CN112638873B - Refining method of indocyanine green - Google Patents
Refining method of indocyanine green Download PDFInfo
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- CN112638873B CN112638873B CN202080003952.2A CN202080003952A CN112638873B CN 112638873 B CN112638873 B CN 112638873B CN 202080003952 A CN202080003952 A CN 202080003952A CN 112638873 B CN112638873 B CN 112638873B
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- C07—ORGANIC CHEMISTRY
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- C07D209/56—Ring systems containing three or more rings
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
- C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
- C09B23/086—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines more than five >CH- groups
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Abstract
The present application provides a method for refining indocyanine green. The method comprises the following steps: at the temperature of 0-80 ℃, the crude indocyanine green is dissolved in a good solvent, then the poor solvent is slowly dripped into the solvent, and the solvent is stirred, fully crystallized and filtered. The method can obtain the medicinal indocyanine green product with high yield and effectively removing inorganic and organic impurities.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a refining method of indocyanine green serving as a medicine.
Background
Fluorescence imaging is an imaging technique based on the injection of fluorescently labeled drugs or other substances into animals or humans and the detection of the localization of fluorescent labels, which has a unique position in modern medical treatment, mainly as a complementary imaging technique to other methods (e.g. MRI, PET, SPECT, echography, radiography or X-tomography), has important research and application values in life sciences and biomedical research. In fluorescence imaging, indocyanine green (chemical name: 2- [7- [1, 3-dihydro-1, 1-dimethyl-3- (4-sulfobutyl) -2H-benzo [ e ] indol-2-ylidene ] -1,3, 5-hept-1-yl ] -1, 1-dimethyl-3- (4-sulfobutyl) -1H-benzo [ e ] indole inner salt, sodium salt (1:1)) is being widely used clinically as a class of near infrared dye with its excellent fluorescence penetrability, good imaging properties and better stability.
As a preparation method of indocyanine green, patent document CN104130178 discloses a synthesis method of indocyanine green, in which triethylamine in triethylamine salt of indocyanine green is replaced with sodium iodide in methanol in the last step, then methanol is evaporated to dryness, acetone is added, reflux is performed, and filtration is performed to obtain indocyanine green. It is stated that pharmaceutically acceptable indocyanine green with 1.0% sodium iodide is obtainable. However, it is known that sodium iodide has limited solubility in acetone and that sodium iodide is coated in the product and cannot be removed effectively by the literature method. The inventors according to the method of this document have failed to obtain a product with a sodium iodide content of less than 5%. Sodium iodide cannot be effectively removed by using acetone alone as a solvent, so that indocyanine green reaching a medicinal grade cannot be obtained. The process in this document only gives crude indocyanine green.
Further, patent documents US2009069573 and WO2017093889 describe a method for removing impurities such as sodium iodide contained in indocyanine green by extraction with an organic solvent in water. However, indocyanine green has better solubility in water, and the extraction method can seriously affect the yield of the product, and is not suitable for industrial production, see comparative example 6 in the specification.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the technical problems that indocyanine green cannot reach the medicinal grade and the yield is low in the purification process in the prior art, the invention aims to provide a refining method capable of efficiently removing inorganic salt impurities and simultaneously obtaining indocyanine green in high yield.
Means for solving the technical problems
In view of the above technical problems, a first aspect of the present application provides a refining method of indocyanine green, the refining method comprising the following steps: at the temperature of 0-80 ℃, the crude indocyanine green is dissolved in a good solvent, then the poor solvent is slowly dripped into the solvent, and the solvent is stirred, fully crystallized and filtered.
According to an embodiment of the present invention, the good solvent may be selected from at least one of methanol, ethanol, and isopropanol; and the poor solvent may be selected from at least one of acetone, methyl t-butyl ether, n-heptane, ethyl acetate, acetonitrile and dichloromethane.
According to a more specific embodiment of the present invention, the poor solvent may be selected from at least one of acetone, methyl t-butyl ether and ethyl acetate.
According to one embodiment of the invention, the mass-to-volume ratio of the crude indocyanine green, the good solvent and the poor solvent can be 1:1-16:1-78.
