Benzobisthiadiazole near-infrared II fluorescent dyes, their preparation methods, and applications

Abstract

This invention provides a benzobisthiadiazole-based near-infrared II fluorescent dye molecule and its preparation method. Using a benzobisthiadiazole derivative as a raw material, 3,4-ethylenedioxythiophene is introduced via a Stille cross-coupling reaction; after bromination with N-bromosuccinimide, a cyano-modified dibenzocarbazole derivative is introduced via a Suzuki cross-coupling reaction; finally, a terminally cyano-functionalized near-infrared II fluorescent small molecule dye is synthesized through reduction and cyclization reactions. The acceptor-donor-acceptor-donor-acceptor (A'-D-A-D-A') structure near-infrared II fluorescent molecule disclosed in this invention possesses properties such as long absorption wavelength, high fluorescence quantum yield, and high fluorescence brightness, and has broad application prospects in the field of near-infrared II fluorescence imaging.

Description

Technical Field

[0001] This invention relates to the field of near-infrared II biofluorescent dye technology, and more specifically to a benzobisthiadiazole near-infrared II fluorescent dye molecule, its preparation method, and its application. Background Technology

[0002] Optical imaging, which involves no ionizing radiation and offers high resolution, is widely used in biomedical imaging. The range of optical imaging can be specifically divided into the visible light region (400–700 nm) and the near-infrared region (NIR, 700–1700 nm). Near-infrared imaging, with its low tissue light scattering and weak autofluorescence, significantly improves penetration depth and imaging quality, and holds great promise in biomedical imaging and life sciences.

[0003] Currently, the near-infrared I fluorescent dye indocyanine green (ICG) has been approved by the U.S. Food and Drug Administration (FDA) for clinical diagnostic applications including angiography and tumor resection. Recent studies have found that with increasing wavelength, the near-infrared II region (NIR-II, 900–1700 nm) exhibits higher resolution and signal-to-noise ratio in in vivo imaging. Therefore, a large number of NIR-II fluorescent probes have been designed and synthesized to date, including rare-earth nanocrystals, inorganic quantum dots, and organic fluorescent dyes.

[0004] Because the combination of strong electron donors and acceptors can reduce the molecular band gap, most NIR-II organic dyes have a donor-acceptor-donor (DAD) structure. These molecules exhibit strong absorption and emission, and their structural units can be arbitrarily combined and controlled in design to obtain fluorescent molecules with longer wavelengths and higher fluorescence quantum yields. However, due to band structure rules, the nonradiative transition rate constant typically increases exponentially as the band between the excited and ground states decreases. Therefore, developing NIR-II organic dyes with high fluorescence quantum yields is a significant challenge.

[0005] Current research focuses on modulating intramolecular interactions within aggregates by introducing rotor structures or employing aggregation-induced emission (AIE) strategies to enhance the fluorescence quantum yield of NIR-II organic dyes. For example, Tang et al. significantly improved the fluorescence quantum yield of aggregates by cleverly incorporating triarylamine or tetraphenylethylene functional groups at both ends of the molecular acceptor (J. Am. Chem. Soc. 2022, 144, 15391; Adv. Mater. 2024, 2411133; Angew. Chem. Int. Ed. 2024, 63, e202318609.). However, these molecularly designed fluorescent dyes often encounter fluorescence quenching challenges in organic solvents, accompanied by low molar extinction coefficients, making it difficult to maintain strong fluorescence properties in organic solvents and limiting their applications. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a benzobisthiadiazole-based near-infrared II fluorescent dye molecule. Through a strategy of finely controlling the molecular charge distribution, the obtained fluorescent dye molecule can not only effectively improve the fluorescence quantum yield, but also significantly enhance the light absorption capacity of the dye molecule, and maintain strong fluorescence properties in organic solvents.

[0007] According to a first aspect of the present invention, a benzobisthiadiazole near-infrared II fluorescent dye molecule is provided, the fluorescent dye molecule being designated BBTPhCzCN, and having a chemical structure as shown in Formula I:

[0008]

[0009] According to a second aspect of the present invention, a method for preparing the aforementioned benzobisthiadiazole near-infrared II fluorescent dye molecule is provided, comprising the following steps:

[0010] S1,5,9-dibromo-7H-dibenzo[C,G]carbazole was reacted with cuprous cyanide in DMF solvent by heating to obtain the first intermediate;

[0011] S2. Under the conditions of potassium acetate as base, DMF as solvent, and palladium dichloride as catalyst, the first intermediate reacts with pinacol diboronate in a Miyaura reaction to obtain the second intermediate.

