Near-infrared fluorescent dye for brain imaging as well as preparation method and application of near-infrared fluorescent dye

Abstract

The invention relates to the field of fluorescent dyes, in particular to a near-infrared fluorescent dye for brain imaging as well as a preparation method and application of the near-infrared fluorescent dye. The near-infrared fluorescent dye for brain imaging has the advantages of novel structure, good biocompatibility, large Stokes shift, high light stability, high fluorescence signal-to-noise ratio and the like; the near-infrared fluorescent dye prepared by the invention is simple in preparation process, mild in reaction condition, short in synthesis path and beneficial to industrial production, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent dye technology for fluorescence analysis and biomedical imaging, specifically relating to a near-infrared fluorescent dye for brain imaging, its preparation method, and its application. Background Technology

[0002] Early diagnosis and monitoring of treatment for brain diseases is a significant challenge for neuroscience and clinical medicine. Due to the presence of the blood-brain barrier (BBB), traditional imaging techniques (such as MRI and CT) have limitations in terms of resolution, real-time performance, and molecular specificity. Optical imaging techniques, especially near-infrared fluorescence (NIR) imaging, have become important tools for brain research due to their high sensitivity, real-time dynamic monitoring capabilities, and low tissue background interference.

[0003] Near-infrared light (700-1700 nm) has deep tissue penetration (up to several centimeters) and low autofluorescence interference, making it suitable for in vivo deep tissue imaging. Currently used near-infrared dyes (such as indocyanine green (ICG) and other Cy series dyes) suffer from the following problems: poor brain penetration, insufficient photostability, susceptibility to photobleaching affecting long-term imaging, and low signal-to-noise ratio. To address these shortcomings, this invention, based on a previously developed near-infrared Rhodol fluorescent dye, introduces fluorine atoms at different sites on its molecular backbone to increase its lipid solubility and reduce its polarity, thereby improving its blood-brain barrier penetration efficiency, signal-to-noise ratio, and stability. This dye can be widely used in intraoperative navigation for brain tumor surgery and in the detection of biomarkers for brain-related neurodegenerative diseases. Summary of the Invention

[0004] One objective of this invention is to provide a novel near-infrared fluorescent dye for brain imaging. By substituting fluorine at different sites on the near-infrared Rhodol fluorescent dye, its lipophilicity is enhanced while its polarity is reduced, promoting BBB penetration. Furthermore, this molecule possesses a large Stokes shift, excellent blood-brain barrier penetration, and dynamic imaging capabilities, providing a promising chemical tool for brain imaging.

[0005] The second objective of this invention is to provide a method for preparing near-infrared fluorescent dyes for brain imaging.

[0006] A third objective of this invention is to provide the application of near-infrared fluorescent dyes for brain imaging.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A near-infrared fluorescent dye for brain imaging, the structural formula of which is shown below: In the formula, R1, R2, R3, R4, R9, R10 It is either hydrogen or fluorine.

[0008] Furthermore, the near-infrared fluorescent dye has the structural formula of any one of the following: , , .

[0009] The preparation method of the near-infrared fluorescent dye for brain imaging described above includes the following steps: In the formula, R1, R2, R3, R4, R9, R 10 It is hydrogen or fluorine; (1) Dissolve compound a in an organic solvent, add glyoxal, heat and stir to react, then separate and purify to obtain intermediate b; mix intermediate b with an organic solvent, add sodium borohydride and glacial acetic acid and heat under reflux, extract after the reaction is complete, then separate and purify to obtain intermediate c; (2) Dissolve compounds d and m in an organic solvent, add anhydrous aluminum chloride, heat and stir to react, then separate and purify to obtain intermediate n; (3) Dissolve intermediate c and intermediate n in an organic solvent, mix them, heat and stir to react, and then separate and purify to obtain the final product.

[0010] Further, in step (1), the heating and stirring reaction temperature is 60-85℃ and the time is 11-13 h; the heating and reflux reaction temperature is 115-125℃ and the time is 11-13 h; in step (2), the heating and stirring reaction temperature is 75-85℃ and the heating and stirring reaction time is 11-13 h; in step (3), the heating and stirring reaction temperature is 110-130℃ and the heating and stirring reaction time is 11-13 h.

