Fluorine-labeled radiopharmaceutical precursor targeting fibroblast activation protein, radiolabeled compound and use thereof

Abstract

Disclosed in the present invention is a fluorine-labeled radioactive drug precursor targeting fibroblast activation protein. The structure is selected from one of the following structures: (I) and (II). The radiolabeled compound of the present invention has the advantages of simple preparation, high labeling rate and good biological performance, and has better in-vivo stability.

Description

Fluorine-labeled radiopharmaceutical precursors targeting fibroblast activation protein, radiolabeled compounds and their applications Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a fluorine-labeled radiopharmaceutical precursor targeting fibroblast activation protein and a radioactively labeled compound and application thereof. Background Art

[0002] Fibroblast activation protein (FAP) is a transmembrane serine protease that is associated with a variety of diseases such as liver fibrosis, pulmonary fibrosis, and osteoarthritis. FAP protein is present in 90% of human tumor stromal cells and epithelial cells, and has important reference value for tumor growth. FAP can be expressed on fibroblasts in the stroma of most epithelial tumors. This specific expression provides a promising target for the diagnosis and treatment of various cancers. Therefore, FAP protein is of great significance as a cancer detection target. In 2019, the Heidelberg Medical School and the German Cancer Research Center in Germany developed 68 Ga-labeled (Fibroblast activating protein inhibitor) FAPI molecular probe, and proved that the radiopharmaceutical molecule can effectively identify nearly 30 types of malignant tumors, fully demonstrating the diagnostic value of FAP protein inhibitors. 68 Ga nuclide, 18 F nuclide has the advantages of longer half-life, better imaging effect, easier mass production, etc. Therefore, subsequent researchers used 18 F nuclide substitution 68 Ga nuclide labeled FAPI molecules also show certain diagnostic value in clinical applications.

[0003] Currently, clinical trials have been carried out 68 The physical half-life (T 1 / 2 68 minutes), which is not conducive to the widespread clinical development. 18 F-glucose-FAPI analogs have extremely poor in vivo stability, making them unsuitable for clinical translation. Furthermore, current molecular probes targeting FAP are rapidly metabolized in the body, resulting in a short retention time at the tumor site.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorine-labeled radiopharmaceutical precursor targeting fibroblast activation protein.

[0006] Another object of the present invention is to provide a radiolabeled compound targeting fibroblast activation protein.

[0007] The third object of the present invention is to provide a use of the radiolabeled compound in the preparation of a tumor imaging agent or a radionuclide targeted therapy agent.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a fluorine-labeled radiopharmaceutical precursor targeting fibroblast activation protein, the structure of which is selected from one of the following structures:

[0010] The second aspect of the present invention provides a radiolabeled compound, which is prepared from the fluorine-labeled radiopharmaceutical precursor targeting fibroblast activation protein and a radionuclide, wherein the radionuclide is selected from 18 F.

[0011] The structure of the radiolabeled compound is selected from one of the following structures:

[0012] The third aspect of the present invention provides a method for preparing a radiolabeled compound, comprising the following steps:

[0013] The fluorine-labeled radioactive drug precursor targeting fibroblast activation protein is dissolved in a mixed solvent of an organic solvent and a buffer solution, and a radionuclide is added. 18 F. Buffer solution containing aluminum chloride, in dry 18 F ion reaction bottle, the temperature is 20-120 ℃ (preferably 90-100 ℃) under the condition of reaction for 5-20 minutes (preferably 7-12 minutes), solid phase extraction or high pressure liquid separation purification, dilution with sodium ascorbate solution, sterilization, to obtain the radiolabeled compound.

[0014] The buffer solution is an acetic acid and sodium acetate buffer solution with a pH of 3.5-5.5 (preferably 4-4.5) and a concentration of 5-15 mmol / L (preferably 10 mmol / L).

[0015] The volume ratio of the organic solvent to the buffer solution is 1:(1-10), preferably 1:(1-5).

[0016] The organic solvent is selected from ethanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, methanol, and tetrahydrofuran.

[0017] The radionuclide 18 Preparation of F:

[0018] Using a 10MV accelerator through nuclear reaction 18 O(p,n) 18 F production 18 F, the target beam current is 60μA, and the bombardment time is 5 to 30min (preferably 20 to 30min) to generate 10 to 40GBq (preferably 25 to 32GBq) 18 F; adsorption by anion exchange solid phase extraction column QMA, elution with a mixed solvent of sodium chloride aqueous solution and acetonitrile in a volume ratio of (1 to 5): 1 (preferably 1: 1, 3: 1) 18 F ions, high temperature drying.

