CN110325502A - Compound and method for detecting hydrogen peroxide - Google Patents

Abstract

Translated fromChinese

本文公开了化合物和用于使用所述化合物原位检测过氧化氢的方法。所述化合物可包括式(I)、式(II)或式(III)的化合物或其盐，其中，X¹为单价前荧光团或前发光团部分；X²是二价前荧光团或前发光团部分；A和A'中的每个独立地由式(IV)或式(V)表示：

Disclosed herein are compounds and methods for in situ detection of hydrogen peroxide using the compounds. The compounds may include compounds of formula (I), formula (II) or formula (III) or salts thereof, wherein X¹ is a monovalent pre-fluorophore or pre-luminophore moiety; X² is a divalent pre-fluorophore or pre-luminophore moiety; each of A and A' is independently represented by formula (IV) or formula (V):

Description

Translated fromChinese

化合物和用于检测过氧化氢的方法Compound and method for detecting hydrogen peroxide

技术领域technical field

本发明属于荧光或发光探针领域，并且涉及化合物和用于检测过氧化氢的方法。The present invention is in the field of fluorescent or luminescent probes and relates to compounds and methods for the detection of hydrogen peroxide.

发明背景Background of the invention

反应性氧物质(ROS)和反应性氮物质(RNS)与老化、炎症和多种疾病如癌症和糖尿病的发展有关。过氧化氢(H₂O₂)是在氧化应激和在生物体中信号传导方面起关键作用的反应性氧物质。为了研究过氧化氢的反应机理，已开发了使用化学发光和荧光探针的分析方法，以检测其细胞内生成。这些探针可有效用于研究氧化应激和各种病理的信号传导。Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in aging, inflammation and the development of various diseases such as cancer and diabetes. Hydrogen peroxide (_H2O2) is a reactive oxygen species that plays a key role in oxidative stress and signaling in living organisms. To study the reaction mechanism of hydrogen peroxide, analytical methods using chemiluminescent and fluorescent probes have been developed to detect its intracellular production. These probes are useful for studying oxidative stress and signaling in various pathologies.

有用于过氧化氢检测分析的可商购荧光探针，例如Amplex Red试剂已被用于检测过氧化氢从活化的人体白细胞的释放。然而，许多可商购荧光探针有诸如缓慢的响应时间和灵敏度等缺点，使得原位跟踪过氧化氢困难。There are commercially available fluorescent probes for hydrogen peroxide detection assays, for example Amplex Red reagent has been used to detect hydrogen peroxide release from activated human leukocytes. However, many commercially available fluorescent probes have disadvantages such as slow response time and sensitivity, making it difficult to track hydrogen peroxide in situ.

发明概述Summary of the invention

因为需要开发新的化学发光和/或荧光探针以监测各种病理的氧化应激和信号传导，本文提供了化合物和用于使用可用于原位检测过氧化氢的化合物的方法。在至少一个具体实施方案中，化合物可包括式(I)、式(II)或式(III)的化合物或其盐：Because of the need to develop new chemiluminescent and/or fluorescent probes to monitor oxidative stress and signaling in various pathologies, provided herein are compounds and methods for using compounds useful for the in situ detection of hydrogen peroxide. In at least one specific embodiment, the compound may comprise a compound of formula (I), formula (II) or formula (III), or a salt thereof:

其中，A和A'中的每个独立地由式(IV)或式(V)表示：Wherein, each of A and A' is independently represented by formula (IV) or formula (V):

其中，所述化合物和三苯基鏻部分之间的连接具有下式(VI)或(VII)：Wherein, the connection between the compound and the triphenylphosphonium moiety has the following formula (VI) or (VII):

其中，n＝1-10；或化合物与吗啉或N，N-二取代胺部分之间的连接具有下式(VIII)或(IX)：Wherein, n=1-10; Or the connection between the compound and morpholine or N,N-disubstituted amine moiety has the following formula (VIII) or (IX):

在另一个具体实施方案中，使用化学发光和/或荧光化合物的方法可以包括使所述化合物与样品接触以形成荧光或发光化合物；和确定所述荧光或发光化合物的荧光或发光性质。In another specific embodiment, a method of using a chemiluminescent and/or fluorescent compound can comprise contacting said compound with a sample to form a fluorescent or luminescent compound; and determining the fluorescent or luminescent property of said fluorescent or luminescent compound.

附图简述Brief description of the drawings

在下面的详述中，参考附图描绘了本发明的示例性、非限制性和非穷尽的实施方案。因此，以可以详细地理解本发明的上述特征的方式，通过参考实施方案可以获得上面简要概述的本发明的更具体的描述，其中一些实施方案在附图中示出。然而，应当注意的是，附图仅示出了本发明的典型实施方案，并且因此不被认为是对其范围的限制，因为本发明可承认其它同样有效的实施方案。In the following detailed description, illustrative, non-limiting and non-exhaustive embodiments of the invention are depicted with reference to the accompanying drawings. So that the manner in which the above recited features of the invention can be understood in detail, a more particular description of the invention briefly summarized above has been had by reference to embodiments, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

图1是提出的YS-4-45和过氧化氢之间的反应机理。Figure 1 is the proposed reaction mechanism between YS-4-45 and hydrogen peroxide.

图2A是示出化合物YS-3-42的荧光强度随过氧化氢的浓度增加变化的图。在图2A中，曲线从上到下分别表示：化合物YS-3-42的10μM溶液+100μM H₂O₂、化合物YS-3-42的10μM溶液+50μM H₂O₂、化合物YS-3-42的10μM溶液+40μM H₂O₂、化合物YS-3-42的10μM溶液+30μMH₂O₂、化合物YS-3-42的10μM溶液+20μM H₂O₂、化合物YS-3-42的10μM溶液+10μM H₂O₂、化合物YS-3-42的10μM溶液+8μM H₂O₂、化合物YS-3-42的10μM溶液+6μM H₂O₂、化合物YS-3-42的10μM溶液+4μM H₂O₂、化合物YS-3-42的10μM溶液+2μM H₂O₂、和仅化合物YS-3-42的10μM溶液。图2B是示出在不同的ROS/RNS的存在下YS-3-42的荧光强度的图。FIG. 2A is a graph showing the change in fluorescence intensity of compound YS-3-42 as the concentration of hydrogen peroxide increases. In Figure 2A, the curves represent from top to bottom: 10 μM solution of compound YS-3-42 + 100 μM H₂ O₂ , 10 μM solution of compound YS-3-42 + 50 μM H₂ O₂ , compound YS-3- 10 μM solution of 42 + 40 μM H₂ O₂ , 10 μM solution of compound YS-3-42 + 30 μM H₂ O₂ , 10 μM solution of compound YS-3-42 + 20 μM H₂ O₂ , 10 μM of compound YS-3-42 Solution+10 μM H₂ O₂ , 10 μM solution of compound YS-3-42+8 μM H₂ O₂ , 10 μM solution of compound YS-3-42+6 μM H₂ O₂ , 10 μM solution of compound YS-3-42+ 4 μM H₂ O₂ , 10 μM solution of compound YS-3-42 + 2 μM H₂ O₂ , and 10 μM solution of compound YS-3-42 only. FIG. 2B is a graph showing the fluorescence intensity of YS-3-42 in the presence of different ROS/RNS.

图3A是示出化合物YS-4-45的荧光强度随过氧化氢的浓度增加变化的图。在图3A中，曲线从上到下分别表示：化合物YS-4-45的10μM溶液+100μM H₂O₂、化合物YS-4-45的10μM溶液+50μM H₂O₂、化合物YS-4-45的10μM溶液+40μM H₂O₂、化合物YS-4-45的10μM溶液+30μMH₂O₂、化合物YS-4-45的10μM溶液+20μM H₂O₂、化合物YS-4-45的10μM溶液+10μM H₂O₂、化合物YS-4-45的10μM溶液+8μM H₂O₂、化合物YS-4-45的10μM溶液+4μM H₂O₂、和仅化合物YS-4-45的10μM溶液。图3B是示出在不同的ROS/RNS的存在下YS-4-45的荧光强度的图。FIG. 3A is a graph showing the change in fluorescence intensity of compound YS-4-45 as the concentration of hydrogen peroxide increases. In Figure 3A, the curves represent from top to bottom: 10 μM solution of compound YS-4-45 + 100 μM H₂ O₂ , 10 μM solution of compound YS-4-45 + 50 μM H₂ O₂ , compound YS-4- 10 μM solution of 45 + 40 μM H₂ O₂ , 10 μM solution of compound YS-4-45 + 30 μM H₂ O₂ , 10 μM solution of compound YS-4-45 + 20 μM H₂ O₂ , 10 μM of compound YS-4-45 solution + 10 μM H₂ O₂ , 10 μM solution of compound YS-4-45 + 8 μM H₂ O₂ , 10 μM solution of compound YS-4-45 + 4 μM H₂ O₂ , and 10 μM of compound YS-4-45 only solution. Fig. 3B is a graph showing the fluorescence intensity of YS-4-45 in the presence of different ROS/RNS.

图4A示出化合物YS-2-172的荧光强度随过氧化氢的浓度增加变化的图。在图4A中，曲线从上到下分别表示：化合物YS-2-172的10μM溶液+500μM H₂O₂、化合物YS-2-172的10μM溶液+100μM H₂O₂、和仅化合物YS-2-172的10μM溶液。图4B是示出在不同的ROS/RNS的存在下YS-2-172的荧光强度的图。Figure 4A shows a graph of the fluorescence intensity of compound YS-2-172 as the concentration of hydrogen peroxide increases. In Fig. 4A, the curves represent from top to bottom: 10 μM solution of compound YS-2-172 + 500 μM H₂ O₂ , 10 μM solution of compound YS-2-172 + 100 μM H₂ O₂ , and only compound YS- 10 μM solution of 2-172. FIG. 4B is a graph showing the fluorescence intensity of YS-2-172 in the presence of different ROS/RNS.

图5示出在有或没有佛波醇12-十四酸酯13-乙酸酯(PMA)(200ng/mL)和DPI(100nM)的情况下与YS-3-42(10μM)共孵育的RAW264.7细胞的共聚焦图象(左)。对每个组中的细胞的相对平均荧光强度进行定量(右)。比例尺代表10μm。数据为平均值±s.e.m.，n＝79-109个细胞；***，相对于未处理的细胞或对照P＜0.001。Figure 5 shows the results of co-incubation with YS-3-42 (10 μM) with or without phorbol 12-myristate 13-acetate (PMA) (200 ng/mL) and DPI (100 nM). Confocal image of RAW264.7 cells (left). The relative mean fluorescence intensity of cells in each group was quantified (right). Scale bar represents 10 μm. Data are mean±s.e.m., n=79-109 cells; ***, P<0.001 vs. untreated cells or control.

图6示出RAW264.7(正常细胞)的共聚焦图像(左)和与YS-3-42(10μM)共孵育的MDA-MB-231(乳腺癌细胞)细胞的共聚焦图像(右)。对每个组中的细胞的相对平均荧光强度进行定量。比例尺代表10μm。数据为平均值±s.e.m.，n＝72-109细胞；**，P＜0.01。Figure 6 shows confocal images of RAW264.7 (normal cells) (left) and MDA-MB-231 (breast cancer cells) cells incubated with YS-3-42 (10 μΜ) (right). The relative mean fluorescence intensity of cells in each group was quantified. Scale bar represents 10 μm. Data are mean±s.e.m., n=72-109 cells; **, P<0.01.

图7示出探针YS-3-42和YS-4-45的细胞毒性。Figure 7 shows the cytotoxicity of probes YS-3-42 and YS-4-45.

图8示出在有或没有佛波醇12-十四酸酯13-乙酸酯(PMA)(200ng/mL)和DPI(100nM)的情况下与YS-4-45(10μM)共孵育的RAW264.7细胞的共聚焦图象。比例尺代表10μm。Figure 8 shows the results of co-incubation with YS-4-45 (10 μM) with or without phorbol 12-myristate 13-acetate (PMA) (200 ng/mL) and DPI (100 nM). Confocal image of RAW264.7 cells. Scale bar represents 10 μm.

图9A是示出化合物YS-4-112的荧光强度随过氧化氢的浓度增加变化的图。在图9A中，曲线从上到下分别表示：化合物YS-4-112的10μM溶液+100μM H₂O₂、化合物YS-4-112的10μM溶液+50μM H₂O₂、化合物YS-4-112的10μM溶液+30μM H₂O₂、化合物YS-4-112的10μM溶液+20μM H₂O₂、化合物YS-4-112的10μM溶液+10μM H₂O₂、化合物YS-4-112的10μM溶液+8μM H₂O₂、化合物YS-4-112的10μM溶液+6μM H₂O₂、化合物YS-4-112的10μM溶液+4μM H₂O₂、化合物YS-4-112的10μM溶液+2μM H₂O₂、和仅化合物YS-4-112的10μM溶液。图9B是用于定量的标准H₂O₂校准曲线。FIG. 9A is a graph showing the change in fluorescence intensity of compound YS-4-112 as the concentration of hydrogen peroxide increases. In Fig. 9A, the curves represent from top to bottom: 10 μM solution of compound YS-4-112 + 100 μM H₂ O₂ , 10 μM solution of compound YS-4-112 + 50 μM H₂ O₂ , compound YS-4- 10 μM solution of 112 + 30 μM H₂ O₂ , 10 μM solution of compound YS-4-112 + 20 μM H₂ O₂ , 10 μM solution of compound YS-4-112 + 10 μM H₂ O₂ , compound YS-4-112 10 μM solution + 8 μM H₂ O₂ , 10 μM solution of compound YS-4-112 + 6 μM H₂ O₂ , 10 μM solution of compound YS-4-112 + 4 μM H₂ O₂ , 10 μM solution of compound YS-4-112 +2 μM H₂ O₂ , and a 10 μM solution of compound YS-4-112 alone. Figure 9B is the standard_{H2O2calibration} curve used for quantification.

图10A示出与YS-4-112(10μM)共孵育的在不同的发育阶段的活斑马鱼的共聚焦图像。图10B示出与YS-3-42(10μM)共孵育的PMA处理的活斑马鱼的共聚焦图像。比例尺代表500μm。Figure 10A shows confocal images of live zebrafish at different developmental stages co-incubated with YS-4-112 (10 μΜ). Figure 10B shows confocal images of live PMA-treated zebrafish co-incubated with YS-3-42 (10 μΜ). Scale bar represents 500 μm.

图11A示出抗坏血酸的H₂O₂清除活性。图11B示出在使用YS-4-112的高通量试验中表没食子儿茶素没食子酸酯的H₂O₂清除活性。Figure 11A shows the_H2O2 scavenging activity of ascorbic_acid . FIG. 11B shows the H₂ O₂ scavenging activity of epigallocatechin gallate in a high-throughput assay using YS-4-112.

发明详述Detailed description of the invention

本文公开的化合物可具有快速的响应，并且是用于原位、在体内和在体外检测过氧化氢的高度选择性的探针。所述化合物可以具有新的和有效的用于过氧化氢检测的机理。它们可以不断提高对过氧化氢的灵敏度和选择性，并避免来自细胞ROS/RNS的干扰。The compounds disclosed herein can have a rapid response and are highly selective probes for the detection of hydrogen peroxide in situ, in vivo and in vitro. The compounds may have novel and efficient mechanisms for hydrogen peroxide detection. They can continuously improve the sensitivity and selectivity to hydrogen peroxide and avoid interference from cellular ROS/RNS.

这些化合物在过氧化氢成像方面的性能在多种细胞类型包括正常细胞和癌细胞中是高度稳健的。另外，这些化合物也可以用于开发用于药物筛选、癌症筛查和疾病诊断的快速、准确、或高通量的检测方法。The performance of these compounds in hydrogen peroxide imaging is highly robust in a variety of cell types, including normal and cancer cells. In addition, these compounds can also be used to develop rapid, accurate, or high-throughput assays for drug screening, cancer screening, and disease diagnosis.

