1-[(3s,4s)-4-[8-(2-chloro-4-pyrimidin-2-yloxyphenyl)-7-fluoro-2-methylimidazo[4,5-c]quinolin-1-yl]-3-fluoropiperidin-1-yl]-2-hydroxyethanone Chemical compound CC1=NC2=CN=C3C=C(F)C(C=4C(=CC(OC=5N=CC=CN=5)=CC=4)Cl)=CC3=C2N1[C@H]1CCN(C(=O)CO)C[C@@H]1F WZZBNLYBHUDSHF-DHLKQENFSA-N 0.000 description 9
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
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- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
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- SZUVGFMDDVSKSI-WIFOCOSTSA-N (1s,2s,3s,5r)-1-(carboxymethyl)-3,5-bis[(4-phenoxyphenyl)methyl-propylcarbamoyl]cyclopentane-1,2-dicarboxylic acid Chemical compound O=C([C@@H]1[C@@H]([C@](CC(O)=O)([C@H](C(=O)N(CCC)CC=2C=CC(OC=3C=CC=CC=3)=CC=2)C1)C(O)=O)C(O)=O)N(CCC)CC(C=C1)=CC=C1OC1=CC=CC=C1 SZUVGFMDDVSKSI-WIFOCOSTSA-N 0.000 description 3
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- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
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Abstract
本发明提供了一种四嗪类化合物及其制备方法和应用，属于化学合成技术领域。首先公开了该四嗪类化合物的结构，包括荧光团R1、连接链R2以及四嗪母核；其次公开了该四嗪类化合物的制备方法，包括以下步骤：(1)将荧光化合物在二价锌离子的催化下与氰基化合物反应，得到产物A；(2)产物A氧化后进行洗脱、纯化，得到四嗪类化合物。该四嗪类化合物的四嗪结构可与超氧阴离子特异性快速反应，生成具有强荧光的产物，且荧光强度与超氧阴离子浓度间存在正相关；随着超氧阴离子浓度增高，荧光强度逐渐增强，从而实现对超氧阴离子的动态检测。
Description
技术领域
本发明属于化学合成技术领域，涉及一种四嗪类化合物及其制备方法和应用。
背景技术
超氧阴离子(O2 ·-)是生物系统中的一种重要的内源性活性氧物质，是其他活性氧物种(ROS)的源头分子，同时也是一种重要的信号分子。由于O2 ·-作为上游的活性氧物种，其参与各种生理过程，但是关于O2 ·-与疾病发生的关系还未阐明，例如衰老、神经退行性疾病、心血管疾病、癌症等。另一方面，O2 ·-在体内浓度较低(10-10M)，半衰期较短(10-6Sec)，很容易转化为其他氧化性物种，所以准确实现O2 ·-的实时检测具有一定的挑战。开发新型的特异性靶向O2 ·-的检测工具对研究其介导的病理发展机制具有重要意义。
