WO2021244345A1 - 一种花氰类衍生化合物及其制备方法和用途 - Google Patents

一种花氰类衍生化合物及其制备方法和用途 Download PDF

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WO2021244345A1
WO2021244345A1 PCT/CN2021/095509 CN2021095509W WO2021244345A1 WO 2021244345 A1 WO2021244345 A1 WO 2021244345A1 CN 2021095509 W CN2021095509 W CN 2021095509W WO 2021244345 A1 WO2021244345 A1 WO 2021244345A1
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formula
compound
reaction
cyanine derivative
substituted
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陈知行
杨中天
李柳菊
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南京浦海景珊生物技术有限公司
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Priority to US17/789,659 priority Critical patent/US20230174479A1/en
Priority to EP21816753.4A priority patent/EP4079723A4/en
Publication of WO2021244345A1 publication Critical patent/WO2021244345A1/zh

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    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/56—Ring systems containing three or more rings
    • C07D209/58—[b]- or [c]-condensed
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

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  • the invention belongs to the field of biological analysis, and specifically relates to a cyanide derivative compound, a preparation method thereof, and application in mitochondrial biological imaging analysis and flow cytometry analysis.
  • Mitochondria are organelles that exist in most eukaryotic cells and are essential energy generators for cells. They also play an important role in other cellular processes (such as cell signal transduction and apoptosis). Mitochondria have a double-layer membrane structure, present different shapes in different cell types and different stages of the cell cycle, and have more complex interactions with other organelles. Therefore, the study of mitochondria depends on the development of live cell imaging and analysis technologies. , And live cell mitochondrial microscopic imaging relies on the development of new mitochondrial fluorescent probes and new microscopic imaging techniques.
  • the present invention proposes a cyanide derivative compound as a mitochondrial fluorescent probe to solve the current problems of mitochondrial fluorescent dyes of high toxicity, high cost and poor imaging effect.
  • n is an integer of at least 1;
  • k is an integer of 1-3;
  • R 1 , R 2 , R 3 , R 4 represent hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted aryl, ester or sulfonic acid group, or R 1 , R 2 , R 3 , R 4 and any two adjacent substituents together with the carbon atoms to which they are connected form an unsubstituted or substituted aliphatic ring, aromatic ring or heteroaromatic ring, and the substituents are selected from: Halogen, alkyl, alkoxy;
  • X is selected from —O— or —NH—
  • Z is selected from —C(O)—or —(CH 2 ) m —;
  • n is an integer of 1-6;
  • Y — is an anion with biocompatibility.
  • the cyanide derivative compound of the present invention has the structure of formula (1a):
  • n is an integer of 1-20, preferably an integer of 1-10, and more preferably an integer of 1-6.
  • R 1 , R 2 , R 3 , R 4 independently represent hydrogen, halogen, substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkoxy, substituted or unsubstituted
  • the C 6-30 aryl group, C 1-20 ester group or sulfonic acid group, or any two adjacent substituents in R 1 , R 2 , R 3 , and R 4 together with the carbon atom to which they are connected A substituted or substituted C 3-10 aliphatic ring, a C 6-30 aromatic ring or a C 1-30 heteroaromatic ring, and the substituent is selected from the group consisting of halogen, C 1-20 alkyl, and C 1-20 alkoxy.
  • R 1 , R 2 , R 3 , R 4 independently represent hydrogen, halogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 1-6 alkoxy, substituted or unsubstituted
  • the C 6-18 aryl group, C 1-6 ester group or sulfonic acid group, or any two adjacent substituents in R 1 , R 2 , R 3 , and R 4 together with the carbon atom to which they are connected A substituted or substituted C 3-10 aliphatic ring, a C 6-18 aromatic ring or a C 1-18 heteroaromatic ring, and the substituent is selected from the group consisting of halogen, C 1-6 alkyl, and C 1-6 alkoxy.
  • the aryl group shown is selected from phenyl.
  • Z represents —C(O)—(carbonyl).
  • Z represents —(CH 2 ) m —, and m is an integer of 1-3.
  • Y - is a bromide, chloride or acetate ions.
  • the cyanine derivatives of the present invention are preferably one of the following structures:
  • the cyanide derivatives of the present invention are preferably selected from one of the following compounds:
  • the present invention also proposes a preparation method of the cyanide-derived mitochondrial fluorescent probe.
  • the compound of formula (2) is used as a raw material, and the compound of formula (4) is obtained through an alkylation reaction with the compound of formula (3), and the compound of formula (5) is then reacted with the compound of formula (5).
