WO2020221217A1 - 一种荧光染料及其制备方法和用途 - Google Patents

一种荧光染料及其制备方法和用途 Download PDF

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WO2020221217A1
WO2020221217A1 PCT/CN2020/087311 CN2020087311W WO2020221217A1 WO 2020221217 A1 WO2020221217 A1 WO 2020221217A1 CN 2020087311 W CN2020087311 W CN 2020087311W WO 2020221217 A1 WO2020221217 A1 WO 2020221217A1
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group
iii
alkyl
compound
fluorescent dye
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PCT/CN2020/087311
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English (en)
French (fr)
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WO2020221217A8 (zh
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朱麟勇
杨弋
张大生
陈显军
林秋宁
苏倪
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上海高驰资产管理有限公司
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Priority to US17/607,005 priority Critical patent/US20220214351A1/en
Priority to EP20798794.2A priority patent/EP3964496A4/en
Priority to JP2021564230A priority patent/JP7321584B2/ja
Publication of WO2020221217A1 publication Critical patent/WO2020221217A1/zh
Publication of WO2020221217A8 publication Critical patent/WO2020221217A8/zh

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    • C—CHEMISTRY; METALLURGY
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    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N11/00—Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties
    • G01N2011/006—Determining flow properties indirectly by measuring other parameters of the system
    • G01N2011/008—Determining flow properties indirectly by measuring other parameters of the system optical properties

Definitions

  • the invention relates to the technical field of fluorescent dyes, in particular to a fluorescent dye with low viscosity response and low background fluorescence, and a preparation method and application thereof.
  • Molecular rotors are a type of dyes whose fluorescence intensity changes with the viscosity of the microenvironment. After they are excited, the molecules undergo conformational distortions to form a charge transfer state in the TICT molecule. The excited state energy is mainly released in the form of non-radiation, and when they are at a higher or higher viscosity In a rigid microenvironment, the distortion of the molecular conformation of this type of molecule is restricted. At this time, the energy of the excited state of the dye is mainly released in the form of radioluminescence, that is, the fluorescent property of this type of molecule is activated. What is important is the fluorescence intensity of this type of molecule Along with the change of the viscosity of the microenvironment, the viscosity change of the microenvironment can be displayed in real time, in situ, sensitively and visually.
  • the conformational distortion based on restricted molecular rotors is also widely used in the construction of fluorescent activation probes.
  • the molecular rotor is combined with BSA, the molecular conformation is restricted by the protein and the fluorescence is lit. The energy of the excited state of the dye not bound to the protein is still dissipated in a non-radiative form, so as to achieve the effect of real-time protein detection and quantification.
  • thiazole orange is in a state of fluorescence quenching before binding to DNA or RNA.
  • the molecular conformation is restricted, so that fluorescence is activated, and it is widely used in DNA and RNA detection and tracing; Malachite Green The molecular rotor is wrapped by antibodies to limit the conformational changes of molecules and is used for protein-activated fluorescence imaging; DHBI is combined with aptamers to construct fluorescent protein mimics for RNA tracing; for example, it is combined with amyloid to limit The conformational changes of molecules are used for the detection and research of AIDS.
  • the purpose of the present invention is to provide a fluorescent dye with viscosity responsiveness and low background fluorescence.
  • a fluorescent dye which is represented by formula (I),
  • D- is HO- or N(X 1 )(X 2 )-, X 1 , X 2 are each independently selected from hydrogen, alkyl and modified alkyl; X 1 , X 2 are optionally connected to each other, and N atom Together form aliphatic heterocycle;
  • R is selected from cyano group, carboxyl group, amide group, ester group, sulfoxide group, sulfone group, sulfonate group or sulfonamide group;
  • Ar 1 and Ar 2 are each independently selected from arylene and heteroarylene; wherein , The hydrogen atoms in Ar 1 and Ar 2 are optionally independently substituted by halogen atoms, hydroxyl groups, aldehyde groups, carboxyl groups, ester groups, amide groups, cyano groups, sulfonic acid groups, phosphoric acid groups, amino groups, primary amino groups, secondary amino groups, Alkyl or modified alkyl substitution;
  • X 1 and X 2 optionally independently form an alicyclic heterocyclic ring with Ar 1 ;
  • the "alkyl” is each independently a C 1 -C 10 linear or branched alkyl; alternatively, a C 1 -C 7 linear or branched alkyl; alternatively, a C 1 -C 5 linear or branched alkyl; alternatively, selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl , 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methyl Pentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl Butyl, 1,3-didi
  • the carbon atom is replaced means that the carbon atom or the carbon atom and the hydrogen atom on it are replaced by the corresponding group;
  • halogen atoms are each independently F, Cl, Br or I;
  • the "aliphatic heterocyclic ring” is a saturated or unsaturated 4-15 membered monocyclic or polycyclic aliphatic heterocyclic ring containing one or more heteroatoms of N, O, S or Si. When the ring contains an S atom, it is -S-, -SO- or -SO 2 -; the aliphatic heterocyclic ring is optionally substituted by a halogen atom, an alkyl group, an aryl group or a modified alkyl group;
  • the "arylene” is a 5- to 13-membered monocyclic or bicyclic or fused bicyclic or fused polycyclic aromatic group
  • heteroarylene is a 5- to 13-membered monocyclic or bicyclic or fused bicyclic or fused polycyclic ring containing one or more heteroatoms selected from N, O, S or Si. Heteroaromatic group;
  • the "amide group” is R'CONR"R"' group
  • the "sulfonic acid group” is R'SO 3 H group
  • the "sulfonate group” is R'SO 2 OR"group
  • the "sulfonamide group” is the R'SO 2 NR"R"'group
  • the "sulfone group” is the R'SO 2 R"group
  • the "sulfoxide group” is an R'SOR" group
  • the "primary amino group” is the R'NH 2 group
  • the "secondary amino group” is the R'NHR" group
  • the "tertiary amino group” is the R'NR"R"' group
  • the "quaternary ammonium salt group" is an R'R"R"'R""N + group
  • R', R", R"', R" is independently a single bond, hydrogen, alkyl, alkylene, modified alkyl or modified alkylene;
  • alkylene group is a C 1 -C 10 linear or branched alkylene group; alternatively, a C 1 -C 7 linear or branched chain alkylene group; alternatively, a C 1 -C 5 linear or branched alkylene;
  • Ar 1 and Ar 2 are each independently a structure selected from the following formulas (II-1) to (II-22):
  • the second aspect of the present invention is to provide a method for preparing the above-mentioned fluorescent dye, which is characterized by comprising the step of aldol condensation reaction between the compound of formula (a) and the compound of formula (b).
  • the third aspect of the present invention is to provide the use of the above-mentioned fluorescent dyes in viscosity testing, protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection, and the use is not a diagnostic method for diseases the use of.
  • the fourth aspect of the present invention is the use of the fluorescent dye provided in the preparation of reagents for viscosity testing, protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection.
  • the fifth aspect of the present invention is to provide a fluorescence activated lighting probe, which includes the above-mentioned fluorescent dye.
  • the sixth aspect of the present invention is to provide the use of the above-mentioned fluorescently activated lighting probe in protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection, and the use is not for disease Use of diagnostic methods.
  • the seventh aspect of the present invention is to provide the use of the above-mentioned fluorescent activated lighting probe in preparing reagents for protein fluorescent labeling, nucleic acid fluorescent labeling, protein quantification or detection, or nucleic acid quantification or detection.
  • the fluorescent dye obtained in the present invention can be used to determine the viscosity of a sample, for example, it is suitable for microscopic viscosity testing.
  • the obtained fluorescent dye can specifically bind to the corresponding antibody, aptamer, or amyloid, or bind to a protein tag or enzyme through a ligand or inhibitor to obtain a series of fluorescent activation points Bright probe for fluorescent labeling, quantification or monitoring of protein, enzyme or nucleic acid.
  • Figure 1 shows the fluorescence emission intensity graph of molecular rotor III-3 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 2 shows the linear relationship between molecular rotor III-3 (1 ⁇ 10 -5 M) viscosity conditions and fluorescence intensity
  • Figure 3 shows the fluorescence emission intensity graph of molecular rotor III-4 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 4 shows the linear relationship between the viscosity of molecular rotor III-4 (1 ⁇ 10 -5 M) and the fluorescence intensity
  • Figure 5 shows the fluorescence emission intensity graph of molecular rotor III-28 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 6 shows the linear relationship between the viscosity of molecular rotor III-28 (1 ⁇ 10 -5 M) and the fluorescence intensity
  • Figure 7 shows the fluorescence emission intensity graph of molecular rotor III-34 (1 ⁇ 10 -5 M) under different viscosity conditions
  • Figure 8 shows the linear relationship between the viscosity of molecular rotor III-34 (1 ⁇ 10 -5 M) and the fluorescence intensity
  • Figure 9 is a comparison diagram of the fluorescence background of molecular rotor III-11 and III-36 (1 ⁇ 10 -6 M) in PBS;
  • Figure 10 is a comparison diagram of the fluorescence background of molecular rotor III-34 and III-37 (1 ⁇ 10 -6 M) in PBS;
  • Figure 11 is a comparison diagram of the fluorescence background of molecular rotors III-31, III-32, III-33 and III-38 (1 ⁇ 10 -6 M) in PBS;
  • Figure 12 is a comparison of the fluorescence background of molecular rotor III-3 and III-39 (1 ⁇ 10 -6 M) in PBS;
  • Figure 13 is a comparison of the fluorescence background of molecular rotor III-21 and III-40 (1 ⁇ 10 -6 M) in PBS;
  • Figure 14 shows the comparison of the fluorescence background of molecular rotors III-28, III-29, III-30 and III-41 (1 ⁇ 10 -6 M) in PBS;
  • Figure 15 is a comparison diagram of the fluorescence background of molecular rotor III-3 and III-42 (1 ⁇ 10 -6 M) in PBS;
  • Figure 16 is a comparison of the fluorescence background of molecular rotor III-3 and III-43 (1 ⁇ 10 -6 M) in PBS;
  • Figure 17 shows molecular rotors III-3, III-4, III-6, III-7, III-8, III-18, III-21 used to label intracellular RNA aptamers, where A is the target RNA expressed Aptamer cells, B is a cell that does not express the target RNA aptamer;
  • Figure 18 shows molecular rotors III-3 and III-43 used to label intracellular mRNA.
