Method for preparing and refining fluorescent dye crude product

Abstract

The invention discloses a preparation method of a fluorescent dye crude product, which is characterized by comprising the following steps: adding 1, 1, 2-trimethyl-3-(4-sulfonic acid butyl)-1H-benzo [e] indole inner salt and 2-[6-(acetanilino)-1, 3, 5-hexantrienyl]-1, 1-dimethyl-3-sulfonic acid butyl-1H-benzo [e] indole inner salt into absolute ethyl alcohol, dropwise adding triethylamine, reacting for 4-5 hours, filtering, washing, and drying to obtain the fluorescent dye crude product, namely the 2-[6-(acetanilino)-1, 3, 5-hexantrienyl]-1, 1-dimethyl-3-sulfonic acid butyl-1H-benzo [e] indole. Dropwise adding an absolute ethyl alcohol solution of sodium iodide, after dropwise adding, adding purified water, heating and stirring to completely dissolve the solid, and then cooling and crystallizing to obtain a fluorescent dye crude product; meanwhile, the invention discloses a refining method of the fluorescent dye crude product. And the yield of the fluorescent dye is ensured, and impurities and solvent residues meet the limit.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for preparing and purifying a fluorescent dye crude product. BACKGROUND

[0002] Indocyanine green (ICG) is a tricarbocyanine dye with unique optical properties, particularly its fluorescence emission in the near-infrared spectral region. The chemical name of indocyanine green is 2-[7-[1,1-dimethyl-3-(4-sulfobutyl)]benzo[e](2-dihydroindolynyl)-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium inner salt, sodium salt, CAS No. 3599-32-4, and the chemical structure is as follows:

[0003]

[0004] Indocyanine green for injection was developed by Akorn and approved for marketing by the US FDA on February 9, 1959, with the trade name: IC-GREEN. Indocyanine green is mainly used for diagnosing various liver diseases and understanding the damage degree and reserve function of the liver. It is used for diagnosing cirrhosis, liver fibrosis, cholestatic hepatitis, occupational and drug toxic liver diseases; it can also be used for choroidal angiography to determine the location of choroidal disorders. Indocyanine green has a wide range of applications in the field of medical imaging and treatment, and is highly valued.

[0005] CN112638873A discloses a method for purifying indocyanine green, which is to dissolve indocyanine green crude product in a good solvent at a temperature of 0℃ to 80℃, and then slowly add a poor solvent to remove inorganic and organic impurities in the pharmaceutical indocyanine green product. However, the sodium iodide content in the indocyanine green of the invention is 1.47-2.46%, and the yield is 81-91%.

[0006] US20190337896A1 uses methanol and isopropyl alcohol to purify indocyanine green crude product, but the yield of this method is only 42%.

[0007] Since sodium iodide is used in the synthesis of ICG, it is difficult to remove sodium iodide. The current Chinese Pharmacopoeia, US Pharmacopoeia and Japanese Pharmacopoeia set the limit of sodium iodide impurity to be not more than 5%, and the existing technology cannot well control the content of sodium iodide. SUMMARY

[0008] The present application aims at the deficiency of the prior art, and provides a preparation method of a fluorescent dye crude product, characterized in that 1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium inner salt shown as chemical formula 2 and 2-[6-(acetanilide)-1,3,5-hexatrienyl]-1,1-dimethyl-3-sulfobutyl-1H-benzo[e]indolium inner salt shown as chemical formula 1 are added into anhydrous ethanol, triethylamine is added dropwise, reaction is carried out for 4-5 hours, a sodium iodide anhydrous ethanol solution is added dropwise, after the dropwise addition is completed, purified water is added and temperature is raised to stir to completely dissolve the solid, then temperature is lowered to crystallize to obtain the fluorescent dye crude product shown as chemical formula 3

[0009]

[0010] In an embodiment, the temperature of the temperature-raising stirring is 65-75℃.

[0011] In an embodiment, the temperature of the temperature-lowering crystallization is 0-5℃.

[0012] In an embodiment, the stirring time is 30-45 minutes.

[0013] In an embodiment, the molar ratio of 1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium inner salt to 2-[6-(acetanilide)-1,3,5-hexatrienyl]-1,1-dimethyl-3-sulfobutyl-1H-benzo[e]indolium inner salt is 1:1.14, and the molar ratio of 1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium inner salt to sodium iodide is 1:1.1.

