CN103242255B - Evans blue complex as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses an Evan's blue complex, a preparation method and application thereof, and its structure contains a macrocyclic polyamine chelating group NOTA. Labeled complexes are obtained after labeling with radionuclides (fluorine-18, copper-64 and gallium-68, etc.). The complex is prepared by wet method or freeze-dry method. The present invention also relates to the use of the complex as an imaging agent in human or animal organs or tissues, especially as a blood pool imaging agent, which has the advantages of simple preparation, low price and high target/non-target ratio advantage.

Description

Evan's blue complex and its preparation method and application

technical field

The invention relates to a class of radioactive complexes, a preparation method thereof and the application of the complexes as blood pool imaging agents.

Background technique

Vascular system diseases seriously affect human health. It is necessary to effectively display blood vessels by imaging means and combine immunohistochemical research to explore the pathogenesis of diseases. At present, the commonly used vascular imaging methods include ink perfusion, immunohistochemistry, tannic acid mordant staining, enzyme histochemistry and so on. However, these methods have their own disadvantages, such as not being able to combine with immunohistochemical research, the process is cumbersome or the fluorescence is easily quenched.

The use of radionuclide-labeled imaging agents to image the vascular system is one of the hotspots in radiopharmaceutical research today. Radionuclide blood pool imaging can non-invasively measure local or overall cardiac function in vivo and provide some cardiac function parameters. It has certain value in the early diagnosis, prognosis and curative effect observation of myocardial diseases such as coronary heart disease, cardiomyopathy and valvular disease. At present, ^99m Tc-RBC (red blood cell) is the most clinically used imaging agent (Wang Xuebin, Ma Xiaoyu, Tang Zhigang, et al. Research on ^99m Tc in vitro labeling of red blood cells. Isotope, 1996, 9(4): 193-199 ). Labeling methods can be divided into in vitro, in vivo and semi-in vitro labeling methods. The in vitro labeling method has a high labeling rate, but the operation is cumbersome, the time is long, and it is difficult to ensure sterility, so it cannot be widely used. The operation of the in vivo labeling method is relatively simple, but its disadvantage is that two injections are required for the patient, and there are many factors affecting the labeling rate, and the labeling rate fluctuates greatly, which limits its clinical application to a certain extent. Although the semi-in vitro labeling method simplifies the operation of the in vitro labeling method to a certain extent, it still needs to collect blood samples from patients, so it is easy to cause cross infection or virus infection, and the labeling rate is lower than that of the in vitro labeling method.

Human serum albumin (HSA) is a natural component in human blood, a single peptide chain macromolecular protein composed of 576 amino acids, including 56 lysine residues, 17 tyrosine phenolic hydroxyl groups and a free sulfhydryl group , with a molecular weight of 68400 and a long biological half-life. ^99m Tc-HSA can be used as a blood pool imaging agent. Generally, the labeling rate of direct labeling method is not high, and the stability of the marker is poor, so that it can be cleared quickly in the blood and the imaging effect is poor. In recent years, there have been many reports on the use of dual-functional linkers coupled to human serum albumin for ^99m Tc labeling, including: DTPA (diethylenetriaminepentaacetic acid), DMP (2,3-dimercaptopropionic acid) and other dual-functional linkers . However, ^99m Tc-DTPA-HSA is cleared from the blood quickly, and it needs to be imaged 5-10 minutes after injection, and there is a certain concentration in the liver, which is not conducive to the diagnosis of some diseases. The more successful ones are ^99m Tc-DMP-HSA (Cambier JP, Verbeke KA, Vanbilloen HP et al. ^99m Tc-dimercaptopropionyl-HSA prepared from a labeling kit: Preliminary investigations as blood pool agent. Nucl Med Commun, 1997, 18:31 -37), the preparation method of the complex has been kitted.

By making some structural modifications to HSA, it is expected to obtain a blood pool imaging agent with excellent performance, but it is a human extract, which is expensive and may also carry some viruses, which limit its application to a certain extent.

Other ^99m Tc radiopharmaceuticals used for blood pool imaging include: ^99m Tc-DX (dextran) and ^99m Tc-labeled liposomes, etc., but no substantial progress has been made clinically.

In addition to the above-mentioned ^99m Tc-labeled imaging agents, ⁶² Cu, ⁶⁷ Cu, and ⁶⁸ Ga radionuclide-labeled HSA and its derivatives are used for blood pool imaging.