[0011] Further, in step (1), the molar ratio of compound a to glyoxal is 1:(1-3), and the molar ratio of intermediate b, sodium borohydride, and glacial acetic acid is 1:10:10; in step (2), the molar ratio of compound d, compound m, and anhydrous aluminum chloride is 1:(1-3):(2-3); and in step (3), the molar ratio of intermediate c to intermediate n is 1:(1-2).

[0012] Further, in steps (1)-(3), the organic solvent is selected from any one or more of anhydrous acetonitrile, anhydrous toluene, nitrobenzene, methanesulfonic acid, and tetrahydrofuran.

[0013] The above describes the application of near-infrared fluorescent dyes for brain imaging.

[0014] Compared with the prior art, the beneficial effects of this invention are as follows: (1) The present invention provides a near-infrared fluorescent dye for brain imaging, which belongs to a novel Rhodol dye molecule. The fluorescent dye of the present invention enhances lipid solubility by fluorine substitution on the benzene ring, while reducing polarity and promoting BBB penetration, which provides a promising prospect for brain imaging and can realize the purpose of brain fluorescence imaging in a direct and dynamic manner.

[0015] (2) The fluorescent dye of the present invention has the advantages of good biocompatibility, large Stokes shift, high photostability, and high fluorescence signal-to-noise ratio.

[0016] (3) The near-infrared fluorescent dye prepared by the present invention has a simple preparation process, mild reaction conditions, and short synthesis route, which is conducive to industrial production. Attached Figure Description

[0017] Figure 1 This is the NMR spectrum of the fluorescent dye prepared for brain imaging in Example 1; Figure 2 This is the NMR spectrum of the fluorescent dye prepared for brain imaging in Example 2; Figure 3 This is the NMR spectrum of the fluorescent dye prepared for brain imaging in Example 3; Figure 4 The image shows the NMR spectrum of the fluorescent dye prepared for brain imaging in Comparative Example 1. Figure 5 The image shows the NMR spectrum of the fluorescent dye prepared for brain imaging in Comparative Example 2. Figure 6 These are the fluorescence spectra of the fluorescent dyes prepared in Examples 1-3 and Comparative Examples 1-2 in PBS; Figure 7 This is a time-tracking imaging image of the fluorescent dye prepared in Example 1; Figure 8 This is a time-tracking imaging image of the fluorescent dye prepared in Example 2; Figure 9 This is a time-tracking image of the fluorescent dye prepared in Comparative Example 1. Figure 10 This is a time-tracking image of the fluorescent dye prepared in Comparative Example 2; Figure 11 This is a time-tracking image of the fluorescent dye prepared in Example 3; Figure 12 These are mouse brain images of the fluorescent dyes prepared in Examples 1-3 and Comparative Examples 1-2; Figure 13 These are brain concentration maps of the fluorescent dyes prepared in Examples 1-3 and Comparative Examples 1-2. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0019] Example 1 A near-infrared fluorescent dye for brain imaging, the structural formula of which is shown in (I-1): (I-1) Example 1 also provides a method for preparing a fluorescent dye for brain imaging, the method comprising the following steps: In formula (I), R1, R2, R3, and R4 are fluorine.

[0020] (1) Compound a, 4-methoxy-o-phenylenediamine (1 mmol), was dissolved in 10 mL of anhydrous acetonitrile solution, followed by the addition of glyoxal (3 mmol) and stirring at 60 °C for 12 h. After the reaction was completed, the solvent was removed under reduced pressure, and the mixture was separated by column chromatography to obtain a yellowish-white solid powder intermediate b with a yield of 72%. Intermediate b (1 mmol) was dissolved in anhydrous toluene (50 mL), and NaBH4 (10 mmol) was added to the toluene over 30 min at 0 °C to obtain a pale yellow slurry. The mixture was stirred for 30 min, and glacial acetic acid (10 mmol) was added dropwise over 1 h at 10 °C. The brown slurry was stirred for another 2 h and then refluxed at 120 °C for 12 h. After the reaction was completed, the mixture was cooled, water was added, and the toluene layer was separated. The aqueous layer was extracted with ethyl acetate, and the combined extract and toluene layer were repeatedly washed with dilute NaCO3 and water. The mixture was dried over Na2SO4, and the solvent was removed by rotary evaporation (a dark brown oily substance). Passing the solution through a silica gel column yielded a golden-yellow oily intermediate c, with a yield of 51.3%.

