A near-infrared methylxanthene dye and its preparation method and application

Abstract

The invention relates to the technical field of fluorescent dyes, and discloses a preparation method and application of a near-infrared methylxanthene dye; the near-infrared methylxanthene dye disclosed by the invention highly targets mitochondria and can realize super-resolution imaging of mitochondria; in particular, when imaging is performed under a structured light illumination microscope, a higher spatial resolution can be displayed; the near-infrared methylxanthene dye disclosed by the invention has a simple synthesis process and low cost, which is far lower than commercially available mitochondrial targeting dyes.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent dyes, and in particular to a near-infrared methylxanthene dye and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging technology has the advantages of simple operation, sensitive imaging, high temporal-spatial resolution, and high safety. It is widely used in clinical diagnosis, new drug development, and contrast agents. However, traditional UV-visible dyes are easily interfered by biological autofluorescence, which limits their further clinical application. Near-infrared organic fluorescent dyes have the advantages of good biocompatibility, low background signal, high imaging resolution, high signal-to-noise ratio, and strong tissue penetration, and have broad application prospects in clinical in vivo biological fluorescence imaging.

[0003] Mitochondria are important organelles present in most eukaryotic cells, providing energy for cell physiological activities and participating in physiological processes such as signal transduction, cell differentiation, growth and death. It is of great significance to use near-infrared fluorescent dyes to perform high-resolution imaging of mitochondria through fluorescence imaging technology.

[0004] In the prior art, the synthesis process of near-infrared methylxanthene dyes suitable for mitochondria is complex, has high requirements on operation methods and equipment, is difficult to achieve batch preparation, and has high cost. Summary of the invention

[0005] In recent years, near-infrared fluorescent dyes have developed rapidly, mainly divided into xanthene, rhodamine, cyanine, boron dipyrrole (BODIPY) and the like. The maximum absorption and emission wavelengths of classic rhodamine dyes are both in the visible light region, which is not suitable for in vivo biological imaging applications. The synthesis method of improving rhodamine dyes to extend the wavelength to the near-infrared region is complicated, and the fluorescence quantum yield of the obtained dye is low, which limits its application in fluorescence imaging. Cyanine dyes have poor stability and are susceptible to photobleaching due to the alternating single and double bond structures in the molecules. The maximum absorption and emission wavelengths of classic BODIPY dyes are also in the visible light region, which cannot meet the needs of near-infrared fluorescence imaging, and the synthesis method of BODIPY dyes is complicated. Xanthene dyes have the advantages of simple synthesis method, high fluorescence quantum yield, good stability, etc., and the molecules have positive charges and can achieve mitochondrial targeting without additional chemical modification. They have important application potential in near-infrared fluorescence imaging of mitochondria, but the oxygen atom in the xanthene molecule is easily attacked by nucleophilic reagents and destroyed, so choosing a suitable group occupancy is the key to solving this problem.

[0006] The present invention provides a near-infrared methylxanthene dye, the chemical formula of the near-infrared methylxanthene dye is:

[0007]

[0008] Preferably, the preparation method of the near-infrared methylxanthene dye is:

[0009] Compound a, ethyl iodide and potassium carbonate are used as raw materials to react to generate intermediate I;

[0010] Compound b and CH 2 (COOPh) 2 As raw materials, react to generate intermediate II;

[0011] The intermediate II is reacted with an inorganic acid as a raw material to generate an intermediate III;

[0012] reacting the intermediate I and the intermediate III in an acid solution to generate the near-infrared methylxanthene dye;

[0013] The structural formula of the compound a is:

[0014] The structural formula of the intermediate I is:

[0015] The structural formula of the compound b is:

[0016] The structural formula of the intermediate II is:

[0017] The structural formula of the intermediate III is:

[0018] Preferably, compound a, ethyl iodide and potassium carbonate are reacted as raw materials to generate intermediate I, including:

[0019] The compound a, iodoethane and potassium carbonate are added to an organic solvent for reaction to obtain a mixture A, the mixture A is extracted to obtain an organic phase B, and the organic phase B is then washed, vacuum evaporated and separated by silica gel column chromatography and concentrated to dryness to obtain the intermediate I.

[0020] More preferably, when the compound a, ethyl iodide and potassium carbonate are added to an organic solvent for reaction, the reaction temperature is 40-100° C. and the reaction time is 12-36 h.