According to a more specific embodiment of the present invention, the mass-to-volume ratio of the crude indocyanine green, the good solvent, and the poor solvent may be 1:5 to 10:10 to 20.
According to the refining method of the present invention, the above steps may be performed at a temperature of 20 to 40 ℃.
According to another embodiment of the invention, after the addition of the poor solvent, a small amount of indocyanine green seed crystals may be added, stirred and filtered again after sufficient crystallization.
According to the refining method of the present invention, crystals can be washed with the same poor solvent after filtration and dried to obtain the final product.
In a second aspect, the present application provides a pharmaceutical indocyanine green product, wherein the content of sodium iodide in the product is below 3.5%; and the content of triethylamine in the product is below 0.5 percent.
More specifically, the sodium iodide content in the product may even be below 2.5%; and the content of triethylamine in the product can be even below 0.2 percent.
In a third aspect, the present application provides a pharmaceutical indocyanine green product, wherein the product is obtained by the above-mentioned refining method of indocyanine green.
Effects of the invention
The purification method can efficiently remove inorganic salts including sodium iodide and stably obtain indocyanine green products meeting the standard of medicinal injection preparations with high yield. The inventors have further found that there is a certain amount of indocyanine green triethylamine salt in indocyanine green raw material synthesized according to the prior method, and the salt has not been reported before. By the method, sodium iodide can be removed, and meanwhile, the salt can be effectively removed, so that inorganic salt impurities in the product are further reduced, and the quality and injection safety of the medicine are improved. In addition, the method of the invention is more preferably carried out at room temperature, is simple to operate and is more suitable for industrialized refining.
Drawings
FIG. 1 is an H NMR spectrum of crude indocyanine green obtained in preparation example; and
FIG. 2 is an H NMR spectrum of indocyanine green obtained by the purification method of example 3 of the present invention.
Detailed Description
Unless otherwise indicated, "%" or "percent" as referred to herein refers to weight percent.
Reference herein to "crude indocyanine green" refers, unless otherwise indicated, to a mixture of unrefined indocyanine green triethylamine salt and indocyanine green obtained by substitution of triethylamine in indocyanine green triethylamine salt with sodium iodide, containing impurities based on sodium iodide and indocyanine green triethylamine salt, wherein the sodium iodide content is greater than 3.5%, in particular greater than 5% specified in pharmacopoeia, and/or the triethylamine content is greater than 0.5%, in particular greater than 2% of the crude product. The source of the "crude indocyanine green" mentioned herein may be prepared by any known method, in particular by the patent application publication No.: the indocyanine green can be prepared by a method disclosed in CN104130178A, and can also be a commercially available indocyanine green raw material.
The "pharmaceutically acceptable"/"injectable" indocyanine green, or "pharmaceutically acceptable"/"injectable" indocyanine green product referred to herein means indocyanine green which is in accordance with the rules of the chinese pharmacopoeia and which can be used for injection into animals or humans for fluorescence imaging detection, wherein the active ingredient is 2- [7- [1, 3-dihydro-1, 1-dimethyl-3- (4-sulfobutyl) -2H-benzo [ e ] indol-2-ylidene ] -1,3, 5-heptatrien-1-yl ] -1, 1-dimethyl-3- (4-sulfobutyl) -1H-benzo [ e ] indol inner salt and sodium salt (1:1).
Indocyanine green is a diagnostic reagent for injection and therefore has high requirements for the impurity content therein. Sodium iodide used in the preparation process of the product is easy to cause sensitization reaction, and the content of sodium iodide in indocyanine green in Chinese pharmacopoeia is required to be not more than 5%. For the preparation of pharmaceutically acceptable indocyanine green, there is a strict limit on the residual sodium iodide. Therefore, the low content of sodium iodide in indocyanine green is one of the core competitiveness of indocyanine green product safety.
The inventors of the present application found that although the known method for removing sodium iodide can effectively remove sodium iodide, the yield of indocyanine green is greatly reduced. The present inventors have made intensive studies to address this phenomenon, and have thus proposed the refining method of the present invention. The present invention does not employ conventional extraction methods, but selects a specific solvent combination to remove inorganic impurities by recrystallization. The method not only removes inorganic salt impurities such as sodium iodide more effectively, but also has high yield.