  • 6-Bromobenzo[b]thiophene-2-carbaldehyde (0.42g, 1.7mmol), dimethylethylamine (40% aqueous solution, 1g, 8.9mmol), CuI (13.9mg, 0.073mmol), K 3 PO 4 ⁇ H 2 O (155.4mg, 0.73mmol) and methylamine (33% aqueous solution, 1g) were placed in a 100ml pressure-resistant bottle, heated in an oil bath at 60°C for 12h under a sealed condition, the system was cooled to room temperature, 50ml water was added, and DCM extracted ( 3 ⁇ 100 ml), the organic phases were combined, dried over Na 2 SO 4 , the organic solvent was removed under reduced pressure, and the residue was separated and purified by column chromatography (0.23 g, 68%).
  • the fluorescent dyes (molecular rotors) prepared in Examples 1-35 were separately dissolved in dimethyl sulfoxide to prepare mother liquors with a concentration of 1 ⁇ 10 -2 M. Each mother liquor was added to glycerol and methanol, and mixed uniformly. , Each prepared solution with a final concentration of 1 ⁇ 10 -5 M, according to different fluorescent dyes, uses the maximum excitation wavelength of each fluorescent dye to detect its fluorescence emission spectrum under the same conditions. The results are shown in Table 1, indicating that the fluorescent dye of the present invention The viscosity changes are sensitive.
  • the fluorescence of the bound compound is significantly activated, and it emits bright fluorescence under the excitation of the excitation light of the appropriate wavelength.
  • the optical properties after binding are shown in Table 2.
  • the compound can also bind to the aptamer in the cell and transcribes The cells of the RNA aptamer have bright fluorescence, as shown in Figure 17A, and the cells that do not express the RNA aptamer have no fluorescence, as shown in Figure 17B, indicating that this series of dyes can be used for nucleic acid labeling.
  • III-6 435 497 54700 0.57 16601 6.7 III-8 458 508 42500 0.30 9091 27.0 III-4 458 514 44100 0.45 4748 12.0 III-3 485 530 65300 0.66 3595 3.5 III-18 515 599 54400 0.43 708 18.0 III-21 577 620 10000 0.58 12600 6.1
  • the III-3 molecular rotor can specifically label the mRNA of the skeleton protein in the cell line expressing the target RNA, and there is no obvious background fluorescence (Figure 18a), while the background fluorescence of the III-43 molecule is higher than that of III. -3, it is impossible to clearly distinguish whether ACTB is expressed ( Figure 18b).

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Abstract

一种荧光染料及其制备方法和用途，所述荧光染料对粘度响应敏感并且特异，本底荧光低等优点，还可以作为荧光激活点亮型探针，用于蛋白、酶或核酸的荧光标记、定量或检测。

Description

一种荧光染料及其制备方法和用途 技术领域
本发明涉及荧光染料技术领域，具体涉及一种粘度响应性、本底荧光低的荧光染料及其制备方法和用途。
背景技术
分子转子是一类荧光强度随微环境粘度变化的染料，它们在激发后分子发生构象扭曲形成TICT分子内电荷转移态，激发态能量主要以非辐射形式释放，而当它们处于粘度较大或者较刚性的微环境时，此类分子的分子构象扭曲被限制，此时染料激发态能量主要以辐射发光的形式释放，即该类分子的荧光性质被激活，重要的是，该类分子的荧光强度随微环境的粘度的变化而变化，能够实时、原位、灵敏、可视化地显示微环境的粘度变化。
目前，基于限制分子转子的构象扭曲除了用于粘度检测领域之外，还被广泛应用于荧光激活探针的构建，如，分子转子与BSA结合后，分子构象被蛋白限制，荧光被点亮，而未与蛋白结合的染料的激发态能量仍然以非辐射形式耗散，从而达到蛋白实时检测、定量的效果。再如，噻唑橙与DNA、RNA结合前处于荧光淬灭的状态，当与DNA或RNA结合后分子构象被限制，从而荧光被激活，被广泛用于DNA、RNA的检测与示踪；孔雀绿等分子转子被抗体包裹，限制分子的构象变化，用于蛋白激活型荧光成像；DHBI与适配体结合，构建用于RNA示踪的荧光蛋白模拟物；再如，与淀粉样蛋白结合，限制分子的构象变化，用于艾滋海默病的检测与研究等。
然而，目前的分子转子普遍存在荧光本底高的缺点，即分子转子在自由状态下的荧光强度较高，较难适用于样本量少、成分复杂、待测物丰度低的样品检测与标记，如生物样品中的内源性蛋白、核酸、代谢物等，因此，发展一类本底荧光低的分子转子能够进一步拓展目前荧光转子的用途。
发明内容
本发明的目的在于提供一种具有粘度响应性、本底荧光低的荧光染料。
本发明一方面，提供一种荧光染料，所述荧光染料如式(Ⅰ)所示，
Figure PCTCN2020087311-appb-000001
其中：
D-为HO-或N(X 1)(X 2)-，X 1、X 2各自独立地选自氢、烷基和改性烷基；X 1，X 2任选相互连接，与N原子一起形成脂杂环；
R选自氰基、羧基、酰胺基、酯基、亚砜基、砜基、磺酸酯基或磺酰胺基；Ar 1和Ar 2各自独立地选自亚芳基，亚杂芳基；其中，Ar 1、Ar 2中的氢原子任选各自独立地被卤原子、羟基、醛基、羧基、酯基、酰胺基、氰基、磺酸基、磷酸基、氨基、伯氨基、仲氨基、烷基或改性烷基取代；
X 1、X 2任选独立地与Ar 1形成脂杂环；
其中：所述“烷基”各自独立地为C 1-C 10直链或支链烷基；可选地，为C 1-C 7直链或支链烷基；可选地，为C 1-C 5直链或支链烷基；可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
所述“改性烷基”各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-S-、-SO 2-、-(S＝O)-、叠氮基、伯氨基、仲氨基、叔氨基、季铵盐基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；
所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
所述“卤原子”各自独立地为F、Cl、Br或I；
所述“脂杂环”为环上含有N、O、S或Si中的一种或多种杂原子的饱和或不饱和的4～15元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO 2-；所述脂杂环任选被卤原子、烷基、芳基或改性烷基取代；
所述“亚芳基”为5～13元单环或双环或稠合双环或稠合多环的亚芳香基团；
所述“亚杂芳基”为环上含有选自N、O、S或Si中的一种或多种杂原子的5～13元单环或双环或稠合双环或稠合多环的亚杂芳香基团；
所述“酯基”为R'(C＝O)OR″基团；
所述“酰胺基”为R′CONR″R″′基团；
所述“磺酸基”为R′SO 3H基团；
所述“磺酸酯基”为R′SO 2OR″基团；
所述“磺酰胺基”为R′SO 2NR″R″′基团；
所述“磷酸基”为R′OP(＝O)(OH) 2基团；
所述“砜基”为R′SO 2R″基团；
所述“亚砜基”为R′SOR″基团；
所述“伯氨基”为R′NH 2基团；
所述“仲氨基”为R′NHR″基团；
所述“叔氨基”为R′NR″R″′基团；
所述“季铵盐基”为R′R″R″′R″″N +基团；
各个R′、R″、R″′、R″″各自独立地为单键、氢、烷基、亚烷基、改性烷基或改性亚烷基；
所述“亚烷基”为C 1-C 10的直链或支链的亚烷基；可选地，为C 1-C 7直链或支链亚烷基；可选地，为C 1-C 5直链或支链亚烷基；
所述“改性亚烷基”为C 1-C 10(优选为C 1-C 6)亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中的基团置换所得的基团；
可选地，所述“改性烷基”为含有选自-OH、-O-、乙二醇单元(-(CH 2CH 2O) n-)、单糖单元、-O-CO-、-NH-CO-、-SO 2-O-、-SO-、Me 2N-、Et 2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、氰基中的一种或多种基团；
可选地，Ar 1和Ar 2各自独立地为选自下式(Ⅱ-1)～(Ⅱ-22)中的结构：
Figure PCTCN2020087311-appb-000002
Figure PCTCN2020087311-appb-000003
可选地，式(I)所示的化合物选自下式化合物：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-1
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-2
本发明的第二个方面是提供制备上述的荧光染料的方法，其特征在于，包括式(a)化合物与式(b)化合物发生羟醛缩合反应的步骤。
Figure PCTCN2020087311-appb-000006
本发明的第三个方面是提供上述的荧光染料在粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
本发明的第四个方面是提供上的荧光染料在制备用于粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
本发明的第五个方面是提供一种荧光激活点亮型探针，其包括上述荧光染料。
本发明的第六个方面是提供上述的荧光激活点亮型探针在蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
本发明的第七个方面是提供上述的荧光激活点亮型探针在制备用于蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
本发明所得荧光染料可用于测定样品的粘度，例如适用于微观粘度的测试。根据另一方面的具体实施方式，所得荧光染料可与对应的抗体、适配体或淀粉样蛋白等特异性结合，或者通过配体或抑制剂与蛋白标签或酶键合，获得系列荧光激活点亮型探针，用于蛋白、酶或核酸的荧光标记、定量或监测。
附图说明
图1为分子转子Ⅲ-3(1×10 -5M)在不同粘度条件下荧光发射强度图；
图2为分子转子Ⅲ-3(1×10 -5M)粘度条件与荧光强度的线性关系图；
图3为分子转子Ⅲ-4(1×10 -5M)在不同粘度条件下荧光发射强度图；
图4为分子转子Ⅲ-4(1×10 -5M)粘度条件与荧光强度的线性关系图；
图5为分子转子Ⅲ-28(1×10 -5M)在不同粘度条件下荧光发射强度图；
图6为分子转子Ⅲ-28(1×10 -5M)粘度条件与荧光强度的线性关系图；
图7为分子转子Ⅲ-34(1×10 -5M)在不同粘度条件下荧光发射强度图；
图8为分子转子Ⅲ-34(1×10 -5M)粘度条件与荧光强度的线性关系图；
图9为分子转子Ⅲ-11与Ⅲ-36(1×10 -6M)在PBS中的荧光本底对比图；
图10为分子转子Ⅲ-34与Ⅲ-37(1×10 -6M)在PBS中的荧光本底对比图；
图11为分子转子Ⅲ-31、Ⅲ-32、Ⅲ-33与Ⅲ-38(1×10 -6M)在PBS中的荧光本底对比图；
图12为分子转子Ⅲ-3与Ⅲ-39(1×10 -6M)在PBS中的荧光本底对比图；
图13为分子转子Ⅲ-21与Ⅲ-40(1×10 -6M)在PBS中的荧光本底对比图；
图14为分子转子Ⅲ-28、Ⅲ-29、Ⅲ-30与Ⅲ-41(1×10 -6M)在PBS中的荧光本底对比图；
图15为分子转子Ⅲ-3与Ⅲ-42(1×10 -6M)在PBS中的荧光本底对比图；
图16为分子转子Ⅲ-3与Ⅲ-43(1×10 -6M)在PBS中的荧光本底对比图；
图17为分子转子Ⅲ-3、Ⅲ-4、Ⅲ-6、Ⅲ-7、Ⅲ-8、Ⅲ-18、Ⅲ-21用于细胞内RNA适配体的标记，其中A是表达了目标RNA适配体的细胞，B是未表达目标RNA适配体的细胞；
图18为分子转子Ⅲ-3、Ⅲ-43用于标记细胞内mRNA。
具体实施方式
实施例1：
化合物Ⅲ-1：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-3
4-N,N-二甲基-苯甲醛(0.35g，2.3mmol)，4-氰基-苯乙腈(0.4g，2.8mmol)于100ml圆底烧瓶中，加入40ml无水乙醇溶解，加入二滴哌啶，Ar保护条件下油浴加热回流2h，反应完毕，冷却至室温，大量固体析出，过滤，冷乙醇冲洗滤饼三次，真空烘干的橙色固体(0.60g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝3.05(s,6H),6.83(d,J＝9.2Hz,2H,),7.84-7.94(m,6H),8.02ppm(s,1H).HRMS(ESI-TOF):Calcd.For C 18H 16O 3[M+H] +:274.1344.Found:274.1345.
