A novel fluorescent compound, a preparation method thereof and applications thereof in mitochondrial fluorescence imaging and viscosity detection

Abstract

The application discloses a novel fluorescent compound, a preparation method thereof and applications of the fluorescent compound in mitochondrial fluorescence imaging and viscosity detection, wherein the fluorescent compound is combined with quinoline or naphthyridine and malonitrile to form an electron-withdrawing part, and is connected with an electron-donating auxiliary chromophore 3-formyl-7-diethylaminocoumarin through a carbon-carbon double bond to form a neutral molecule with a typical D-pi-A structure. The fluorescent compound prolongs the conjugation degree of quinoline or naphthyridine to reach the red channel, has an aggregation-induced emission phenomenon, has small cytotoxicity, can be used for labeling mitochondria in biological imaging, and has an obvious viscosity response in optical testing, and can be used for mitochondrial viscosity detection. In addition, the preparation method of the fluorescent compound is simple, and raw materials are easy to obtain, so that the fluorescent compound provides a low-cost option for mitochondrial red fluorescence labeling, and has a good application prospect in mitochondrial fluorescence imaging and mitochondrial viscosity detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis and fluorescent marker preparation, and particularly relates to a novel fluorescent compound, a preparation method thereof and applications thereof in online mitochondrial fluorescence imaging and viscosity detection. BACKGROUND

[0002] The abnormality of intracellular microenvironment (viscosity, polarity, pH, etc.) is related to many physiological processes such as inflammation, neurodegenerative diseases and cancer (see: Chem. Soc. Rev., 2021, 50, 8887-8902). Studies have shown that abnormal changes in the viscosity of cell mitochondria are related to Alzheimer's disease, atherosclerosis and diabetes (see: Anal. Chem. 2019, 91, 10302-10309). Organelle mitochondrial autophagy removes dysfunctional mitochondria; and recycles its components in the lysosomal degradation pathway, which plays a crucial role in maintaining intracellular homeostasis, which will lead to changes in the microenvironment in mitochondria, such as pH, viscosity and polarity. (see: Anal. Chem. 2021, 93, 3241-3249). Since the viscosity in cells is an important indicator of the physiological state of the cell, the viscosity detection in cells is of great significance in diagnosing and tracking physiological processes.

[0003] In the reports so far, based on the twisted intramolecular charge transfer (TICT) mechanism, a large number of small molecule fluorescent viscosity probes have been reported; in a viscous environment, intramolecular rotation is inhibited, so non-radiative decay is weakened, and fluorescence is significantly enhanced (see: Anal. Chem. 2020, 92, (5) 3517-3521). However, there is still a lot of room for improvement in these molecules. First, most of the viscosity probes have a short emission wavelength and cannot reach the near-infrared region; and the viscosity probes that basically reach the near-infrared region are salt compounds, which have the disadvantage of high toxicity; secondly, many viscosity probes are easily interfered by nucleophilic reagents, and the selectivity is not ideal. At present, the viscosity probes with clear organelle targeting are mostly cationic viscosity probes. Taking mitochondria as an example: these probes enter the mitochondria due to the negative charge potential of the mitochondria, which makes it easier for cationic probes to enter the mitochondria, however, this inevitably destroys the microenvironment of the organism (see: Anal. Chem. 2019, 91, (13), 8415-8421).

[0004] In 1989, Deng Qingyun of Kodak first reported the electroluminescent device using pyrromethene derivative (DCM) as red light dye (see: J. Appl. Phys., 1989, 65, 3610-3616). And the red light material in solid form, it is extremely easy to cause fluorescence quenching; although so far, people through the modification of DCM, get a series of substituted derivatives, but its synthesis method is not only cumbersome, and the fluorescence quantum yield is still not high. Fortunately, Professor Tang Benzhong of Hong Kong University of Science and Technology found the compound with aggregation induced emission phenomenon in 2001 (see: Chem. Commun., 2001, 1740-1741). The compound presents weak fluorescence in solution, but has high fluorescence quantum yield in solid state, which provides a new way for the development of solid fluorescent materials. In addition, the near-infrared viscosity probe based on the aggregation induced property is used for imaging of mitochondrial autophagy, and there are few reports in recent years, and basically all are cationic probes, therefore, it is crucial to develop specific near-infrared probes with mitochondrial aggregation induced property to monitor the viscosity in the process of mitochondrial autophagy. SUMMARY

[0005] The technical problem solved by the present application is to provide a new fluorescent compound, a preparation method thereof and an application thereof in mitochondrial fluorescence imaging and viscosity detection. The new fluorescent compound is a neutral molecule with typical D-π-A structure, has small cytotoxicity, has weak fluorescence in solution, has strong fluorescence in aggregation or solid state, has an emission wavelength reaching the near-infrared light region, and can target mitochondria, and has good application prospect in mitochondrial fluorescence imaging and viscosity detection.

