WO2022151981A1 - Indotricarbocyanine dye compound, preparation method therefor and use thereof - Google Patents

Abstract

The present invention belongs to the field of medicines, and in particular relates to an indotricarbocyanine dye compound, a preparation method therefor and the use thereof. A large-conjugate water-soluble indotricarbocyanine dye compound is synthesized and prepared in the present invention, and the compound can be used as a fluorescent dye and has potential development and application prospects. In the present invention, a related analysis method of the compound is also developed, which can provide a research basis for the research into impurities in indocyanine green medicaments and can provide a basis for the safety research into the indocyanine green medicaments.

Description

A kind of indole tricarbocyanine dye compound and preparation method and application thereof technical field

The invention belongs to the field of medicine, and in particular relates to an indole tricarbocyanine dye compound, a preparation method thereof, and an application in pharmacy.

Background technique

Indocyanine dyes are a very important class of near-infrared dyes and a very important branch of cyanine dyes. According to the number of carbon atoms on the conjugated methine chain of the dye molecule, indocyanine dyes can be divided into monomethine dyes. , Sanjiachuan dye, Wujiachuan dye and Qijiachuan dye, etc.

Indocyanine green (ICG) is a fluorescent tricarbocyanine dye developed by Kodak Laboratories in 1955 and used in the diagnosis of human diseases since 1956. Most clinical applications of ICG take advantage of its fluorescence function. Indocyanine green has the characteristics of strong penetrating power and high binding rate to plasma proteins, and can clearly display choroid images. The early application of ICG molecular fluorescence imaging technology in the field of ophthalmology, the technology began to be used for intraoperative navigation, identification of key structures and guidance of solid tumor resection, vascular imaging in neurosurgery, cardiac surgery, vascular surgery, and breast cancer. In recent years, with the continuous development of indocyanine green imaging technology, the clinical application of indocyanine green has also been expanded. ICG is widely used today in most surgical fields such as ophthalmology, urology, and general surgery.

Intravenously injected ICG is mainly bound to serum proteins (mainly globulin) and transported in the blood in two different forms, bound and unbound, and the concentration of ICG in plasma decreases exponentially after injection. Unbound ICG is rapidly cleared from the vasculature by the liver and transported unmodified into bile by the protein glutathione S-transferase. Freely soluble ICG can localize to the desired target, but its relatively short retention time within the target limits its potential for diagnostic and therapeutic applications.

In addition, the stability and optical properties of ICG molecules are related to factors such as concentration, temperature, solvent medium, and pH, which affect the effectiveness of ICG molecules. The Pharmacopoeia clearly stipulates that the content of sodium iodide in indocyanine green should not exceed 5%. The role of sodium iodide in indocyanine green is to promote solubility on the one hand and increase the stability of ICG on the one hand. The absorption spectrum of ICG varies greatly with concentration, the ICG molecules in solution remain as monomers at concentrations below about 400 ug/ml, and the absorption spectrum peaks between 780 and 810 nm, depending on the solvent medium. With prolonged storage time, colorless conjugates (a less conjugated molecule) are formed in the ICG solution, leading to degradation of the ICG, resulting in a decrease in absorbance in the IR range and a change in absorption peaks. The degradation rate in water is significantly faster than that of organic solvents such as alcohols. In addition, the degradation process is more likely to occur under conditions of continuous light or elevated temperature.

Relevant studies have shown that ICG has cytotoxicity after decomposition (eg: RPE cells). Due to the differences in intraoperative ICG dose and conditions of use, the precise safety threshold for ICG application has not been determined. Therefore, the research on ICG degradants is conducive to the research on the clinically relevant side effects of ICG, and ensures a more scientific use of the drug.

In the past few years, qualitative and quantitative methods such as high pressure liquid chromatography combined with UV detector (HPLC-UV) or liquid chromatography combined with mass spectrometry (LC-MS) have been developed to analyze the stability of ICG. However, most of these methods focus on the analysis of intact ICG compounds, and there are few studies on ICG degradation and decomposition products.