实施例2：
化合物Ⅲ-2：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-4
参照化合物Ⅲ-1的合成方法，(0.34g，89％)。 1H NMR(400MHz,DMSO-d 6):δ＝1.23(t，J＝7.60Hz,6H),3.05(t，J＝7.60Hz,4H),6.84(d,J＝9.2Hz,2H,),7.84-7.95(m,6H),8.09ppm(s,1H).HRMS(ESI-TOF):Calcd.For C 20H 20O 3[M+H] +:302.1657.Found:302.1658.
实施例3：
化合物Ⅲ-3：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-5
参照化合物Ⅲ-1的合成方法，(0.33g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝7.96(s,1H),7.85(d,J＝16.0Hz,6H),6.81(d,J＝8.0Hz,2H),4.77(s,1H),3.55(d,J＝28.0Hz,4H),3.04(s,1H).HRMS(ESI-TOF):Calcd.For C 19H 18N 3O[M+H] +:304.1450.Found:304.1451.
实施例4：
化合物Ⅲ-4：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-6
化合物Ⅲ-3(0.61g，2.0mmol)于40mL干燥DCM中，加入TEA(0.25g，2.2mmol)，0℃条件下缓慢加入对甲苯磺酰氯的(0.38g，2.0mmol)的10ml DCM溶液，Ar保护条件下缓慢升至室温，反应完毕，加入2mL水淬灭反应，分出有机相，Na 2SO 4干燥，减压条件下除去有机溶剂，残余物不经进一步处理，直接用于下一步。
残余物溶于20ml乙腈中，加入1ml甲胺的甲醇溶液，体系在Ar保护条件下油浴加热回流过夜，反应完毕，加压除去溶剂，体系溶于50ml DCM中，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na 2SO 4干燥，加压除去溶剂，残余物经柱色谱分离得橙红色固体(0.54g,82％)。 1H NMR(400MHz,CDCl 3):δ＝7.88(d,J＝9.0Hz,2H),7.74–7.65(m,4H),7.48(s,1H),6.73(d,J＝9.1Hz,2H),3.60–3.55(m,2H),3.08(s,3H),2.57–2.52(m,2H),2.34(s,6H).HRMS(ESI-TOF):Calcd.For C 21H 23N 4[M+H] +:331.1923.Found:331.1925.
实施例5：
化合物Ⅲ-5：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-7
4-羟基-3,5-二氟-苯甲醛(0.32g，2.0mmol)，4-氰基-苯乙腈(0.35g，2.4mmol)于100ml圆底烧瓶中，加入40ml无水乙醇溶解，加入二滴哌啶，Ar保护条件下油浴加热回流2h，反应完毕，冷却至室温，大量固体析出，过滤，冷乙醇冲洗滤饼三次，真空烘干的橙色固体。 1H NMR(400MHz,CDCl 3):δ＝7.80(d,J＝9.0Hz,2H),7.74–7.66(m,4H),7.48(s,1H).HRMS(ESI-TOF):Calcd.For C 16H 9F 2N 2O[M+H] +:283.0683.Found:283.0684.
实施例6：
化合物5-(N-甲基-N-羟乙基)氨基-吡嗪-2-甲醛：
Figure PCTCN2020087311-appb-000012
4-N-甲基-N-羟乙基胺(2.6g，35mmol)，5-氯-吡嗪-2-甲醛(0.50g，3.5mmol) 于100ml圆底烧瓶中，加入20ml无水乙腈溶解，加入K 2CO 3(0.71g，5.3mmol)，Ar保护条件下油浴加热回流24h，反应完毕，冷却至室温，过滤，真空除去溶剂，残余物溶于100ml DCM中，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na2SO4干燥，除去有机溶剂，残余物经柱色谱分离得5-(N-甲基-N-羟乙基)-吡嗪-2-醛(0.48g，76％)。 1H NMR(400MHz,CDCl 3):δ9.88(s,1H),8.62(d,J＝1.2Hz,1H),8.14(d,J＝1.1Hz,1H),3.92(m,2H),3.88–3.83(m,2H),3.28(s,3H).HRMS(ESI-TOF):Calcd.For C 8H 12N 3O 2[M+H] +:182.1.Found:182.1.
化合物Ⅲ-6：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-8
参照化合物Ⅲ-1的合成方法，(0.36g，96％)。 1H NMR(400MHz,CDCl 3):δ8.39(s,1H),8.30(s,1H),7.80(d,J＝8.5Hz,2H),7.72(d,J＝8.4Hz,2H),7.51(s,1H),3.93(t,J＝4.9Hz,2H),3.88–3.83(m,2H),3.29(s,3H).HRMS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1355.Found:306.1357.
实施例7：
化合物Ⅲ-7：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-9
参照化合物Ⅲ-4的合成方法，(0.21g，67％)。 1H NMR(400MHz,DMSO-d 6):δ8.37(d,J＝5.2Hz,2H),8.06(s,1H),8.00–7.85(m,4H),3.77(t,J＝6.5Hz,2H),3.20(s,3H),2.56(m,2H),2.23(s,6H).HRMS(ESI-TOF):Calcd.For C 19H 21N 6[M+H] +:333.1828.Found:333.1829.
实施例8：
化合物6-(N-甲基-N-羟乙基)氨基-吡嗪-3-醛：
Figure PCTCN2020087311-appb-000015
按照化合物5-(N-甲基-N-羟乙基)氨基-吡嗪-2-醛的合成方法，(0.45g，68％)。 1H NMR(400MHz,CDCl 3):δ＝9.69(s,1H),8.43(d,J＝2.1Hz,1H),7.86(dd,J＝9.0,2.3Hz,1H),6.56(d,J＝9.1Hz,1H),3.86–3.79(m,4H),3.15(s,3H).HRMS(ESI-TOF):Calcd.For C 9H 13O 2N 2[M+H] +:181.1.Found:181.1.
化合物Ⅲ-8：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-10
参照化合物Ⅲ-1的合成方法，(0.39g，89％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.54(d,J＝4.0Hz,1H),8.30(dd,J＝9.3,2.5Hz,1H),8.03(s,1H),7.92(d,J＝8.0Hz,2H),7.85(d,J＝8.0Hz,2H),6.84(d,J＝8.0Hz,1H),4.77(t,J＝5.4Hz,1H),3.67(t,J＝5.3Hz,2H),3.60(q,J＝5.4Hz,2H),3.15(s,3H).HRMS(ESI-TOF):Calcd.For C 18H 27N 4O[M+H] +:305.1402.Found:305.1401.
实施例9：
化合物Ⅲ-9：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-11
参照化合物Ⅲ-4的合成方法，(0.31g,92％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.55(d,J＝4.0Hz,1H),8.31(dd,J＝9.3,2.5Hz,1H),8.05(s,1H),7.93(d,J＝8.0Hz,2H),7.84(d,J＝8.0Hz,2H),6.85(d,J＝8.0Hz,1H),4.78(t,J＝5.4Hz,1H),3.67(t,J＝5.3Hz,2H),3.60(q,J＝5.4Hz,2H),3.17(t,J＝8.0Hz,4H),1.17(t,J＝8.0Hz,6H).HRMS(ESI-TOF):Calcd.For C 22H 26N 5[M+H] +:360.2188.Found:360.2187.
实施例10：
4-N,N-二甲基-6醛基-吡啶：
Figure PCTCN2020087311-appb-000018
参照化合物4-N-甲基-N-(2-N’,N’-二甲基-乙基)-苯甲醛的合成方法，(0.31g，49％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.86(d,J＝0.6Hz,1H),8.17(d,J＝2.9Hz,1H),7.83(d,J＝8.9Hz,1H),6.94(dd,J＝8.8,2.9Hz,1H),3.10(s,6H).HRMS(ESI-TOF):Calcd.For C 8H 11N 2O[M+H] +:151.1.Found:151.1.
化合物Ⅲ-10：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-12
参照化合物Ⅲ-1的合成方法，(0.36g，96％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.86(d,J＝0.6Hz,1H),8.26(s，1H),8.17(d,J＝2.9Hz,1H),7.83(d,J＝8.9Hz,1H),7.46(m,4H),6.94(dd,J＝8.8,2.9Hz,1H),3.10(s,6H).HRMS(ESI-TOF):Calcd.For C 17H 15N 4[M+H] +:275.1297.Found:275.1298.
实施例11：
2-(N-甲基-N-羟乙基)氨基-5-醛基-嘧啶：
Figure PCTCN2020087311-appb-000020
参照化合物4-N-甲基-N-(2-N’,N’-二甲基-乙基)-苯甲醛的合成方法，(0.42g，72％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.89(s,1H),8.73(s，2H),3.64(t,J＝8.9Hz,2H),3.45(t,J＝8.8Hz,2H),3.10(s,3H).HRMS(ESI-TOF):Calcd.For C 8H 12N 3O[M+H] +:182.1.Found:182.1.