  • the compound undergoes condensation reaction to obtain the compound of formula (6), and finally the compound of formula (6) undergoes the removal of the acetyl group and the esterification reaction with cyclooctatetraene carboxylic acid to obtain the bicyclooctatetraene derivatized cyanine compound, that is, the formula (1 ) Compound, as shown in the following reaction scheme:
  • the reaction solvent of the alkylation reaction is acetonitrile or toluene.
  • the reaction temperature of the alkylation reaction is 100-150°C, more preferably 110°C.
  • the molar ratio of the compound of formula (2) to the compound of formula (3) is 1:1.5.
  • the reaction solvent of the condensation reaction is acetic anhydride
  • the catalyst is sodium acetate
  • the reaction temperature of the condensation reaction is 100-150°C, more preferably 140°C.
  • the molar ratio of the compound of formula (4) to the compound of formula (5) is 2:1.
  • the compound of formula (4) is subjected to the removal of the acetyl group in methanol and sodium hydroxide.
  • esterification reaction is catalyzed by 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and triethylamine conduct.
  • HATU 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • the reaction solvent of the esterification reaction is DMF.
  • the reaction temperature of the esterification reaction is 15-40°C, more preferably room temperature.
  • the molar ratio of the compound of formula (6) to cyclooctatetraenecarboxylic acid in the esterification reaction is 1:2.5.
  • the alkylation reaction temperature is 110°C
  • the reaction solvent is acetonitrile or toluene
  • the reaction is carried out in a sealed tube.
  • the molar ratio of the compound of formula (2) to the compound of formula (3) is 1:1.5.
  • the solvent for the condensation reaction is acetic anhydride
  • the catalyst is sodium acetate
  • the reaction temperature is 140°C.
  • the molar ratio of the compound of formula (4) to the compound of formula (5) is 2:1.
  • the compound of formula (4) is deacetylated in methanol-sodium hydroxide, and then in 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate
  • the ester (HATU) and triethylamine are catalyzed by the esterification reaction with cyclooctatetraene carboxylic acid.
  • the reaction solvent is DMF, and the reaction temperature is room temperature.
  • the molar ratio of the compound of formula (6) to cyclooctatetraenecarboxylic acid is 1:2.5.
  • the present invention also proposes the application of the compound of formula (1) as a fluorescent marker for mitochondria.
  • the compound of formula (1) can be incubated into living cells (including subcultured cells such as HeLa cells and primary cells such as cardiomyocytes).
  • living cells including subcultured cells such as HeLa cells and primary cells such as cardiomyocytes.
  • fluorescence is emitted in the presence of excitation light, which serves as a probe for live cell fluorescence imaging analysis or flow cytometry analysis.
  • Typical application steps include:
  • the probe concentration is 50-250 nM, and the incubation time is 10-20 min.
  • the phototoxicity of the probes 1-4, 1-5, 1-6 of the present invention and the existing commercial probes was determined by measuring the cell survival rate after continuous irradiation of the cells under high light intensity. The results show that the probes of the present invention are relatively The existing commercial mitochondrial probes MTR CMXRos and MTDR have significantly lower phototoxicity. Compared with the existing commercial mitochondrial probes MTR CMXRos and MTDR, the phototoxicity of the probes 1-4, 1-5, 1-6 of the present invention is 5 times lower than that of non-optimized compounds of MTR CMXRos, MTDR and 1-6 about.
  • the present invention has the following advantages:
  • the present invention has less toxicity and phototoxicity to cells, the overall biocompatibility is optimal, and it can allow more harsher conditions, longer time, and stronger light intensity. High imaging experiment.
  • the present invention can reduce cell damage caused by the toxicity and phototoxicity of previous commercial dyes, and maintain the physiological activity of the sorted cells.
  • Figure 1 is the normalized absorption and emission spectra of the compounds of formula (1-4) and formula (1-5) synthesized in Example 1 of the present invention in methanol;
  • Figure 2 is a comparison data chart of the phototoxicity of the compound of formula (1-4) and the commercial dye MitoTracker Red CMXRos (MTR CMXRos) on HeLa cells;
  • Figure 3 is a comparison diagram of the phototoxicity of the compound of formula (1-5) and the commercial dye MitoTracker Deep Red FM (MTDR) on HeLa cells.
  • MTDR MitoTracker Deep Red FM
  • Figure 4 is a co-localization experiment of the compound of formula (1-4) and formula (1-5) and the commercial dye MitoTracker green FM on the mitochondrial labeling of HeLa cells;
  • Figure 5 is a comparison diagram of the phototoxicity of compounds of formula (1-4) and formula (1-5) and commercial dyes on rat cardiomyocytes;
  • Figure 6 is the experimental comparison data of 3D-laser confocal time series imaging of rat cardiomyocytes labeled with the compound of formula (1-4) and the commercial dye MTR CMXRos;.