Furthermore, the present inventors have found that, when analyzing crude indocyanine green, a substance similar to indocyanine green structure is contained in the crude indocyanine green. The H NMR analysis shows that the substance is triethylamine salt of indocyanine green, and is an intermediate in the preparation process of indocyanine green. That is, in the crude indocyanine green before purification, impurities generally contained include not only sodium iodide and a small amount of free triethylamine but also a content of indocyanine green triethylamine salt (see the following structural formula) as an impurity, which may contribute up to about 5% of triethylamine, and there is a certain safety hazard, so that it is necessary to remove the triethylamine salt.
Indocyanine green has two sulfonate groups and is therefore easier to combine with Yi Yang ionized triethylamine to form a salt. In the reported literature, this impurity is not mentioned, nor is a method for removing this impurity proposed. Because of the similar structure of indocyanine green triethylamine salt and indocyanine green sodium salt, and thus similar solubility, the known purification process of extraction from water with organic solvents is not effective in removing the impurity.
Furthermore, triethylamine in the triethylamine salt of indocyanine green acid cannot be detected by conventional gas chromatography methods, and thus conventional product inspection tends to ignore the presence of the impurity.
The inventors of the present application have found that the process of the present invention surprisingly enables the simultaneous and efficient removal of sodium iodide and triethylamine salts from crude indocyanine green, thereby obtaining indocyanine green for injection with further improved quality. In addition, the method obviously improves the final yield of the product, is simple to operate and is suitable for industrial production.
The method of the invention is to dissolve indocyanine green in good solvent at 0-80 ℃, then slowly drop poor solvent into the solvent, stir and filter.
According to the method of the present invention, the good solvent is methanol, ethanol and/or isopropanol. The poor solvent is acetone, methyl tert-butyl ether, n-heptane, ethyl acetate, acetonitrile and/or dichloromethane. The good solvent and the poor solvent may be combined in any manner. That is, one or more of the good solvents may be selected, and one or more of the same poor solvents may be selected. Among them, methanol is a preferable good solvent. Preferred poor solvents are acetone, methyl tert-butyl ether and/or ethyl acetate.
A more preferred combination is a combination in which methanol, ethanol and/or isopropanol are used as good solvents and acetone, methyl tert-butyl ether and/or ethyl acetate are used as poor solvents. Among the most preferred combinations are methanol-acetone, methanol-ethyl acetate, methanol-methyl t-butyl ether, ethanol-acetone, ethanol-methyl t-butyl ether, isopropanol-acetone, and isopropanol-ethyl acetate.
The good solvent and the poor solvent and the indocyanine green crude product are matched in proper proportion, otherwise, the ideal impurity removing effect is not achieved, or the yield is reduced to the extent that the industrial production is not acceptable. According to the invention, the mass-to-volume ratio (wt/v/v: g: ml) of the crude indocyanine green, the good solvent and the poor solvent is 1:1-16:1-78. Within this ratio range, the effect of satisfying the injection preparation requirement and having a yield of 70% or more can be obtained.
Preferably, the mass-to-volume ratio (wt/v/v: g: ml: ml) of crude indocyanine green, good solvent and poor solvent is 1: 5-10: 10 to 20. Within this preferred ratio range, the beneficial effects of sodium iodide residues below 3.0%, even below 2.5%, triethylamine residues below 0.2% and even below 0.15% can be obtained while maintaining yields above 80%, even above 85%.
The purification method of the present invention can be carried out in a wide temperature range, but is preferably carried out at 20 to 40 ℃, more preferably at ambient temperature. This can further reduce the energy consumption and can even obtain effects superior to impurity removal and high yield under heating conditions.
The ambient temperature referred to herein refers to 20-28 c, with the most common ambient temperature being room temperature (i.e., about 25 c).
According to another embodiment, the method of the present invention may also include a step of adding a small amount of seed crystal after dropping the poor solvent to accelerate crystallization. For example, the mixture may be stirred for 1 hour or more after adding a small amount of seed crystals to complete crystallization. The seed crystal is obtained according to the above method of the invention.
The indocyanine green medicinal product with lower impurity content can be obtained in high yield by the method.