[0012] S3. Under the conditions of potassium carbonate as base, THF as solvent, and tetra(triphenylphosphine)palladium as catalyst, the second intermediate is coupled with a benzobisthiadiazole derivative via Suzuki coupling to obtain the third intermediate.

[0013] S4. The third intermediate undergoes a reduction reaction with iron powder in acetic acid medium. After the reaction is complete, the product is extracted and dried. The crude product obtained after drying is then dissolved in chloroform solvent and cyclized with N-sulfinylaniline using triethylamine as base and trimethylchlorosilane as catalyst. After extraction and column chromatography, a benzobisthiadiazole near-infrared II fluorescent dye molecule is obtained, denoted as BBTPhCzCN.

[0014] As an optional implementation, the chemical structure of the first intermediate is shown in Formula II:

[0015]

[0016] As an optional implementation, the chemical structure of the second intermediate is shown in Formula III:

[0017]

[0018] As an optional implementation, the chemical structure of the third intermediate is shown in Formula IV:

[0019]

[0020] As an optional implementation, 5,9-dibromo-7H-dibenzo[C,G]carbazole is heated in DMF solvent with cuprous cyanide under reflux at 140°C to 180°C for 8 to 12 hours, and column chromatography is used to obtain the first intermediate; wherein the molar ratio of 5,9-dibromo-7H-dibenzo[C,G]carbazole to cuprous cyanide is 1:(1.2 to 2).

[0021] As an optional implementation, under the conditions of potassium acetate as a base and DMF as a solvent, the first intermediate and pinacol diborate are heated and reacted at 100℃~120℃ for 12h~18h, extracted, dried, and subjected to column chromatography to obtain the second intermediate; wherein, the molar ratio of the first intermediate, pinacol diborate, potassium acetate, and bis(triphenylphosphine)palladium dichloride is 1:(1.2~2):(2.5~4):0.1.

[0022] As an optional implementation, under the conditions of potassium carbonate as base, THF as solvent, and tetra(triphenylphosphine)palladium as catalyst, the second intermediate and the benzobisthiadiazole derivative are refluxed at 65℃~75℃ for 36h~48h, extracted, dried, and subjected to column chromatography to obtain the third intermediate; wherein, the molar ratio of the second intermediate, potassium carbonate, tetra(triphenylphosphine)palladium, and benzobisthiadiazole derivative is (2~3):(6~8):0.1:1.

[0023] As an optional implementation, the third intermediate is reacted with iron powder in acetic acid medium at 70°C to 85°C for 6 to 8 hours, extracted, and dried to obtain a crude product; wherein the molar ratio of the third intermediate to iron powder is 1:(8 to 12).

[0024] The dried crude product was dissolved in chloroform solvent, and N-sulfinyl aniline was reacted with triethylamine as base and trimethylchlorosilane as catalyst at 45℃~55℃ for 12h~18h. After extraction, drying, and column chromatography, benzobisthiadiazole near-infrared II fluorescent dye molecules were obtained. The molar ratio of the dried crude product, N-sulfinyl aniline, triethylamine, and trimethylchlorosilane was 1:(4~6):(8~12):(4~6).

[0025] According to a third aspect of the present invention, the aforementioned benzobisthiadiazole near-infrared II fluorescent dye molecules are provided for use in the preparation of near-infrared II fluorescent imaging reagents, fluorescent probes, fluorescent sensors, photothermal agents for tumor photothermal therapy, surgical navigation imaging reagents, and optoelectronic devices.

[0026] As can be seen from the above technical solutions of the present invention, the benzobisthiadiazole near-infrared II fluorescent dye molecule proposed in this invention uses terminal cyano functionalization to modify the benzobisthiadiazole dye, obtaining a special acceptor-donor-donor-acceptor (A'-DAD-A') electronic structure. This structure can effectively adjust the molecular charge distribution, reduce the nonradiative transition rate of the molecule, and improve the fluorescence quantum yield. In addition, the strong chemical bond stretching vibration in the C≡N group of the fluorescent dye molecule can effectively enhance the structural rigidity of the molecule, thereby enhancing the molar extinction coefficient, suppressing the fluorescence quenching phenomenon caused by the expansion of the π conjugated system, and resulting in strong fluorescence brightness.