In the present invention, a large conjugated and water-soluble indole tricarbocyanine dye compound is synthesized and prepared, and the compound can be used as a fluorescent dye and has potential development and application prospects. The invention simultaneously develops the relevant analysis method of the compound, which can provide a research basis for the research of impurities in the indocyanine green medicine, and provide a basis for the safety research of the indocyanine green medicine.

SUMMARY OF THE INVENTION

The present invention has synthesized and prepared an indole tricarbocyanine dye compound, which has the following structural features:

In the above structure, it is characterized in that Compound I is in the form of sodium iodide salt, wherein the number of sodium iodide is 0<X≦2.

In the above structure, it is characterized in that the sodium iodide content of compound I is 0.01% to 5%, preferably 3.0% to 5.0%.

Another aspect of the present invention provides a method for the preparation of compound I.

The specific method is as follows:

The preparation method is characterized in that indocyanine green or a pharmaceutically acceptable salt thereof is prepared by high performance liquid chromatography after reacting with an oxidant under a suitable solvent and temperature. The above-mentioned preparation method comprises the steps:

In the step (1), indocyanine green or a pharmaceutically acceptable salt thereof is used in a suitable solvent and temperature to react with an oxidant to prepare a solution to be separated, and high performance liquid chromatography is used to separate the solution to be separated, Collect the free form of the compound I;

In the step (2), compound I is prepared by using the free form of compound I as a raw material, and adding quantitative sodium iodide to form a salt.

The above-mentioned preparation step (1) is characterized in that indocyanine green or a pharmaceutically acceptable salt thereof is used in a suitable solvent and temperature to react with an oxidant to prepare a solution to be separated, separate by liquid chromatography, and collect the obtained solution. the free form of the compound represented by the formula I;

The above-mentioned preparation step (1) is characterized in that the reaction solvent is selected from one or more of water, alcohol, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, acetone, and acetonitrile, and the reaction The solvent is preferably water;

The above preparation step (1) is characterized in that the reaction temperature is selected from 0°C to 100°C.

Above-mentioned preparation step (1) is characterized in that oxidant is selected from air, oxygen, ozone, hydrogen peroxide, chlorate, perchlorate, nitrate, permanganate, dichromate, organic peroxide etc. One or more, the oxidant is preferably hydrogen peroxide;

The above-mentioned preparation step (1) is characterized in that, one or more of the mobile phase water, acetonitrile, methanol, and ethanol used in the high-performance liquid chromatography, preferably water and acetonitrile, and the ultraviolet detection wavelength is 10-400 nm, 263nm is preferred, and the elution mode is gradient elution.

Above-mentioned preparation step (1) is characterized in that the method for described gradient elution is as follows:

0～30min, keep the volume percentage of mobile phase A at 85%;

30-40min, the volume percentage of mobile phase A decreased from 85% to 70%;

40～42min, the volume percentage of mobile phase A decreased from 70% to 45%;

For 42-50 min, keep the volume percentage of mobile phase A at 85%.

The above-mentioned preparation step (2) is characterized in that the salt-forming solvent is selected from the reaction solvent and is selected from one or more of water, alcohol, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, acetone, and acetonitrile. species; the salt-forming solvent is preferably ethanol.

The above preparation step (2) is characterized in that the salt-forming reaction temperature is selected from 0°C to 100°C.

The above preparation step (2) is characterized in that the sodium iodide feeding amount is 0.01% to 32.46%, preferably 6.0% to 10.0%;

Another aspect of the present invention provides an amorphous form of Compound I, and Compound I exists in two forms: a crystalline state and an amorphous state, and the amorphous state has better water solubility than the crystalline state, and is more suitable for formulation requirements.