It can be seen from this that the existing relatively successful blood pool imaging agents are limited to technetium-99m-labeled red blood cells or radiolabeled biological extract HSA, and there are still complex preparation methods, high prices, easy virus infection, and imaging effects. It is easy to be affected by other factors and other disadvantages. If a new type of radiolabeled blood pool imaging agent with the characteristics of simple preparation method, low cost and good imaging effect can be provided, it will have broad application prospects.

Evan's blue is a fluorescent dye that is blue under white light and bright red under the green excitation light of a fluorescent microscope. Using Evan's blue for vascular imaging can be used to study vascular permeability and other characteristics. It can combine with plasma albumin to form Evan's blue-albumin complex, and then combine with plasma albumin receptors on the vascular endothelial cell membrane, so in theory it should be possible to display the shape of blood vessels by perfusion.

The schematic structural formula of Evans blue:

Based on the above considerations, the applicant proposed a new class of radiolabeled Evans blue derivatives as blood pool imaging agents. Preliminary research results show that this new type of blood pool imaging agent has excellent biological properties, and the preparation method is simple and low cost, which is expected to be popularized and applied clinically.

Contents of the invention

The primary purpose of the present invention is to provide a new class of radioactively labeled Evan's blue complexes with excellent imaging properties;

Another object of the present invention is to provide a method for preparing a new radiolabeled Evan's blue complex;

Another object of the present invention is to provide an application of the complex as an imaging agent in heart blood pool and tumor blood pool.

The present invention provides an Evans blue derivative (compound 6) and its radiolabeled complexes (compounds 7, 8 and 9).

Compound 6

The Evans blue derivative ligand (compound 6) of the present invention can be synthesized in the following manner:

1) Mix appropriate amount of compound 1 and compound 2, under the action of diethyl cyanophosphate, use amino condensation reaction to obtain compound 3.

2) The amino group of compound 3 was reduced with NaNO ₂ under the condition of hydrochloric acid (HCl) to obtain azo compound 4.

3) Compound 4 was reacted with compound 5 (1-amino-8-naphthol-2,4-disulfonic acid) to obtain target compound 6.

Its synthetic route is:

The present invention provides a method for preparing the radiolabeled Evans blue complex: radionuclide ( ¹⁸ F, ⁶⁴ Cu, ⁶⁸ Ga, ⁶² Cu, ⁶⁷ Cu, ⁸⁶ Y or ⁸⁹ Zr, etc., preferably ¹⁸ F, ⁶⁴ Cu and ⁶⁸ Ga) react with the Evans blue derivative ligand (compound 6) under appropriate conditions to prepare the radiolabeled Evans blue complex.

Preferred preparation methods are the following wet or lyophilized methods:

1. Synthesis of Radioactive ¹⁸ F Labeled Evans Blue Complex (Compound 7)

Wet labeling scheme:

1) Dissolving an appropriate amount of compound 6 in buffer solution or deionized water;

2) Dissolve aluminum chloride in buffer solution or deionized water;

3) Mix the above two solutions evenly;

4) Add acetonitrile or ethanol and freshly prepared ¹⁸ F ionic aqueous solution, seal the reaction at 70-120°C for 5-30 minutes, and cool down;

5) After diluting the reaction solution with water, it was separated and purified on a Sep-Pak C18 chromatographic column, the chromatographic column was washed with buffer or water to remove unreacted ¹⁸ F ions, and compound 7 was obtained by eluting with hydrochloric acid ethanol solution or ethanol solution. Compound 7 injection was obtained by sterile filtration after dilution with normal saline.

Its synthetic route is:

Labeling scheme for lyophilization:

1) The solution in step 3) of the wet method is aseptically filtered, divided into containers, freeze-dried, and then sealed with a stopper to obtain a freeze-dried kit. The dispensed containers are preferably regulatory antibiotic bottles.

2) Add an appropriate amount of acetic acid solution or buffer solution to the above kit to dissolve, then add acetonitrile or ethanol and freshly prepared ¹⁸ F ionic water solution, seal it at 70-120°C for 5-30 minutes, and cool;

3) After diluting the reaction solution with water, it was separated and purified on a Sep-Pak C18 chromatographic column, the chromatographic column was washed with buffer or water to remove unreacted ¹⁸ F ions, and compound 7 was obtained by eluting with hydrochloric acid ethanol solution or ethanol solution. Compound 7 injection was obtained by sterile filtration after dilution with normal saline.

