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

Abstract

The present invention discloses a fluorine-labeled radioactive drug precursor targeting fibroblast activation protein, the structure of which is selected from one of the following structures: #imgabs0# The radioactive labeled compound of the present invention is simple to prepare, has a high labeling rate, good biological properties, and better in vivo stability.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a fluorine-labeled radioactive drug precursor targeting fibroblast activation protein and a radioactive 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 FAPI molecular probes have been developed, and it has been demonstrated that this radioactive drug molecule can effectively identify nearly 30 types of malignant tumors, fully demonstrating the diagnostic value of FAP protein inhibitors. 68 Ga nuclide, 18 F nuclides have the advantages of longer half-life, better imaging effect, and easier mass production. 18 F nuclide substitution 68 Ga nuclide labeled FAPI molecules also showed 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 stability in vivo and cannot meet clinical transformation requirements. At the same time, the current molecular probes targeting FAP are metabolized quickly in the body and the probes have a short retention time in the tumor. Summary of the invention

[0004] The object of the present invention is to provide a fluorine-labeled radioactive drug precursor targeting fibroblast activation protein.

[0005] Another object of the present invention is to provide a radiolabeled compound targeting fibroblast activation protein.

[0006] The third object of the present invention is to provide a use of the radioactive labeled compound in the preparation of a tumor imaging agent or a radionuclide targeted therapy agent.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] 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:

[0009]

[0010] The second aspect of the present invention provides a radioactive labeled compound, which is prepared by the fluorine-labeled radioactive drug 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]

[0013] The third aspect of the present invention provides a method for preparing a radiolabeled compound, comprising the following steps:

[0014] 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 F ion reaction bottle, react at a temperature of 20-120° C. (preferably 90-100° C.) for 5-20 minutes (preferably 7-12 minutes), purify by solid phase extraction or high pressure liquid separation, dilute with a sodium ascorbate solution, and sterilize to obtain the radioactive labeled compound.

[0015] 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).

[0016] The volume ratio of the organic solvent to the buffer solution is 1:(1-10), preferably 1:(1-5).

[0017] The organic solvent is selected from ethanol, dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, acetone, methanol, and tetrahydrofuran.

[0018] The radionuclide 18 Preparation of F:

[0019] Using a 10 MV accelerator through a nuclear reaction18 O(p,n) 18 F Production 18 F, the target beam current is 60μA, the bombardment time is 5 to 30min (preferably 20 to 30min) to generate 10 to 40GBq (preferably 25 to 32GBq) 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 to 5): 1 (preferably 1: 1, 3: 1) 18 F ions, high temperature drying.

[0020] The mass concentration of the sodium chloride aqueous solution is 0.1-2% (preferably 0.9%).

[0021] The buffer solution containing aluminum chloride: the concentration of the aluminum chloride is 5-15 mmol / L (preferably 5, 10 mmol / L); 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).

[0022] 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, and the solid phase extraction column is used for separation and purification (the reaction solution is directly loaded), and the product on the solid phase extraction column is 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.

[0023] 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.

[0024] The sodium ascorbate solution is prepared by dissolving sodium ascorbate in an ethanol solution with a concentration of 1 to 10% (preferably 5%), with a concentration of 1 to 1000 mg / mL (preferably 8.33 mg / mL); or, by dissolving sodium ascorbate in water, with a concentration of 1 to 1500 mg / mL (preferably 12.5 mg / mL).

[0025] The sterilization is performed using a 0.22 micron sterilization filter membrane.

[0026] 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).

[0027] The fourth aspect of the present invention provides a use of the radioactive labeled compound in the preparation of a tumor imaging agent or a radionuclide targeted therapy agent.

[0028] 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, atherosclerosis, etc.

[0029] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0030] The present invention develops a novel labeling precursor NOTA-De-FAPI or NOTA-Glu-FAPI and adopts 18 F-Al labeling method for radioactive labeling, due to 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.

[0031] 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%.

[0032] The present invention synthesizes two types of precursor reagents, NOTA-De-FAPI or NOTA-Glu-FAPI, targeting fibroblast activation protein (FAP), for the first time. 18 The F-Al method for labeling 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.