[0014] In another aspect, the present application provides a refining method of a fluorescent dye, comprising the following steps

[0015] (1) primary refining: the fluorescent dye crude product is added into a first solvent to be heated and dissolved, then temperature is lowered to crystallize, to obtain a fluorescent dye primary refined product;

[0016] (2) secondary refining: the primary refined product obtained in the primary refining is added into a second solvent to be heated and dissolved, and is filtered while hot, the filtrate is cooled and crystallized, the solid is collected, and after vacuum drying, a fluorescent dye refined product is obtained.

[0017] The indocyanine green crude product is obtained by the method in any one of claims 1-5.

[0018] In an embodiment, the solid-liquid ratio of the indocyanine green crude product to the first solvent is 1:(16-20).

[0019] In an embodiment, the solid-liquid ratio of the indocyanine green crude product to the second solvent is 1:(6-7.5).

[0020] In an embodiment, the first solvent is selected from at least one of methanol, anhydrous ethanol, isopropanol and water.

[0021] In an embodiment, the second solvent is selected from at least one of methanol, anhydrous ethanol, isopropanol and water.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The present application effectively improves the yield and purity of the indocyanine green crude product by precisely controlling the reaction conditions in the preparation process of the fluorescent dye (hereinafter referred to as indocyanine green) crude product. The entire operation process is simple and controllable, and is very suitable for large-scale industrial production.

[0024] In addition, the present application also extensively screens the refining conditions of the indocyanine green crude product, aiming to guarantee the yield of indocyanine green, and effectively controls the content of sodium iodide which is difficult to remove and the solvent residue. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly described below. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art belong to the scope of protection of the present application.

[0026] The terms "first", "second", and the like in the specification and claims of the present application are used to distinguish similar objects, and are not used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged under appropriate circumstances, so that the embodiments of the present application can be implemented in an order other than those described herein, and the objects distinguished by "first", "second", etc. are usually a class, not limited to the number of objects, for example, the first object can be one or more. In addition, "and / or" in the specification and claims means at least one of the connected objects, and the character " / ", generally indicates that the front and rear associated objects are in an "or" relationship.

[0027] The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0028] The preparation method of the indocyanine green crude product of the present application comprises:

[0029] 1,1,2-trimethyl-3-(4-sulfobutyl)-1H-benzo[e]indolium inner salt (hereinafter referred to as "YYL-1") and 2-[6-(acetanilino)-1,3,5-hexatrienyl]-1,1-dimethyl-3-sulfobutyl-1H-benzo[e]indolium inner salt (hereinafter referred to as "YYL-2") are added to anhydrous ethanol, triethylamine is added dropwise, and the reaction is carried out for 4 to 5 hours. Then, a solution of sodium iodide in anhydrous ethanol is added dropwise, and after the dropwise addition is completed, purified water is added and the temperature is raised to stir the solution until the solid is completely dissolved. Then, the temperature is lowered to precipitate crystals to obtain crude indocyanine green (hereinafter referred to as "YYL-CP").

[0030] In one embodiment of the present application, the temperature of the temperature-raising stirring is 65 to 75°C, preferably 65 to 70°C or 70 to 75°C, and in a specific embodiment, for example, it can be 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C. However, it is not limited to the listed values, and other values not listed within the range of the values are also applicable.

[0031] In one embodiment of the present application, the temperature of the temperature-lowering crystallization is 0 to 5°C, and in a specific embodiment, for example, it can be 0°C, 1°C, 2°C, 3°C, 4°C or 5°C. However, it is not limited to the listed values, and other values not listed within the range of the values are also applicable.

[0032] In the preparation of crude indocyanine green, when the reaction solution is added dropwise with sodium iodide to form a salt, fine solid particles are precipitated, the time for suction filtration is long, the filter cake is in a slurry state, and the content of impurities and solvent residue is high. However, by raising the temperature to 65 to 75°C and then lowering the temperature to 0 to 5°C, the problem of difficult suction filtration is effectively solved, the yield and purity of crude indocyanine green are improved, and the content of sodium iodide which is difficult to remove is significantly reduced.

[0033] In one embodiment of the present application, the temperature of the temperature-raising stirring is 65 to 75°C, preferably 65 to 70°C or 70 to 75°C, and in a specific embodiment, for example, it can be 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C or 75°C. However, it is not limited to the listed values, and other values not listed within the range of the values are also applicable.

[0034] In one embodiment of the present application, the stirring time is 30 to 45 minutes, and in a specific embodiment, for example, it can be 30 minutes, 35 minutes, 40 minutes or 45 minutes. However, it is not limited to the listed values, and other values not listed within the range of the values are also applicable.

