WO2013139006A1

WO2013139006A1 - Anthrapyridone fluorescent dye n-substituted in position 4, preparation method and use thereof

Info

Publication number: WO2013139006A1
Application number: PCT/CN2012/072715
Authority: WO
Inventors: 彭孝军; 强新新; 樊江莉; 王静云; 宋锋玲; 孙世国
Original assignee: 大连理工大学; 大连科荣生物技术有限公司
Priority date: 2012-03-21
Filing date: 2012-03-21
Publication date: 2013-09-26

Abstract

Disclosed are an anthrapyridone fluorescent dye N-substituted in position 4, the preparation method and use thereof, the compound having a structure as shown in general formula I. In general formula I, n is an integer of 1-10; R is selected from a hydrogen atom, methyl, hydroxyl, amino, dimethylamino, a trimethylammonium halide group and a guanidine hydrochloride group. The anthrapyridone fluorescent dye N-substituted in position 4 in the present invention has a simple synthesis process, good light stability and a relatively long emission wavelength, and has good cell membrane permeability. Thus, another object of the present invention is to provide use of the anthrapyridone fluorescent dye N-substituted in position 4 in the present invention for biological dyeing, such as dyeing in fixed cells, living cells and biological tissues.

Description

4-position N-substituted anthrapyridone fluorescent dye, preparation method and application thereof

The present invention relates to a new class of fluorescent dyes in the field of fine chemicals, a preparation method and application thereof, in particular to a dye of a type of anthrapyridone structure, a preparation method thereof, and the use of the fluorescent dye, and a conjugate thereof or The use of its composition in biological dyeing.

Say

Background technique

Fluorescent dyes have been widely used in various fields of science and technology as functional indicators, especially in the practical applications of life sciences, clinical medical diagnosis, and fluorescent immunoassay, which have attracted wide attention from scientists all over the world. In the field of cell biology, fluorescent dyes are used for the location and content of intracellular components and other changes, and cell identification and classification also rely on flow cytometry with fluorescence technology as the core.

At present, commercial fluorescent dyes such as phenanthridines, acridines, and cyanines play an important role in genomics, nucleic acid detection, and cell analysis. However, these dyes each have their own application limitations. This is mainly manifested in the fact that most fluorescent dyes are limited to fixed cell samples. For example, ethidium bromide (EB), propidium iodide (PI), etc., can be applied to fluorescent labeling of biological samples by cell fixation, etc., but this method tends to be a cell or organism. The true form of the organization has a destructive effect. At the same time, acridine and phenanthridine dyes such as EB have great toxicity and carcinogenic effects.

In addition, some of the fluorescent dyes have an excitation wavelength in the ultraviolet region. Due to the high energy of ultraviolet light, the biological components such as nucleic acids and proteins in the cells are damaged. At the same time, long-term photoexcitation also causes photobleaching of fluorescent dyes. . These all limit the use of such dyes in fluorescence imaging. In addition, when ultraviolet light is used as the excitation wavelength, the absorption of the biological sample itself in this interval makes it difficult for ultraviolet light to penetrate the inside of the biological tissue sample, and some biological molecules in the biological sample are excited by ultraviolet light to generate a strong fluorescent background. To make fluorescence detection difficult. Therefore, it is required to develop a fluorescent dye having good fluorescence properties and living cell permeability. This is the key to the development of disciplines such as fluorescence detection technology and life sciences. Summary of the invention

The object of the first aspect of the present invention is to provide a compound which is simple for synthesis, good in photostability, long in emission wavelength, and has good cell membrane permeability for fixing cells, living cells and biological tissues. The 4-position N-substituted anthrapyridone fluorescent dye of the present invention has the following structural formula I:

In the formula I, n is an integer from 1 to 10;

R is selected from the group consisting of a hydrogen atom, a methyl group, an ethyl group, a hydroxyl group, an amino group, a dimethylamino group, a diethylamino group, a dipropylamino group, a dibutylamino group, a dihydroxyethylamino group, a trimethylammonium halide group, and a guanidinium hydrochloride group.

Among them, the ammonium trimethylammonium halide is ammonium trimethyl ammonium iodide, aluminum trimethyl ammonium chloride or ammonium trimethyl ammonium fluoride.