所述化合物可以包括但不限于式(I)、式(II)或式(III)的化合物或其盐：The compounds may include, but are not limited to, compounds of formula (I), formula (II) or formula (III) or salts thereof:

其中：in:

R¹、R²、R³、R⁴和R⁵独立地选自H、F、Cl、Br、I、CN、烷基、卤代烷基、杂烷基、链烯基、炔基、芳烷基、芳基、烷芳基、杂环基、环烷基、环烯基、环炔基、羟烷基、氨基烷基、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、酰基氨基、羟基、硫醇、硫代烷基、烷氧基、烷硫基、烷氧基烷基、芳氧基、芳基烷氧基、酰氧基、硝基、氨基甲酰基、三氟甲基、苯氧基、苄氧基、膦酸、磷酸酯、磺酸(-SO₃H)、磺酸酯、磺酰胺、-C(＝O)-P¹和-C(＝O)-M-P²，其中，P¹和P²选自氢、卤素、烷氧基、羟基、硫醇、烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基、芳烷基、氨基甲酸酯基、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、烷硫基、杂烷基、烷基三苯基鏻和具有3至7个环原子的杂环基，或R²和R³一起形成选自芳基、杂环基、杂芳基和杂芳族基团的5、6或7元环，或R⁴和R⁵一起形成选自芳基、杂环基、杂芳基和杂芳族基团的5、6或7元环，R¹ , R² , R³ , R⁴ and R⁵ are independently selected from H, F, Cl, Br, I, CN, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aralkyl , aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino, alkylaryl ylamino, diarylamino, acylamino, hydroxy, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, nitro group, carbamoyl group, trifluoromethyl group, phenoxy group, benzyloxy group, phosphonic acid, phosphoric acid ester, sulfonic acid (-SO₃ H), sulfonate ester, sulfonamide, -C(=O)-P¹ and -C(=O)-MP² , wherein P¹ and P² are selected from hydrogen, halogen, alkoxy, hydroxyl, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl , cycloalkynyl, aryl, alkaryl, aralkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio radical, heteroalkyl, alkyltriphenylphosphonium and heterocyclyl having³ to 7 ring atoms, or R and^R together form a group selected from aryl, heterocyclyl, heteroaryl and heteroaromatic A^{5, 6 or 7 membered ring of a group, or R and R together form a 5,} 6 or 7 membered ring selected from aryl, heterocyclyl, heteroaryl and heteroaromatic groups,

M选自亚烷基、亚烯基、亚炔基、亚芳基、亚芳烷基和亚烷芳基；M is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, aralkylene and alkarylene;

R⁶选自氢、烷基、烷氧基烷基、烷酰基、-CF₃、卤素取代的低级烷基和(C＝O)-O-Z¹，其中，Z¹是选自烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基和芳基烷基的基团；R⁶ is selected from hydrogen, alkyl, alkoxyalkyl, alkanoyl, -CF₃ , lower alkyl substituted with halogen and (C=O)-OZ¹ , wherein Z¹ is selected from alkyl, alkene radical, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl and arylalkyl;

X¹是单价前荧光团或前发光团部分；X is^a monovalent profluorophore or proluminophore moiety;

X²是二价前荧光团或前发光团部分；X is a bivalent^{profluorophore} or proluminophore moiety;

A和A'中的每个独立地由式(IV)或式(V)表示：Each of A and A' is independently represented by formula (IV) or formula (V):

其中，R⁷和R⁹中的至少一个是(C＝O)-W¹，其中，W¹选自氢、烷基、烷氧基烷基、烷酰基、CF₃、卤素取代的低级烷基和(C＝O)-O-Z²，其中，Z²是选自烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基或芳基烷基的基团，和R⁷和R⁹的另一个、R⁸、R¹⁰和R¹¹独立地选自H、F、Cl、Br、I、CN、烷基、卤代烷基、杂烷基、链烯基、炔基、芳烷基、芳基、烷芳基、杂环基、环烷基、环烯基、环炔基、羟烷基、氨基烷基、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、酰基氨基、羟基、硫醇、硫代烷基、烷氧基、烷硫基、烷氧基烷基、芳氧基、芳基烷氧基、酰氧基、硝基、氨基甲酰基、三氟甲基、苯氧基、苄氧基、膦酸、磷酸酯、磺酸(-SO₃H)、磺酸酯、磺酰胺、-C(＝O)-P³和-C(＝O)-M-P⁴，其中P³和P⁴独立地选自氢、卤素、烷氧基、羟基、硫醇、烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基、芳基烷基、氨基甲酸酯、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、烷硫基、杂烷基、烷基三苯基鏻或具有3至7个环原子的杂环基；Wherein, at least one of R⁷ and R⁹ is (C=O)-W¹ , wherein W¹ is selected from hydrogen, alkyl, alkoxyalkyl, alkanoyl, CF₃ , lower alkyl substituted with halogen and (C=O)-OZ² , wherein Z² is selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl , and the other of R⁷ and R⁹ , R⁸ , R¹⁰ and R¹¹ are independently selected from H, F, Cl, Br, I, CN, alkyl, haloalkyl, heteroalkyl, alkenes radical, alkynyl, aralkyl, aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, Dialkylamino, alkylarylamino, diarylamino, acylamino, hydroxyl, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkane Oxy, acyloxy, nitro, carbamoyl, trifluoromethyl, phenoxy, benzyloxy, phosphonic acid, phosphate, sulfonic acid (-SO₃ H), sulfonate, sulfonamide, - C(=O)-P³ and -C(=O)-MP⁴ , wherein P³ and P⁴ are independently selected from hydrogen, halogen, alkoxy, hydroxyl, thiol, alkyl, alkenyl, alkyne radical, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl, arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylaryl Amino, diarylamino, alkylthio, heteroalkyl, alkyltriphenylphosphonium or heterocyclyl having 3 to 7 ring atoms;

M是亚烷基、亚烯基、亚炔基、亚芳基、亚芳烷基或亚烷芳基；M is alkylene, alkenylene, alkynylene, arylene, aralkylene or alkarylene;

R⁸和R¹⁰中的至少一个优选是羟基、烷氧基、或选自氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基的给电子基团；^At least^one of R and R is preferably hydroxyl, alkoxy, or an electron-donating group selected from amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino ;

或R⁷和R⁸一起形成选自芳基、杂环基、杂芳基或杂芳族基团的5、6或7元环；or R and R together form a⁵ , 6 or⁷ membered ring selected from aryl, heterocyclyl, heteroaryl or heteroaromatic;

或R¹⁰和R¹¹一起形成选自芳基、杂环基、杂芳基或杂芳族基团的5、6或7元环。Or R¹⁰ and R¹¹ together form a 5, 6 or 7 membered ring selected from aryl, heterocyclyl, heteroaryl or heteroaromatic.

单价前荧光团和/或前发光团部分可以包括但不限于：单价荧光素(fluorescein)(CAS号2321-07-5)、单价香豆素(CAS号91-64-5)、单价胺萘二甲酰亚胺、单价丹酰、单价二胺(bimane)(CAS号79769-56-5)、单价曙红、单价罗丹明(CAS号81-88-9；989-38-8；62669-70-9)、单价对甲氨基酚(CAS号3086-44-0)、单价花青、单价尼罗红(CAS号7385-67-3)、单价呫吨酮(CAS号90-47-1)、单价呫吨(CAS号92-83-1)、单价flazo橙(CAS号3566-94-7)、单价SNARF-1、单价荧光黄(CAS号71206-95-6；67769-47-5)、单价6-十二酰基-N,N-二甲基-2-萘胺(laurdan)(CAS号74515-25-6)、单价2-萘胺(CAS号91-59-8)、单价试卤灵(CAS号635-78-9)和单价荧光素(luciferin)(例如CAS号2591-17-5)。Monovalent profluorophore and/or proluminophore moieties may include, but are not limited to: monovalent fluorescein (CAS No. 2321-07-5), monovalent coumarin (CAS No. 91-64-5), monovalent amine naphthalene Dicarboximide, monovalent dansyl, monovalent diamine (bimane) (CAS No. 79769-56-5), monovalent eosin, monovalent rhodamine (CAS No. 81-88-9; 989-38-8; 62669- 70-9), monovalent rhodol (CAS No. 3086-44-0), monovalent cyanine, monovalent Nile Red (CAS No. 7385-67-3), monovalent xanthone (CAS No. 90-47-1 ), monovalent xanthene (CAS No. 92-83-1), monovalent flazo orange (CAS No. 3566-94-7), monovalent SNARF-1, monovalent fluorescent yellow (CAS No. 71206-95-6; 67769-47-5 ), monovalent 6-lauroyl-N,N-dimethyl-2-naphthylamine (laurdan) (CAS No. 74515-25-6), monovalent 2-naphthylamine (CAS No. 91-59-8), monovalent Resorufin (CAS No. 635-78-9) and monovalent luciferin (eg CAS No. 2591-17-5).

二价前荧光团或/和前发光团部分可以包括但不限于：二价荧光素(CAS号2321-07-5)、二价香豆素(CAS号91-64-5)、二价胺萘二甲酰亚胺、二价丹酰、二价二胺(CAS号79769-56-5)、二价曙红、二价罗丹明(CAS号81-88-9；989-38-8；62669-70-9)、二价对甲氨基酚(CAS号3086-44-0)、二价花青、二价尼罗红(CAS号7385-67-3)、二价呫吨酮(CAS号90-47-1)、二价呫吨(CAS号92-83-1)、二价flazo橙(CAS号3566-94-7)、二价SNARF-1、二价荧光黄(CAS号71206-95-6；67769-47-5)、二价6-十二酰基-N,N-二甲基-2-萘胺(CAS号74515-25-6)、二价2-萘胺(CAS号91-59-8)、二价试卤灵(CAS号635-78-9)和二价荧光素(如CAS号2591-17-5)。Divalent profluorophore or/and proluminophore moieties may include, but are not limited to: divalent fluorescein (CAS No. 2321-07-5), divalent coumarin (CAS No. 91-64-5), divalent amine Naphthalimide, divalent dansyl, divalent diamine (CAS No. 79769-56-5), divalent eosin, divalent rhodamine (CAS No. 81-88-9; 989-38-8; 62669-70-9), divalent rhodol (CAS No. 3086-44-0), divalent cyanine, divalent Nile Red (CAS No. 7385-67-3), divalent xanthone (CAS No. 90-47-1), divalent xanthene (CAS No. 92-83-1), divalent flazo orange (CAS No. 3566-94-7), divalent SNARF-1, divalent fluorescent yellow (CAS No. 71206 -95-6; 67769-47-5), divalent 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (CAS No. 74515-25-6), divalent 2-naphthylamine (CAS No. 91-59-8), divalent resorufin (CAS No. 635-78-9) and divalent fluorescein (such as CAS No. 2591-17-5).

所述化合物可以包括但不限于化合物1-28和57：Such compounds may include, but are not limited to, compounds 1-28 and 57:

其中，R＝H或CF₃。Wherein, R=H or CF₃ .

所述化合物可以包括但不限于化合物29-44：Such compounds may include, but are not limited to, compounds 29-44:

其中，R＝H或CF₃。Wherein, R=H or CF₃ .

所述化合物可以包含但不限于一个或多个游离羧基基团，其中，至少一个羧基基团通过酰胺键连接与带正电荷的线粒体靶向的三苯基鏻部分或溶酶体靶向的吗啉或N，N-二取代的胺部分缀合，其中，所述化合物与三苯基鏻部分之间的连接具有下式(VI)或(VII)：The compound may comprise, but is not limited to, one or more free carboxyl groups, wherein at least one carboxyl group is linked via an amide bond to a positively charged mitochondrial-targeting triphenylphosphonium moiety or a lysosome-targeting moiety. A morphine or N, N-disubstituted amine moiety is conjugated, wherein the link between the compound and the triphenylphosphonium moiety has the following formula (VI) or (VII):

其中n＝1-10；where n=1-10;

或所述化合物与吗啉或N，N-二取代胺部分之间的连接具有下式(VIII)或(IX)：Or the link between the compound and the morpholine or N,N-disubstituted amine moiety has the following formula (VIII) or (IX):

其中，n＝1-10；式(IX)中的R¹²或R¹³独立地为C_1-10烷基或烯烃。Wherein, n=1-10; R¹² or R¹³ in formula (IX) is independently C_1-10 alkyl or alkene.

所述化合物可以包括但不限于化合物45-56：Said compounds may include, but are not limited to compounds 45-56:

其中，R＝H或CF₃。Wherein, R=H or CF₃ .

如本文中所使用的，术语“烷基”包括含有直链和支链的饱和脂肪族烃。在一些实施方案中，烷基基团具有1至20个碳原子、1至10个碳原子、1至6个碳原子、或1至4个碳原子。例如，术语“C_1-6烷基”，以及本文提及的其它基团的烷基部分(例如，C_1-6烷氧基)是指1至6个碳原子的直链或支链基团(例如，甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基或正己基)。对于又一个实例，术语“C_1-4烷基”是指1至4个碳原子的直链或支链的脂肪族烃链；术语“C_1-3烷基”是指1至3个碳原子的直链或支链的脂肪族烃链；术语“C_1-2烷基”是指1至2个碳原子的直链或支链的脂肪族烃链；并且术语“C₁烷基”是指甲基。术语“低级烷基”是指1至6个碳原子的直链或支链基团。烷基基团任选地可被一个或多个(例如1至5个)合适的取代基取代。As used herein, the term "alkyl" includes both straight and branched chain saturated aliphatic hydrocarbons. In some embodiments, an alkyl group has 1 to 20 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. For example, the term "C_1-6 alkyl", as well as the alkyl portion of other groups referred to herein (e.g., C_1-6 alkoxy), refers to straight or branched chain groups of 1 to 6 carbon atoms group (eg, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl). For yet another example, the term "C_1-4 alkyl" refers to a straight or branched aliphatic hydrocarbon chain of 1 to 4 carbon atoms; the term "C_1-3 alkyl" refers to a 1 to 3 carbon atom straight or branched aliphatic hydrocarbon chain; the term "C_1-2 alkyl" means a straight or branched aliphatic hydrocarbon chain of 1 to 2 carbon atoms; and the term "C₁ alkyl" means methyl. The term "lower alkyl" refers to a straight or branched chain group of 1 to 6 carbon atoms. Alkyl groups are optionally substituted with one or more (eg 1 to 5) suitable substituents.

如本文所使用的，术语“链烯基”包括具有至少一个碳碳双键的脂肪族烃，包括具有至少一个碳碳双键的直链和支链。在一些实施方案中，链烯基基团具有2至20个碳原子、2至10个碳原子、2至6个碳原子、3至6个碳原子、或2至4个碳原子。例如，如本文所用，术语“C_2-6链烯基”意指2至6个碳原子的直链或支链的不饱和基团(具有至少一个碳碳双键)，包括但不限于：乙烯基、1-丙烯基、2-丙烯基(烯丙基)、异丙烯基、2-甲基-1-丙烯基、1-丁烯基、2-丁烯基等。链烯基基团可以任选地被一个或多个(例如1至5个)合适的取代基取代。当化合物含有链烯基基团时，该链烯基基团可以作为纯E型、纯Z型或其任何混合物存在。As used herein, the term "alkenyl" includes aliphatic hydrocarbons having at least one carbon-carbon double bond, including straight and branched chains having at least one carbon-carbon double bond. In some embodiments, an alkenyl group has 2 to 20 carbon atoms, 2 to 10 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 2 to 4 carbon atoms. For example, as used herein, the term "C alkenyl" means a linear or branched unsaturated group (having at least one carbon-carbon double bond) of 2 to₆ carbon atoms, including but not limited to: Vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like. An alkenyl group may be optionally substituted with one or more (eg 1 to 5) suitable substituents. When a compound contains an alkenyl group, the alkenyl group may exist as pure E form, pure Z form, or any mixture thereof.

如本文中所使用的，术语“炔基”包括具有至少一个碳碳三键的脂肪族烃，包括具有至少一个碳碳三键的直链和支链。在一些实施方案中，炔基基团具有2至20个、2至10个、2至6个、或3至6个碳原子。例如，如本文所用，术语“C_2-6炔基”指具有2至6个碳原子的如上文所定义的直链或支链烃链炔基基团。炔基基团可以任选地被一个或多个(例如1至5个)合适的取代基取代。As used herein, the term "alkynyl" includes aliphatic hydrocarbons having at least one carbon-carbon triple bond, including straight and branched chains having at least one carbon-carbon triple bond. In some embodiments, an alkynyl group has 2 to 20, 2 to 10, 2 to 6, or 3 to 6 carbon atoms. For example, the term "C alkynyl" as used herein refers to a straight or branched chain alkynyl group as defined above having 2 to₆ carbon atoms. An alkynyl group may be optionally substituted with one or more (eg 1 to 5) suitable substituents.