[0027] The benzobisthiadiazole near-infrared II fluorescent dye molecule proposed in this invention has excellent photophysical properties. In tetrahydrofuran solution, its ultraviolet absorption spectrum has a maximum absorption wavelength of 700 nm, and it exhibits strong fluorescence emission between 900 and 1200 nm, with a fluorescence emission tail peak exceeding 1300 nm. It also has good optical absorption performance, high fluorescence quantum yield, strong fluorescence brightness, large imaging depth, and good biocompatibility in the near-infrared region. It has great application prospects in bioimaging, fluorescent probes, fluorescent sensors, tumor therapy, surgical navigation, and optoelectronic devices. Attached Figure Description

[0028] Figure 1 This is a synthetic circuit diagram of the benzobisthiadiazole near-infrared II fluorescent dye molecule of the present invention.

[0029] Figure 2 This is the 1H-NMR spectrum of compound BBTPhCzCN in the example of this invention.

[0030] Figure 3 This is the 13C-NMR spectrum of compound BBTPhCzCN in the example of this invention.

[0031] Figure 4 This is the MALDI-TOF-Mass spectrum of compound BBTPhCzCN in the example of this invention.

[0032] Figure 5 This is a fluorescence quantum yield test graph of compound BBTPhCzCN in tetrahydrofuran, as described in this invention example.

[0033] Figure 6 This is a dynamic light scattering test image of the BBTPhCzCN nanoparticles in the example of this invention.

[0034] Figure 7 This is the ultraviolet absorption spectrum of the BBTPhCzCN nanoparticles in the example of this invention.

[0035] Figure 8 This is the fluorescence emission spectrum of the BBTPhCzCN nanoparticles in the example of this invention.

[0036] Figure 9 This is a near-infrared II fluorescence imaging depth test image of the BBTPhCzCN nanoparticles in the example of this invention.

[0037] Figure 10 These are near-infrared II region in vivo vascular fluorescence imaging test images of the BBTPhCzCN nanoparticles in this invention example; wherein, 10a is a fluorescence imaging image of the hind limb blood vessels of a mouse, 10b is a fluorescence imaging image of the brain blood vessels of a mouse, 10c is a fluorescence intensity analysis image of the red line region of the hind limb blood vessels of a mouse, and 10d is a fluorescence intensity analysis image of the red line region of the brain blood vessels of a mouse.

[0038] Figure 11 This is a near-infrared II region fluorescence imaging test image of the leg lymph nodes of the BBTPhCzCN nanoparticles in this invention example; wherein, 11a is a fluorescence imaging image of the mouse leg lymph nodes, and 11b is a fluorescence intensity analysis image of the red line region of the leg lymphatic vessels.

[0039] Figure 12 This is a fluorescence imaging image of the BBTPhCzCN nanoparticles in the example of this invention at the tumor site in a breast cancer model nude mouse. Detailed Implementation

[0040] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0041] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0042] This invention constructs a terminal cyano-functionalized benzobisthiadiazole NIR-II small molecule fluorescent dye through a strategy of finely controlling molecular charge distribution. Using a benzobisthiadiazole derivative as a raw material, 3,4-ethylenedioxythiophene is introduced via a Stille cross-coupling reaction; after bromination with N-bromosuccinimide, a cyano-modified dibenzocarbazole derivative is introduced via a Suzuki cross-coupling reaction; finally, a terminal cyano-functionalized near-infrared II fluorescent small molecule dye is synthesized through reduction and cyclization reactions. The synthesis process is simple, easy to purify, and yields a high amount of dye.

[0043] The obtained fluorescent dye small molecule has a well-defined structure with a unique A'-DAD-A' electronic structure. The near-infrared II fluorescent molecule has properties such as long absorption wavelength, high fluorescence quantum yield, and high fluorescence brightness, and has broad application prospects in the field of near-infrared II fluorescence imaging.

[0044] The fluorescent dye synthesized using this novel molecular design strategy can still maintain strong fluorescence properties in organic solvents, opening up a new path for the design of NIR-II fluorescent molecules.