[0006] In order to solve the above technical problem, the present application provides the following technical solutions.

[0007] The first aspect of the present application provides a new fluorescent compound, and the fluorescent compound has the following structural general formula:

[0008]

[0009] In the formula, X is C or N.

[0010] The present application uses nitrogen-containing heterocyclic quinoline and [1,8]-naphthyridine and malononitrile to build the electron-withdrawing part of the molecular structure, and connects the electron-donating auxiliary chromophore 3-formyl-7-diethylaminocoumarin through carbon-carbon double bond, to form a neutral molecule with typical D-π-A structure.

[0011] The second aspect of the present application provides a preparation method of the fluorescent compound of the first aspect, comprising the following steps:

[0012] (1) reacting a compound represented by formula (2) with a methylating agent in the presence of a solvent to obtain a compound represented by formula (3);

[0013] (2) reacting a compound represented by formula (3) with malononitrile in the presence of a base reagent and a solvent to obtain a compound represented by formula (4);

[0014] (3) reacting a compound represented by formula (4) with 3-formyl-7-diethylaminocoumarin in the presence of a basic catalyst and a solvent to obtain the fluorescent compound;

[0015] The structures of the above-mentioned formula (2) to formula (4) are as follows:

[0016]

[0017] wherein X is C or N;

[0018] When X is C, the fluorescent compound prepared by the above preparation method is a quinoline nitrile derivative represented by formula (I);

[0019] When X is N, the fluorescent compound prepared by the above preparation method is a naphthyridine nitrile derivative represented by formula (II);

[0020]

[0021] Further, in step (1), the methylating agent is preferably methyl iodide.

[0022] Further, in step (1), the temperature of the reaction is preferably 70-100°C, and the reaction time is preferably 10-15H.

[0023] Further, in step (2), the base reagent is preferably one or more of sodium tert-butoxide, sodium hydride, and potassium tert-butoxide.

[0024] Further, in step (2), the temperature of the reaction is preferably 10-30°C, and the reaction time is preferably 4-6H.

[0025] Further, in step (3), the molar ratio of the compound represented by formula (4) to 3-formyl-7-diethylaminocoumarin is 1:1-1.5, for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc., without being limited to the above-mentioned listed molar ratios.

[0026] Further, in step (3), the basic catalyst is piperidine and / or 4-methylpiperidine.

[0027] Further, in step (3), the temperature of the reaction is 78-130°C, and the reaction time is 6-15H.

[0028] Furthermore, in steps (1) to (3), the solvent is one or more of ethanol, acetonitrile, N,N-dimethylformamide, dimethyl sulfoxide, and N,N-dimethylacetamide.

[0029] The third aspect of the present invention further provides a use of the fluorescent compound described in the first aspect as a fluorescent marker in mitochondrial fluorescence imaging.

[0030] Furthermore, the fluorescent marker is a red fluorescent marker of mitochondria.

[0031] Furthermore, the fluorescent marker is co-cultured with the cells to be tested in a culture medium to perform cell imaging; wherein the concentration of the fluorescent marker in the culture medium is 1 to 3 μM.

[0032] In some preferred embodiments of the present invention, in order to verify whether the fluorescent compound can be used as a fluorescent marker for fluorescent imaging of mitochondria, the cells to be tested are co-cultured with a green commercial marker containing the fluorescent compound and mitochondria in a culture medium, and cell imaging is performed to test the fluorescence intensity of the fluorescent compound and the colocalization coefficient with the green commercial marker.

[0033] The fourth aspect of the present invention provides a use of the fluorescent compound described in the first aspect as a fluorescent probe in detecting mitochondrial viscosity.

[0034] Furthermore, the fluorescent probe is co-cultured with the cells to be tested in a culture medium, and cell imaging is performed to reflect changes in mitochondrial viscosity of the cells to be tested by detecting changes in the fluorescence intensity of the fluorescent probe; wherein the concentration of the fluorescent probe in the culture medium is 1 to 3 μM.

[0035] Furthermore, the co-culture conditions are as follows: the cells to be tested are cultured in a saturated humidity, 37° C., 5% CO 2 incubator for 10 minutes, and then quinolinecarbonitrile or naphthyridinecarbonitrile derivatives are added and co-cultured for 10 minutes.