如本文所用，术语“环烷基”包括饱和的或不饱和的非芳族单环或多环(如双环)的烃环(例如，单环如环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基、或双环包括螺环、稠合或桥接体系(例如双环[1.1.1]戊基、双环[2.2.1]庚烷基、双环[3.2.1]辛基或双环[5.2.0]壬基、十氢萘基等)。所述环烷基基团可具有3至15个碳原子。在一些实施方案中，环烷基可任选地含有一个、两个或更多个非累积的非芳族双键或三键和/或1至3个氧代基团。在一些实施方案中，双环烷基基团有6至14个碳原子。例如，术语“C_3-14环烷基”包括3至14个成环碳原子的饱和或不饱和非芳族单环或多环(例如双环)烃环(例如，环丙基、环丁基、环戊基、环己基、双环[1.1.1]戊基、或环癸基)；和术语“C_3-7环烷基”包括3至7个成环碳原子的饱和或不饱和的非芳族单环或多环(如双环)(例如，环丙基、环丁基、环戊基、环己基、双环[1.1.1]戊-1-基、或双环[1.1.1]戊-2-基)。对于另一个实例，术语“C_3-6环烷基”包括3至6个成环碳原子的饱和或不饱和非芳族单环或多环(如双环)烃环。对于又一个实例，术语“C_3-4环烷基”是指环丙基或环丁基。也包括在术语“环烷基”中的是具有一个或多个稠合到环烷基环的芳族环(包括芳基和杂芳基)的部分，例如，环戊烷、环戊烯、环己烷等的苯并或噻吩基衍生物(例如，2，3-二氢-1H-茚-1-基、或1H-茚-2(3H)-酮-1-基)。环烷基基团可以任选地被1个或多个(例如，1至5个)合适的取代基取代。As used herein, the term "cycloalkyl" includes saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon rings (for example, monocyclic such as cyclopropyl, cyclobutyl, cyclopentyl, Cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic rings including spiro, fused or bridged systems (e.g. bicyclo[1.1.1]pentyl, bicyclo[2.2.1]heptyl, bicyclo[3.2 .1] octyl or bicyclo[5.2.0] nonyl, decahydronaphthyl, etc.). The cycloalkyl group can have from 3 to 15 carbon atoms. In some embodiments, the cycloalkyl group can optionally contain one, two or more non-cumulative non-aromatic double or triple bonds and/or 1 to 3 oxo groups. In some embodiments, bicycloalkyl groups have 6 to 14 carbons Atoms. For example, the term "C_3-14 cycloalkyl" includes saturated or unsaturated non-aromatic monocyclic or polycyclic (eg bicyclic) hydrocarbon rings (eg, cyclopropyl, cyclo butyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pentyl, or cyclodecyl); and the term "C_3-7 cycloalkyl" includes saturated or unsaturated Non-aromatic monocyclic or polycyclic (such as bicyclic) (for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[1.1.1]pent-1-yl, or bicyclo[1.1.1] Pent-2-yl). For another example, the term "C_3-6 cycloalkyl" includes saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon rings of 3 to 6 ring-forming carbon atoms For yet another example, the term "C_3-4 cycloalkyl" refers to cyclopropyl or cyclobutyl. Also included in the term "cycloalkyl" are those having one or more rings fused to the cycloalkyl Moieties of aromatic rings (including aryl and heteroaryl), for example, benzo or thienyl derivatives of cyclopentane, cyclopentene, cyclohexane, etc. (for example, 2,3-dihydro-1H-indene -1-yl, or 1H-inden-2(3H)-one-1-yl). The cycloalkyl group can be optionally substituted by one or more (for example, 1 to 5) suitable substituents .

如本文中所使用的，术语“芳基”可以包括具有共轭π电子体系的所有碳单环或稠环多环芳族基团。芳基基团在环(多个环)中具有6或10个碳原子。最常见地，芳基基团在环中具有6个碳原子。例如，如本文中所使用的，术语“C_6-10芳基”是指含有6至10个碳原子的芳族基团，例如苯基或萘基。芳基基团可任选地被一个或多个(例如，1至5个)合适的取代基取代。术语“亚芳基”是指二价芳基部分。As used herein, the term "aryl" may include all carbon monocyclic or fused-ring polycyclic aromatic groups having a conjugated π-electron system. An aryl group has 6 or 10 carbon atoms in the ring(s). Most commonly, aryl groups have 6 carbon atoms in the ring. For example, as used herein, the term "C_6-10 aryl" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl or naphthyl. An aryl group can be optionally substituted with one or more (eg, 1 to 5) suitable substituents. The term "arylene" refers to a divalent aryl moiety.

如本文中所使用的，术语“杂芳基”包括具有一个或多个杂原子环成员(成环原子)的单环或稠环多环芳族杂环基团，在至少一个环中所述一个或多个杂原子环成员独立地选自O、S和N。杂芳基基团具有5至14个成环原子，包括1至13个碳原子，和1至8个选自O、S和N的杂原子。在一些实施方案中，杂芳基基团具有5至10个成环原子，包括一至四个杂原子。杂芳基基团还可以含有一至三个氧代或硫羰(即＝S)基团。在一些实施方案中，杂芳基基团具有5至8个成环原子，包含一个、两个或三个杂原子。例如，术语“5元杂芳基”是指如上所述在单环杂芳基环中具有5个成环原子的单环杂芳基基团；术语“6元杂芳基”包括在单环杂芳基环中具有6个成环原子的如上定义的单环杂芳基基团；并且术语“5或6元杂芳基”包括在单环杂芳基环中具有5或6个成环原子的如上所定义的单环杂芳基基团。对于另一个实例，术语“5或10元杂芳基”包括在单环或双环杂芳基环中具有5、6、7、8、9或10个成环原子的如上所定义的单环或双环杂芳基基团。杂芳基基团可以任选地被1或多个(例如，1至5个)合适的取代基取代。单环杂芳基的实例包括具有5个成环原子(包括1-3个杂原子)的那些或具有6个成环原子(包括一个、两个或三个氮杂原子)的那些。稠合的双环杂芳基的实例包括两个稠合的5和/或6元单环环(包括一至四个杂原子)。As used herein, the term "heteroaryl" includes monocyclic or fused-ring polycyclic aromatic heterocyclic groups having one or more heteroatom ring members (ring-forming atoms), in at least one ring The one or more heteroatom ring members are independently selected from O, S and N. A heteroaryl group has 5 to 14 ring-forming atoms, including 1 to 13 carbon atoms, and 1 to 8 heteroatoms selected from O, S and N. In some embodiments, heteroaryl groups have 5 to 10 ring-forming atoms, including one to four heteroatoms. A heteroaryl group may also contain one to three oxo or thiocarbonyl (ie =S) groups. In some embodiments, heteroaryl groups have 5 to 8 ring atoms, including one, two, or three heteroatoms. For example, the term "5-membered heteroaryl" refers to a monocyclic heteroaryl group having 5 ring-forming atoms in the monocyclic heteroaryl ring as described above; the term "6-membered heteroaryl" includes A monocyclic heteroaryl group as defined above having 6 ring-forming atoms in the heteroaryl ring; and the term "5 or 6 membered heteroaryl" includes 5 or 6 ring-forming atoms in the monocyclic heteroaryl ring Atoms of a monocyclic heteroaryl group as defined above. For another example, the term "5- or 10-membered heteroaryl" includes monocyclic or bicyclic heteroaryl rings as defined above having 5, 6, 7, 8, 9 or 10 ring-forming atoms in the monocyclic or bicyclic heteroaryl ring. Bicyclic heteroaryl groups. A heteroaryl group can be optionally substituted with one or more (eg, 1 to 5) suitable substituents. Examples of monocyclic heteroaryl groups include those having 5 ring atoms (including 1-3 heteroatoms) or those having 6 ring atoms (including one, two or three nitrogen heteroatoms). Examples of fused bicyclic heteroaryl groups include two fused 5 and/or 6 membered monocyclic rings (including one to four heteroatoms).

如本文中所使用的，术语“杂环基”包括饱和的和部分饱和的具有5至15个选自碳、氮、硫和氧的环成员的含杂原子的环形基团，其中至少一个环原子是杂原子。杂环基基团可以含有一个、两个或三个环，其中，这种环可以以悬垂的方式连接或可以稠合。饱和杂环基团的实例包括饱和的含有1至4个氮原子的3至6元杂单环基团[例如吡咯烷基、咪唑烷基、哌啶基、哌嗪基等]；饱和的含有1至2个氧原子和1至3个氮原子的3至6元杂单环基团[例如吗啉基等]；含有1至2个硫原子和1至3个氮原子的饱和的3至6元杂单环基团[例如，噻唑烷基等]。部分饱和的杂环基基团的实例包括二氢噻吩、二氢吡喃、二氢呋喃和二氢噻唑。杂环基基团的非限制性实例包括2-吡咯啉基、3-吡咯啉基、吡咯烷基、1，3-二氧戊环基、2H-吡喃基、4H-吡喃基、哌啶基、1,4-二噁烷基、吗啉基、1,4-二噻烷基、硫代吗啉基等。As used herein, the term "heterocyclyl" includes saturated and partially saturated heteroatom-containing ring groups having 5 to 15 ring members selected from carbon, nitrogen, sulfur, and oxygen, wherein at least one ring Atoms are heteroatoms. A heterocyclyl group may contain one, two or three rings, wherein such rings may be attached in a pendant fashion or may be fused. Examples of saturated heterocyclic groups include saturated 3- to 6-membered heteromonocyclic groups [such as pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, etc.] containing 1 to 4 nitrogen atoms; 3 to 6-membered heteromonocyclic groups [such as morpholinyl, etc.] with 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms; saturated 3 to 3 nitrogen atoms containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms 6-membered heteromonocyclic group [for example, thiazolidinyl, etc.]. Examples of partially saturated heterocyclyl groups include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole. Non-limiting examples of heterocyclyl groups include 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl, 2H-pyranyl, 4H-pyranyl, piper Pyridyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, etc.

如本文所用，术语“烷氧基”或“烷基氧基”包括-O-烷基基团。例如，术语“C_1-6烷氧基”或“C_1-6烷基氧基”包括-O-(C_1-6烷基)基团；和术语“C_1-4烷氧基”或“C_1-4烷基氧基”可以包括-O-(C_1-4烷基)基团。对于另一个实例，术语“C_1-2烷氧基”或“C_1-2烷基氧基”是指-O-(C_1-2烷基)基团。烷氧基的实例包括甲氧基、乙氧基、丙氧基(例如正丙氧基和异丙氧基)、叔丁氧基等。所述烷氧基或烷基氧基基团可以任选地被1或多个(例如，1至5个)合适的取代基取代。As used herein, the term "alkoxy" or "alkyloxy" includes the -O-alkyl group. For example, the term "C_1-6 alkoxy" or "C_1-6 alkyloxy" includes a -O-(C_1-6 alkyl) group; and the term "C_1-4 alkoxy" or "C_1-4 alkyloxy" may include -O-(C_1-4 alkyl) groups. For another example, the term "C_1-2 alkoxy" or "C_1-2 alkyloxy" refers to a -O-(C_1-2 alkyl) group. Examples of alkoxy groups include methoxy, ethoxy, propoxy (eg, n-propoxy and isopropoxy), tert-butoxy, and the like. The alkoxy or alkyloxy groups may be optionally substituted with one or more (eg, 1 to 5) suitable substituents.

如这里所使用的，术语“C_6-10芳氧基”包括-O-(C_6-10芳基)基团。C_6-10芳氧基基团的实例是-O-苯基[即，苯氧基]。C_6-10芳氧基基团可以任选地被一个或多个(例如，1至5个)合适的取代基取代。As used herein, the term "C_6-10 aryloxy" includes -O-(C_6-10 aryl) groups. An example of a C_6-10 aryloxy group is -O-phenyl [ie, phenoxy]. A C_6-10 aryloxy group may be optionally substituted with one or more (eg, 1 to 5) suitable substituents.

如本文所用，术语“氨基烷基”包括具有1至约10个碳原子的直链和/或支链烷基基团，所述1至约10个碳原子中的任何一个可被一个或多个氨基基团取代。这类基团的实例包括氨基甲基、氨基乙基、氨基丙基、氨基丁基和氨基己基。As used herein, the term "aminoalkyl" includes linear and/or branched chain alkyl groups having from 1 to about 10 carbon atoms, any of which may be replaced by one or more Amino group substitution. Examples of such groups include aminomethyl, aminoethyl, aminopropyl, aminobutyl and aminohexyl.

如本文所使用的，“氧代”是指＝O。当氧代在碳原子上取代时，它们一起形成羰基部分[-C(＝O)-]。当氧代在硫原子上取代时，它们一起形成亚磺酰基部分[-S(＝O)-]；当两个氧代基团在硫原子上取代时，它们一起形成磺酰基部分[-S(＝O)₂-]。As used herein, "oxo" refers to =O. When oxo is substituted on a carbon atom, they together form the carbonyl moiety [-C(=O)-]. When oxo groups are substituted on a sulfur atom, they together form a sulfinyl moiety [-S(=O)-]; when two oxo groups are substituted on a sulfur atom, they together form a sulfonyl moiety [-S (=0)_2- ].

如本文中所使用的，术语“任选地取代”指取代是任选的，且因此包括未取代和取代的原子和部分。“取代”的原子或部分表示，在指定原子或部分上的任意氢可以用选自指定的取代基团取代(最多在所指定原子或部分上的每个氢原子用选自指定的取代基团取代)，条件是不超过所指定原子或部分的正常化合价，并且取代得到稳定的化合物。例如，如果甲基基团(即，CH₃)是任选取代的，那么碳原子上的最多达3个氢原子可以用取代基团取代。As used herein, the term "optionally substituted" means that substitution is optional, and thus includes unsubstituted and substituted atoms and moieties. A "substituted" atom or moiety means that any hydrogen on the specified atom or moiety may be replaced with a substituent selected from the specified substituents (up to each hydrogen atom on the specified atom or moiety with a substituent selected from the Substitution), provided that the normal valence of the designated atom or moiety is not exceeded, and that the substitution results in a stable compound. For example, if a methyl group (ie,_CH3 ) is optionally substituted, up to 3 hydrogen atoms on the carbon atoms may be replaced with substituent groups.

所述化合物可被用作用于测量、检测和/或筛选过氧化氢的试剂。这些化合物可以产生荧光或发光的颜色，如蓝色、绿色、黄色、红色、或远红色。这些化合物可用于直接或间接地测量化学样品、生物样品和病理样品中过氧化氢的存在和/或过氧化氢的量。所述化合物可以用于原位、在体内和在体外检测过氧化氢的存在或确定过氧化氢的水平。Said compounds can be used as reagents for measuring, detecting and/or screening hydrogen peroxide. These compounds can produce fluorescent or luminescent colors such as blue, green, yellow, red, or far red. These compounds can be used to directly or indirectly measure the presence and/or amount of hydrogen peroxide in chemical, biological and pathological samples. The compounds can be used to detect the presence or determine the level of hydrogen peroxide in situ, in vivo and in vitro.

使用该化合物检测样品中过氧化氢的存在和/或确定样品中过氧化氢的水平的方法可以包括但不限于：使式(I)、式(II)、式(III)的化合物或其盐与样品接触以形成荧光和/或发光化合物；和确定所述荧光或发光化合物的荧光和/或发光性质。Methods of using the compound to detect the presence of hydrogen peroxide in a sample and/or determine the level of hydrogen peroxide in a sample may include, but are not limited to: making a compound of formula (I), formula (II), formula (III) or a salt thereof contacting a sample to form a fluorescent and/or luminescent compound; and determining the fluorescent and/or luminescent properties of the fluorescent or luminescent compound.

使用所述化合物用于检测生物体的体内过氧化氢的存在或确定生物体的体内过氧化氢的水平的方法可以包括但不限于：将式(I)、式(II)、式(III)的化合物或其盐给予生物体以形成荧光和/或发光化合物；和确定所述荧光和/或发光化合物的荧光和/或发光性质。The method of using the compound for detecting the presence of hydrogen peroxide in an organism or determining the level of hydrogen peroxide in an organism may include, but is not limited to: formula (I), formula (II), formula (III) administering a compound or a salt thereof to an organism to form a fluorescent and/or luminescent compound; and determining the fluorescent and/or luminescent properties of the fluorescent and/or luminescent compound.

使用所述化合物用于在体外检测过氧化氢的存在或确定过氧化氢的水平的方法可以包括但不限于：将式(I)、式(II)、式(III)的化合物或其盐给予体外样品以形成荧光和/或发光化合物；和确定所述荧光和/或发光化合物的荧光和/或发光。Methods of using the compounds for detecting the presence of hydrogen peroxide or determining the level of hydrogen peroxide in vitro may include, but are not limited to: administering a compound of formula (I), formula (II), formula (III), or a salt thereof an in vitro sample to form a fluorescent and/or luminescent compound; and determining the fluorescence and/or luminescence of said fluorescent and/or luminescent compound.

