Synthetic method of fluorescent dye

Abstract

The invention discloses a synthetic method of a fluorescent dye, which comprises the following steps: A, dropwise adding POCl3 into DMF (Dimethyl Formamide) at the temperature of 0 DEG C under the condition of protective atmosphere and stirring, and fully reacting; continuously dropwise adding cyclohexanone, heating, stirring, fully reacting, adding the reaction liquid into ice water, and separating out a solid first compound; b, under the protective atmosphere, stirring and refluxing p-sulfonyl phenylhydrazine, 3-methyl-2-butanone and acetate in acetic acid to react completely, cooling the reaction liquid to-10 to 5 DEG C, stirring, and separating to obtain a second compound; c, under the protective atmosphere, after the second compound and 1, 4-butanesultone are stirred, refluxed and completely reacted in xylene, separating to obtain a third compound; and D, stirring and refluxing the third compound, the first compound and acetate in methanol at the temperature of 80 DEG C under the conditions of protective atmosphere and light shielding, simultaneously dropwise adding acetic anhydride, cooling to room temperature after full reaction, and separating and purifying. According to the scheme, the synthesis process is simple and efficient, and high-quality synthesis of the target dye S0456 is effectively realized.

Description

Technical Field

[0001] The invention relates to an organic intermediate synthesis technology, in particular to a method for synthesizing a fluorescent dye. Background Art

[0002] Near-infrared fluorescence imaging is a highly sensitive molecular imaging technology. Compared with traditional CT imaging, magnetic resonance imaging (MRI), and positron emission tomography (PET) / CT imaging, it has the advantages of low cost, ease of use, no ionizing radiation, and the ability to provide real-time image information. It can be used to guide tumor resection in real time during surgery. Near-infrared fluorescent contrast agents are injected intravenously or locally. When they accumulate at the tumor site, they can generate longer emission light signals under the irradiation of excitation light with a wavelength of 700-900nm. The signals are captured by the fluorescence imaging system to provide real-time tumor information to the surgeon. Autologous tissue has low light absorption in this band, and the generated autofluorescence signal is weak, which increases the specificity and penetration of imaging (about 1 cm). Near-infrared fluorescence imaging has high sensitivity and intuitive imaging. It can be used to identify tumor metastases that are invisible to the naked eye during surgery and is not limited by the surgeon's experience. Indocyanine green (ICG), a commonly used near-infrared fluorescent dye, is metabolized by the liver. Clinical studies have shown that it can be used to show tiny metastases that cannot be detected before surgery during liver cancer surgery, and can be used to show sentinel lymph nodes for metastasis during breast cancer, cervical cancer, and endometrial cancer surgery. However, when ICG is used for intraoperative imaging of ovarian cancer patients, a high number of false positives occur due to the lack of tumor targeting. Therefore, how to improve the fluorescence signal-to-noise ratio (tumor-to-back ratio, TBR) between tumor tissue and surrounding normal tissue is currently the biggest challenge for near-infrared fluorescence imaging during ovarian cancer surgery.

[0003] Indocyanine green (ICG) is a cyanine dye discovered by Alan Waggoner and his colleagues at Carnegie Mellon University in the early 1990s. ICG dye is less toxic than CY series cyanine dyes, has higher safety and better photostability, and is approved by the U.S. Food and Drug Administration (FDA) as a near-infrared imaging agent for clinical use. ICG dye has the following main advantages: (1) ICG is a cyanine dye with a near-infrared characteristic absorption peak. Its absorption wave is in the near-infrared range of 800-835nm. The near-infrared light has a large penetration depth in tissues and is less affected by the background of biological tissues. Since ICG has near-infrared absorption and fluorescence emission characteristics, it can be used as an excellent in vivo tissue penetrant. (2) ICG can almost completely bind to plasma proteins in plasma and whole blood. It almost completely remains in blood vessels and is not easy to diffuse outward. Therefore, it is used as a commonly used vascular contrast agent. (3) ICG is also commonly used to measure cardiac output, liver function, hepatic blood flow and ophthalmic angiography. It has a maximum absorption spectrum near 800nm. These infrared bands can pass through the retinal layer, allowing ICG angiography images to enter deeper blood circulation than fluorescein angiography. (4) ICG has a half-life of 150 to 180 seconds, has no known metabolites, is excreted only through the liver and bile duct, and completely exits the circulation by liver bile. Because it is not absorbed by the intestinal mucosa, it can be classified as low toxicity. (5) ICG can also strongly absorb light energy and convert it into heat energy or produce singlet oxygen, which can be used for photothermal therapy (PTT) or photodynamic therapy (PDT). There are also reports in the literature that ICG can be used for fluorescence imaging during surgery to locate certain in situ and metastatic tumors and their sentinel lymph nodes. In summary, ICG has been widely used in clinical practice.