The above-mentioned preparation method of the amorphous compound I is characterized in that the compound I is prepared into an aqueous solution of a suitable concentration by using a freeze-drying method, and is prepared by freeze-drying at a low temperature.

In addition, the present invention also provides an analysis and detection method for the compound I. The content of this impurity in indocyanine green is greatly affected by the preparation process of the drug, storage conditions, etc. Therefore, it is necessary to develop a fast and efficient analysis and detection method, Assess product quality and provide medication safety guidance.

The above analysis and detection method is characterized in that the mobile phase A used in the high performance liquid chromatography is a sodium dihydrogen phosphate buffer solution, the mobile phase B is a methanol-acetonitrile mixed solution, the ultraviolet detection wavelength is 263 nm, and the elution mode is a gradient elute. The detection method mainly includes the following steps:

Described step (1) gets the reference substance of described compound I, dissolves with solvent, prepares reference substance solution;

The step (2) takes the indocyanine green test substance, dissolves or extracts it with a solvent, and prepares a test solution;

Described step (3) adopts high performance liquid chromatography to detect described reference substance solution and need testing solution

The above detection method is characterized in that the filler of the chromatographic column used in the high performance liquid chromatography is octadecylsilane bonded silica gel.

The above detection method is characterized in that the pH value of the mobile phase A is 4.4-5.2.

The above-mentioned detection method is characterized in that the ratio of methanol and acetonitrile in the mobile phase B is 2:48;

Above-mentioned detection method is characterized in that the method for described gradient elution is as follows:

From 0 to 15min, the volume percentage of mobile phase A decreased from 83% to 80%;

15～40min, the volume percentage of mobile phase A decreased from 80% to 50%;

40～55min, the volume percentage of mobile phase A decreased from 50% to 45%;

55～60min, the volume percentage of mobile phase A increased from 45% to 83%;

For 60-70 min, keep the volume percentage of mobile phase A at 83%.

beneficial effect

Compared with the prior art, the present invention has the following beneficial effects:

1. Compound I was prepared for the first time in the present invention, which is the largest single impurity of the indocyanine green bulk drug and is a dimer compound. It can be used as an indole tricarbocyanine dye, expanding the application of indocyanine dyes.

2. Compound I is the main impurity (more than 3%) of the original research drug of indocyanine green and domestic commercial products. The compound can provide a research basis for the degradation products of indocyanine green drugs, and provide a basis for the safety research of indocyanine green drugs. There are few studies on ICG degradation products and clinical side effects, and the study of this compound is conducive to a comprehensive and in-depth understanding of ICG.

3. The present invention separates, prepares, concentrates, purifies, detects and identifies compound I through various technical means such as semi-preparative liquid chromatography, high performance liquid chromatography, high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, and the raw materials are common and It is easy to obtain, and the synthesis and separation steps are simple and controllable, which can provide a reference substance for the research of ICG impurities, facilitate the quality control of ICG, and improve its drug safety.

4. The present invention prepares the amorphous compound I with better water solubility by freeze-drying method, which is beneficial to the development and research of indocyanine green preparation products and facilitates comprehensive research on the quality control method of indocyanine green.

5. The present invention has developed a detection method for the compound, which can detect the content of compound I in the ICG bulk drug and preparation by using high performance liquid chromatography with a suitable mobile phase setting. The detection method is simple in operation and reliable in results, and can realize rapid and low-cost detection of compound I in ICG raw materials and preparations.

Description of drawings

Accompanying drawing 1 I MS spectrum of the compound of the present invention.

Fig. 2 I ¹ H-NMR spectrum of the compound of the present invention.

Fig. 3 I ¹³ C-NMR spectrum of the compound of the present invention.

The HPLC chromatograms of the indocyanine green analyte sample and the compound when the pH values of mobile phase A are 3.6 and 4.0 under the conditions of Example 13 of accompanying drawing 4.