[0035] In one embodiment of the present application, the molar ratio of YYL-1 to YYL-2 is 1:1.14, and the molar ratio of YYL-1 to sodium iodide is 1:1.1.

[0036] In another aspect, the present application provides a method for purifying indocyanine green, comprising the steps of:

[0037] (1) First purification: dissolving indocyanine green crude product in a first solvent by heating, and then cooling to induce crystallization, thereby obtaining a first purified product;

[0038] (2) Second purification: dissolving the first purified product obtained in the first purification step in a second solvent by heating, filtering the hot solution, and then cooling the filtrate to induce crystallization, thereby obtaining a purified product.

[0039] In the present application, the indocyanine green crude product is obtained by the above method.

[0040] In one embodiment of the present application, the solid-liquid ratio of the indocyanine green crude product to the first solvent is 1: (16-20), for example, it can be 1:16, 1:17, 1:18, 1:19, or 1:20. However, it is not limited to the listed values, and other unlisted values within this range are also applicable.

[0041] In one embodiment of the present application, the solid-liquid ratio of the indocyanine green crude product to the second solvent is 1: (6-7.5). For example, it can be 1:6, 1:6.5, 1:7, or 1:7.5. However, it is not limited to the listed values, and other unlisted values within this range are also applicable.

[0042] In one embodiment of the present application, the first solvent is selected from at least one of methanol, anhydrous ethanol, isopropanol, and water, for example, it can be methanol, anhydrous ethanol, isopropanol, water, methanol and water (e.g., methanol / water = 9 / 1, 10 / 1, 8 / 2, 5 / 1, 1 / 1), anhydrous ethanol and water (e.g., anhydrous ethanol / water = 9 / 1, 10 / 1, 8 / 2, 5 / 1, 2 / 1, 1 / 1), isopropanol and water (e.g., isopropanol / water = 9 / 1, 10 / 1, 8 / 2, 7 / 3, 5 / 1, 1 / 1), methanol and anhydrous ethanol (e.g., methanol / anhydrous ethanol = 5 / 5), methanol and anhydrous ethanol and water (e.g., 30% methanol / 30% ethanol / 40% water), isopropanol and anhydrous ethanol and water (e.g., 40% isopropanol / 30% ethanol / 30% water), methanol and isopropanol and water (e.g., 25% methanol / 25% isopropanol / 50% water), methanol and anhydrous ethanol and isopropanol and water (e.g., 20% methanol / 20% ethanol / 20% isopropanol / 40% water). However, it is not limited to the listed combinations and ratios, and other unlisted combinations and ratios are also applicable.

[0043] In an embodiment of the present application, the second solvent is selected from at least one of methanol, anhydrous ethanol, isopropanol and water, for example, can be methanol, anhydrous ethanol, isopropanol, water, methanol and water (for example, methanol / water = 9 / 1, 10 / 1, 8 / 2, 5 / 1, 1 / 1), anhydrous ethanol and water (for example, anhydrous ethanol / water = 9 / 1, 10 / 1, 8 / 2, 5 / 1, 2 / 1, 1 / 1), isopropanol and water (for example, isopropanol / water = 9 / 1, 10 / 1, 8 / 2, 7 / 3, 5 / 1, 1 / 1), methanol and anhydrous ethanol (for example, methanol / anhydrous ethanol = 5 / 5), methanol and anhydrous ethanol and water (for example, 30% methanol / 30% ethanol / 40% water), isopropanol and anhydrous ethanol and water (for example, 40% isopropanol / 30% ethanol / 30% water), methanol and isopropanol and water (for example, 25% methanol / 25% isopropanol / 50% water), methanol and anhydrous ethanol and isopropanol and water (for example, 20% methanol / 20% ethanol / 20% isopropanol / 40% water). But not limited to the listed combinations and proportions, other combinations and ratios not listed are also applicable.

[0044] In the refining process of indocyanine green, the selection and amount of solvent have a significant impact on the solubility of indocyanine green, which is directly related to the yield of the final product and the content of impurities and residual solvent. Selecting the appropriate solvent can ensure that the compound is fully dissolved, thereby facilitating the improvement of the yield of indocyanine green. However, the use of excessive solvent can lead to excessive solubility, making it difficult to achieve supersaturation, which not only reduces the yield of indocyanine green, but also can cause an increase in the content of impurities and residual solvent. Therefore, in the refining process, the selection and amount of solvent need to be carefully balanced to achieve efficient separation and purification of indocyanine green.

[0045] The specific embodiments of the present application are further described below in conjunction with examples, which do not limit the present application to the described examples.