  • Figure 7 is a comparative data diagram of Hessian-SIM super-resolution mitochondrial imaging of COS7 cells labeled with the compound of formula (1-5) and the commercial dye MTDR;
  • the compound of formula (7) is prepared by the same method as above, 100 mg of the compound of formula (7) and 50 mg of N-(3-(phenylamino)allyl)aniline hydrochloride are mixed in 5 ml of acetic anhydride The reaction mixture was heated to 110° C. and stirred for 2 hours. The reaction mixture was spin-dried and purified by HPLC to obtain 71 mg of the compound of formula (9).
  • Figure 1 is the normalized spectra of the absorption and emission of the compounds of formula (1-4) and formula (1-5) in methanol;
  • Example 1-1 The compound of formula (7) in the preparation method of Example 1-1 was replaced with a compound of formula (7-1) to prepare a compound of formula (1-6), namely probe 1-6.
  • the HeLa cells were stained with 250nM probe 1-4 or probed 1-5 and commercial dye MitoTracker Green FM for 15 minutes. After the dye was washed off, the co-localization detection was performed. The results show that, as shown in Figure 4, the present invention has excellent co-localization with commercial dyes and can effectively mark mitochondria.
  • the HeLa cells were irradiated with corresponding LED light (using the same light intensity for the same channel) for different times under a wide-field fluorescence microscope.
  • the irradiated cells were placed in a cell incubator and incubated at 37°C for 2 hours.
  • the cells were then stained with propidium iodide (PI) for viability counts.
  • PI propidium iodide
  • the rat cardiomyocytes were stained with 250 nM of the present invention or commercial dye for 15 minutes, and then the dye was washed off, and the cells were covered with medium again.
  • the cells were continuously irradiated and imaged with the corresponding laser. Analyze the imaging results and count the time of irreversible contraction of cardiomyocytes. The results show that, as shown in Figure 5, probes 1-4 and 1-5 of the present invention are about five times less phototoxic than commercial dyes in rat cardiomyocytes.
  • the probe 1-6 of the present invention and its control compound of formula (1-6-1) are also subjected to phototoxicity comparison determination according to the above steps.
  • the results show that the probe 1-6 of the present invention has the same characteristics as the probe 1-4 and the probe Needles 1-5 have similar effects and also have lower phototoxicity.