[0019] The mass concentration of the sodium chloride aqueous solution is 0.1-2% (preferably 0.9%).

[0020] The aluminum chloride-containing buffer solution: the concentration of the aluminum chloride is 5 to 15 mmol / L (preferably 5 or 10 mmol / L); the buffer solution is an acetic acid and sodium acetate buffer solution with a pH of 3.5 to 5.5 (preferably 4 to 4.5), and the concentration is 5 to 15 mmol / L (preferably 10 mmol / L).

[0021] The solid phase extraction separation and purification steps are as follows: the solid phase extraction chromatographic column is selected from C-18, tC-18, HLB column, etc.; the completely reacted solution is dried, 5% ethanol solution is added to dissolve it, and a solid phase extraction column is used for separation and purification (the reaction solution is directly loaded), rinsed with sterile water for injection, and the product on the solid phase extraction column is rinsed into a transfer bottle with 50% ethanol solution.

[0022] Before the high-pressure liquid phase separation and purification, the reaction solution was dried, 15% ethanol solution was added, and then the high-pressure liquid phase separation and purification was carried out; the conditions of the high-pressure liquid phase separation and purification were as follows: the mobile phase was 15% ethanol solution, the flow rate was 3 mL / min, the ultraviolet absorption wavelength was 254 nm, and the chromatographic column was a commercially available C18-B, 250 mm × 10 mm, 5 μm, Semi-preparative chromatography column, product elution time is 9-12 minutes, and product collection time is 2 minutes.

[0023] The sodium ascorbate solution is prepared by dissolving sodium ascorbate in an ethanol solution having a concentration of 1 to 10% (preferably 5%) to a concentration of 1 to 1000 mg / mL (preferably 8.33 mg / mL); or by dissolving sodium ascorbate in water to a concentration of 1 to 1500 mg / mL (preferably 12.5 mg / mL).

[0024] The sterilization is carried out using a 0.22 micron sterilization filter membrane.

[0025] The radioactive labeled compound has an undecay-corrected radiochemical yield of 10-40% (preferably 16.2%, 26.6%), and a radioactivity specific activity of 30-90 GBq / uM (preferably 52-80 GBq / uM).

[0026] The fourth aspect of the present invention provides a use of the radiolabeled compound in the preparation of a tumor imaging agent or a radionuclide targeted therapy agent.

[0027] The tumor is selected from sarcoma, esophageal cancer, breast cancer, bile duct cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, head and neck cancer, ovarian cancer, glioma, pancreatic cancer, prostate cancer, pheochromocytoma, renal cell carcinoma, differentiated thyroid cancer, adenoid cystic carcinoma, gastric cancer, myocardial infarction, sarcoidosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, rheumatoid arthritis, atherosclerosis, etc.

[0028] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0029] The present invention develops a new type of labeling precursor NOTA-De-FAPI or NOTA-Glu-FAPI, and adopts 18 F-Al labeling method is used for radioactive labeling. 18 The physical half-life of F (110 min) is greater than 68 The physical half-life of Ga (68min) can meet the routine distribution of clinical drugs.

[0030] The radioactive labeled compound of the invention is simple to prepare, has a high labeling rate, and has good in vivo and in vitro stability. After the molecular probe is incubated in fetal bovine serum for 2 hours, the prototype retention rate is greater than 95%.

[0031] The present invention synthesized two types of precursor reagents targeting fibroblast activation protein (FAP) for the first time, NOTA-De-FAPI or NOTA-Glu-FAPI, which can be used 18 The F-Al method is used for labeling, which has good stability and biological activity in vivo and in vitro, has good targeting to fibroblasts, can reflect the status of cancer or fibrosis, and is very suitable for PET imaging.

[0032] In the preparation process of the radiolabeled compound, the radiolabeling is rapid and efficient; the target compound is conveniently and simply separated; the radioactive product has a very high radioactivity; and the marker has value for clinical examination and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a schematic diagram of the mass spectra of compounds NOTA-De-FAPI and NOTA-Glu-FAPI.

[0034] Figure 2 is 18 Distribution of FAl-NOTA-De-FAPI in normal mice.

[0035] Figure 3 is 18 Distribution of FAl-NOTA-Glu-FAPI in normal mice.

[0036] Figure 4 is 18 In vivo distribution of FAl-NOTA-De-FAPI in tumor models.