[0046] In the examples of the present application, the purity checking method is HPLC method, and the residual solvent detection method is residual solvent determination method in Chinese Pharmacopoeia 2020 edition Volume III General Chapter 0861.

[0047] The yield calculation formula of YYL-CP is: m YYL-CP / M YYL-CP ×M YYL-1 / m YYL-1 × 100%

[0048] m YYL-CP is the mass of YYL-CP, M YYL-CP is the molecular weight of YYL-CP, m YYL-1 is the mass of YYL-1, M YYL-1 is the molecular weight of YYL-1

[0049] The yield calculation formula of YYL is: mYYL / m YYL-CP x 100%

[0050] m YYL mass of YYL, m YYL-CP mass of YYL-CP

[0051] Example 1

[0052] (1) Preparation of YYL-CP

[0053] The reaction equation is as follows:

[0054]

[0055] YYL-1 1.4 kg and YYL-2 5 kg were weighed into a 100 L glass reactor, 28 L anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a uniform speed at 20-30 °C, and after the addition was completed, the temperature was controlled at 20-30 °C for 4-5 hours of reaction. After the reaction was completed, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide dissolved in 2 L of anhydrous ethanol) was added dropwise, and after the addition was completed, stirring was continued for 30 minutes. 6 L of purified water was added, the system was heated to 70-75 °C and stirred for 30 minutes, and then the temperature was lowered to 0-5 °C and stirring was continued for 2 hours of crystallization. Filtration was performed, the filter cake was washed with about 5000 ml of cold anhydrous ethanol (0-10 °C) and dried, and the solid was dried at 50 °C under a blast of air for 8 hours. YYL-CP 2.5 kg was obtained, with a yield of 80% and a purity of 98.75%.

[0056] (2) Preparation of YYL-refined product

[0057] First refinement: 41.7 L of anhydrous ethanol and 8.3 L of purified water were added to a 100 L reactor, stirring was started, 2.5 kg of YYL-CP was added, the temperature was raised to 70-75 °C, and stirring was continued for 30 minutes, and then the temperature was lowered to 15-20 °C and stirring was continued for 2 hours of crystallization. Filtration was performed, the filter cake was washed with cold anhydrous ethanol, dried, and the solid was dried at 50 °C under vacuum for 12 hours to obtain 2.3 kg of the first refined product.

[0058] Second refinement: 13.6 L of methanol and 1.36 L of purified water were added to a 20 L reactor, stirring was started, and the YYL first refined product obtained in the above first refinement (about 2.3 kg) was added. The temperature was raised to 70-75 °C, and stirring was continued for 30 minutes. Hot filtration was performed, the filtrate was cooled to 0-5 °C and stirred for 2 hours of crystallization. Filtration was performed, and the solid was dried at 50 °C under vacuum for 8 hours to obtain 2.2 kg of YYL refined product.

[0059] Example 2

[0060] The temperature was raised to 65-70 °C after the addition of sodium iodide was adjusted, and the YYL-CP was prepared as follows, with the remaining steps and conditions being the same as in Example 1.

[0061] YYL-1 1.4 kg, YYL-2 2.5 kg were added into a 100 L glass reactor, then 28 L anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a constant speed at 20-30 °C, and after the addition was completed, the temperature was controlled at 20-30 °C for 4-5 hours. After the reaction was completed, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide was dissolved in 2 L of anhydrous ethanol) was added dropwise, and stirring was continued for 30 minutes. Then 6 L of purified water was added, the temperature was raised to 65-70 °C, and stirring was continued for 45 minutes. Then the temperature was lowered to 0-5 °C, and stirring was continued for 2 hours to obtain crystals. The crystals were filtered, washed with about 5000 ml of cold anhydrous ethanol (0-10 °C), and dried at 50 °C for 8 hours. YYL-CP 2.49 kg was obtained, with a yield of 79.3 % and a purity of 98.74 %.

[0062] Comparative Example 1

[0063] The temperature was raised to 50-60 °C after the addition of sodium iodide was completed. The preparation of YYL-CP was as follows, and the other steps and conditions were the same as in Example 1.

[0064] YYL-1 1.4 kg, YYL-2 2.5 kg were added into a 100 L glass reactor, then 28 L anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a constant speed at 20-30 °C, and after the addition was completed, the temperature was controlled at 20-30 °C for 4-5 hours. After the reaction was completed, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide was dissolved in 2 L of anhydrous ethanol) was added dropwise, and stirring was continued for 30 minutes. Then 6 L of purified water was added, the temperature was raised to 65-70 °C, and stirring was continued for 45 minutes. Then the temperature was lowered to 0-5 °C, and stirring was continued for 2 hours to obtain crystals. The crystals were filtered, washed with about 5000 ml of cold anhydrous ethanol (0-10 °C), and dried at 50 °C for 8 hours. YYL-CP 2.49 kg was obtained, with a yield of 79.3 % and a purity of 98.74 %.