  • the phototoxicity data are listed in the table below.
  • FIG. 6 is an experimental comparison of 3D-laser confocal time series imaging of rat cardiomyocytes labeled with probes 1-5 and commercial dye MTDR. Commercial dye-labeled cells violently shrink and die after 8 time series, while the present invention is labeled The same process of the cells occurred after 23 time series.
  • Figure 7 is a comparison of Hessian-SIM super-resolution mitochondrial imaging of COS7 cells labeled with probes 1-4 and commercial dye MTDR.
  • the MTDR-labeled mitochondria have been severely deformed and rounded at 200 frames of imaging, and the similar process of probes 1-4 is still less severe than MTDR after 2000 frames of imaging.

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Abstract

本发明公开一种花氰类衍生化合物及其制备方法和用途，该化合物具有式(1)的结构，并且公开了该化合物的制备方法，本发明系列化合物可作为线粒体的荧光标记物进行活细胞成像分析或者流式细胞分析的用途，解决目前线粒体荧光染料毒性大、成本高和成像效果差的问题。

Description

一种花氰类衍生化合物及其制备方法和用途
本申请要求于2020年06月02日提交中国专利局、申请号为202010492298.8，方面名称为“一种花氰类衍生化合物及其制备方法和用途”的中国专利申请优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物分析领域，具体涉及一种花氰类衍生化合物、其制备方法及其在线粒体生物成像分析和流式细胞分析中的应用。
背景技术
线粒体是一种存在于绝大多数真核细胞中的细胞器，是细胞必不可少的能量发生器，同时也在其它细胞过程(如细胞信号转导和细胞凋亡)中发挥重要作用。线粒体具有双层膜结构，在不同细胞类型和细胞周期的不同阶段中呈现不同的形状，并且和其它细胞器进行较为复杂的相互作用，因此，对线粒体的研究依赖于活细胞成像和分析技术的发展，而活细胞线粒体显微成像又依赖于新型线粒体荧光探针和新型显微成像技术的发展。近年来，随着超分辨成像技术应用于生命科学研究，各种超分辨显微镜，如STED、SIM和STORM显微镜已经被用于活体线粒体成像，取得了一系列成果，同时也凸显了现有的商用线粒体荧光染料的一些缺陷。
目前主流的商业可得线粒体荧光染料，以Molecular
Figure PCTCN2021095509-appb-000001
公司开发的Mito
Figure PCTCN2021095509-appb-000002
系列染料(US6291203)为代表，多为花氰类(cyanine)结构衍生物，染料依靠本身带有的正电荷在线粒体中累积，并在成像条件下产生荧光。而在超分辨成像或者更为敏感的细胞成像当中，这些染料往往在高光强下产生大量三线态，诱导产生活性氧物种(ROS)，进而干扰细胞正常的生理活动，在线粒体成像当中，表现为线粒体变形、变圆和膨胀，导致这些染料在活细胞成像中的应用受到制约。因此，超分辨活细胞成像对线粒体荧光染料提出了更高的要求，现有的线粒体染料存在毒性大、成本高和成像效果差等多种缺陷，无法满足条件更为苛刻、时间更长、光强更高的成像实验。
发明内容
针对现有技术的以上缺陷，本发明提出一种花氰衍生化合物作为线粒体荧光探针，解决目前线粒体荧光染料毒性大、成本高和成像效果差的问题。
本发明的目的是通过以下技术方案实现的，本发明具有式(1)的结构：
Figure PCTCN2021095509-appb-000003
其中，
n为至少为1的整数；
k为1-3的整数；
R 1，R 2，R 3，R 4表示氢，卤素，取代或未取代的烷基，取代或未取代的烷氧基，取代或未取代的芳基，酯基或者磺酸基，或者R 1，R 2，R 3，R 4中任意两个相邻的取代基与它们所连接的碳原子一起形成未取代或取代的脂肪环、芳环或杂芳环，所述取代基选自：卤素、烷基、烷氧基；
X选自—O—或—NH—；
Z选自—C(O)—或—(CH 2) m—；
m为1-6的整数；
Y —是具有生物相容性的阴离子。
优选地，本发明花氰类衍生化合物具有式(1a)的结构：
Figure PCTCN2021095509-appb-000004
优选地，n为1～20的整数，优选为1～10的整数，更优选为1～6的整数。
优选地，R 1，R 2，R 3，R 4独立地表示氢、卤素、取代或未取代的C 1-20烷基、取代或未取代的C 1-20烷氧基、取代或未取代的C 6-30芳基、C 1-20酯基或者磺酸基，或者R 1，R 2，R 3，R 4中任意两个相邻的取代基与它们所连接的碳原子一起形成未取代或取代的C 3-10脂肪环、C 6-30 芳环或C 1-30杂芳环，所述取代基选自：卤素、C 1-20烷基、C 1-20烷氧基。