[0036] In some preferred embodiments of the present invention, in order to verify whether the fluorescent compound can be used to detect mitochondrial viscosity, cells are placed in different culture media containing the fluorescent compound and different concentrations of drugs that induce changes in mitochondrial viscosity, and cell imaging is performed to test the change in fluorescence intensity of the fluorescent compound in the cells before and after the addition of the drug.

[0037] Beneficial effects of the present invention:

[0038] 1.The present application provides a new type of fluorescent compound, first quinoline or [1,8]-naphthyridine containing nitrogen heterocycle and malononitrile through Michael addition elimination to form quinoline nitrile and naphthyridine nitrile derivatives, and then connected with auxiliary chromophore 3-formyl-7-diethylaminocoumarin through Knoevenagel condensation reaction to obtain the target product, which expands the conjugation degree of quinoline nitrile or naphthyridine nitrile to the red channel.

[0039] 2.The fluorescent compound provided by the present application has a typical D-π-A structure, and the structure of the neutral molecule makes it have relatively small cytotoxicity, good light stability, aggregation-induced emission performance, and mitochondrion organelle targeting ability, in addition, the synthesis method of the fluorescent compound is simple, the raw materials are cheap and easy to obtain, and the mitochondrion red fluorescent marker can be prepared at low cost; in addition, the fluorescent compound has obvious viscosity response in optical test, and can be used for detecting the viscosity of mitochondrion in cells, which has important scientific significance and commercial value in organelle labeling and organelle viscosity detection. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 It is a synthesis route of the new fluorescent compound;

[0041] Figure 2 It is the ultraviolet-visible absorption spectrum and fluorescence spectrum of the new fluorescent compound in glycerol and water, respectively; wherein (a) is the ultraviolet-visible absorption spectrum and fluorescence spectrum of quinoline nitrile derivative 1a in glycerol and water, respectively, (b) is the ultraviolet-visible absorption spectrum and fluorescence spectrum of naphthyridine nitrile derivative 1b in glycerol and water, respectively;

[0042] Figure 3 It is the fluorescence emission spectrum of the new fluorescent compound in different proportions of tetrahydrofuran aqueous solution; wherein (a) is the fluorescence emission spectrum of quinoline nitrile derivative 1a in different proportions of tetrahydrofuran aqueous solution, (b) is the fluorescence emission spectrum of naphthyridine nitrile derivative 1b in different proportions of tetrahydrofuran aqueous solution;

[0043] Figure 4 It is the residual absorption rate of quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b after irradiation for different times;

[0044] Figure 5 It is the cell survival rate of cells cultured in culture medium containing different concentrations of quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b;

[0045] Figure 6 It is the cell imaging diagram of the new fluorescent compound in HeLa cells, respectively; wherein (a1-a6) is the cell imaging diagram of quinoline nitrile derivative 1a in HeLa cells, (b1-b6) is the cell imaging diagram of naphthyridine nitrile derivative 1b in HeLa cells.

[0046] Figure 7 Cell imaging images of a new fluorescent compound, quinoline nitrile derivative 1a, in HeLa cells before and after adding a drug to induce cell viscosity changes; wherein (A) is the cell imaging image of quinoline nitrile derivative 1a in HeLa cells before and after adding a drug, (B) is the cell imaging image of naphthyridine nitrile derivative 1b in HeLa cells before and after adding a drug. DETAILED DESCRIPTION

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0048] The present application is further described by the following examples and comparative examples, and with reference to the accompanying drawings. These examples do not, however, limit or define the scope of the application.

[0049] Example 1

[0050] This example relates to the preparation of a new fluorescent compound, naphthyridine nitrile derivative 1a, and the synthetic route is shown in Figure 1 The specific preparation process is as follows:

[0051] (1) 2-methylquinoline (14.9 g, 0.1 mol) and iodomethane (21.2 g, 0.15 mol) were dissolved in 30 mL of acetonitrile and protected with nitrogen, and stirred at 80°C under reflux for 12 h. The reaction progress was monitored using TLC during the reaction. After the reaction was completed, it was cooled to room temperature, and a dark yellow solid was produced, which was collected by suction filtration. The crude product was washed with petroleum ether or ethyl ether to obtain yellow solid N-methyl-2-methylquinoline iodide about 27.0 g, with a yield of 95%.

[0052] (2) Sodium tert-butoxide (7.07 g, 73.6 mmol), malononitrile (4.86 g, 73.6 mmol), acetonitrile (30 mL), N-methyl-2-methylquinoline iodide (5.24 g, 18.4 mmol) were added to a 100 mL flask and stirred at room temperature for 5 h, and then suction filtration was performed to obtain the above compound N-methyl-2-methyl-4-(α,α-dicyano)methylene-1,4-dihydroquinoline, which was a golden yellow solid 3.6 g, with a yield of 71%.

