A polycyanine structure near-infrared fluorescent dye, and a preparation method and application thereof

Abstract

The application discloses a multi-cyan structure near-infrared fluorescent dye, by changing the structure of end group and intermediate condensing agent, the accurate control of dye electron donating type, charge separation degree and electron mobility is realized. The multi-cyan structure near-infrared fluorescent dye compound prepared by the application has significant advantages in spectral characteristics, the molar extinction coefficient is higher than that of traditional multi-cyan dye, the light energy absorption capacity is strong, which is helpful to improve the light energy utilization rate and reduce the dye amount; the absolute fluorescence quantum yield reaches a high level, the imaging signal intensity and sensitivity are significantly enhanced, a more sensitive and reliable tool is provided for biological imaging and molecular detection; the fluorescent dye compound has excellent biocompatibility and phototoxicity, the safety of the dye in the application in the biological and medical fields is ensured, and strong support is provided for the application of the dye in the field of cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent dyes, in particular to a polycyanine structure near-infrared fluorescent dye and a preparation method and application thereof. BACKGROUND

[0002] With the deepening of scientific research, biological fluorescent staining technology has become an indispensable tool for exploring the mystery of life and analyzing biological structure and function. This technology ingeniously combines fluorescent dye molecules with target biomolecules (such as proteins, antibodies, cell membranes, etc.) through covalent or non-covalent bonding, achieving precise labeling and efficient detection of specific components in biological samples, and providing strong support for the visualization analysis of intracellular dynamic processes. Photodynamic therapy, as a new cancer treatment strategy, has a unique advantage over traditional chemotherapy by inducing a series of active factors that kill cancer cells through energy conversion of dye molecules under light excitation, thereby inhibiting tumor growth.

[0003] In the field of polycyanine structure research, as a powerful electron-withdrawing group, it has been widely used in the innovative design of dye molecules. This structure not only exhibits red-shifted emission and absorption spectrum characteristics due to its extended conjugated chain, but also is known for its excellent stability, which can significantly improve the overall performance of the dye, effectively resist the interference of the body's own fluorescence, and enhance the targeted killing ability of deep tumor cells.

[0004] However, the current development of polycyanine dyes also faces many challenges, which are as follows: the low molar extinction coefficient limits the efficiency of light energy capture and utilization of dye molecules, affecting their performance in photodynamic therapy and fluorescence imaging; the insufficient fluorescence quantum yield restricts the potential of dyes in high-efficiency imaging applications, making it difficult to meet the needs of high-resolution and high-sensitivity imaging; the limited structural diversity and relatively single synthesis path lack diversified structural changes, limiting the modifiability and functional expansion of dye molecules; the solubility problem is poor in non-polar solvents, hindering the widespread application of dyes in biological systems. SUMMARY

[0005] In view of the technical problems existing in the prior art, the present application provides a polycyanine structure near-infrared fluorescent dye and a preparation method and application thereof.

[0006] In order to achieve the above-mentioned purpose, the present application provides a polycyanine structure near-infrared fluorescent dye, which has the structure of general formula I:

[0007]

[0008] wherein R1 is selected from one of O, NH, and NCH3;

[0009] R2 and R3 are each independently selected from one of methyl, hydroxyl, phenyl, trifluoromethyl, alkoxy having 1-18 carbons;

[0010] R4 is selected from one of hydrogen, halogen, aldehyde group, phenyl, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, alkylsulfonic acid group having 1-18 carbons, ester group having 1-18 carbons, alkylsulfonate having 1-18 carbons, hydroxyalkyl having 1-18 carbons.

[0011] To achieve the above object, the present application further provides a preparation method of the multi-cyan structure near-infrared fluorescent dye, characterized in that the preparation method comprises the following steps:

[0012]

[0013] S1: Compound Y4 and hexamethylenetetramine are dissolved in trifluoroacetic acid, stirred at 80-120°C under inert gas protection for 24-36 h, then cooled to 60-70°C, and continuously stirred under acidic condition for 3-6 h, cooled to room temperature, extracted by organic solvent, concentrated, filtered and purified by silica gel column to obtain compound Y5;

[0014] S2: Compound Y3 and Y5 are dissolved in an organic solvent, a basic catalyst is added, and refluxed under inert gas protection for 1-5 h, and after the reaction is completed, concentrated and purified by silica gel column to obtain the multi-cyan structure near-infrared fluorescent dye I.

[0015] Further, the molar ratio of Y4 and hexamethylenetetramine is 1:2-4.

[0016] Further, the preparation method of compound Y3 is:

[0017]

[0018] Compound Y1 and malononitrile are added in an organic solvent in a molar ratio of 1:2-4, after stirring, a basic catalyst is added, the molar ratio of Y1 and the basic catalyst is 1:0.1-0.3, after refluxing for 0.5-2 h, cooling, filtering, washing and drying, compound Y3 with R1 being O is obtained, which is denoted as compound Y3-1.

[0019] Further, the preparation method of compound Y3 is:

[0020]

[0021] The compound Y2 and malononitrile dimer are added in a molar ratio of 1:1-3, the mixture is dissolved in an organic solvent, stirred under alkaline conditions for 2-4 h, cooled to 0-5°C, and then filtered, washed, and dried to obtain a compound Y3 in which R1 is NH and R3 is a hydroxyl group, denoted as compound Y3-2.

[0022] Further, the preparation method of the compound Y3 is:

[0023]

[0024] The compound Y3-2 and the compound Y01 are dissolved in an organic solvent in a molar ratio of 1:1-3, uniformly mixed, and cooled to 0-5°C, trifluoroacetic acid is slowly added dropwise, stirred for 10-14 h, and then stirred for 24-36 h after being raised to room temperature, and then recrystallized in methyl tert-butyl ether at 0-5°C, filtered, washed, and purified by a silica gel column to obtain a compound Y3 in which R1 is NH, R3 is one of a methyl group, a phenyl group, and a trifluoromethyl group, denoted as compound Y3-3.

[0025] Further, the preparation method of the compound Y3 is:

[0026]

[0027] The compound Y3-2 and the compound Y02 are dissolved in an organic solvent in a molar ratio of 1:1-1:3, uniformly mixed, and cooled to 0-5°C, trifluoroacetic acid is slowly added dropwise, stirred for 10-14 h, and then stirred for 24-36 h after being raised to room temperature, and then recrystallized in methyl tert-butyl ether at 0-5°C, filtered, washed, and purified by a silica gel column to obtain a compound Y3 in which R1 is NH, R3 is one of a methoxy group and an alkoxy group having 1-18 carbons, denoted as compound Y3-3.

[0028] Further, the preparation method of the compound Y3 is:

[0029]

[0030] The compound Y3-3 is added to an organic solvent, and a basic catalyst is added in a molar ratio of 1:0.1-0.3, stirred under inert gas protection for 1-3 h, iodomethane is added, stirred at 40-70°C for 24-32 h, cooled to room temperature, concentrated, filtered, and purified by a silica gel column to obtain a compound Y3 in which R1 is NCH3, denoted as compound Y3-4.