[0021] More preferably, the compound a, ethyl iodide and potassium carbonate are added to an organic solvent for reaction, cooled to room temperature, and water is added to terminate the reaction to obtain a mixture.

[0022] More preferably, the organic solvent to which the compound a, ethyl iodide and potassium carbonate are added is methanol, ethanol, acetonitrile, N,N-dimethylformamide or dimethyl sulfoxide.

[0023] More preferably, the organic solvent used when extracting the mixture A is dichloromethane, ethyl acetate, chloroform, toluene or n-hexane.

[0024] More preferably, the organic phase B is washed with water and saturated brine respectively.

[0025] More preferably, the mobile phase ratio of the organic phase B for the silica gel column chromatography separation is petroleum ether:ethyl acetate=80:1-8:1.

[0026] Preferably, compound b and CH 2 (COOPh) 2 As raw materials, the reaction is carried out to generate intermediate II, including:

[0027] The raw material b and CH 2 (COOPh) 2 After being dissolved in an organic solvent for reaction, the mixture is filtered to obtain a filter cake, and the filter cake is washed and dried to obtain the intermediate II.

[0028] More preferably, the raw materials b and CH 2 (COOPh) 2 The organic solvent is methanol, ethanol, acetic acid, dichloromethane, ethyl acetate, chloroform, toluene, chlorobenzene, nitrobenzene or n-hexane.

[0029] More preferably, the filter cake is washed with n-hexane, toluene or dichloromethane.

[0030] More preferably, the raw materials b and CH 2 (COOPh) 2 When dissolved in an organic solvent for reaction, the reaction temperature is 70-140°C and the reaction time is 4-36h.

[0031] More preferably, the raw materials b and CH 2 (COOPh) 2 After being dissolved in an organic solvent and reacted, the solid was cooled to room temperature to precipitate, and then filtered.

[0032] Preferably, the intermediate II is reacted with an inorganic acid as a raw material to generate an intermediate III, comprising:

[0033] The intermediate II is added to the inorganic acid for reaction to obtain a reaction solution, the pH of the reaction solution is adjusted to 4-9 with an inorganic base to obtain a mixture C, the mixture C is extracted to obtain an organic phase D, the organic phase D is washed, and the organic phase D is evaporated under vacuum to obtain a crude intermediate III, the crude intermediate III is separated by silica gel column chromatography and then concentrated to dryness to obtain the intermediate III.

[0034] More preferably, the inorganic acid is hydrochloric acid, sulfuric acid, perchloric acid, nitric acid or phosphoric acid.

[0035] More preferably, the reaction temperature of the reaction is 80-150° C., and the reaction time is 1-12 h.

[0036] More preferably, the inorganic base is sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate or ammonia water.

[0037] More preferably, the organic solvent used when extracting the mixture C is dichloromethane, ethyl acetate, chloroform, toluene or n-hexane.

[0038] More preferably, the organic phase D is washed with water and saturated brine respectively.

[0039] More preferably, the crude intermediate III is separated by silica gel column chromatography with a mobile phase ratio of petroleum ether:ethyl acetate = 30:1-3:1.

[0040] Preferably, the intermediate I and the intermediate III are reacted in an acid solution to generate the near-infrared methylxanthene dye, comprising:

[0041] The intermediate I and the intermediate III are dissolved in an organic acid, and then an inorganic acid is added to react, water is added to the reaction solution to precipitate a solid, and then the solid is filtered and dried to obtain a crude near-infrared methylxanthene dye. The crude near-infrared methylxanthene dye is separated by silica gel column chromatography and concentrated to dryness to obtain the near-infrared methylxanthene dye.

[0042] More preferably, the organic acid in which the intermediate I and the intermediate III are dissolved is formic acid, acetic acid, propionic acid, butyric acid, valeric acid, benzoic acid, phenylacetic acid or phenylpropionic acid.

[0043] More preferably, the intermediate I and the intermediate III are dissolved in an organic acid, and the inorganic acid added is hydrochloric acid, sulfuric acid, perchloric acid, nitric acid or phosphoric acid.

[0044] More preferably, the intermediate I and the intermediate III are dissolved in an organic acid, and then an inorganic acid is added to react at a reaction temperature of 60-150° C. and a reaction time of 12-48 h.