[0004] In order to improve the targeting of near-infrared fluorescent molecules, some enzyme-activated or ligand-targeted molecular probes have been used in clinical or clinical trials. Among them, indocyanine green-like dyes (S0456) are linked to OTL38 synthesized from folic acid (FA) to target the folate receptor, which has the greatest potential for near-infrared fluorescence imaging to display tumors during surgery for ovarian cancer, prostate cancer, etc. Clinical phase II trials have shown that at least one additional lesion was detected in 48.3% of patients through near-infrared fluorescence imaging, and the sensitivity for folate receptor-positive tumors reached 85.9%, but the TBR of OTL38 was only 4.4±1.4. Some researchers have proposed that tumors can only be identified absolutely reliably when the TBR is greater than 5. Therefore, it is not enough to obtain the accuracy of near-infrared fluorescence imaging by simply improving the TBR of near-infrared fluorescence imaging by actively targeting fluorescent molecules to target tumor ligands. The preparation methods disclosed in the prior art, such as CN113861717A, etc., are complicated in process and easily introduce impurity elements including metal elements, which is not conducive to high-purity and high-efficiency production and preparation, and easily affects the safety of application.

[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a method for synthesizing a fluorescent dye which is simple and efficient, and effectively realizes the high-quality synthesis of the target dye S0456.

[0007] To achieve the above object, the embodiment of the present invention provides a method for synthesizing a fluorescent dye. The fluorescent dye is The method comprises the following steps: A. adding POCl3 to DMF at 0°C, under a protective atmosphere and under stirring conditions, and fully reacting; continuing to add cyclohexanone, stirring and fully reacting after heating, and then adding the reaction solution to ice water to precipitate the first solid compound; B. stirring and refluxing sulfonic acid phenylhydrazine, 3-methyl 2-butanone, and acetate in acetic acid under a protective atmosphere until the reaction is complete, and then cooling the reaction solution and stirring to separate and obtain the second compound; C. stirring and refluxing the second compound and 1,4-butane sultone in xylene under a protective atmosphere after the reaction is complete, and then separating and obtaining the third compound; D. stirring and refluxing the third compound, the first compound, and the acetate in methanol at 80°C under a protective atmosphere and under light-proof conditions, and simultaneously adding acetic anhydride, and cooling to room temperature after sufficient reaction, and then separating and purifying to obtain the target product. The light-proof conditions can be achieved in a dark room, under aluminum foil shielding protection, and other conditions to avoid side reactions under light and improve the yield and purity of the product.

[0008] In one or more embodiments of the present invention, the protective gas of the protective atmosphere is selected from nitrogen, helium, and argon.

[0009] In one or more embodiments of the present invention, the final temperature of the heating in step A is 45-50°C.

[0010] In one or more embodiments of the present invention, the acetate is selected from sodium acetate and potassium acetate.

[0011] In one or more embodiments of the present invention, the stirring in steps AD is magnetic stirring, and the stirring speed is 30-180 rpm. Preferably, the synthesis method of the fluorescent dye comprises the following steps: A. adding POCl3 to DMF at 0°C, a protective atmosphere and stirring at 30 rpm to fully react; continuing to add cyclohexanone, heating and stirring at 60 rpm to fully react, and then adding the reaction solution to ice water to precipitate a solid first compound; B. stirring and refluxing sulfonic acid phenylhydrazine, 3-methyl 2-butanone and acetate in acetic acid at 80 rpm to react completely under a protective atmosphere, cooling the reaction solution (preferably to -10 to 5°C) and stirring at 100 rpm to separate and obtain a second compound; C. stirring and refluxing the second compound and 1,4-butanesulfonate in xylene at 80 rpm to fully react under a protective atmosphere to separate and obtain a third compound; D. stirring and refluxing the third compound, the first compound and the acetate in methanol at 80°C and 100 rpm under a protective atmosphere, and simultaneously adding acetic anhydride, and cooling to room temperature after sufficient reaction, separating and purifying to obtain the target product. Here, by appropriately adjusting different stirring speeds in each step or even in different stages of the same step, the reaction efficiency can be ensured while ensuring the stability of the reaction, avoiding the occurrence of overheating and the like which may lead to an increase in by-products. Adjusting the reaction speed is beneficial to improving the overall yield.

[0012] In one or more embodiments of the present invention, the dropping speed of cyclohexanone in step A is 1-5 seconds per drop to prevent overheating.

[0013] In one or more embodiments of the present invention, the temperature of the reflux reaction in step B is 105-120°C.

[0014] In one or more embodiments of the present invention, the temperature of the reflux reaction in step C is 135-150°C.

[0015] In one or more embodiments of the present invention, the dripping speed of acetic anhydride in step D is 1-3 drops per second.

[0016] In one or more embodiments of the present invention, the cooling end point of the cooling in step D is 15-25°C.

[0017] Compared with the prior art, the method for synthesizing fluorescent dyes according to the embodiment of the present invention effectively improves the preparation efficiency and yield by optimizing the preparation process of the product, thereby greatly improving the production efficiency and optimizing the feasibility of large-scale promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a mass spectrum of a dye according to an embodiment of the present invention;

[0019] Figure 2 is a fluorescence comparison diagram of a probe including a dye according to an embodiment of the present invention;

[0020] Figure 3 is a nuclear magnetic resonance image of the dye according to one embodiment of the present invention. DETAILED DESCRIPTION

[0021] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0022] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising”, etc., will be understood to include the stated elements or components but not to exclude other elements or components.