[0031] Further, the organic solvent is selected from at least one of toluene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran; and the basic catalyst is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia, sodium bicarbonate, sodium ethoxide, sodium methoxide, pyridine, piperidine, triethylamine, trimethylamine, diisopropylethylamine.

[0032] To achieve the above object, the application further provides a use of the multi-cyan structure near-infrared fluorescent dye in the preparation of cell and tumor imaging agents or photodynamic and photothermal therapy drugs.

[0033] In summary, the application has the following advantages:

[0034] The multi-cyan structure near-infrared fluorescent dye compound provided by the application has significant advantages in spectral properties. Different compounds exhibit diverse absorption and emission spectra in acetonitrile solvent, covering a wide range, and the molar extinction coefficient is higher than that of traditional multi-cyan dyes, which can reach 42552 L / mol -1 ·cm -1 , indicating that it has strong absorption capacity for light energy, which helps to improve the utilization rate of light energy and reduce the amount of dye.

[0035] The absolute fluorescence quantum yield of the fluorescent dye compound reaches a high level, which can reach 0.103. High fluorescence quantum yield means that the dye molecules can be more effectively converted into fluorescence emission after absorbing light energy, thereby significantly enhancing the intensity and sensitivity of the imaging signal, providing a more sensitive and reliable tool for biological imaging and molecular detection.

[0036] The fluorescent dye compound has structural diversity and modifiability. By changing the structure of the end group and the intermediate condensing agent, the application successfully realizes the precise control of the electron donation type, charge separation degree and electron mobility of the dye molecule. This structural diversity and modifiability not only enriches the types of dyes, but also provides a broad space for further optimizing the spectral properties and application performance of the dyes.

[0037] The fluorescent dye compound has excellent biocompatibility and phototoxicity. The results of the cytotoxicity experiment show that even at a higher concentration, the dye has little effect on the survival rate of MCF-7 cells, showing good biocompatibility. This property ensures the safety of the dye in biological and medical applications. At the same time, the phototoxicity experiment further reveals the killing effect of the dye on cells under specific light conditions, providing strong support for its application in the field of cancer treatment and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0039] Figure 1 is a high-resolution mass spectrum of the compound 1 disclosed by the present application.

[0040] Figure 2 is a high-resolution mass spectrum of the compound 2 disclosed by the present application.

[0041] Figure 3 is the ultraviolet-visible absorption spectrum of the compound 1 disclosed by the present application in acetonitrile solvent.

[0042] Figure 4 is the ultraviolet-visible absorption spectrum of the compound 1 disclosed by the present application in acetonitrile solvent.

[0043] Figure 5 is the ultraviolet-visible absorption spectrum of the compound 2 disclosed by the present application in acetonitrile solvent.

[0044] Figure 6 is the ultraviolet-visible absorption spectrum of the compound 2 disclosed by the present application in acetonitrile solvent.

[0045] Figure 7 is the confocal image of the compound 2 disclosed by the present application on MCF-7 cells.

[0046] Figure 8 is the lysosome targeting co-localization analysis diagram of the compound 2 disclosed by the present application on MCF-7 cells.

[0047] Figure 9 is the MTT dark toxicity experiment diagram of the compound 2 disclosed by the present application.

[0048] Figure 10 is the MTT light toxicity experiment diagram of the compound 2 disclosed by the present application. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0050] Unless otherwise defined, all terms used in the present disclosure have the following meanings. The term "halogen" used in the present disclosure includes fluorine, chlorine, bromine and iodine; "MTT" refers to a method for detecting cell survival and growth; "alkyl" includes straight-chain alkyl and branched alkyl; "room temperature" refers to a temperature range of 20-25℃.

[0051] The present disclosure provides a polycyanine structure near-infrared fluorescent dye having a general formula I:

[0052]

[0053] wherein R1 is selected from one of O, NH, NCH3;

[0054] R2 and R3 are each independently selected from one of methyl, hydroxyl, phenyl, trifluoromethyl, alkoxy having 1-18 carbons;

[0055] R4 is selected from one of hydrogen, halogen, aldehyde group, phenyl, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, alkylsulfonic acid group having 1-18 carbons, ester group having 1-18 carbons, alkylsulfonate having 1-18 carbons, hydroxyalkyl having 1-18 carbons.

[0056] To achieve the above-mentioned purpose, the present disclosure further provides a preparation method of a polycyanine structure near-infrared fluorescent dye, characterized in that the preparation method comprises the following steps:

[0057]

[0058] S1: Compound Y4 and hexamethylenetetramine are dissolved in trifluoroacetic acid, stirred at 80-120℃ under inert gas protection for 24-36 h, then cooled to 60-70℃, and continue to stir under acidic conditions for 3-6 h, cooled to room temperature, extracted with organic solvent, concentrated, filtered and purified by silica gel column to obtain compound Y5;

[0059] S2: Compound Y3 and Y5 are dissolved in an organic solvent, a basic catalyst is added, and the reaction is carried out under reflux for 1-5 h under inert gas protection, and after the reaction is completed, it is concentrated and purified by silica gel column to obtain a polycyanine structure near-infrared fluorescent dye I.

[0060] Further, the molar ratio of Y4 and hexamethylenetetramine is 1:2-4.

[0061] Further, the preparation method of compound Y3 is:

[0062]

[0063] In an organic solvent, compound Y1 and malononitrile are added in a molar ratio of 1:2-4, after stirring, a basic catalyst is added, the molar ratio of Y1 to the basic catalyst is 1:0.1-0.3, after refluxing for 0.5-2 h, cooling, filtering, washing and drying, a compound Y3 with R1 being O is obtained, which is denoted as compound Y3-1.

[0064] Further, the preparation method of the compound Y3 is:

[0065]

[0066] Compound Y2 and malononitrile dimer are added in a molar ratio of 1:1-3, the mixture is dissolved in an organic solvent, after stirring for 2-4 h under basic conditions, cooling to 0-5℃, filtering, washing and drying, a compound Y3 with R1 being NH and R3 being hydroxyl is obtained, which is denoted as compound Y3-2.

[0067] Further, the preparation method of the compound Y3 is:

[0068]

[0069] Compound Y3-2 and compound Y01 are dissolved in an organic solvent in a molar ratio of 1:1-3, uniformly mixed and cooled to 0-5℃, trifluoroacetic acid is slowly added dropwise, stirring for 10-14 h, continuing to stir for 24-36 h after rising to room temperature, then recrystallized in methyl tert-butyl ether at 0-5℃, filtering, washing and purifying by a silica gel column, a compound Y3 with R1 being NH, R3 being one of methyl, phenyl and trifluoromethyl is obtained, which is denoted as compound Y3-3.

[0070] Further, the preparation method of the compound Y3 is:

[0071]

[0072] Compound Y3-2 and compound Y02 are dissolved in an organic solvent in a molar ratio of 1:1-1:3, uniformly mixed and cooled to 0-5℃, trifluoroacetic acid is slowly added dropwise, stirring for 10-14 h, continuing to stir for 24-36 h after rising to room temperature, then recrystallized in methyl tert-butyl ether at 0-5℃, filtering, washing and purifying by a silica gel column, a compound Y3 with R1 being NH, R3 being one of methoxy and alkoxy with 1-18 carbons is obtained, which is denoted as compound Y3-3.