Figure 5 shows the HPLC chromatograms of the indocyanine green analyte sample and the compound I prepared in Example 4 under the conditions of Example 13, when the pH value of mobile phase A is 5.8.

Figure 6 shows the HPLC chart of the compound I of the formula prepared by the sample and the compound I prepared in Example 4 under the conditions of Example 14.

7 is the chromatogram of the reference solution, one batch of homemade preparations and the original product (IC-Green) under the conditions of Example 15.

Detailed ways

The specific steps of the present invention are illustrated by the following examples, but are not limited by the examples.

Terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

The present invention will be described in further detail below with reference to specific examples and data. It should be understood that these examples are intended to illustrate the invention only and not to limit the scope of the invention in any way.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

In the embodiment of the present invention, indocyanine green is prepared with reference to patent US2895955A; the HPLC analysis method of indocyanine green is:

In the embodiment of the present invention, adopt high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition four general rules 0512) to detect this product sodium iodide, and the chromatographic conditions are as follows:

Chromatographic column: Thermo BDS HypersilTM C18, 4.6×250mm, 5μm or equivalent column. Mobile phase A: tetrabutylammonium hydroxide solution (take 12.5 ml of 10% tetrabutylammonium hydroxide solution, add 1000 ml of water, shake well, and adjust pH to 6.5 with phosphoric acid solution); mobile phase B: acetonitrile. Solvent: water; column temperature: 25℃; flow rate: 1ml/min; detection wavelength: 227nm; injection volume: 10μl

Gradient elution was performed according to the following table:

The present invention will be further described below in conjunction with specific embodiments.

Example 1

Dissolve 5.0 g of indocyanine green self-made raw material drug (compound I content of 3.0%) in 5 mL of methanol, let stand, filter, and collect the filtrate. The obtained filtrate is separated through a high-pressure reversed-phase preparative column, and the specific steps are as follows:

The high-pressure preparative liquid phase is Hanbang Technology AS20005 binary analysis liquid chromatography system (including NU3000UV/VIS detector and NP7000 analytical liquid chromatography pump); chromatography column: Morphling WD-C18 column (20mm×250mm, 5μm); mobile phase : gradient elution with water/acetonitrile mixed solution; column temperature: 25°C; detection wavelength: 263 nm; flow rate: 5-10 mL/min; injection volume: 5 mL.

Mobile phase A: water;

Mobile phase B: acetonitrile;

Gradient elution was performed according to the following table:

The eluate was collected and freeze-dried to obtain a dark blue-green solid, namely the free form of compound I (50 mg), with a yield of 33% and a sodium iodide content of 0.02%. The MS spectrum is shown in FIG. 1 , the ¹ H-NMR spectrum is shown in FIG. 2 , and the ¹³ C-NMR spectrum is shown in FIG. 3 .

MS: 752.2[1/2M+H] ⁺

¹ HNMR(400MHz,DMSO-d6)δ8.21-8.29(m,10H),8.05-8.07(d,4H),7.97-8.01(m,4H),7.77-7.79(d,2H),7.62-7.70 (m,6H),7.51-7.55(m,2H),7.45-7.49(m,2H),6.63-6.66(m,2H),5.76-5.79(d,2H),4.23(m,4H),4.00 (m, 4H), 2.34-2.48 (m, 8H), 1.96-2.03 (m, 24H), 1.67-1.77 (m, 16H)

¹³ CNMR(400MHz,DMSO-d6))δ175.02,171.54,156.70,153.03,148.21,140.54,139.98,134.59,132.80,131.99,131.48,130.80,130.62,130.34,128.88,128.14,128.01,127.84,125.55,124.85, 123.73, 122.78, 122.56, 112.48, 111.87, 105.90, 99.64, 51.43, 51.13, 51.05, 50.62, 44.56, 43.93, 27.84, 27.27, 27.13, 26.42, 22.93, 22.84.