2. Synthesis of radioactive ⁶⁴ Cu-labeled Evans blue complex (compound 8):

Wet labeling scheme:

1) Dissolving an appropriate amount of compound 6 in buffer solution or deionized water;

2) Add ⁶⁴ Cu ²⁺ (CuCl ₂ ) sodium acetate solution to the above solution, seal it at 40-80°C for 5-100 minutes, and cool down;

3) After separation and purification by semi-preparative HPLC (high performance liquid chromatography), the solvent was removed by rotary evaporation, the residue was dissolved in phosphate buffer saline (PBS) or water, and compound 8 injection was obtained by sterile filtration.

Its synthetic route is:

Labeling scheme for lyophilization:

1) The solution in step 1) of the wet method is aseptically filtered, then divided into containers, freeze-dried, then sealed with a stopper, and wait until the freeze-dried kit. The dispensed containers are preferably regulatory antibiotic bottles. According to the formation of the freeze-dried powder of the kit, excipients such as mannitol and ascorbic acid can be added to the kit, and the dosage of compound 6 and excipients can be adjusted to achieve the best molding of the kit.

2) Add an appropriate amount of acetic acid solution or buffer solution to the above kit to dissolve, then add ⁶⁴ Cu ²⁺ (CuCl ₂ ) sodium acetate solution, seal the reaction at 70-120°C for 5-30 minutes, and cool;

3) After separation and purification by semi-preparative HPLC (high performance liquid chromatography), the solvent was removed by rotary evaporation, the residue was dissolved in phosphate buffer saline (PBS) or water, and compound 8 injection was obtained by sterile filtration.

3. Synthesis of radioactive ⁶⁸ Ga-labeled Evans blue complex (compound 9):

Wet labeling scheme:

1) Dissolving an appropriate amount of compound 6 in buffer solution or deionized water;

2) Add ⁶⁸ Ga ³⁺ (GaCl ₃ ) hydrochloric acid eluent to the above solution, seal it at 25-80°C for 5-40 minutes, and cool down;

3) Separation and purification by semi-preparative HPLC (high performance liquid chromatography), rotary evaporation to remove the solvent, dissolving the residue in phosphate buffer saline (PBS) or water or saline, and sterile filtration to obtain compound 9 injection.

Its synthetic route is:

Labeling scheme for lyophilization:

1) The solution in step 1) of the wet method is aseptically filtered, then divided into containers, freeze-dried, then sealed with a stopper, and wait until the freeze-dried kit. The dispensed containers are preferably regulatory antibiotic bottles. According to the formation of the freeze-dried powder of the kit, excipients such as mannitol and ascorbic acid can be added to the kit, and the dosage of compound 6 and excipients can be adjusted to achieve the best molding of the kit.

2) Add an appropriate amount of acetic acid solution or buffer solution to the above kit to dissolve, then add ⁶⁸ Ga ³⁺ (GaCl ₃ ) hydrochloric acid eluent to the above solution, seal it at 25-80°C for 5-40 minutes, and cool down;

3) Separation and purification by semi-preparative HPLC (high performance liquid chromatography), rotary evaporation to remove the solvent, dissolving the residue in phosphate buffer saline (PBS) or water or saline, and sterile filtration to obtain compound 9 injection.

Other chemical substances used in the above synthesis steps are commercially available.

In the above method, the radionuclide is the imaging part of nuclear medicine, in addition to ¹⁸ F, ⁶⁴ Cu and ⁶⁸ Ga, it may also be ¹¹¹ In, ⁶² Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁸⁶ Y and ⁸⁹ Zr; The buffer solution is a material for stabilizing the pH value of the reaction solution, which can be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate and phosphate, and their mixture etc.

The preparation method of the radiolabeled Evan's blue complex provided by the invention is simple and the cost is low. Biological test results show that it has high uptake and good retention in blood, and has a high target/non-target ratio, and is suitable for use as a blood pool imaging agent.