化合物Ⅲ-11：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-13
参照化合物Ⅲ-1的合成方法，(0.36g，96％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.26(s,1H),8.73(s，2H),7.64(m,4H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.11(s,3H).HRMS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1355.Found:306.1356.
实施例12：
5-(N-甲基-N-羟乙基)氨基-2-醛基-嘧啶：
Figure PCTCN2020087311-appb-000022
参照化合物4-N-甲基-N-(2-N’,N’-二甲基-乙基)-苯甲醛的合成方法，(0.42g，72％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.98(s,1H),8.21(s，2H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H).HRMS(ESI-TOF):Calcd.For C 8H 12N 3O 2[M+H] +:182.1.Found:182.1.
1-氰基-1-(4-苯乙腈)-2-2-(5-(N-甲基-N-羟乙基)氨基-)嘧啶-乙烯：
Figure PCTCN2020087311-appb-000023
参照化合物Ⅲ-1的合成方法，(0.56g，89％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.21(s，2H),7.99(s,1H),7.64(s,4H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H).HRMS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1.Found:306.1.
化合物Ⅲ-12：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-14
参照化合物Ⅲ-4的合成方法，(0.36g，96％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.21(s，2H),7.99(s,1H),7.64(s,4H),3.77(t,J＝6.5Hz,2H),3.20(s,3H),2.56(m,2H),2.23(s,6H).HRMS(ESI-TOF):Calcd.For C 19H 21N 6[M+H] +:333.1828.Found:333.1829.
实施例13：
2-乙腈-5-氰基-吡啶：
Figure PCTCN2020087311-appb-000025
2-溴甲基-5-氰基吡啶(0.50g，2.5mmol)于100ml圆底烧瓶中，加入50ml THF溶解，Ar保护条件下加入10ml NaCN的2M的水溶液，油浴加热回流12h，反应完毕，体系冷却至室温，DCM萃取(3×100ml)，合并有机相，分别用水、饱和食盐水洗涤(2×100ml)，有机相用NaSO4干燥，减压除去溶剂，残余物经柱色谱纯化分离得2-乙腈-5-氰基吡啶(0.19g，56％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.78(s,1H),7.95(m,1H),7.56(m,1H),4.01(s,2H).HRMS(ESI-TOF):Calcd.For C 8H 6N 3[M+H] +:144.1.Found:144.1.
化合物Ⅲ-13：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-15
参照化合物Ⅲ-1的合成方法，(0.45g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.78(s,1H),8.21(s,1H),7.94(m,1H),7.86(d,J＝8.0Hz,2H),7.57(m,1H),6.80(d,J＝8.0Hz,2H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H). HRMS(ESI-TOF):Calcd.For C 18H 17N 4O[M+H] +:305.1402.Found:305.1403.
实施例14：
2-氰基-5-乙腈-吡嗪：
Figure PCTCN2020087311-appb-000027
2-氯-吡嗪-5-乙腈(0.32g,2.0mmol)、CuCN(0.93g，10.0mmol)于100ml圆底烧瓶中，加入30ml干燥DMSO溶解，Ar保护条件下80℃油浴加热12h，反应完毕，体系倒入100ml水中，DCM萃取(4×50ml)，合并有机相，分别用水、饱和食盐水洗涤(2×100ml)，有机相用Na2SO4干燥，减压除去有机溶剂，残余物经柱色谱分离得2-氰基-吡嗪-5-乙腈(0.20g，69％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.60(s,1H),8.48(s,1H),3.92(s,2H).HRMS(ESI-TOF):Calcd.For C 7H 5N 4[M+H] +:145.1.Found:145.1.
化合物Ⅲ-14：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-16
参照化合物Ⅲ-1的合成方法，(0.25g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.60(s,1H),8.48(s,1H),8.11(s,1H),7.81(d,J＝8.2Hz,2H),6.84(d,J＝8.2Hz,2H),3.60(t,J＝9.2Hz,2H),3.46(t,J＝9.2Hz,2H),3.12(s,3H).HRMS(ESI-TOF):Calcd.For C 17H 16N 5O[M+H] +:306.1355.Found:306.1354.
实施例15：
化合物Ⅲ-15：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-17
参照化合物Ⅲ-1的合成方法，(0.25g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.22(s,1H),8.00(d,J＝9.1Hz,1H),7.77–7.69(m,1H),7.43–7.34(m,1H),6.88(d,J＝9.1Hz,1H),4.81(t,J＝5.2Hz,1H),3.31–3.25(m,4H),2.66-2.63(m,4H),1.89-1.81(m,4H).HRMS(ESI-TOF):Calcd.For C 22H 20N 3[M+H] +:326.1657.Found:326.1658.
实施例16：
化合物Ⅲ-16：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-18
参照化合物Ⅲ-1的合成方法，(0.29g，94％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.11(2H,d,J＝10.4Hz),7.99(3H,dd,J＝8.6,3.0Hz),7.54(1H,dd,J＝8.0,8.0Hz),7.44(1H,dd,J＝8.0,8.0Hz),6.88(2H,d,J＝9.2Hz),4.82(1H,bt,t,J＝5.2Hz),3.01-3.08(m,2H),3,53-3.60(m,2H),2.89(s,3H).HRMS(ESI-TOF):Calcd.For C 19H 16N 3[M+H] +:286.1344.Found:286.1345.
实施例17：
6-甲胺-苯并[b]噻吩-2-甲醛：
Figure PCTCN2020087311-appb-000031
6-溴苯并[b]噻吩-2-甲醛(0.42g，1.7mmol)、二甲基乙胺(40％水溶液，1g，8.9mmol)、CuI(13.9mg，0.073mmol)、K 3PO 4·H 2O(155.4mg，0.73mmol)、甲胺(33％水溶液，1g)于100ml耐压瓶中，密封条件下60℃油浴加热12h，体系冷却至室温，加入50ml水，DCM萃取(3×100ml)，合并有机相，Na 2SO 4干燥，减压除去有机溶剂，残余物经柱色谱分离纯化(0.23g，68％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.92(1H,s),8.14(1H,s),7.82(1H,d,J＝9.1Hz),7.18(1H,d,J＝2.1Hz),7.01(1H,dd,J＝9.1,2.3Hz),3.05(3H,s).HRMS(ESI-TOF):Calcd.For C 10H 10NOS[M+H] +:192.0.Found:192.0.
化合物Ⅲ-17：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-19
参照化合物Ⅲ-1的合成方法，(0.29g，94％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.45(s,1H),7.92(d,J＝8.6Hz,2H),7.85(d,J＝8.3Hz,3H),7.73(dd,J＝8.6,3.9Hz,1H),7.21(d,J＝1.9Hz,1H),7.21(d,J＝1.9Hz,1H),6.96(dd,J＝9.1,2.3Hz,1H),3.05(s,3H).HRMS(ESI-TOF):Calcd.For C 19H 14N 3S[M+H] +:360.1171. Found:360.1173.
实施例18:
6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛：
Figure PCTCN2020087311-appb-000033
参照化合物6-甲胺-苯并[b]噻吩-2-甲醛的合成方法，(0.54g，79％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.91(s,1H),8.14(s,1H),7.81(d,J＝5.2Hz,1H),7.17(d,J＝2.0Hz,1H),7.01(dd,J＝2.0,8.8Hz,1H),4.76(t,J＝5.6Hz,1H),3.58(t,J＝4.2Hz,2H),3.52(t,J＝4.2Hz,2H),3.04(s,3H).HRMS(ESI-TOF):m/z Calcd.For C 12H 14NO 2S,[M+H] +:235.1.Found 236.1.
化合物Ⅲ-18：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-20
参照化合物Ⅲ-1的合成方法，(0.21g，95％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.45(s,1H),7.92(d,J＝8.6Hz,2H),7.85(d,J＝8.3Hz,3H),7.73(dd,J＝8.6,3.9Hz,1H),7.21(d,J＝1.9Hz,1H),7.21(d,J＝1.9Hz,1H),6.96(dd,J＝9.1,2.3Hz,1H),3.63–3.57(m,2H),3.52(t,J＝5.7Hz,2H),3.05(s,3H).HRMS(ESI-TOF):Calcd.For C 21H 19N 3OS[M+H] +:360.1171.Found:360.1173.
实施例19:
5-N,N-二甲胺-2-醛基并二噻吩：
Figure PCTCN2020087311-appb-000035
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.54g，79％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.66(s,1H),8.05(s,1H),6.30(s,1H),4.88(bt,1H),3.07(s,6H).HRMS(ESI-TOF):m/z Calcd.For C 9H 12NOS 2[M+H] +:214.0；found 214.0.
化合物Ⅲ-19：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-21
参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.34(s,1H),7.86(d,J＝8.0Hz,2H),7.81(s,1H),7.77(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.08(s,6H).HRMS(ESI-TOF):Calcd.For C 18H 14N 3S 2[M+H] +:336.0629.Found:336.0630.
实施例20：
5-N,N-二乙胺-2-醛基并二噻吩：
Figure PCTCN2020087311-appb-000037
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.44g，75％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.78(s,1H),8.09(s,1H),6.30(s,1H),4.87(bt,1H),3.27(t,J＝8.4Hz,4H),1.26(t,J＝8.4Hz,4H).HRMS(ESI-TOF):m/z Calcd.For C 9H 12NOS 2[M+H] +:214.0；found 214.0.
化合物Ⅲ-20：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-22
参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.34(s,1H),7.86(d,J＝8.0Hz,2H),7.81(s,1H),7.77(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.27(t,J＝8.4Hz,4H),1.26(t,J＝8.4Hz,4H).HRMS(ESI-TOF):Calcd.For C 20H 18N 3S 2[M+H] +:364.0942.Found:364.0943.
实施例21:
5-(N-甲基-N-羟乙基)氨基-2-醛基并二噻吩：
Figure PCTCN2020087311-appb-000039
参照化合物6-N-甲基-N-羟乙基-苯并[b]噻吩-2-甲醛的合成方法，(0.44g，75％)。 1H NMR(400MHz,DMSO-d 6):δ＝9.66(s,1H),8.05(s,1H),6.30(s,1H),4.88(bt,1H),3.64(t,J＝5.6Hz,2H),3.44(t,J＝5.6Hz,2H),3.07(s,3H).HRMS(ESI-TOF):m/z Calcd.For C 10H 12NO 2S 2[M+H] +:241.0；found 242.0.
化合物Ⅲ-21：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-23
参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ8.34(s,1H),7.86(d,J＝8.0Hz,2H),7.81(s,1H),7.77(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.65(q,J＝5.5Hz,2H),3.44(t,J＝5.5Hz,2H),3.34(s,1H),3.08(s,3H).HRMS(ESI-TOF):Calcd.For C 19H 16N 3OS 2[M+H] +:366.0735.Found:366.0736.