优选地，R 1，R 2，R 3，R 4独立地表示氢，卤素，取代或未取代的C 1-6烷基，取代或未取代的C 1-6烷氧基，取代或未取代的C 6-18芳基，C 1-6酯基或者磺酸基，或者R 1,R 2,R 3,R 4中任意两个相邻的取代基与它们所连接的碳原子一起形成未取代或取代的C 3-10脂肪环、C 6-18芳环或C 1-18杂芳环，所述取代基选自：卤素、C 1-6烷基、C 1-6烷氧基。
优选地，所示芳基选自苯基。
优选地，Z表示—C(O)—(羰基)。
优选地，Z表示—(CH 2) m—，m为1-3的整数。
优选地，Y —是溴离子，氯离子或乙酸根离子。
本发明所述花氰类衍生物优选为下述结构之一：
Figure PCTCN2021095509-appb-000005
其中Y —如前述定义。
本发明所述花氰类衍生物优选自如下化合物之一：
Figure PCTCN2021095509-appb-000006
本发明还提出了所述花氰衍生线粒体荧光探针的制备方法，以式(2)化合物为原料，经过与式(3)化合物烷基化反应得到式(4)化合物，再与式(5)化合物进行缩合反应得到式(6)化合物，最后式(6)化合物进行乙酰基的脱除以及与环辛四烯甲酸的酯化反应得到双环辛四烯衍生化花氰化合物，即式(1)化合物，如下反应路线所示：
Figure PCTCN2021095509-appb-000007
其中，当k＝1时，式(5)所示化合物为原甲酸三乙酯；当k＝2或3时，化合物(5)为：
Figure PCTCN2021095509-appb-000008
优选地，所示烷基化反应的反应溶剂为乙腈或甲苯。
优选地，所述烷基化反应的反应温度为100-150℃，更优选为110℃。
优选地，所述烷基化反应中，式(2)化合物与式(3)化合物的摩尔比为1:1.5。
优选地，所述缩合反应的反应溶剂是乙酸酐，催化剂是乙酸钠。
优选地，所述缩合反应的反应温度为为100-150℃，更优选为140℃。
优选地，所述缩合反应中，式(4)化合物与式(5)化合物的摩尔比为2:1。
优选地，将式(4)化合物在甲醇和氢氧化钠中进行所述乙酰基的脱除。
优选地，所述酯化反应在2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)与三乙胺催化下进行。
优选地，所述酯化反应的反应溶剂为DMF。
优选地，所述酯化反应的反应温度为15-40℃，更优选为室温。
优选地，所述酯化反应中式(6)化合物与环辛四烯甲酸的摩尔比为1:2.5。
具体包括：
(1)烷基化反应
所述烷基化反应温度为110℃，反应溶剂为乙腈或甲苯，反应在封管中进行。反应中式(2)化合物与式(3)化合物的摩尔比为1:1.5。
(2)缩合反应
缩合反应的溶剂是乙酸酐，催化剂是乙酸钠，反应温度为140℃。反应中式(4)化合物与式(5)化合物的摩尔比为2:1。
(3)脱乙酰基与酯化反应
式(4)化合物在甲醇-氢氧化钠中脱去乙酰基，然后在2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)与三乙胺催化下与环辛四烯甲酸进行酯化反应。反应溶剂为DMF，反应温度为室温。酯化反应中式(6)化合物与环辛四烯甲酸的摩尔比为1:2.5。
本发明还提出了式(1)所述的化合物作为线粒体的荧光标记物的应用，式(1)化合物可经孵育进入活细胞(包括传代培养细胞如HeLa细胞和原代细胞如心肌细胞)的线粒体当中，在激发光存在时发出荧光，从而作为活细胞荧光成像分析或者流式细胞分析的探针。
典型的应用步骤包括：
(1)细胞与探针的孵育
配制一定浓度的探针在细胞培养基当中的溶液，以此溶液在标准细胞培养条件下孵育细胞一定时间。孵育一定时间后去掉探针溶液，以新培养基洗去残余。
(2)进行成像实验或流式分析实验
其中在步骤(1)中探针浓度为50-250nM，孵育时间为10-20min。
通过在高光强下持续照射细胞后测定细胞存活率测定了本发明所述探针1-4、1-5、1-6和已有商业探针的光毒性，结果表明本发明所述探针相对于已有商业线粒体探针MTR CMXRos和MTDR具有显著更低的光毒性。与已有商业线粒体探针MTR CMXRos和MTDR相比，本发明探针1-4、1-5、1-6的光毒性与MTR CMXRos、MTDR和1-6的非优化化合物相比低5倍左右。
相比于现有技术，本发明的有益之处在于：
(1)、通过双保护基团的三线态猝灭作用，显著减少各种显微成像过程中显微镜光源经由染料三线态产生的毒性物种，最接近真实地呈现细胞本身的生理活动。
(2)、与已有的商业线粒体探针相比，本发明对细胞的毒性和光毒性均更小，整体生物相容性最优，可以允许更多条件更苛刻、时间更长、光强更高的成像实验。
(3)、在以线粒体为荧光标记物的流式细胞分析和分选中，本发明可以减小以往商业染料的毒性和光毒性造成的细胞损伤，维持被分选细胞的生理活性。
(4)、原料易得，成本低，反应条件较为简单，具备使其商业化的优势。
附图说明
图1是本发明实施例1中合成的式(1-4)和式(1-5)的化合物在甲醇中的吸收和发射归一化光谱；
图2是式(1-4)的化合物与商业染料MitoTracker Red CMXRos(MTR CMXRos)对HeLa细胞的光毒性对比数据图；
图3是式(1-5)的化合物与商业染料MitoTracker Deep Red FM(MTDR)对HeLa细胞的光毒性对比图。
图4是式(1-4)和式(1-5)的化合物与商业染料MitoTracker green FM对HeLa细胞线粒体标记的共定位实验；