[0073] Further, the preparation method of the compound Y3 is:

[0074]

[0075] The compound Y3-3 is added to an organic solvent, and a basic catalyst is added, the molar ratio of Y3-3 to the basic catalyst is 1:0.1-0.3, after stirring under inert gas protection for 1-3 h, iodomethane is added, stirring at 40-70 DEG C for 24-32 h, after cooling to room temperature, concentration, filtration and silica gel column purification, the compound Y3 with R1 as NCH3 is obtained, which is denoted as compound Y3-4.

[0076] Further, the organic solvent is selected from at least one of toluene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran; the basic catalyst is selected from at least one of sodium hydroxide, potassium hydroxide, ammonia, sodium bicarbonate, sodium ethoxide, sodium methoxide, pyridine, piperidine, triethylamine, trimethylamine, diisopropylethylamine.

[0077] To achieve the above object, the application further provides application of the multi-cyano structure near-infrared fluorescent dye in preparation of cell and tumor imaging preparation or photodynamic and photothermal therapy drugs.

[0078] In summary, the application has the following beneficial effects:

[0079] The multi-cyano structure near-infrared fluorescent dye compound provided by the application has significant advantages in spectral properties. Different compounds exhibit diversified absorption and emission spectra in acetonitrile solvent, covering a wide range, and the molar extinction coefficient is higher than that of traditional multi-cyano dyes, which can reach 42552 L / mol-1·cm-1, indicating that the dye has strong absorption capacity for light energy, which helps to improve the utilization rate of light energy and reduce the amount of dye.

[0080] The absolute fluorescence quantum yield of the fluorescent dye compound reaches a high level, which can reach 0.103. High fluorescence quantum yield means that the dye molecules can be more effectively converted into fluorescence emission after absorbing light energy, thereby significantly enhancing the intensity and sensitivity of the imaging signal, providing a more sensitive and reliable tool for biological imaging and molecular detection.

[0081] The fluorescent dye compound has structural diversity and modifiability. The application successfully realizes the precise regulation of the electron donation type, charge separation degree and electron mobility of the dye molecule by changing the structure of the terminal group and the intermediate condensing agent. This structural diversity and modifiability not only enriches the types of dyes, but also provides a broad space for further optimizing the spectral properties and application performance of the dyes.

[0082] The fluorescent dye compound has excellent biocompatibility and phototoxicity. The cytotoxicity experiment results show that the dye has little effect on the survival rate of MCF-7 cells even at a higher concentration, which shows good biocompatibility. This property ensures the safety of the dye in the application of biological and medical fields. At the same time, the phototoxicity experiment further reveals the killing effect of the dye on cells under specific light conditions, which provides strong support for its application in the field of cancer treatment and the like.

[0083] Example 1

[0084] Preparation of polycyanine structure near-infrared fluorescent dye compound 1

[0085] S1 Preparation of compound 1.1

[0086]

[0087] In 20 mL of ethanol, 3-hydroxy-3-methyl-2-butanone (1.00 g, 9.8 mmol) and malononitrile (1.29 g, 19.6 mmol) were added, and after stirring, sodium ethoxide (106 mg, 1.9 mmol) was added, and refluxed for 2 h. After cooling to room temperature, the cold ethanol was filtered, washed, and then dried in vacuum to obtain yellow-gray compound 1.1 (1.40 g, 7.0 mmol, Y=72%).

[0088] S2 Preparation of compound 1.2

[0089]

[0090] p-Hydroxybenzaldehyde (1.00 g, 8.19 mmol) and hexamethylenetetramine (3.44 g, 24.57 mmol) were dissolved in 30 mL of trifluoroacetic acid, stirred at 100°C under nitrogen protection for 24 h, then cooled to 70°C, 50 mL of HCl (4 M) was added, and stirred for another 3 h. After cooling to room temperature, it was extracted with dichloromethane, concentrated, filtered and purified by silica gel column to obtain yellow solid compound 1.2 (450 mg, 2.53 mmol, Y=35%).

[0091] S3 Preparation of compound 1

[0092]

[0093] Compound 1.1 (1 g, 5.02 mmol) and compound 1.2 (358 mg, 2.01 mmol) were dissolved in toluene solution, 3 drops of triethylamine was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature after concentration, and the obtained crude product was purified by silica gel column to obtain compound 1 (310 mg, 0.57 mmol, Y = 28%). High resolution mass spectrum is shown in Figure 1 .1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.15 (s, 2H), 8.00 (d, J = 15.8 Hz, 2H), 7.92 (d, J = 15.9 Hz, 2H), 1.78 (s, 12H).

[0094] Example 2

[0095] Preparation of polycyanine structure near-infrared fluorescent dye compound 2:

[0096] S1 Preparation of compound 2.1

[0097]

[0098] Ethyl p-hydroxybenzoate (1.00 g, 6.02 mmol) and hexamethylenetetramine (2.53 g, 18.05 mmol) were dissolved in 30 mL of trifluoroacetic acid, stirred at 100°C for 24 h under nitrogen protection, then cooled to 70°C, 50 mL of HCl (4 M) was added, and stirred for another 3 h, extracted with dichloromethane after cooling to room temperature, concentrated, filtered and purified by silica gel column to obtain compound 2.1 (610 mg, 2.75 mmol, Y = 45%) as a yellow solid.

[0099] S2 Preparation of compound 2

[0100]

[0101] Compound 1.1 (1 g, 5.02 mmol) and compound 2.1 (446 mg, 2.01 mmol) were dissolved in ethanol solution, 3 drops of ammonia water was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature after concentration, and the obtained crude product was purified by silica gel column to obtain compound 2 (580 mg, 0.99 mmol, Y = 49%). High resolution mass spectrum is shown in Figure 21H NMR (400 MHz, DMSO-d6) δ 8.16 (s, 2H), 8.02 (d, J = 15.7 Hz, 2H), 7.90 (d, J = 15.7 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 1.79 (s, 12H), 1.31 (t, J = 7.1 Hz, 3H).

[0102] Example 3

[0103] Preparation of polycyanostucture near-infrared fluorescent dye compound 3

[0104] S1 Preparation of compound 3.1

[0105]

[0106] P-methoxyphenol (1.00 g, 8.05 mmol) and hexamethylenetetramine (3.38 g, 24.16 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100 °C for 24 h under nitrogen protection, then cooled to 70 °C, 50 mL HCl (4 M) was added, and stirring was continued for 3 h. After cooling to room temperature, it was extracted with dichloromethane, concentrated, filtered and purified by silica gel column to obtain compound 3.1 (680 mg, 37.74 mmol, Y = 46%) as a yellow solid.

[0107] S2 Preparation of compound 3

[0108]

[0109] Compound 1.1 (1 g, 5.02 mmol) and compound 3.1 (362 mg, 2.01 mmol) were dissolved in isopropanol solution, 10 mg sodium hydroxide was added to catalyze the reaction, and stirring was refluxed for 4 h. After cooling the reaction to room temperature, it was concentrated, and the obtained crude product was purified by silica gel column to obtain compound 3 (372 mg, 0.68 mmol, Y = 34%).1H NMR (400 MHz, Chloroform-d) δ 7.16 (d, J = 1.0 Hz, 4H), 6.84 (s, 2H), 3.80 (s, 3H), 1.50 (s, 12H).