Example 2

Dissolve 4g of indocyanine green product in 2.0L of water, under the condition of 20-30℃, stir to ensure full dissolution, continue to introduce oxygen into the reaction solution, make the reaction solution fully contact with oxygen, stir and react for 3 days, the content of oxidized impurities about 50.0%. Concentrate, take 200 mg of the concentrate, add 5 mL of water to dissolve, separate according to the method of Example 1, collect the eluate, and freeze-dry to obtain a dark blue-green solid, that is, compound I free form (250 mg). The spectral data are the same as those in Example 1. The rate is 12.5%, and the sodium iodide content is 0.02%.

Example 3

Dissolve 200 mg of indocyanine green product in 5 mL of methanol, add 1 mL of 3% hydrogen peroxide solution, stir at room temperature for 10 minutes, stand, filter, and collect the filtrate to obtain a liquid to be separated. The obtained separation liquid was separated according to the method described in Example 1, and the eluate was collected and freeze-dried to obtain a dark blue-green solid, namely Compound I free form (5mg). Content 0.01%.

Example 4

Dissolve 200 mg of indocyanine green product in 5 mL of water, add the prepared aqueous sodium perchlorate solution (500 mg in 5 mL of water), stir at room temperature for 1 hour, stand, filter, and collect the filtrate to obtain the liquid to be separated. The obtained separation liquid was separated according to the method described in Example 1, and the eluate was collected and lyophilized to obtain a dark blue-green solid, namely Compound I free form (10 mg). Content 0.01%.

Example 5

Dissolve 200 mg of indocyanine green product in 5 mL of water, add potassium nitrate aqueous solution (500 mg in 5 mL of water), stir at room temperature for 1 hour, stand, filter, and collect the filtrate to obtain a liquid to be separated. The obtained separation liquid was separated according to the method described in Example 1, and the eluate was collected and freeze-dried to obtain a dark blue-green solid, namely Compound I free form (8mg). Content 0.01%.

Example 6

Dissolve 200 mg of indocyanine green product in 5 mL of methanol, add potassium permanganate aqueous solution (500 mg in 10 mL of water), stir at room temperature for 1 hour, let stand, filter, and collect the filtrate to obtain the liquid to be separated. The obtained separation liquid was separated according to the method described in Example 1, and the eluate was collected and freeze-dried to obtain a dark blue-green solid, namely Compound I free form (6mg). Content 0.01%.

Example 7

Dissolve 200 mg of indocyanine green product in 5 mL of methanol, add potassium dichromate aqueous solution (500 mg in 10 mL of water), stir at room temperature for 1 hour, let stand, filter, and collect the filtrate to obtain the liquid to be separated. The obtained separation liquid was separated according to the method described in Example 1, and the eluate was collected and lyophilized to obtain a dark blue-green solid, namely Compound I free form (12 mg). Content 0.01%.

Example 8

Dissolve 200 mg of indocyanine green product in 5 mL of ethanol, add ethanol solution of peroxybenzoic acid (1 ml of peroxybenzoic acid is dissolved in 5 ml of ethanol), stir at room temperature for 2 hours, let stand, filter, and collect the filtrate to obtain the desired solution. Separation liquid. The obtained separation liquid was separated according to the method described in Example 1, and the eluate was collected and freeze-dried to obtain a dark blue-green solid, namely Compound I free form (5mg). Content 0.01%.

The preparation of embodiment 9 sodium iodide salt

Take 200 mg of compound I free form prepared in Example 2, dissolve it in 3 ml of absolute ethanol, stir to dissolve it at room temperature, add an ethanolic solution of sodium iodide (35 mg of sodium iodide is dissolved in 1 ml of ethanol), and react 1 Hour, a large amount of solid was precipitated, filtered, and the filter cake was washed with acetone, and the filter cake was dried under reduced pressure at 50 ° C for 8 hours, and the collection was a dark blue-green solid, that is, compound I (125mg). The spectral data is the same as in Example 1, and the yield is 60.7. %, the sodium iodide content is 4.25%.