[0037] Figure 5 is 18 In vivo distribution of FAl-NOTA-Glu-FAPI in tumor models. DETAILED DESCRIPTION

[0038] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0039] (S)-N1-(2-aminoethyl)-N 4 -(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethylcarbamoyl)quinolin-8-ylsuccinamide and N5-(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)glutamine were purchased from Jiangxi Nanchang Probe Biotechnology Co., Ltd.

[0040] Example 1

[0041] (S)-N1-(2-aminoethyl)-N 4 1-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethylcarbamoyl)quinolin-8-ylsuccinamide (10 mg, 20 μmol, 1 eq) was dissolved in 2 mL of anhydrous dimethylformamide, and 1-hydroxybenzotriazole (2.6 mg, 1 eq), N,N-diisopropylethylamine (10.0 mg, 4 eq), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (3.1 mg, 1 eq) and 2,2,2-triazaalkane-1,4,7-triyltriacetic acid (6.0 mg, 1 eq) were added. The mixture was reacted at room temperature overnight. The reaction solution was purified by semi-HPLC (0.1% trifluoroacetic acid in water / acetonitrile = 70 / 30, v / v) to give 2.3 mg of a white solid (yield: 15%); MS (ESI) theoretical molecular weight [M+H] + 787.33, measured molecular weight 787.1 [M+H] + .

[0042] Example 2

[0043] N5-(4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)glutamine (9.8 mg, 20 μmol, 1 equivalent) was dissolved in 2 mL of anhydrous dimethylformamide, and 1-hydroxybenzotriazole (2.7 mg, 1 equivalent), N,N-diisopropylethylamine (10.2 mg, 4 equivalents), 1-ethyl-(3-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)glutamine (9.8 mg, 20 μmol, 1 equivalent) was added. A mixture of 2,2,2-triazol-1,4,7-triyltriacetic acid (6.1 mg, 1 equivalent) and 1,4,7-dimethylaminopropylcarbodiimide (3.0 mg, 1 equivalent) was reacted at room temperature overnight. The reaction solution was purified by semi-HPLC (0.1% trifluoroacetic acid in water / acetonitrile = 75 / 25, v / v) to obtain 2.1 mg of a white solid (yield: 13.5%); MS (ESI) theoretical molecular weight [M+H] + 773.29, measured molecular weight 774.2 [M+H] + ; Figure 1 is a schematic diagram of the mass spectrum of compounds NOTA-De-FAP and NOTA-Glu-FAPI.

[0044] Example 3

[0045] A radiolabeled compound 18 The preparation method of FAl-NOTA-De-FAPI comprises the following steps:

[0046] Radionuclides 18 The preparation method of F comprises the following steps:

[0047] Using Japan's Sumitomo 10MV accelerator through nuclear reaction 18 O(p,n) 18 F production 18 F, the target beam current is 60μA, and the bombardment for 20min generates 25GBq 18 F; adsorbed by anion exchange solid phase extraction column QMA, 1mL of a mixed solvent of 0.9% sodium chloride aqueous solution and acetonitrile with a volume ratio of 1:1 18 The F ions are eluted into the reaction bottle and the solvent is dried at high temperature (greater than 90 degrees).

[0048] 100 μg of NOTA-De-FAPI was dissolved in 500 μl of acetic acid / sodium acetate buffer solution with a pH of 4.0 and a concentration of 10 mmol / L and 500 μl of acetonitrile, and the radionuclide was added. 18F, add 5 μl of 10 mmol / L aluminum chloride in acetic acid / sodium acetate buffer solution, the buffer solution is acetic acid and sodium acetate buffer solution with a pH of 4.0, the concentration is 10 mmol / L, and transfer to dry 18 The F ion reaction bottle was reacted at 90°C for 12 minutes and then separated and purified by solid phase extraction using a C-18 column. The reaction solution was dried and dissolved in 10 ml of 5% ethanol solution. The reaction solution was separated and purified using a solid phase extraction column (the reaction solution was directly loaded). The product was rinsed with 10 ml of sterile water for injection (commercially purchased) and washed from the solid phase extraction column with 2 ml of 50% ethanol solution into a transfer bottle. 8 ml of an aqueous solution containing 100 mg of sodium ascorbate was added for dilution and sterilized with a 0.22 μm sterilizing filter membrane to obtain 6.66 GBq of radiolabeled compound. 18 FAl-NOTA-De-FAPI. The undecay-corrected radiochemical yield is 26.6% and the radioactivity is 52 GBq / uM. 18 The distribution diagram of FAl-NOTA-De-FAPI in normal mice shows that it is primarily eliminated through renal metabolism. Over time, the uptake of the molecular probe in the kidneys first increases and then decreases. The uptake pattern in the liver is similar to that in the kidneys, with the total uptake value being much lower than that in the kidneys.