图5是式(1-4)和式(1-5)的化合物与商业染料对大鼠心肌细胞造成的光毒性对比图；
图6是式(1-4)的化合物和商业染料MTR CMXRos标记的大鼠心肌细胞3D-激光共聚焦时间序列成像的实验对比数据；。
图7是式(1-5)的化合物和商业染料MTDR标记的COS7细胞的Hessian-SIM超分辨线粒体成像的对比数据图；
具体实施方式
实施例1荧光探针的合成
所有对水和空气敏感的反应均在氮气气氛、无水条件下进行。通过薄层色谱层析法(TLC，GF254)监测反应，使用紫外光以及磷钼酸和硫酸铈的乙醇溶液作为可视化剂。如果没有特别说明，使用硅胶快速柱色谱法分离化合物。使用Bruker Advance 400(1H 400MHz)核磁共振波谱仪测量化合物的核磁共振谱，并使用残留的未氘化溶剂进行校准(在1H NMR中7.26ppm中为氘代氯仿；3.31ppm为甲醇-d 4)。以下缩写用于解释多重性：s＝单重峰，d＝双重峰，t＝三重峰，q＝四重峰，m＝多重峰，br＝宽峰。使用Acquity I class UPLC synapt G2-SI和ESI(电喷雾电离)获得质谱数据。
实施例1-1、当本发明的通式(1)中，n＝2，k＝1，X为O，R 1-R 4为氢时，本实施例的化合物结构为式(1-4)，即探针1-4，其制备方法如下：
取7.1克2-溴乙醇和3克2,3,3-三甲基吲哚混合溶于50毫升DMF中，于110℃加热搅拌12小时，反应混合物冷却至室温后有白色固体析出，抽滤得到白色固体并乙醚洗涤得到2.5克纯度为46％结构为式(7)的化合物。
1H NMR(400MHz,Methanol-d4)δ7.90–7.82(m,1H),7.82–7.74(m,1H),7.70–7.59(m,2H),4.70–4.63(t,J＝5.1Hz,2H,),4.08–4.01(t,J＝5.1Hz,2H),1.62(s,6H).
Figure PCTCN2021095509-appb-000009
取200毫克式(7)的化合物与133毫克原甲酸三乙酯在5毫升乙酸酐中加热至110℃，搅拌反应2小时，反应混合物旋干后用HPLC纯化，得到180mg式(8)的化合物。
1H NMR(400MHz,Methanol-d 4)δ8.60(t,J＝13.4Hz,1H),7.58–7.51(m,2H),7.45(ddd,J＝8.3,7.1,1.2Hz,2H),7.44–7.37(m,2H),7.32(td,J＝7.3,1.2Hz,2H),6.60(d,J＝13.4Hz,2H),4.60–4.54(m,4H),4.54–4.46(m,4H),1.83(s,6H),1.77(s,12H).
13C NMR(101MHz,Methanol-d 4)δ175.63,170.84,151.24,142.20,140.66,128.46,125.49,122.14,111.21,102.84,60.23,49.39,43.22,26.81,19.16.
HRMS(ESI)calcd for C31H37N2O4+[M+]501.2748,found 501.2753.
Figure PCTCN2021095509-appb-000010
取30毫克式(8)化合物溶于2毫升甲醇，加入15毫克氢氧化钠，搅拌反应2小时。旋蒸得到固体，该固体与50毫克2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，10毫克环辛四烯甲酸(COTCOOH)和20微升的三乙胺在DMSO中室温混合反应12小时，反应混合物以水稀释，用二氯甲烷萃取三次，以HPLC纯化得到12mg式(1-4)的化合物。
1H NMR(400MHz,Methanol-d4)δ8.59(t,J＝13.4Hz,1H),7.56(d,J＝7.5Hz,2H),7.51–7.38(m,4H),7.35(t,J＝7.4Hz,2H),6.88(s,1H),6.53(d,J＝13.4Hz,2H),5.95–5.66(m,12H),4.65(t,J＝5.0Hz,4H),4.54(t,J＝5.2Hz,4H),1.78(s,12H).
13C NMR(101MHz,Chloroform-d)δ174.60,165.25,151.16,143.54,142.44,140.28,133.94,132.94,132.62,132.02,131.35,130.04,129.30,128.88,125.45,122.02,111.30,104.52,60.96,49.15,43.37,28.09.
HRMS(ESI)calcd for C 45H 45N 2O 4 +[M +]677.3374,found 677.3377。
Figure PCTCN2021095509-appb-000011
实施例1-2、当本发明的通式(1)中，n＝2，k＝2，X为O时，本实施例的化合物结构为式(1-5)，探针1-5，其制备方法如下
采用上述同样的方法制得式(7)的化合物，取100毫克式(7)化合物与50毫克N-(3-(苯基氨基)亚烯丙基)苯胺盐酸盐在5毫升乙酸酐中加热至110℃，搅拌反应2小时，反应混合物旋干后用HPLC纯化，得到71mg式(9)的化合物。
1H NMR(400MHz,Methanol-d 4)δ8.32(t,J＝13.0Hz,2H),7.49(dd,J＝7.5,1.2Hz,2H),7.42(td,J＝7.7,1.2Hz,2H),7.34(d,J＝7.9Hz,2H),7.27(td,J＝7.4,1.0Hz,2H),6.65(t,J＝12.4Hz,1H),6.39(d,J＝13.7Hz,2H),4.52(t,J＝5.1Hz,4H),4.42(t,J＝5.1Hz,4H),1.85(s,6H),1.73(s,12H).