[0045] Benzobisthiadiazole near-infrared II fluorescent dye molecules

[0046] In an exemplary embodiment of the present invention, a benzobisthiadiazole near-infrared II fluorescent dye molecule is provided, denoted as BBTPhCzCN, and its chemical structure is shown in Formula I:

[0047]

[0048] Preparation method

[0049] Combination Figure 1 The reaction route shown illustrates the exemplary method for preparing benzobisthiadiazole near-infrared II fluorescent dye molecules of this invention, comprising the following steps:

[0050] Step (1): Dissolve 5,9-dibromo-7H-dibenzo[C,G]carbazole (compound 1) and cuprous cyanide in N,N-dimethyldiamide (DMF), heat under reflux, and purify to obtain the first intermediate (compound 2).

[0051] Step (2): Compound 2 and pinacol diboronate were dissolved in DMF under a nitrogen atmosphere and heated under reflux with potassium acetate and palladium dichloride as catalysts to purify and obtain the second intermediate (compound 3).

[0052] Step (3): Compound 3 and benzobisthiadiazole derivative (compound 4) were dissolved in tetrahydrofuran (THF) under a nitrogen atmosphere, and then heated under reflux in potassium carbonate solution and tetra(triphenylphosphine)palladium catalysis to purify and obtain the third intermediate (compound 5).

[0053] Step (4): Dissolve compound 5 in acetic acid, add iron powder as a reducing agent under a nitrogen atmosphere, heat the reaction to obtain crude product, further dissolve it with N-sulfinylaniline in chloroform, heat the reaction under the catalysis of triethylamine and trimethylchlorosilane, and purify to obtain the final product (BBTPhCzCN).

[0054] The chemical structure of the first intermediate is shown in Formula II:

[0055]

[0056] The chemical structure of the second intermediate is shown in Formula III:

[0057]

[0058] The chemical structure of the third intermediate is shown in Formula IV:

[0059]

[0060] The chemical structural formula of compound 4 is shown in formula V:

[0061]

[0062] As an optional example, 5,9-dibromo-7H-dibenzo[C,G]carbazole was heated with cuprous cyanide in DMF solvent under reflux at 140°C to 180°C for 8 to 12 hours, and column chromatography was used to obtain the first intermediate; wherein the molar ratio of 5,9-dibromo-7H-dibenzo[C,G]carbazole to cuprous cyanide was 1:(1.2 to 2).

[0063] In a more specific example, 5,9-dibromo-7H-dibenzo[C,G]carbazole was heated with cuprous cyanide in DMF solvent under reflux at 160°C for 12 h, and column chromatography was used to obtain the first intermediate; wherein the molar ratio of 5,9-dibromo-7H-dibenzo[C,G]carbazole to cuprous cyanide was 1:1.5.

[0064] As an optional example, under the conditions of potassium acetate as base, DMF as solvent, and bis(triphenylphosphine)palladium dichloride as catalyst, the first intermediate and pinacol diborate are heated and reacted at 100℃~120℃ for 12h~18h, extracted, dried, and subjected to column chromatography to obtain the second intermediate; wherein the molar ratio of the first intermediate, pinacol diborate, potassium acetate, and bis(triphenylphosphine)palladium dichloride is 1:(1.2~2):(2.5~4):0.1.

[0065] In a more specific example, under the conditions of potassium acetate as a base and DMF as a solvent, the first intermediate and pinacol diborate were heated and reacted at 100°C for 12 h, extracted, dried, and subjected to column chromatography to obtain the second intermediate; wherein the molar ratio of the first intermediate, pinacol diborate, potassium acetate, and bis(triphenylphosphine)palladium dichloride was 1:1.5:3:0.1.

[0066] As an optional example, under the conditions of potassium carbonate as base, THF as solvent, and tetra(triphenylphosphine)palladium as catalyst, the second intermediate and the benzobisthiadiazole derivative are refluxed at 65℃~75℃ for 36h~48h, extracted, dried, and subjected to column chromatography to obtain the third intermediate; wherein the molar ratio of the second intermediate, potassium carbonate, tetra(triphenylphosphine)palladium, and benzobisthiadiazole derivative is (2~3):(6~8):0.1:1.

[0067] In a more specific example, under the conditions of potassium carbonate as base, THF as solvent, and tetra(triphenylphosphine)palladium as catalyst, the second intermediate and the benzobisthiadiazole derivative were refluxed at 70°C for 48 h, extracted, dried, and subjected to column chromatography to obtain the third intermediate; wherein the molar ratio of the second intermediate, potassium carbonate, tetra(triphenylphosphine)palladium, and benzobisthiadiazole derivative was 2.5:7.5:0.1:1.