The technical scheme of the invention will be clearly and completely described below in connection with the specific embodiments of the invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All modifications attainable by one versed in the art from the embodiments according to the present invention without further creative efforts are within the scope of the present invention as claimed.
The content of sodium iodide and triethylamine (including triethylamine in free form and triethylamine in the form of indocyanine green acid triethylamine salt) was measured by the following titration method:
sodium iodide titration method
About 0.1g of a sample is taken, precisely weighed, 100ml of water is added for dissolution, 1ml of nitric acid is added for shaking, and titration is carried out by using silver nitrate titration solution (0.01 mol/L) according to a potentiometric titration method (general rule 0701). Each 1ml of silver nitrate titrant (0.01 mol/L) corresponds to 1.499mg of NaI. The content of sodium iodide is less than 5.0% calculated on dry product.
Triethylamine titration method
Taking a sample, precisely weighing, placing the sample into a separating funnel filled with water, shaking to dissolve the sample, adding a sodium hydroxide solution with a certain concentration, and uniformly mixing. And (3) measuring chloroform for extraction, collecting a lower solution after layering, precisely measuring the solution, adding the solution into an ethanol water solution, titrating with hydrochloric acid titration solution with a certain concentration, and calculating the content of triethylamine in the sample according to the consumption volume of the hydrochloric acid titration solution.
Indocyanine green purity detection method
The purity of the indocyanine green crude product and the refined product is detected by HPLC, and the indocyanine green crude product and the refined product are calculated by adding correction factors. The specific method comprises the following steps:
HPLC chromatograph: agilent 1260 type high performance liquid chromatograph
Chromatographic column: waters C18 μm, 4.6X1250 mm
Gradient elution was performed with phosphate buffer (pH 8.0) as mobile phase A and phosphate buffer (pH 8.0) -organic phase (30:70) as mobile phase B.
The purity calculation method comprises the following steps: and calculating the content of related impurities under different wavelengths according to respective correction factors to obtain the purity data of the indocyanine green product.
Preparation example: preparation of indocyanine green crude product
The present invention provides crude indocyanine green according to the method disclosed in patent document CN104130178A (i.e. the method of example 1 therein), which is incorporated herein by reference in its entirety.
Specifically, first, 2, 3-trimethyl-1- (4-sulfobutyl) -4, 5-benzindolbetamine is synthesized: 2, 3-trimethyl-4, 5-benzoindolebenzene and 1, 4-butyl sodium sulfonate lactone are mixed according to the weight ratio of 1:1.6, reflux is carried out for 3 hours at 140 ℃ in the presence of dimethylbenzene, the mixture is cooled to 50 ℃, acetone with 6 times volume of dimethylbenzene is added, reflux is carried out for half an hour at 55 ℃, the mixture is cooled to 20 ℃, and filtration and drying are carried out. A pale blue solid was obtained as 2, 3-trimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine, with xylene added in a weight ratio of 2, 3-trimethyl-4, 5-benzindole benzene to xylene of 1:0.8.
Then synthesizing 2- [6- (N-ethylbenzene amino) -1,3, 5-hexatrien-1-yl ] -3, 3-dimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine: the 2, 3-trimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine obtained in the previous step is mixed with 2-pentenedicarboxydi-anilide hydrochloride in a weight ratio of 1:0.9, condensed in the presence of acetic anhydride at 140 ℃ for 5 minutes, then cooled to 0 ℃, filtered and the filter cake is washed with acetone to obtain a blue-black solid which is 2- [6- (N-ethylbenzene-amino) -1,3, 5-hexatrien-1-yl ] -3, 3-dimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine, wherein acetic anhydride is added in 5 times by weight of 2, 3-trimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine.