Wherein R is preferably dimethylamino, diethylamino, dipropylamino, dibutylamino, dihydroxyethylamino, trimethylammonium halide, amino and guanidinium hydrochloride. Particularly preferred are diethylamino, dipropylamino, dibutylamino, dihydroxyethylamino, trimethylammonium iodide and guanidinium hydrochloride. n is preferably an integer of 2-6.

As one of the preferred technical solutions, the 4-position N-substituted anthrapyridone fluorescent dye of the present invention is C, D or F, wherein: C specificity for fluorescent staining of living cells, D-specificity for living cells Outer membrane fluorescent staining, D-specificity staining of living cell Golgi apparatus.

In a further preferred embodiment, the 4-position N-substituted anthrapyridone fluorescent dye is A, E or G, and is used for specificity to fluorescent staining of living cells nucleoli and lysosomal.

In another preferred embodiment, the 4-position N-substituted anthrapyridone fluorescent dye is B for specificity to fluorescent staining of fixed cell nucleoli.

Another object of the present invention is to provide a method for preparing the above 4-substituted N-substituted anthrapyridone fluorescent dye: using the compound of the formula i and the compound of the formula ii as a raw material, reacting in the presence of a catalyst and an acid binding agent to prepare the formula I compound of;

In the formula i, X is F, Cl, Br or I;

Wherein the catalyst is a monovalent copper salt, a divalent copper salt or an iodide salt;

The reaction solvent is selected from the group consisting of water, hydrazine, hydrazine-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethylene glycol monomethyl ether, and ethylene glycol.

Where, corresponding to the compound,

In the formula ii, n is an integer of 1 to 10, preferably an integer of 2 to 6; R is a hydrogen atom, a methyl group, a hydroxyl group, an amino group, Dimethylamino, diethylamino, dipropylamino, dibutylamino, dihydroxyethylamino, trimethylammonium or guanidine hydrochloride, preferably a trimethylammonium halide, an amino group or a guanidine hydrochloride group. Ammonium trimethyl iodide, an amino group or a guanidinium hydrochloride group is particularly preferred.

In the preparation method of the 4-position N-substituted anthrapyridone fluorescent dye, X is preferably Br.

In the method for producing a 4-position N-substituted anthrapyridone fluorescent dye of the present invention, the catalyst is a monovalent copper salt, a divalent copper salt or an iodide salt. Preference is given to cuprous iodide, copper sulphate, copper acetate, potassium iodide or copper iodide. Copper iodide, copper sulfate or copper iodide is particularly preferred.

In the preparation method of the 4-position N-substituted anthrapyridone fluorescent dye of the present invention, the acid binding agent is an organic base or an inorganic base, including triethylamine, trimethylamine, potassium carbonate, sodium carbonate, sodium hydrogencarbonate, acetic acid. Sodium, sodium hydroxide or potassium hydroxide. Preference is given to potassium carbonate or sodium carbonate.

The 4-position N-substituted anthrapyridone fluorescent dye according to the present invention has a simple synthesis method, good light stability, long emission wavelength, and good cell membrane permeability, and therefore, the object of still another aspect of the present invention The use of the above-described 4-position N-substituted anthrapyridone fluorescent dye of the present invention for biological staining can be used for staining of fixed cells, living cells and biological tissues. DRAWINGS

Figure 7 of the accompanying drawings, wherein:

Figure 1 is a laser confocal fluorescence imaging image of HeLa cells of dye A;

Figure 2 is a laser confocal fluorescence imaging image of HeLa cells of dye B;

Figure 3 is a laser confocal fluorescence imaging image of HeLa cells of dye C;

Figure 4 is a laser confocal fluorescence imaging image of HeLa cells of dye D;

Figure 5 is a laser confocal fluorescence imaging image of HeLa cells of dye E;

Figure 6 is a laser confocal fluorescence imaging image of dyed F and commercial Golgi fluorescent dye NBD C6-ceramide counterstained HeLa cells. The left side of the picture is the staining result of dye F, and the picture on the right is the NBD C6-ceramide staining result;

Figure 7 is a laser confocal fluorescence imaging image of HeLa cells of dye G. Detailed ways