实施例22：
化合物Ⅲ-22：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-24
参照化合物Ⅲ-1的合成方法，(0.31g，90％)。 1H NMR(400MHz,DMSO-d 6):δ＝3.04(s,6H),6.82(d,J＝9.2Hz,2H,),7.59(d,J＝9.1Hz,2H),7.84-7.94(m,6H),8.02ppm(s,1H).HRMS(ESI-TOF):Calcd.For C 24H 19O 3[M+H] +:350.1657.Found:350.1656.
实施例23：
化合物Ⅲ-23：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-25
参照化合物Ⅲ-1： 1H NMR(400MHz,DMSO-d 6):δ＝3.02(s,6H),6.72(d,J＝8.0Hz,2H,),7.24(d,J＝4.0Hz,1H),7.49(d,J＝8.8Hz,2H),7.55(d,J＝8.0Hz,1H),7.69(d,J＝8.8Hz，2H),8.02ppm(s,1H).HRMS(ESI-TOF):Calcd.For C 22H 18N 3S[M+H] +:356.1221.Found:356.1220.
实施例24：
化合物Ⅲ-24：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-26
参照化合物化合物Ⅲ-1的合成方法，其中化合物1(参照Chem.Commun.2011,47,985-987.的合成方法)： 1H NMR(400MHz,DMSO-d 6):δ＝3.63(m,16H),3.77(m,4H),6.76(d,J＝8.8Hz,2H,),7.38(d,J＝4.0Hz,2H),7.49(d,J＝8.8Hz,2H),7.59(d,J＝8.8Hz,2H),7.72(m,4H),8.28(s,1H).HRMS(ESI-TOF):Calcd.For C 30H 32O 3N 4S[M+H] +:530.2114.Found:530.2115.
实施例25：
化合物Ⅲ-25：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-27
参照化合物化合物Ⅲ-1的合成方法，其中化合物2(参照J.Org.Chem.2008,73,6587-6594.的合成方法)： 1H NMR(400MHz,DMSO-d 6):δ＝1.23(t,J＝7.2Hz,6H),3.35(m,J＝7.2Hz,4H),5.78(d,J＝4.0Hz,1H),6.92(d,J＝4.0Hz,1H),7.12(d,J＝4.0Hz,1H),7.49(d,J＝8.8Hz,2H),7.56(d,J＝4.0Hz,1H),7.69(d,J＝8.8Hz,2H),8.28(s,1H).HRMS(ESI-TOF):Calcd.For C 30H 32O 3N 4S[M+H] +:390.1099.Found:390.1097.
实施例26：
化合物Ⅲ-26：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-28
参照化合物化合物Ⅲ-1的合成方法， 1H NMR(400MHz,DMSO-d 6):δ＝3.30(s,6H),5.71(d,J＝4.0Hz,1H),6.93(d,J＝4.0Hz,1H),7.15(d,J＝4.0Hz,1H),7.47(d,J＝8.8Hz,2H),7.56(d,J＝4.0Hz,1H),7.64(d,J＝8.8Hz,2H),8.28(s,1H).HRMS(ESI-TOF):Calcd.For C 20H 17O 2N 2S 2[M+H] +:381.0731.Found:381.0730.
实施例27：
化合物Ⅲ-27：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-29
参照化合物Ⅲ-1的合成方法，其中化合物4(参照Heterocycles,1997,46,489-501.) 1H NMR(400MHz,CDCl 3):δ2.07(m,4H),3.33(t,J＝6.6Hz,4H),4.2(s,3H),5.70(d,J＝4.4Hz,1H),6.92(d,J＝4.0Hz,1H),7.15(d,J＝4.0Hz,1H),7.43(d,J＝8.2Hz,2H),7.51(d,J＝8.2Hz,2H),7.57(d,J＝4.0Hz,1H),8.10(s,1H).HRMS(ESI-TOF):Calcd.For C 23H 21O 2N 2S 2[M+H] +:421.1044.Found:521.1042.
实施例28：
化合物Ⅲ-28：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-30
参照化合物Ⅲ-1的合成方法，其中化合物5(参照WO2018014821). 1H-NMR(400MHz，DMSO-d 6)：δ＝7.84(s，1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.24(s，1H),3.78(t,2H,J＝4.80Hz),3.44(t,2H,J＝4.80Hz),3.02(s,3H)。HRMS(ESI-TOF):Calcd.For C 21H 16ON 3S 3.[M+H] +:422.0455.Found:422.0456.
实施例29：
化合物Ⅲ-29：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-31
参照化合物Ⅲ-1的合成方法，其中化合物6(参照WO2018014821)。 1H-NMR(400MHz,DMSO-d 6):δ＝7.84(s,1H)7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.24(s,1H),3.56(q,J＝4.0Hz,2H),3.01(s,6H),1.21(t,J＝4.0Hz,3H).HRMS(ESI-TOF):Calcd.For C 22H 19O 2N 2S 3.[M+H] +:439.0609.Found:439.0610.
实施例30：
化合物Ⅲ-30：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-32
参照化合物Ⅲ-1的合成方法，其中化合物7(WO 2014048547)。 1H-NMR(400MHz,DMSO-d 6)：δ＝7.84(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.24(s,1H),3.10(s,3H),3.01(s,6H).HRMS(ESI-TOF):Calcd.For C 21H 17O 1N 2S 4.[M+H] +:429.0024.Found:429.0026.
实施例31：
化合物Ⅲ-31：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-33
参照化合物Ⅲ-1的合成方法，其中化合物9(J.Chem.Pharm.Res.,2012,4,1661-1669.)。 1H-NMR(400MHz,DMSO-d 6):δ＝7.84(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.24(s,1H),3.14(s,3H),3.01(s,6H).HRMS(ESI-TOF):Calcd.For C 22H 23O 2N 2S 3Si .[M+H] +:471.0691.Found:471.0690.
实施例32：
化合物Ⅲ-32：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-34
参照化合物Ⅲ-1的合成方法。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.84(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.24(s,1H),3.77(t,2H,J＝4.80Hz),3.41(t,2H,J＝4.80Hz)，3.00(s,3H).HRMS(ESI-TOF):Calcd.For C 22H 24O 3N 3S 3Si .[M+H] +:502.0749.Found:502.0752.
实施例33：
化合物Ⅲ-33：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-35
参照化合物Ⅲ-1的合成方法。 1H-NMR(400MHz,CDCl 3)：δ＝7.89(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.18(s,1H),6.96(d,2H,J＝5.6Hz),3.85(t,2H,J＝4.80Hz),3.46(t,2H,J＝4.80Hz),3.06(s,3H),0.46(s,6H).Calcd.For C 23H 22ON 3S 2Si .[M+H] +:448.0974.Found:448.0972.
实施例34：
化合物Ⅲ-34：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-36
参照化合物Ⅲ-1的合成方法。 1H-NMR(400MHz,CDCl 3)：δ＝7.83(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.11(s,1H),3.85(t,2H,J＝4.80Hz),3.46(t,2H,J＝4.80Hz),3.06(s,3H),1.46(s,6H).HRMS(ESI-TOF):Calcd.For C 24H 24O 2N 3S 2[M+H] +:450.1310.Found:450.1311.
实施例35：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-37
化合物12：
参照文献公开方法(K.T.Arun et.al.J.Phys.Chem.A.2005,109,5571-5578.) 1H-NMR(400MHz,CDCl3)：δ＝10.01(s,1H),7.89(s,1H),7.18(s,1H),6.96(d,2H,J＝5.6Hz),3.52-3.65(m,20H),3.37(s,3H),2.97(s,3H).HRMS(ESI-TOF):Calcd.For C 24H 22ON 3S 2Si .[M+H] +:432.1204.Found:432.1203.Calcd.For C 24H 36O 6N 1S 2.[M+H] +:497.3.Found:497.3.
化合物Ⅲ-35:
参照化合物Ⅲ-1的合成方法。 1H-NMR(400MHz,CDCl 3)：δ＝7.89(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.18(s,1H),6.96(d,2H,J＝5.6Hz),3.52-3.65(m,20H),3.37(s,3H),2.97(s,3H).HRMS(ESI-TOF):Calcd.For C 33H 39O 5N 3S 2.[M+H] +:622.2409.Found:622.2409.
对比例1：
化合物Ⅲ-36：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-38
参照化合物Ⅲ-1的合成方法，(0.25g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.21(s，2H),7.99(s,1H),7.64(s,4H),3.64(t,J＝8.9Hz,2H),3.44(t,J＝8.8Hz,2H),3.12(s,3H).HRMS(ESI-TOF):Calcd.For C 16H 17N 4O 4S[M+H] +:361.0971.Found:361.0970
对比例2：
化合物Ⅲ-37：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-39
参照化合物Ⅲ-1的合成方法，(0.39g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝7.83(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.11(s,1H),3.85(t,2H,J＝4.80Hz),3.46(t,2H,J＝4.80Hz),3.05(s,3H),1.46(s,6H).HRMS(ESI-TOF):Calcd.For C 23H 23N 2O 4S 3[M+H] +:487.0820.Found:487.0821.
对比例3：
化合物Ⅲ-38：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-40
参照化合物Ⅲ-1的合成方法，其中化合物11(CN 106349105.)。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.84(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),7.24(s,1H),3.78(t,2H,J＝4.80Hz),3.44(t,2H,J＝4.80Hz),3.01(s,3H).HRMS (ESI-TOF):Calcd.For C 22H 23O 4N 2S 3Si .[M+H] +:503.0589.Found:203.0588.
对比例4：
化合物Ⅲ-39：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-41
参照化合物Ⅲ-1的合成方法。 1H-NMR(400MHz，DMSO-d 6)：δ＝7.84(s,1H),7.59(d,J＝8.8Hz,2H),7.49(d,J＝8.8Hz,2H),3.78(t,2H,J＝4.80Hz),3.44(t,2H,J＝4.80Hz),3.01(s,3H).HRMS(ESI-TOF):Calcd.For C 18H 19O 4N 2S .[M+H] +:359.1066.Found:359.1065.