Example 10 Preparation of Amorphous Compounds

Take 100 mg of Compound I prepared in Example 9, add 10 ml of water for injection to dissolve, filter with a filter membrane to remove insolubles, and freeze the prepared filtrate with a freeze dryer for 24 hours to obtain a dark blue-green solid, that is, amorphous Compound I. (95mg), the spectral data is the same as that of Example 1, and the yield is 95.0%.

Example 11: Solubility Comparison Test

Weigh 10 mg each of compound I and amorphous compound I solid, add a certain amount of water or ethanol at 25°C, shake for 30 seconds every 5 minutes, and observe the solubility after 30 minutes. The results are shown in Table 1. Compared with compound I, amorphous compound I has a slightly better solubility in water, but has a significant increase in solubility in ethanol. After compound I is formed into a salt, its water solubility can be further enhanced.

Table 1 Compound solubility comparison test

compound	temperature	medium	Solubility
Compound I	25℃	water	2.50mg/mL
Compound I	25℃	Ethanol	0.18mg/mL
Amorphous Compound I	25℃	water	>20.0mg/mL
Amorphous Compound I	25℃	Ethanol	2.6mg/mL

Example 12: High temperature stability comparison test

According to literature reports, sodium iodide in indocyanine green has the effect of enhancing stability, but excessive iodide ions can cause some clinical side effects, so the content of sodium iodide in the Pharmacopoeia is set as no more than 5.0%. 10 mg of compound I solids (sodium iodide content from 0.01% to 4.5%) were weighed, respectively, and kept at 40°C/60°C for 10 days. The high temperature stability results of each group are shown in Table 2. With the increase of sodium iodide content, compound I has a significant high temperature stability advantage.

Table 2 Compound high temperature stability comparison test

Example 13:

Sample: indocyanine green API, batch number 20032701-5.

The indocyanine green raw material drug and the compound I prepared in Example 10 were used as reference substances to carry out the following research. The chromatographic conditions are as follows:

Instrument: high performance liquid chromatography-ultraviolet detector;

Chromatographic column: Welch Xtimate C18, 4.6×250mm, 5μm;

Mobile phase A: Phosphate buffers with different pH values;

Mobile phase A-1: 20 mmol/L sodium dihydrogen phosphate solution, adjusted to pH 3.6 with phosphoric acid;

Mobile phase A-2: 10 mmol/L sodium dihydrogen phosphate solution, adjusted to pH 4.0 with phosphoric acid;

Mobile phase A-3: 10mmol/L sodium dihydrogen phosphate-10mmol/L disodium hydrogen phosphate (94:6, pH value is about 5.8);

Mobile phase B: acetonitrile;

Gradient elution was performed according to Table 3 below:

Table 3 Elution procedure

Column temperature: 30℃;

Flow rate: 1.0ml/min;

UV detection wavelength: 263nm;

Injection volume: 10 μl;

Autosampler temperature: 5℃

Solvent: methanol.

Preparation of reference solution: take an appropriate amount of compound I prepared in Example 10, accurately weigh it, dissolve it in a solvent, and dilute it quantitatively to prepare a solution containing about 0.5 μg per 1 ml.

Preparation of sample solution: take an appropriate amount of the sample, accurately weigh it, dissolve it in a solvent and quantitatively dilute it to make a solution containing about 0.25mg per 1ml.

The mobile phase A with different pH values was injected and analyzed according to the above conditions. The chromatograms are shown in Figure 4 and Figure 5. Among them, the chromatographic peaks in the sample with the same retention time as the reference substance were normalized according to the peak area. Calculate, compare with the following table 4:

Table 4. Mobile phase A comparison experiments with different pH values

Mobile Phase A - pH	detection solution	Impurity content (%)
3.6	sample solution	0.845
4.0	sample solution	0.153
5.8	sample solution	0.009

The results showed that the indocyanine green bulk drug, batch number 20032701-5, contained the same impurity (compound I). And when the test solution is detected, the pH value of mobile phase A has a significant impact on the detection results, indicating that the pH value of mobile phase A should be strictly controlled to prevent false positive detection results of compound I.