[0068] As an optional example, the third intermediate is reacted with iron powder in acetic acid medium at 70℃~85℃ for 6h~8h, extracted, and dried to obtain crude product; wherein the molar ratio of the third intermediate to iron powder is 1:(8~12).

[0069] The dried crude product was dissolved in chloroform solvent, and N-sulfinyl aniline was reacted with triethylamine as base and trimethylchlorosilane as catalyst at 45℃~55℃ for 12h~18h. After extraction, drying, and column chromatography, benzobisthiadiazole near-infrared II fluorescent dye molecules were obtained. The molar ratio of the dried crude product, N-sulfinyl aniline, triethylamine, and trimethylchlorosilane was 1:(4~6):(8~12):(4~6).

[0070] In a more specific example, the third intermediate was reacted with iron powder in acetic acid medium at 85°C for 8 hours, extracted, and dried to obtain the crude product; wherein the molar ratio of the third intermediate to iron powder was 1:10.

[0071] The dried crude product was dissolved in chloroform solvent, and reacted with N-sulfinyl aniline at 50 °C for 12 h using triethylamine as base and trimethylchlorosilane as catalyst. After extraction, drying, and column chromatography, benzobisthiadiazole near-infrared II fluorescent dye molecules were obtained. The molar ratio of the dried crude product, N-sulfinyl aniline, triethylamine, and trimethylchlorosilane was 1:5:10:5.

[0072] application

[0073] In another exemplary embodiment of the present invention, the application of the aforementioned benzobisthiadiazole near-infrared II fluorescent dye molecules in the preparation of near-infrared II fluorescent imaging reagents, fluorescent probes, fluorescent sensors, photothermal agents for tumor photothermal therapy, surgical navigation imaging reagents, and optoelectronic devices is also provided.

[0074] In one preferred embodiment, the aforementioned benzobisthiadiazole near-infrared II fluorescent dye molecules are prepared into nanoparticles by nanoprecipitation and used as reagents for bioimaging.

[0075] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0076] Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0077] Example 1

[0078] [according to Figure 1 The synthetic route was used to synthesize BBTPhCzCN]

[0079] (1) Compound 1 (4.15 g, 7 mmol), cuprous cyanide (0.95 g, 10.5 mmol), and N,N-dimethyldiamide (30 mL) were added to a dry 250 mL double-necked flask. The mixture was heated to 150 °C under a nitrogen atmosphere and reacted for 12 hours. The mixture was then cooled to room temperature, filtered with diatomaceous earth, and the filtrate was collected. The mixture was extracted with ethyl acetate, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:15) to obtain a pale yellow oily substance, compound 2 (1.43 g, yield 38%).

[0080] (2) Compound 2 (1.08 g, 2 mmol), pinacol diborate (0.76 g, 3 mmol), potassium acetate (0.59 g, 6 mmol), and anhydrous N,N-dimethyldiamide (20 mL) were added to a dry 100 mL double-necked flask and bubbled for 5 minutes under a nitrogen atmosphere. Then, the catalyst bis(triphenylphosphine)palladium dichloride (140 mg, 0.2 mmol) was added under a nitrogen atmosphere and the temperature was raised to 100 °C. After reacting for 12 hours, the mixture was cooled to room temperature, extracted with ethyl acetate, and the solvent was removed by vacuum distillation. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 1:15) to give compound 3 (0.91 g, yield 78%) as a yellow oil.

[0081] (3) Compound 3 (586 mg, 1 mmol), compound 4 (265 mg, 0.4 mmol), potassium carbonate aqueous solution (1 M, 3 mL), and tetrahydrofuran (20 mL) were added to a dry 100 mL double-necked flask. The mixture was bubbled under a nitrogen atmosphere for 5 minutes. Then, the catalyst tetra(triphenylphosphine)palladium (28 mg, 0.04 mmol) was added under a nitrogen atmosphere and the temperature was raised to 70 °C. After reacting for 48 hours, the mixture was cooled to room temperature and extracted with dichloromethane. The solvent was removed by vacuum distillation. The crude product was purified by column chromatography (dichloromethane: petroleum ether = 2:1) to obtain a black solid compound 5 (170 mg, yield 30%).