[0021] (2) Compound d, tetrafluorophthalic anhydride (1 mmol), was dissolved in nitrobenzene (50 mL), followed by the addition of compound e, resorcinol (1 mmol) and anhydrous aluminum chloride (2.2 mmol). The mixture was stirred under nitrogen protection at 80 °C for 12 h. After the reaction was completed, the reaction mixture was poured into a two-phase solution of 30 mL of n-hexane and 40 mL of 0.5 M HCl under vigorous stirring. After stirring for 2 h, an orange-yellow precipitate was obtained, which was filtered to obtain the crude product. The product was then passed through a silica gel column to obtain an orange solid intermediate f, with a yield of 54%.

[0022] (3) Dissolve intermediate c (1 mmol) in 10 mL of methanesulfonic acid, then add intermediate f (1 mmol), stir and reflux at 120 °C for 12 h. After the reaction is complete, cool to room temperature, pour ice water into the reaction solution, then add a small amount of perchloric acid. A precipitate is formed. Filter to obtain crude product, pass through silica gel column to obtain purple compound (I-1), with a yield of 42%.

[0023] (4) The nuclear magnetic resonance spectrum of a fluorescent dye for brain imaging provided in Example 1 is as follows: Figure 1 As shown, the structural characterization results are as follows: 1 H NMR (300 MHz, DMSO-d6) δ 7.58 (d, J = 9.0 Hz, 1H), 7.44 (s, 1H), 7.35 (s, 1H), 7.21 (s, 1H), 6.23 (s, 1H), 4.01 (s, 4H), 3.56 (s, 4H), 1.44(s, 3H), 1.16(s, 3H).

[0024] Example 2 A near-infrared fluorescent dye for brain imaging, with the following structural formula: (I-2) This embodiment also provides a method for preparing a near-infrared fluorescent dye for brain imaging, the preparation method comprising the following steps: In formula (I), R1, R2, R3, and R4 are hydrogen.

[0025] (1) This step is the same as step (1) in Example 1, and the product obtained is denoted as intermediate c; (2) Intermediate d phthalic anhydride (1 mmol) was dissolved in nitrobenzene (50 mL), followed by the addition of compound m resorcinol (1 mmol) and anhydrous aluminum chloride (2.2 mmol). The mixture was stirred under nitrogen protection at 80 °C for 12 h. After the reaction was completed, the reaction mixture was poured into a two-phase solution of 30 mL n-hexane and 40 mL 0.5 M HCl under vigorous stirring. After stirring for 2 h, an orange-yellow precipitate was obtained, which was filtered to obtain the crude product. The product was then passed through a silica gel column to obtain intermediate f, with a yield of 48%.

[0026] (3) Dissolve intermediate c (1 mmol) in 10 mL of methanesulfonic acid, then add intermediate n (1 mmol), stir and reflux at 120 °C for 12 h. After the reaction is complete, cool to room temperature, pour ice water into the reaction solution, then add a small amount of perchloric acid. A precipitate is formed. Filter to obtain crude product, pass through silica gel column to obtain purple compound (I-2), with a yield of 61%.

[0027] (4) The NMR spectrum of a fluorescent dye used for brain imaging in this embodiment is as follows: Figure 2 As shown, the structural characterization results are as follows: 1 H NMR (300 MHz, DMSO-d6) δ 8.08 (d, J = 7.6 Hz, 1H), 7.82 (t, J = 7.4 Hz,2H), 7.33 (d, J = 7.6 Hz, 1H), 6.84 (s, 2H), 6.73 (d, J = 11.0 Hz, 2H), 5.62(s, 1H), 3.52 (s, 4H), 3.21 (s, 4H), 1.17 (s, 3H), 0.79 (t, J = 7.0 Hz, 3H).

[0028] Example 3 A near-infrared fluorescent dye for brain imaging, the structural formula of which is shown in (III-1): (III-1) Example 3 also provides a method for preparing a near-infrared fluorescent dye for brain imaging, the method comprising the following steps: In equation (III), R9, R 10 It is fluorine.

[0029] (1) This step is the same as step (1) in Example 1, and the product obtained is denoted as intermediate c; (2) Compound l, phthalic anhydride (1 mmol), was dissolved in nitrobenzene (50 mL), followed by the addition of compound m2,4-fluororesorcinol (1 mmol) and anhydrous aluminum chloride (2.2 mmol). The mixture was stirred under nitrogen protection at 80 °C for 12 h. After the reaction was completed, the reaction mixture was poured into a two-phase solution of 30 mL of n-hexane and 40 mL of 0.5 M HCl under vigorous stirring. After stirring for 2 h, an orange-yellow precipitate was obtained, which was filtered to obtain the crude product. The product was then passed through a silica gel column to obtain an orange solid intermediate n, with a yield of 57%.