四嗪类化合物(tetrazine)又名四氮杂苯，含4个氮原子的六元杂环化合物。有1,2,3,4-、1,2,4,5-和1,2,3,5-四嗪三种异构体。其中较重要的是1,2,4,5-四嗪，它溶于水、醇和醚。在农药、染料、炸药及液晶等领域具有广泛应用。近年来，发现1,2,4,5-四嗪类化合物及其衍生物具有较多的生理活性，如抗炎、镇痛、抗病毒、抗菌及抗癌等。另外，随着四嗪类化合物在生物正交化学中的独特性，使其在生物正交荧光探针、药物的靶向给药、特殊蛋白的标定以及生物材料的前体等有广泛的应用价值。
中国专利申请CN 106317112 A公开了一种四嗪类化合物及其制备方法、应用。其中，四嗪类化合物为四嗪磷酸酯，通过该发明所述制备方法制备的四嗪磷酸酯能够应用于制备四嗪类衍生物，含有荧光基团的四嗪类衍生物可作为生物正交荧光探针。但该发明所述的方法涉及含四嗪结构化合物的磷酸酯化衍生，制备方法相对复杂。
中国专利申请CN 112010817 A公开了一种新型制备四嗪类化合物的方法，该方法使用腈和水合肼，在硫醇类催化剂的存在下可以进行各种取代，后经亚硝酸钠氧化，获取带有各种官能的四嗪化合物，方便四嗪化合物进一步的修饰和改造，从而得到更多有特殊功能的四嗪类衍生物。该方法第一步中需用到有刺鼻气味的硫醇化合物为催化剂，第二步用到有致癌性的亚硝酸钠为氧化剂，反应方法的环保性较差。
发明内容
针对现有技术存在的问题，本发明提供了一种结构新颖、能实现对超氧阴离子的动态检测的四嗪类化合物及其制备方法和应用。
为实现上述目的，本发明采用的技术方案如下：
首先，提供了一种四嗪类化合物，具有式I的结构，或其互变异构体、药学上可接受的盐；
其中：
R1选自萘胺、喹啉、磺酰萘胺、1,8萘二酰亚胺、氧杂蒽、4-硝基苯并噁二唑、氟硼二吡咯、香豆素、碳杂蒽、硅杂蒽或花菁染料；所述R1为荧光团，优选地，为发射波长400nm-800nm的荧光团；
R2选自单键或二价基团，所述二价基团选自亚烷基、亚芳基、亚杂芳基、含O的二价基团、含N的二价基团、含酰胺键的二价基团或含酯键的二价基团；
R选自H、C1-6直链或支链烷基、C3-6环烷基、杂环烷基、苯基或杂芳基，所述R可被一个或多个取代基所取代。
进一步地，所述R1选自以下结构的一种：
进一步地，所述R2为二价基团，所述二价基团选自C1-6亚烷基，亚芳基、亚芳基氧基、亚芳基胺基，亚芳基-C1-3烷基-氧基，亚芳基-C1-3烷基-胺基，亚芳基-C1-3烷基-胺基-C(O)基、亚C1-2-C(O)-C1-2-胺基或亚C1-2-C(O)-C1-2-氧基，所述二价基团被1-2个C1-3烷基所取代；
优选地，所述R2选自单键或以下结构中的一种：
优选地，所述R2选自单键或以下结构中的一种：
进一步地，所述R选自C1-4直链或C1-4支链烷基、C4-6环烷基、C2-5杂环烷基、苯基、五元-六元杂芳基；所述R可被一个或多个取代基所取代；
优选地，所述R选自以下结构的一种：
更优选地，所述R选自甲基。
进一步地，所述的四嗪类化合物，或其互变异构体、药学上可接受的盐，选自以下结构中的一种：
其次，提供了一种所述的四嗪类化合物，或其互变异构体、药学上可接受的盐的制备方法，包括以下步骤：
(1)将R1CN与RCN在二价锌离子的催化下反应，得到产物A；
(2)产物A进行氧化，后洗脱、纯化，得到四嗪类化合物；
其中，R1CN为含氰基的荧光化合物，RCN为含R基团的氰基化合物；
所述四嗪类化合物的制备方法的化学反应方程式如下：
进一步地，步骤(1)中所述R1CN与RCN的摩尔比为1：10-50。
进一步地，步骤(2)中所述氧化所用氧化剂为空气或氧气。
进一步地，步骤(2)中所述洗脱、纯化的方式如下：以含乙酸乙酯的石油醚溶液(20％或33％)作为洗脱剂进行硅胶柱层析纯化；以含甲醇的二氯甲烷溶液(1％、3％或10％)作为洗脱剂进行硅胶柱层析纯化。
最后，提供了一种所述的四嗪类化合物，或其互变异构体、药学上可接受的盐或所述的制备方法得到的四嗪类化合物，或其互变异构体、药学上可接受的盐在超氧阴离子检测中的应用。