[0049] Establishment of tumor model U87MG mice: Resuscitate the U87MG cell line that highly expresses FAP, add CHO D02 medium to the T75 flask, inoculate the cells and place in a 37℃ 5% CO2 incubator for culture. When the cell density reaches the requirement, collect the cells by centrifugation, dilute and resuspend the cells with sterile PBS, count the cells, aspirate the corresponding volume of cell suspension according to the inoculation density of 500,000 cells per mouse, store it on ice, and transfer it to the animal operation room. Select 5-week-old Balb / c nude mice, fix the mice, use a pipette to blow the cell suspension thoroughly and evenly before injecting the cells, use a sterile syringe to aspirate the cell suspension, and slowly inject the cell suspension containing the target cell amount into the subcutaneous position of the mouse armpit at a liquid volume of 100-150 microliters per mouse. Observe the vital signs and movement of the mouse after the injection. When the tumor volume is greater than 300mm 3 Then used in subsequent experiments.

[0050] Figure 4 shows the injection of 3.2 MBq of probe into the tumor model U87MG mice. 18 Small animal PET / CT images 1 hour after FAl-NOTA-De-FAPI metabolism, showing that the molecular probe 18 FAl-NOTA-De-FAPI has a high uptake in tumors, and the radiolabeled compound itself is used as a molecular probe.

[0051] Example 4

[0052] A radiolabeled compound 18 The preparation method of FAl-NOTA-Glu-FAPI comprises the following steps:

[0053] Radionuclides 18 Preparation of F:

[0054] Using Japan's Sumitomo 10MV accelerator through nuclear reaction 18 O(p,n) 18 F production 18 F, the target beam current is 60μA, and the bombardment for 30min generates 32GBq 18 F; adsorption by anion exchange solid phase extraction column QMA, with 1mL volume ratio of 3:1 0.9% sodium chloride aqueous solution and acetonitrile mixed solvent 18 The F ions are eluted into the reaction bottle and the solvent is dried at high temperature.

[0055] 50 μg of NOTA-Glu-FAPI was dissolved in 1000 μl of acetic acid / sodium acetate buffer solution with a pH of 4.2 and a concentration of 10 mmol / L and 500 μl of acetonitrile, and the radionuclide was added. 18 F, add 5 μl of acetic acid / sodium acetate buffer solution with a concentration of 5 mmol / L aluminum chloride, the buffer solution is acetic acid and sodium acetate buffer solution with a pH of 4.2, the concentration is 10 mmol / L, and transfer to dry 18 F ion reaction bottle, the temperature was 100 ℃ under the condition of reaction for 7 minutes, after the above solution was dried, 2 ml of 15% ethanol solution was added, separated and purified by HPLC, and diluted with 6 ml of 5% ethanol solution containing 50 mg of sodium ascorbate, and sterilized with 0.22 micron sterilizing filter membrane to obtain 5.18 GBq of radiolabeled compound. 18 The undecay-corrected radiochemical yield of FAl-NOTA-Glu-FAPI was 16.2%, and the specific radioactivity was 80 GBq / uM.

[0056] HPLC separation and purification conditions: mobile phase is 15% ethanol solution, flow rate is 3 mL / min, UV absorption wavelength is 254 nm, chromatographic column is commercially available C18-B, 250 mm × 10 mm, 5 μm, Semi-preparative chromatography column, product elution time is 9-12 minutes, and product collection time is 2 minutes.

[0057] Figure 3 is 18The distribution of FAl-NOTA-Glu-FAPI in normal mice. This figure shows that the probe is primarily eliminated through renal metabolism. Over time, renal uptake of the molecular probe initially increases and then decreases. The uptake pattern in the liver is similar to that in the kidneys, with the total uptake value being much lower than that in the kidneys.

[0058] Figure 5 shows the injection of 4.1MBq of probe into the tumor model U87MG mice. 18 Small animal PET / CT images of FAl-NOTA-Glu-FAPI after 1 hour of metabolism, showing that the molecular probe 18 FAl-NOTA-Glu-FAPI also has a high uptake in tumors.