Finally synthesizing indocyanine green: the 2, 3-trimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine obtained in the above two steps was mixed with 2- [6- (N-ethylbenzene amino) -1,3, 5-hexatrien-1-yl ] -3, 3-dimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine in a weight ratio of 1:1.3, 3% triethylamine by weight of the absolute ethyl alcohol (8 times by weight based on 2, 3-trimethyl-1- (4-sulfobutyl) -4, 5-benzindole betaine) was added under stirring, reacted at reflux temperature for 15 minutes, then cooled to 20 ℃, diethyl ether was added in an absolute ethyl alcohol volume of 1.37 times, the supernatant was poured out, the extraction was repeated three times, the residue was dissolved with methanol, refluxed at 68 for 5 minutes, the filtrate was collected, a sodium iodide methanol solution (55 g of sodium iodide was dissolved in 1000mL of methanol, 1/3% by volume of sodium iodide was prepared in water, and dried, and the aqueous phase was dried to obtain a crude product of the dried and distilled off, and the dried product was obtained after the reflux was dried.
The crude product obtained was used for further purification of the following comparative examples and examples.
H NMR (BRUKER 400M nuclear magnetic resonance spectrometer) was performed on the crude product, and the obtained spectrum is shown in FIG. 1. As can be seen from fig. 1: the two sets of peaks at chemical shifts of about 1.2ppm and 3.1ppm are generated by hydrogen on methyl and methylene groups in the triethylamine structure, demonstrating that triethylamine is contained in the crude product. In addition, 2.1ppm corresponds to the residual acetone.
In addition, the contents of sodium iodide and triethylamine in the obtained products were measured according to the above methods, as shown in the following table 1.
The purity of indocyanine green in the crude product was determined to be 98.63% by the method of HPLC described above.
Other reagents and materials used in the present invention are commercially available. The process according to the invention and its advantages will be further illustrated by the following comparative examples and examples. It will be appreciated by those skilled in the art that the following examples are provided for illustration only and are not intended to limit the scope of the invention. The sodium iodide and triethylamine contents of the products obtained in the following comparative examples and examples were measured by the methods described above, respectively.
Comparative example 1:
adding 2.01g of the crude indocyanine green product into 12ml of acetone, stirring for 3h at 20-30 ℃, filtering, leaching a filter cake with 2ml of acetone, and drying to obtain 1.89g of indocyanine green product 1.
Comparative example 2:
adding 2.02g of crude indocyanine green into 12ml of acetone, stirring uniformly, adding 0.4g of purified water, stirring continuously for 3 hours, filtering, leaching a filter cake with 2ml of acetone, and drying to obtain 1.78g of indocyanine green product 2.
Comparative example 3:
2.02g of crude indocyanine green is added into 12ml of acetone, after being stirred uniformly, 1.2g of purified water is added, the mixture is stirred for 3 hours, filtration is carried out, a filter cake is rinsed with 2ml of acetone, and 1.25g of indocyanine green product 3 is obtained after drying.
The relevant detection data of the indocyanine green crude products and indocyanine green products 1-3 are shown in the following table 1.
TABLE 1
| Batch of | Residual sodium iodide | Residual triethylamine | Yield is good |
| Crude product (preparation)Alternate examples | 7.20％ | 5.60％ | N/A |
| Product 1 (comparative example 1) | 6.96％ | 4.32％ | 94.03％ |
| Product 2 (comparative example 2) | 6.18％ | 4.26％ | 88.12％ |
| Product 3 (comparative example 3) | 4.61％ | 3.06％ | 61.88％ |
As is clear from the above experimental results, unlike the indocyanine green product reported in patent document CN104130178A in which the sodium iodide content was 1.0%, the present inventors found that the removal effect of acetone on sodium iodide and indocyanine green triethylamine salt was not very remarkable through the above experiments. The above results are practically consistent with the low solubility of sodium iodide in acetone, which is not favored by the use of less hydrophilic organic solvents. The inventors further tried to add water to acetone to mix and slurry the refining method, the effect of removing impurities was not significantly improved, and the added water amount was increased, which resulted in a significant decrease in yield.
Specifically, the sodium iodide content in the crude product prepared according to the method of example 1 in patent document CN104130178A was 7.20%, the total amount of triethylamine was 5.60%, which is far from the sodium iodide content of 1.0% and the content of the related substances in the product obtained in example 1 reported in this document. This means that the process in this document only gives crude indocyanine green and does not give a product for injection which meets the pharmacopoeia requirements.