进一步地，所述的四嗪类化合物，或其互变异构体、药学上可接受的盐还可应用在检测细胞不同氧化损伤程度中，其中检测是识别细胞内不同浓度超氧阴离子以区分细胞不同程度的氧化应激损伤。
进一步地，所述超氧阴离子为溶液、细胞、组织或活体动物中的超氧阴离子。
进一步地，所述超氧阴离子的检测方法包括以下步骤：将四嗪类化合物用二甲基亚砜溶解制备储备液，然后用PBS或细胞培养基稀释至四嗪类化合物的终浓度为1-10μM，最后加入含有超氧阴离子的体系中，25-35min后，对超氧阴离子进行检测。其中所述储备液中四嗪类化合物的浓度为1-10mM，所述细胞培养基为DMEM。
本发明超氧自由基检测机理为：探针结构中的四嗪基为良好的荧光淬灭基团，因而探针本底荧光呈淬灭状态，强度弱；但该四嗪基团可与超氧发生特异性高效转化反应，随着四嗪结构被超氧自由基破坏，探针荧光强度可以恢复。该恢复的荧光强度与超氧呈剂量依赖关系，因而可被用于定性、定量地指征超氧。
经过进一步研究，发现四嗪结构和超氧自由基接触后，经过质谱分析，荧光恢复状态下的探针分子量比荧光淬灭状态下的探针分子量减少了12，判断其中的四嗪结构转化为了恶二唑结构，由于恶二唑不具有荧光淬灭能力，因此可实现荧光探针的荧光恢复。具体过程如下：
相对于现有技术，本发明具有以下有益效果：
(1)本发明提供了一类结构新颖的四嗪类化合物；
(2)该四嗪类化合物的四嗪结构可与超氧阴离子特异性快速反应，生成具有强荧光的产物，且荧光强度与超氧阴离子浓度间存在正相关；随着超氧阴离子浓度增高，荧光强度逐渐增强，从而实现对超氧阴离子的动态检测。
附图说明
图1为化合物的结构通式图；
图2：A)为化合物22和30在HepG 2细胞中响应不同浓度过氧化氢诱导所产生的超氧阴离子的荧光显微镜成像图，B)为荧光强度的定量结果；
图3：A)为化合物22和30在HepG 2细胞中响应2mM过氧化氢诱导不同时间所产生的超氧阴离子的荧光显微镜成像图，B)为荧光强度的定量结果；
图4：A)为化合物22和30、DCFHDA、DHE在HepG 2细胞中响应不同设置条件(1-4组)诱导产生的超氧阴离子图的荧光显微镜成像图，B)为荧光强度的定量结果；
图5是化合物30在心肌组织切片中成像心脏缺血再灌注诱导产生的超氧阴离子；
图6是化合物30在活体动物中检测心脏缺血再灌注诱导产生的超氧阴离子的心脏解剖成像图。
具体实施方式
值得说明的是，本发明中使用的原料均为普通市售产品，对其来源不做具体限定。
实施例化合物的合成
化合物22，23，30，31的合成方法
带氰基的荧光团(1eq)、乙腈(10eq)、三氟甲磺酸锌(0.5eq)、80％水合肼(50eq)置于压力闷罐中，油浴70℃反应12h，反应结束后，加入乙酸乙酯提取有机相，水相依次用水、饱和食盐水洗涤三次，有机相用无水硫酸钠干燥，旋干，以含乙酸乙酯的石油醚溶液(50％)作为洗脱剂进行硅胶柱层析纯化得到中间体二氢四嗪衍生物。将中间体二氢四嗪衍生物溶于5mL甲醇溶液中，不断鼓入空气进行氧化，待反应结束后，旋干，以含乙酸乙酯的石油醚溶液(20％)或含甲醇的二氯甲烷溶液(10％)作为洗脱剂进行硅胶柱层析纯化得到化合物22(ESI-MS(m/z):[M+H]+calc’d.for C18H20N3:306.1719；found:306.25)；化合物23(ESI-MS(m/z):[M+H]+calc’d.for C24H31N6O2:435.2508；found:435.10)；化合物30(ESI-MS(m/z):[M+H]+calc’d.for C23H30N7O2:436.2461；found:436.25)；化合物31(ESI-MS(m/z):[M+H]+calc’d.for C17H19N6:307.1617；found:307.20。化合物22、23、30、31对应的荧光团的结构如下：