The following non-limiting examples are provided to enable a person of ordinary skill in the art to understand the invention, but not to limit the invention in any way. Example 1. Synthesis of Dye A and Live Cell Fluorescence Imaging

(1) Synthesis of dye A

4-bromo-N-methylpyridinone (13 g, 38.2 mmol), ethylenediamine (5 mL, 83.3 mmol), potassium carbonate (5.3 g, 38.2 mmol) and anhydrous copper sulfate (1.0 g 6.3 mmol) Dissolved in 150 mL of ethylene glycol methyl ether, refluxing for 20 h, TLC detection material 4-bromomethylpyridinone, filtered to remove insolubles, the filtrate was evaporated under reduced pressure to give a dark red solid. The ester was washed (3 x 50 mL) and dried to give the desired product l lO g, yield 89%.

¹ H NMR (400 MHz, DMSO) δ 10.47 (t, J = 9.6 Hz, 1H, NH), 8.55 (d, J = 7.9 Hz, 1H, ArH), 8.36 (d, / = 7.7 Hz, 1H, ArH ), 8.23 (t, J = 6.0 Hz, 1H, NH), 7.94 (d, / = 9.7 Hz, 1H, ArH), 7.87 - 7.70 (m, 3H, ArH, CH), 7.55 (d, J = 9.7 Hz, 1H, ArH), 5.69 (s, 1H, CH), 5.35 (s, 1H, CH), 3.77 (s, 3H, CH ₃ ), 3.66 - 3.55 (m, 2H, CH ₂ ), 3.42 (dt , J = 11.5, 5.6 Hz, 2H, CH ₂ ), 1.88 (s, 3H, CH ₃ ). MS (TOF MS ES+) calculated for [C ₁₉ H ₁₈ N ₃ 0 ₂ ] ⁺ : 320.1394, measured: 320.1388.

(2) Observation of the staining of living cells HeLa by compound A under confocal laser scanning microscopy:

Compound A was added to the cultured HeLa cells in a 2 mL cell culture medium to a final concentration of 10 μM. Incubate for 1 hour in a 37 V, 5% CO 2 cell incubator. Then, 0.01 M of PBS was vortexed and rinsed for 5 minutes χ3, and then added to the medium, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, excited by a 514 nm channel, and the receiving wavelength was 560-620 nm, which was observed three times with an oil mirror (ΙΟΟΟχ). Figure 1 is a fluorescence micrograph of Compound A staining live cells HeLa. As shown in the figure, Compound A was stained for nucleoli and lysosomes of HeLa cells. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm. Example 2. Synthesis of Dye B and Live Cell Fluorescence Imaging

(1) Synthesis of dye B

Dye B is synthesized in a similar manner to Dye A, and the main raw materials used are 4-bromomethylpyridinone and ethanolamine. Crude product silica gel column separation, yield: 83%

1H NMR (400 MHz, DMSO) δ 10.53 (s, 1H, NH), 8.53 (d, / = 3.2 Ηζ, ΙΗ, ), 8.37 (d, J = 5.6 Ηζ, ΙΗ, ArH), 7.76-7.94 (m , 4H, ArH, CH), 7.41 (d, / = 3·2 Ηζ, ΙΗ), 5.02 (s, 1H), 3.74 (m, 5H), 3.51 (s, 2H).MS (TOF MS ES+) calculated For [C ₁₉ H ₁₇ N ₂ 0 ₃ ]+: 321.1234, measured: 321.1238.

(2) Observation of the staining of living cells HeLa by compound B under confocal laser scanning microscopy:

Compound B was added to the cultured HeLa cells in 2 mL of cell culture medium to a final concentration of 5 μM. Incubate for 30 minutes in a 37 ° C, 5% CO 2 cell incubator. Then, 0.01 M of PBS was vortexed and rinsed for 5 minutes x3, and then added to the medium, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, excited by a 514 nm channel, and the receiving wavelength was 560-620 nm, which was observed three times with an oil mirror (ΙΟΟΟχ). Figure 2 is a fluorescence micrograph of Compound B staining of living cells HeLa. Compound B was specifically stained for nucleoli in HeLa cells as shown. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm. Example 3. Synthesis of Dye C and Live Cell Fluorescence Imaging

(1) Synthesis of dye C

The synthesis method of dye C is similar to dye A, and the main raw materials used are 4-bromomethylpyridinone and hydrazine, hydrazine-two. Methyl propylene diamine. The crude product was isolated on silica gel column, yield: 60%.