对比例5：
化合物Ⅲ-40：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-42
参照化合物Ⅲ-1的合成方法。 1H-NMR(400MHz，DMSO-d 6)：δ＝8.34(s,1H),7.59(d,J＝8.0Hz,2H),7.81(s,1H),7.49(d,J＝8.0Hz,2H),6.32(s,1H),4.88(t,J＝4.0Hz,1H),3.65(q,J＝5.5Hz,2H),3.44(t,J＝5.5Hz,2H),3.34(s,1H),3.08(s,3H).HRMS(ESI-TOF):Calcd.For C 18H 17O 4N 2S 3.[M+H] +:421.0350.Found:421.0351.
对比例6：
化合物Ⅲ-41：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-43
参照化合物Ⅲ-1的合成方法. 1H-NMR(400MHz，DMSO-d 6)：δ＝7.85(s，1H),7.59(d,J＝8.8Hz,2H),7.47(d,J＝8.8Hz,2H),7.24(s，1H),3.79(t,2H,J＝4.80Hz),3.43(t,2H,J＝4.80Hz),3.01(s,3H)。HRMS(ESI-TOF):Calcd.For C 20H 17O 4N 2S 4.[M+H] +:477.0071.Found:477.0070.
对比例7：
化合物Ⅲ-42：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-44
参照化合物Ⅲ-1的合成方法，(0.25g，91％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.22(s,1H),8.00(d,J＝9.1Hz,1H),7.77–7.69(m,1H),7.43–7.34(m,1H),6.88(d,J＝9.1Hz,1H),4.81(t,J＝5.2Hz,1H),3.64–3.52(m,3H),3.09(s,1H).LR-HRMS(ESI-TOF):Calcd.For C 19H 18N 3O 2[M+H] +:320.1399.Found:320.1397.对比例8：
化合物Ⅲ-43：
[根据细则91更正 19.05.2020]　
Figure WO-DOC-FIGURE-45
参照化合物Ⅲ-1的合成方法，(0.29g，94％)。 1H NMR(400MHz,DMSO-d 6):δ＝8.11(2H,d,J＝10.4Hz),7.99(3H,dd,J＝8.6,3.0Hz),7.54(1H,dd,J＝8.0,8.0Hz),7.44(1H,dd,J＝8.0,8.0Hz),6.88(2H,d,J＝9.2Hz),4.82(1H,bt,t,J＝5.2Hz),3.60(2H,t,J＝5.2Hz),3.56(2H,t,J＝5.2Hz),3.09(3H,s).LR-HRMS(ESI-TOF):Calcd.For C 19H 18N 3OS[M+H] +:336.1171.Found:336.1170.
试验例1：
将实施例1-35制备的荧光染料(分子转子)分别溶于二甲基亚砜中，各自制得浓度为1×10 -2M的母液，将各母液分别加入甘油和甲醇中，混合均匀，各配制终浓度为1×10 -5M的溶液，根据荧光染料不同，依次用各荧光染料最大激发波长在相同条件下检测其荧光发射图谱，结果如表1所示，表明本发明荧光染料粘度变化响应灵敏。
表1
化合物名称 最大发射波长(nm) 甘油/甲醇荧光强度比
Ⅲ-1 530 990
Ⅲ-2 530 870
Ⅲ-3 530 1025
Ⅲ-4 521 892
Ⅲ-5 525 1028
Ⅲ-6 490 1148
Ⅲ-7 485 977
Ⅲ-8 495 1168
Ⅲ-9 490 920
Ⅲ-10 520 1620
Ⅲ-11 470 869
Ⅲ-12 542 855
Ⅲ-13 545 752
Ⅲ-14 550 785
Ⅲ-15 561 1011
Ⅲ-16 555 491
Ⅲ-17 587 828
Ⅲ-18 595 978
Ⅲ-19 620 991
Ⅲ-20 620 836
Ⅲ-21 620 544
Ⅲ-22 650 989
Ⅲ-23 661 687
Ⅲ-24 662 596
Ⅲ-25 678 783
Ⅲ-26 676 368
Ⅲ-27 678 486
Ⅲ-28 662 559
Ⅲ-29 665 684
Ⅲ-30 660 756
Ⅲ-31 687 624
Ⅲ-32 690 817
Ⅲ-33 705 691
Ⅲ-34 689 489
Ⅲ-35 690 710
试验例2：
分子转子Ⅲ-3、Ⅲ-4、Ⅲ-28、Ⅲ-34加入二乙醇-甘油混合溶液中，配制成终浓度为1×10 -5M的溶液，480nm激发，不同粘度条件下荧光发射光谱如图1、3、5、7所示，相同浓度的分子转子在不同粘度条件下荧光强度逐渐增大，说明 分子转子荧光强度随环境粘度的增大荧光增强，荧光强度的对数和溶剂粘度的对数关系符合哈夫曼方程，具有很好的线性关系，如图2、4、6、8所示，证明分子转子对粘度反应灵敏，并且可以用于未知样品的粘度测试。
试验例3：
分子转子Ⅲ-11与Ⅲ-36；Ⅲ-34与Ⅲ-37；Ⅲ-31、Ⅲ-32、Ⅲ-33与Ⅲ-38；Ⅲ-3与Ⅲ-39；Ⅲ-21与Ⅲ-40；Ⅲ-28、Ⅲ-29、Ⅲ-30与Ⅲ-41；Ⅲ-3与Ⅲ-42；Ⅲ-3与Ⅲ-43加入PBS溶液中，配置终浓度为1×10 -6M的溶液，分别用各化合物最大激发波长激发，检测他们在PBS中的荧光强度，以各组最强荧光为100对各样品进行归一化，分别如图9、图10、图11、图12、图13、图14、图15和图16所示。结果表明：与拉电子基团上的芳香环上有磺酸基取代的分子转子以及没有取代的分子转子相比较，本申请拉电子基团上的芳香环上具有氰基、酯基、亚砜、砜、磺酰胺取代的分子转子具有更低的本底荧光。
试验例4：
化合物Ⅲ-3、Ⅲ-4、Ⅲ-6、Ⅲ-7、Ⅲ-8、Ⅲ-18、Ⅲ-21与RNA适配体(序列10：F30-8Pepper-5RNA适配体序列
Figure PCTCN2020087311-appb-000063
特异性结合，结合后的化合物荧光被显著激活，在合适波长激发光的激发下发射明亮的荧光，结合后的光学性质见表2，化合物还可以与细胞内与该适配体结合，转录了该RNA适配体的细胞具有明亮的荧光，如图17A，未表达该RNA适配体的细胞无荧光，如图17B，说明该系列染料可以用于核酸的标记。
表2
名称 Ex/nm Em/nm ε(M -1cm -1) QY(-) 激活倍数 K d(nM)
Ⅲ-7 443 485 49100 0.42 691 8.0
Ⅲ-6 435 497 54700 0.57 16601 6.7
Ⅲ-8 458 508 42500 0.30 9091 27.0
Ⅲ-4 458 514 44100 0.45 4748 12.0
Ⅲ-3 485 530 65300 0.66 3595 3.5
Ⅲ-18 515 599 54400 0.43 708 18.0
Ⅲ-21 577 620 10000 0.58 12600 6.1
注：荧光量子产率以罗丹明6G为标准(QY＝0.94)，采用相对法测得。
试验例5：
构建了以适配体(ACTB-4Pepper RNA适配体序列
AUGGAUGAUGAUAUCGCCGCGCUCGUCGUCGACAACGGCUCCGGCAUGUGCAAGGCCGGCUUCGCGGGCGACGAUGCCCCCCGGGCCGUCUUCCCCUCCAUCGUGGGGCGCCCCAGGCACCAGGGCGUGAUGGUGGGCAUGGGUCAGAAGGAUUCCUAUGUGGGCGACGAGGCCCAGAGCAAGAGAGGCAUCCUCACCCUGAAGUACCCCAUCGAGCACGGCAUCGUCACCAACUGGGACGACAUGGAGAAAAUCUGGCACCACACCUUCUACAAUGAGCUGCGUGUGGCUCCCGAGGAGCACCCCGUGCUGCUGACCGAGGCCCCCCUGAACCCCAAGGCCAACCGCGAGAAGAUGACCCAGAUCAUGUUUGAGACCUUCAACACCCCAGCCAUGUACGUUGCUAUCCAGGCUGUGCUAUCCCUGUACGCCUCUGGCCGUACCACUGGCAUCGUGAUGGACUCCGGUGACGGGGUCACCCACACUGUGCCCAUCUACGAGGGGUAUGCCCUCCCCCAUGCCAUCCUGCGUCUGGACCUGGCUGGCCGGGACCUGACUGACUACCUCAUGAAGAUCCUCACCGAGCGCGGCUACAGCUUCACCACCACGGCCGAGCGGGAAAUCGUGCGUGACAUUAAGGAGAAGCUGUGCUACGUCGCCCUGGACUUCGAGCAAGAGAUGGCCACGGCUGCUUCCAGCUCCUCCCUGGAGAAGAGCUACGAGCUGCCUGACGGCCAGGUCAUCACCAUUGGCAAUGAGCGGUUCCGCUGCCCUGAGGCACUCUUCCAGCCUUCCUUCCUGGGCAUGGAGUCCUGUGGCAUCCACGAAACUACCUUCAACUCCAUCAUGAAGUGUGACGUGGACAUCCGCAAAGACCUGUACGCCAACACAGUGCUGUCUGGCGGCACCACCAUGUACCCUGGCAUUGCCGACAGGAUGCAGAAGGAGAUCACUGCCCUGGCACCCAGCACAAUG AAGAUCAAGAUCAUUGCUCCUCCUGAGCGCAAGUACUCCGUGUGGAUCGGCGGCUCCAUCCUGGCCUCGCUGUCCACCUUCCAGCAGAUGUGGAUCAGCAAGCAGGAGUAUGACGAGUCCGGCCCCUCCAUCGUCCACCGCAAAUGCUUCUAGCACUCGCUAGAGCAUGGUUAAGCUUCCCACGGAGGAUCCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCCCAAUCGUGGCGUGUCGGCCUCUCUUCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGAGAGGCACUGGCGCCGGGAUCCUCCGUGGG)融合骨架蛋白mRNA的稳定细胞株(293T/17细胞株)，在常规哺乳动物细胞培养条件下(37℃，5％二氧化碳，100％相对湿度)，待该细胞株与对照细胞(293T/17)生长至细胞汇合度达90％后，将细胞消化下来，800rpm离心，并使用含有0.2μMⅢ-3和0.2μMⅢ-43分子的PBS重悬细胞并孵育5分钟后进行流式检测，检测结果可见图18，Ⅲ-3分子转子在表达了目标RNA的细胞株中可特异性标记骨架蛋白的mRNA，且无明显本底荧光(如图18a)，而Ⅲ-43分子本底荧光高于Ⅲ-3，无法清晰区分ACTB是否表达(如图18b)。

Claims (10)

  1. 一种荧光染料，其结构式如式(Ⅰ)所示，
    Figure PCTCN2020087311-appb-100001
    其中：
    D-为HO-或N(X 1)(X 2)-，X 1、X 2各自独立地选自氢、烷基和改性烷基；X 1，X 2任选相互连接，与N原子一起形成脂杂环；
    R选自氰基、羧基、酰胺基、酯基、亚砜基、砜基、磺酸酯基或磺酰胺基；
    Ar 1和Ar 2各自独立地选自亚芳基，亚杂芳基；其中，Ar 1、Ar 2中的氢原子任选各自独立地被卤原子、羟基、醛基、羧基、酯基、酰胺基、氰基、磺酸基、磷酸基、氨基、伯氨基、仲氨基、烷基或改性烷基取代；
    X 1、X 2任选独立地与Ar 1形成脂杂环；
    其中：
    所述“烷基”各自独立地为C 1-C 10直链或支链烷基；可选地，为C 1-C 7直链或支链烷基；可选地，为C 1-C 5直链或支链烷基；可选地，选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基，1-甲基丁基、2-甲基丁基、3-甲基丁基、异戊基、1-乙基丙基、新戊基、正己基、1-甲基戊基、2-甲基戊基、3-甲基戊基、异己基、1,1-二甲基丁基、2,2-二甲基丁基、3,3-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、正庚基、2-甲基己基、3-甲基己基、2,2-二甲基戊基、3,3-二甲基戊基、2,3-二甲基戊基、2,4-二甲基戊基、3-乙基戊基或2,2,3-三甲基丁基；
    所述“改性烷基”各自独立地为烷基的任意碳原子被选自卤原子、-OH、-CO-、-O-、-CN、-S-、-SO 2-、-(S＝O)-、叠氮基、伯氨基、仲氨基、叔氨基、季铵盐基的一种或多种基团置换所得的基团，所述改性烷基具有1-10个碳原子，其中碳碳单键任选独立地被碳碳双键或碳碳三键置换；
    所述的碳原子被置换，是指碳原子或碳原子与其上的氢原子一起被相应的基团置换；
    所述“卤原子”各自独立地为F、Cl、Br或I；
    所述“脂杂环”为环上含有N、O、S或Si中的一种或多种杂原子的饱和或
    不饱和的4～15元单环或多环脂杂环，所述脂杂环上含有S原子时，其为-S-、-SO-或-SO 2-；所述脂杂环任选被卤原子、烷基、芳基或改性烷基取代；
    所述“亚芳基”为5～13元单环或双环或稠合双环或稠合多环的亚芳香基团；
    所述“亚杂芳基”为环上含有选自N、O、S或Si中的一种或多种杂原子的5～13元单环或双环或稠合双环或稠合多环的亚杂芳香基团；
    所述“酯基”为R'(C＝O)OR”基团；
    所述“酰胺基”为R'CONR”R”'基团；
    所述“磺酸基”为R'SO 3H基团；
    所述“磺酸酯基”为R'SO 2OR”基团；
    所述“磺酰胺基”为R'SO 2NR”R”'基团；
    所述“磷酸基”为R'OP(＝O)(OH) 2基团；
    所述“砜基”为R'SO 2R”基团；
    所述“亚砜基”为R'SOR”基团；
    所述“伯氨基”为R'NH 2基团；
    所述“仲氨基”为R'NHR”基团；
    所述“叔氨基”为R'NR”R”'基团；
    所述“季铵盐基”为R'R”R”'R””N +基团；
    各个R'、R”、R”'、R””各自独立地为单键、氢、烷基、亚烷基、改性烷基或改性亚烷基；
    所述“亚烷基”为C 1-C 10的直链或支链的亚烷基；可选地，为C 1-C 7直链或支链亚烷基；可选地，为C 1-C 5直链或支链亚烷基；
    所述“改性亚烷基”为C 1-C 10(优选为C 1-C 6)亚烷基的任意碳原子被选自-O-、-OH、-CO-、-CS-、-(S＝O)-中的基团置换所得的基团。
  2. 根据权利要求1所述的荧光染料，其特征在于，
    所述“改性烷基”为含有选自-OH、-O-、乙二醇单元、单糖单元、-O-CO-、-NH-CO-、-SO 2-O-、-SO-、Me 2N-、Et 2N-、-S-S-、-CH＝CH-、F、Cl、Br、I、氰基中的一种或多种基团。
  3. 根据权利要求1或2所述的荧光染料，其特征在于，
    Ar 1和Ar 2各自独立地为选自下式(Ⅱ-1)～(Ⅱ-22)中的结构：
    Figure PCTCN2020087311-appb-100002
  4. 根据权利要求1-3任一项所述的荧光染料，其特征在于，式(I)所示的化合物选自下式化合物：
    Figure PCTCN2020087311-appb-100003
    Figure PCTCN2020087311-appb-100004
  5. 制备权利要求1-4任一项所述的荧光染料的方法，其特征在于，包括式(a)化合物与式(b)化合物发生羟醛缩合反应的步骤，
    Figure PCTCN2020087311-appb-100005