使用该化合物检测样品中过氧化氢的存在或测定样品中过氧化氢的水平的高通量方法可以包括但不限于：使式(I)、式(II)、式(III)的化合物或其盐与样品接触以形成一种或多种荧光或发光化合物；和确定所述荧光和/或发光化合物的荧光和/或发光性质以确定样品中过氧化氢的存在和/或过氧化氢的量。High-throughput methods for detecting the presence of hydrogen peroxide in a sample or determining the level of hydrogen peroxide in a sample using the compound may include, but are not limited to: making a compound of formula (I), formula (II), formula (III) or contacting the salt with the sample to form one or more fluorescent or luminescent compounds; and determining the fluorescent and/or luminescent properties of the fluorescent and/or luminescent compounds to determine the presence and/or amount of hydrogen peroxide in the sample .

使用所述化合物用于筛选提高或降低过氧化氢水平的一种或多种目标化合物的高通量方法可包括：使式(I)、式(II)、式(III)的化合物或其盐与目标化合物接触以形成一种或多种荧光或发光化合物；和测量所述荧光或发光化合物的荧光或发光性质，以确定目标化合物的存在和/或目标化合物的量。A high-throughput method of using the compound for screening one or more target compounds that increase or decrease hydrogen peroxide levels may comprise: making a compound of formula (I), formula (II), formula (III), or a salt thereof contacting a target compound to form one or more fluorescent or luminescent compounds; and measuring the fluorescent or luminescent properties of the fluorescent or luminescent compounds to determine the presence and/or amount of the target compound.

用于任何使用所述化合物的方法的样品可以包括但不限于化学样品、生物样品和病理样品。生物样品可以包括但不限于微生物、细胞、组织、器官、来自植物或动物的部分、整个植物或动物以及它们的提取物。病理样品可以包括但不限于血液、尿、唾液、血清、呼吸气体、呼出气冷凝物、关节液、以及它们的提取物。Samples for any method using the compounds may include, but are not limited to, chemical samples, biological samples, and pathological samples. Biological samples may include, but are not limited to, microorganisms, cells, tissues, organs, parts from plants or animals, whole plants or animals, and extracts thereof. Pathological samples may include, but are not limited to, blood, urine, saliva, serum, breath gas, exhaled breath condensate, joint fluid, and extracts thereof.

使用化合物的方法可以包括将化合物用于荧光和/或发光探针组合物中。荧光和/或发光探针组合物可以包括但不限于一种或多种载体、一种或多种溶剂、一种或多种酸、一种或多种碱、一种或多种缓冲剂、及其混合物。Methods of using the compounds can include using the compounds in fluorescent and/or luminescent probe compositions. Fluorescent and/or luminescent probe compositions may include, but are not limited to, one or more carriers, one or more solvents, one or more acids, one or more bases, one or more buffers, and mixtures thereof.

在一些实施方案中，本发明涉及本发明的化合物或本发明的荧光或发光探针组合物用于在生物体的体内或在体外检测样品中过氧化氢的存在和/或确定其中过氧化氢的水平的用途；或本发明的化合物或本发明的荧光或发光探针组合物用于筛选提高或降低过氧化氢的水平的一种或多种目标化合物的用途。In some embodiments, the invention relates to the use of compounds of the invention or fluorescent or luminescent probe compositions of the invention for detecting the presence and/or determining the presence of hydrogen peroxide in a sample in vivo or in vitro in an organism. or the use of the compound of the present invention or the fluorescent or luminescent probe composition of the present invention for screening one or more target compounds that increase or decrease the level of hydrogen peroxide.

材料和方法Materials and methods

本文描述的实施例和实施方案仅用于说明目的，并且将给本领域技术人员建议其各种修改或改变并且各种修改或改变包括在本申请的精神和范围内。此外，本文公开的任何发明或其实施方案的任何要素或限制可以与本文公开的任何和/或所有其他要素或限制(单独地或以任何组合)或其任何其他发明或实施方案组合，并且所有这些组合预期在本发明的范围但不限于此范围。The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes thereof will be suggested to those skilled in the art and are included within the spirit and scope of the present application. Furthermore, any element or limitation of any invention or embodiment disclosed herein may be combined with any and/or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all These combinations are contemplated but not limited to the scope of the invention.

实施例1-绿色荧光化合物YS-3-42、YS-4-45、YS-2-172和红色荧光化合物YS-4-Embodiment 1-green fluorescent compound YS-3-42, YS-4-45, YS-2-172 and red fluorescent compound YS-4-112的合成Synthesis of 112

在氩气下在0℃向NaH(在矿物油中60wt％，44mg，1.10mmol)的DMF(2mL，无水)搅拌溶液中加入2-羟基-4-(羟甲基)苯甲醛(152mg，1.00mmol)的DMF(2mL，无水)溶液。搅拌15分钟后，逐滴加入氯甲基甲醚(91μL，1.20mmol)，然后再搅拌30分钟。然后用乙酸乙酯稀释反应混合物，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-3-1分离为浅黄色油状物，使用乙酸乙酯：己烷(1：1)作为洗脱剂。产率：174mg(89％)。¹H NMR(300MHz,CDCl₃)δ10.39(s,1H),7.75(d,J＝8.0Hz,1H),7.19(s,1H),7.00(d,J＝8.0Hz,1H),5.27(s,2H),4.69(s,2H),3.48(s,3H)；¹³C NMR(75MHz,CDCl₃)δ189.7,159.9,150.1,128.6,124.4,119.7,112.6,94.5,64.4,56.6。To a stirred solution of NaH (60 wt % in mineral oil, 44 mg, 1.10 mmol) in DMF (2 mL, anhydrous) was added 2-hydroxy-4-(hydroxymethyl)benzaldehyde (152 mg, 1.00 mmol) in DMF (2 mL, anhydrous). After stirring for 15 minutes, chloromethyl methyl ether (91 μL, 1.20 mmol) was added dropwise, followed by stirring for another 30 minutes. The reaction mixture was then diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-3-1 was isolated as light yellow oil by flash chromatography on silica gel using ethyl acetate:hexane (1:1) as eluent. Yield: 174 mg (89%).¹ H NMR (300MHz, CDCl₃ )δ10.39(s,1H),7.75(d,J=8.0Hz,1H),7.19(s,1H),7.00(d,J=8.0Hz,1H),5.27 (s, 2H), 4.69 (s, 2H), 3.48 (s, 3H);¹³ C NMR (75 MHz, CDCl₃ ) δ 189.7, 159.9, 150.1, 128.6, 124.4, 119.7, 112.6, 94.5, 64.4, 56.6.

向YS-3-1(130mg，0.662mmol)和氨基甲酰基咪唑(100mg，0.796mmol)的DMF(4mL，无水)搅拌溶液中加入NaH(在矿物油中60wt％，30mg，0.728mmol)。将悬浮液在室温下搅拌18小时，然后在空气流下浓缩。通过快速色谱法在硅胶上将化合物YS-3-2分离为白色粘性固体，使用乙酸乙酯：己烷(1：1)作为洗脱剂。产率：54mg(32％)。To a stirred solution of YS-3-1 (130 mg, 0.662 mmol) and carbamoyl imidazole (100 mg, 0.796 mmol) in DMF (4 mL, anhydrous) was added NaH (60 wt % in mineral oil, 30 mg, 0.728 mmol). The suspension was stirred at room temperature for 18 hours, then concentrated under a stream of air. Compound YS-3-2 was isolated as a white sticky solid by flash chromatography on silica gel using ethyl acetate:hexane (1:1) as eluent. Yield: 54 mg (32%).

¹H NMR(300MHz,CDCl₃)δ10.41(s,1H),7.75(d,J＝7.9Hz,1H),7.13(s,1H),6.99(d,J＝7.9Hz,1H),5.25(s,2H),5.07(s,2H),3.47(s,3H),2.77(d,J＝4.9Hz,3H)；¹³C NMR(75MHz,CDCl₃)δ189.4,159.7,156.7,145.4,128.6,124.8,120.6,113.7,94.6,65.7,56.6,27.6。¹ H NMR (300MHz, CDCl₃ ) δ10.41(s, 1H), 7.75(d, J=7.9Hz, 1H), 7.13(s, 1H), 6.99(d, J=7.9Hz, 1H), 5.25 (s,2H),5.07(s,2H),3.47(s,3H),2.77(d,J=4.9Hz,3H);¹³ C NMR(75MHz,CDCl₃ )δ189.4,159.7,156.7,145.4,128.6 , 124.8, 120.6, 113.7, 94.6, 65.7, 56.6, 27.6.

向YS-3-3(200mg，0.478mmol)的甲醇(4mL)溶液中加入NaOH(191mg，4.78mmol)的水(2mL)溶液。将得到的混合物在室温下搅拌1小时，然后蒸发有机溶剂，用1N HCl中和直到形成大量沉淀。将悬浮液过滤，用水洗涤并在真空中干燥，以得到粗荧光素衍生物。在氩气下在室温下向粗产物的DMF(5mL)溶液加入Et₃N(201μL，1.43mmol)。搅拌10分钟后，将PhNTf₂(205mg，0.574mmol)加入到得到的混合物中，将其在室温下搅拌2小时。用乙酸乙酯稀释反应混合物，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩，以得到粗品荧光素三氟甲烷磺酸酯衍生物。在氩气下室温下向粗产物的DMF(5mL)溶液中加入Cs₂CO₃(188mg，0.526mmol)，搅拌30分钟后，加入MeI(60μL,0.956mmol)。将得到的混合物搅拌2小时，然后用乙酸乙酯稀释，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-3-9分离为白色粘性固体，使用乙酸乙酯：己烷(1：4)作为洗脱剂。产率：77mg(31％)。To a solution of YS-3-3 (200 mg, 0.478 mmol) in methanol (4 mL) was added a solution of NaOH (191 mg, 4.78 mmol) in water (2 mL). The resulting mixture was stirred at room temperature for 1 hour, then the organic solvent was evaporated and neutralized with 1N HCl until a large precipitate formed. The suspension was filtered, washed with water and dried in vacuo to obtain crude fluorescein derivatives. To a solution of the crude product in DMF (5 mL) was added_Et3N (201 μL, 1.43 mmol) at room temperature under argon. After stirring for 10 minutes, PhNTf₂ (205 mg, 0.574 mmol) was added to the resulting mixture, which was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated in vacuo to give crude fluorescein triflate derivative. To a solution of the crude product in DMF (5 mL) under argon at room temperature was added Cs₂ CO₃ (188 mg, 0.526 mmol), and after stirring for 30 minutes, MeI (60 μL, 0.956 mmol) was added. The resulting mixture was stirred for 2 hours, then diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-3-9 was isolated as a white sticky solid by flash chromatography on silica gel using ethyl acetate:hexane (1:4) as eluent. Yield: 77 mg (31%).

¹H NMR(400MHz,CDCl₃)δ8.69(s,0.5H),8.36(d,J＝8.0Hz,0.5H),8.31(d,J＝8.0Hz,0.5H),8.10(d,J＝8.0Hz,0.5H),7.81(s,0.5H),6.96(d,J＝8.8Hz,1H),6.88(d,J＝8.8Hz,1H),6.81(s,1H),6.72–6.63(m,2H),3.99(s,1.5H),3.89(s,1.5H),3.84(s,3H)。¹ H NMR (400MHz, CDCl₃ ) δ8.69(s, 0.5H), 8.36(d, J=8.0Hz, 0.5H), 8.31(d, J=8.0Hz, 0.5H), 8.10(d, J =8.0Hz,0.5H),7.81(s,0.5H),6.96(d,J=8.8Hz,1H),6.88(d,J=8.8Hz,1H),6.81(s,1H),6.72–6.63 (m,2H), 3.99(s,1.5H), 3.89(s,1.5H), 3.84(s,3H).

¹³C NMR(100MHz,CDCl₃)δ168.02,165.42,161.97,156.46,152.91,151.89,150.26,136.86,136.53,132.80,131.54,130.12,130.08,129.75,129.03,128.98,127.06,126.82,125.58,125.29,124.27,119.18,116.98,112.77,110.78,101.19,101.16,82.06,81.78,55.80,52.93。¹⁹F NMR(376MHz,CDCl₃)δ–72.6(m,3F)。¹³ C NMR(100MHz,CDCl₃ )δ168.02,165.42,161.97,156.46,152.91,151.89,150.26,136.86,136.53,132.80,131.54,130.12,130.08,129.75,129.03,128.98,127.06,126.82,125.58,125.29,124.27 , 119.18, 116.98, 112.77, 110.78, 101.19, 101.16, 82.06, 81.78, 55.80, 52.93.¹⁹ F NMR (376 MHz, CDCl₃ ) δ - 72.6 (m, 3F).

给烘箱干燥的圆底烧瓶中装入Pd₂(dba)₃(12mg，0.013mmol)、Xantphos(23mg，0.039mmol)和Cs₂CO₃(59mg，0.182mmol)，并用氩气冲洗5分钟。加入YS-3-9(70mg,0.13mmol)和YS-3-2(40mg,0.156mmol)的无水二噁烷(5mL)溶液，并将所得混合物在室温在氩气下搅拌30分钟，加热至100℃并再搅拌20小时。使反应混合物冷却至室温，用DCM稀释并通过硅藻土垫过滤。然后将滤液在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-3-36分离为白色粘性固体，通过使用EtOAc：DCM(1：5)作为洗脱剂。产率：38mg(46％)。An oven-dried round bottom flask was charged with Pd2(dba₎₃₍ 12mg, 0.013mmol), Xantphos (23mg, 0.039mmol) and_Cs2CO3 (_59mg , 0.182mmol) and flushed with argon for 5 minutes. A solution of YS-3-9 (70 mg, 0.13 mmol) and YS-3-2 (40 mg, 0.156 mmol) in anhydrous dioxane (5 mL) was added, and the resulting mixture was stirred at room temperature under argon for 30 minutes, heated to 100°C and stirred for another 20 hours. The reaction mixture was cooled to room temperature, diluted with DCM and filtered through a pad of celite. The filtrate was then concentrated in vacuo. Compound YS-3-36 was isolated as a white sticky solid by flash chromatography on silica gel by using EtOAc:DCM (1:5) as eluent. Yield: 38 mg (46%).

¹H NMR(400MHz,CDCl₃)δ10.44(s,1H),8.69(s,0.5H),8.37–8.32(m,0.5H),8.31–8.27(m,0.5H),8.09(d,J＝8.0Hz,0.5H),7.82(s,0.5H),7.80(d,J＝8.0Hz,1H),7.25–7.23(m,1H),7.11(s,1H),7.02–6.96(m,2H),6.80–6.75(m,2H),6.70–6.60(m,2H),5.22(s,2H),5.20(s,2H),4.00(s,1.5H),3.89(s,1.5H),3.84(s,3H),3.46(s,3H),3.37(s,3H)；¹³C NMR(100MHz,CDCl₃)δ189.39,168.42,168.35,165.59,161.83,159.91,156.92,154.77,153.34,152.41,152.34,151.61,151.54,145.23,145.18,144.82,136.62,136.28,132.53,131.25,130.04,129.11,129.07,128.75,128.56,128.52,127.11,126.90,125.43,125.38,124.98,124.36,121.29,120.34,116.16,113.99,113.45,112.19,110.31,110.28,101.15,101.12,94.64,82.94,82.74,66.89,56.63,55.77,52.89,37.69。¹ H NMR (400MHz, CDCl₃ )δ10.44(s,1H),8.69(s,0.5H),8.37–8.32(m,0.5H),8.31–8.27(m,0.5H),8.09(d, J=8.0Hz,0.5H),7.82(s,0.5H),7.80(d,J=8.0Hz,1H),7.25–7.23(m,1H),7.11(s,1H),7.02–6.96(m ,2H),6.80–6.75(m,2H),6.70–6.60(m,2H),5.22(s,2H),5.20(s,2H),4.00(s,1.5H),3.89(s,1.5H ),3.84(s,3H),3.46(s,3H),3.37(s,3H);¹³ C NMR(100MHz,CDCl₃ )δ189.39,168.42,168.35,165.59,161.83,159.91,156.92,154.77,153.34, 152.41,152.34,151.61,151.54,145.23,145.18,144.82,136.62,136.28,132.53,131.25,130.04,129.11,129.07,128.75,128.56,128.52,127.11,126.90,125.43,125.38,124.98,124.36,121.29,120.34, 116.16, 113.99, 113.45, 112.19, 110.31, 110.28, 101.15, 101.12, 94.64, 82.94, 82.74, 66.89, 56.63, 55.77, 52.89, 37.69.