[0110] Example 4

[0111] Preparation of polycyanostucture near-infrared fluorescent dye compound 4:

[0112] S1 Preparation of compound 4.1

[0113]

[0114] Compound 4.1 was prepared by dissolving 4-fluorophenol ethyl ester (1.00 g, 8.92 mmol) and hexamethylenetetramine (3.75 g, 26.76 mmol) in 30 mL trifluoroacetic acid, stirring at 100 °C for 24 h under nitrogen protection, then cooling to 70 °C, adding 50 mL HCl (4 M), continuing stirring for 3 h, cooling to room temperature, extracting with dichloromethane, concentrating, filtering and purifying with silica gel column to give yellow solid compound 4.1 (542 mg, 32.24 mmol, Y = 36%).

[0115] S2 Preparation of compound 4

[0116]

[0117] Compound 4 was prepared by dissolving compound 1.1 (1 g, 5.02 mmol) and compound 4.1 (338 mg, 2.08 mmol) in acetonitrile solution, catalyzing the reaction by adding 10 mg potassium hydroxide, stirring at reflux for 4 h, cooling the reaction to room temperature, concentrating, and purifying the obtained crude product with silica gel column to give compound 4 (251 mg, 0.47 mmol, Y = 24%).1H NMR (400 MHz, Chloroform-d) δ 7.18 (s, 4H), 7.10 (d, J = 8.0 Hz, 2H), 1.50 (s, 12H).

[0118] Example 5

[0119] Preparation of polycyanine near-infrared fluorescent dye compound 5

[0120] S1 Preparation of compound 5.1

[0121]

[0122] Compound 5.1 was prepared by dissolving p-methylphenol (1.00 g, 9.24 mmol) and hexamethylenetetramine (3.89 g, 27.74 mmol) in 30 mL trifluoroacetic acid, stirring at 100 °C for 24 h under nitrogen protection, then cooling to 70 °C, adding 50 mL HCl (4 M), continuing stirring for 3 h, cooling to room temperature, extracting with dichloromethane, concentrating, filtering and purifying with silica gel column to give yellow solid compound 5.1 (945 mg, 5.76 mmol, Y = 62%).

[0123] S2 Preparation of compound 5

[0124]

[0125] Compound 1.1 (1 g, 5.02 mmol) and compound 5.1 (330 mg, 2.08 mmol) were dissolved in tetrahydrofuran solution, 10 mg sodium bicarbonate was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 5 (457 mg, 0.87 mmol, Y=43%).1H NMR (400MHz, DMSO-d6) δ 8.25 (d, J = 16.3 Hz, 1H), 7.94 (s, 1H), 7.30 (d, J = 16.3 Hz, 1H), 3.17 (s, 1H), 1.79 (s, 6H).

[0126] Example 6

[0127] Preparation of polycyanine structure near-infrared fluorescent dye compound 6

[0128] S1 Preparation of compound 6.1

[0129]

[0130] p-Methylphenol (1.00 g, 5.87 mmol) and hexamethylenetetramine (2.47 g, 17.63 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100°C for 24 h under nitrogen protection, then cooled to 70°C, 50 mL HCl (4 M) was added, and stirring was continued for 3 h, then extracted with dichloromethane after cooling to room temperature, concentrated, filtered and purified by silica gel column to obtain compound 6.1 (1.14 g, 5.04 mmol, Y=86%) as a yellow solid.

[0131] S2 Preparation of compound 6

[0132]

[0133] Compound 1.1 (1 g, 5.02 mmol) and compound 6.1 (454 mg, 2.08 mmol) were dissolved in methanol solution, 10 mg sodium methoxide was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature after concentration, and the obtained crude product was purified by silica gel column to obtain compound 6 (786 mg, 1.33 mmol, Y = 67%).1H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 16.2 Hz, 1H), 8.27 (s, 1H), 7.78 (d, J = 7.3 Hz, 1H), 7.57 (d, J = 16.2 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 0H), 1.81 (s, 4H).

[0134] Example 7

[0135] Preparation of polycyanostucture near-infrared fluorescent dye compound 7

[0136]

[0137] Compound 1.1 (1 g, 5.02 mmol) and 2-hydroxyisophthalaldehyde (301 mg, 2.08 mmol) were dissolved in ethanol solution, 10 mg sodium ethoxide was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature after concentration, and the obtained crude product was purified by silica gel column to obtain compound 7 (295 mg, 0.576 mmol, Y = 29%).1H NMR (400 MHz, Chloroform-d) δ 7.63 (m, 2H), 7.28 (d, J = 0.8 Hz, 2H), 7.25 - 7.20 (m, 1H), 7.17 (d, J = 15.1 Hz, 2H), 1.50 (s, 12H).

[0138] Example 8

[0139] Preparation of polycyanostucture near-infrared fluorescent dye compound 8

[0140] S1 Preparation of compound 8.1

[0141]

[0142] 2-(trifluoromethyl)but-1,3-diene (1.00 g, 6.40 mmol) and malononitrile (846 mg, 12.8 mmol) were added to 20 mL of ethanol, stirred, and then sodium ethoxide (1.05 g, 12.86 mmol) was added. The mixture was then refluxed for 2 h. After cooling to room temperature, the mixture was filtered through cold ethanol, washed, and dried under vacuum to give a yellowish-gray compound 8.1 (1.21 g, 4.78 mmol, Y=74%).

[0143] Preparation of S2 compound 8

[0144]

[0145] Compound 8.1 (1 g, 3.95 mmol) and compound 3.1 (285 mg, 1.58 mmol) were dissolved in methanol solution. Three drops of pyridine were added to catalyze the reaction, which was stirred and refluxed for 4 h. After cooling to room temperature, the mixture was concentrated, and the crude product was purified by silica gel column chromatography to give compound 8 (212 mg, 0.32 mmol, Y=21%). ¹H NMR (400 MHz, Chloroform-d) δ 7.24 (d, J = 15.1 Hz, 2H), 7.17 (d, J = 15.1 Hz, 2H), 6.84 (s, 2H), 3.81 (s, 3H), 1.47 (s, 6H).

[0146] Example 9

[0147] Preparation of near-infrared fluorescent dye compound 9 with polycyano structure

[0148] Preparation of compound 9.1 (S1)

[0149]

[0150] 2-(trifluoromethyl)-2-phenyl-but-1,3-diene (1.00 g, 4.58 mmol) and malononitrile (606 mg, 9.17 mmol) were added to 20 mL of ethanol, stirred, and then sodium ethoxide (752 mg, 9.17 mmol) was added. The mixture was then refluxed for 2 h. After cooling to room temperature, the mixture was filtered through cold ethanol, washed, and dried under vacuum to give a yellowish-gray compound 9.1 (946 mg, 3.00 mmol, Y=65%).