[0053] (3) Compound N-methyl-2-methyl-4-(a,a-dicyano)methylene-l,4-dihydroquinoline (221.28 mg, 1 mmol) was dissolved in 10 mL of ethanol, and 200 μL of piperidine was added thereto. After refluxing at 80°C for 12 hours under a nitrogen atmosphere, the reaction was cooled to room temperature, and the organic solvent was removed by a rotary evaporator. After separation by column chromatography (eluent: dichloromethane), a brownish red solid dye la, 258.1 mg, was obtained at a yield of 54%.

[0054] NMR of quinoline nitrile derivative la 1 H NMR (400 MHz, CDC13) δ (ppm) 9.11 (d, J = 7.3 Hz, 1H, Ar-H), 8.00 (d, J = 15.3 Hz, 1H, Ar-H), 7.78 (t, J = 9.8 Hz, 2H, Ar-H), 7.62 (d, J = 8.2 Hz, 1H, Ar-H), 7.48 (t, J = 7.2 Hz, 1H, Ar-H), 7.36 (d, J = 8.9 Hz, 1H, Ar-H), 7.14 (t, J = 16.4 Hz, 2H, CH=CH), 6.64-6.66 (q, J = 6.5 Hz, 1H, Ar-H), 6.51 (d, J = 2.4 Hz, 1H, Ar-H), 3.93 (s, 3H, N-CH3), 3.44-3.49 (q, J = 6.8 Hz 4H, 2xCH2), 1.23-1.27 (t, J = 7.1 Hz 6H, 2xCH3).

[0055] NMR of quinoline nitrile derivative la 13 CNMR (151 MHz, CDC13) δ (ppm) 160.4, 156.3, 153.3, 151.8, 149.5, 145.5, 139.3, 135.4, 133.0, 129.9, 126.6, 124.6, 121.7, 121.3, 120.4, 119.4, 116.2, 114.4, 109.7, 108.7, 106.8, 96.82, 45.0, 36.8, 12.4.

[0056] HRMS (ESI) of quinoline nitrile derivative la + ) : m / z calcd for C 28 H 25 N4O2 + [M+H] + : 449.1973, found: 449.1978.

[0057] Example 2

[0058] This example relates to the preparation of a novel fluorescent compound, naphthyridine nitrile derivative 1b, the synthetic route is shown below Figure 1 The specific preparation process is as follows:

[0059] (1) 2-methyl-[1,8]-naphthyridine (2.9 g, 20 mmol) and iodomethane (4.3 g, 30 mol) were dissolved in 30 mL of acetonitrile and protected with nitrogen or argon, and the reaction was refluxed at 80°C for 12H under magnetic stirring. The reaction progress was monitored by TLC during the reaction. After the reaction was completed, it was cooled to room temperature, and dark green solid was produced, which was collected by suction filtration. The crude product was washed with petroleum ether or ether to obtain dark green solid 1,2-dimethyl-[1,8]-naphthyridine iodide about 5.8 g, yield 97%.

[0060] (2) Sodium tert-butoxide (7.07 g, 73.6 mmol), malononitrile (4.86 g, 73.6 mmol), acetonitrile (30 mL), 1,2-dimethyl-[1,8]-naphthyridine iodide (5.26 g, 18.4 mmol) were added to a 100 mL flask and stirred at room temperature for 5H, and the above compound 2-(1,2-dimethyl-[1,8]-naphthyridine-4(1H)-ylidene)malononitrile was obtained by suction filtration, which was a dark green solid 2.4 g, yield 58%. The proton nuclear magnetic resonance spectrum of the product 1 HNMR (300 MHz, DMSO-d6) δ (ppm) 9.10 (d, J = 8.6 Hz, 1H, Ar-H), 8.23 (d, J = 7.6 Hz 1H, Ar-H), 7.57 (d, J = 8.5 Hz, 1H, Ar-H), 6.79 (d, J = 7.3 Hz, 1H, Ar-H), 3.94 (s, 3H, N-CH3), 3.71 (s, 3H, CH3). Carbon nuclear magnetic resonance spectrum 13 C NMR (151 MHz, DMSO-d6) δ (ppm) 154.3, 146.2, 133.9, 132.2, 130.4, 124.1, 120.2, 102.7, 44.4, 12.9. HRMS (ESI + ) : m / z calculated value: C 13 H 11 N4 + [M+H] + : 223.0979, found: 223.0977.