在冰/水浴中向YS-3-36(11mg,0.017mmol)的DCM(3mL)溶液中逐滴加入TFA(3mL)，然后将溶液在室温下搅拌2小时。将反应混合物在真空中浓缩，与甲苯共沸3次，然后溶解在THF(4.5mL)中。在室温下逐滴加入LiOH(2.5mg,0.104mmol)的水(1.5mL)溶液。将得到的混合物在室温下搅拌2小时。将反应混合物用乙酸乙酯稀释，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-3-42分离为白色粘性固体，使用乙醇：DCM(1：9)作为洗脱剂。产率：7.8mg(79％).¹H NMR(400MHz,丙酮-d6)δ10.01(s,1H),8.55(s,0.5H),8.42(d,J＝7.9Hz,0.5H),8.36(d,J＝7.5Hz,0.5H),8.15(d,J＝8.2Hz,0.5H),7.88(s,0,5H),7.78–7.71(m,1H),7.48(d,J＝7.5Hz,0.5H),7.46(d,J＝8.6Hz,1H),7.20(d,J＝8.6Hz,H),7.05(d,J＝9.0Hz,2H),6.98–6.88(m,3H),6.88–6.82(m,1H),6.74(d,J＝8.7Hz,1H),5.23(s,1H),3.89(s,3H),3.41(s,3H)；¹³C NMR(100MHz,CDCl₃)δ196.25,168.46,165.64,161.95,161.85,156.94,154.81,153.31,152.47,152.40,151.65,146.36,145.18,136.65,136.32,134.14,132.54,131.27,130.08,129.12,129.08,128.56,128.51,127.12,126.93,125.49,125.40,124.43,120.26,118.50,115.96,113.95,112.24,110.34,101.18,101.15,66.68,55.78,52.91。To a solution of YS-3-36 (11 mg, 0.017 mmol) in DCM (3 mL) was added TFA (3 mL) dropwise in an ice/water bath, and the solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo, azeotroped 3 times with toluene, then dissolved in THF (4.5 mL). A solution of LiOH (2.5 mg, 0.104 mmol) in water (1.5 mL) was added dropwise at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-3-42 was isolated as a white sticky solid by flash chromatography on silica gel using ethanol:DCM (1:9) as eluent. Yield: 7.8 mg (79%).¹ H NMR (400MHz, acetone-d6) δ10.01 (s, 1H), 8.55 (s, 0.5H), 8.42 (d, J = 7.9Hz, 0.5H), 8.36(d,J=7.5Hz,0.5H),8.15(d,J=8.2Hz,0.5H),7.88(s,0,5H),7.78–7.71(m,1H),7.48(d,J= 7.5Hz, 0.5H), 7.46(d, J=8.6Hz, 1H), 7.20(d, J=8.6Hz, H), 7.05(d, J=9.0Hz, 2H), 6.98–6.88(m, 3H ),6.88–6.82(m,1H),6.74(d,J＝8.7Hz,1H),5.23(s,1H),3.89(s,3H),3.41(s,3H);¹³ C NMR(100MHz, CDCl₃ )δ196.25,168.46,165.64,161.95,161.85,156.94,154.81,153.31,152.47,152.40,151.65,146.36,145.18,136.65,136.32,134.14,132.54,131.27,130.08,129.12,129.08,128.56,128.51,127.12 , 126.93, 125.49, 125.40, 124.43, 120.26, 118.50, 115.96, 113.95, 112.24, 110.34, 101.18, 101.15, 66.68, 55.78, 52.91.

将3-碘苯酚(1.1g,5.0mmol)和邻苯二甲酸酐(370mg,2.50mmol)在甲磺酸(3mL)中的混合物在135℃在氩气下搅拌48小时。在冷却至室温后，将反应混合物倒入50mL冰/水混合物中，并搅拌以沉淀出灰色固体。将固体通过过滤收集并溶解在氯仿中，然后通过硅胶垫以得到无色溶液。将溶液浓缩，以得到YS-4-42[817170-65-3]，为白色固体。产率：629mg(45％)。A mixture of 3-iodophenol (1.1 g, 5.0 mmol) and phthalic anhydride (370 mg, 2.50 mmol) in methanesulfonic acid (3 mL) was stirred at 135 °C under argon for 48 hours. After cooling to room temperature, the reaction mixture was poured into 50 mL of ice/water mixture and stirred to precipitate a gray solid. The solid was collected by filtration and dissolved in chloroform, then passed through a pad of silica gel to give a colorless solution. The solution was concentrated to give YS-4-42 [817170-65-3] as a white solid. Yield: 629 mg (45%).

¹H NMR(300MHz,CDCl₃)δ8.07–8.00(m,1H),7.69–7.62(m,4H),7.38(d,J＝1.6Hz,1H),7.36(d,J＝1.6Hz,1H),7.16–7.08(m,1H),6.55(s,1H),6.52(s,1H)。¹ H NMR (300MHz, CDCl₃ ) δ8.07–8.00(m,1H),7.69–7.62(m,4H),7.38(d,J=1.6Hz,1H),7.36(d,J=1.6Hz, 1H), 7.16–7.08(m,1H), 6.55(s,1H), 6.52(s,1H).

给烘箱干燥的圆底烧瓶装入Pd₂(dba)₃(12mg,0.013mmol)、Xantphos(23mg,0.039mmol)和Cs₂CO₃(59mg,0.182mmol)，并用氩气冲洗5分钟。加入YS-4-42(36mg,0.066mmol)和YS-3-2(50mg,0.197mmol)的二噁烷(5mL)溶液，并将得到的混合物首先在氩气下在室温下搅拌30分钟，加热至100℃并再搅拌20小时。使反应混合物冷却至室温，用DCM稀释并通过硅藻土垫过滤。然后将滤液在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-4-44分离为白色粘性固体，使用乙酸乙酯：己烷(3：2)作为洗脱剂。产率：18mg(34％)。An oven-dried round bottom flask was charged with Pd2(dba₎₃₍ 12mg, 0.013mmol), Xantphos (23mg, 0.039mmol) and_Cs2CO3 (_59mg , 0.182mmol) and flushed with argon for 5 minutes. A solution of YS-4-42 (36 mg, 0.066 mmol) and YS-3-2 (50 mg, 0.197 mmol) in dioxane (5 mL) was added, and the resulting mixture was first stirred at room temperature under argon for 30 minutes, Heat to 100°C and stir for a further 20 hours. The reaction mixture was cooled to room temperature, diluted with DCM and filtered through a pad of celite. The filtrate was then concentrated in vacuo. Compound YS-4-44 was isolated as a white sticky solid by flash chromatography on silica gel using ethyl acetate:hexane (3:2) as eluent. Yield: 18 mg (34%).

¹H NMR(300MHz,CDCl₃)δ10.44(s,2H),8.05(d,J＝6.8Hz,1H),7.79(d,J＝8.0Hz,2H),7.73–7.63(m,2H),7.25(d,J＝1.9Hz,2H),7.19(d,J＝7.1Hz,1H),7.11(s,2H),7.05–6.96(m,4H),6.84(s,1H),6.81(s,1H),5.22(s,4H),5.20(s,4H),3.46(s,6H),3.37(s,6H)；¹ H NMR (300MHz, CDCl₃ ) δ10.44(s, 2H), 8.05(d, J=6.8Hz, 1H), 7.79(d, J=8.0Hz, 2H), 7.73–7.63(m, 2H) ,7.25(d,J=1.9Hz,2H),7.19(d,J=7.1Hz,1H),7.11(s,2H),7.05–6.96(m,4H),6.84(s,1H),6.81( s,1H),5.22(s,4H),5.20(s,4H),3.46(s,6H),3.37(s,6H);

¹³C NMR(75MHz,CDCl₃)δ189.3,169.2,159.8,154.7,152.9,151.3,145.1,144.7,135.3,130.1,128.7,128.5,126.3,125.3,124.9,124.0,121.3,120.3,116.6,113.8,113.4,94.6,81.8,66.8,56.5,37.6。¹³ C NMR (75MHz, CDCl₃ ) δ189.3, 169.2, 159.8, 154.7, 152.9, 151.3, 145.1, 144.7, 135.3, 130.1, 128.7, 128.5, 126.3, 125.3, 124.9, 124.0, 121.3, 1113.64. , 94.6, 81.8, 66.8, 56.5, 37.6.

在冰/水浴中向YS-4-44(12mg,0.015mmol)的DCM(3mL)溶液中逐滴加入TFA(3mL)，然后将溶液在室温下搅拌2小时。将反应混合物在真空中浓缩，并与甲苯共沸3次。通过快速色谱法在硅胶上将化合物YS-4-45分离为白色粘性固体，使用乙酸乙酯：己烷(3：2)作为洗脱剂。产率：10mg(99％)。To a solution of YS-4-44 (12 mg, 0.015 mmol) in DCM (3 mL) was added dropwise TFA (3 mL) in an ice/water bath, and the solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and azeotroped 3 times with toluene. Compound YS-4-45 was isolated as a white sticky solid by flash chromatography on silica gel using ethyl acetate:hexane (3:2) as eluent. Yield: 10 mg (99%).

¹H NMR(400MHz,CDCl₃)δ11.04(s,2H),9.87(s,2H),8.05(d,J＝7.4Hz,1H),7.74–7.62(m,2H),7.53(d,J＝7.9Hz,2H),7.22(d,J＝7.5Hz,1H),7.01(d,J＝8.7Hz,2H),6.93(d,J＝8.0Hz,2H),6.90(s,2H),6.82(s,1H),6.80(s,1H),5.19(s,4H),3.38(s,6H)；¹³C NMR(150MHz,CDCl3)δ196.1,169.2,161.8,154.71,152.8,151.4,146.2,145.0,135.3,134.0,130.1,128.5,126.3,125.3,124.1,121.1,120.2,118.4,116.6,115.9,113.8,81.9,66.6,37.6。¹ H NMR (400MHz, CDCl₃ ) δ11.04(s, 2H), 9.87(s, 2H), 8.05(d, J=7.4Hz, 1H), 7.74–7.62(m, 2H), 7.53(d, J=7.9Hz, 2H), 7.22(d, J=7.5Hz, 1H), 7.01(d, J=8.7Hz, 2H), 6.93(d, J=8.0Hz, 2H), 6.90(s, 2H) ,6.82(s,1H),6.80(s,1H),5.19(s,4H),3.38(s,6H);¹³ C NMR(150MHz,CDCl3)δ196.1,169.2,161.8,154.71,152.8,151.4,146.2 , 145.0, 135.3, 134.0, 130.1, 128.5, 126.3, 125.3, 124.1, 121.1, 120.2, 118.4, 116.6, 115.9, 113.8, 81.9, 66.6, 37.6.

将4-溴-1,8-萘二甲酸酐(1.229g,4.40mmol)和丙胺(0.400mL,4.90mmol)在乙醇(200mL)中的悬浮液在50℃下搅拌1小时，然后加热至回流再持续1小时。将反应混合物冷却至室温，然后在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-2-166[100865-05-2]分离为白色粘性固体，使用己烷：DCM(1：1)作为洗脱剂。产率：1.268g(90％)。A suspension of 4-bromo-1,8-naphthalic anhydride (1.229 g, 4.40 mmol) and propylamine (0.400 mL, 4.90 mmol) in ethanol (200 mL) was stirred at 50 °C for 1 h, then heated to reflux Continue for 1 more hour. The reaction mixture was cooled to room temperature, then concentrated in vacuo. Compound YS-2-166 [100865-05-2] was isolated as a white sticky solid by flash chromatography on silica gel using hexane:DCM (1:1) as eluent. Yield: 1.268 g (90%).

¹H NMR(300MHz,CDCl₃)δ8.65(d,J＝7.3Hz,1H),8.55(d,J＝8.5Hz,1H),8.40(d,J＝7.9Hz,1H),8.03(d,J＝7.9Hz,1H),7.84(dd,J＝8.5,7.3Hz,1H),4.17–4.10(m,2H),1.83–1.69(m,2H),1.01(t,J＝7.4Hz,3H)。¹ H NMR (300MHz, CDCl₃ ) δ8.65(d, J=7.3Hz, 1H), 8.55(d, J=8.5Hz, 1H), 8.40(d, J=7.9Hz, 1H), 8.03(d ,J=7.9Hz,1H),7.84(dd,J=8.5,7.3Hz,1H),4.17–4.10(m,2H),1.83–1.69(m,2H),1.01(t,J=7.4Hz, 3H).

将YS-2-166溶液(250mg,0.789mmol)、Cu粉(5mg,0.0789mmol)、氢氧化铵溶液(32wt.％NH₃水溶液,5mL)和N-甲基-2-吡咯烷酮(5mL)在密封管中在100℃下搅拌12小时。然后将反应混合物冷却至室温，用乙酸乙酯稀释，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-2-167[94860-68-1]分离为黄色粘性固体，使用丙酮：氯仿(1：6)作为洗脱液。产率：74mg(37％)。YS-2-166 solution (250mg, 0.789mmol), Cu powder (5mg, 0.0789mmol), ammonium hydroxide solution (32wt.% NH₃ aqueous solution, 5mL) and N-methyl-2-pyrrolidone (5mL) in Stir at 100°C for 12 hours in a sealed tube. The reaction mixture was then cooled to room temperature, diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-2-167[94860-68-1] was isolated as a yellow sticky solid by flash chromatography on silica gel using acetone:chloroform (1:6) as eluent. Yield: 74 mg (37%).

¹H NMR(300MHz,CDCl₃)δ8.59(d,J＝7.3Hz,1H),8.41(d,J＝8.2Hz,1H),8.10(d,J＝8.4Hz,1H),7.65(dd,J＝8.4,7.3Hz,1H),6.88(d,J＝8.2Hz,1H),4.17–4.10(m,2H),1.81–1.63(m,2H),1.00(t,J＝7.4Hz,3H)。¹ H NMR (300MHz, CDCl₃ ) δ8.59(d, J=7.3Hz, 1H), 8.41(d, J=8.2Hz, 1H), 8.10(d, J=8.4Hz, 1H), 7.65(dd ,J=8.4,7.3Hz,1H),6.88(d,J=8.2Hz,1H),4.17–4.10(m,2H),1.81–1.63(m,2H),1.00(t,J=7.4Hz, 3H).

在室温下在氩气下向2-羟基-4-(羟甲基)苯甲醛(40mg,0.263mmol)和K₂CO₃(72mg,0.526mmol)的THF(5mL无水)溶液中加入烯丙基溴(173μL,0.526mmol)。将该溶液在回流下搅拌12小时。用乙酸乙酯稀释反应混合物，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-2-165分离为无色油状物，使用乙酸乙酯：己烷(1：2)作为洗脱剂。产率：31mg(70％)。To a solution of 2-hydroxy-4-(hydroxymethyl)benzaldehyde (40 mg, 0.263 mmol) and K₂ CO₃ (72 mg, 0.526 mmol) in THF (5 mL of anhydrous) at room temperature under argon was added allyl bromide (173 μL, 0.526 mmol). The solution was stirred at reflux for 12 hours. The reaction mixture was diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-2-165 was isolated as a colorless oil by flash chromatography on silica gel using ethyl acetate:hexane (1:2) as eluent. Yield: 31 mg (70%).

¹H NMR(400MHz,CDCl₃)δ10.46(s,1H),7.79(d,J＝7.9Hz,1H),7.03(s,1H),6.96(d,J＝7.9Hz,1H),6.13–6.01(m,1H),5.48–5.41(m,1H),5.33(d,J＝10.6Hz,1H),4.73(s,2H),4.66(d,J＝5.1Hz,2H)；¹ H NMR (400MHz, CDCl₃ ) δ10.46(s, 1H), 7.79(d, J=7.9Hz, 1H), 7.03(s, 1H), 6.96(d, J=7.9Hz, 1H), 6.13 –6.01(m,1H),5.48–5.41(m,1H),5.33(d,J=10.6Hz,1H),4.73(s,2H),4.66(d,J=5.1Hz,2H);

¹³C NMR(100MHz,CDCl₃)δ189.7,161.4,149.9,132.4,128.8,124.3,118.8,118.3,110.8,69.3,64.8。¹³ C NMR (100 MHz, CDCl₃ ) δ 189.7, 161.4, 149.9, 132.4, 128.8, 124.3, 118.8, 118.3, 110.8, 69.3, 64.8.