[0045] More preferably, the intermediate I and the intermediate III are dissolved in an organic acid, and then an inorganic acid is added to react. After the reaction is completed, the mixture is cooled to room temperature, and then water is added to the reaction solution to precipitate a solid.

[0046] More preferably, the mobile phase ratio of dichloromethane:methanol is 100:1-10:1 for separating the crude near-infrared methylxanthene dye by silica gel column chromatography.

[0047] The present invention also provides an application of any of the near-infrared methylxanthene dyes, wherein the application comprises at least one of the following:

[0048] ① Application of the near-infrared methylxanthene dye in cell confocal imaging;

[0049] ② Application of the near-infrared methylxanthene dye in organelle localization;

[0050] ③ Application of the near-infrared methylxanthene dye in culturing fluorescent cells;

[0051] ④ Use of the near-infrared methylxanthene dye in preparing a culture medium for culturing fluorescent cells.

[0052] Preferably, the cell confocal imaging is CLSM super-resolution fluorescence imaging or SIM super-resolution fluorescence imaging.

[0053] More preferably, the organelle is mitochondria.

[0054] More preferably, the targeted organelle of the cell confocal imaging is mitochondria.

[0055] Compared with the prior art, the invention has the following beneficial effects:

[0056] The invention provides a preparation method and application of a near-infrared methylxanthene dye.

[0057] The near-infrared methylxanthene dye disclosed in the present invention is highly targeted to mitochondria and can achieve super-resolution imaging of mitochondria. In particular, when imaging is performed under a structured illumination microscope (SIM), a higher spatial resolution can be displayed. The near-infrared methylxanthene dye disclosed in the present invention has a simple synthesis process and low cost, which is far lower than commercially available mitochondrial targeting dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The photophysical properties of the near-infrared methylxanthene dye synthesized in Example 1 in different solvents are shown in Figure 1, wherein (a) is the normalized ultraviolet absorption spectrum and (b) is the normalized fluorescence spectrum;

[0059] Figure 2 The ultraviolet absorption spectra of the near-infrared methylxanthene dye synthesized in Example 1 under different pH conditions;

[0060] Figure 3 The ultraviolet absorption spectrum of the near-infrared methylxanthene dye synthesized in Example 1 under continuous irradiation of 605 nm laser;

[0061] Figure 4After the near-infrared methylxanthene dye synthesized in Example 1 was co-incubated with MCF-7, Hela, 4T1 and LO2, the cell activity was detected by CCK-8;

[0062] Figure 5 CLSM images of MCF-7 cells after incubation with the near-infrared methylxanthene dye synthesized in Example 1 and MTG, scale bar 25 μm;

[0063] Figure 6 CLSM images of MCF-7 cells cultured with the near-infrared methylxanthene dye and MTG synthesized in Example 1 at different concentrations, with a scale of 25 μm; wherein (a) is the near-infrared methylxanthene dye MTG synthesized in Example 1, (b) is the CLSM image of MCF-7 cells cultured, and (b) is the CLSM image of MCF-7 cells cultured with MTG(b);

[0064] Figure 7 The photostability results of the near-infrared methylxanthene dye and MTG synthesized in Example 1 in MCF-7 cells fixed with pre-cooled polymethanol;

[0065] Figure 8 The CLSM and SIM imaging comparison of the near-infrared methylxanthene dye (25 nM) synthesized in Example 1 in MCF-7 cells, wherein (a) is the CLSM of mitochondria infected with the near-infrared methylxanthene dye synthesized in Example 1, with a scale bar of 10 um; (b) is the SIM imaging of mitochondria infected with the near-infrared methylxanthene dye synthesized in Example 1, with a scale bar of 10 um; (c) is the CLSM and SIM imaging of the enlarged ROI 1 in (a) and (b), with a scale bar of 1 μm; (d) is the normalized fluorescence intensity distribution along the white dotted line in (c); (e) is the CLSM and SIM imaging of the enlarged ROI 2 in (a) and (b), with a scale bar of 1 μm; (f) is the normalized fluorescence intensity distribution along the white dotted line in (e);

[0066] Fig. 9 This is the synthesis route of the near-infrared methylxanthene dye of Example 1. DETAILED DESCRIPTION

[0067] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0068] It should be noted that the reagents used in the following examples are all commercially available products.