  6. 权利要求1-4任一项所述的荧光染料在粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
  7. 权利要求1-4任一项所述的荧光染料在制备用于粘度测试、蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
  8. 一种荧光激活点亮型探针，其包括权利要求1-4任一项所述的荧光染料。
  9. 权利要求8所述的荧光激活点亮型探针在蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测中的用途，所述用途为非用于疾病的诊断方法的用途。
  10. 权利要求8所述的荧光激活点亮型探针在制备用于蛋白荧光标记、核酸荧光标记、蛋白定量或检测、或者核酸定量或检测的试剂中的用途。
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DATABASE REGISTRY 10 August 2001 (2001-08-10), ANONYMOUS: "3,5-Pyridinedicarbonitrile, 2,4-diamino-6-[1-cyano-2-[4-[methyl(3- methylbutyl)amino]phenyl]ethenyl]- (CA INDEX NAME)", XP055858751, retrieved from STN Database accession no. 351065-38-8 *
DATABASE REGISTRY 17 February 2005 (2005-02-17), ANONYMOUS: "Benzeneacetonitrile, .alpha.-[(3-chloro-5-ethoxy-4- hydroxyphenyl)methylene]-4-cyano- (CA INDEX NAME)", XP055858741, retrieved from STN Database accession no. 832683-13-3 *
DATABASE REGISTRY 2 June 2008 (2008-06-02), ANONYMOUS: "Benzoic acid, 4-[2-(3-bromo-4-hydroxy-5-methoxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858729, retrieved from STN Database accession no. 1024709-74-7 *
DATABASE REGISTRY 2 June 2008 (2008-06-02), ANONYMOUS: "Benzoic acid, 4-[2-(3-bromo-5-ethoxy-4-hydroxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858733, retrieved from STN Database accession no. 1024672-75-0 *
DATABASE REGISTRY 20 July 2004 (2004-07-20), ANONYMOUS: "Benzeneacetonitrile, .alpha.-[[3-bromo-4-(dimethylamino)phenyl]methylene]- 4-cyano- (CA INDEX NAME)", XP055858750, retrieved from STN Database accession no. 713104-90-6 *
DATABASE REGISTRY 23 February 2005 (2005-02-23), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-[4-(diethylamino)-2-methoxyphenyl]ethenyl]- (CA INDEX NAME)", XP055858738, retrieved from STN Database accession no. 835894-06-9 *
DATABASE REGISTRY 24 April 2018 (2018-04-24), ANONYMOUS: "- Benzeneacetonitrile, 4-cyano-.alpha.-[(4-hydroxy-3,5- diiodophenyl)methylene]- (CA INDEX NAME)", XP055858707, retrieved from STN Database accession no. 2218151-87-0 *
DATABASE REGISTRY 24 April 2018 (2018-04-24), ANONYMOUS: " Benzeneacetonitrile, 4-cyano-.alpha.-[(4-hydroxy-3-iodo-5- methoxyphenyl)methylene]- (CA INDEX NAME)", XP055858713, retrieved from STN Database accession no. 2217738-28-6 *
DATABASE REGISTRY 24 April 2018 (2018-04-24), ANONYMOUS: "Benzeneacetonitrile, 4-cyano-.alpha.-[(3,5-dibromo-4- hydroxyphenyl)methylene]- (CA INDEX NAME)", XP055858708, retrieved from STN Database accession no. 2217743-67-2 *
DATABASE REGISTRY 24 January 2006 (2006-01-24), ANONYMOUS: "Benzoic acid, 4-[2-[3-bromo-4-(dimethylamino)phenyl]-1-cyanoethenyl]- (CA INDEX NAME)", XP055858736, retrieved from STN Database accession no. 872546-69-5 *
DATABASE REGISTRY 25 April 2018 (2018-04-25), ANONYMOUS, XP055858697, retrieved from STN Database accession no. 2219015-05-9 *
DATABASE REGISTRY 25 April 2018 (2018-04-25), ANONYMOUS: "Benzeneacetonitrile, .alpha.-[(3-bromo-4-hydroxyphenyl)methylene]-4-cyano- (CA INDEX NAME)", XP055858698, retrieved from STN Database accession no. 2218889-27-9 *
DATABASE REGISTRY 25 April 2018 (2018-04-25), ANONYMOUS: "Benzeneacetonitrile, .alpha.-[(3-bromo-5-ethoxy-4-hydroxyphenyl)methylene]- 4-cyano- (CA INDEX NAME)", XP055858706, retrieved from STN Database accession no. 2218860-32-1 *
DATABASE REGISTRY 25 April 2018 (2018-04-25), ANONYMOUS: "Benzeneacetonitrile, 4-cyano-.alpha.-[(3-ethoxy-4-hydroxy-5- iodophenyl)methylene]- (CA INDEX NAME)", XP055858701, retrieved from STN Database accession no. 2218864-24-3 *
DATABASE REGISTRY 26 April 2018 (2018-04-26), ANONYMOUS: " Benzoic acid, 4-[1-cyano-2-[4-hydroxy-3-methoxy-5-(2-propen-1- yl)phenyl]ethenyl]- (CA INDEX NAME)", XP055858687, retrieved from STN Database accession no. 2219463-03-1 *
DATABASE REGISTRY 26 April 2018 (2018-04-26), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(2,3-dibromo-4-hydroxy-5-methoxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858694, retrieved from STN Database accession no. 2219461-36-4 *
DATABASE REGISTRY 26 April 2018 (2018-04-26), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(3-ethoxy-4-hydroxy-5-iodophenyl)ethenyl]- (CA INDEX NAME)", XP055858685, retrieved from STN Database accession no. 2219482-61-6 *
DATABASE REGISTRY 26 April 2018 (2018-04-26), ANONYMOUS: "Benzoic acid, 4-[2-(3-chloro-5-ethoxy-4-hydroxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858692, retrieved from STN Database accession no. 2219461-41-1 *
DATABASE REGISTRY 26 April 2018 (2018-04-26), ANONYMOUS: "Benzoic acid, 4-[2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-1- cyanoethenyl]- (CA INDEX NAME)", XP055858684, retrieved from STN Database accession no. 2219483-47-1 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(2,3-dibromo-5-ethoxy-4-hydroxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858665, retrieved from STN Database accession no. 2220839-27-8 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(3-ethoxy-4-hydroxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858659, retrieved from STN Database accession no. 2220911-46-4 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(4-hydroxy-3-iodo-5-methoxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858663, retrieved from STN Database accession no. 2220883-25-8 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(4-hydroxy-3-iodophenyl)ethenyl]- (CA INDEX NAME)", XP055858661, retrieved from STN Database accession no. 2220885-30-1 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(4-hydroxy-3-methoxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858663, retrieved from STN Database accession no. 2220833-71-4 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(4-hydroxy-3-methoxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858677, retrieved from STN Database accession no. 2220685-06-1 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(4-hydroxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858669, retrieved from STN Database accession no. 2220836-02-0 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[2-(2-bromo-5-ethoxy-4-hydroxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858668, retrieved from STN Database accession no. 2220836-50-8 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[2-(3-bromo-4-hydroxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858671, retrieved from STN Database accession no. 2220834-27-3 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[2-(3-bromo-5-chloro-4-hydroxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858680, retrieved from STN Database accession no. 2220684-82-0 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[2-(3-chloro-4-hydroxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858666, retrieved from STN Database accession no. 2220838-14-0 *
