CN114315795A

CN114315795A - 68Ga-labeled inhibitor radioactive probe of targeted fibroblast activation protein and preparation method thereof

Info

Publication number: CN114315795A
Application number: CN202111437982.7A
Authority: CN
Inventors: 朱霖; 洪海燕; 查志豪; 赵睿玥; 孔繁渊
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-04-12
Anticipated expiration: 2041-11-30
Also published as: CN114315795B

Abstract

The invention relates to a⁶⁸Ga-labeled inhibitor radioactive probes of targeted fibroblast activation protein and a preparation method thereof, belonging to the technical field of radiopharmaceutical chemistry; the structural formula of the radioactive probe of the invention is as follows:

labeling of the Radioactive Probe of the inventionThe recording time is 5 minutes, the radiochemical yield is more than 99 percent, the radiochemical purity of the product measured by radio-HPLC is more than 99 percent, and the product shows high affinity and high specificity to the fibroblast activation protein, has high in vitro cell uptake value and is a potential fibroblast activation protein PET imaging agent.

Description

68Ga-labeled inhibitor radioactive probe of targeted fibroblast activation protein and preparation method thereof

Technical Field

The invention relates to⁶⁸Ga-labeled inhibitor radioprobe ([ 2 ]) targeting Fibroblast Activation Protein (FAP)⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer、 [⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer) and a preparation method thereof, belonging to the technical field of radiopharmaceutical chemistry.

Background

Tumor masses consist of tumor cells and tumor stroma, which accounts for more than 90% of tumor masses in highly desmoplastic tumors such as breast, colon and pancreatic cancers. Cancer-associated fibroblasts (CAFs), also known as tumor-associated fibroblasts (TAFs) or activated fibroblasts, are important components of the tumor stroma and participate in the growth, migration and development of tumors. CAFs express different markers such as Fibroblast Activation Protein (FAP), alpha-smooth muscle actin (alpha-SMA), vimentin (vimentin), and the like.

Since alpha-SMA and vimentin are expressed in resting fibroblasts, pericytes and vascular smooth muscle cells, while FAP is highly specifically expressed only in activated fibroblasts and is not expressed in benign tumor or normal adult tissues, FAP is the most potential molecular marker for CAFs. In addition, FAP is also an essential factor for promoting tumor cell proliferation and metastasis, remodeling extracellular matrix, inducing angiogenesis, mediating immunosuppression, participating in tumor cell energy metabolism, and the like. Therefore, FAP becomes a potential target for tumor imaging and treatment, and further, the deep research on FAP has great significance for the diagnosis, treatment and prognosis judgment of malignant tumors.

Notably, activated fibroblasts are present not only in tumors, but also in wound healing and matrix remodeling diseases such as chronic inflammation, myocardial infarction and liver, lung or kidney fibrosis, and thus FAP is highly expressed not only in tumor interstitial CAFs, but also in many tissue remodeling processes. Therefore, FAP expression is not tumor specific. At the same time, however, FAP-targeted radiopharmaceuticals are not only useful for imaging and treatment of tumors, but also for imaging of many non-tumor diseases such as myocardial infarction, chronic inflammatory diseases, and pulmonary, hepatic, or renal fibrosis.

FAP is a 97kDa type II transmembrane protein which is active only when present as a dimer of 170kDa (FAP-FAP). A great deal of research reports on the application of FAP targeted inhibitor radiopharmaceuticals in tumor imaging and treatment are hot spots in recent years.

Loktev et al, 2018 report⁶⁸Ga]Ga-FAPI-02。[⁶⁸Ga]Ga-FAPI-02 shows high FAP specificity and binding affinity in vitro and in vivo, and can be rapidly taken up and internalized by FAP-highly expressing cells. In patients with metastasis of breast cancer, lung cancer and pancreatic cancer⁶⁸Ga]Ga-FAPI-02 is rapidly cleared, mainly by renal excretion, low uptake in normal tissues, large accumulation of radioactivity observed in primary tumors, lymph nodes and metastatic foci of bone, and high image contrast obtained. Comparison [2 ]⁶⁸Ga]Ga-FAPI-02 and [2 ]¹⁸F]The imaging of FDG in a patient with locally advanced lung adenocarcinoma was found⁶⁸Ga]Ga-FAPI-02 is obviously superior to [, ]¹⁸F]FDG：[⁶⁸Ga]The intake of Ga-FAPI-02 in a metastasis is high, the background is low, the contrast of a focus is high, and the visibility is good; and 2¹⁸F]The difference in the strong uptake of FDG in a high glucose metabolism tissue such as the brain⁶⁸Ga]Ga-FAPI-02 selectively targets FAP highly expressed tissues. Although⁶⁸Ga]Ga-FAPI-02 shows primary good tumor imaging properties, but the rapid elimination speed of the Ga-FAPI-02 probably cannot well reflect the conditions of tumors such as head and neck cancer, ovarian cancer, liver cancer and the like, and in addition, the short tumor retention time is not suitable for treatment, so FAP targeted inhibitor radiopharmaceuticals with longer tumor retention time need to be developed.