[0033] In the preparation process of the radioactive labeled compound, the radioactive labeling is fast and efficient; the target compound is convenient and simple to separate; the radioactive product has a very high radioactive specific activity; and the label has the value of being used for clinical examination and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the mass spectrum of compounds NOTA-De-FAPI and NOTA-Glu-FAPI.

[0035] Figure 2 yes 18 The body distribution of FAl-NOTA-De-FAPI in normal mice.

[0036] Figure 3 yes 18 The body distribution of FAl-NOTA-Glu-FAPI in normal mice.

[0037] Figure 4 yes 18 In vivo distribution of FAl-NOTA-De-FAPI in tumor models.

[0038] Figure 5 yes 18 In vivo distribution of FAl-NOTA-Glu-FAPI in tumor models. DETAILED DESCRIPTION

[0039] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art 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.

[0040] (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.

[0041] Example 1

[0042]

[0043] (S)-N1-(2-aminoethyl)-N 4-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethylcarbamoyl)quinolin-8-ylsuccinamide (10 mg, 20 μmol, 1 equivalent) was dissolved in 2 mL of anhydrous dimethylformamide, and 1-hydroxybenzotriazole (2.6 mg, 1 equivalent), N,N-diisopropylethylamine (10.0 mg, 4 equivalents), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (3.1 mg, 1 equivalent) and 2,2,2-triazaalkane-1,4,7-triyltriacetic acid (6.0 mg, 1 equivalent) were added and reacted at room temperature. After overnight, the reaction solution was purified by Semi-HPLC (0.1% trifluoroacetic acid water / acetonitrile = 70 / 30, v / v) to obtain 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] + .

[0044] Example 2

[0045]

[0046] 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. 1-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-(2-2-2-(2-(2-(2-2-2-(2-2-2-(2-(2-(2-2-2-2-(2-2-2-(2-2-2-(2-2-2-(2-2-2-(2-2-2- + 773.29, measured molecular weight 774.2 [M+H] + ; Figure 1 It is a schematic diagram of the mass spectrum of compounds NOTA-De-FAP and NOTA-Glu-FAPI.

[0047] Example 3

[0048] A radiolabeled compound 18 The preparation method of FAl-NOTA-De-FAPI comprises the following steps:

[0049]

[0050] Radionuclides 18The preparation method of F comprises the following steps:

[0051] 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 min generates 25GBq 18 F; adsorbed by anion exchange solid phase extraction column QMA, and 1 mL of a mixed solvent of 0.9% sodium chloride aqueous solution and acetonitrile in a volume ratio of 1:1 was used to 18 The F ions are eluted into the reaction bottle and the solvent is dried at high temperature (temperature greater than 90 degrees).

[0052] 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. 18 F, 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 and a concentration of 10 mmol / L, and transfer to dry 18 F ion reaction bottle, react at 90°C for 12 minutes, separate and purify by solid phase extraction, solid phase extraction chromatographic column uses C-18 column, dry the reaction solution, add 10 ml of 5% ethanol solution to dissolve, separate and purify by solid phase extraction column (reaction solution is directly loaded), rinse with 10 ml of sterile injection water (commercially purchased), and rinse the product on the solid phase extraction column into the transfer bottle with 2 ml of 50% ethanol solution. Add 8 ml of aqueous solution containing 100 mg of sodium ascorbate to dilute, sterilize with 0.22 micron sterilizing filter membrane, and obtain 6.66 GBq of radioactive labeled compound 18 The undecay-corrected radiochemical yield of FAl-NOTA-De-FAPI was 26.6% and the specific radioactivity was 52 GBq / uM. Figure 2 yes 18 The distribution diagram of FAl-NOTA-De-FAPI in normal mice. The figure shows that it is mainly cleared through kidney metabolism. As time goes by, the uptake of molecular probes in the kidneys first increases and then decreases. The uptake change pattern of the drug in the liver is similar to that in the kidneys, and the total uptake value is much lower than that in the kidneys.