[0065] Comparative Example 2

[0066] The temperature was raised to 80-90 °C after the addition of sodium iodide was completed. The preparation of YYL-CP was as follows, and the other steps and conditions were the same as in Example 1.

[0067] YYL-11.4 kg, YYL-22.5 kg were added into a 100 L glass reactor, then 28 L anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a constant speed at 20-30 °C, and after the addition was completed, the temperature was controlled at 20-30 °C for 4-5 hours of reaction. After the reaction was completed, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide was dissolved in 2 L of ethanol) was added dropwise, and stirring was continued for 30 minutes. Then 6 L of purified water was added, the system was heated to 80-90 °C and stirred for 30 minutes, then cooled to 0-5 °C and stirred for 2 hours of crystallization. Filtration was performed, the filter cake was washed with about 5000 ml of cold anhydrous ethanol (0-10 °C) and dried, and the solid was dried at 50 °C for 8 hours. YYL-CP 2.1 kg was obtained, with a yield of 66.9% and a purity of 96.5%.

[0068] Comparative Example 3

[0069] The crystallization temperature was adjusted to -10 to -5 °C, and the preparation method of YYL-CP was as follows, and the other steps and conditions were the same as in Example 1.

[0070] YYL-11.4 kg, YYL-22.5 kg were added into a 100 L glass reactor, then 28 L anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a constant speed at 20-30 °C, and after the addition was completed, the temperature was controlled at 20-30 °C for 4-5 hours of reaction. After the reaction was completed, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide was dissolved in 2 L of ethanol) was added dropwise, and stirring was continued for 30 minutes. Then 6 L of purified water was added, the system was heated to 80-90 °C and stirred for 30 minutes, then cooled to 0-5 °C and stirred for 2 hours of crystallization. Filtration was performed, the filter cake was washed with about 5000 ml of cold anhydrous ethanol (0-10 °C) and dried, and the solid was dried at 50 °C for 8 hours. YYL-CP 2.1 kg was obtained, with a yield of 66.9% and a purity of 96.5%.

[0071] Comparative Example 4

[0072] The crystallization temperature was adjusted to 10-15 °C, and the preparation method of YYL-CP was as follows, and the other steps and conditions were the same as in Example 1.

[0073] YYL-11.4 kg, YYL-22.5 kg were weighed into a 100 L glass reaction kettle, then 28 L of anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a uniform speed at 20-30 °C, and after the addition was complete, the temperature was controlled at 20-30 °C for 4-5 hours of reaction. After the reaction was complete, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide dissolved in 2 L of anhydrous ethanol) was added dropwise, and stirring was continued for 30 minutes. 6 L of purified water was then added, the system was heated to 65-70 °C and stirred for 45 minutes, then cooled to 10-15 °C and stirred for 2 hours of crystallization. Filtration was performed, the filter cake was rinsed with about 5000 ml of cold anhydrous ethanol (0-10 °C) and dried, and the solid was dried at 50 °C with air blowing for 8 hours. YYL-CP 1.9 kg was obtained, with a yield of 60.5% and a purity of 97.2%.

[0074] Comparative Example 5

[0075] After adding sodium iodide, the temperature was not raised, and the YYL-CP was prepared as follows, with the remaining steps and conditions being the same as in Example 1.

[0076] YYL-11.4 kg, YYL-22.5 kg were weighed into a 100 L glass reaction kettle, then 28 L of anhydrous ethanol was added, and stirring was started. 1077 ml of triethylamine was added at a uniform speed at 20-30 °C, and after the addition was complete, the temperature was controlled at 20-30 °C for 4-5 hours of reaction. After the reaction was complete, a solution of sodium iodide in anhydrous ethanol (667.7 g of sodium iodide dissolved in 2 L of anhydrous ethanol) was added dropwise, and stirring was continued for 30 minutes. The temperature was then lowered to 0-5 °C and stirring was continued for 2 hours of crystallization. Filtration was performed, the filter cake was rinsed with about 5000 ml of cold anhydrous ethanol (0-10 °C) and dried, and the solid was dried at 50 °C with air blowing for 8 hours. YYL-CP 20.1 kg was obtained, with a yield of 64% and a purity of 86.7%.