化合物5，7，8，10，12的合成方法
将带羧基的荧光素/罗丹明/硅罗丹明/碳罗丹明(1eq)、6-甲基-3-(4-甲基苄氨)1,2,4,5-四嗪氨基(1.2eq)、EDCI(2.5eq)、HOBT(2.5eq)溶于10mL无水二氯甲烷溶液中，冰浴下加入DIPEA(3.0eq)，反应液在氮气氛围下搅拌12h。反应完后，反应液中依此加入水、饱和盐水、无水硫酸钠萃取并干燥有机相，旋干溶剂，以含甲醇的二氯甲烷溶液(3％)作为洗脱剂进行硅胶柱层析纯化得到化合物5(ESI-MS(m/z):[M+H]+calc’d.for C31H24F2N5O4:568.1796；found:568.25)；化合物7(ESI-MS(m/z):[M+H]+calc’d.for C36H34N7O4 +:628.2667；found:628.20)；化合物8(ESI-MS(m/z):[M+H]+calc’d.for C39H42N7O2 +:640.3395；found:640.10)；化合物10(ESI-MS(m/z):[M+H]+calc’d.for C40H40N7O3Si+:694.2956；found:694.50)；化合物12(ESI-MS(m/z):[M+H]+calc’d.for C38H40N7O+:610.3289；found:610.50)。化合物5、7、8、10、12对应的荧光团的结构如下：
化合物32，33的合成方法
将4位溴取代的萘酰亚胺衍生物(1eq)、6-甲基-3-苄氨-1,2,4,5-四嗪(1.2eq)、Pd(OAc)2(0.1eq)、BINAP(0.2eq)和碳酸铯(2.5eq)溶于10mL无水甲苯溶液中，80℃油浴加热，反应液在氮气氛围下搅拌12h。反应完后，旋干溶剂，以含甲醇的二氯甲烷溶液(1％)作为洗脱剂进行硅胶柱层析纯化得到化合物32(ESI-MS(m/z):[M+H]+calc’d.for C28H28N7O3:510.2254；found:510.25)；化合物33(ESI-MS(m/z):[M+H]+calc’d.for C29H30N7O4:540.2359；found:540.50)。化合物32，33对应的荧光团的结构如下：
化合物2的合成方法
将Cy7-Cl(1eq)和6-甲基-3-(4-甲基苄氨)1,2,4,5-四嗪(5eq)溶于10毫升无水二氯甲烷中，加入DIPEA(5eq)室温下搅拌24h，待荧光团消耗完全后，旋干溶剂，以含甲醇的二氯甲烷溶液(10％)作为洗脱剂进行硅胶柱层析纯化得到化合物2(ESI-MS(m/z):[M+H]+calc’d.for C43H48IN7:789.3016；found:789.10)。化合物2对应的荧光团的结构如下：
化合物14的合成方法
将9-丁酸乙酯-6，8，8-三甲基-2-氧代-6，7，8，9-四氢-2H-吡喃酮[3，2-g]喹啉-3-羧酸(1eq)、6-甲基-3-苄氨-1,2,4,5-四嗪(1.2eq)、EDCI(2.5eq)、HOBT(2.5eq)溶于10mL无水二氯甲烷溶液中，冰浴下加入DIPEA(3.0eq)，反应液在氮气氛围下搅拌12h。反应完后，反应液中依此加入水、饱和盐水、无水硫酸钠萃取并干燥有机相，旋干溶剂，以含乙酸乙酯的石油醚溶液(33％)作为洗脱剂进行硅胶柱层析纯化得到化合物14(ESI-MS(m/z):[M+H]+calc’d.for C32H37N6O5:585.2825；found:585.10)。化合物14对应的荧光团的结构如下：
化合物18的合成方法