在室温下在氩气下向YS-2-167(46mg,0.182mmol)的甲苯(5mL，无水)溶液中加入DIPEA(90μL，0.546mmol)和三光气(65mg,0.218mmol)。将溶液加热至回流持续1小时。冷却至室温后，将YS-2-165(35mg,0.182mmol)的无水DCM(2mL)的溶液加入至反应混合物中，再将其搅拌3小时，在真空中浓缩，并溶解在乙醇(5mL)中。将所得溶液用氩气脱气30分钟，并在氩气下加入Pd(PPh₃)₄(21mg,0.0182mmol)。将所得混合物在回流下加热12小时。将反应混合物冷却至室温，通过硅藻土垫过滤，并在真空中浓缩。通过快速色谱法在硅胶上将化合物YS-2-172分离为黄色粘性固体，使用乙酸乙酯：己烷(1：2)作为洗脱剂。产率：12mg(15％)。To a solution of YS-2-167 (46 mg, 0.182 mmol) in toluene (5 mL, anhydrous) was added DIPEA (90 μL, 0.546 mmol) and triphosgene (65 mg, 0.218 mmol) at room temperature under argon. The solution was heated to reflux for 1 hour. After cooling to room temperature, a solution of YS-2-165 (35 mg, 0.182 mmol) in anhydrous DCM (2 mL) was added to the reaction mixture, which was stirred for 3 hours, concentrated in vacuo, and dissolved in ethanol (5 mL )middle. The resulting solution was degassed with argon for 30 minutes, and Pd(PPh₃ )₄ (21 mg, 0.0182 mmol) was added under argon. The resulting mixture was heated at reflux for 12 hours. The reaction mixture was cooled to room temperature, filtered through a pad of celite, and concentrated in vacuo. Compound YS-2-172 was isolated as a yellow sticky solid by flash chromatography on silica gel using ethyl acetate:hexane (1:2) as eluent. Yield: 12 mg (15%).

¹H NMR(300MHz,CDCl₃)δ11.09(s,1H),9.91(s,1H),8.64(d,J＝7.1Hz,1H),8.61(d,J＝8.3Hz,1H),8.36(d,J＝8.3Hz,1H),8.20(d,J＝8.4Hz,1H),7.83–7.75(m,1H),7.60(d,J＝8.2Hz,1H),7.55(s,1H),7.09–7.01(m,2H),5.31(s,2H),4.17–4.10(m,2H),1.81–1.72(m,2H),1.01(t,J＝7.4Hz,3H)；¹ H NMR (300MHz, CDCl₃ ) δ11.09(s, 1H), 9.91(s, 1H), 8.64(d, J=7.1Hz, 1H), 8.61(d, J=8.3Hz, 1H), 8.36 (d,J=8.3Hz,1H),8.20(d,J=8.4Hz,1H),7.83–7.75(m,1H),7.60(d,J=8.2Hz,1H),7.55(s,1H) ,7.09–7.01(m,2H),5.31(s,2H),4.17–4.10(m,2H),1.81–1.72(m,2H),1.01(t,J＝7.4Hz,3H);

¹³C NMR(125MHz,CDCl₃)δ196.2,164.2,163.8,152.8,145.3,138.6,134.3,132.6,131.5,129.1,127.0,125.8,123.8,120.6,119.0,117.2,116.6,66.8,42.1,21.5,11.7。¹³ C NMR (125MHz, CDCl₃ ) δ196.2, 164.2, 163.8, 152.8, 145.3, 138.6, 134.3, 132.6, 131.5, 129.1, 127.0, 125.8, 123.8, 120.6, 119.0, 117.2, 116.427, 61.8, .

在冰/水浴中向试卤灵(250mg,1.17mmol)的DMF(12mL,无水)溶液中加入NaH(在矿物油中60wt％，59mg,1.41mmol)，并将该混合物搅拌30分钟。然后，加入PhNTf₂(500mg,1.41mmol)，并使反应混合物温热至室温，并再搅拌8小时。用乙酸乙酯稀释反应混合物，用1N HCl、水和盐水洗涤。将有机层用无水硫酸镁干燥，并在真空中浓缩。通过快速柱色谱法在硅胶上将化合物YS-4-110[941601-75-8]分离为黄色粘性固体，使用乙酸乙酯：己烷(1：2)作为洗脱剂。产率：300mg(74％)。To a solution of resorufin (250 mg, 1.17 mmol) in DMF (12 mL, anhydrous) was added NaH (60 wt% in mineral oil, 59 mg, 1.41 mmol) in an ice/water bath, and the mixture was stirred for 30 minutes. Then,_PhNTf2 (500 mg, 1.41 mmol) was added, and the reaction mixture was allowed to warm to room temperature and stirred for a further 8 hours. The reaction mixture was diluted with ethyl acetate, washed with 1N HCl, water and brine. The organic layer was dried over anhydrous magnesium sulfate, and concentrated in vacuo. Compound YS-4-110[941601-75-8] was isolated as a yellow sticky solid by flash column chromatography on silica gel using ethyl acetate:hexane (1:2) as eluent. Yield: 300 mg (74%).

¹H NMR(300MHz,CDCl₃)δ7.85–7.75(m,1H),7.36(d,J＝9.9Hz,1H),7.23–7.18(m,2H),6.81(dd,J＝9.9,2.0Hz,1H),6.27(d,J＝2.0Hz,1H)。¹ H NMR (300MHz, CDCl₃ ) δ7.85–7.75 (m, 1H), 7.36 (d, J=9.9Hz, 1H), 7.23–7.18 (m, 2H), 6.81 (dd, J=9.9, 2.0 Hz, 1H), 6.27 (d, J=2.0Hz, 1H).

给烘箱干燥的圆底烧瓶装入Pd₂(dba)₃(18mg,0.020mmol)、Xantphos(34mg,0.059mmol)和Cs₂CO₃(59mg,0.182mmol)，并用氩气冲洗5分钟。加入YS-4-110(83mg,0.24mmol)和YS-3-2(56mg,0.20mmol)的二噁烷(5mL)溶液，并将得到的混合物在氩气下在室温下首先搅拌30分钟，然后加热至100℃并再搅拌24小时。使反应混合物冷却至室温，用DCM稀释并通过硅藻土垫过滤。然后将滤液在真空中浓缩。通过快速柱色谱法在硅胶上将化合物YS-4-111分离为红色粘性固体，使用EtOAc：DCM(1：4)作为洗脱剂。产率：51mg(57％)。An oven-dried round bottom flask was charged with Pd₂ (dba)₃ (18 mg, 0.020 mmol), Xantphos (34 mg, 0.059 mmol) and Cs₂ CO₃ (59 mg, 0.182 mmol) and flushed with argon for 5 minutes. A solution of YS-4-110 (83 mg, 0.24 mmol) and YS-3-2 (56 mg, 0.20 mmol) in dioxane (5 mL) was added, and the resulting mixture was first stirred at room temperature under argon for 30 minutes, It was then heated to 100°C and stirred for a further 24 hours. The reaction mixture was cooled to room temperature, diluted with DCM and filtered through a pad of celite. The filtrate was then concentrated in vacuo. Compound YS-4-111 was isolated as a red sticky solid by flash column chromatography on silica gel using EtOAc:DCM (1:4) as eluent. Yield: 51 mg (57%).

¹H NMR(300MHz,CD₂Cl₂)δ7.83–7.71(m,2H),7.43(d,J＝9.8Hz,1H),7.40–7.32(m,2H),7.17(s,1H),7.04(d,J＝7.9Hz,1H),6.80(dd,J＝9.8,2.0Hz,1H),6.27(d,J＝2.0Hz,1H),5.26(s,2H),5.23(s,2H),3.47(s,3H),3.42(s,3H)；¹ H NMR (300MHz, CD₂ Cl₂ )δ7.83–7.71(m,2H),7.43(d,J＝9.8Hz,1H),7.40–7.32(m,2H),7.17(s,1H), 7.04(d, J=7.9Hz, 1H), 6.80(dd, J=9.8, 2.0Hz, 1H), 6.27(d, J=2.0Hz, 1H), 5.26(s, 2H), 5.23(s, 2H ),3.47(s,3H),3.42(s,3H);

¹³C NMR(75MHz,CD₂Cl₂)δ189.35,186.41,160.19,154.70,150.07,148.64,147.28,144.96,144.49,135.23,135.19,131.43,130.70,128.67,125.40,122.20,120.68,113.98,112.39,107.14,95.05,67.39,56.79,37.56。¹³ C NMR(75MHz,CD₂ Cl₂ )δ189.35,186.41,160.19,154.70,150.07,148.64,147.28,144.96,144.49,135.23,135.19,131.43,130.70,128.67,125.40,122.20,120.68,113.98,112.39,107.14 , 95.05, 67.39, 56.79, 37.56.

在冰/水浴中向YS-4-111(12mg,0.027mmol)的DCM(3mL)溶液中逐滴加入TFA(3mL)，然后将溶液在室温下搅拌2小时。将反应混合物在真空中浓缩，并与甲苯共沸3次。通过快速柱色谱在硅胶上将化合物YS-4-112分离为红色粘性固体，使用EtOAc：DCM(1：4)作为洗脱剂。产率：10mg(92％)。To a solution of YS-4-111 (12 mg, 0.027 mmol) in DCM (3 mL) was added TFA (3 mL) dropwise in an ice/water bath, and the solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and azeotroped 3 times with toluene. Compound YS-4-112 was isolated as a red sticky solid by flash column chromatography on silica gel using EtOAc:DCM (1:4) as eluent. Yield: 10 mg (92%).

¹H NMR(300MHz,CDCl₃)δ11.08(s,1H),9.89(s,1H),7.77(d,J＝9.2Hz,1H),7.56(d,J＝7.8Hz,1H),7.44(d,J＝9.8Hz,1H),7.36(d,J＝7.7Hz,2H),6.97(d,J＝7.9Hz,2H),6.86(dd,J＝9.8,2.0Hz,1H),6.33(d,J＝2.0Hz,1H),5.24(s,2H),3.46(s,3H)；¹ H NMR (300MHz, CDCl₃ ) δ11.08(s, 1H), 9.89(s, 1H), 7.77(d, J=9.2Hz, 1H), 7.56(d, J=7.8Hz, 1H), 7.44 (d,J=9.8Hz,1H),7.36(d,J=7.7Hz,2H),6.97(d,J=7.9Hz,2H),6.86(dd,J=9.8,2.0Hz,1H),6.33 (d,J=2.0Hz,1H),5.24(s,2H),3.46(s,3H);

¹³C NMR(100MHz,CDCl₃)δ196.2,186.5,162.0,154.5,149.7,148.3,146.8,145.8,135.2,134.9,134.2,131.1,130.6,121.8,120.4,118.6,116.1,112.0,107.2,67.1,37.4。¹³ C NMR (100MHz, CDCl₃ ) δ196.2, 186.5, 162.0, 154.5, 149.7, 148.3, 146.8, 145.8, 135.2, 134.9, 134.2, 131.1, 130.6, 121.8, 120.4, 118.6, 116.1, 1407.2, 3, 0 .

实施例2-用绿色荧光化合物YS-3-42对过氧化氢的灵敏性和选择性检测Embodiment 2-use green fluorescent compound YS-3-42 to the sensitivity and selectivity detection of hydrogen peroxide

本实施例显示，绿色荧光化合物YS-3-42灵敏地和选择性地检测过氧化氢。具体地，将化合物YS-3-42溶解在pH为7.4的0.1M磷酸钾缓冲液中以形成10μM的溶液(含有0.1％DMF和100μM CCl₃CN)，其中激发光谱和发射光谱分别在480nm和527nm处。将化合物YS-3-42的10μM溶液用各种浓度的过氧化氢处理。图2A示出，化合物YS-3-42的荧光强度随过氧化氢浓度的增加而增加。This example shows that the green fluorescent compound YS-3-42 detects hydrogen peroxide sensitively and selectively. Specifically, the compound YS-3-42 was dissolved in 0.1 M potassium phosphate buffer at pH 7.4 to form a 10 μM solution (containing 0.1% DMF and 100 μM CCl₃ CN), where the excitation and emission spectra were at 480 nm and 527nm. A 10 μM solution of compound YS-3-42 was treated with various concentrations of hydrogen peroxide. Figure 2A shows that the fluorescence intensity of compound YS-3-42 increases with the increase of hydrogen peroxide concentration.

对不同的反应性氧物质(ROS)和反应性氮物质(RNS)比较化合物YS-3-42的反应性。具体地，将化合物YS-3-42的10μM溶液用各种ROS/RNS(100μM)处理。图2B示出，用过氧化氢处理比用其它ROS和RNS处理导致化合物YS-3-42的高得多的荧光强度增加。The reactivity of compound YS-3-42 was compared against different reactive oxygen species (ROS) and reactive nitrogen species (RNS). Specifically, a 10 μM solution of compound YS-3-42 was treated with various ROS/RNS (100 μM). Figure 2B shows that treatment with hydrogen peroxide resulted in a much higher increase in fluorescence intensity for compound YS-3-42 than treatment with other ROS and RNS.

实施例3-用绿色荧光化合物YS-4-45对过氧化氢的灵敏性和选择性检测Example 3 - Sensitivity and selectivity detection of hydrogen peroxide with green fluorescent compound YS-4-45

本实施例显示，绿色荧光化合物YS-4-45灵敏地和选择性地检测过氧化氢。具体地，将化合物YS-4-45溶解在pH为7.4的0.1M磷酸盐缓冲液中，以形成10μM的溶液(含有0.1％DMF和100μM CCl₃CN)，其中激发光谱和发射光谱分别在520nm和543nm处。将化合物YS-4-45的10μM溶液用不同浓度的过氧化氢处理30分钟。图3A示出，化合物YS-4-45的荧光强度随过氧化氢的浓度增加而增加。This example shows that the green fluorescent compound YS-4-45 detects hydrogen peroxide sensitively and selectively. Specifically, compound YS-4-45 was dissolved in 0.1 M phosphate buffer at pH 7.4 to form a 10 μM solution (containing 0.1% DMF and 100 μM CCl₃ CN), where the excitation and emission spectra were respectively at 520 nm and 543nm. A 10 μM solution of compound YS-4-45 was treated with different concentrations of hydrogen peroxide for 30 minutes. Figure 3A shows that the fluorescence intensity of compound YS-4-45 increases with the concentration of hydrogen peroxide.

对不同的反应性氧物质(ROS)和反应性氮物质(RNS)比较化合物YS-4-45的反应性。具体地，将化合物YS-4-45的10μM溶液用各种ROS/RNS(100μM)处理。图3B示出，用过氧化氢处理30min(图3B中左边柱状图)或60min(图3B中右边柱状图)比用其它ROS和RNS处理导致化合物YS-4-45的高得多的荧光强度增加。The reactivity of compound YS-4-45 was compared against different reactive oxygen species (ROS) and reactive nitrogen species (RNS). Specifically, a 10 μM solution of compound YS-4-45 was treated with various ROS/RNS (100 μM). Figure 3B shows that treatment with hydrogen peroxide for 30 min (left histogram in Figure 3B) or 60 min (right histogram in Figure 3B) resulted in much higher fluorescence intensity of compound YS-4-45 than other ROS and RNS treatments Increase.

实施例4-用绿色荧光化合物YS-2-172对过氧化氢的灵敏性和选择性检测Example 4- Sensitivity and selectivity detection of hydrogen peroxide with green fluorescent compound YS-2-172

本实施例显示，绿色荧光化合物YS-2-172灵敏地和选择性地检测过氧化氢。具体地，将化合物YS-2-172溶解在pH为7.4的0.1M磷酸盐缓冲液/CH₃CN(v/v＝1/1)中，以形成10μM的溶液(含有0.1％DMF)，其中激发光谱和发射光谱分别在429nm和530nm处。将化合物YS-2-172的10μM溶液用不同浓度的过氧化氢处理60分钟。图4A示出，化合物YS-2-172的荧光强度随过氧化氢的浓度增加而增加。This example shows that the green fluorescent compound YS-2-172 detects hydrogen peroxide sensitively and selectively. Specifically, compound YS-2-172 was dissolved in 0.1 M phosphate buffer/CH₃ CN (v/v=1/1) at pH 7.4 to form a 10 μM solution (containing 0.1% DMF), in which Excitation and emission spectra are at 429 nm and 530 nm, respectively. A 10 μM solution of compound YS-2-172 was treated with different concentrations of hydrogen peroxide for 60 minutes. Figure 4A shows that the fluorescence intensity of compound YS-2-172 increases with the concentration of hydrogen peroxide.

对不同的反应性氧物质(ROS)和反应性氮物质(RNS)比较化合物YS-2-172的反应性。具体地，将化合物YS-2-172的10μM溶液用各种ROS/RNS(100μM)处理。图4B示出，用过氧化氢处理30min(图4B中左边柱状图)或60min(图4B中右边柱状图)比用其它ROS和RNS处理导致化合物YS-2-172的高得多的荧光强度增加。The reactivity of compound YS-2-172 was compared against different reactive oxygen species (ROS) and reactive nitrogen species (RNS). Specifically, a 10 μM solution of compound YS-2-172 was treated with various ROS/RNS (100 μM). Figure 4B shows that treatment with hydrogen peroxide for 30 min (left histogram in Figure 4B) or 60 min (right histogram in Figure 4B) resulted in much higher fluorescence intensity of compound YS-2-172 than other ROS and RNS treatments Increase.

