Preparation method and application of an indocyanine green dimer compound

Abstract

The application discloses a preparation method of an indocyanine green dimer. The method comprises the following steps: taking indocyanine green as a starting material, and dimerizing in a solvent under light-proof conditions and in a temperature range of 0-20 DEG C. The method has high conversion rate of target product, simple subsequent collection and purification steps, and can easily obtain high-purity indocyanine green dimer. The method can be used for industrial production of indocyanine green dimer and safety and impurity research of the drug indocyanine green.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal chemistry, and particularly relates to a preparation method and application of an indocyanine green dimer compound. BACKGROUND

[0002] Indocyanine dyes are very important near-infrared dyes, and are a very important branch of cyanine dyes. According to the number of carbon atoms in the conjugated methine chain of the dye molecule, indocyanine dyes can be divided into monomethine dyes, trimethine dyes, pentamethine dyes and heptamethine dyes.

[0003] Indocyanine green (ICG, as shown below) is a fluorescent tricarbocyanine dye, which was developed by Kodak Laboratories in 1955. Indocyanine green is a contrast agent, which has strong penetration and high plasma protein binding rate by using its fluorescence function, and can clearly display the choroid image.

[0004]

[0005] The common preparation of indocyanine green is indocyanine green for injection, which is clinically used for checking liver function and liver effective blood flow. After intravenous injection into the body, it immediately binds with plasma proteins, rapidly distributes in the blood vessels of the whole body, is efficiently and selectively taken up by hepatocytes, and is excreted from the hepatocytes into the bile in a free form, enters the intestine through the biliary tract, and is excreted out of the body with feces. Due to fast excretion, about 97% of indocyanine green is excreted from the blood 20 minutes after intravenous injection in normal people, does not participate in chemical reactions in the body, does not participate in enterohepatic circulation (ICG entering the intestinal tract is not absorbed into the blood), does not have lymphatic reflux, and is not excreted from other extrahepatic organs such as the kidney.

[0006] Impurities in drugs can cause adverse side effects and must be controlled. At present, the quality standards of indocyanine green and indocyanine green for injection are collected in the United States Pharmacopoeia (USP) and the People's Republic of China Pharmacopoeia (ChP), and no specific process impurities and degradation impurities are collected.

[0007] The same maximum single impurity can be detected in the indocyanine green for injection on the market, which is shown as formula I below and is called indocyanine green dimer. For example, the indocyanine green for injection marketed by First Chemical Co., Ltd. is the HPLC maximum single impurity: 1.2% (see figure: 1). Since the content of this impurity in the drug is large, it must be studied. Therefore, a certain amount of high-purity impurity is needed to be prepared, which is used as an impurity reference substance for quality control of indocyanine green raw material drugs, and is used for safety research of indocyanine green drugs.

[0008] SUMMARY

[0009] The present application aims to provide a preparation method of indocyanine green dimer with simple steps, easy purification and high purity.

[0010] A preparation method of indocyanine green dimer (Formula I) is provided, which uses indocyanine green as starting material, and dimerizes in a solvent under light-proof condition at a temperature of 0-20℃.

[0011] According to the preparation method of the present application, the solvent is a single solvent or a mixed solvent selected from purified water, or a combination of methanol or ethanol and purified water.

[0012] According to the preparation method of the present application, the reaction temperature is 5-10℃.

[0013] According to the preparation method of the present application, the mass / volume ratio of indocyanine green to solvent is 1g:30-200ml; preferably 1g:50-70ml.

[0014] According to the preparation method of the present application, the reaction is carried out for 6-12 days, preferably 9-10 days.

[0015] According to the preparation method of the present application, the solvent is purified water, and the reaction temperature is 5-10℃, and the reaction equation is shown as follows:

[0016]

[0017] According to the preparation method of the present application, after the reaction is completed, the reaction solution is freeze-dried to obtain a solid crude product.

[0018] According to the preparation method of the present application, the obtained crude product is dissolved in methanol, and then pure product is precipitated after adding acetone, and the volume ratio of methanol to acetone is 1:2.

[0019] According to the preparation method of the present application, the purified solid is washed and vacuum dried to obtain the pure product.

[0020] Advantages

[0021] As a listed drug, it is necessary to study the impurities of indocyanine green. The indocyanine green dimer is the main impurity of the drug. In the prior art, it is known that indocyanine green dimer can be detected in indocyanine green aqueous solution after standing for a period of time. However, due to the reasons such as too low conversion rate of the target compound indocyanine green dimer, too many components in the solution, difficult separation, complicated steps and poor separation effect, the process cannot be used as an industrialization method of indocyanine green dimer. It is unexpectedly found in the present application that indocyanine green is completely converted into indocyanine green dimer in a solvent, especially in purified water, at a lower temperature in a dark environment, and the subsequent collection and purification steps of the method are simple, and high-purity indocyanine green dimer can be easily obtained. The purity of the product can reach more than 99%. In the present application, light protection and low temperature are the key conditions for preparing high-conversion-rate and high-purity products. If either condition is not met, the conversion rate of the target product will be greatly reduced, and the impurities will be complicated, making the subsequent collection and purification steps complicated. Therefore, the present application provides a preparation method of indocyanine green dimer which can be used as a reference substance for impurity research. The method is simple, easy to obtain, and has high yield and purity. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 HPLC related substance spectrum of indocyanine green for injection listed by Shionogi & Co., Ltd.