[0082] (4) Compound 5 (71 mg, 0.05 mmol), reduced iron powder (28 mg, 0.5 mmol), and anhydrous acetic acid (10 mL) were added to a dry 100 mL double-necked flask. The reaction was heated to 85 °C and stirred vigorously for 8 hours under a nitrogen atmosphere. After the reaction was completed, the reaction was cooled to room temperature, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain an orange solid product, which could be used in the next step without further purification.

[0083] The above-mentioned orange solid (55 mg, 0.04 mmol), N-sulfinylaniline (28 mg, 0.2 mmol), trimethylchlorosilane (22 mg, 0.2 mmol), triethylamine (40 mg, 0.4 mmol), and anhydrous chloroform (10 mL) were added to a dry 100 mL double-necked flask. The mixture was refluxed and heated for 12 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was cooled to room temperature and extracted with dichloromethane. The solvent was removed by vacuum distillation. The crude product was purified by column chromatography (dichloromethane: petroleum ether = 1:1) to obtain the green product BBTPhCzCN (35 mg, yield 62.8%).

[0084] like Figure 2 As shown, 1H NMR (400MHz, CDCl3): δppm 9.21-9.12(m,4H),8.46(t,J=8.0Hz,4H),8.14(s,2H),7.97(s,2H),7.77-7.62(m,8H),4.54 (t,J=7.2Hz,4H),4.45(s,8H),1.99-1.92(m,4H),1.46-1.23(m,36H),0.84(t,J=7.2Hz,6H).

[0085] like Figure 3 As shown, 13 C NMR (101MHz, CDCl3): δppm 152.06,141.24,139.34,138.32,135.29,130.24,129.61,128.71,128.2 3,128.16,126.75,126.47,126.39,125.57,125.47,125.19,124.54,120. 99,120.02,117.83,117.46,113.95,113.39,110.95,106.74,64.93,42.43,31.97,30.22,29.69,29.66,29.61,29.49,29.40,27.35,22.76,14.23.

[0086] like Figure 4 As shown, MALDI-TOF MS Calculation for [M] + C 84 H 78 N8O4S4:1390.5029,found1391.3356.

[0087] Combination Figure 2-4 The chemical structure of BBTPhCzCN can be determined as shown in Formula I:

[0088]

[0089] Example 2

[0090] [Fluorescence quantum yield of BBTPhCzCN in tetrahydrofuran solution]

[0091] Add 2.5 mL of a tetrahydrofuran solution of BBTPhCzCN (prepared according to the method in Example 1) to a quartz cuvette, control its absorbance at 808 nm wavelength to be less than 0.1, and test its fluorescence spectrum.

[0092] like Figure 5As shown, using dye IR-1061 as a reference, its fluorescence quantum yield was measured to be 12.4%, indicating that it has a high fluorescence quantum yield in the organic phase, demonstrating that the BBTPhCzCN of the present invention can still maintain strong fluorescence characteristics in organic solvents.

[0093] Example 3

[0094] Preparation of BBTPhCzCN nanoparticles

[0095] BBTPhCzCN nanoparticles were prepared by a nanoprecipitation method, as detailed below:

[0096] BBTPhCzCN (1 mg) (prepared according to the method in Example 1) and DSPE-PEG 2000 Dissolve 10 mg of the solution in 1 mL of tetrahydrofuran. After sonicating for 5 minutes, quickly inject the solution into 10 mL of deionized water and sonicate for 30 minutes. After stirring and evaporating to completely remove the tetrahydrofuran, filter the solution through a 0.22 μm filter to obtain a clear and transparent green BBTPhCzCN nanoparticle solution. Store at 4 °C for later use.

[0097] like Figure 6 As shown, the dynamic light scattering particle size distribution test results of BBTPhCzCN nanoparticles show that their hydrated particle size is 80 nm, which meets the requirements of enhanced permeability and retention (EPR) effect.

[0098] Example 4

[0099] [UV absorption and fluorescence emission of BBTPhCzCN nanoparticles]

[0100] A solution of BBTPhCzCN nanoparticles (2.5 mL, prepared by the method in Example 2) was added to a quartz cuvette, and its ultraviolet absorption and fluorescence emission spectra were tested.