Example 14

In view of the fact that under the chromatographic conditions of Example 13, the pH value of mobile phase A has a significant influence on the detection results, the chromatographic conditions are therefore optimized.

Sample: indocyanine green API, batch number 20051103.

The chromatographic conditions adopted in this example are as follows:

Instrument: high performance liquid chromatography-ultraviolet detector;

Chromatographic column: Welch Xtimate C18, 4.6×250mm, 5μm;

Mobile phase A: Phosphate buffers with different pH values;

Mobile phase A-1: 10mmol/L sodium dihydrogen phosphate solution-10mmol/L disodium hydrogen phosphate (498:2; pH value is about 5.2);

Mobile phase A-2: 10mmol/L sodium dihydrogen phosphate solution-10mmol/L disodium hydrogen phosphate (998:2; pH value is about 5.0);

Mobile phase A-3: 10mmol/L sodium dihydrogen phosphate solution, phosphoric acid adjusts the pH value to 4.8;

Mobile phase A-4: 10mmol/L sodium dihydrogen phosphate solution, phosphoric acid adjusts the pH value to 4.6;

Mobile phase A-5: 10mmol/L sodium dihydrogen phosphate solution, phosphoric acid adjusts the pH value to 4.4;

Mobile phase B: methanol-acetonitrile (3:47);

Carry out gradient elution according to Table 5 below;

Table 5 Elution program

Column temperature: 30℃;

Flow rate: 1.0ml/min;

Ultraviolet wavelength: 263nm;

Injection volume: 10 μl;

Solvent: methanol.

Test solution: take an appropriate amount of the sample, accurately weigh it, dissolve it in the solvent, place it under light conditions (4500±500lux, 90μW/cm ² ) for 6.5h, add the solvent and quantitatively dilute it to make about 0.25mg per 1ml of the sample. solution.

The chromatogram is shown in Figure 6; the detected amount of impurity I is shown in Table 6 below.

Table 6 Comparison test of mobile phase A light conditions with different pH values

Mobile Phase A - pH	detection solution	Compound I content (%)
4.4	light damage solution	0.537
4.6	light damage solution	0.602
4.8	light damage solution	0.516
5.0	light damage solution	0.525
5.2	light damage solution	0.512

Example 15 Detection method and method application of compound I.

Samples: indocyanine green raw materials, made in the laboratory, batch numbers 20090301 and 20092701; indocyanine green for injection: made in the laboratory, in batches 20092105 and 20073001; foreign products: IC-Green, AKRON INC, USA, Batch 061179A; Diagnogreen, Daiichi Sankyo Co., Ltd., batch QHA0133. Domestic marketed preparations: Ruidu, Dandong Yichuang Pharmaceutical Co., Ltd., batch 19061213;

Reference substance: prepared by the method described in Example 4, batch 20120401.

Among them, the content of the reference substance was calibrated by mass balance method, and it was 93.12%.

Chromatographic conditions:

Instrument: high performance liquid chromatography-ultraviolet detector;

Chromatographic column: Welch Xtimate C18, 4.6×250mm, 5μm;

Mobile phase A: 10 mmol/L sodium dihydrogen phosphate solution, adjusted to pH 4.6 with phosphoric acid;

Mobile phase B: methanol-acetonitrile (2:48);

Gradient elution was performed according to Table 7 below:

Table 7 Elution program

Column temperature: 30°C; flow rate: 1.0 ml/min; UV wavelength: 263 nm; injection volume: 10 μl; solvent: methanol.

Reference solution: take an appropriate amount of compound I, accurately weigh it, dissolve it in a solvent and dilute it quantitatively to make a solution containing about 5μg per 1ml.