Description of drawings

Fig. 1 is the HPLC analysis figure of compound 7;

Figure 2 is the PET images of compound 7 in normal nude mice at different times, the uptake values of different tissues and organs, and the time-radiation curve (0-50min) after administration;

Figure 3 is the PET dynamic imaging of compound 7 in nude mice bearing INS-1 insulinoma and U87MG glioma respectively (A) and its time-radioactive uptake curve within 60 minutes after administration based on PET imaging (B), CD-31 immunostaining of tumor sections (C), comparison of uptake in different tumor vessels (D) and quantitative comparison of tumor blood volume, blood flow and vascular permeability (E);

Figure 4 shows the PET imaging (A) and dynamic time-radioactivity curve (B) of compound 8 in 22B tumor-bearing nude mice.

Detailed ways

Can make the present invention be illustrated more clearly below by specific preparation example and embodiment:

Example 1

1. Preparation of Evans Blue Derivative Ligand (Compound 6)

1) Synthesis of compound 3:

Mix and dissolve 20.0 mg of compound 1 (ortho-dimethylbenzidine, 94 μmol) and 20.0 mg of compound 2 (NOTA-3HCl, 48 μmol) in 1 mL of DMSO, then add 3.6 μL of diethyl cyanophosphate (24 μmol) and 25 μL Diisopropylethylamine (DIPEA) was added to the above mixture, and after stirring at room temperature for 40 min, 3.6 μL of diethyl cyanophosphate (24 μmol) was added again, and the reaction mixture was kept stirring overnight at room temperature. The product (compound 3) was separated and purified by semi-preparative HPLC, and the fraction with a retention time of 10.0 min was collected and freeze-dried to obtain about 12.2 mg of pure compound 3 with a yield of 26.4%. The intermediate product was identified by liquid chromatography-mass spectrometry (LC-MS): [MH] ⁺ =498.2462, and the calculated value (m/z) was 497.2638 (C ₂₆ H ₃₅ N ₅ O ₅ ).

2) Synthesis of compound 6:

Add 2.5 mg of compound 3 (5.0 μmol) and 0.1 mL of hydrochloric acid solution (18 μmol HCl) dissolved in 0.3 mL of water into a 20 mL glass container, and cool the reaction mixture in an ice-water bath, then add 0.5 mg of sodium nitrite ( 7.2μmol, dissolved in 0.1mL water). The mixture was stirred for 20 min under ice-water bath conditions to obtain a yellow azo salt solution (azo compound 4). Without further purification, the above solution was added dropwise to another reaction tube in an ice-water bath, which contained 4.0 mg of compound 5 (1-amino-8-naphthol-2,4-disulfo acid, 10 μmol) and 2.4 mg sodium bicarbonate (28.5 μmol), and the final reaction mixture was stirred in an ice-water bath for 2 h to obtain the target compound 6. The product (compound 6) was separated and purified by semi-preparative HPLC, and the fraction with a retention time of 19.0 min was collected and freeze-dried to obtain about 1.4 mg of pure compound 6 with a yield of 46.6%. The product was identified by liquid chromatography-mass spectrometry (LC-MS): [MH] ^- =826.2415, and the calculated value (m/z) was 827.2255 (C ₃₆ H ₄₁ N ₇ O ₁₂ S ₂ ).

Example 2

1. Preparation of radioactive ¹⁸ F-labeled freeze-dried kit (take the preparation of 100 tubes as an example)

Weigh 8 mg of compound 6 and dissolve in 10 mL of 0.5 mol/L acetic acid-sodium acetate buffer solution (pH4), then dissolve 0.08 mg of aluminum chloride (AlCl ₃ ) in 10 mL of 0.5 mol/L acetic acid-sodium acetate buffer solution ( pH4), mix the two evenly. After sterile filtration, it is divided into 100 control antibiotic vials, then placed in a freeze dryer for 24 hours, and then sealed with a stopper to obtain a freeze-dried medicine box. According to the output of the kit and the requirements for the content of the components in each kit, the amount of compound 6 and aluminum chloride can be adjusted so that their weight ratio falls within the range of (20-100):1.