[0030] (3) Dissolve intermediate c (1 mmol) in 10 mL of methanesulfonic acid, then add intermediate n (1 mmol), stir and reflux at 120 °C for 12 h. After the reaction is complete, cool to room temperature, pour ice water into the reaction solution, then add a small amount of perchloric acid. A precipitate is formed. Filter to obtain crude product, pass through silica gel column to obtain purple compound (III-1) with a yield of 72%.

[0031] (4) The NMR spectrum of a fluorescent dye for brain imaging provided in Example 3 is shown below. Figure 3 As shown, the structural characterization results are as follows: 1 H NMR (300 MHz, Methanol-d4) δ 8.25 – 8.17 (m, 1H), 7.73 – 7.62 (m,2H), 7.29 (d, J = 6.6 Hz, 1H), 6.90 (s, 1H), 6.58 (d, J = 11.3 Hz, 1H), 6.04(s, 1H), 3.63 (d, J = 5.6 Hz, 4H), 3.07 (p, J = 6.8 Hz, 3H), 2.68 (s, 2H), 1.26 (d, J = 5.4 Hz, 6H). Comparative Example 1 A fluorescent dye for brain imaging, the structural formula of which is shown in (II-1): (II-1) This comparative example describes a method for preparing a fluorescent dye for brain imaging, which includes the following steps: In formula (II), R1, R2, R3, and R4 are hydrogen.

[0032] (a) Compound g, trifluorotetranitroanisole (1 mmol), was dissolved in 20 mL of anhydrous acetonitrile solution. L-proline methyl ester hydrochloride (2.5 mmol) and triethylamine (6 mmol) were then added, and the mixture was stirred at 80 °C for 12 h. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was separated by column chromatography to collect a yellow oily intermediate, h, with a yield of 73%. Intermediate h (1 mmol) was dissolved in formic acid (10 mL), followed by the addition of zinc powder (2.1 mmol) and ammonium chloride (2.2 mmol). The mixture was stirred at room temperature for 12 h. After the reaction was complete, the solvent was removed under reduced pressure, and the starting material was dissolved in ethyl acetate, washed with water, dried over Na₂SO₄, and passed through a silica gel column to obtain intermediate i, with a yield of 43%. Intermediate i (1 mmol) was dissolved in 10 mL of tetrahydrofuran, followed by the addition of sodium borohydride (5 mmol) and boron trifluoride diethyl ether (4 mmol), and refluxed at 60 °C for 1 h. After the reaction was complete, the mixture was cooled to room temperature, the pH was adjusted to 12, and the reaction solution was extracted with ethyl acetate. The solvent was removed under reduced pressure, and the solution was passed through a silica gel column to give intermediate j in 67% yield. Compound j (1.5 mmol) was dissolved in 10 mL of tetrahydrofuran, and trifluoroacetic anhydride (1.5 mmol) was added dropwise. The mixture was reacted at room temperature for 10 min, and after the reaction was complete, the solution was evaporated to dryness, dissolved again in tetrahydrofuran, followed by the addition of sodium borohydride (7.5 mmol) and boron trifluoride diethyl ether (7.5 mmol), and refluxed for 1.5 h. After the reaction was complete, the mixture was cooled to room temperature, quenched with water, extracted with ethyl acetate, dried over the organic phase, and the solvent was removed under reduced pressure. The solution was passed through a silica gel column to give intermediate k in 71% yield.

[0033] (b) This step is the same as step (2) in Example 2, and the product obtained is denoted as intermediate f; (c) Intermediate k (1 mmol) was dissolved in 10 mL of methanesulfonic acid, followed by intermediate f (1 mmol). The mixture was stirred and refluxed at 120 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and ice water was poured into the reaction solution. Then a small amount of perchloric acid was added dropwise, and a precipitate was formed. The crude product was filtered and passed through a silica gel column to give the purple compound (II-1) with a yield of 48%.

[0034] Comparative Example 1 provides an NMR spectrum of a fluorescent dye used for brain imaging, as shown below. Figure 4 As shown, the structural characterization results are as follows: 1 H NMR (300 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.77 (d, J 11.1 Hz, 2H), 7.26 (d,J 7.7 Hz, 1H), 6.67 (t, J 45.6 Hz, 4H), 5.89 (s, 1H), 4.11 (s, 7H), 1.23 (s,4H).