[0077] Table 1 YYL-CP yield and purity

[0078]

[0079] During the preparation of YYL-CP crude product, the inventors observed through extensive experiments that when sodium iodide was added to the reaction solution to form salts, the precipitated solid particles were very fine, which led to a prolonged filtration process and the filter cake was in a slurry state, affecting the quality of YYL-CP crude product, as shown in Comparative Example 5. To solve this problem, the inventors adopted an improved method, i.e., first heating the mixture to a higher temperature to completely dissolve it, and then cooling it to promote crystallization. This method effectively alleviated the difficulties encountered during the filtration process, and by carefully adjusting the dissolution temperature range (65-75 °C) and controlling the temperature range for temperature reduction and crystallization (0-5 °C), the purity and yield of YYL-CP crude product were successfully improved. This series of optimization measures not only improved the quality of the product, but also improved the production efficiency.

[0080] Example 3

[0081] The first refining method for YYL-refined products is as follows, and the remaining steps and conditions are the same as in Example 1.

[0082] Primary purification: Add 33.33L of anhydrous ethanol and 6.67L of purified water to a 100L reactor, start stirring, add 2.5kg of YYL-CP, heat to 70-75℃, stir for 30 minutes, then cool to 15-20℃ to crystallize for 2 hours, filter, wash the filter cake with cold anhydrous ethanol, dry under vacuum, and dry the solid at 50℃ under vacuum for 12 hours to obtain 2.26kg of the primary purified product.

[0083] After secondary refining, 2.15 kg of refined YYL product was obtained.

[0084] Example 4

[0085] The first refining method for YYL-refined products is as follows, and the remaining steps and conditions are the same as in Example 1.

[0086] Primary purification: Add 26.67L of anhydrous ethanol and 13.33L of purified water to a 100L reactor, start stirring, add 2.5kg of YYL-CP, heat to 70-75℃, stir for 30 minutes, then cool to 15-20℃ to crystallize for 2 hours, filter, wash the filter cake with cold anhydrous ethanol, dry under vacuum, and dry the solid at 50℃ under vacuum for 12 hours to obtain 2.28kg of the primary purified product.

[0087] After secondary refining, 2.19 kg of refined YYL product was obtained.

[0088] Example 5

[0089] The secondary refining method for YYL-refined products is as follows, with the remaining steps and conditions being the same as in Example 1.

[0090] Secondary purification: Add 17.05L of methanol and 1.7L of purified water to a 20L reactor, start stirring, add the YYL primary purified product (about 2.3kg) obtained from the first purification above, heat to 70-75℃, keep warm and stir for 30 minutes, filter while hot, cool the filtrate to 0-5℃ to crystallize for 2 hours, filter by suction, and dry the solid under vacuum at 50℃ for 8 hours.

[0091] 2.17 kg of YYL refined products were obtained.

[0092] Comparative Example 6

[0093] Step 4: The first refining method of YYL-refined product is as follows, and the remaining steps and conditions are the same as in Example 1.

[0094] Primary refining: 45.83 L of anhydrous ethanol and 9.17 L of purified water were added to a 100 L reaction kettle, and stirring was started. 2.5 kg of YYL-CP was added, and the temperature was raised to 70-75 °C. After stirring for 30 minutes, the temperature was lowered to 15-20 °C for crystallization for 2 hours. Filtration was performed, the filter cake was rinsed with cold anhydrous ethanol, and then dried to obtain a solid. The solid was vacuum dried at 50 °C for 12 hours to obtain 2 kg of the primary refined YYL product.

[0095] After secondary refining, 1.7 kg of YYL refined product was obtained.

[0096] Comparative Example 7

[0097] Step four: the primary refining method of YYL-refined product is as follows, and the remaining steps and conditions are the same as in Example 1.

[0098] Primary refining: 45.83 L of anhydrous ethanol and 9.17 L of purified water were added to a 100 L reaction kettle, and stirring was started. 2.5 kg of YYL-CP was added, and the temperature was raised to 70-75 °C. After stirring for 30 minutes, the temperature was lowered to 15-20 °C for crystallization for 2 hours. Filtration was performed, the filter cake was rinsed with cold anhydrous ethanol, and then dried to obtain a solid. The solid was vacuum dried at 50 °C for 12 hours to obtain 2 kg of the primary refined YYL product.

[0099] After secondary refining, 1.7 kg of YYL refined product was obtained.

[0100] Comparative Example 8

[0101] Step four: the primary refining method of YYL-refined product is as follows, and the remaining steps and conditions are the same as in Example 1.