13C NMR(101MHz,Methanol-d 4)δ174.30,170.85,154.67,144.93,142.28,141.11,128.23,124.97,122.03,110.70,103.40,60.26,49.30,42.84,26.43,19.16.
HRMS(ESI)calcd for C 33H 39N 2O 4 +[M +]527.2904,found 527.2915
Figure PCTCN2021095509-appb-000012
取91毫克式(9)化合物溶于5毫升甲醇，加入45毫克氢氧化钠，搅拌反应2小时。旋蒸得到固体，该固体与50毫克2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HATU)，13毫克环辛四烯甲酸(COTCOOH)和30微升的三乙胺在DMSO中室温混合反应12小时，反应混合物以水稀释，用二氯甲烷萃取三次，以HPLC纯化得到17mg式(1-5)的化合物。
1H NMR(400MHz,Methanol-d 4)δ8.30(t,J＝13.1Hz,2H),7.50(d,J＝7.5Hz,2H),7.43(t,J＝7.7Hz,2H),7.35(d,J＝8.0Hz,2H),7.29(t,J＝7.4Hz,2H),6.89(s,2H),6.61(t,J＝12.4Hz,1H),6.39(d,J＝13.7Hz,2H),5.92–5.70(m,12H),4.62(t,J＝5.0Hz,4H),4.49(t,J＝5.1Hz,4H),1.74(s,12H).
13C NMR(101MHz,Chloroform-d)δ173.97,165.30,153.95,143.97,142.01,141.09,134.29,133.38,132.46,132.03,131.44,129.94,129.02,128.59,126.57,125.37,122.32,60.61,49.61,43.01,27.87.
HRMS(ESI)calcd for C 47H 47N 2O 4 +[M+]703.3530,found 703.3520.
Figure PCTCN2021095509-appb-000013
图1是为式(1-4)和式(1-5)的化合物在甲醇中的吸收和发射归一化光谱；
实施例1-3的制备
将实施例1-1制备方法中式(7)的化合物替换为式(7-1)的化合物，制得式(1-6)的化合物，即探针1-6。
Figure PCTCN2021095509-appb-000014
1H NMR(400MHz,Chloroform-d)δ8.68(t,J＝13.1Hz,1H),8.12(d,J＝8.4Hz,qH),7.94(dd,J＝17.4,8.5Hz,4H),7.64(ddd,J＝8.3,6.8,1.3Hz,2H),7.56(d,J＝13.2Hz,2H),7.51(ddd,J＝8.0,6.9,1.0Hz,2H),7.46(d,J＝8.8Hz,2H),6.83(s,2H),5.78(s,1H),5.69–5.55(m,12H),5.32((d,J＝13.2Hz,4H),4.84(d,J＝13.2Hz,4H),2.06(s,12H).
HRMS(ESI C 53H 49N 2O 4 +found 777.5654.
同时合成了作为光毒性实验对照的式(1-6-1)的化合物：
Figure PCTCN2021095509-appb-000015
HRMS(ESI C 49H 45N 2O 4 +found 725.4713.