1H NMR (400 MHz, CDC13) δ 10.53 (s, 1H, H), 8.52 (d, J = 6.8 Hz, 1H, ArH), 8.24 (d. J = 7.8 Hz, 1H, ArH), 7.82 - 7.62 ( m, 4H, ArH, CH), 7.30 (d, 1H, ArH), 3.88 (s, 3H, CH3), 3.52 (dd, J = 12.5, 6.8 Hz, 2H, CH2), 2.53 (t, J = 7.0 Hz, 2H, CH2), 2.33 (s, 6H, CH3), 2.01 (dd, J = 14.1, 7.0 Hz, 2H, CH2). MS (TOF MS ES+) calculated for [C22H24N302] ⁺ : 362.1863, measured: 362.1869 .

(2) Observation of the staining of living cells HeLa by compound C under confocal laser scanning microscopy:

Compound C was added to the cultured HeLa cells in 2 mL of cell culture medium to a final concentration of 5 μM. Incubate for 30 minutes in a 37 ° C, 5% CO 2 cell incubator. Then, 0.01 M of PBS was vortexed and rinsed for 5 minutes x3, and then added to the medium, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, excited by a 514 nm channel, and the receiving wavelength was 560-620 nm, which was observed three times with an oil mirror (ΙΟΟΟχ). Figure 3 is a fluorescence micrograph of Compound C staining live cells HeLa. As shown in the figure, Compound C was specifically stained for nucleoli of HeLa cells. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm. Example 4. Synthesis of Dye D and Live Cell Fluorescence Imaging

( 1 )

The synthetic starting material of Dye D was Dye A and Compound d (CAS: 4338-95-8) prepared in Example 1. 1 g of dye A, 0.68 g of compound d, dissolved in 50 mL of absolute ethanol, refluxed for 5 hours, TLC test material A, the solvent was distilled off under reduced pressure, and the obtained red solid was washed with 5% aqueous sodium carbonate, and then It was washed with distilled water until neutral, and dried under vacuum to give a dye D 0.9 g, yield 72%.

1H NMR (400 MHz, DMSO) δ 10.46 (s, 2H), 8.58 (s, H), 8.36 (s, H), 7.91-7.78 (m, 3H), 7.60 (s, H), 7.46 (s, H), 3.77 (d, J = 12.1 Hz, 2H), 3.63 (s, 2H), 3.34 (m, 3H).MS (TOF MS ES+) calculated for [C22H24N302] ⁺ : 362.1863, measured: 362.1869. (2) The staining of live cells HeLa by Compound D was observed under a confocal laser scanning microscope: Compound C was added to the cultured HeLa cells in a 2 mL cell culture medium to a final concentration of 5 μM. Incubate for 1 hour in a 37 ° C, 5% CO 2 cell incubator. Then, 0.01 M of PBS was shaken and rinsed for 5 minutes x3, and then the medium was added, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, excited at 514 nm channel, and the receiving wavelength was 560-620 nm, which was observed three times with an oil mirror (ΙΟΟΟχ). Figure 4 is a fluorescence micrograph of Compound D staining live cell HeLa. As shown in the figure, Compound D was specifically stained for the plasma membrane of HeLa cells. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm. Example 5. Synthesis and fineness of dye E

(1) Synthesis of dye E

4-Bromo-N-methylpyridinone (4.9 g, 14.4 mmol), anhydrous copper sulfate (0.2 g, 10 mol%) and 1,6-hexanediamine (2 g, 17.2 mmol) were dissolved in 100 Anhydrous potassium carbonate (3 g, 21.6 mmol) was added as an acid binding agent to mL ethylene glycol monomethyl ether. After refluxing for 24 h, potassium carbonate and copper sulfate were removed by filtration. The filtrate was removed by rotary evaporation to give a red solid. The solid was washed with ethanol to remove excess 1,6-hexanediamine. Separation on a silica gel column (ethyl acetate: methanol = 10:1 as eluent), yield 51%.