[0101] like Figure 7 , 8 As shown, BBTPhCzCN nanoparticles exhibit a maximum absorption peak of 700 nm and a maximum emission peak of 995 nm in water, with an emission tail exceeding 1300 nm. Using IR-1061 as a reference, their fluorescence quantum yield was measured to be 2.9%, and their fluorescence intensity was 279.04 MΩ. -1 cm -1 It has good potential for near-infrared II fluorescence imaging.

[0102] Example 5

[0103] [Near-infrared II fluorescence imaging depth measurement of BBTPhCzCN nanoparticles]

[0104] A 1% fat emulsion injection was used to simulate biological tissue, as shown in the attached... Figure 9 As shown, a solution of compound I BBTPhCzCN nanoparticles (100 μg / mL) was filled into a capillary, and fluorescence imaging was performed using a near-infrared II real-time imaging system.

[0105] As can be seen from the figure, as the thickness of the fat emulsion injection gradually increases to 6 mm, the capillary with BBTPhCzCN nanoparticles still maintains a signal-to-noise ratio of 2.7 in the fat emulsion injection, which is suitable for in vivo fluorescence imaging in the near-infrared II imaging window.

[0106] Example 6

[0107] [Near-infrared II fluorescence imaging of BBTPhCzCN nanoparticles in vivo]

[0108] like Figure 10 As shown, to investigate the vascular imaging capability of BBTPhCzCN nanoparticles in vivo, fluorescence imaging was performed using a near-infrared II real-time imaging system after BBTPhCzCN nanoparticles were injected into the tail vein.

[0109] As can be seen from the figure, the blood vessels in the hind limbs of the mouse ( Figure 10 a) and cerebral blood vessels ( Figure 10 b) can be clearly observed, indicating that BBTPhCzCN nanoparticles have good near-infrared II in vivo fluorescence imaging effects; fluorescence intensity analysis of the red line region shows that hind limb blood vessels ( Figure 10 c) and cerebral blood vessels ( Figure 10 d) The signal-to-noise ratios compared to muscle tissue were 2.4 and 2.7, respectively, indicating that the BBTPhCzCN nanoparticles of the present invention have good near-infrared II in vivo fluorescence imaging resolution.

[0110] Example 7

[0111] [Near-infrared II fluorescence imaging of leg lymph nodes using BBTPhCzCN nanoparticles]

[0112] The fluorescence imaging capability of BBTPhCzCN nanoparticles in leg lymph nodes was investigated by injecting them into the footpads of mice. Figure 11 As shown, the lymphatic vessels in the mouse's legs and the popliteal lymph nodes can be clearly observed. Figure 11 a) Fluorescence intensity analysis showed ( Figure 11b) The half-peak widths of the leg lymphatic vessels were 294, 187, 224, and 243 μm, respectively, indicating that the BBTPhCzCN nanoparticles of the present invention can perform high-resolution near-infrared II fluorescence imaging of fine lymphatic vessels.

[0113] Example 8

[0114] [Fluorescence imaging of BBTPhCzCN nanoparticles at tumor sites in breast cancer model nude mice]

[0115] A 4T1 tumor-bearing mouse model was established by subcutaneous injection of 4T1 cancer cells. BBTPhCzCN nanoparticles (200 μL, 200 μg / mL) were injected into mice via the tail vein, and fluorescence images of the tumor site were recorded at different time points (1, 2, 4, 8, 12, 24 hours).

[0116] like Figure 12 As shown, after intravenous injection of BBTPhCzCN nanoparticles into mice, the fluorescence signal at the tumor site gradually increased over time, reaching a maximum value at 12 hours, and then gradually weakened. This indicates that the BBTPhCzCN nanoparticles of the present invention can effectively accumulate at the tumor site and be used for tumor diagnosis.

[0117] As can be seen from the above tests, the benzobisthiadiazole near-infrared II fluorescent dye molecules of the present invention can suppress the fluorescence quenching phenomenon caused by the expansion of the π-conjugated system, maintain strong fluorescence characteristics in organic solvents, and have good optical absorption performance in the near-infrared region. They also have high fluorescence quantum yield, strong fluorescence brightness, large imaging depth, and good biocompatibility.

[0118] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A benzobisthiadiazole near-infrared II fluorescent dye molecule, characterized in that, The fluorescent dye molecule is designated BBTPhCzCN, and its chemical structure is shown in Formula I: Formula I.