[0023] Figure 2 HPLC related substance spectrum of the compound of formula I prepared in Example 1;

[0024] Figure 3 LC-HRMS spectrum of the compound of formula I prepared in Example 1;

[0025] Figure 4 1H-NMR spectrum of the compound of formula I prepared in Example 1. DETAILED DESCRIPTION

[0026] The preparation method of the compound of formula I of the present application will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0027] All reagents and raw materials of the present application are commercially available or self-made according to the methods of the prior art.

[0028] Chromatographic conditions in the examples:

[0029] Chromatographic column: MidX Spectrum Blue XR-C18 4.6x250mm 5μm

[0030] Flow rate: 1.0 ml / min

[0031] Column temperature: 30℃

[0032] Injection volume: 10 μl

[0033] Wavelength: 263nm

[0034] Mobile phase A: 0.03% aqueous phosphoric acid solution (adjusted to pH 4.0 with triethylamine)

[0035] Mobile phase B: Acetonitrile: Methanol (40:10)

[0036] gradient:

[0037] Time (min) A％ B％ 0 75 25 5 75 25 30 20 80 35 20 80 35.1 75 25 45 75 25

[0038] Example 1

[0039] Add 10g of indocyanine green to a 1000ml reaction flask, then add 600ml of purified water. Stir and react for 10 days at 5-10℃ in the dark. (The reaction was monitored by HPLC, and the indocyanine green reaction was complete).

[0040] After freeze-drying the reaction solution, 10g of crude reddish-brown solid was obtained.

[0041] Add 10g of the crude product to a 500ml single-necked flask, then add 100ml of methanol. Stir at room temperature for 30 minutes until the solid is completely dissolved. Add 200ml of acetone dropwise and continue stirring for another 30 minutes. Filter by suction. Wash the filter cake with 10ml x 2 of a methanol / acetone mixture (1 / 2). Dry the wet product under vacuum at 40-50℃ for 10 hours to obtain 8.7g of a dark green solid, yield 87.0%, purity HPLC: 99.2%.

[0042] (See Figure 2 )

[0043] Figure 1 The HPLC chromatogram of indocyanine green is known in the prior art. The largest single impurity peak (1.2%) at 25 min is the indocyanine green dimer, and at 30 min it is the indocyanine green. Figure 2 This is the HPLC chromatogram of the product prepared in Example 1 of this application. At 25 min, it is an indocyanine green dimer. As can be seen from the figure, the impurity content of this product is very low.

[0044] Comparative Example 1:

[0045] Add 10g of indocyanine green to a 1000ml reaction flask, then add 600ml of purified water. Stir and react for 8 days at 25-30℃ in the dark (HPLC monitoring showed that the indocyanine green reaction was complete).

[0046] After freeze-drying the reaction solution, 10g of crude reddish-brown solid was obtained.

[0047] The crude product 10 g was added to a 500 ml single-neck flask, then 100 ml of methanol was added, and stirring was carried out at room temperature for 30 minutes until the solid was completely dissolved. Then 200 ml of acetone was added dropwise, and stirring was continued for 30 minutes. Filtration was carried out, the filter cake was washed with 10 ml of a mixed solution of methanol / acetone = 1 / 2 twice, and the wet product was vacuum dried at 40-50°C for 10 hours to obtain 8.1 g, with a yield of 81.0%, and HPLC: 81.6%.

[0048] Comparative Example 2:

[0049] The crude product 10 g was added to a 500 ml single-neck flask, then 100 ml of methanol was added, and stirring was carried out at room temperature for 30 minutes until the solid was completely dissolved. Then 200 ml of acetone was added dropwise, and stirring was continued for 30 minutes. Filtration was carried out, the filter cake was washed with 10 ml of a mixed solution of methanol / acetone = 1 / 2 twice, and the wet product was vacuum dried at 40-50°C for 10 hours to obtain 8.1 g, with a yield of 81.0%, and HPLC: 81.6%.

[0050] After the reaction solution was freeze-dried, a red-brown solid crude product 10 g was obtained.

[0051] The crude product 10 g was added to a 500 ml single-neck flask, then 100 ml of methanol was added, and stirring was carried out at room temperature for 30 minutes until the solid was completely dissolved. Then 200 ml of acetone was added dropwise, and stirring was continued for 30 minutes. Filtration was carried out, the filter cake was washed with 10 ml of a mixed solution of methanol / acetone = 1 / 2 twice, and the wet product was vacuum dried at 40-50°C for 10 hours to obtain 8.1 g, with a yield of 81.0%, and HPLC: 81.6%.

[0052] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing indocyanine green dimer as shown in formula I, which is prepared from indocyanine green as starting material, in the presence of light-avoiding condition, in the temperature range of 0-20℃, in solvent purified water, and for 6-12 days to form dimer. 。 2. The method according to claim 1, wherein the temperature of the reaction is 5-10℃.

3. The method according to claim 1, wherein the mass / volume ratio of indocyanine green to solvent is 1g:30-200ml.

4. The method according to claim 2, wherein the reaction equation is shown as follows: 。 5. The method according to claim 1, wherein after the reaction is completed, the reaction solution is subjected to freeze-drying to obtain solid crude product.

6. The method according to claim 5, wherein the obtained crude product is dissolved in methanol, and then pure product is precipitated after addition of acetone, and the volume ratio of methanol to acetone is 1:

2.

7. The method according to claim 6, wherein the purified solid is washed and vacuum-dried to obtain pure product.

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