[0102] Primary refining: 45.83 L of anhydrous ethanol and 9.17 L of purified water were added to a 100 L reaction kettle, and stirring was started. 2.5 kg of YYL-CP was added, and the temperature was raised to 70-75 °C. After stirring for 30 minutes, the temperature was lowered to 15-20 °C for crystallization for 2 hours. Filtration was performed, the filter cake was rinsed with cold anhydrous ethanol, and then dried to obtain a solid. The solid was vacuum dried at 50 °C for 12 hours to obtain 2 kg of the primary refined YYL product.

[0103] After secondary refining, 1.6 kg of YYL refined product was obtained.

[0104] Comparative Example 9

[0105] YYL crude product was refined according to the conditions of Example 6 of the reference patent US20190337896A1, and the preparation method of YYL crude product was the same as in Example 1. The specific refining method is as follows:

[0106] YYL crude product 100 g was added in a mixture of 400 ml of methanol and 600 ml of isopropyl alcohol. The reaction mixture was heated at 60-80 °C for 1 hour and the solid was filtered under hot condition at 60-75 °C. The filtered solid was washed with 200 ml of isopropyl alcohol and dried under vacuum to obtain YYL refined product 66.8 g.

[0107] Comparative Example 10

[0108] YYL crude product was refined by referring to the conditions of patent CN112638873A example 3, and the preparation method of YYL crude product was the same as example 1. The specific refining method was as follows:

[0109] Indocyanine green crude product 2.00 g was added in 15 ml of methanol, stirred and dissolved at room temperature, then 20 ml of acetone was added, stirred for 2 h after completion of addition, filtered, the filter cake was washed with 2 ml of acetone, and dried to obtain 1.6 g of indocyanine green product.

[0110] Comparative Example 11

[0111] Step four: the secondary refining method of YYL-refined product was as follows, and the remaining steps and conditions were the same as example 1.

[0112] Secondary refining: 15 L of ethyl acetate was added to a 20 L reaction kettle, stirring was started, YYL primary refined product (about 2.3 kg) obtained by the above primary refining was added, the temperature was raised to 70-75 °C, and stirring was maintained for 30 minutes. The filtrate was filtered while hot, the filtrate was cooled to 0-5 °C for crystallization for 2 hours, suction filtration was performed, and the solid was dried under vacuum at 50 °C for 8 hours.

[0113] YYL refined product 2 kg was obtained.

[0114] Comparative Example 12

[0115] Step four: the secondary refining method of YYL-refined product was as follows, and the remaining steps and conditions were the same as example 1.

[0116] Secondary refining: 15 L of ethyl acetate was added to a 20 L reaction kettle, stirring was started, YYL primary refined product (about 2.3 kg) obtained by the above primary refining was added, the temperature was raised to 70-75 °C, and stirring was maintained for 30 minutes. The filtrate was filtered while hot, the filtrate was cooled to 0-5 °C for crystallization for 2 hours, suction filtration was performed, and the solid was dried under vacuum at 50 °C for 8 hours.

[0117] YYL refined product 1.98 kg was obtained.

[0118] Comparative Example 13

[0119] Step four: the secondary refining method of YYL-refined product was as follows, and the remaining steps and conditions were the same as example 1.

[0120] Secondary refining: 15 L of acetone was added into a 20 L reactor, and stirring was started. The YYL primary refined product (about 2.3 kg) obtained in the above step was added, and the temperature was raised to 70-75 °C. After 30 minutes of stirring, the mixture was filtered while hot. The filtrate was cooled to 0-5 °C for 2 hours to precipitate crystals. The crystals were filtered and dried at 50 °C under vacuum for 8 hours.

[0121] YYL refined product 2.06 kg was obtained.

[0122] Table 2 YYL refined product yield, purity, sodium iodide and residual solvent data

[0123]

[0124] According to the data in Table 2, in the first step of refining indocyanine green (YYL), using ethanol / water solution as the refining solvent, and the solid-liquid ratio of YYL-CP is (16-20):1, the yield of YYL is higher, and the residual amount of sodium iodide is effectively controlled. When other solvents are used (as shown in Comparative Examples 8-10), the yield of YYL is lower, and the residual amount of sodium iodide is too high. Comparative Example 10 refers to the technical solution of the existing patent, which found that after completely dissolving indocyanine green with methanol, 60 times or more acetone is needed for crystallization to effectively remove impurities, but the yield is low. According to the amount of acetone used in Comparative Example 10, the quality of YYL cannot reach the best state. In Comparative Example 7, although too much ethanol / water solution is used, and not enough solution is used in Comparative Example 6, the purity of YYL obtained in both cases is still acceptable, but the yield and residual sodium iodide are not stable.