In comparative example 1, the crude product obtained by the preparation is further refined by pulping with acetone, and the removal effect on sodium iodide and indocyanine green triethylamine salt is still poor. In comparative example 2, the sodium iodide removal effect is limited by beating with a mixed system of purified water and acetone, the sodium iodide removal effect can be reduced by about 1% by one-time refining, and the indocyanine green triethylamine salt removal is not significantly improved. In comparative example 3, the yield was significantly reduced by 20% or more by increasing the purified water ratio, and the improvement in purification effect was not significant.
The indocyanine green triethylamine salt and indocyanine green sodium salt have similar structure and related physicochemical properties, are not easy to remove, can not detect the triethylamine salified in the product by adopting a gas phase method, have higher residue when detecting the triethylamine by adopting a titration method, and greatly exceed the limit requirement of 0.5 percent of the triethylamine in the medicine requirement in ICH Q3C (namely, ICH Q3C (R7) impurity: guiding principle of residual solvent), thereby seriously affecting the product quality.
Example 1:
2.02g of crude indocyanine green is added into 14ml of methanol, after stirring and dissolving at room temperature, 40ml of ethyl acetate is added, after the addition is finished, stirring is carried out for 2 hours, filtering is carried out, the filter cake is rinsed with 2ml of ethyl acetate, and drying is carried out, thus obtaining 1.76g of indocyanine green product 4.
Example 2:
2.02g of crude indocyanine green is added into 17ml of ethanol, after stirring and dissolving at room temperature, 20ml of n-heptane is added, after the addition is finished, stirring is carried out for 2 hours, filtering is carried out, the filter cake is rinsed with 2ml of n-heptane, and drying is carried out, thus obtaining 1.68g of indocyanine green product 5.
Example 3:
2.00g of crude indocyanine green is added into 15ml of methanol, after stirring and dissolving at room temperature, 20ml of acetone is added, after the addition is finished, stirring is carried out for 2 hours, filtering is carried out, a filter cake is rinsed by 2ml of acetone, and drying is carried out, thus obtaining 1.82g of indocyanine green product 6.
The H NMR of the product was measured in small amounts and the results are shown in FIG. 2. As can be seen from fig. 2: after refining, the two sets of peaks at chemical shifts of about 1.2ppm and 3.1ppm disappeared and triethylamine was substantially removed.
Furthermore, the purity of indocyanine green in indocyanine green product 6 obtained in this example was determined to be 99.78% according to the HLPC method described above.
Example 4:
2.00g of crude indocyanine green is added into 20ml of isopropanol, after stirring and dissolving at room temperature, 35ml of acetonitrile is added, after adding, stirring is carried out for 2 hours, filtering is carried out, the filter cake is rinsed with 2ml of acetonitrile, and drying is carried out, thus obtaining 1.64g of indocyanine green product 7.
Example 5:
2.00g of crude indocyanine green is added into 10ml of methanol, after stirring and dissolving at room temperature, 30ml of dichloromethane is added, after adding, stirring is carried out for 2 hours, filtering is carried out, a filter cake is rinsed with 2ml of dichloromethane, and drying is carried out, thus obtaining 1.62g of indocyanine green product 8.
Example 6:
2.03g of crude indocyanine green is added into 10ml of methanol, after stirring and dissolving at 50 ℃, 30ml of methyl tertiary butyl ether is added, after the addition is finished, stirring is carried out for 2 hours, filtering is carried out, the filter cake is leached by 2ml of methyl tertiary butyl ether, and drying is carried out, thus obtaining 1.66g of indocyanine green product 9.
Example 7:
2.00g of crude indocyanine green is added into 17ml of ethanol, after stirring and dissolving at room temperature, 30ml of acetone is added, after adding, stirring is carried out for 2 hours, filtering is carried out, a filter cake is rinsed by 2ml of acetone, and drying is carried out, thus obtaining 1.78g of indocyanine green product 10.
Example 8:
2.00g of crude indocyanine green is added into 12ml of ethanol, after stirring and dissolving at 50 ℃, 30ml of methyl tertiary butyl ether is added, after the addition is finished, stirring is carried out for 2 hours, filtering is carried out, the filter cake is leached by 2ml of methyl tertiary butyl ether, and drying is carried out, thus obtaining 1.76g of indocyanine green product 11.