DATABASE REGISTRY 27 April 2018 (2018-04-27), ANONYMOUS: "Benzoic acid, 4-[2-[3-bromo-4-(diethylamino)phenyl]-1-cyanoethenyl]- (CA INDEX NAME)", XP55858657, retrieved from STN Database accession no. 2221047-68-1 *
DATABASE REGISTRY 29 April 2018 (2018-04-29), ANONYMOUS: "Benzeneacetonitrile, .alpha.-[(3-bromo-4-hydroxy-5- methoxyphenyl)methylene]-4-cyano- (CA INDEX NAME)", XP055858643, retrieved from STN Database accession no. 2221753-28-0 *
DATABASE REGISTRY 29 April 2018 (2018-04-29), ANONYMOUS: "Benzeneacetonitrile, 4-cyano-.alpha.-[(4-hydroxy-3- methoxyphenyl)methylene]- (CA INDEX NAME)", XP055858645, retrieved from STN Database accession no. 2221651-87-0 *
DATABASE REGISTRY 29 April 2018 (2018-04-29), ANONYMOUS: "Benzoic acid, 4-[2-(2-bromo-4-hydroxy-5-methoxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME) ", XP055858652, retrieved from STN Database accession no. 2221154-44-3 *
DATABASE REGISTRY 29 April 2018 (2018-04-29), ANONYMOUS: "Benzoic acid, 4-[2-(3-bromo-4-hydroxy-5-iodophenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858651, retrieved from STN Database accession no. 2221523-52-8 *
DATABASE REGISTRY 3 February 2005 (2005-02-03), ANONYMOUS: "Benzeneacetonitrile, .alpha.-[(3-chloro-4-hydroxy-5- methoxyphenyl)methylene]-4-cyano- (CA INDEX NAME)", XP055858747, retrieved from STN Database accession no. 824974-06-3 *
DATABASE REGISTRY 3 June 2008 (2008-06-03), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(3,5-dibromo-4-hydroxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858722, retrieved from STN Database accession no. 1025124-96-2 *
DATABASE REGISTRY 3 June 2008 (2008-06-03), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(3,5-dichloro-4-hydroxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858725, retrieved from STN Database accession no. 1024752-75-7 *
DATABASE REGISTRY 4 June 2008 (2008-06-04), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-[3-ethoxy-4-hydroxy-5-(2-propen-1- yl)phenyl]ethenyl]- (CA INDEX NAME)", XP055858715, retrieved from STN Database accession no. 1025322-92-2 *
DATABASE REGISTRY 4 June 2008 (2008-06-04), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-[4-(diethylamino)phenyl]ethenyl]- (CA INDEX NAME)", XP055858717, retrieved from STN Database accession no. 1025246-06-3 *
DATABASE REGISTRY 4 June 2008 (2008-06-04), ANONYMOUS: "Benzoic acid, 4-[2-(3-chloro-4-hydroxy-5-methoxyphenyl)-1-cyanoethenyl]- (CA INDEX NAME)", XP055858720, retrieved from STN Database accession no. 1025227-78-4 *
DATABASE REGISTRY 4 March 2014 (2014-03-04), ANONYMOUS, retrieved from STN Database accession no. 156223-81-2 *
DATABASE REGISTRY 4 March 2014 (2014-03-04), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-(2,4-dihydroxyphenyl)ethenyl]-, 1-methylethyl ester (CA INDEX NAME)", XP055858763, retrieved from STN Database accession no. 1562326-45-7 *
DATABASE REGISTRY 4 March 2014 (2014-03-04), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-(4-hydroxyphenyl)ethenyl]-, 1-methylethyl ester (CA INDEX NAME)", XP055858755, retrieved from STN Database accession no. 1562283-43-5 *
DATABASE REGISTRY 4 March 2014 (2014-03-04), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-[4-(dimethylamino)phenyl]ethenyl]-, 1-methylethyl ester (CA INDEX NAME)", XP055858758, retrieved from STN Database accession no. 1562311-50-5 *
DATABASE REGISTRY 5 June 2008 (2008-06-05), ANONYMOUS: "Benzoic acid, 4-[1-cyano-2-(4-hydroxy-3,5-dimethoxyphenyl)ethenyl]- (CA INDEX NAME)", XP055858714, retrieved from STN Database accession no. 1025666-91-4 *
DATABASE REGISTRY 6 March 2014 (2014-03-06), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-(2,4-dihydroxyphenyl)ethenyl]-, methyl ester (CA INDEX NAME)", XP055858791, retrieved from STN Database accession no. 1563471-00-0 *
DATABASE REGISTRY 6 March 2014 (2014-03-06), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-(4-hydroxy-3-methoxyphenyl)ethenyl]-, 1-methylethyl ester (CA INDEX NAME)", XP055858798, retrieved from STN Database accession no. 1563491-13-3 *
DATABASE REGISTRY 6 March 2014 (2014-03-06), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-(4-hydroxy-3-methoxyphenyl)ethenyl]-, methyl ester (CA INDEX NAME)", XP055858775, retrieved from STN Database accession no. 1563251-01-3 *
DATABASE REGISTRY 6 March 2014 (2014-03-06), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-(4-hydroxyphenyl)ethenyl]-, methyl ester (CA INDEX NAME)", XP055858778, retrieved from STN Database accession no. 1563281-43-5 *
DATABASE REGISTRY 6 March 2014 (2014-03-06), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-[4-(diethylamino)phenyl]ethenyl]-, methyl ester (CA INDEX NAME)", XP055858783, retrieved from STN Database accession no. 1563300-10-6 *
DATABASE REGISTRY 6 March 2014 (2014-03-06), ANONYMOUS: "2-Furancarboxylic acid, 5-[1-cyano-2-[4-(dimethylamino)phenyl]ethenyl]-, methyl ester (CA INDEX NAME)", XP055858789, retrieved from STN Database accession no. 1563438-71-0 *
DATABASE REGISTRY 7 February 2005 (2005-02-07), ANONYMOUS: "Benzeneacetonitrile, 4-cyano-.alpha.-[(4-hydroxyphenyl)methylene]- (CA INDEX NAME)", XP055858743, retrieved from STN Database accession no. 827011-74-5 *
E.FEYTMANS-DE MEDICIS : "Constantes d'acidité, à l'état excité, des a-cyanostilbènes et des a-cyanodiphénylazomethines paradiméthylamines - [Acidity Constants in the Excited State of p-Dimethylamino=α-Cyanostilbenes and α-Cyanodiphenylazomethines] ", BULLETIN DE LA CLASSE DES SCIENCES, ACADEMIE ROYALE DE BELGIQUE, vol. 53, no. 11, 31 December 1967 (1967-12-31), pages 1259 - 1265, XP009531157, ISSN: 0001-4141 *
E.FEYTMANS-DE MEDICIS ET AL.: "Spectrographic Detection Method for Active Methylenes and the Synthesis of Some Styrene, Cyanostilbene, and Cyanazomethine Derivatives. X. Kinetics of Stilbene and Azomethine Condensations", BULLETIN DES SOCIETES CHIMIQUES BELGES, vol. 75, no. 11-12, 31 December 1966 (1966-12-31), XP009531156, ISSN: 0037-9646 *
E.FEYTMANS-DE MEDICIS: "Acid-Base Equilibria of 4'-Dimethylamino-α-Cyanostilbene and Derivatives. II. m- and p-Cyano Compounds = Équilibre acide-base du diméthylamino-4',alpha-cyanostilbène et de ses dérivés de substitution. II. Dérives méta et para-cyanes", BULLETIN DES SOCIETES CHIMIQUES BELGES, vol. 72, 31 December 1963 (1963-12-31), pages 642 - 643, XP009531155, ISSN: 0037-9646 *
G. S'HEEREN ET AL.: "Synthesis of Frequency Doubling Nonlinear Optical Polymers, Functionalized with Aminocyano- and Alkoxycyano-Stilbene Dyes. Second Harmonic Generation in Corona Poled Thin Films", EUROPEAN POLYMER JOURNAL, vol. 30, no. 7, 31 July 1994 (1994-07-31), XP024052622, ISSN: 0014-3057, DOI: 20200621135645X *
H.MUSTROPH : "UV/VIS Spectral Behavior of Azo Dyes. IX. The Absorption Behavior of Azo Dyes of 4’-Diethylamino-α-Cyanostilbene-4-Diazonium Ions", JOURNAL FUER SIGNALAUFZEICHNUNGS MATERIALIEN, vol. 12, no. 2, 31 December 1984 (1984-12-31), pages 115 - 120, XP009531154, ISSN: 0323-598X *
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