2. Preparation of ¹⁸ F-labeled Evans blue complex (compound 7):

1) Wet method: In a 1 mL plastic tube, add 3 μL of 2 mM AlCl ₃ acetic acid-acetate buffer solution (0.5 mol/L, pH=4) and 6 μL of 3 mM compound 6 acetic acid-acetate buffer solution (0.5 mol/L , pH=4), then add 0.13mL of acetonitrile and 0.05mL of about 370 MBq (MBq) of ¹⁸ F ^- water solution, mix well and place in a boiling water bath for 10 minutes to react to obtain the target compound 7 complex. After the reaction solution was cooled, it was diluted with water to 10mL volume, separated and purified by VarianBond Elut C18 column (100mg), washed with 10mL water to remove unreacted ¹⁸ F ions, and washed out with 0.3mL 80% ethanol aqueous solution (containing 1mM HCl). Compound 7 was spin-dried with ethanol under the protection of argon. The final product was dissolved in phosphate buffer (0.5mol/L, pH=7.4) and filtered sterilely. The obtained Compound 7 injection was identified by analytical HPLC.

2) Freeze-drying method: Add 0.5 mL of 0.5 mol/L acetic acid-acetate buffer solution (pH=4) to the freeze-dried kit, add about 37 to 3700 megabeq (MBq) ¹⁸ F ^- Acetonitrile eluent (obtained from anion trapping column QMA), sealed at 120°C for 5 minutes, cooled; dilute the reaction solution with water, separated and purified by Sep-Pak C18 chromatographic column, with 0.5mol/L phosphate buffer (pH7.4 ) to wash the chromatographic column to remove unreacted ¹⁸ F ions, rinse with ethanol hydrochloric acid solution to obtain compound 7, dilute with normal saline and filter aseptically to obtain compound 7 injection.

3. Preparation of radioactive ⁶⁴ Cu and ⁶⁸ Ga labeled freeze-dried kits (take the preparation of 100 tubes as an example)

Weigh 8 mg of compound 6 and dissolve it in 10 mL of 0.5 mol/L tartaric acid-potassium sodium tartrate buffer solution (pH4). After sterile filtration, it is divided into 100 control antibiotic vials, and then placed in a freeze dryer to freeze dry for 24 hour, stoppered and sealed to obtain a freeze-dried medicine box. According to the formation of the freeze-dried powder injection of the kit, excipients, such as mannitol, ascorbic acid, etc., can be added to the kit, and the dosage of compound 6 and excipients can be adjusted to achieve the best molding of the kit.

4. Preparation of ⁶⁴ Cu-labeled Evans blue complex (compound 8):

1) Wet method: add about 18.5-1850 megabeq (MBq) ⁶⁴ CuCl ₂ sodium acetate solution into a controlled antibiotic bottle containing 0.5 mL of compound 6 in acetic acid-acetate solution (4.0 g/L), and place in The target compound 8 was obtained by reacting in a water bath at 60°C for 20 minutes. After separation and purification by semi-preparative HPLC (high performance liquid chromatography), the solvent was removed by rotary evaporation, the residue was dissolved in water, and compound 8 injection was obtained by sterile filtration.

2) Freeze-drying method: add about 18.5-1850 megabeq (MBq) ⁶⁴ CuCl ₂ sodium acetate solution into the freeze-dried kit, mix well and heat in a water bath at 60°C for 20 minutes to obtain the target compound 8. After separation and purification by semi-preparative HPLC (high performance liquid chromatography), the solvent was removed by rotary evaporation, the residue was dissolved in phosphate buffer saline (PBS), and compound 8 injection was obtained by sterile filtration.

5. Preparation of ⁶⁸ Ga-labeled Evans blue complex (compound 9):

1) Wet method: add about 18.5-1850 megabeq (MBq) ⁶⁸ GaCl ₃ hydrochloric acid solution (leached from the gallium generator) to the acetic acid-acetate solution (4.0g/L) containing 0.5mL compound 6 Put it in an antibiotic bottle and react at room temperature for 20 minutes to obtain the target compound 9. After separation and purification by semi-preparative HPLC (high performance liquid chromatography), the solvent was removed by rotary evaporation, the residue was dissolved in phosphate buffer saline (PBS), and compound 9 injection was obtained by sterile filtration.

2) Freeze-drying method: add about 18.5-1850 megabeq (MBq) ⁶⁸ GaCl ₃ hydrochloric acid solution (leached from the gallium generator) into the freeze-dried kit, mix well and react at room temperature for 20 minutes to obtain the target Compound 9. After separation and purification by semi-preparative HPLC (high performance liquid chromatography), the solvent was removed by rotary evaporation, the residue was dissolved in phosphate buffer saline (PBS), and compound 9 injection was obtained by sterile filtration.