Example 9:
2.01g of crude indocyanine green is added into 20ml of isopropanol, after stirring and dissolving at room temperature, 30ml of acetone is added, after adding, stirring is carried out for 2 hours, filtering is carried out, the filter cake is rinsed by 2ml of acetone, and drying is carried out, thus obtaining 1.75g of indocyanine green product 12.
Example 10:
2.00g of indocyanine green crude product is added into 12ml of isopropanol, after stirring and dissolving at 50 ℃, 30ml of ethyl acetate is added, after adding, stirring is carried out for 2 hours, filtering is carried out, the filter cake is rinsed with 2ml of ethyl acetate, and drying is carried out, thus obtaining 1.80g of indocyanine green product 13.
Comparative example 4:
adding 2.00g of indocyanine green crude product into 10ml of dimethyl sulfoxide, stirring at room temperature for dissolution, adding 14ml of acetone, cooling to 20-30 ℃ after the addition, stirring for 2 hours, filtering, leaching a filter cake with 2ml of acetone, and drying to obtain 1.45g of indocyanine green product 14.
Comparative example 5:
adding 2.01g of indocyanine green crude product into 14ml of methanol, stirring at room temperature for dissolution, adding 14ml of N-methylpyrrolidone, cooling to 20-30 ℃ after the addition, stirring for 2 hours, filtering, leaching a filter cake with 2ml of N-methylpyrrolidone, and drying to obtain 1.30g of indocyanine green product 15.
TABLE 2 removal effect of different solvent combinations on sodium iodide and indocyanine green triethylamine salt
| Batch of | Residual sodium iodide | Residual triethylamine | Yield is good |
| Crude product (preparation example) | 7.20％ | 5.60％ | N/A |
| Product 4 (example 1) | 2.10％ | 0.10％ | 87.13％ |
| Product 5 (example 2) | 2.46％ | 0.20％ | 83.17％ |
| Product 6 (example 3) | 1.62％ | 0.08％ | 91.00％ |
| Product 7 (example 4) | 2.20％ | 0.18％ | 82.00％ |
| Product 8 (example 5) | 2.52％ | 0.17％ | 81.00％ |
| Product 9 (example 6) | 2.16％ | 0.14％ | 81.77％ |
| Product 10 (example 7) | 1.60％ | 0.06％ | 89.00％ |
| Product 11 (example 8) | 1.64％ | 0.08％ | 88.00％ |
| Product 12 (example 9) | 1.57％ | 0.07％ | 87.06％ |
| Product 13 (example 10) | 1.47％ | 0.06％ | 90.00％ |
| Product 14 (comparative example 4) | 5.10％ | 2.76％ | 72.50％ |
| Product 15 (comparative example 5) | 4.80％ | 0.92％ | 64.68％ |
The above data indicate that indocyanine green of high purity can be obtained in high yield by recrystallization using a specific good solvent (methanol) and a poor solvent (ethyl acetate, n-heptane, acetone, acetonitrile, dichloromethane, methyl tert-butyl ether).
Examples 11 to 14 below take methanol and acetone solvent combinations as examples, and examine the effect of the solvent amount ratio on removal of sodium iodide and indocyanine green triethylamine salt.
Example 11:
2.03g of crude indocyanine green is added into 10ml of methanol, after stirring and dissolving at room temperature, 20ml of acetone is added, after adding, stirring is carried out for 2 hours, filtering is carried out, a filter cake is rinsed by 2ml of acetone, and drying is carried out, thus obtaining 1.84g of indocyanine green product 16.
Example 12:
2.00g of crude indocyanine green is added into 20ml of methanol, 50ml of acetone is added after stirring and dissolving at room temperature, stirring is carried out for 2 hours after adding, filtering is carried out, a filter cake is rinsed by 2ml of acetone, and drying is carried out, thus obtaining 1.70g of indocyanine green product 17.
Example 13:
2.00g of crude indocyanine green is added into 32ml of methanol, after stirring and dissolving at room temperature, 150ml of acetone is added, after the addition is finished, stirring is carried out for 2 hours, filtering is carried out, the filter cake is rinsed by 2ml of acetone, and drying is carried out, thus obtaining 1.50g of indocyanine green product 18.