[0053] Establishment of tumor model U87MG mouse: Resuscitate U87MG cell line with high expression of FAP, add CHOD02 medium to T75 flask, inoculate cells and place in 37℃ 5% CO2 incubator for culture. When the cell density reaches the requirement, collect cells by centrifugation, dilute and resuspend cells with sterile PBS, count cells, aspirate corresponding volume of cell suspension according to the inoculation density of 500,000 cells per mouse, store on ice, and transfer 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 before injecting cells, use a sterile syringe to aspirate the cell suspension, and slowly inject the cell suspension containing the target amount of cells 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.

[0054] Figure 4 The tumor model U87MG mice were injected with 3.2MBq of probe 18 Small animal PET / CT images after 1 hour of FAl-NOTA-De-FAPI metabolism, indicating 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.

[0055] Example 4

[0056] A radiolabeled compound 18 The preparation method of FAl-NOTA-Glu-FAPI comprises the following steps:

[0057]

[0058] Radionuclides 18 Preparation of F:

[0059] 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 min generates 32GBq 18 F; adsorbed by anion exchange solid phase extraction column QMA, and 1 mL of a mixed solvent of 0.9% sodium chloride aqueous solution and acetonitrile in a volume ratio of 3:1 was used to 18 The F ions are eluted into the reaction bottle and the solvent is dried at high temperature.

[0060] 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, react at 100 ° C for 7 minutes, wait for the above solution to dry, add 2 ml of 15% ethanol solution, separate and purify by HPLC, add 6 ml of 5% ethanol solution containing 50 mg of sodium ascorbate to dilute, sterilize with 0.22 micron sterilizing filter membrane, and obtain 5.18 GBq of radioactive labeled compound 18 FAl-NOTA-Glu-FAPI. The undecay-corrected radiochemical yield was 16.2% and the specific radioactivity was 80 GBq / uM.

[0061] HPLC separation and purification conditions: mobile phase is 15% ethanol solution, flow rate is 3mL / min, UV absorption wavelength is 254nm, chromatographic column is commercially available C18-B, 250mm×10mm, 5μm, Semi-preparative column, product elution time is 9-12 minutes, product collection time is 2 minutes.

[0062] Figure 3 yes 18 The distribution diagram of FAl-NOTA-Glu-FAPI in normal mice. The figure shows that this probe is mainly cleared through kidney metabolism. Over time, the uptake of the molecular probe in the kidney first increases and then decreases. The uptake change pattern of the drug in the liver is similar to that in the kidney, but the total uptake value is much lower than that in the kidney.

[0063] Figure 5 The tumor model U87MG mice were injected with 4.1MBq of probe 18 Small animal PET / CT images after 1 hour of FAl-NOTA-Glu-FAPI metabolism, indicating that the molecular probe 18 FAl-NOTA-Glu-FAPI also has a high uptake in tumors.

[0064] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

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

2. A radiolabeled compound, characterized in that The method is prepared by 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; The structure of the radiolabeled compound is selected from one of the following structures:

3. A method for preparing a radiolabeled compound according to claim 2, 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 F ion reaction bottle, reacting at a temperature of 20-120° C. for 5-20 minutes, purifying by solid phase extraction or high pressure liquid separation, diluting with a sodium ascorbate solution, and sterilizing to obtain the radiolabeled compound; 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 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; The solid phase extraction separation and purification step: the solid phase extraction chromatographic column is selected from C-18, tC-18, and HLB small column; the completely reacted solution is dried, 5% ethanol solution is added to dissolve, and the solid phase extraction column is used for separation and purification, and the product on the solid phase extraction column is rinsed with sterile injection water, and 50% ethanol solution is used to elute the product into the transfer bottle; 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; 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.

4. The method for preparing a radiolabeled compound according to claim 3, characterized in that: 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.

5. Use of a radiolabeled compound prepared by the method of claim 3 or 4 in the preparation of a tumor imaging agent, 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, differentiated thyroid cancer, adenoid cystic carcinoma, and gastric cancer.

6. Use of a radiolabeled compound prepared by the method of claim 3 or 4 in preparing a radionuclide targeted therapy agent, characterized in that: The radionuclide targeted therapy reagent is used for treating myocardial infarction, sarcoidosis, pulmonary fibrosis, liver fibrosis, kidney fibrosis, rheumatoid arthritis and atherosclerosis.

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