1H-NMR (400 MHz, CDC13) δ 10.49 (m, 1H, H), 8.46 (d, J = 7.7 Hz, 1H, ArH,), 8.30 (d, J = 7.9 Hz, 1H, ArH), 8.20 ( d, J = 7.9 Hz, 1H, ArH), 7.83 - 7.62 (m, 4H, ArH, CH2), 3.86 (s, 3H, CH3), 1.71 - 1.60 (nb, 12H, CH2). MS (TOF MS ES+ ) calculated for [C23H26N302] ⁺ : 376.2020, measured: 376.2022.

(2) Observation of the staining of living cells HeLa by compound E under confocal laser scanning microscopy:

Compound E was added to the cultured HeLa cells in a 2 mL cell culture medium to a final concentration of 10 μM. Incubate for 1 hour in a 37 ° C, 5% CO 2 cell incubator. Then, 0.01 M PBS was shaken for 5 minutes. X3, medium was added, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, excited at 514 nm channel, and the receiving wavelength was 560-620 nm, which was observed three times with an oil mirror (ΙΟΟΟχ). Figure 5 is a fluorescence micrograph of Compound E staining live cell HeLa. As shown in the figure, Compound E was stained for nucleoli and lysosomes of HeLa cells. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm. Example 6. Synthesis of dye F and live cell fluorescence

(1) Synthesis of dye F

4-bromo-N-methylpyridinone (4.9 g, 14.4 mmol), anhydrous copper sulfate (0.2 g, 10 mol%) and 65-70% aqueous ethylamine (8 mL, ~100 mmol) Anhydrous potassium carbonate (3 g, 21.6 mmol) was added as an acid binding agent to 100 mL of ethylene glycol monomethyl ether. After reacting at 100 ° C for 24 h, potassium carbonate and copper sulfate were removed by filtration. The filtrate was evaporated using rotary evaporation to give a red solid. The crude product was separated on silica gel column (ethyl acetate elute), yield 56%.

1H NMR (400 MHz, CDC13) δ 10.29 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.68 (dt, J = 14,6, 7.1 Hz, 2H), 7.61 - 7.45 (m, 2H), 7.10 (d, J = 9.5 Hz, 1H), 3.79 (s, 3H), 3.51 - 3.29 (m, 2H), 1.43 (t, J = 7.2 Hz, 3H). MS (TOF MS ES+) calculated for [C19H16N202] ⁺ : 305.1285, measured: 305.12077.

(2) Observation of the staining of living cells HeLa by compound F under confocal laser scanning microscopy:

Compound F was added to the cultured HeLa cells in a 2 mL cell culture medium to a final concentration of 10 μM. Incubate for 30 minutes in a 37 ° C, 5% CO 2 cell incubator. Then, 0.01 M of PBS was shaken and rinsed for 5 minutes x3, and then the medium was added, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, compound F: 514 nm channel excitation, receiving wavelength 570-620 nm; Golgi fluorescent dye NBD C6-ceramide: 458 nm channel excitation, receiving wavelength 500-550 nm observed with oil mirror (ΙΟΟΟχ), repeat three times. Figure 6 is a fluorescence micrograph of Compound F staining live cells HeLa. Comparing the commercial Golgi fluorescent dye NBD C6-ceramide, it was found that Compound F specifically stained the HeLa Golgi. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm, 458nm. Example 7. Synthesis of dye G and live cell fluorescence imaging

(1) Synthesis of dye G

The synthetic raw material of the dye G was the compound A and methyl iodide prepared in Example 1. Take 1 gA (3.1 mmol), iodomethane 2.5 g (19.5 mmol), dissolve in 50 mL of ethylene glycol monomethyl ether, reflux for 8 hours, TLC test material A, distill off the solvent under reduced pressure to give a dark red solid It was washed with methanol several times with ultrasonic vibration, filtered, and dried under vacuum to obtain a dye G 0.5 g, yield 32%.

1H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 8.54 (d, J = 7.5 Hz, 1H, ArH), 8.33 (d, J = 7.5 Hz, 1H, ArH), 7.93 (d, J = 9.8 Hz, 1H, ArH), 7.79 (m, 3H, ArH), 7.48 (d, J = 9.5 Hz, 1H, ArH), 4.01 (d, J = 4.8 Hz, 2H, CH2), 3.75 (s, 3H , CH3), 3.69 (t, J = 6.5 Hz, 2H, CH2), 3.66 (s, 9H, CH3).MS (TOF MS ES+) calculated for [C22H24N302] ⁺ : 362.1863, measured: 362.1868.