实施例5-将主题化合物应用在细胞试验中Example 5 - Application of subject compounds in cell assays

RAW264.7细胞，一种小鼠单核细胞巨噬细胞系，自ATCC(美国典型培养物保藏)获得和在37℃含5％CO₂下保持在补充有10％热灭活的胎牛血清(Gibco)和1％青霉素/链霉素的DMEM(Dulbecco的改良Eagle培养基)中。生长培养基每两至三天更新。在80％汇合处，通过刮擦将细胞剥离，用新鲜培养基洗涤并离心(spin down)(500rpm Eppendorf微量离心机)用于细胞计数。为了共聚焦成像，将细胞以2×10⁴个细胞/mL的密度典型地接种在35mm共聚焦培养皿(Mat-Tek：MA，USA)中。RAW264.7 cells, a mouse monocyte-macrophage cell line, were obtained from ATCC (American Type Culture Collection) and maintained at 37°C with 5%_CO2 supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin/streptomycin in DMEM (Dulbecco's Modified Eagle Medium). The growth medium was refreshed every two to three days. At 80% confluence, cells were detached by scraping, washed with fresh medium and spun down (500 rpm Eppendorf microcentrifuge) for cell counts. For confocal imaging, cells were typically seeded in 35 mm confocal dishes (Mat-Tek: MA, USA) at a density of² x 104 cells/mL.

对于急性过氧化氢诱导(30min)，以指定的剂量将佛波醇12-十四酸酯13-乙酸酯(PMA)加入到HBSS(Hank平衡盐溶液)和与YS-3-42共孵育直至成像(见图5)。在过氧化氢诱导期间将酶抑制剂(NOX抑制剂DPI)与PMA一起加入。已知的是，癌细胞，与正常细胞相比，是在增加的氧化应激和增加的ROS产生下。因此，癌症和正常细胞之间的过氧化氢的基础水平的差异用YS-3-42进行测试(见图6)。另外，主题化合物在其应用浓度下显示无细胞毒性(见图7)。图7示出RAW264.7细胞中探针YS-3-42和YS-4-45的细胞毒性。使RAW264.7细胞随着增加的探针浓度孵育24小时。在孵育24h后探针显示可忽略不计的毒性或没有毒性。数据表示平均值±s.e.m，一式三份进行Cell-Titer Glo测定。For acute hydrogen peroxide induction (30min), phorbol 12-myristate 13-acetate (PMA) was added to HBSS (Hank's Balanced Salt Solution) at the indicated doses and incubated with YS-3-42 until imaging (see Figure 5). An enzyme inhibitor (NOX inhibitor DPI) was added along with PMA during hydrogen peroxide induction. It is known that cancer cells, compared to normal cells, are under increased oxidative stress and increased ROS production. Therefore, the difference in basal levels of hydrogen peroxide between cancer and normal cells was tested with YS-3-42 (see Figure 6). In addition, the subject compound showed no cytotoxicity at its applied concentration (see Figure 7). Figure 7 shows the cytotoxicity of probes YS-3-42 and YS-4-45 in RAW264.7 cells. RAW264.7 cells were incubated with increasing probe concentrations for 24 hours. Probes showed negligible or no toxicity after 24 h of incubation. Data represent mean ± s.e.m, Cell-Titer Glo assay performed in triplicate.

类似地，YS-4-45也成功地应用在RAW264.7细胞中内源性H₂O₂的共聚焦成像(见图8)。PMA挑战的H₂O₂产生可以稳健地显现，这可以通过添加DPI被有效衰减。Similarly, YS-4-45 was also successfully applied to confocal imaging of endogenous H₂ O₂ in RAW264.7 cells (see Figure 8). PMA_- challenged H2O2 production can be_robustly visualized, which can be effectively attenuated by adding DPI.

实施例6-用红色荧光化合物YS-4-112定量过氧化氢Embodiment 6-quantitative hydrogen peroxide with red fluorescent compound YS-4-112

该实施例表明，红色荧光化合物YS-4-112可定量地检测过氧化氢。具体地，将化合物YS-4-112溶解在pH为7.4的0.1M磷酸钾缓冲液中以形成10μM的溶液(含有0.5％DMF和100μM CCl₃CN)，其中，激发光谱和发射光谱分别在565nm和602nm处。将化合物YS-4-112的10μM溶液用各种浓度的过氧化氢处理。图9A示出，化合物YS-4-112的荧光强度随过氧化氢浓度的增加而增加。如图9B中所描绘的，观察到YS-4-112测试溶液的荧光强度在602nm处与H₂O₂(0-30μM)的浓度的线性关系，并且估计检测限低至4.8nM(3σ/K)。该标准校准曲线可以用于定量各种样品和高通量试验中的H₂O₂浓度。This example shows that the red fluorescent compound YS-4-112 can quantitatively detect hydrogen peroxide. Specifically, compound YS-4-112 was dissolved in 0.1 M potassium phosphate buffer at pH 7.4 to form a 10 μM solution (containing 0.5% DMF and 100 μM CCl₃ CN), wherein the excitation spectrum and emission spectrum were respectively at 565 nm and 602nm. A 10 μM solution of compound YS-4-112 was treated with various concentrations of hydrogen peroxide. Figure 9A shows that the fluorescence intensity of compound YS-4-112 increases with the increase of hydrogen peroxide concentration. As depicted in Figure 9B, a linear relationship was observed between the fluorescence intensity of the YS-4-112 test solution at 602 nm and the concentration of H₂ O₂ (0-30 μM), and the estimated detection limit was as low as 4.8 nM (3σ/ K). This standard calibration curve can be used to quantify_H2O2 concentrations in various samples and high_- throughput experiments.

实施例7-将主题化合物应用在活体动物试验中Embodiment 7-application of subject compound in living animal experiment

该实施例显示，YS-3-42和YS-4-112可检测活斑马鱼中的过氧化氢。成鱼(HKWT)的交配和斑马鱼胚胎的选择在香港大学斑马鱼核心设施中进行。收集卵并将其置于有E-3培养基的90mm培养皿中，并在28℃孵育直至胚胎发育至期望阶段(例如24hpf，48hpf，72hpf；hpf：受精后小时)。在显微镜下通过Dumont Tweezers小心地将24hpf胚胎的绒毛膜(卵壳)除去以释放胚胎。在有或无PMA(500ng/mL)的情况下将胚胎在室温下经10μM的YS-3-42或YS-4-112(在1mL E3缓冲液中含有100μM的CCl₃CN)处理30分钟，然后用1mL E-3培养基洗涤两次，然后在LSM 710上成像。在不同的发育阶段的斑马鱼中过氧化氢的产生和贡献可以用YS-4-112可视化(图10A)。斑马鱼中PMA挑战的H₂O₂产生也成功地用YS-3-42检测到(图10B)。This example shows that YS-3-42 and YS-4-112 can detect hydrogen peroxide in live zebrafish. Mating of adults (HKWT) and selection of zebrafish embryos were performed at the Zebrafish Core Facility of the University of Hong Kong. Eggs were collected and placed in 90mm Petri dishes with E-3 medium and incubated at 28°C until the embryos developed to the desired stage (eg 24hpf, 48hpf, 72hpf; hpf: hours post fertilization). The chorion (egg shell) of the 24hpf embryos was carefully removed under a microscope by Dumont Tweezers to release the embryos. Embryos were treated with 10 μM YS-3-42 or YS-4-112 (100 μM CCl3CN in₁ mL E3 buffer) for 30 min at room temperature with or without PMA (500 ng/mL), They were then washed twice with 1 mL of E-3 medium before imaging on an LSM 710. The production and contribution of hydrogen peroxide in zebrafish at different developmental stages can be visualized with YS-4-112 (Fig. 10A). PMA-challenged H₂ O₂ production in zebrafish was also successfully detected with YS-3-42 ( FIG. 10B ).

实施例8-主题化合物在高通量试验中的应用Example 8 - Application of subject compounds in high-throughput assays

该实施例表明主题化合物可用于开发高通量试验。首先，将0.125、0.25、0.5、1.0、2.0mM(终浓度)的抗氧化剂加入到200μL的1.0mM的H₂O₂的pH为7.4的0.1M磷酸盐缓冲液溶液中，并将该溶液在96孔板上在37℃下孵育30分钟。然后，将2μL的得到的溶液加入到YS-4-112的溶液(10μM，在pH为7.4的0.1M磷酸盐缓冲液中，含有100μM CCl₃CN，每个孔200μL)中，并且将溶液在96孔板上在37℃下孵育30分钟，以测定剩余的H₂O₂浓度。可以将该96孔板放置在读板器上，以确定在用565nm激发的每个孔在602nm处的荧光发射。抗氧化能力可以通过H₂O₂清除率评估，其通过下式计算：This example demonstrates that the subject compounds can be used to develop high throughput assays. First, 0.125, 0.25, 0.5, 1.0,_2.0 mM (final concentration) of antioxidants were added to₂₀₀ μL of 1.0 mM H2O2 pH 7.4 0.1M phosphate buffer solution, and the solution was Incubate the 96-well plate at 37°C for 30 minutes. Then, 2 μL of the obtained solution was added to a solution of YS-4-112 (10 μM in 0.1 M phosphate buffer at pH 7.4 containing 100 μM CCl₃ CN, 200 μL per well), and the solution was Incubate the 96_- well plate at 37 °C for 30 min to determine the remaining_H2O2 concentration. The 96-well plate can be placed in a plate reader to determine the fluorescence emission at 602 nm for each well excited with 565 nm. Antioxidant capacity can be evaluated by_H2O2 scavenging rate, which is calculated by the following_formula :

H＝(F₀-F₁)/(F₀-F_空白)*100％H=(F₀ -F₁ )/(F₀ -F_blank )*100%

F₀，没有抗氧化剂的荧光强度；F₁，具有各种浓度的抗氧化剂的荧光强度；F_空白，YS-4-112的荧光背景。F₀ , fluorescence intensity without antioxidant; F₁ , fluorescence intensity with various concentrations of antioxidant; F_blank , fluorescence background of YS-4-112.

抗坏血酸和表没食子儿茶素没食子酸酯(EGCG)的H₂O₂清除活性通过该高通量试验(图11A和图11B)确定。那些数据对于药物筛选和评估是非常有用的。The H₂ O₂ scavenging activity of ascorbic acid and epigallocatechin gallate (EGCG) was determined by this high-throughput assay ( FIG. 11A and FIG. 11B ). Those data are very useful for drug screening and evaluation.

尽管本文参照用于特定应用的说明性实施方案对本发明进行描述，但是应当理解，本发明不限于此。那些具有本领域普通技术和能获得本文提供的教导的人将认识到另外的修改、应用和其范围内的实施方案和另外的领域，在其中本发明将具有显著的效用。因此，通过所附权利要求旨在覆盖本发明的范围内的任何和所有这种应用、修改和实施方案。While the invention is described herein with reference to illustrative embodiments for particular applications, it should be understood that the invention is not limited thereto. Those having ordinary skill in the art and having access to the teachings provided herein will recognize additional modifications, applications, and embodiments within its scope and additional fields in which the present invention will have significant utility. It is therefore intended by the appended claims to cover any and all such applications, modifications and implementations which are within the scope of this invention.

具有结构1-6、8-21、23-28的化合物，其中R＝H或CF₃、和具有结构7和22的化合物，其中R＝CF₃，可以用用于制备化合物YS-3-42(即，具有结构7的化合物，其中R＝H)和化合物YS-2-172(即，具有结构22的化合物，其中R＝H)的类似方法制备，其中，使用用于相应单价前荧光团或前发光团部分的起始材料和/或R＝CF₃的起始材料。这些化合物的结构被¹HHMR和MS确证。Compounds with structures 1-6, 8-21, 23-28, where R=H or CF₃ , and compounds with structures 7 and 22, where R=CF₃ , can be used to prepare compound YS-3-42 (i.e., a compound of structure 7, wherein R=H) and compound YS-2-172 (i.e., a compound of structure 22, wherein R=H) were prepared in a similar manner, wherein, using the corresponding monovalent profluorophore Or the starting material for the pro-luminophore moiety and/or the starting material for R=_CF3 . The structures of these compounds were confirmed by¹ HHMR and MS.

具有结构30-44的化合物，其中R＝H或CF₃、和具有结构29的化合物，其中R＝CF₃，可以用用于制备化合物YS-4-45(即，具有结构29的化合物，其中R＝H)的类似方法制备，其中，使用相应二价前荧光团或前发光团部分的起始材料和/或R＝CF₃的起始材料。这些化合物的结构被¹HHMR和MS确证。Compounds of structure 30-44, wherein R = H or CF₃ , and compounds of structure 29, wherein R = CF₃ , can be used to prepare compound YS-4-45 (ie, compounds of structure 29, wherein R=H) is prepared analogously, wherein the starting material of the corresponding divalent profluorophore or proluminophore moiety and/or the starting material of R=_CF3 is used. The structures of these compounds were confirmed by¹ HHMR and MS.

本发明的化合物可以具有如在图1中提出的用于与过氧化氢的反应的相同或类似的机理。因此，对于本发明的化合物也可以获得类似的技术效果和应用，包括但不限于过氧化氢的灵敏性和选择性检测。The compounds of the invention may have the same or similar mechanism as proposed in Figure 1 for the reaction with hydrogen peroxide. Accordingly, similar technical effects and applications, including but not limited to sensitive and selective detection of hydrogen peroxide, can also be obtained for the compounds of the present invention.

如本文所使用的，单数形式“一(a)”、“一个(an)”和“所述(the)”旨在也包括复数形式，除非上下文另有明确指示。此外，就术语“包括(including)”、“包含(includes)”、“具有(having)”、“有(has)”、“含有(with)”或其变型在详述和/或权利要求书中使用的范畴而言，此类术语旨在是以类似于术语“包含(comprising)”的方式包括的。过渡性术语/短语(及其任何语法变型)、“包含(comprising)”、“包括(comprises)”、“包括(comprise)”、“基本由...组成(consisting essentially of)”、“基本由......组成(consistsessentially of)”和“由......组成(consisting)”和“由……组成(consists)”可以互换使用。As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. In addition, the terms "including", "includes", "having", "has", "with" or variations thereof in the detailed description and/or claims Such terms are intended to be inclusive in a manner similar to the term "comprising" as used in . Transitional terms/phrases (and any grammatical variants thereof), "comprising", "comprises", "comprise", "consisting essentially of", "essentially Consistsessentially of" and "consisting" and "consists" are used interchangeably.

本文提到或引用的所有专利、专利申请、临时申请和公开，包括所有附图和表格通过引用整体并入，至它们不与本说明书的明确教导相矛盾的程度。All patents, patent applications, provisional applications and publications, including all figures and tables, mentioned or cited herein are incorporated by reference in their entirety to the extent they do not contradict the express teachings of this specification.