[0035] Comparative Example 2 A fluorescent dye for brain imaging, the structural formula of which is shown in (II-2): (II-2) This comparative example provides a method for preparing a fluorescent dye for brain imaging, comprising the following steps: In formula (II), R1, R2, R3, and R4 are fluorine.

[0036] (a) This step is the same as step (a) of Comparative Example 1, and the product obtained is denoted as compound k.

[0037] (b) This step is the same as step (2) in Example 1, and the product obtained is denoted as compound f.

[0038] (c) Intermediate k (1 mmol) was dissolved in 10 mL of methanesulfonic acid, followed by the addition of intermediate f (1 mmol). The mixture was stirred and refluxed at 120 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature, and ice water was poured into the reaction solution. A small amount of perchloric acid was then added dropwise, and a precipitate was formed. The crude product was obtained by filtration and passed through a silica gel column to give the purple compound (II-2) with a yield of 55%.

[0039] Comparative Example 1 provides an NMR spectrum of a fluorescent dye used for brain imaging, as shown below. Figure 5 As shown, the structural characterization results are as follows: 1 H NMR (300 MHz, DMSO-d6) δ 6.95 (s, 1H), 6.66 (s, 2H), 6.33 (s, 1H), 6.15 (d, J = 9.7 Hz, 1H), 4.07 (s, 4H), 3.15 (s, 3H), 2.80 (d, J = 41.4 Hz, 2H), 0.83 (s, 4H).

[0040] Experimental Example 1 To evaluate the optical properties of the fluorescent dyes prepared in Comparative Examples 1-2 and Examples 1-3, ultraviolet absorption and fluorescence spectra were measured in dimethyl sulfoxide (DMSO). The specific steps are as follows: (1) Preparation of mother liquor: The fluorescent dyes obtained in the above examples and comparative examples are dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mM dye mother liquor for later use; (2) Testing: The dye stock solution obtained above was diluted with an appropriate amount of PBS to 10 μM, and the fluorescence spectral properties of the fluorescent probe were tested using a fluorescence spectrometer. The results are as follows: Figure 6As shown.

[0041] The results are as follows Figure 6 As shown, Figure 6 In the image, A, B, C, D, and E represent the fluorescence spectra of the fluorescent dyes prepared in PBS in Examples 1, 2, 3, Comparative Example 1, and Comparative Example 2, respectively. Figure 6 It can be seen that the maximum excitation wavelength of the fluorescent dye in Example 1 is 564 nm, the maximum emission wavelength is 719 nm, and the Stokes shift is 155 nm; the maximum excitation wavelength of the fluorescent dye in Example 2 is 542 nm, the maximum emission wavelength is 691 nm, and the Stokes shift is 149 nm; the maximum excitation wavelength of the fluorescent dye in Example 3 is 548 nm, the maximum emission wavelength is 698 nm, and the Stokes shift is 150 nm; the maximum excitation wavelength of the fluorescent dye in Comparative Example 1 is 547 nm, the maximum emission wavelength is 608 nm, and the Stokes shift is 61 nm; the maximum excitation wavelength of the fluorescent dye in Comparative Example 2 is 570 nm, the maximum emission wavelength is 624 nm, and the Stokes shift is 54 nm.

[0042] This invention modifies the molecular structure of neutral near-infrared dyes to improve their blood-brain barrier permeability through fluorination. Brain imaging is an in vivo application, and whether the emission wavelength can reach the near-infrared region (>650nm) is an important parameter. However, near-infrared dyes generally have large molecular weights and carry a charge, making it difficult for them to penetrate the blood-brain barrier. Therefore, these spectral data are insufficient as screening criteria; actual imaging results and the amount of brain tissue preserved should be the primary criteria. Therefore, this invention further investigates intracranial imaging in mice, as detailed in Experimental Example 2.

[0043] Experimental Example 2 To assess the brain retention of the five fluorescent dyes in Comparative Examples 1-2 and Examples 1-3, we conducted brain imaging studies in mice, as follows: (1) Solution preparation: Four fluorescent dyes were prepared into solutions with a concentration of 100 μM. The dynamic changes of fluorescence in the mouse brain were monitored by injecting 200 μL into the mice.