(2) Observation of the staining of living cells HeLa by compound G under confocal laser scanning microscopy:

Compound G was added to the cultured HeLa cells in a 2 mL cell culture medium to a final concentration of 10 μM. Incubate for 1 hour in a 37 ° C, 5% CO 2 cell incubator. Then, 0.01 M of PBS was vortexed for 5 minutes x3, and the medium was added, and the morphology of the cells was observed by confocal laser scanning microscopy (Leica, TCS-SP2, Germany). A representative region was selected, excited at 514 nm channel, and the receiving wavelength was 560-620 nm, which was observed three times with an oil mirror (ΙΟΟΟχ). Figure 7 is a fluorescence micrograph of Compound E staining live cells HeLa. As shown in the figure, Compound G was stained for nucleoli and lysosomes of HeLa cells. The instrument used was a confocal laser scanning microscope, model: Leica, TCS-SP2, excitation channel: 514 nm.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. For those of ordinary skill in the art to which the present invention pertains, It is also possible to make a number of simple derivations or substitutions without departing from the spirit and scope of the invention. As a fluorescent dye is a use of the novel compounds of the present invention, it is not believed that the compounds of the present invention are used only for fluorescent dyes, and the same effect of the compounds of the present invention as fluorescent dyes is known to those of ordinary skill in the art to which the present invention pertains. Under the consideration of the mechanism, a number of simple inferences can also be made, and other application uses of the compounds of the present invention are considered to be within the scope of the present invention.

Claims

Claim

1. A class of 4-substituted N-substituted pyridines has the following structural formula I:

(I)

In the formula I, n is an integer from 1 to 10;

The 4-position N-substituted anthrapyridone fluorescent dye according to claim 1, wherein said R is selected from the group consisting of dimethylamino, diethylamino, dipropylamino, dibutylamino and dihydroxyethylamino. , trimethylammonium halide, amino and guanidinium hydrochloride.

The 4-position N-substituted anthrapyridone fluorescent dye according to claim 2, wherein said R is selected from the group consisting of diethylamino, dipropylamino, dibutylamino, dihydroxyethylamino, and trimethyl iodide. Ammonium and guanidinium hydrochloride.

The 4-position N-substituted anthrapyridone fluorescent dye according to Claim 1, 2 or 3, wherein the n is an integer of 2 to 6.

The method for preparing a 4-position N-substituted anthrapyridone fluorescent dye according to claim 1, wherein the compound of the formula i and the compound of the formula ii are used as a raw material, and the reaction of the catalyst of the formula I and the acid-binding agent is carried out to prepare the formula I. Compound

In the formula i, is ? , Cl, Br or I;

The reaction solvent is selected from the group consisting of water, hydrazine, hydrazine-dimethylformamide, dimethyl sulfoxide, ethylene glycol monomethyl ether and ethylene glycol; the acid binding agent is selected from the group consisting of triethylamine, trimethylamine, potassium carbonate, and sodium carbonate. , sodium bicarbonate, sodium acetate, sodium hydroxide and potassium hydroxide.

6. The use of the 4-position anthracene-substituted anthrapyridone fluorescent dye according to claim 1 for biological staining.

7. The use of a 4-position N-substituted anthrapyridone fluorescent dye according to claim 6 for biological dyeing, characterized in that

Among them: C-specific fluorescence staining of living cell nucleoli, D-specific fluorescence staining of living cell outer membrane, D-specific staining of living cell Golgi apparatus.

The use of the 4-position N-substituted anthrapyridone fluorescent dye according to claim 6, in the biological dyeing, characterized in that the fluorescent dye is A, E or G, and is used for specificity on living cell nucleolus and Lysosomal fluorescence staining.

The use of the 4-position N-substituted anthrapyridone fluorescent dye according to claim 6 for biological staining, characterized in that the fluorescent dye is B, which is used for specificity to fluorescent staining of fixed cell nucleoli.