[0151] Preparation of S2 compound 9

[0152]

[0153] Compound 9.1 (1 g, 3.17 mmol) and compound 5.1 (208 mg, 1.27 mmol) were dissolved in toluene, 3 drops of triethylamine was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 9 (308 mg, 0.41 mmol, Y = 32%).1H NMR (400 MHz, Chloroform-d) δ 7.52 - 7.45 (m, 2H), 7.42 (dd, J = 7.6, 1.6 Hz, 2H), 7.33 - 7.27 (m, 2H), 7.27 (d, J = 15.1 Hz, 2H), 7.19 (d, J = 15.2 Hz, 2H), 7.05 (d, J = 0.8 Hz, 2H), 2.34 (d, J = 0.7 Hz, 3H).

[0154] Example 10

[0155] Preparation of polycyanine near-infrared fluorescent dye compound 10

[0156] S1 Preparation of compound 10.1

[0157]

[0158] Diacyl (1.72 g, 20 mmol) and 2% NaOH aqueous solution (0.6 g) were added to malononitrile dimer (2.64 g, 20 mmol) dissolved in 25 mL of aqueous ethanol solution (20%, v / v), stirred at room temperature for 2 h, then filtered, the solution was adjusted to 0°C and filtered, washed with cold ethyl acetate, dried to obtain intermediate 10.1 (2.9 g, 15 mmol, Y = 73%).

[0159] S2 Preparation of compound 10

[0160]

[0161] Compound 10.1 (1 g, 4.99 mmol) and compound 4.1 (335 mg, 2.00 mmol) were dissolved in isopropanol solution, 3 drops of diisopropylethylamine was added to catalyze the reaction, stirred at reflux for 4 h, after the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 10 (350 mg, 0.62 mmol, Y=31%).1H NMR (400MHz, Chloroform-d) δ 7.16 (d, 2H), 7.13 (d, 2H), 5.65 (s, 2H), 1.64 (s, 6H).

[0162] Example 11 Preparation of polycyanine structure near-infrared fluorescent dye compound 11

[0163] S1 Preparation of compound 11.1

[0164]

[0165] Compound 10.1 (1.00 g, 20 mmol) was added to 15 mL of benzyl alcohol solution and cooled to 0°C, 2 mL of trifluoroacetic acid was slowly added to the above solution, and then stirred for 12 h, and continued to stir for 24 h after being raised to room temperature, after the reaction was completed, the above solution was slowly added to methyl tert-butyl ether at 0°C for recrystallization. Filtration, washing and purification by silica gel column to obtain 11.1 (950 mg, 32.72 mmol, Y=65%).

[0166] S2 Preparation of compound 11.2

[0167]

[0168] Compound 11.1 (1.00 g, 3.44 mmol) was added to 10 mL of acetonitrile solution and 0.5 g of potassium carbonate solid was added, stirred for 1 h under nitrogen, then iodomethane (1.46 g, 10.33 mmol) was slowly added and stirred at 50°C for 24 h, after being cooled to room temperature, concentrated, filtered and purified by silica gel column to obtain yellow solid compound 11.2 (875 mg, 2.87 mmol, Y=83%).

[0169] S3 Preparation of compound 11

[0170]

[0171] Compound 11.2 (1 g, 3.28 mmol) and compound 5.1 (216 mg, 1.31 mmol) were dissolved in methanol solution, 3 drops of triethylamine were added to catalyze the reaction, stirred at reflux for 4 h, after the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 11 (215 mg, 0.29 mmol, Y = 22%).1H NMR (400 MHz, Chloroform-d) δ 9.93 (s, 1H), 7.38 - 7.25 (m, 10H), 7.19 (d, J = 15.1 Hz, 2H), 7.08 (d, J = 15.1 Hz, 2H), 7.03 (s, 2H), 4.66 (dt, J = 12.5, 0.9 Hz, 2H), 4.52 (dt, J = 12.6, 0.9 Hz, 2H), 3.00 (s, 6H), 2.34 (d, J = 0.8 Hz, 3H), 1.46 (s, 6H).

[0172] Example 12

[0173] Preparation of polycyanine structure near-infrared fluorescent dye compound 12

[0174] S1 Preparation of compound 12.1

[0175]

[0176] 10.1 (1.00 g, 5.00 mmol) was added to a 15 mL solution of n-decanol and cooled to 0 °C, to the above solution, 2 mL of trifluoroacetic acid was slowly added dropwise, and then stirred for 12 h, after being raised to room temperature, continue to stir for 24 h, after the reaction was completed, the above solution was slowly added dropwise into methyl tert-butyl ether at 0 °C for recrystallization. Filtration, washing and purification by silica gel column to obtain 12.1 (1.15 g, 3.38 mmol, Y = 67%).

[0177] S2 Preparation of compound 12

[0178]

[0179] Compound 12.1 (1 g, 2.94 mmol) and compound 6.1 (266 mg, 1.18 mmol) were dissolved in methanol solution, 3 drops of piperidine catalytic reaction were added, stirred at reflux for 4 h, after the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 12 (372 mg, 0.43 mmol, Y = 36%).1H NMR (400 MHz, Chloroform-d) δ 7.61 - 7.53 (m, 2H), 7.45 - 7.33 (m, 5H), 7.17 (d, J = 3.1 Hz, 4H), 6.83 (s, 2H), 3.54 - 3.36 (m, 4H), 1.71 - 1.52 (m, 10H), 1.42 - 1.32 (m, 4H), 1.32 - 1.18 (m, 25H), 0.95 - 0.83 (m, 6H).

[0180] Example 13

[0181] Preparation of polycyanine structure near-infrared fluorescent dye compound 13

[0182] S1 Preparation of compound 13.1

[0183]

[0184] Compound 10.1 (1.00 g, 5.00 mmol) was added to a 15 mL n-octadecanol solution and cooled to 0°C, 2 mL of trifluoroacetic acid was slowly added to the above solution, and then stirred for 12 h, and continued to stir for 24 h after rising to room temperature, after the reaction was completed, the above solution was slowly added to methyl tert-butyl ether at 0°C for recrystallization. Filtration, washing and purification by silica gel column to obtain 13.1 (1.27 g, 2.81 mmol, Y = 56%).

[0185] S2 Preparation of compound 13

[0186]

[0187] Compound 13.1 (1 g, 2.21 mmol) and compound 6.1 (227 mg, 1.00 mmol) were dissolved in methanol solution, 3 drops of piperidine catalytic reaction was added, stirred at reflux for 4 h, the reaction was cooled to room temperature after concentration, and the obtained crude product was purified by silica gel column to obtain compound 13 (210 mg, 0.19 mmol, Y = 19%).1H NMR (400 MHz, Chloroform-d) δ 7.60 - 7.53 (m, 1H), 7.46 - 7.31 (m, 3H), 7.17 (d, J = 3.1 Hz, 2H), 6.83 (s, 1H), 3.54 - 3.36 (m, 2H), 1.69 - 1.52 (m, 5H), 1.44 - 1.19 (m, 27H), 0.95 - 0.84 (m, 3H).

[0188] Example 14

[0189] Preparation of polycyanine structure near-infrared fluorescent dye compound 14:

[0190] S1 Preparation of compound 14.1

[0191]

[0192] p-Hydroxyphenylpropionic acid (1.00 g, 6.02 mmol) and hexamethylenetetramine (2.53 g, 18.05 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100°C for 24 h under nitrogen protection, then cooled to 70°C, 50 mL HCl (4 M) was added, and stirred for another 3 h, extracted with dichloromethane after cooling to room temperature, concentrated, filtered and purified by silica gel column to obtain compound 14.1 (1.14 g, 5.31 mmol, Y = 85%) as a yellow solid.