2. A method for preparing the benzobisthiadiazole near-infrared II fluorescent dye molecule as described in claim 1, characterized in that, Includes the following steps: S1, 5,9-dibromo-7H-dibenzo[C,G]carbazole is reacted with cuprous cyanide in DMF solvent by heating to obtain a first intermediate; wherein, the expression of the 5,9-dibromo-7H-dibenzo[C,G]carbazole is: ； S2. Under the conditions of potassium acetate as base, DMF as solvent, and palladium dichloride as catalyst, the first intermediate reacts with pinacol diboronate in a Miyaura reaction to obtain the second intermediate. S3. Under the conditions of potassium carbonate as base, THF as solvent, and tetra(triphenylphosphine)palladium as catalyst, the second intermediate is coupled with a benzobisthiadiazole derivative via Suzuki coupling to obtain the third intermediate. S4. The third intermediate undergoes a reduction reaction with iron powder in acetic acid medium. After the reaction is complete, extraction and drying are performed. Subsequently, the crude product obtained after drying is dissolved in chloroform solvent, using triethylamine as the base and trimethylchlorosilane as the catalyst, and... N - Sulfinyl aniline undergoes a cyclization reaction, followed by extraction and column chromatography to obtain a benzobisthiadiazole near-infrared II fluorescent dye molecule, denoted as BBTPhCzCN.

3. The preparation method according to claim 2, characterized in that, The chemical structure of the first intermediate is shown in Formula II: Formula II.

4. The preparation method according to claim 2, characterized in that, The chemical structure of the second intermediate is shown in Formula III: Formula III.

5. The preparation method according to claim 2, characterized in that, The chemical structure of the third intermediate is shown in Formula IV: Formula IV.

6. The preparation method according to claim 2, characterized in that, 5,9-Dibromo-7H-dibenzo[C,G]carbazole was heated with cuprous cyanide in DMF solvent under reflux at 140 ℃~180 ℃ for 8 h~12 h, and column chromatography was used to obtain the first intermediate; wherein the molar ratio of 5,9-dibromo-7H-dibenzo[C,G]carbazole to cuprous cyanide was 1:(1.2~2).

7. The preparation method according to claim 2, characterized in that, Under the conditions of potassium acetate as base, DMF as solvent, and bis(triphenylphosphine)palladium dichloride as catalyst, the first intermediate and pinacol diboronate were heated and reacted at 100 ℃~120 ℃ for 12 h~18 h, extracted, dried, and subjected to column chromatography to obtain the second intermediate; wherein the molar ratio of the first intermediate, pinacol diboronate, potassium acetate, and bis(triphenylphosphine)palladium dichloride was 1:(1.2~2):(2.5~4):0.

1.

8. The preparation method according to claim 2, characterized in that, Under the conditions of potassium carbonate as base, THF as solvent, and tetra(triphenylphosphine)palladium as catalyst, the second intermediate and the benzobisthiadiazole derivative were refluxed at 65 ℃~75 ℃ for 36 h~48 h, extracted, dried, and subjected to column chromatography to obtain the third intermediate; wherein the molar ratio of the second intermediate, potassium carbonate, tetra(triphenylphosphine)palladium, and benzobisthiadiazole derivative was (2~3):(6~8):0.1:

1.

9. The preparation method according to claim 2, characterized in that, The third intermediate was reacted with iron powder in acetic acid medium at 70℃~85℃ for 6 h~8 h, extracted, and dried to obtain crude product; wherein the molar ratio of the third intermediate to iron powder was 1:(8~12). The dried crude product was dissolved in chloroform solvent, using triethylamine as a base and trimethylchlorosilane as a catalyst, and... N -Sylenylaniline was reacted at 45 ℃~55 ℃ for 12 h~18 h, extracted, dried, and subjected to column chromatography to obtain benzobisthiadiazole near-infrared II fluorescent dye molecules; among which, the crude product obtained after drying, N The molar ratio of sulfinyl aniline, triethylamine, and trimethylchlorosilane is 1:(4~6):(8~12):(4~6).

10. The application of the benzobisthiadiazole near-infrared II fluorescent dye molecule of claim 1 in the preparation of near-infrared II fluorescent imaging reagents, fluorescent probes, fluorescent sensors, photothermal agents for tumor photothermal therapy, surgical navigation imaging reagents, and optoelectronic devices.

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