[0069] Example 1

[0070] Synthesis of Near-Infrared Methylxanthene Dyes:

[0071] Synthesis of intermediate I: 2.5 g of compound a, 7.25 g of iodoethane, and 12.88 g of potassium carbonate were added to 25 mL of ethanol, reacted at 40°C for 12 h, cooled to room temperature, and water was added to terminate the reaction to obtain a mixture, which was extracted with toluene (100 mL × 3 times), then washed with water and saturated brine respectively, and the organic phase was evaporated to dryness under vacuum, and the organic phase was separated by silica gel column chromatography, with the mobile phase ratio of petroleum ether: ethyl acetate = 20:1, and the organic phase was concentrated to dryness to obtain 3.3 g of intermediate I, with a yield of 98%. HR-ESI: 217.14506.

[0072] Synthesis of intermediate II: 2.22 g of compound b and 3.45 g of CH 2 (COOPh) 2 Dissolve in 10 mL of acetic acid, react at 90°C for 8 h, cool to room temperature, precipitate solid, filter, wash the filter cake with dichloromethane, and dry to obtain 1.9 g of light yellow solid, Intermediate II, with a yield of 63%. HR-ESI: 234.11284

[0073] Synthesis of intermediate III: 1.18 g of intermediate II was added to 10 mL of hydrochloric acid, reacted at 80°C for 5 h, cooled to room temperature, the reaction solution was adjusted to pH 9 with sodium hydroxide, the mixture was extracted with ethyl acetate (100 mL × 3 times), the organic phase was washed with water and saturated brine respectively, the organic phase was evaporated to dryness under vacuum, the crude product was separated by silica gel column chromatography, the mobile phase ratio of petroleum ether: ethyl acetate = 10:1, the organic phase was concentrated to dryness to obtain 1.01 g of intermediate I, with a yield of 96%. HR-ESI: 207.12376.

[0074] Synthesis of near-infrared methylxanthene dye: 0.52g of intermediate I and 0.49g of intermediate III were dissolved in 10mL of benzoic acid, and then 10mL of perchloric acid was added. The mixture was reacted at 100℃ for 15h, cooled to room temperature, and 500mL of water was slowly added to the reaction solution. Solids were precipitated, filtered, and dried. The crude product was separated by silica gel column chromatography, and the mobile phase ratio was dichloromethane: methanol = 10:1. The organic phase was concentrated to dryness to obtain 0.77g of near-infrared methylxanthene dye, with a yield of 72%. HR-ESI: 389.25879.

[0075] The structural formula of compound a is:

[0076] The structural formula of intermediate I is:

[0077] The structural formula of compound b is:

[0078] The structural formula of intermediate II is:

[0079] The structural formula of intermediate III is:

[0080] The structural formula of near-infrared methylxanthene dye is:

[0081] Example 2

[0082] The photophysical properties of the near-infrared methylxanthene dye synthesized in Example 1 were tested:

[0083] In view of the obvious conjugated structure of the dye, we measured the photophysical properties of methylxanthene dye in different solvents. Figure 1 As shown, we detected the UV-visible absorption spectrum and fluorescence emission spectrum of the dye; when dichloromethane was used as the solvent, the absorption peak was 600nm and the emission peak was 640nm. In methanol, the absorption peak was 591nm and the emission peak was 639nm. In acetonitrile, the absorption peak was 588nm and the emission peak was 641nm. In dimethyl sulfoxide, the absorption peak was about 596nm and the emission peak was about 648nm. Table 1 collects the maximum absorption wavelength and emission wavelength of different solvents for near-infrared methylxanthene dyes, the molar extinction coefficient and the corresponding fluorescence quantum yield (QY).

[0084] Table 1 Photophysical properties of near-infrared methylxanthene dyes

[0085] Solvents λabs(nm) λem(nm) ε(M-1cm-1) ФY(%) Brightness (ε×ΦY) PBS 591 628 12000 51 6120 Dichloromethane 600 640 23000 97 22310 Methanol 591 639 20000 92 18400 Acetonitrile 588 641 21000 87 18270 Dimethyl sulfoxide 596 648 17000 74 12580

[0086] Where, λabs = absorption spectrum peak; λem = fluorescence spectrum peak; ε = molar extinction coefficient; ΦY = fluorescence quantum yield.