Test solution: Take an appropriate amount of indocyanine green to be tested, accurately weigh it, dissolve it in a solvent and quantitatively dilute it to make a solution of about 0.25 mg of indocyanine green per 1 ml.

Calculation formula:

(1) Correction factor

In the formula: F—correction factor;

C _{reference substance} —concentration of reference substance solution (mg/ml);

A _{reference substance} —the peak area of the reference substance solution.

(2) Impurity I content

In the formula: F _average - the average value of the correction factor;

A _{test sample} —the peak area of impurity I in the test solution;

D—the dilution factor of the test product;

W _{Test sample} —weighed sample weight (mg) of the test sample.

The chromatograms of the reference solution, one batch of self-made preparations and the original product (IC-Green) are shown in Figure 7: according to the external standard method, the detection results of compound I in the test solution are shown in Table 8 below;

Table 8 Comparison test of self-made preparations and original products

Claims (6)

1. A compound of structural formula as shown in formula I

   

   where 0<X≤2.
2. The compound of claim 1, wherein the sodium iodide content of compound I is 0.01% to 5%.
3. The preparation method of the compound according to claim 1 or 2, characterized in that the compound I is prepared by using indocyanine green or a pharmaceutically acceptable salt thereof as a raw material, undergoing an oxidation reaction, and then adding sodium iodide.
4. The preparation method of claim 3, characterized in that it comprises:

   Step (1): prepare and separate the free form of compound I by reacting indocyanine green or a pharmaceutically acceptable salt thereof with an oxidizing agent;

   Step (2): using the free form of the compound of formula I as a raw material, adding quantitative sodium iodide for reaction, and preparing the compound represented by formula I.

   Wherein the solvent used in the step (1) is one or more of water, alcohol, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, acetone, acetonitrile;

   Wherein the reaction temperature in step (1) is 0 ℃ to 100 ℃;

   In step (1), the oxidant is air, oxygen, ozone, hydrogen peroxide, chlorate, perchlorate, nitrate, permanganate, dichromate, one or more in organic peroxide;

   Wherein the reaction solvent in the step (2) is one or more of water, alcohol, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, acetone, and acetonitrile;

   In step (2), the reaction temperature is 0°C to 100°C;

   In step (2), the sodium iodide feeding amount is 0.01% to 5% of the free body feeding amount.
5. A method for preparing an amorphous compound I, characterized in that the compound I described in claim 1 or 2 is prepared into an aqueous solution, and the amorphous compound I is prepared by freeze-drying.
6. A kind of detection method of I compound as claimed in claim 1 or 2, is characterized in that, comprises the steps:

   Get the reference substance of the compound of formula I, dissolve with solvent, prepare reference substance solution;

   Take the indocyanine green test substance, dissolve or extract it with a solvent, and prepare a test solution;

   The reference substance solution and the test solution are detected by high performance liquid chromatography;

   The specific detection method is as follows:

   The mobile phase A used in the high performance liquid chromatography is sodium dihydrogen phosphate buffer, the mobile phase B is a methanol-acetonitrile mixed solution, the ultraviolet detection wavelength is 263 nm, and the elution mode is gradient elution;

   The filler of the chromatographic column used in the high performance liquid chromatography is octadecylsilane bonded silica gel;

   The pH value of the mobile phase A is 4.4 to 5.2;

   In the mobile phase B, the mixing ratio of methanol-acetonitrile is 2:48;

   The gradient elution method is as follows:

   From 0 to 15min, the volume percentage of mobile phase A decreased from 83% to 80%;

   15～40min, the volume percentage of mobile phase A decreased from 80% to 50%;

   40～55min, the volume percentage of mobile phase A decreased from 50% to 45%;

   55～60min, the volume percentage of mobile phase A increased from 45% to 83%;

   For 60-70 min, keep the volume percentage of mobile phase A at 83%.

Images