[0044] (2) Dynamic monitoring of fluorescence in the mouse brain: Animals were anesthetized in oxygen with 1.5% isoflurane. To avoid light absorption by the black hair, the hair on the animal's head was shaved. Dye was injected into the tail vein of the mouse, and imaging of the mouse brain was performed at 1 h, 2 h, and 3 h. The results are as follows: Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown: Figure 7 , Figure 8 , Figure 9 , Figure 10 , Figure 11 The images shown are tracking time points after the injection of fluorescent dye for Examples 1, 2, 1 (Comparative Example), 2 (Comparative Example), and 3 (Example 3). Figure 12 This is an anatomical diagram of the mouse brain 3 hours after injection of fluorescent dyes. As shown in the figure, the fluorescence growth trends of the five fluorescent dyes in the brain were basically consistent after intravenous injection via the tail vein, and all stabilized after 3 hours. The results from the isolated brain fluorescence show that… Figure 12 Example 1 has the best imaging effect, Example 2 and Example 3 have slightly worse imaging effects, and Comparative Example 1 and Comparative Example 2 have the worst effects.

[0045] (3) Measurement of brain retention: Through time-point imaging of mouse brains, we learned that the five fluorescent dyes tend to stabilize in vivo after 3 hours. Dissection was performed at 3 hours, and intracranial fluorescence imaging was conducted. The fluorescence intensity in the mouse brain was analyzed using ImageJ, and the retention amount was calculated by substituting the data into a standard curve. The calculation results are as follows: Figure 13 As shown.

[0046] Figure 13 This is a comparison chart of drug administration amounts to the mouse brain. As shown in the figure, after the mice were injected with the drug and the drug stabilized in vivo, the fluorescent dye of Example 1 had the highest brain retention amount compared to Example 2, Example 3, Comparative Example 1, and Comparative Example 2, indicating that it has the strongest blood-brain barrier penetration.

Claims

1. A near-infrared fluorescent dye for brain imaging, characterized in that, The structural formula of the near-infrared fluorescent dye is shown below: In the formula, R1, R2, R3, R4, R9, R 10 It is either hydrogen or fluorine.

2. The near-infrared fluorescent dye for brain imaging according to claim 1, characterized in that, The near-infrared fluorescent dye has any one of the following structural formulas: 、 、 。 3. The method for preparing a near-infrared fluorescent dye for brain imaging according to claim 1 or 2, characterized in that, The preparation method includes the following steps: In the formula, R1, R2, R3, R4, R9, R 10 It is hydrogen or fluorine; (1) Dissolve compound a in an organic solvent, add glyoxal, heat and stir to react, then separate and purify to obtain intermediate b; mix intermediate b with an organic solvent, add sodium borohydride and glacial acetic acid and heat under reflux, extract after the reaction is complete, then separate and purify to obtain intermediate c; (2) Dissolve compounds d and m in an organic solvent, add anhydrous aluminum chloride, heat and stir to react, then separate and purify to obtain intermediate n; (3) Dissolve intermediate c and intermediate n in an organic solvent, mix them, heat and stir to react, and then separate and purify to obtain the final product.

4. The method for preparing the near-infrared fluorescent dye for brain imaging according to claim 3, characterized in that, The heating and stirring reaction in step (1) is carried out at a temperature of 60-85℃ for 11-13 h; the heating and reflux reaction is carried out at a temperature of 115-125℃ for 11-13 h; the heating and stirring reaction in step (2) is carried out at a temperature of 75-85℃ for 11-13 h; the heating and stirring reaction in step (3) is carried out at a temperature of 110-130℃ for 11-13 h.

5. The method for preparing a near-infrared fluorescent dye for brain imaging according to claim 3, characterized in that, In step (1), the molar ratio of compound a to glyoxal is 1:(1-3), and the molar ratio of intermediate b, sodium borohydride, and glacial acetic acid is 1:10:10; in step (2), the molar ratio of compound d, compound m, and anhydrous aluminum chloride is 1:(1-3):(2-3); in step (3), the molar ratio of intermediate c and intermediate n is 1:(1-2).

6. The method for preparing a near-infrared fluorescent dye for brain imaging according to claim 3, characterized in that, In steps (1)-(3), the organic solvent is selected from any one or more of anhydrous acetonitrile, anhydrous toluene, nitrobenzene, methanesulfonic acid, and tetrahydrofuran.

7. The application of the near-infrared fluorescent dye for brain imaging according to claim 1 or 2 in brain imaging.

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