[0193] S2 Preparation of compound 14

[0194]

[0195] Compound 8.1 (1 g, 3.95 mmol) and compound 14.1 (399 mg, 1.80 mmol) were dissolved in ethanol solution, 3 drops of ammonia water were added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 14 (451 mg, 0.65 mmol, Y = 36%).1H NMR (400 MHz, Chloroform-d) δ 9.97 (s, 1H), 7.26 - 7.13 (m, 4H), 7.04 (t, J = 1.0 Hz, 2H), 2.98 - 2.81 (m, 2H), 2.62 (dt, J = 12.3, 7.0 Hz, 1H), 2.51 (dt, J = 12.4, 7.2 Hz, 1H), 1.47 (s, 6H).

[0196] Example 15

[0197] Preparation of polycyanine near-infrared fluorescent dye compound 15:

[0198] S1 Preparation of compound 15.1

[0199]

[0200] P-hydroxyphenyl valeric acid butyl ester (1.00 g, 4.00 mmol) and hexamethylenetetramine (1.68 g, 11.98 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100 °C for 24 h under nitrogen protection, then cooled to 70 °C, 50 mL HCl (4 M) was added, and stirring was continued for 3 h, after cooling to room temperature, dichloromethane extraction, concentration, filtration and silica gel column purification, compound 15.1 (0.94 g, 3.07 mmol, Y = 77%) was obtained as a yellow solid.

[0201] S2 Preparation of compound 15

[0202]

[0203] Compound 8.1 (1 g, 3.95 mmol) and compound 15.1 (550 mg, 1.80 mmol) were dissolved in ethanol solution, 3 drops of ammonia water were added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 15 (341 mg, 0.44 mmol, Y = 24%).1H NMR (400 MHz, Chloroform-d) δ 9.97 (s, 1H), 7.20 (d, J = 4.1 Hz, 4H), 7.06 (t, J = 1.0 Hz, 2H), 4.08 (t, J = 7.0 Hz, 2H), 2.73 - 2.54 (m, 2H), 2.33 (t, J = 7.0 Hz, 2H), 1.77 - 1.62 (m, 2H), 1.62 - 1.48 (m, 5H), 1.48 - 1.26 (m, 7H), 0.90 (t, J = 7.9 Hz, 3H).

[0204] Example 16

[0205] Preparation of polycyanine near-infrared fluorescent dye compound 16

[0206] S1 Preparation of compound 16.1

[0207]

[0208] Dodecyl oxyphenol (1.00 g, 3.59 mmol) and hexamethylenetetramine (1.51 g, 10.77 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100 °C for 24 h under nitrogen protection, then cooled to 70 °C, 50 mL HCl (4 M) was added, and stirring was continued for 3 h, after cooling to room temperature, dichloromethane extraction, concentration, filtration and silica gel column purification to obtain compound 16.1 (954 mg, 2.85 mmol, Y = 79%) as a yellow solid.

[0209] S2 Preparation of compound 16

[0210]

[0211] Compound 1.1 (1 g, 5.02 mmol) and compound 16.1 (763 mg, 2.28 mmol) were dissolved in isopropanol solution, 10 mg sodium hydroxide was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 16 (514 mg, 0.74 mmol, Y=32%).1H NMR (400MHz, Chloroform-d) δ 7.17 (s, 4H), 6.88 (s, 2H), 4.04 (t, J = 7.1 Hz, 2H), 1.76 (p, J = 7.1 Hz, 2H), 1.50 (s, 12H), 1.42 (dq, J = 7.6, 6.7 Hz, 2H), 1.32 - 1.18 (m, 17H), 0.94 - 0.84 (m, 3H).

[0212] Example 17

[0213] Preparation of polycyanine near-infrared fluorescent dye compound 17

[0214] S1 Preparation of compound 17.1

[0215]

[0216] Octadecylphenol (1.00 g, 2.88 mmol) and hexamethylenetetramine (1.21 g, 8.66 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100 °C for 24 h under nitrogen protection, then cooled to 70 °C, 50 mL HCl (4M) was added, and stirred for another 3 h, extracted with dichloromethane after cooling to room temperature, concentrated, filtered and purified by silica gel column to obtain compound 17.1 (576 mg, 1.43 mmol, Y=50%) as a yellow solid.

[0217] S2 Preparation of compound 17

[0218]

[0219] Compound 1.1 (1 g, 5.02 mmol) and compound 17.1 (919 mg, 2.28 mmol) were dissolved in isopropanol solution, 10 mg sodium hydroxide was added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, concentrated, and the obtained crude product was purified by silica gel column to obtain compound 17 (431 mg, 0.56 mmol, Y=24%).1H NMR (400MHz, Chloroform-d) δ 7.17 (s, 4H), 7.13 (t, J = 1.0 Hz, 2H), 2.59 (tt, J = 7.1, 1.1 Hz, 2H), 1.60 (p, J = 7.1 Hz, 2H), 1.50 (s, 11H), 1.37 - 1.28 (m, 2H), 1.32 - 1.25 (m, 2H), 1.28 - 1.18 (m, 26H), 0.95 - 0.84 (m, 3H).

[0220] Example 18

[0221] Preparation of polycyanine near-infrared fluorescent dye compound 18

[0222] S1 Preparation of compound 18.1

[0223]

[0224] p-Hydroxyphenylacetic acid (1.00 g, 4.94 mmol) and hexamethylenetetramine (2.08 g, 14.83 mmol) were dissolved in 30 mL trifluoroacetic acid, stirred at 100 °C for 24 h under nitrogen protection, then cooled to 70 °C, 50 mL HCl (4M) was added, and stirred for another 3 h, cooled to room temperature, extracted with dichloromethane, concentrated, filtered and purified by silica gel column to obtain compound 18.1 (682 mg, 2.64 mmol, Y=53%) as a yellow solid.

[0225] S2 Preparation of compound 18

[0226]

[0227] Compound 11.2 (1 g, 3.28 mmol) and compound 18.1 (386 mg, 1.49 mmol) were dissolved in methanol solution, 3 drops of triethylamine were added to catalyze the reaction, stirred at reflux for 4 h, the reaction was cooled to room temperature, after concentration, the obtained crude product was purified by silica gel column to obtain compound 18 (395 mg, 0.47 mmol, Y = 32%).1H NMR (400 MHz, Chloroform-d) δ 7.39 - 7.25 (m, 11H), 7.18 (d, J = 15.1 Hz, 2H), 7.13 - 7.05 (m, 4H), 4.66 (dt, J = 12.4, 0.9 Hz, 2H), 4.52 (dt, J = 12.4, 0.9 Hz, 2H), 3.29 - 3.10 (m, 2H), 3.09 - 2.92 (m, 2H), 3.00 (s, 7H), 1.45 (s, 5H).