[0023]

[0024] Including but not limited to the following embodiments, the light-proof condition can be achieved in a dark room, under aluminum foil shielding protection, etc., and the following reaction system is subjected to the reaction in a three-necked flask by completely wrapping the three-necked flask with a commercially available 0.1 mm thick aluminum foil.

[0025] Example 1

[0026] Step 1: Synthesis of C221211A-02:

[0027]

[0028] 0℃, N2 protection, 30rpm magnetic stirring, POCl3 (207.90g, 1.3562mol, 3.8eq) was added dropwise to DMF (175ml, 5V) (addition was completed in 20min), and stirred at 60rpm for 30min. Cyclohexanone C221211A-01 (35.00g, 0.3569mol, 1.0eq) was added dropwise to the reaction solution, 3 seconds per drop, and the temperature was raised to 50℃ and stirred for 5h. The reaction solution was added to ice water (700ml, 20V) and stirred to precipitate solids, which were filtered and the filter cake was concentrated under reduced pressure to obtain 46.86g of the first compound, [M+H] + =172.98.

[0029] Step 2: Synthesis of C221211A-05:

[0030]

[0031] Under N2 protection, sulfonic acid phenylhydrazine C221211A-03 (350.00 g, 1.86 mol, 1.0 eq), 3-methyl 2-butanone C221211A-04 (472.60 g, 5.487 mol, 2.95 eq), sodium acetate (506.22 g, 3.72 mol, 2.0 eq) were stirred and refluxed at 110 ° C and 80 rpm in acetic acid (2.1 L, 6 V) for 4 h. After HPLC reaction, the reaction solution was cooled to room temperature and stirred at 100 rpm in an ice water bath to precipitate solids, which were filtered to obtain a filter cake. MTBE (3.5 L, 10 V) was added to the filter cake and stirred and slurried, and the filter cake was filtered to obtain a filter cake. T = 35 ° C vacuum drying gave the second product compound 337.80 g, [M+H] + =239.98.

[0032] Step 3: Synthesis of C221211A-07:

[0033]

[0034] Under N2 protection, the second compound C221211A-05 (140.00 g, 0.5851 mol, 1.0 eq) and 1,4-butanesulfonic acid lactone C221211A-06 (183.2 g, 1.3456 mol, 2.3 eq) were stirred and refluxed at 145 ° C and 80 rpm in xylene (700 ml, 5 V) for 5 h. The raw materials were sampled and sent to HPLC. The reaction was complete and the product was obtained at 1.749 min. After the reaction solution was cooled to room temperature, the upper clear liquid was poured out, and the lower solid was slurried with MTBE (700 ml, 5 V), filtered, and the filter cake was vacuum dried to obtain 280.00 g of the product (overweight, containing xylene), [M+H] + =380.3. Step 4: Synthesis of product C221211A-int1:

[0035]

[0036] Under N2 protection and aluminum foil light protection (aluminum foil covers to form a dark room, the same below), the third compound C221211A-07 (97.88g, 0.2607mol, 3.0eq), the first compound C221211A-02 (15.00g, 0.0869mol, 1.0eq) and sodium acetate (23.65g, 0.1738mol, 2.0eq) were stirred and refluxed at 80°C and 100rpm in methanol (2.5L, 25V), and acetic anhydride (2.5L, 25V) was slowly added dropwise, 2 drops per second, and the reaction reflux was obvious. After adding, continue to stir and react for 15h. The HPLC control showed that the raw material C221211A-07 had reacted, and C221211A-02 remained more. The reaction solution was cooled to room temperature, concentrated under reduced pressure to remove methanol, and then added dropwise to MTBE (25 L, 10 V) at room temperature, stirred to precipitate solids, centrifuged to collect the solids, added to purified water (2.5 L) and stirred to dissolve, then added dropwise to isopropanol (25 L), stirred to precipitate solids, centrifuged to collect the solids, and dried in vacuo at T = 40 ° C to obtain 36.92 g of crude product. HPLC: 74.17%. Figure 1 As shown, [M+H] + =888.4. NMR spectrum Figure 3 .

[0037] The samples of this embodiment were prepared in the application to obtain the following probes for application testing:

[0038]

[0039] 1. In vitro flow cytometry to detect receptor binding:

[0040] Experimental Principle

[0041] Prostate-specific membrane antigen (PSMA) is overexpressed on prostate cancer cells. Both DUPA-FITC and the probe can target the PSMA receptors in prostate cancer cells. The difference is that DUPA-FITC is connected to a visible light dye (Ex=495nm, Em=520nm), while the probe is connected to a near-infrared dye (Ex=776nm, Em=793nm). Since most of the laser types equipped in flow cytometers are within the visible light range, it is impossible to directly measure the binding force between the probe and the receptor. Therefore, the probe is used to compete with DUPA-FITC for binding to PSMA to indirectly react to the binding force between the test compound and the receptor.