实施例2与已有商业探针的共定位实验
将HeLa细胞用250nM探针1-4或探针化1-5与商业染料MitoTracker Green FM进行染色15分钟，洗去染料之后进行共定位检测。结果表明，如图4所示，本发明与商业染料有极好的共定位，能对线粒体进行有效标记。
实施例3对本发明的探针和已有商业探针的光毒性测定
将HeLa细胞在宽场荧光显微镜下，用对应LED光照射细胞(对相同通道使用相同光强)不同的时间。将照射之后的细胞置于细胞培养箱中37℃下孵育2小时。之后将细胞用碘化丙啶(PI)进行染色，进行存活率计数。结果表明，如图2和图3所示实施例1中的两种探针1-4，探针1-5在HeLa细胞中比商业染料光毒性低五倍左右。
将大鼠心肌细胞用250nM本发明或者商业染料染色15分钟之后洗去染料，重新用培养基覆盖细胞，在高通量成像系统中，用对应激光对细胞进行持续照射与成像。对成像结果进行分析，统计心肌细胞发生不可逆收缩的时间。结果表明，如图5所示，本发明探针1-4,探针1-5在大鼠心肌细胞中比商业染料光毒性低五倍左右。
本发明探针1-6，和其对照式(1-6-1)化合物也按照上述步骤进行光毒性比对测定，结果显示，本发明探针1-6具有与探针1-4和探针1-5相似的效果，也具有较低的光毒性，光毒性数据列于下表中。
  1分钟照射细胞存活率 5分钟照射细胞存活率 15分钟照射细胞存活率
探针1-4 99.0％ 94.0％ 48.9％
MitoTracker Red CMXRos 86.9％ 0％ 0％
探针1-5 100％ 86％ 69％
MitoTracker Deep Red FM 100％ 0％ 0％
探针1-6 100％ 99％ 95％
1-6-1 100％ 95％ 25％
实施例4使用本探针对细胞进行成像实验
将目的细胞用250nM本发明染料进行染色10-15分钟，洗去染料之后加入相应的培养基，之后进行不同的显微成像实验。图6是以探针1-5和商业染料MTDR标记的大鼠心肌细胞3D-激光共聚焦时间序列成像的实验对比，商业染料标记的细胞在8个时间序列后剧烈收缩死亡，而本发明标记的细胞的同样过程发生在23个时间序列后。
图7是以探针1-4和商业染料MTDR标记的COS7细胞的Hessian-SIM超分辨线粒体成像的对比。MTDR标记的线粒体在200帧成像时已经剧烈形变，变圆，而探针1-4的类似过 程在成像2000帧后程度仍比MTDR剧烈程度较轻。

Claims (8)

  1. 一种花氰类衍生物，其特征在于具有式(1)的结构：
    Figure PCTCN2021095509-appb-100001
    其中，
    X选自—O—或—NH—；
    Z选自—C(O)—或—(CH 2) m—；
    Y —是具有生物相容性的阴离子；
    R 1，R 2，R 3，R 4选自氢、卤素、取代或未取代的C 1-20烷基、取代或未取代的C 1-20烷氧基、取代或未取代的C 6-30芳基、C 1-20酯基或者磺酸基，或者R 1，R 2，R 3，R 4中的任意两个相邻基团与其所连接的碳原子共同形成未取代或取代的C 3-10脂肪环、C 6-30芳环或C 1-30杂芳环，其中所述取代基选自卤素、烷基、烷氧基；
    n为不小于1的整数；
    k为1-3的整数；
    m为1-6的整数。
  2. 如权利要求1所述的一种花氰衍生物，其特征在于Z选自—C(O)—；
  3. 如权利要求1或2所述的一种花氰类衍生物，其特征在于R 1，R 2，R 3，R 4均选自氢，n＝2，X为—O—，Y为Br；
  4. 如权利要求1或2所述的一种花氰类衍生物，其特征在于R 1,R 2均选自氢，R 3，R 4与它们所连接的碳原子形成苯基，Y为Br；
  5. 如权利要求1所述的一种花氰类衍生物的制备方法，其特征在于，该制备方法包含如下步骤：
    步骤1、以式(2)化合物为原料，经过与式(3)化合物烷基化反应得到式(4)化合物；
    Figure PCTCN2021095509-appb-100002
    步骤2、式(4)化合物与式(5)化合物进行缩合反应得到式(6)化合物；
    Figure PCTCN2021095509-appb-100003
    其中，当k＝1时，式(5)所示化合物为原甲酸三乙酯；当k＝2或3时，化合物(5)为：
    Figure PCTCN2021095509-appb-100004
    步骤3、式(6)化合物进行乙酰基的脱除，并与环辛四烯甲酸进行酯化反应得到式(1)的花氰类衍生物；
    Figure PCTCN2021095509-appb-100005
  6. 如权利要求5所述的一种花氰类衍生物的制备方法，其特征在于，所述步骤1的烷基化反应中使用的溶剂是甲苯或者乙腈；所述步骤2的缩合反应中使用的溶剂是乙酸酐，催化剂是 乙酸钠；所述步骤3的酯化反应的催化剂为HATU和三乙胺，溶剂为DMF；
  7. 如权利要求5所述的一种花氰类衍生物的制备方法，其特征在于，所述烷基化反应中式(2)化合物与式(3)化合物的摩尔比为1：1.5；所述缩合反应中式(4)化合物与式(5)化合物的摩尔比为2：1；所述酯化反应中式(6)化合物与环辛四烯甲酸的摩尔比为1：2.5；
  8. 如权利要求1-4中任一项所述的花氰类衍生物作为线粒体的荧光标记物进行活细胞成像分析或者流式细胞分析的用途。
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