[0059] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A fluorine-labeled radiopharmaceutical precursor targeting fibroblast activation protein, It is characterized in that The structure is selected from one of the following structures:

2. A radioactively labeled compound prepared from the fluorine-labeled radioactive drug precursor targeting fibroblast activation protein according to claim 1 and a radionuclide, wherein the radionuclide is selected from 18 F.

3. The radiolabeled compound according to claim 2, It is characterized in that The structure of the radiolabeled compound is selected from one of the following structures:

4. A method for preparing the radiolabeled compound according to claim 2 or 3, It is characterized in that The following steps are involved: The fluorine-labeled radioactive drug precursor targeting fibroblast activation protein is dissolved in a mixed solvent of an organic solvent and a buffer solution, and a radioactive nuclide is added. 18 F. Buffer solution containing aluminum chloride, in dry 18 In the F ion reaction bottle, the temperature The reaction is carried out at 20-120° C. for 5-20 minutes, and the radioactive labeled compound is obtained by solid phase extraction or high pressure liquid separation and purification, and diluted with a sodium ascorbate solution and sterilized.

5. The method for preparing a radiolabeled compound according to claim 4, It is characterized in that The buffer solution is an acetic acid and sodium acetate buffer solution with a pH of 3.5-5.5 and a concentration of 5-15 mmol / L; The volume ratio of the organic solvent to the buffer solution is 1:(1-10); The organic solvent is selected from ethanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, methanol, and tetrahydrofuran; The radionuclide 18 Preparation of F: Using a 10 MV accelerator through a nuclear reaction 18 O(p,n) 18 F Production 18 F, the target beam current is 60μA, and the bombardment time is 5 to 30 minutes to generate 10 to 40 GBq 18 F; adsorbed by anion exchange solid phase extraction column QMA, eluted with a mixed solvent of sodium chloride aqueous solution and acetonitrile in a volume ratio of (1-5):1 18 F ion, high temperature drying; The buffer solution containing aluminum chloride: the concentration of the aluminum chloride is 5 to 15 mmol / L; the buffer solution is an acetic acid and sodium acetate buffer solution with a pH of 3.5-5.5 and a concentration of 5 to 15 mmol / L.

6. The method for preparing a radiolabeled compound according to claim 3, It is characterized in that The solid phase extraction separation and purification steps are as follows: the solid phase extraction chromatographic column is selected from C-18, tC-18, and HLB columns; the completely reacted solution is dried, dissolved in 5% ethanol solution, separated and purified with a solid phase extraction column, rinsed with sterile water for injection, and the product on the solid phase extraction column is rinsed into a transfer bottle with 50% ethanol solution.

7. The method for preparing a radiolabeled compound according to claim 3, It is characterized in that Before the high-pressure liquid phase separation and purification, the reaction solution is dried, a 15% ethanol solution is added, and then the high-pressure liquid phase separation and purification is performed; the conditions of the high-pressure liquid phase separation and purification are as follows: the mobile phase is a 15% ethanol solution, the flow rate is 3 mL / min, the ultraviolet absorption wavelength is 254 nm, and the chromatographic column is a commercially available C18-B, 250 mm×10 mm, 5 μm, Semi-preparative column, product elution time is 9-12 minutes, product collection time is 2 minutes.

8. The method for preparing a radiolabeled compound according to claim 3, It is characterized in that The sodium ascorbate solution is prepared by dissolving sodium ascorbate in an ethanol solution with a concentration of 1 to 10%, with a concentration of 1 to 1000 mg / mL; or, by dissolving sodium ascorbate in water, with a concentration of 1 to 1500 mg / mL; The sterilization is carried out using a 0.22 micron sterilization filter membrane; The radiochemical yield of the radiolabeled compound that is not corrected for decay is 10-40%, and the radioactivity specific activity is 30-90 GBq / uM.

9. Use of a radiolabeled compound prepared by the method according to any one of claims 3 to 8 in the preparation of a tumor imaging agent or a radionuclide targeted therapy agent.

10. The use according to claim 9, It is characterized in that The tumor is selected from sarcoma, esophageal cancer, breast cancer, bile duct cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, head and neck cancer, ovarian cancer, glioma, pancreatic cancer, prostate cancer, pheochromocytoma, renal cell, differentiated thyroid cancer, adenoid cystic carcinoma, gastric cancer, myocardial infarction, sarcoidosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, rheumatoid arthritis, and atherosclerosis.

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