[0042] Experimental Materials

[0043] Probe, DUPA-FITC, 22RV1 (PSMA+), PC3 (PSMA-), 1640 culture medium, FBS, double antibody, trypsin, centrifuge, 4°C constant temperature incubator, flow cytometer.

[0044] Experimental methods:

[0045] Drug preparation: 100 nmol / L DUPA-FITC was prepared in culture medium, and the test compound was diluted to 0.00098 μM, 0.002 μM, 0.008 μM, 0.01 μM, 0.03 μM, 0.05 μM, 0.09 μM, 0.1 μM, and 0.125 μM with culture medium containing 100 nmol / L DUPA-FITC, for a total of nine concentrations, with three replicate wells for each concentration.

[0046] Cell culture: cells were inoculated in T75 culture flasks. After 48 hours, cells in logarithmic growth phase were obtained and digested with trypsin for 1-2 minutes. Fresh culture medium was added to terminate digestion. After counting the cells, the cells were transferred into 96-well plates (1*10 5 Cells / well), 27 wells in total. Centrifuge (800rpm, 5min) to discard the culture medium, rinse the cells twice with PBS and collect the cell precipitate by centrifugation. Add the prepared 9 concentrations of culture medium to the wells with cells, 3 replicates for each concentration. Incubate at 4℃ in the dark for 30min, discard the culture medium in the wells, add 300μL fresh culture medium (2 times) and saline (1 time) to each well to wash the cells, then resuspend the cells with 300μL saline for flow cytometry detection.

[0047] The experiment showed that the negative cell line PC3 had no binding ability to the PSMA receptor, while the positive cell line 22RV1 had a certain binding ability to the PSMA receptor, and the probe showed good affinity.

[0048] 2. In vivo imaging drug efficacy verification experiment

[0049] Experimental Principle

[0050] Prostate-specific membrane antigen (PSMA) is overexpressed on prostate cancer cells. The test compound is a PSMA-targeting molecule with a near-infrared I dye attached to the molecule. The test compound can bind to PSMA and "light up" cancer cells under the stimulation of near-infrared light sources. This experiment uses in vivo live imaging to detect the tissue distribution of the test compound in mice.

[0051] Experimental Materials

[0052] Probe, 22RV1 (PSMA+), PC3 (PSMA-), 1640 culture medium, FBS, double antibody, trypsin, Matrigel (BD), vernier caliper, centrifuge, IVIS live imager.

[0053] Experimental methods

[0054] Cell line: 22RV1 (PSMA+)

[0055] Place Matrigel on ice to melt in advance, and pre-cool the culture medium on ice. After centrifugation of the cells, resuspend the cells in pre-cooled culture medium and add HC Matrigel gel in an equal volume to the culture medium. Place the centrifuge tube containing the cells on ice and transfer it to the animal room. Blow the cells thoroughly before inoculation. Disinfect the right shoulder of the mouse with an alcohol cotton ball and administer a single intravenous dose with an inoculation volume of 200 μL / mouse. All operations are performed on ice. The tumor volume reaches 300-400mm 3 The drugs were administered in groups. Three mice with tumors that were in good growth condition and regular in shape were selected for inclusion in the group. The drug was administered at 10 nmol / mouse. The scanning time was 2 h.

[0056] 2h in vitro tissue distribution results (rats were used as experimental subjects in this part):

[0057] Fluorescence ratio of tumor and normal prostate in situ:

[0058]

[0059] Fluorescence Units:

[0060] photon / second

[0061] From the above table and Figure 2 It can be seen that when the compound of this example is used as an identification aid, there is an obvious signal intensity ratio in tumor / normal prostate tissue, which can fully reflect the ability of the drug to distinguish normal tissue from diseased tissue, such as including but not limited to the ability to distinguish normal prostate from diseased prostate.

[0062] Example 2

[0063] Step 1: Synthesis of C221211A-02:

[0064]

[0065] 0℃, N2 protection, 30rpm magnetic stirring, add POCl3 (207.90g, 1.3562mol, 3.8eq) to DMF (175ml, 5V) dropwise (addition completed in 20min), stir at 60rpm for 30min. Add cyclohexanone C221211A-01 (35.00g, 0.3569mol, 1.0eq) to the reaction solution, 1 second per drop, heat to 50℃, stir and react for 5h. Add the reaction solution to ice water (700ml, 20V), stir to precipitate solid, filter, and concentrate the filter cake under reduced pressure to obtain 45.86g of the first compound, [M+H] + =172.98.