[0087] Example 3

[0088] Experiment on the stability of the near-infrared methylxanthene dye synthesized in Example 1:

[0089] Prepare PBS solutions of different pH values, weigh a certain amount of near-infrared methylxanthene dye and dissolve it in the pH solution, so that the final concentration of the dye in the PBS solution is 10uM. Perform UV absorption spectrometry on PBS solutions of different pH values, such as Figure 2 As shown, the dye is relatively stable in the pH range of 3-9.

[0090] A PBS (pH = 7.4) solution containing 10uM near-infrared methylxanthene dye was prepared, and the test solution was continuously irradiated with a 605nm laser. After each irradiation for 5 minutes, the solution was tested for ultraviolet absorption. Figure 3As shown, under continuous laser irradiation for 30 minutes, the attenuation of the ultraviolet absorption intensity of the solution is negligible, indicating that the near-infrared methylxanthene dye molecules have good photostability under continuous laser irradiation.

[0091] Example 4

[0092] Example 1 Cytotoxicity experiment of the synthesized near-infrared methylxanthene dye:

[0093] The present invention studied the cytotoxicity of methylxanthene dyes to human breast cancer cells (MCF-7), human cervical cancer cells (Hela), mouse breast cancer cells (4T1) and human normal liver cells (LO2) by the standard CCK-8 method. MCF-7, Hela, 4T1 and LO2 cells were inoculated into 96-well plates at a cell density of 8×10 4 / well, and cultured in a 37°C constant temperature incubator for 24 hours. After the cells adhered to the wall, discard the old cell culture medium. Add fresh culture medium containing different concentrations of methylxanthene dye to the 96-well plate and continue to culture. The concentration gradient of methylxanthene dye is 0.00, 0.10, 0.25, 0.50, 1.00 and 2.00 μM, and continue to incubate for 72 hours. After the incubation, the survival rate of cells in different groups was detected by microplate reader. The results are as follows Figure 4 As shown, when the dye concentration was 2.00 μM, the survival rates of the four cell types were above 80%, indicating that the toxicity of the dye molecules to the cells was negligible.

[0094] Example 5

[0095] Example 1 Organelle localization experiment of the synthesized near-infrared methylxanthene dye:

[0096] To determine the precise localization of the dye in cells, we performed co-staining experiments with the commercially available mitochondrial visualization tool Mito-Tracker Green FM (MTG). Figure 5 The staining pattern of the dye is shown. The degree of correlation was assessed using the Pearson correlation coefficient (Rr), ranging from 1 (high correlation) to 0 (no correlation), to quantify the degree of overlap of the stained area between the dye and MTG. The colocalization curves show that the signal from MTG mostly overlaps with that of the dye, with a Pearson's correlation coefficient of 0.82. These data show that this fluorescent dye specifically localizes to mitochondria with high imaging contrast.

[0097] Example 6

[0098] Confocal imaging comparison experiment of the synthesized near-infrared methylxanthene dye and commercial dye in Example 1:

[0099] Cell imaging experiments were performed using different concentrations of methylxanthene dye and MTG. After incubating the cells with the dye for 15 min, the dye-containing medium was removed and confocal fluorescence imaging was performed. Figure 6 As shown in the figure, from the confocal imaging results, the fluorescence of the dye infecting mitochondria in the cytoplasm can be detected at a concentration as low as 5nM. At the same time, MTG was subjected to confocal imaging of cell uptake at the same time gradient as the corresponding concentration of the dye. The confocal results showed that when the cells were infected at the same time, MTG had weak fluorescence at a concentration of 25nM, while the fluorescence of the experimental dye could be detected at a lower concentration.

[0100] Example 7

[0101] Experimental comparison of photostability of the synthesized near-infrared methylxanthene dye and commercial dyes in Example 1:

[0102] Inoculate and incubate MCF-7 cells at a certain density in a confocal dish, prepare culture media with a concentration of 25nM methylxanthene dye and commercially available MTG dye, replace the culture media of the confocal dish with the dye-containing culture media, incubate at 37°C for 15 minutes, remove the culture media, wash with PBS, and perform confocal imaging under continuous laser irradiation of a confocal laser scanning microscope. MTG uses the FITC channel: 488nm excitation, laser intensity: 0.2%; methylxanthene dye uses the TRITC channel: 561nm excitation, laser intensity: 0.2%. Take a confocal picture every 5 minutes, and shoot continuously for 30 minutes. The obtained confocal pictures were analyzed for fluorescence intensity using ImageJ software, as shown in Figure 7 As shown in the figure, after 30 minutes of continuous irradiation, the initial fluorescence intensity of the commercial Mito-Tracker Green FM (MTG) used for comparison showed a significant attenuation within 30 minutes as the laser irradiation time increased, and the intensity dropped to 43%. The fluorescence intensity of the NIR-IDye probe decayed slowly, with the intensity only dropping to 91.7%. Compared with MTG, this dye showed satisfactory photostability.