[0061] (3) Compound 2-(1,2-dimethyl-[1,8]-naphthyridin-4(1H)-ylidene)malononitrile (4b) (222.25 mg, 1 mmol) was dissolved in 10 mL of ethanol, and 200 μL of piperidine was added thereto. After refluxing at 80 °C for 12 hours under a nitrogen atmosphere, the reaction was cooled to room temperature, and the organic solvent was removed by a rotary evaporator. After separation by column chromatography (eluent: dichloromethane), the brownish red solid dye 1b was obtained in a yield of 218.1 mg, 48%.

[0062] NMR spectrum of naphthyridine nitrile derivative 1b 1 H NMR (300 MHz, CDC13) δ (ppm) 9.47 (d, J = 8.2 Hz, 1H, Ar-H), 8.80 (d, J = 2.8 Hz, 1H, Ar-H), 8.09 (d, J = 15.3 Hz, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.37 (d, J = 8.9 Hz, 2H, Ar-H), 7.20 (d, J = 6.5 Hz, 2H, Ar-H), 6.67 (d, J = 8.0 Hz, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 4.11 (s, 3H, N-CH3), 3.44-3.51 (q, J = 6.8 Hz 4H, 2 x CH2), 1.23-1.28 (t, J = 6.9 Hz 6H, 2 x CH3).

[0063] NMR spectrum of naphthyridine nitrile derivative 1b 13 C NMR (151 MHz, CDC13) δ (ppm) 160.3, 156.4, 152.9, 152.6, 152.0, 150.6, 148.2, 145.8, 135.9, 135.3, 130.0, 121.0, 120.0, 118.7, 116.5, 114.4, 109.8, 108.8, 106.2, 96.8, 51.8, 45.0, 34.1, 12.4.

[0064] HRMS (ESI) of naphthyridine nitrile derivative 1b + : m / z calcd for C 27 H 25 N5O2 + [M+H] + : 450.1925, found: 450.1924.

[0065] Performance test and application

[0066] (1) Fluorescence performance test

[0067] The quinoline nitrile derivative 1a and the naphthyridine nitrile derivative 1b prepared in the above example were added to glycerol and water respectively to prepare a solution with a compound concentration of 10 μM. The ultraviolet absorption and fluorescence emission spectra of the two compounds in different reagents were tested. The test results are as follows: Figure 2 As shown in the figure, the horizontal axis is the wavelength, and the vertical axis is the absorbance (dashed line) or fluorescence intensity (solid line).

[0068] Figure 2 (a) are the ultraviolet absorption and fluorescence emission spectra of quinoline nitrile derivative 1a in glycerol and water, respectively. It can be seen from the figure that quinoline nitrile derivative 1a has maximum absorption at 485nm; under the conditions of excitation wavelength of 485nm and slit width of 5 / 3nm, the emission wavelength corresponding to the highest fluorescence intensity of quinoline nitrile derivative 1a in glycerol is at 620nm, and the emission wavelength in water reaches 700nm, which has reached the near-infrared light region.

[0069] Figure 2 (b) are the ultraviolet absorption and fluorescence emission spectra of naphthyridinium nitrile derivative 1b in glycerol and water, respectively. It can be seen from the figure that naphthyridinium nitrile derivative 1b has maximum absorption at 512nm; under the conditions of excitation wavelength of 512nm and slit width of 5 / 3nm, the emission wavelength corresponding to the highest fluorescence intensity of naphthyridinium nitrile derivative 1b in glycerol is at 650nm, and the emission wavelength in water reaches 720nm, which has reached the near-infrared light region.

[0070] In summary, the quinolinecarbonitrile derivative 1a and the naphthyridinecarbonitrile derivative 1b prepared in the present invention have emission wavelengths in the near-infrared region in both the aqueous phase and the oil phase.

[0071] (2) Aggregation-induced emission performance test

[0072] The quinoline nitrile derivative 1a and the naphthyridine nitrile derivative 1b prepared in the above examples were added to tetrahydrofuran aqueous solution at different ratios to prepare a solution with a compound concentration of 10 μM. The fluorescence emission spectra were tested. The test results are as follows: Figure 3 Therefore, the horizontal axis in the figure is the wavelength and the vertical axis is the fluorescence intensity.

[0073] Figure 3 (a) Fluorescence emission spectra of quinoline nitrile derivative 1a in aqueous tetrahydrofuran solutions at varying water contents (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%), with water acting as a poor solvent and tetrahydrofuran acting as a benign solvent. The figure shows that the fluorescence intensity of quinoline nitrile derivative 1a increases as the water content increases from 0% to 80%, reaching its highest intensity at 80% water content, indicating that quinoline nitrile derivative 1a exhibits aggregation-induced emission. The slit width is 5 / 3 nm.