将8-(3-羟基苯基)氟硼吡咯(1eq)和6-甲基-3-氯-1,2,4,5-四嗪(5eq)溶于10毫升无水二氯甲烷中，加入DIPEA(5eq)室温下搅拌24h，待荧光团消耗完全后，旋干溶剂，以含甲醇的二氯甲烷溶液(10％)作为洗脱剂进行硅胶柱层析纯化得到化合物18(ESI-MS(m/z):[M+H]+calc’d.for C22H21BF2N6O:434.1838；found:434.20)。化合物18对应的荧光团的结构如下：
实验例1化合物对超氧自由基的响应性
实验方法
将1mg上述化合物(结构通式图见图1)分别用作探针分子，分别以少量二甲基亚砜(DMSO)溶解作为5mM母液。对照组：将探针母液用PBS稀释至终浓度为5μM，记录其在最大发射波长下的荧光强度。实验组：另取探针母液分别与20当量超氧阴离子(KO2溶于含有1％18-冠醚-6的乙腈)反应30min后用PBS稀释至探针终浓度为5μM，记录其在最大发射波长下的荧光强度。将对照组与实验组最大波长处的荧光强度进行比较，化合物对超氧阴离子呈荧光显著增强。实验结果如表1所示。
表1
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实验例2化合物22和30检测HepG2细胞中不同浓度造模剂所产生超氧阴离子的荧光变化
将化合物22和30用作探针分子，将探针分子以少量二甲基亚砜(DMSO)溶解，配制成探针溶液。细胞预先用不同浓度的造模剂过氧化氢(0-2mM)孵育2h，然后用培养基稀释探针至5μM，加入HepG2细胞中孵育30min后，利用荧光显微镜进行成像。成像结果见图2A)所示，且如图2B)所示，化合物30可在活细胞中检测低浓度造模剂所诱导的超氧阴离子，化合物22在活细胞中只可检测高浓度造模剂所诱导的超氧阴离子。说明化合物30比化合物22对低浓度的超氧阴离子更灵敏。
实验例3化合物22和30检测HepG2细胞中同浓度造模剂造模不同时间所产生超氧阴离子的荧光变化
将化合物22和30用作探针分子。将探针分子以少量DMSO溶解，配制成探针溶液。细胞预先用2mM造模剂过氧化氢孵育不同时间(0-4h)，然后用培养基稀释探针至5μM，加入HepG2细胞中孵育30min后，利用荧光显微镜镜进行成像。成像结果见图3A)所示，且如图3B)所示，随着造模时间延长，化合物22和30可在活细胞中检测超氧阴离子的浓度变化。
实验例4化合物22和30检测HepG2细胞中造模剂诱导产生超氧阴离子的荧光变化及预先加入超氧阴离子清除剂后对造模剂诱导产生超氧阴离子的荧光变化
将化合物22和30用作探针分子，市售探针DCFHDA和DHE作为对照分子。将探针分子以少量DMSO溶解，配制成探针溶液。细胞分为4组，第一组(Control)不进行预处理然后用培养基稀释探针至5μM，加入HepG2细胞中孵育30min；第二组(H2O2)用2mM造模剂过氧化氢孵育2h然后用培养基稀释探针至5μM，加入HepG2细胞中孵育30min；第三组(Tiron+H2O2)预先孵育100μM Tiron 1h，然后再用2mM造模剂过氧化氢孵育2h，然后用培养基稀释探针至5μM，加入HepG2细胞中孵育30min；第四组(TEMPO+H2O2)预先孵育300μM TEMPO 1h，然后再用2mM造模剂过氧化氢孵育2h，用培养基稀释探针至5μM，加入HepG2细胞中孵育30min。利用荧光显微镜镜进行成像，成像结果见图4A)所示，且如图4B)所示，第二组细胞即造模组荧光强度均比其余一、三、四组强，但是化合物4和5在预先孵育超氧阴离子清除剂Tiron和TEMPO后，荧光强度能够显著降低，而市售探针DHE荧光未明显降低，说明化合物22和30对超氧阴离子的特异性更高。DCFHDA对细胞内超氧阴离子的响应倍数较低，说明化合物22和30对超氧阴离子的灵敏性更高。
实验例5化合物30检测缺血再灌注小鼠心肌组织的超氧阴离子