[0228] Test and result and analysis

[0229] (1) Identification of polycyanine structure near-infrared fluorescent dye compounds

[0230] High-precision, high-sensitivity high-resolution mass spectrometry analysis technology was used to identify the compounds. The results are shown in Figure 1 and Figure 2 .

[0231] The high-resolution mass spectrometry analysis results of compound 1 show that the mass spectrum ( Figure 1 ) exhibits a clear and sharp main peak, the position of which is highly consistent with the molecular weight calculated by the theoretical calculation of compound 1, and no obvious impurity peaks or fragment peaks are observed, which fully proves the purity of compound 1 and the correctness of its structure, indicating the successful preparation of compound 1. At the same time, the characteristic peak distribution in the mass spectrum is consistent with the functional groups and connection modes in the expected structure, further verifying the specified structure of compound 1. Similarly, Figure 2 shows the high-resolution mass spectrometry analysis results of compound 2. Similar to compound 1, the mass spectrum of compound 2 also presents a main peak, the position of which accurately corresponds to the theoretical molecular weight of compound 2, and the spectrum is clear and free of interference, reflecting the high purity and accuracy of the structure of compound 2.

[0232] (2) Test of ultraviolet-visible absorption spectrum and fluorescence spectrum and photophysical properties of fluorescent dye compounds

[0233] Dye preparation method

[0234] The dyes were accurately weighed using a balance with a precision of 1 / 10,000 after being subjected to strict vacuum drying. Subsequently, the weighed dyes were dissolved in dimethyl sulfoxide (DMSO) to prepare a dye stock solution with a concentration of 2 mmol / L. The stock solution was carefully aliquoted into brown sample bottles to prevent light interference with its stability and stored in a 4°C refrigerator to ensure chemical stability and activity during long-term storage. Before performing ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy tests, test solutions needed to be prepared. The specific steps were as follows: 12 μL of the dye stock solution was accurately measured using a high-precision micropipette, and it was slowly injected into a quartz cuvette containing 3 mL of the predetermined solvent (such as acetonitrile), and the solution was mixed evenly by gentle shaking. This operation diluted the dye concentration to 7.0 μmol / L, which was suitable for subsequent spectroscopic analysis.

[0235] Spectroscopic conditions and photophysical property test conditions

[0236] All spectroscopic tests were performed in a strictly controlled constant temperature environment of 25°C to eliminate the potential impact of temperature fluctuations on experimental results and ensure the repeatability and accuracy of the obtained data. During the photophysical property test, the accurately prepared dye stock solution was used as the test solution. According to the principle of Beer's law, the molar extinction coefficient of each dye was calculated by measuring the absorbance at a specific wavelength combined with the molar extinction coefficient calculation formula. The absolute fluorescence quantum yield of each dye sample was measured under the same conditions using a high-precision absolute fluorescence quantum yield instrument (Hamamatsu, model C11347) to comprehensively evaluate its fluorescence emission efficiency.

[0237] Results analysis

[0238] Figure 3 With Figure 5 The absorption spectra of compound 1 and compound 2 in acetonitrile solvent are shown respectively, and the results show that they have significantly different maximum absorption peaks, which are located at 685 nm and 645 nm respectively. Further, Figure 4 With Figure 6 The characteristics of the two compounds in fluorescence emission are revealed, and the maximum emission peaks of compound 1 and compound 2 are located at 772 nm and 659 nm respectively. It is worth noting that compound 1 exhibits a larger Stokes shift (87 nm), while the Stokes shift of compound 2 is relatively small (11 nm), and this significant difference is due to the different sensitivity of different substituents to the solvent environment, which in turn affects the energy loss in the electronic transition process.

[0239] It is particularly noted that the maximum emission wavelength of compound 2 does not change significantly even after specific activation treatment, indicating its good structural stability. Through targeted chemical modification, the spectral properties of such dyes, including absorption and emission wavelengths, can be finely tuned, providing broad possibilities for their application in cutting-edge technology fields such as near-infrared biofluorescence imaging, protein marker detection, etc.

[0240] The results of the photophysical property tests of compounds 1-18 are shown in Table 1:

[0241] Table 1: Photophysical property parameters of compounds 1-18

[0242]

[0243] Table 1 lists the key photophysical property parameters of compounds 1-18 as polycyanine structure near-infrared fluorescent dyes, including absorption wavelength, emission wavelength, molar extinction coefficient (ε), and absolute fluorescence quantum yield (φ). These data not only reveal the diversity and superiority of this class of dyes in spectral characteristics, but also reflect the significant achievements of the present solution in solving the problems of the prior art.

[0244] The absorption wavelengths of compounds 1-18 cover the near-infrared region from 617 nm to 692 nm, while the emission wavelengths are distributed between 647 nm and 772 nm. The wide wavelength coverage range allows these dyes to flexibly select the most suitable excitation and emission wavelengths for imaging and photodynamic therapy, optimizing the imaging and treatment effects. The significant Stokes shift exhibited by compound 1 helps to reduce self-absorption and scattering effects, improving the clarity and depth of imaging.

[0245] The molar extinction coefficients of compounds 1-18 are significantly higher than those of traditional polycyanine dyes, reaching 42552 L / mol -1 ·cm -1 (compound 8). High molar extinction coefficient means that the dye molecule has stronger light absorption ability, which can achieve effective light energy capture and conversion at lower concentration, thereby improving light energy utilization, reducing dye usage, and reducing potential biological toxicity. All compounds exhibit high fluorescence quantum yield, reaching 0.104 (compound 13). High fluorescence quantum yield means that the dye molecule can more effectively convert absorbed light energy into fluorescence emission, thereby enhancing the intensity and sensitivity of the imaging signal and improving the imaging quality.

[0246] (3) Fluorescent labeling of lysosomes in cells by activated compounds

[0247] In the cell experiment, MCF-7 cells were first seeded in a confocal culture dish and cultured to the logarithmic growth phase. Then, 2 μM concentration of compound 2 was added to the culture system, and after 10 minutes of incubation, a commercial lysosome localization dye was added for co-incubation for 15 minutes. The cells were observed using a confocal microscope, and the fluorescence channels of compound 2 and the commercial dye were overlaid and analyzed by software. As shown in Figure 7 , the green channel represents the fluorescence signal of the commercial lysosome probe, and the red channel represents the fluorescence signal of compound 2. The observation of the overlaid channel shows that there is a high degree of colocalization between compound 2 and lysosomes, and further quantitative analysis Figure 8 indicates that the colocalization coefficient is as high as 0.89, confirming the excellent performance of compound 2 as a lysosome-specific fluorescent labeling dye.

[0248] (4) Toxicity experiment of polycyanine structure near-infrared fluorescent dye compound

[0249] In order to comprehensively evaluate the biological safety and potential application value of compound 2, the cell toxicity and phototoxicity of compound 2 were systematically analyzed by MTT colorimetric method. MTT experiment is based on the principle that living cells can reduce MTT to form insoluble formazan crystals. By measuring the light absorption value of formazan dissolution products at 570 nm wavelength, the number of living cells is indirectly reflected.