[0074] Figure 3 (b) The fluorescence emission spectra of naphthyridine nitrile derivative 1b in different proportions of tetrahydrofuran aqueous solution (water content is 0%, 10%, 20%, 30%, 40%, 50%, 60%, respectively), water is a poor solvent, and tetrahydrofuran is a good solvent. As can be seen from the figure, the fluorescence intensity of naphthyridine nitrile derivative 1b increases with the increase of water content from 0% to 60%, and the fluorescence intensity is the highest when the water content is 60%, indicating that naphthyridine nitrile derivative 1b has the performance of aggregation-induced emission, and the slit width is 5 / 3 nm.

[0075] (3) Light stability test

[0076] The light stability test was performed on the quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b prepared in the above examples. The specific operation is as follows: the corresponding mass of quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b was weighed, and the quinoline nitrile or naphthyridine nitrile derivative was respectively dissolved in acetonitrile to prepare a solution with a compound concentration of 10 μM, and all the samples were irradiated with a Philips iodine tungsten lamp (500 W), and the distance between the lamp and the sample was set to 25 cm. An 8 cm thick NaNO2 (60 g / L) cold trap was placed between the lamp and the sample to eliminate heat and short-wavelength light. Continuous irradiation for 6 hours, and every half hour, an ultraviolet fluorescence test was performed, and after six hours, the light stability was calculated according to the residual absorption rate of the absorption intensity after irradiation at different times and the initial absorption intensity.

[0077] The test results are shown in Figure 4 As shown in the table, the residual absorption rate of quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b decreases slowly with the increase of irradiation time, but after continuous irradiation for 6 hours, the residual absorption of quinoline nitrile derivative 1a can still reach 92%, and the residual absorption of naphthyridine nitrile derivative 1b also remains 90% of the initial absorption intensity, so it can be known that the quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b prepared by the present application have relatively good light stability.

[0078] (4) Cytotoxicity test

[0079] The cytotoxicity test was performed on the quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b prepared in the above examples. The CCK-8 method was used to measure the cell survival rate of HeLa cells in the presence of different concentrations of quinoline nitrile derivative 1a or naphthyridine nitrile derivative 1b. The specific operation is as follows: HeLa cells were incubated with different concentrations (0, 2, 4, 6, 8 and 10 μM) of dyes (quinoline nitrile derivative 1a, naphthyridine nitrile derivative 1b) for 6 hours.

[0080] The test results of cytotoxicity are shown in Figure 5 The horizontal axis of the figure represents the concentration of the dye, and the vertical axis represents the cell survival rate of HeLa cells. Among them, the cell survival rate (%) = (Asample –A b ) / (A c –A b ), wherein A c : negative control (including medium and cells, without addition of the dye to be tested), A b : blank (including the dye to be tested and medium, without addition of cells), A sample : test group (including medium, cells and the dye to be tested).

[0081] As can be seen from Figure 5 , the cell survival rate decreases with the increase of the dye concentration, and the cell survival rate is 1a: 90%, 1b: 85% when the concentration of quinoline nitrile derivative 1a or naphthyridine nitrile derivative 1b in the medium is 10 μM after incubation for 6 hours, which shows that the quinoline nitrile and naphthyridine nitrile derivatives prepared in the present application have low cytotoxicity and are suitable for live cell imaging.

[0082] (5) Organelle fluorescence imaging

[0083] a. Targeted imaging of mitochondria by quinoline nitrile derivative 1a

[0084] Quinoline nitrile derivative 1a was configured into a stock solution using DMSO (dimethyl sulfoxide), and then added to the conventional cell culture medium to make the concentration of quinoline nitrile derivative 1a in the cell culture medium 3 μM, and then incubated with HeLa cells in a saturated humidity, 37°C, 5% CO2 incubator for 10 minutes, and then added with commercial mitochondrial green marker Mito Tracker Green C1048 (3 μM) and incubated for another 10 minutes; then washed with PBS buffer for three times, and then cell imaging was performed by laser confocal microscope. The green light channel was excited by 488 nm wavelength, and the fluorescence signal in the range of 500-580 nm was collected. The red light channel was excited by 561 nm wavelength, and the fluorescence signal in the range of 600-800 nm was collected.