[0066] Step 2: Synthesis of C221211A-05:

[0067]

[0068] Under N2 protection, sulfonic acid phenylhydrazine C221211A-03 (350.00 g, 1.86 mol, 1.0 eq), 3-methyl 2-butanone C221211A-04 (472.60 g, 5.487 mol, 2.95 eq), sodium acetate (506.22 g, 3.72 mol, 2.0 eq) were stirred and refluxed at 105 ° C and 80 rpm in acetic acid (2.1 L, 6 V) for 4 h. After HPLC reaction, the reaction solution was cooled to room temperature and stirred at -10 ° C and 100 rpm to precipitate solids, which were filtered to obtain a filter cake. MTBE (3.5 L, 10 V) was added to the filter cake and stirred and slurried, and the filter cake was filtered to obtain a filter cake, which was dried in vacuum at T = 35 ° C to obtain the second product compound 334.80 g, [M+H] + =239.98.

[0069] Step 3: Synthesis of C221211A-07:

[0070]

[0071] Under N2 protection, the second compound C221211A-05 (140.00 g, 0.5851 mol, 1.0 eq) and 1,4-butanesulfonic acid lactone C221211A-06 (183.2 g, 1.3456 mol, 2.3 eq) were stirred and refluxed at 135 ° C and 80 rpm in xylene (700 ml, 5 V) for 5 h. The samples were sent to HPLC. The raw materials reacted completely and the product was obtained at 1.749 min. After the reaction solution was cooled to room temperature, the upper clear liquid was poured out, and the lower solid was slurried with MTBE (700 ml, 5 V), filtered, and the filter cake was vacuum dried to obtain 282.00 g of the product (overweight, containing xylene), [M+H] + =380.3. Step 4: Synthesis of product C221211A-int1:

[0072]

[0073] Under N2 protection and aluminum foil light protection, the third compound C221211A-07 (97.88g, 0.2607mol, 3.0eq), the first compound C221211A-02 (15.00g, 0.0869mol, 1.0eq) and sodium acetate (23.65g, 0.1738mol, 2.0eq) were stirred and refluxed at 80℃100rpm in methanol (2.5L, 25V), and acetic anhydride (2.5L, 25V) was slowly added dropwise, 1 drop per second, and the reaction reflux was obvious. After adding, the reaction was stirred and continued for 15h. The HPLC control showed that the raw material C221211A-07 had reacted, and C221211A-02 remained more. The reaction solution was cooled to 18°C, concentrated under reduced pressure to remove methanol, and added dropwise to MTBE (25L, 10V) at room temperature. Solids were precipitated by stirring. The solids were collected by centrifugation. Purified water (2.5L) was added and stirred to dissolve. The solution was added dropwise to isopropanol (25L). Solids were precipitated by stirring. The solids were collected by centrifugation. The crude product (33.05g) was obtained by vacuum drying at T = 40°C. HPLC: 72.23%. [M+H] + =888.4.

[0074] Example 3

[0075] Step 1: Synthesis of C221211A-02:

[0076]

[0077] 0℃, N2 protection, 30rpm magnetic stirring, POCl3 (207.90g, 1.3562mol, 3.8eq) was added dropwise to DMF (175ml, 5V) (addition was completed in 20min), and stirred at 60rpm for 30min. Cyclohexanone C221211A-01 (35.00g, 0.3569mol, 1.0eq) was added dropwise to the reaction solution, 2 seconds per drop, and the temperature was raised to 50℃ and stirred for 5h. The reaction solution was added to ice water (700ml, 20V) and stirred to precipitate solids, which were filtered and the filter cake was concentrated under reduced pressure to obtain 47.36g of the first compound, [M+H] + =172.98.

[0078] Step 2: Synthesis of C221211A-05:

[0079]

[0080] Under N2 protection, sulfonic acid phenylhydrazine C221211A-03 (350.00 g, 1.86 mol, 1.0 eq), 3-methyl 2-butanone C221211A-04 (472.60 g, 5.487 mol, 2.95 eq), sodium acetate (506.22 g, 3.72 mol, 2.0 eq) were stirred and refluxed at 115 ° C and 80 rpm in acetic acid (2.1 L, 6 V) for 4 h. After HPLC reaction, the reaction solution was cooled to room temperature and stirred at -5 ° C and 100 rpm to precipitate solids, which were filtered to obtain a filter cake. MTBE (3.5 L, 10 V) was added to the filter cake and stirred and slurried, and the filter cake was filtered to obtain a filter cake, which was dried in vacuo at 35 ° C to obtain 340.20 g of the product, [M+H] + =239.98.

[0081] Step 3: Synthesis of C221211A-07:

[0082]

[0083] Under N2 protection, the second compound C221211A-05 (140.00 g, 0.5851 mol, 1.0 eq) and 1,4-butanesulfonic acid lactone C221211A-06 (183.2 g, 1.3456 mol, 2.3 eq) were stirred and refluxed at 140 ° C and 80 rpm in xylene (700 ml, 5 V) for 5 h. The raw materials were completely reacted and the product was obtained at 1.749 min. After the reaction solution was cooled to room temperature, the upper clear liquid was poured out, and the lower solid was slurried with MTBE (700 ml, 5 V), filtered, and the filter cake was vacuum dried to obtain 282.00 g of the product (overweight, containing xylene), [M+H] + =380.3. Step 4: Synthesis of product C221211A-int1:

[0084]

[0085] Under N2 protection and aluminum foil light protection, the third compound C221211A-07 (97.88g, 0.2607mol, 3.0eq), the first compound C221211A-02 (15.00g, 0.0869mol, 1.0eq) and sodium acetate (23.65g, 0.1738mol, 2.0eq) were stirred and refluxed at 80℃100rpm in methanol (2.5L, 25V), and acetic anhydride (2.5L, 25V) was slowly added dropwise, 2 drops per second, and the reaction reflux was obvious. After adding, continue stirring and reacting for 15h. HPLC control showed that the raw material C221211A-07 had reacted, and C221211A-02 remained more. The reaction solution was cooled to 22°C, concentrated under reduced pressure to remove methanol, and added dropwise to MTBE (25L, 10V) at room temperature. Solids were precipitated by stirring. The solids were collected by centrifugation. Purified water (2.5L) was added and stirred to dissolve. The solution was added dropwise to isopropanol (25L). Solids were precipitated by stirring. The solids were collected by centrifugation. The crude product (30.63g) was obtained by vacuum drying at 40°C. HPLC: 72.36%. [M+H] + =888.4.

[0086] Example 4

[0087] Step 1: Synthesis of C221211A-02:

[0088]

[0089] 0℃, N2 protection, 30rpm magnetic stirring, POCl3 (207.90g, 1.3562mol, 3.8eq) was added dropwise to DMF (175ml, 5V) (addition was completed in 20min), and stirred at 60rpm for 30min. Cyclohexanone C221211A-01 (35.00g, 0.3569mol, 1.0eq) was added dropwise to the reaction solution, 4 seconds per drop, and the temperature was raised to 50℃ and stirred for 5h. The reaction solution was added to ice water (700ml, 20V) and stirred to precipitate solids, which were filtered and the filter cake was concentrated under reduced pressure to obtain 45.93g of the first compound, [M+H] + =172.98.

[0090] Step 2: Synthesis of C221211A-05:

[0091]

[0092] Under N2 protection, sulfonic acid phenylhydrazine C221211A-03 (350.00 g, 1.86 mol, 1.0 eq), 3-methyl 2-butanone C221211A-04 (472.60 g, 5.487 mol, 2.95 eq), sodium acetate (506.22 g, 3.72 mol, 2.0 eq) were stirred and refluxed at 120 ° C and 80 rpm in acetic acid (2.1 L, 6 V) for 4 h. After HPLC reaction, the reaction solution was cooled to room temperature and stirred at 0 ° C and 100 rpm to precipitate solids, which were filtered to obtain a filter cake. MTBE (3.5 L, 10 V) was added to the filter cake and stirred and slurried, and the filter cake was filtered to obtain a filter cake, which was dried in vacuum at T = 35 ° C to obtain the second product compound 328.70 g, [M+H] + =239.98.

[0093] Step 3: Synthesis of C221211A-07:

[0094]

[0095] Under N2 protection, the second compound C221211A-05 (140.00 g, 0.5851 mol, 1.0 eq) and 1,4-butanesulfonic acid lactone C221211A-06 (183.2 g, 1.3456 mol, 2.3 eq) were stirred and refluxed at 150 ° C and 80 rpm in xylene (700 ml, 5 V) for 5 h. The raw materials were completely reacted and the product was obtained at 1.749 min. After the reaction solution was cooled to room temperature, the upper clear liquid was poured out, and the lower solid was slurried with MTBE (700 ml, 5 V), filtered, and the filter cake was vacuum dried to obtain 276.00 g of the product (overweight, containing xylene), [M+H] + =380.3. Step 4: Synthesis of product C221211A-int1:

[0096]

[0097] Under N2 protection and aluminum foil light protection, the third compound C221211A-07 (97.88g, 0.2607mol, 3.0eq), the first compound C221211A-02 (15.00g, 0.0869mol, 1.0eq) and sodium acetate (23.65g, 0.1738mol, 2.0eq) were stirred and refluxed at 80°C and 100rpm in methanol (2.5L, 25V), and acetic anhydride (2.5L, 25V) was slowly added dropwise at 3 drops per second, and the reaction reflux was obvious. After adding, the reaction was stirred and continued for 15h. The HPLC control showed that the raw material C221211A-07 had reacted and C221211A-02 remained more. The reaction solution was cooled to 15°C, concentrated under reduced pressure to remove methanol, and added dropwise to MTBE (25L, 10V) at room temperature. Solids were precipitated by stirring. The solids were collected by centrifugation. Purified water (2.5L) was added and stirred to dissolve. The solution was added dropwise to isopropanol (25L). Solids were precipitated by stirring. The solids were collected by centrifugation. The crude product (35.15g) was obtained by vacuum drying at T = 40°C. HPLC: 69.83%. [M+H] + =888.4.