[0250] Cell toxicity experiment: MCF-7 cells were evenly seeded in a 96-well plate, and after the cells adhered and grew stably, a series of concentration gradients (0-12 μmol / L) of compound 2 were added to different wells, and incubated for 24 hours. Then MTT experiment was performed, and the results showed Figure 9 that even at the highest test concentration (12 μmol / L), the cell survival rate remained at a high level, indicating that the polycyanine structure near-infrared fluorescent dye had good biocompatibility and no significant toxic side effects on cells within the working concentration range, suitable for application in biological and medical fields.

[0251] Phototoxicity experiment: Similarly, MCF-7 cells were cultured in a 96-well plate to a stable state, and then different concentrations of compound 2 (0-12 μmol / L) were added for 2 hours. Then the cells were irradiated with 660 nm laser, and incubated for 24 hours. The cell activity was detected by MTT experiment, and the results showed Figure 10 that with the increase of the concentration of compound 2, the cell survival rate decreased significantly, especially at the highest concentration, the cell survival rate decreased to about 50%. This result shows that the dye can effectively induce cell death under specific light conditions, showing good phototoxicity and potential application prospects for cancer treatment.

[0252] In summary, by changing the structure of the end group and the intermediate condensing agent, the present application successfully realizes the precise control of the dye's electron donating type, charge separation degree and electron mobility. The prepared polycyanine structure near-infrared fluorescent dye compound exhibits significant advantages in spectral characteristics. The molar extinction coefficient is higher than that of traditional polycyanine dyes, and the absorption capacity of light energy is strong, which helps to improve the light energy utilization rate and reduce the dye dosage. The absolute fluorescence quantum yield reaches a high level, significantly enhances the intensity and sensitivity of the imaging signal, and provides a more sensitive and reliable tool for biological imaging and molecular detection. The fluorescent dye compound has excellent biocompatibility and phototoxicity, ensuring the safety of the dye in the application of biological and medical fields and providing strong support for its application in the field of cancer treatment.

[0253] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A polycyanino structured near-infrared fluorescent dye, characterized by, It has the following structure: Or Or Or has the structure of general formula I: Wherein, R1 is selected from one of O, NH, NCH3; R2 and R3 are each independently selected from one of methyl, hydroxyl, phenyl, trifluoromethyl, alkoxy with 1~18 carbons; R4 is selected from one of hydrogen, halogen, aldehyde group, phenyl, alkyl with 1~18 carbons, carboxyalkyl with 1~18 carbons, alkyl sulfonic acid group with 1~18 carbons, ester group with 1~18 carbons, alkyl sulfonate with 1~18 carbons, hydroxyl alkyl with 1~18 carbons.

2. A method for preparing a polycyanino structured near infrared fluorescent dye, characterized by, The polycyanine structure near-infrared fluorescent dye has the structure of general formula I, wherein the selection of R1, R2, R3 and R4 is the same as that in claim 1, and the preparation method comprises the following steps: S1: Compound Y4 and hexamethylenetetramine are dissolved in trifluoroacetic acid, stirred at 80~120℃ under inert gas protection for 24~36h, then cooled to 60~70℃, continue to stir under acidic conditions for 3~6h, cooled to room temperature, extracted with organic solvent, concentrated, filtered and purified by silica gel column to obtain compound Y5; S2: Compound Y3 and Y5 are dissolved in an organic solvent, a basic catalyst is added, and reflux reaction is carried out under inert gas protection for 1~5h, after the reaction is completed, concentrated and purified by silica gel column to obtain the polycyanine structure near-infrared fluorescent dye I.

3. The method for preparing a near-infrared fluorescent dye with a polycyano structure according to claim 2, characterized in that, The molar ratio of Y4 and hexamethylenetetramine is 1:2~4.

4. The method for preparing a near-infrared fluorescent dye with a polycyano structure according to claim 3, characterized in that, The preparation method of the compound Y3: In an organic solvent, compound Y1 and malononitrile are added in a molar ratio of 1:2~4, after stirring, a basic catalyst is added, the molar ratio of Y1 to the basic catalyst is 1:0.1~0.3, after reflux reaction for 0.5~2h, cooling, filtering, washing and drying, the compound Y3 with R1 being O is obtained, which is denoted as compound Y3-1.

5. The method for preparing a near-infrared fluorescent dye with a polycyano structure according to claim 3, characterized in that, The preparation method of the compound Y3: Compound Y2 and malononitrile dimer are added, the molar ratio of Y2 to malononitrile dimer is 1:1~3, the mixture is dissolved in an organic solvent, stirred under basic conditions for 2~4h, after cooling to 0~5℃, filtering, washing and drying, the compound Y3 with R1 being NH and R3 being hydroxyl is obtained, which is denoted as compound Y3-2.

6. The method for preparing a near-infrared fluorescent dye with a polycyano structure according to claim 5, characterized in that, The preparation method of the compound Y3: Compound Y3-2 and compound Y01 are dissolved in an organic solvent in a molar ratio of 1:1~3, uniformly mixed and cooled to 0~5℃, trifluoroacetic acid is slowly added dropwise, stirred for 10~14h, after rising to room temperature, continue to stir for 24~36h, then recrystallized in methyl tert-butyl ether at 0~5℃, filtered, washed and purified by silica gel column to obtain the compound Y3 with R1 being NH, R3 being one of methyl, phenyl and trifluoromethyl, which is denoted as compound Y3-3.

7. The method for preparing a near-infrared fluorescent dye with a polycyano structure according to claim 5, characterized in that, The preparation method of the compound Y3: The compound Y3-2 and the compound Y02 in a molar ratio of 1:1 to 1:3 are dissolved in an organic solvent, mixed uniformly, cooled to 0 to 5℃, slowly added with trifluoroacetic acid, stirred for 10 to 14 h, continuously stirred for 24 to 36 h after being raised to room temperature, then recrystallized in methyl tert-butyl ether at 0 to 5℃, filtered, washed and purified by a silica gel column to obtain a compound Y3 with R1 being NH, R3 being methoxy and one of alkoxy groups with 1 to 18 carbons, denoted as compound Y3-3.

8. The process for preparing a polycyano structure near infrared fluorescent dye according to claim 6 or 7, characterized by, The preparation method of the compound Y3: The compound Y3-3 is added to an organic solvent, and a basic catalyst is added, the molar ratio of the Y3-3 to the basic catalyst being 1:0.1 to 0.3, stirred for 1 to 3 h under inert gas protection, then added with iodomethane, stirred for 24 to 32 h at 40 to 70℃, cooled to room temperature, concentrated, filtered and purified by a silica gel column to obtain a compound Y3 with R1 being NCH3, denoted as compound Y3-4.

9. The process for preparing a polycyanine near-infrared fluorescent dye according to any one of claims 2 to 7, characterized by, The organic solvent is at least one selected from toluene, ethanol, methanol, isopropanol, acetonitrile, tetrahydrofuran; the basic catalyst is at least one selected from sodium hydroxide, potassium hydroxide, ammonia, sodium bicarbonate, sodium ethoxide, sodium methoxide, pyridine, piperidine, triethylamine, trimethylamine, diisopropylethylamine.

10. Use of the polycyanine structure near-infrared fluorescent dye according to claim 1 in the preparation of a cell, tumor imaging preparation or a photodynamic and photothermal treatment drug.

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