[0103] Example 8

[0104] Example 1 Comparative experiment of CLSM and SIM imaging of synthesized near-infrared methylxanthene dye (25 nM) in MCF-7 cells:

[0105] The medium in the confocal dish inoculated with appropriate density of MCF-7 cells was replaced with a medium containing 25 nM methylxanthene dye, incubated at 37°C for 15 min, then the medium was removed, washed with PBS, and imaged and compared under confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM). Figure 8As shown in Figure 2, mitochondria captured by SIM clearly show higher spatial resolution compared to CLSM. Figure 8 In ROI 1, we can see that SIM imaging shows sharp signals, while CLSM imaging shows blurred signals ( Figure 8 c). Figure 8 The fluorescence intensity distribution along the white dashed line in c shows that the narrow half-peak width of mitochondria in SIM imaging is 174 nm, compared with a half-peak width of 309 nm in CLSM imaging ( Figure 8 d). In addition, two adjacent spherical mitochondria (ROI 2) can be clearly separated from each other using SIM super-resolution imaging. The half-peak width of the two adjacent mitochondria in CLSM imaging is 1750nm, in sharp contrast to the two narrow peaks collected in SIM imaging, with half-peak widths of 563nm and 500nm, respectively. This shows that SIM can provide us with better imaging resolution than CLSM based on the use of this near-infrared methylxanthene dye to infect mitochondria.

[0106] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a near-infrared methylxanthene dye, characterized in that: The structural formula of the near-infrared methylxanthene dye is: The preparation method of the near-infrared methylxanthene dye is: Compound a, ethyl iodide and potassium carbonate are used as raw materials to react to generate intermediate I; Compound b and CH2(COOPh)2 are used as raw materials to react to generate intermediate II; The intermediate II is reacted with an inorganic acid as a raw material to generate an intermediate III; reacting the intermediate I and the intermediate III in an acid solution to generate the near-infrared methylxanthene dye; The structural formula of the compound a is: The structural formula of the intermediate I is: The structural formula of the compound b is: The structural formula of the intermediate II is: The structural formula of the intermediate III is: Compound a, ethyl iodide and potassium carbonate are used as raw materials to react to generate intermediate I, including: The compound a, iodoethane and potassium carbonate are added to an organic solvent for reaction to obtain a mixture A, the mixture A is extracted to obtain an organic phase B, the organic phase B is then washed, vacuum evaporated and separated by silica gel column chromatography, and then concentrated to dryness to obtain the intermediate I; wherein the organic solvent is ethanol; Compound b and CH2(COOPh)2 are used as raw materials to react to generate intermediate II, including: The raw material b and CH2(COOPh)2 are dissolved in an organic solvent, reacted, and then filtered to obtain a filter cake, and the filter cake is washed and dried to obtain the intermediate II; wherein the organic solvent is acetic acid; The intermediate II is reacted with an inorganic acid as a raw material to generate an intermediate III, comprising: The intermediate II is added to the inorganic acid for reaction to obtain a reaction solution, the reaction solution is adjusted to pH 4-9 with an inorganic base to obtain a mixture C, the mixture C is extracted to obtain an organic phase D, the organic phase D is washed, and the organic phase D is evaporated under vacuum to obtain a crude intermediate III, the crude intermediate III is separated by silica gel column chromatography and then concentrated to dryness to obtain the intermediate III; wherein the inorganic acid is hydrochloric acid; The intermediate I and the intermediate III are reacted in an acid solution to generate the near-infrared methylxanthene dye, comprising: The intermediate I and the intermediate III are dissolved in an organic acid, and then an inorganic acid is added to react, water is added to the reaction solution to precipitate a solid, and then the solid is filtered and dried to obtain a crude near-infrared methylxanthene dye. The crude near-infrared methylxanthene dye is separated by silica gel column chromatography and concentrated to dryness to obtain the near-infrared methylxanthene dye; wherein the organic acid is benzoic acid; and the inorganic acid is perchloric acid.

Images