将化合物30用作探针分子，以含少量DMSO的生理盐水溶解配制成探针溶液。小鼠造心脏缺血再灌注(IR)模型，缺血45min，再灌15min，然后心内注射20μL探针溶液，将小鼠处死后，取心脏组织进行切片，然后进行共聚焦成像。结果如图5所示，对照组不进行处理，心肌组织几乎无荧光，缺血再灌注造模组的组织切片荧光显著增强。
实验例6化合物30检测缺血再灌注小鼠活体心脏组织的超氧阴离子
将化合物30用作探针分子，以含少量DMSO的生理盐水溶解配制成探针溶液。小鼠造心脏缺血再灌注(IR)模型，缺血45min，再灌15min，然后心内注射20μL探针溶液，将小鼠处死后，取心脏组织用小动物成像仪成像。结果如图6所示，对照组不进行处理，心脏组织几乎无荧光，缺血再灌注造模组的心脏组织荧光显著增强。
实验例7反应机理研究
将上述化合物分别溶解于DMSO中，制备10mM的储备液。取少量储备液，加入色谱纯乙腈稀释至40μM浓度；取超氧化钾固体以干燥色谱纯乙腈溶解，加入少量18-冠醚-6助溶，配制饱和溶液，并利用紫外分光光度计测定其在255nm下吸光度，利用摩尔吸光系数测定其浓度(ε＝1460M-1cm-1)。取20当量超氧溶液加入探针溶液中，并通过补足乙腈的方式将探针终浓度稀释至20μM，利用LCMS表征超氧与探针间的反应情况。LCMS条件，流动相A：含0.1％甲酸的纯水，流动性B：甲醇。流速：0.3mL/min。流动相比例：0-3min 20％流动相B→95％流动相B；3-8min 95％流动相B→20％流动相B；8-10min 20％流动相B。检测波长：254nm。检测结果见表2。
表2
| 化合物编号 | 超氧处理前分子量 | 超氧处理后分子量 | 转化率/％ |
| 2 | 662 | 650 | 100 |
| 5 | 567 | 555 | 100 |
| 7 | 628 | 616 | 100 |
| 8 | 640 | 628 | 100 |
| 10 | 694 | 682 | 100 |
| 12 | 610 | 598 | 100 |
| 14 | 584 | 572 | 100 |
| 18 | 434 | 422 | 100 |
| 22 | 305 | 293 | 100 |
| 23 | 434 | 422 | 100 |
| 30 | 435 | 423 | 100 |
| 31 | 306 | 294 | 100 |
| 32 | 509 | 497 | 100 |
| 33 | 539 | 527 | 100 |
最后应当说明的是，以上内容仅用以说明本发明的技术方案，而非对本发明保护范围的限制，本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换，均不脱离本发明技术方案的实质和范围。
Claims (6)
1.一种四嗪类化合物，或其互变异构体、药学上可接受的盐，其特征在于，选自以下结构中的一种：
2.权利要求1所述的四嗪类化合物，或其互变异构体、药学上可接受的盐的制备方法，其特征在于，包括以下步骤：
(1)将R1CN与乙腈、水合肼在二价锌离子的催化下反应，得到产物A；
(2)产物A进行氧化，后洗脱、纯化，得到四嗪类化合物；
所述四嗪类化合物的制备方法的化学反应方程式如下：
其中，所述R1CN选自如下结构的化合物：
3.根据权利要求2所述的制备方法，其特征在于，步骤(1)中所述R1CN与乙腈的摩尔比为1：10-50。
4.根据权利要求2所述的制备方法，其特征在于，步骤(2)中所述氧化所用氧化剂为空气或氧气。
5.权利要求1所述的四嗪类化合物，或其互变异构体、药学上可接受的盐或权利要求2-4任一项所述的制备方法得到的四嗪类化合物，或其互变异构体、药学上可接受的盐在超氧阴离子检测中的应用。
6.根据权利要求5所述的应用，其特征在于，所述超氧阴离子为溶液、细胞、组织或活体动物中的超氧阴离子。
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