Taking radioactive fluorine-18-labeled Evans blue complex (compound 7) as an example, its performance measurement is described as follows:

1. HPLC analysis and identification

The semi-preparative HPLC system is as follows: Waters600 type (Waters996PDA detector); Waters Nova-PakHR C18 column (6μm, 7.8×300mm). The flow rate was 6 mL/min.

The analytical HPLC system is as follows: Perkin-Elmer Series200LC equipped with Waters2784 dual absorption wavelength ultraviolet detector and Bioscan radioactive detector, Waters Symmetry analytical column (5μm, 150x3.9mm). The flow rate is 1mL/min, the retention time of compound 7 is about 16min and the radiochemical purity is greater than 95%. The HPLC results are shown in Figure 1.

Elution gradient: 0 to 5 minutes: 5% acetonitrile (0.1% TFA) and 95% water (0.1% TFA) remain unchanged; 5 to 35 minutes: increase to 65% acetonitrile (0.1% TFA) and 35% water ( 0.1% TFA).

2. MicroPET imaging of compound 7 in normal nude mice

A solution of Compound 7 with a radiochemical purity greater than 95% was prepared according to the examples. Inject 0.1 mL (about 3.7 MBq) of Compound 7 solution into the tail vein of normal nude mice (about 20 grams in weight), and then perform dynamic MicroPET static imaging at 0 to 50 minutes after administration under isoflurane anesthesia. The results are shown in figure 2. It can be seen that compound 7 has a relatively high uptake in the blood pool of mice, and the non-target background uptake is low, so it can be used for heart blood pool imaging.

3. MicroPET imaging of compound 7 in tumor model mice

1) The compound 7 solution with a radiochemical purity greater than 95% was prepared according to the example, and 0.1 mL (about 3.7 MBq) was injected into the tail vein of nude mice bearing INS-1 insulinoma (body weight about 20 g), and dynamic PET was performed immediately Images were collected until 60 min after administration. Regions of interest (ROI) were delineated on whole-body decay-corrected coronal images obtained from MicroPET scans. The radioactivity in organs such as tumor, muscle, liver, kidney, and urine was obtained from the average pixel values of multiple ROIs and converted into MBq/mL, and the obtained value was divided by the injected dose to obtain %ID/g (assuming a tissue density of 1 g/mL) mL). The imaging results and the dynamic time-radioactivity curve are shown in Figure 3. It can be seen that compound 7 has obvious uptake in the tumor (retention in tumor blood vessels), and reaches the maximum value 10-20 minutes after administration, and there is no obvious change thereafter. Urinary uptake gradually increased, possibly because the compound was metabolized by the kidneys.

2) Under the same conditions as 1), MicroPET dynamic imaging was performed on U87MG glioma-bearing nude mice. See Figure 3 for imaging results. It can be seen that compound 7 can be retained in tumor blood vessels, but the retention effect varies with different tumor types. It can be used to display the shape of tumor blood vessels and obtain the following parameters of tumor blood vessels: blood flow velocity (mL/min/ g tissue), blood volume (mL/g tissue) and permeability (mL/g tissue), etc., provide the possibility for tumor diagnosis, curative effect and prognosis evaluation.

4. MicroPET imaging of compound 8 in 22B tumor-bearing nude mice

Compound 8 ( ⁶⁴ Cu-NOTA-EB) was prepared according to the freeze-drying method in Preparation Example 4, and PET imaging was performed using the same method as in the above 3. Dynamic PET image acquisition was performed within the first 60 min after administration, and then static PET image acquisition was performed at 120 min, 240 min and 24 h after administration, respectively. Draw region of interest (ROI) for heart and tumor, tomographic image and dynamic time-radioactivity curve are shown in Figure 4. It can be seen that compound 8 can be used as a blood pool imaging agent. 24 hours after administration, only the blood uptake in the tumor is obvious, while the uptake in other tissues or organs has been basically eliminated. The advantage of ⁶⁴ Cu nuclide is that it has a longer half-life (12.7h) and can observe the distribution in the body for a longer time. In addition, ⁶⁴ Cu can also emit electrons and have therapeutic effects, and can also be used for single photon emission computed tomography (SPECT) imaging. It has many uses.