[0085] As shown in Figure 6 (a1)-(a5), (a1) is a bright field imaging image, (a2) is a cell imaging image of the commercial mitochondrial green marker, (a3) is a cell imaging image of quinoline nitrile derivative 1a, (a4) is a superimposed image of the green light channel and the red light channel, (a5) is a colocalization experiment, and the colocalization coefficient reaches 0.93, (a6) is the fluorescence intensity of the ROI line in the superimposed image, and the fluorescence intensity of quinoline nitrile derivative 1a labeled mitochondria is similar to that of the commercial mitochondrial green marker. The above cell imaging results show that quinoline nitrile derivative 1a can well label mitochondria in HeLa cells and can be used as a mitochondrial red marker.

[0086] b. Targeted imaging of mitochondria by naphthyridine nitrile derivative 1b

[0087] Naphthyridine nitrile derivative 1b was configured into mother liquor using DMSO (dimethyl sulfoxide), then added into normal cell culture medium, so that the concentration of naphthyridine nitrile derivative 1b in the cell culture medium was 1 μM, and then co-cultured with HeLa cells in a saturated humidity, 37°C, 5% CO2 incubator for 10 minutes, then added with commercial mitochondrial green marker Mito Tracker Green C1048 (3 μM) and cultured for another 10 minutes; then washed with PBS buffer for three times, and then cell imaging was performed using laser confocal microscope. The green light channel was excited at 488 nm wavelength, and the fluorescence signal in the range of 500-580 nm was collected. The red light channel was excited at 561 nm wavelength, and the fluorescence signal in the range of 600-800 nm was collected.

[0088] As shown in (b1)-(b5), (b1) is a bright field imaging image, (b2) is a cell imaging image of commercial mitochondrial green marker, (b3) is a cell imaging image of naphthyridine nitrile derivative 1b, (b4) is an overlay image of green light channel and red light channel, (b5) is a colocalization experiment, and the colocalization coefficient reaches 0.94, (b6) is the fluorescence intensity of ROI line in the overlay image, and the fluorescence intensity of naphthyridine nitrile derivative 1b for labeling mitochondria is similar to that of commercial mitochondrial green marker, but the concentration of naphthyridine nitrile derivative 1b is lower. The above cell imaging results show that naphthyridine nitrile derivative 1b can well label mitochondria in HeLa cells, and the fluorescence intensity is high, so that it can be used as a mitochondrial red marker. Figure 6 (b1)~(b5), (b1) is a bright field imaging image, (b2) is a cell imaging image of commercial mitochondrial green marker, (b3) is a cell imaging image of naphthyridine nitrile derivative 1b, (b4) is an overlay image of green light channel and red light channel, (b5) is a colocalization experiment, and the colocalization coefficient reaches 0.94, (b6) is the fluorescence intensity of ROI line in the overlay image, and the fluorescence intensity of naphthyridine nitrile derivative 1b for labeling mitochondria is similar to that of commercial mitochondrial green marker, but the concentration of naphthyridine nitrile derivative 1b is lower. The above cell imaging results show that naphthyridine nitrile derivative 1b can well label mitochondria in HeLa cells, and the fluorescence intensity is high, so that it can be used as a mitochondrial red marker.

[0089] (6) Organelle viscosity detection

[0090] a. Quinoline nitrile derivative 1a for detecting mitochondrial viscosity

[0091] Quinoline nitrile derivative 1a was configured into mother liquor using DMSO (dimethyl sulfoxide), then added into normal cell culture medium, so that the concentration of quinoline nitrile derivative 1a in the cell culture medium was 6 μM, and then co-cultured with HeLa cells in a saturated humidity, 37°C, 5% CO2 incubator for 10 minutes, then added with ionophore nystatin so that the concentration of nystatin in the cell culture medium was 5 μM, and co-cultured for 30 minutes, then washed with PBS buffer for three times, and then cell imaging was performed using laser confocal microscope. The red light channel was excited at 561 nm wavelength, and the fluorescence signal in the range of 600-800 nm was collected.

[0092] As shown in (b1)-(b5), (b1) is a bright field imaging image, (b2) is a cell imaging image of commercial mitochondrial green marker, (b3) is a cell imaging image of naphthyridine nitrile derivative 1b, (b4) is an overlay image of green light channel and red light channel, (b5) is a colocalization experiment, and the colocalization coefficient reaches 0.94, (b6) is the fluorescence intensity of ROI line in the overlay image, and the fluorescence intensity of naphthyridine nitrile derivative 1b for labeling mitochondria is similar to that of commercial mitochondrial green marker, but the concentration of naphthyridine nitrile derivative 1b is lower. The above cell imaging results show that naphthyridine nitrile derivative 1b can well label mitochondria in HeLa cells, and the fluorescence intensity is high, so that it can be used as a mitochondrial red marker. Figure 7(a1) is bright field imaging, (a2) is cell imaging of quinoline nitrile derivative 1a, (a3) is the superposition of (a1) and (a2); (b1) is bright field imaging, (b2) is cell imaging of quinoline nitrile derivative 1a with nystatin, (b3) is the superposition of (b1) and (b2), (c) is the average fluorescence intensity. The cell imaging results show that the fluorescence intensity of quinoline nitrile derivative 1a is significantly increased after the viscosity of mitochondria is increased by adding nystatin.