[0125] In the second step of refining, the inventors compared a variety of different solvents. Comparative Example 11 used ethyl acetate, although the yield and purity of ICG were good, the residual amounts of sodium iodide and ethyl acetate were both beyond the acceptable limit, and were difficult to remove in subsequent processing. Comparative Example 12 used methyl tert-butyl ether, which had a similar problem, with the residual amounts of sodium iodide and ethanol also exceeding the specified limit. Comparative Example 13 used acetone, which also had the problem of excessive residual amounts of sodium iodide and acetone.

[0126] In the refining process of YYL, the choice and amount of solvent have a significant impact on the yield, purity, residual amount of sodium iodide, and amount of residual solvent of the final product. Since sodium iodide is used in the synthesis of YYL, and it is difficult to remove, the current Chinese Pharmacopoeia, United States Pharmacopoeia, and Japanese Pharmacopoeia all set the limit of sodium iodide impurities to not more than 5%. Through optimization of the process, the present application effectively reduces the content of sodium iodide in YYL, and ensures that the yield and purity of the product reach a high level, while the residual solvent is also within an acceptable range. This achievement not only improves the quality of the product, but also meets the strict pharmacopoeia standards.

[0127] The above describes the embodiments of the present application, but the present application is not limited to the above-described specific embodiments, which are merely illustrative but not restrictive, and a person of ordinary skill in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims.

Claims

1. A method for preparing a crude fluorescent dye, characterized in that, 1,1,2-trimethyl-3-(4-sulfonyl)-1H-benzo[e]indole inner salt (as shown in Formula 2) and 2-[6-(acetanilide)-1,3,5-hextrienyl]-1,1-dimethyl-3-sulfonyl-1H-benzo[e]indole inner salt (as shown in Formula 1) were added to anhydrous ethanol, and triethylamine was added dropwise. The reaction was carried out for 4-5 hours, and an anhydrous ethanol solution containing sodium iodide was added dropwise. After the addition was complete, purified water was added, and the mixture was heated and stirred until the solid was completely dissolved. Then, the mixture was cooled and crystallized to obtain the crude fluorescent dye (as shown in Formula 3).

2. The method for preparing the crude fluorescent dye according to claim 1, characterized in that, The temperature for heating and stirring is 65–75°C.

3. The method for preparing the crude fluorescent dye according to claim 2, characterized in that, The cooling and crystallization temperature is 0–5°C.

4. The method for preparing the crude fluorescent dye according to claim 3, characterized in that, The stirring time is 30 to 45 minutes.

5. The method for preparing the crude fluorescent dye according to any one of claims 1 to 4, characterized in that, The molar ratio of the 1,1,2-trimethyl-3-(4-sulfonyl)-1H-benzo[e]indole inner salt to the 2-[6-(acetanilide)-1,3,5-hextrienyl]-1,1-dimethyl-3-sulfonyl-1H-benzo[e]indole inner salt is 1:1.14; the molar ratio of the 1,1,2-trimethyl-3-(4-sulfonyl)-1H-benzo[e]indole inner salt to the sodium iodide is 1:1.

1.

6. A method for purifying a fluorescent dye, characterized in that, Includes the following steps (1) Primary purification: The crude fluorescent dye is added to the first solvent and heated to dissolve, and then cooled to crystallize, thus obtaining the primary purified indocyanine green product; (2) Secondary refining: The primary refined product obtained from the primary refining is added to the second solvent and heated to dissolve. The mixture is filtered while hot, the filtrate is cooled to crystallize, the solid is collected, and then vacuum dried to obtain the fluorescent dye refined product. The crude fluorescent dye is obtained by the method described in any one of claims 1 to 5.

7. The method for purifying fluorescent dyes according to claim 6, characterized in that, The solid-liquid ratio of the crude fluorescent dye to the first solvent is 1:(16-20).

8. The method for purifying fluorescent dye according to claim 6, characterized in that, The solid-liquid ratio of the crude fluorescent dye to the second solvent is 1:(6-7.5).

9. The method for purifying fluorescent dyes according to claim 7, characterized in that, The first solvent is selected from at least one of methanol, anhydrous ethanol, isopropanol, and water.

10. The method for purifying fluorescent dye according to claim 7, characterized in that, The second solvent is selected from at least one of methanol, anhydrous ethanol, isopropanol and water.

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