参考文献references

www.thermofisher.com/order/catalog/product/A22177www.thermofisher.com/order/catalog/product/A22177

www.thermofisher.com/hk/en/home/references/molecular-probes-the-handbook/probes-for-reactive-oxygen-species-including-nitric-oxide/generating-and-detecting-reactive-oxygen-species.htmlwww.thermofisher.com/hk/en/home/references/molecular-probes-the-handbook/probes-for-reactive-oxygen-species-including-nitric-oxide/generating-and-detecting-reactive-oxygen-species. html

Claims (22)

Translated fromChinese

1.一种式(I)、式(II)或式(III)的化合物或其盐：1. A compound of formula (I), formula (II) or formula (III) or a salt thereof:A-X¹ (II)，AX¹ (II),A-X²-A′ (III)，AX² -A′ (III),其中：in:R¹、R²、R³、R⁴和R⁵独立地选自H、F、Cl、Br、I、CN、烷基、卤代烷基、杂烷基、链烯基、炔基、芳烷基、芳基、烷芳基、杂环基、环烷基、环烯基、环炔基、羟烷基、氨基烷基、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、酰基氨基、羟基、硫醇、硫代烷基、烷氧基、烷硫基、烷氧基烷基、芳氧基、芳基烷氧基、酰氧基、硝基、氨基甲酰基、三氟甲基、苯氧基、苄氧基、膦酸、磷酸酯、磺酸(-SO₃H)、磺酸酯、磺酰胺、-C(＝O)-P¹和-C(＝O)-M-P²，R¹ , R² , R³ , R⁴ and R⁵ are independently selected from H, F, Cl, Br, I, CN, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aralkyl , aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino, alkylaryl ylamino, diarylamino, acylamino, hydroxy, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyloxy, nitro group, carbamoyl group, trifluoromethyl group, phenoxy group, benzyloxy group, phosphonic acid, phosphoric acid ester, sulfonic acid (-SO₃ H), sulfonate ester, sulfonamide, -C(=O)-P¹ and -C(=O)-MP² ,其中，P¹和P²选自氢、卤素、烷氧基、羟基、硫醇、烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基、芳烷基、氨基甲酸酯基、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、烷硫基、杂烷基、烷基三苯基鏻和具有3至7个环原子的杂环基，^Wherein , P and P are selected from^hydrogen , halogen, alkoxy, hydroxyl, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl , aralkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio, heteroalkyl, alkyltriphenyl Phosphonium and heterocyclic groups having 3 to 7 ring atoms,或R²和R³一起形成选自芳基、杂环基、杂芳基和杂芳族基团的5、6或7元环，or R and^R together form a⁵ , 6 or 7 membered ring selected from aryl, heterocyclyl, heteroaryl and heteroaromatic groups,或R⁴和R⁵一起形成选自芳基、杂环基、杂芳基和杂芳族基团的5、6或7元环，or R and R together form a⁵ ,⁶ or 7 membered ring selected from aryl, heterocyclyl, heteroaryl and heteroaromatic groups,M选自亚烷基、亚烯基、亚炔基、亚芳基、亚芳烷基和亚烷芳基；M is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, aralkylene and alkarylene;R⁶选自氢、烷基、烷氧基烷基、烷酰基、-CF₃、卤素取代的低级烷基和(C＝O)-O-Z¹，R6 is selected from the group consisting of hydrogen, alkyl, alkoxyalkyl, alkanoyl,_-CF3 , halogen^- substituted lower alkyl and (C=O)^-OZ1 ,其中，Z¹是选自烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基和芳基烷基的基团；Wherein, Z is^a group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl and arylalkyl;X¹是单价前荧光团或前发光团部分；X is^a monovalent profluorophore or proluminophore moiety;X²是二价前荧光团或前发光团部分；X is a bivalent^{profluorophore} or proluminophore moiety;A和A'中的每个独立地由式(IV)或式(V)表示：Each of A and A' is independently represented by formula (IV) or formula (V):其中，R⁷和R⁹中的至少一个是(C＝O)-W¹，Wherein, at least one of R⁷ and R⁹ is (C=O)-W¹ ,其中，W¹选自氢、烷基、烷氧基烷基、烷酰基、CF₃、卤素取代的低级烷基和(C＝O)-O-Z²，Wherein, W¹ is selected from hydrogen, alkyl, alkoxyalkyl, alkanoyl, CF₃ , lower alkyl substituted with halogen and (C=O)-OZ² ,其中Z²是选自烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基或芳基烷基的基团，wherein^Z is a group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl,和R⁷和R⁹的另一个、R⁸、R¹⁰和R¹¹独立地选自H、F、Cl、Br、I、CN、烷基、卤代烷基、杂烷基、链烯基、炔基、芳烷基、芳基、烷芳基、杂环基、环烷基、环烯基、环炔基、羟烷基、氨基烷基、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、酰基氨基、羟基、硫醇、硫代烷基、烷氧基、烷硫基、烷氧基烷基、芳氧基、芳基烷氧基、酰氧基、硝基、氨基甲酰基、三氟甲基、苯氧基、苄氧基、膦酸、磷酸酯、磺酸(-SO₃H)、磺酸酯、磺酰胺、-C(＝O)-P³和-C(＝O)–M-P⁴，and the other of^R7 and^R9 ,^R8 ,^R10 and^R11 are independently selected from H, F, Cl, Br, I, CN, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl , aralkyl, aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, amino, alkylamino, arylamino, dialkylamino , alkylarylamino, diarylamino, acylamino, hydroxyl, thiol, thioalkyl, alkoxy, alkylthio, alkoxyalkyl, aryloxy, arylalkoxy, acyl Oxygen, nitro, carbamoyl, trifluoromethyl, phenoxy, benzyloxy, phosphonic acid, phosphoric acid ester, sulfonic acid (-SO₃ H), sulfonate, sulfonamide, -C(=O )-P³ and -C(=O)-MP⁴ ,其中P³和P⁴独立地选自氢、卤素、烷氧基、羟基、硫醇、烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基、芳基烷基、氨基甲酸酯、氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基、烷硫基、杂烷基、烷基三苯基鏻或具有3至7个环原子的杂环基；M是亚烷基、亚烯基、亚炔基、亚芳基、亚芳烷基或亚烷芳基；wherein^P and P^are independently selected from hydrogen, halogen, alkoxy, hydroxyl, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl radical, arylalkyl, carbamate, amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino, alkylthio, heteroalkyl, alkyltriphenyl A base phosphonium or a heterocyclic group with 3 to 7 ring atoms; M is an alkylene, alkenylene, alkynylene, arylene, aralkylene or alkarylene;R⁸和R¹⁰中的至少一个优选是羟基、烷氧基、或选自氨基、烷基氨基、芳基氨基、二烷基氨基、烷基芳基氨基、二芳基氨基的给电子基团；^At least^one of R and R is preferably hydroxyl, alkoxy, or an electron-donating group selected from amino, alkylamino, arylamino, dialkylamino, alkylarylamino, diarylamino ;或R⁷和R⁸一起形成选自芳基、杂环基、杂芳基或杂芳族基团的5、6或7元环；or R and R together form a⁵ , 6 or⁷ membered ring selected from aryl, heterocyclyl, heteroaryl or heteroaromatic;或R¹⁰和R¹¹一起形成选自芳基、杂环基、杂芳基或杂芳族基团的5、6或7元环。Or R¹⁰ and R¹¹ together form a 5, 6 or 7 membered ring selected from aryl, heterocyclyl, heteroaryl or heteroaromatic.2.权利要求1所述的化合物，其中，式(I)中R¹、R³和R⁵中的至少一个是羟基基团。2. The compound of claim 1, wherein at least^one of R1,^R3 and R5 in^formula (I) is a hydroxyl group.3.权利要求2所述的化合物，其中，式(I)中R⁶选自氢、CF₃、卤素取代的低级烷基或(C＝O)-O-Z³，其中，Z³是烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基或芳基烷基。3. The compound of claim 2, wherein, in formula (I), R⁶ is selected from hydrogen, CF₃ , lower alkyl substituted by halogen or (C=O)-OZ³ , wherein Z³ is alkyl, Alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl.4.权利要求1所述的化合物，其中，式(IV)和式(V)中R⁸和R¹⁰中的至少一个是羟基基团。4. The compound of claim 1, wherein at least one of R⁸ and R¹⁰ in formula (IV) and formula (V) is a hydroxyl group.5.权利要求4所述的化合物，其中，R⁷和R⁹中的至少一个是(C＝O)-W²，其中，W²为氢、CF₃、卤素取代的低级烷基或(C＝O)-O-Z⁴，其中，Z⁴是选自烷基、链烯基、炔基、环烷基、环烯基、环炔基、芳基、烷芳基或芳基烷基的基团。5. The compound of claim 4, wherein at least one of R⁷ and R⁹ is (C=O)-W² , wherein W² is hydrogen, CF₃ , lower alkyl substituted with halogen, or (C =O)-OZ⁴ , wherein Z⁴ is a group selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, alkaryl or arylalkyl .6.权利要求1所述的化合物，其中，X¹选自单价前荧光素、单价前香豆素、单价前胺萘二甲酰亚胺、单价前丹酰、单价前二胺(pro-bimane)、单价前曙红、单价前罗丹明、单价前对甲氨基酚、单价前花青、单价尼罗红、单价呫吨酮、单价呫吨、单价flazo橙、单价SNARF-1、单价前荧光黄、单价前6-十二酰基-N,N-二甲基-2-萘胺、单价前2-萘胺、单价试卤灵和单价荧光素。6. The compound of claim¹ , wherein X is selected from the group consisting of monovalent profluorescein, monovalent procoumarin, monovalent proamine naphthalimide, monovalent predansyl, monovalent pro-bimane ), monovalent proeosin, monovalent prorhodamine, monovalent prorhodamine, monovalent procyanine, monovalent Nile red, monovalent xanthone, monovalent xanthene, monovalent flazo orange, monovalent SNARF-1, monovalent profluoro Yellow, monovalent pro-6-dodecanoyl-N,N-dimethyl-2-naphthylamine, monovalent pro-2-naphthylamine, monovalent resorufin, and monovalent fluorescein.7.权利要求1或权利要求6所述的化合物，其中，所述化合物具有结构1-28和57之一：7. The compound of claim 1 or claim 6, wherein the compound has one of structures 1-28 and 57:其中，R＝H或CF₃。Wherein, R=H or CF₃ .8.权利要求1所述的化合物，其中X²选自前荧光素、二价前香豆素、二价前胺萘二甲酰亚胺、二价前丹酰、二价前二胺、二价前曙红、二价前罗丹明、二价前对甲氨基酚、二价前花青、二价尼罗红、二价呫吨酮、二价呫吨、二价flazo橙、二价SNARF-1和二价试卤灵，包括缀合物。8. The compound of claim¹ , wherein X is selected from prefluorescein, divalent procoumarin, divalent proamine naphthalimide, divalent predansyl, divalent prediamine, divalent Prevalent eosin, divalent prorhodamine, divalent prorhodamine, divalent procyanine, divalent Nile red, divalent xanthone, divalent xanthene, divalent flazo orange, divalent SNARF -1 and divalent resorufin, including conjugates.9.权利要求1或权利要求8所述的化合物，其中，所述化合物具有结构29-44之一：9. The compound of claim 1 or claim 8, wherein the compound has one of structures 29-44:其中，R＝H或CF₃。Wherein, R=H or CF₃ .10.权利要求1所述的化合物，其中，所述化合物包含一个或多个游离羧基基团，其中，至少一个羧基基团通过酰胺键连接与带正电荷的线粒体靶向的三苯基鏻部分或溶酶体靶向的吗啉或N，N-二取代的胺部分缀合，其中，所述化合物与三苯基鏻部分之间的连接具有下式(VI)或(VII)：10. The compound of claim 1, wherein the compound comprises one or more free carboxyl groups, wherein at least one carboxyl group is linked via an amide bond to a positively charged mitochondria-targeting triphenylphosphonium moiety or lysosome-targeted morpholine or N,N-disubstituted amine moiety conjugates, wherein the link between the compound and the triphenylphosphonium moiety has the following formula (VI) or (VII):其中，n＝1-10；Wherein, n=1-10;或所述化合物与吗啉或N，N-二取代胺部分之间的连接具有下式(VIII)或(IX)：Or the link between the compound and the morpholine or N,N-disubstituted amine moiety has the following formula (VIII) or (IX):其中，n＝1-10；式(IX)中的R¹²或R¹³独立地为C_1-10烷基或烯烃。Wherein, n=1-10; R¹² or R¹³ in formula (IX) is independently C_1-10 alkyl or alkene.11.权利要求10所述的化合物，其中，所述化合物具有结构45-56之一：11. The compound of claim 10, wherein the compound has one of structures 45-56:其中，R＝H或CF₃。Wherein, R=H or CF₃ .12.一种荧光或发光探针组合物，其包含权利要求1至11中任一项所述的化合物和载体。12. A fluorescent or luminescent probe composition comprising the compound according to any one of claims 1 to 11 and a carrier.13.权利要求12所述的荧光或发光探针组合物，其中，所述荧光或发光探针组合物还包含溶剂、酸、碱、缓冲溶液或其组合。13. The fluorescent or luminescent probe composition of claim 12, wherein the fluorescent or luminescent probe composition further comprises a solvent, an acid, a base, a buffer solution, or a combination thereof.14.一种用于检测样品中过氧化氢的存在和/或确定样品中过氧化氢的水平的方法，其包括：14. A method for detecting the presence of and/or determining the level of hydrogen peroxide in a sample comprising:使权利要求1至11中任一项的化合物与样品接触，以形成荧光或发光化合物；和contacting a compound according to any one of claims 1 to 11 with a sample to form a fluorescent or luminescent compound; and确定荧光或发光化合物的荧光或发光性质。Determine the fluorescent or luminescent properties of fluorescent or luminescent compounds.15.权利要求14所述的方法，其中，所述样品是化学样品或生物样品或病理样品。15. The method of claim 14, wherein the sample is a chemical sample or a biological sample or a pathological sample.16.权利要求15所述的方法，其中，所述样品是包括微生物、细胞、组织、器官、来自植物或动物的部分、整个植物或动物或它们的提取物的生物样品。16. The method of claim 15, wherein the sample is a biological sample comprising microorganisms, cells, tissues, organs, parts from plants or animals, whole plants or animals or extracts thereof.17.权利要求15所述的方法，其中，所述样品是包括血液、尿、唾液、血清、呼吸气体、呼出气冷凝物、关节液、或它们的提取物的病理样品。17. The method of claim 15, wherein the sample is a pathological sample comprising blood, urine, saliva, serum, respiratory gas, exhaled breath condensate, joint fluid, or extracts thereof.18.一种用于在体内检测生物体的过氧化氢的存在或在体内确定生物体的过氧化氢的水平的方法，其包括：18. A method for detecting the presence of hydrogen peroxide in an organism or determining the level of hydrogen peroxide in an organism in vivo, comprising:将权利要求1至11中任一项的化合物给予所述生物体以形成荧光或发光化合物；和administering a compound of any one of claims 1 to 11 to said organism to form a fluorescent or luminescent compound; and确定荧光或发光化合物的荧光或发光性质。Determine the fluorescent or luminescent properties of fluorescent or luminescent compounds.19.一种用于在体外检测过氧化氢的存在或在体外确定过氧化氢的水平的方法，其包括：19. A method for detecting the presence of hydrogen peroxide in vitro or determining the level of hydrogen peroxide in vitro comprising:将权利要求1至11中任一项的化合物给予体外样品以形成荧光或发光化合物；和administering a compound according to any one of claims 1 to 11 to an in vitro sample to form a fluorescent or luminescent compound; and确定荧光或发光化合物的荧光或发光性质。Determine the fluorescent or luminescent properties of fluorescent or luminescent compounds.20.一种用于检测样品中过氧化氢的存在或确定样品中过氧化氢的水平的高通量筛选方法，其中，所述高通量方法包括以下步骤：20. A high throughput screening method for detecting the presence of hydrogen peroxide in a sample or determining the level of hydrogen peroxide in a sample, wherein said high throughput method comprises the steps of:使权利要求1至11中任一项的化合物与样品接触以形成一种或多种荧光或发光化合物；和contacting a compound of any one of claims 1 to 11 with a sample to form one or more fluorescent or luminescent compounds; and确定荧光或发光化合物的荧光或发光性质，以确定样品中过氧化氢的存在和/或过氧化氢的量。Determine the fluorescent or luminescent properties of fluorescent or luminescent compounds to determine the presence and/or amount of hydrogen peroxide in a sample.21.一种用于筛选提高或降低过氧化氢水平的一种或多种目标化合物的高通量方法，其中，所述高通量方法包括以下步骤：21. A high-throughput method for screening one or more target compounds that increase or decrease hydrogen peroxide levels, wherein the high-throughput method comprises the steps of:使权利要求1至11中任一项的化合物与目标化合物接触以形成一种或多种荧光或发光化合物；和contacting a compound of any one of claims 1 to 11 with a target compound to form one or more fluorescent or luminescent compounds; and测量荧光或发光化合物的荧光或发光性质以确定目标化合物的存在和/或目标化合物的量。Fluorescent or luminescent properties of fluorescent or luminescent compounds are measured to determine the presence and/or amount of the target compound.22.权利要求1至11中任一项的化合物或权利要求12或13的荧光或发光探针组合物用于检测生物体的体内或在体外的样品中过氧化氢的存在和/或确定生物体的体内或在体外的样品中过氧化氢的水平的用途；或权利要求1至11中任一项的化合物或权利要求12或13的荧光或发光探针组合物用于筛选提高或降低过氧化氢的水平的一种或多种目标化合物的用途。22. The compound of any one of claims 1 to 11 or the fluorescent or luminescent probe composition of claim 12 or 13 is used to detect the presence of hydrogen peroxide in the body of an organism or in a sample in vitro and/or to determine the presence of biological or the use of the level of hydrogen peroxide in a sample in vivo or in vitro; or a compound according to any one of claims 1 to 11 or a fluorescent or luminescent probe composition according to claim 12 or 13 for screening for increased or decreased peroxide Use of one or more target compounds at the level of hydrogen peroxide.