[0093] b. Detection of mitochondrial viscosity by naphthyridine nitrile derivative 1b

[0094] Naphthyridine nitrile derivative 1b was configured into a mother liquor using DMSO (dimethyl sulfoxide), then added into the conventional cell culture medium to make the concentration of naphthyridine nitrile derivative 1b in the cell culture medium be 3 μM, and then cultured with HeLa cells in a 37℃, 5% CO2 incubator with saturated humidity for 10 minutes, then ionophore nystatin was added to make the concentration of nystatin in the cell culture medium be 5 μM, and then cultured for 30 minutes, and then washed with PBS buffer for three times, and then cell imaging was performed using a laser confocal microscope. The red light channel was excited using a 561 nm wavelength, and the fluorescence signal in the range of 600-800 nm was collected.

[0095] As shown in Figure 7 (a1) is bright field imaging, (a2) is cell imaging of naphthyridine nitrile derivative 1b, (a3) is the superposition of (a1) and (a2); (b1) is bright field imaging, (b2) is cell imaging of naphthyridine nitrile derivative 1b with nystatin, (b3) is the superposition of (b1) and (b2), (c) is the average fluorescence intensity. The cell imaging results show that the fluorescence intensity of naphthyridine nitrile derivative 1b is significantly increased after the viscosity of mitochondria is increased by adding nystatin.

[0096] In summary, the prepared quinoline nitrile derivative 1a and naphthyridine nitrile derivative 1b can be used for targeting mitochondria and as a fluorescent marker for fluorescent imaging of mitochondria, and the fluorescence intensity of the quinoline nitrile / naphthyridine nitrile derivative after targeting mitochondria is increased with the increase of the viscosity of mitochondria, and thus can be used for detecting the viscosity change of mitochondria.

[0097] The above-described embodiments are only the preferred examples for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by the person skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A fluorescent compound, characterized in that The fluorescent compound has the following general structural formula: ， Wherein, X is C or N; The fluorescent compound has aggregation-induced emission properties.

2. A method for preparing the fluorescent compound according to claim 1, comprising the following steps: (1) reacting the compound represented by formula (2) with iodomethane in the presence of a solvent to obtain the compound represented by formula (3); (2) reacting the compound represented by formula (3) with malononitrile in the presence of an alkaline reagent and a solvent to obtain the compound represented by formula (4); (3) reacting the compound represented by formula (4) with 3-formyl-7-diethylaminocoumarin in the presence of a basic catalyst and a solvent to obtain the fluorescent compound; The structures of the above formulas (2) to (4) are as follows: 、 、 ， Wherein, X is C or N.

3. The preparation method according to claim 2, characterized in that In step (1), the reaction temperature is 70-100°C and the reaction time is 10-15 h.

4. The preparation method according to claim 2, characterized in that In step (2), the alkaline reagent is selected from one or more of sodium tert-butoxide, sodium hydride, and potassium tert-butoxide; the reaction temperature is 10-30°C, and the reaction time is 4-6 hours.

5. The preparation method according to claim 2, characterized in that In step (3), the molar ratio of the compound represented by formula (4) to 3-formyl-7-diethylaminocoumarin is 1:1-1.

5.

6. The preparation method according to claim 2, characterized in that In step (3), the alkaline catalyst is piperidine and / or 4-methylpiperidine; the reaction temperature is 78-130°C, and the reaction time is 6-15 hours.

7. Use of the fluorescent compound according to claim 1 in preparing a fluorescent marker for mitochondrial fluorescence imaging.

8. The use according to claim 7, characterized in that The fluorescent marker is a red fluorescent marker of mitochondria.

9. Use of the fluorescent compound according to claim 1 in preparing a fluorescent probe for detecting mitochondrial viscosity.

10. The use according to claim 9, characterized in that The fluorescent probe and the cells to be tested are co-cultured in a culture medium, and cell imaging is performed. The change in the mitochondrial viscosity of the cells to be tested is reflected by detecting the change in the fluorescence intensity of the fluorescent probe.

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