Compound 7 and Compound 8 are a new radiolabeled Evans blue complexes developed by the inventors. Compared with the existing commonly used blood vessel imaging agents, their radioactive rays have strong penetrability, and can be used for non-invasive blood pool imaging in vivo. picture. The obvious difference from the existing radioactive blood pool imaging agents ^99m Tc-RBC and ^99m Tc-HSA is that the latter two are biological extracts, the labeling process is complicated, the price is expensive, and they are also prone to virus infection. Compound 7 of the present invention overcomes the shortcomings of existing blood pool imaging agents: Evans blue derivatives are small organic molecules, simple to synthesize, mature in labeling methods and easy to obtain complexes with high radiochemical purity, and have a long storage time. Long, and the price is far lower than the existing blood pool imaging agent.

The preparation process of compounds 7 and 8 is very simple after being kitted, especially the cheap price is more conducive to clinical application.

The radionuclide used in the present invention may also include: ⁶⁸ Ga nuclide to obtain compound 9. Other more optional radionuclides include but not limited to ⁶² Cu, ⁶⁷ Cu, ⁸⁹ Zr, etc., and the preparation method is similar.

Claims (8)

1. A kind of Evan's blue derivative, structural formula is: 2. the synthetic method of a kind of Evans blue derivative as claimed in claim 1, comprises the steps: 1) Mix compound 1 and compound 2 at a molar ratio of 1-10:1-10, and use amino condensation reaction under the action of diethyl cyanophosphate to obtain compound 3; 2) Compound 3 was reduced to the amino group with NaNO ₂ under hydrochloric acid conditions to obtain azo compound 4; 3) react compound 4 with compound 5 (1-amino-8-naphthol-2,4-disulfonic acid) to obtain the target compound; Its synthetic route is: . 3. A radiolabeled complex, characterized in that the radiolabeled complex is obtained by labeling the Evans blue derivative according to claim 1 with a radionuclide. 4. A radiolabeled complex as claimed in claim 3, wherein the radionuclide includes ¹⁸ F, ⁶⁴ Cu, ⁶⁸ Ga, ⁶² Cu, ⁶⁷ Cu, ⁸⁶ Y or ⁸⁹ Zr. 5. A radiolabeled complex, characterized in that it has one of the following structures: or or 6. Use of the radiolabeled complex according to any one of claims 3 to 5 in the preparation of an organ or tissue imaging agent for humans or animals. 7. The preparation method of the radiolabeled complex according to any one of claims 3 to 5, characterized in that, the complex is prepared by a wet method or a freeze-drying method; The wet preparation of the radiolabeled complex comprises the following steps: 1) Evans blue derivatives according to claim 1 are dissolved in buffer solution or deionized water; 2) Add the corresponding radionuclide solution to the above solution, seal the reaction for 5-40 minutes, and cool down; 3) Separation and purification by high performance liquid chromatography, rotary evaporation to remove the solvent, dissolving the residue with phosphate buffer, water or saline, and sterile filtration to obtain the corresponding radiolabeled complex injection; The lyophilization method of the radiolabeled complex comprises the following steps: 1) Evans blue derivatives according to claim 1 are dissolved in buffer solution or deionized water; 2) After the solution in step 1) is sterile-filtered, it is divided into containers, and after freeze-drying, it is sealed with a stopper and waited until the freeze-dried medicine box; 3) Add an appropriate amount of acetic acid solution or buffer solution to the above kit to dissolve, then add the corresponding radionuclide solution, seal the reaction for 5-40 minutes, and cool down; 4) Separation and purification by high-performance liquid chromatography, rotary evaporation to remove the solvent, dissolving the residue with phosphate buffer or water, and sterile filtration to obtain the corresponding radioactive injection. 8. A ¹⁸ F-labeled complex, its wet preparation method is: 1) Evans blue derivatives according to claim 1 are dissolved in buffer solution or deionized water; 2) aluminum chloride is dissolved in buffer solution or deionized water; 3) mix the above two solutions of 1) and 2) evenly; 4) Add acetonitrile or ethanol and freshly prepared ¹⁸ F ionic water solution, seal at 70-120°C for 5-30 minutes, and cool; 5) Add water to dilute the reaction solution and then separate and purify it on a Sep-Pak C18 chromatographic column, wash the chromatographic column with buffer or water to remove unreacted ¹⁸ F ions, rinse with hydrochloric acid ethanol solution or ethanol solution, and dilute with normal saline The ¹⁸ F-labeled Evans blue derivative injection was obtained by bacterial filtration.