Example 14:
2.00g of crude indocyanine green is added into 4ml of methanol, stirring is carried out at room temperature, incomplete dissolution is carried out, 12ml of acetone is added, stirring is carried out for 2 hours after the addition is finished, filtration is carried out, the filter cake is rinsed with 2ml of acetone, and drying is carried out, thus obtaining 1.86g of indocyanine green product 19.
TABLE 3 influence of solvent dosage on removal of sodium iodide and indocyanine green triethylamine salt
| Batch of | Sodium iodide | Triethylamine | Yield is good |
| Crude product (preparation example) | 7.20％ | 5.60％ | N/A |
| Product 16 (example 11) | 1.50％ | 0.09％ | 90.64％ |
| Product 17 (example 12) | 1.35％ | 0.06％ | 85.00％ |
| Product 18 (example 13) | 1.32％ | 0.07％ | 75.00％ |
| Product 19 (example 14) | 3.10％ | 0.87％ | 93.00％ |
According to the experimental results of the above-mentioned different solvent combinations, according to the solvent amount ratio range of the patent, for example 11-14, the indocyanine green product with high quality can be obtained in high yield. The solvent amount is less than the preferred range of this patent, as in example 14, the sodium iodide and triethylamine residual levels are significantly increased, but still able to meet pharmacopoeia requirements.
Comparative example 6:
according to the method disclosed in US2009069573, 2.01g of crude indocyanine green is added to 20ml of purified water, then extracted 3 times with 20ml of dichloromethane, and the organic phases are combined and evaporated to dryness to give 1.13 g of indocyanine green product 20. The test shows that the sodium iodide content is 3.89%, the residual triethylamine content is 1.15%, and the yield is 56.22%.
According to the method disclosed in the patent document US2009069573, the extraction method has a certain removal effect on sodium iodide and indocyanine green triethylamine salt, but the yield is too low (56.22%), and the removal efficiency of sodium iodide and residual indocyanine green triethylamine salt is still obviously poorer than that of the method disclosed by the invention.
Test case
The above product purified by the method of the present invention was measured for sodium iodide and triethylamine content in the above test method and compared with foreign source developer product (first Sanyo Co., ltd., batch number: QHB 0132) as shown in Table 4 below.
TABLE 4 comparison of impurity levels of the products obtained by the process of the invention and of the original developer
| Sodium iodide | Triethylamine × | |
| Standard requirements | ≤5.0％ | ≤0.5％ |
| Original developer | 3.84％ | 0.20％ |
| Products prepared in examples 1 to 14 | 1.32％～2.52％ | 0.04％～0.20％ |
* According to the chinese pharmacopoeia (2020); * According to guidelines for drug impurities by ICH.
The residual quantity of sodium iodide and triethylamine in the indocyanine green product obtained by the refining method is far lower than the pharmacopoeia standard, and is obviously superior to the original developer product. Meanwhile, compared with the known refining method, the yield of the indocyanine green prepared by the method is obviously improved, the operation process is simple, and the method is suitable for industrial production.
Claims (4)
1. The refining method of indocyanine green is characterized by comprising the following steps:
dissolving the crude indocyanine green product in a good solvent at the temperature of 0-80 ℃, slowly dripping a poor solvent into the solvent, stirring, fully crystallizing and filtering;
wherein the good solvent is selected from at least one of methanol, ethanol and isopropanol; and the poor solvent is at least one selected from acetone, methyl tertiary butyl ether, n-heptane, ethyl acetate, acetonitrile and dichloromethane;
the mass volume ratio of the crude indocyanine green, the good solvent and the poor solvent is 1:5-10:10-20.
2. The method for purifying indocyanine green according to claim 1, wherein the poor solvent is at least one selected from the group consisting of acetone, methyl tert-butyl ether, and ethyl acetate.
3. The method for refining indocyanine green according to claim 1, wherein the temperature is 20-40 ℃.
4. The method for purifying indocyanine green according to claim 1 or 2, wherein a small amount of indocyanine green seed crystal is added after dropping the poor solvent, and stirring is performed.
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