[0098] Example 5

[0099] Step 1: Synthesis of C221211A-02:

[0100]

[0101] 0℃, N2 protection, 30rpm magnetic stirring, POCl3 (207.90g, 1.3562mol, 3.8eq) was added dropwise to DMF (175ml, 5V) (addition completed in 20min), and stirred at 60rpm for 30min. Cyclohexanone C221211A-01 (35.00g, 0.3569mol, 1.0eq) was added dropwise to the reaction solution, 5 seconds per drop, and the temperature was raised to 50℃ and stirred for 5h. The reaction solution was added to ice water (700ml, 20V) and stirred to precipitate solids, which were filtered and the filter cake was concentrated under reduced pressure to obtain 45.06g of the first compound, [M+H] + =172.98.

[0102] Step 2: Synthesis of C221211A-05:

[0103]

[0104] Under N2 protection, sulfonic acid phenylhydrazine C221211A-03 (350.00 g, 1.86 mol, 1.0 eq), 3-methyl 2-butanone C221211A-04 (472.60 g, 5.487 mol, 2.95 eq), sodium acetate (506.22 g, 3.72 mol, 2.0 eq) were stirred and refluxed at 108 ° C and 80 rpm in acetic acid (2.1 L, 6 V) for 4 h. After HPLC reaction, the reaction solution was cooled to room temperature and stirred at 5 ° C and 100 rpm to precipitate solids, which were filtered to obtain a filter cake. MTBE (3.5 L, 10 V) was added to the filter cake and stirred and slurried, and the filter cake was filtered to obtain a filter cake, which was dried in vacuum at T = 35 ° C to obtain the second product compound 338.10 g, [M+H] + =239.98.

[0105] Step 3: Synthesis of C221211A-07:

[0106]

[0107] Under N2 protection, the second compound C221211A-05 (140.00 g, 0.5851 mol, 1.0 eq) and 1,4-butanesulfonic acid lactone C221211A-06 (183.2 g, 1.3456 mol, 2.3 eq) were stirred and refluxed at 138 ° C and 80 rpm in xylene (700 ml, 5 V) for 5 h. The raw materials were sampled and sent to HPLC. The reaction was complete and the product was obtained at 1.749 min. After the reaction solution was cooled to room temperature, the upper clear liquid was poured out, and the lower solid was slurried with MTBE (700 ml, 5 V), filtered, and the filter cake was vacuum dried to obtain 282.00 g of the product (overweight, containing xylene), [M+H] + =380.3. Step 4: Synthesis of product C221211A-int1:

[0108]

[0109] Under N2 protection and aluminum foil light protection, the third compound C221211A-07 (97.88g, 0.2607mol, 3.0eq), the first compound C221211A-02 (15.00g, 0.0869mol, 1.0eq) and sodium acetate (23.65g, 0.1738mol, 2.0eq) were stirred and refluxed at 80°C and 100rpm in methanol (2.5L, 25V), and acetic anhydride (2.5L, 25V) was slowly added dropwise, 1 drop per second, and the reaction reflux was obvious. After adding, the reaction was stirred and continued for 15h. The HPLC control showed that the raw material C221211A-07 had reacted, and C221211A-02 remained more. The reaction solution was cooled to 20°C, concentrated under reduced pressure to remove methanol, and added dropwise to MTBE (25L, 10V) at room temperature. Solids were precipitated by stirring. The solids were collected by centrifugation. Purified water (2.5L) was added and stirred to dissolve. The solution was added dropwise to isopropanol (25L). Solids were precipitated by stirring. The solids were collected by centrifugation. The crude product (39.35g) was obtained by vacuum drying at T = 40°C. HPLC: 72.13%. [M+H] + =888.4.

[0110] Example 6

[0111] Step 1: Synthesis of C221211A-02:

[0112]

[0113] 0℃, N2 protection, 30rpm magnetic stirring, POCl3 (207.90g, 1.3562mol, 3.8eq) was added dropwise to DMF (175ml, 5V) (addition was completed in 20min), and stirred at 60rpm for 30min. Cyclohexanone C221211A-01 (35.00g, 0.3569mol, 1.0eq) was added dropwise to the reaction solution, 2.5 seconds per drop, and the temperature was raised to 50℃ and stirred for 5h. The reaction solution was added to ice water (700ml, 20V) and stirred to precipitate solids, which were filtered and the filter cake was concentrated under reduced pressure to obtain 45.96g of the first compound, [M+H] + =172.98.

[0114] Step 2: Synthesis of C221211A-05:

[0115]

[0116] Under N2 protection, sulfonic acid phenylhydrazine C221211A-03 (350.00 g, 1.86 mol, 1.0 eq), 3-methyl 2-butanone C221211A-04 (472.60 g, 5.487 mol, 2.95 eq), sodium acetate (506.22 g, 3.72 mol, 2.0 eq) were stirred and refluxed at 110 ° C and 80 rpm in acetic acid (2.1 L, 6 V) for 4 h. After HPLC reaction, the reaction solution was cooled to room temperature and stirred at 3 ° C and 100 rpm to precipitate solids, which were filtered to obtain a filter cake. MTBE (3.5 L, 10 V) was added to the filter cake and stirred and slurried, and the filter cake was filtered to obtain a filter cake, which was dried in vacuum at T = 35 ° C to obtain the second product compound 334.85 g, [M+H] + =239.98.

[0117] Step 3: Synthesis of C221211A-07:

[0118]

[0119] Under N2 protection, the second compound C221211A-05 (140.00 g, 0.5851 mol, 1.0 eq) and 1,4-butanesulfonic acid lactone C221211A-06 (183.2 g, 1.3456 mol, 2.3 eq) were stirred and refluxed at 143 ° C and 80 rpm in xylene (700 ml, 5 V) for 5 h. The raw materials were completely reacted and the product was obtained at 1.749 min. After the reaction solution was cooled to room temperature, the upper clear liquid was poured out, and the lower solid was slurried with MTBE (700 ml, 5 V), filtered, and the filter cake was vacuum dried to obtain 262.00 g of the product (overweight, containing xylene), [M+H] + =380.3. Step 4: Synthesis of product C221211A-int1:

[0120]

[0121] Under N2 protection and aluminum foil light protection, the third compound C221211A-07 (97.88g, 0.2607mol, 3.0eq), the first compound C221211A-02 (15.00g, 0.0869mol, 1.0eq) and sodium acetate (23.65g, 0.1738mol, 2.0eq) were stirred and refluxed at 80°C and 100rpm in methanol (2.5L, 25V), and acetic anhydride (2.5L, 25V) was slowly added dropwise at 3 drops per second, and the reaction reflux was obvious. After adding, the reaction was stirred and continued for 15h. The HPLC control showed that the raw material C221211A-07 had reacted and C221211A-02 remained more. The reaction solution was cooled to 25°C, concentrated under reduced pressure to remove methanol, and added dropwise to MTBE (25L, 10V) at room temperature. Solids were precipitated by stirring. The solids were collected by centrifugation. Purified water (2.5L) was added and stirred to dissolve. The solution was added dropwise to isopropanol (25L). Solids were precipitated by stirring. The solids were collected by centrifugation. The crude product (33.75g) was obtained by vacuum drying at T = 40°C. HPLC: 72.24%. [M+H] + =888.4.

[0122] Comparative Example 1

[0123] The difference between this comparative example and Example 1 is that the reaction conditions are: N2 protection, the third compound C221211A-07 (97.88 g, 0.2607 mol, 3.0 eq), the first compound C221211A-02 (15.00 g, 0.0869 mol, 1.0 eq) and sodium acetate (23.65 g, 0.1738 mol, 2.0 eq) are refluxed with magnetic stirring at 80°C and 100 rpm in methanol (2.5 L, 25 V). Vacuum drying is performed to obtain 31.05 g of crude product. HPLC: 50.23%.

[0124] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A method for synthesizing a fluorescent dye, wherein the fluorescent dye is The steps include: A. Add POCl3 dropwise to DMF at 0°C under protective atmosphere and stirring conditions to fully react; continue to add cyclohexanone dropwise, stir after heating and fully react, and then add the reaction solution into ice water to precipitate the first compound as a solid; B. Under a protective atmosphere, sulfonic acid phenylhydrazine, 3-methyl 2-butanone and acetate are stirred and refluxed in acetic acid until the reaction is complete, and the reaction solution is cooled to -10 to 5°C and stirred to separate and obtain a second compound; C. Under a protective atmosphere, the second compound and 1,4-butanesulfone are stirred and refluxed in xylene to react completely, and then the third compound is separated; D. Under protective atmosphere and light-proof conditions, the third compound, the first compound and the acetate are stirred and refluxed in methanol at 80° C., and acetic anhydride is added dropwise. After sufficient reaction, the mixture is cooled to room temperature, and the target product is obtained by separation and purification.

2. The method for synthesizing a fluorescent dye according to claim 1, wherein The protective gas of the protective atmosphere is selected from nitrogen, helium and argon.

3. The method for synthesizing a fluorescent dye according to claim 1, wherein: The final temperature of the heating in step A is 45-50°C.

4. The method for synthesizing a fluorescent dye according to claim 1, wherein The acetate is selected from sodium acetate and potassium acetate.

5. The method for synthesizing a fluorescent dye according to claim 1, wherein: The stirring in steps AD is magnetic stirring, and the stirring speed is 30-180 rpm.

6. The method for synthesizing a fluorescent dye according to claim 1, wherein: The dropping speed of cyclohexanone in step A is 1-5 seconds per drop.

7. The method for synthesizing a fluorescent dye according to claim 1, wherein: The temperature of the reflux reaction in step B is 105-120°C.

8. The method for synthesizing a fluorescent dye according to claim 1, wherein: The temperature of the reflux reaction in step C is 135-150°C.

9. The method for synthesizing a fluorescent dye according to claim 1, wherein: The dropping speed of the acetic anhydride in step D is 1-3 drops per second.

10. The method for synthesizing a fluorescent dye according to claim 1, characterized in that: The cooling end point of the cooling in step D is 15-25°C.

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