For this purpose, Lindner et al are in the same yearFAPI-04 is reported and applied to⁶⁸Ga and¹⁷⁷designation of Lu. [⁶⁸Ga]Ga-FAPI-04 exhibits a sum [ alpha ]⁶⁸Ga]Ga-FAPI-02 has similarly rapid renal clearance, low background and high tumor uptake. Use by researchers¹⁷⁷The FAPI-04 is marked by Lu to obtain the [ alpha ], [ alpha ] and [ alpha ], [ alpha ] or a¹⁷⁷Lu]The results of biodistribution experiments on Lu-FAPI-04, HT1080-FAP tumor-bearing mice show that¹⁷⁷Lu]Lu-FAPI-04 ratio¹⁷⁷Lu]Lu-FAPI-02 has higher tumor uptake and longer tumor retention, and both have very low background, and after 24 hours, the value of¹⁷⁷Lu]Effective tumor uptake ratio of Lu-FAPI-04 [2 ]¹⁷⁷Lu]The Lu-FAPI-02 is higher than 100 percent, which indicates that FAPI-04 has potential in the aspect of treatment. The term "used in one example of the patient⁹⁰Y]Treatment with Y-FAPI-04 showed significant reduction in patient pain, metastatic lesions were observed at 24 hours and low background, no side effects such as hematologic toxicity were observed, but clinical data were limited and more clinical trials were performed to verify their feasibility.

To further increase tumor uptake and tumor retention time, FAPI-21 and FAPI-46 were reported by Loktev et al in 2019. The biodistribution result of HT1080-FAP tumor-bearing mice shows that⁶⁸Ga]Ga-FAPI-21 and [ [ alpha ] ]⁶⁸Ga]Ga-FAPI-46 tumor uptake is higher than that of [2 ]⁶⁸Ga]Ga-FAPI-04, but⁶⁸Ga]The liver uptake and muscle uptake of Ga-FAPI-21 are higher than those of [2 ], [⁶⁸Ga]Ga-FAPI-04；[⁶⁸Ga]The tumor/blood, tumor/muscle and tumor/liver ratios of Ga-FAPI-46 are all higher than that of the product⁶⁸Ga]Ga-FAPI-04 and [2 ]⁶⁸Ga]Ga-FAPI-21。[¹⁷⁷Lu]Lu-FAPI-21 and¹⁷⁷Lu]the tumor accumulation of Lu-FAPI-46 is higher than that of the [ Lu-FAPI-46 ] 1-4 hours after the injection¹⁷⁷Lu]Lu-FAPI-04; tumor retention rate [ 24 hours after injection ]¹⁷⁷Lu]Lu-FAPI-21>[¹⁷⁷Lu]Lu-FAPI-04>[¹⁷⁷Lu]Lu-FAPI-46; the blood radioactivity levels of the three compounds are equivalent¹⁷⁷Lu]The tumor/liver, tumor/kidney and tumor/brain ratios of Lu-FAPI-46 are improved. Intravenous injection in patients with mucoepidermoid carcinoma, oropharyngeal cancer, ovarian cancer, and colorectal cancer⁶⁸Ga]Ga-FAPI-21 and [ [ alpha ] ]⁶⁸Ga]Ga-FAPI-46, both in primary tumors and metastasesRapidly accumulates, 1 hour after administration, the SUVmax is 11.9 +/-3.33 and 12.76 +/-0.90 respectively, the radioactive uptake of normal tissues is low, and the radioactivity is rapidly cleared from blood and is mainly excreted through kidney; but [2 ]⁶⁸Ga]Uptake of Ga-FAPI-21 in the oral mucosa, thyroid, parotid and submandibular glands was increased, suggesting that FAPI-21 may not be suitable as a therapeutic drug. Notably, tumor accumulation is highly dependent on tumor type. Lindner et al found that injection⁶⁸Ga]Ga-FAPI-21 or [ Ga-FAPI ]⁶⁸Ga]After Ga-FAPI-46, the radioactivity of the tumor remains relatively stable in colorectal, ovarian, oropharyngeal and pancreatic cancers within 3 hours, while in breast cancers, the radioactivity of the tumor continues to decrease; in addition, one patient with an unknown tumor had a sustained increase in tumor uptake within 1-3 hours after dosing.

Human tumors form a complex heterogeneous structure, and the number and distribution of FAP-expressing CAFs and the number of FAP molecules per cell may differ, leading to radiopharmaceuticals with different pharmacokinetic properties in different tumor types. In the clinical study, it was observed that⁶⁸Ga]Ga-FAPI-21 or [ Ga-FAPI ]⁶⁸Ga]Differences in uptake of Ga-FAPI-46 in different types of tumors: uptake in colorectal, ovarian, oropharyngeal and pancreatic cancers is relatively stable, while uptake in breast cancer continues to decline. This may be due to a source of heterogeneity in the CAFs. Due to the fact thatOrigin of originThese CAFs may show a different proteome, have strong variation, and even lack the expression of FAP. Thus, radiopharmaceuticals with longer tumor residence times may better reflect the true condition of the tumor than those that clear quickly. In addition, radiopharmaceuticals with longer tumor residence times and high tumor uptake are also clinically desirable as therapeutic agents. At present, the most successful FAP-targeted inhibitor imaging drugs are FAPI-02, FAPI-04 and FAPI-46, and the same⁶⁸Ga-labeled drugs are most clinically used, but there is still room for improvement in these drugs to achieve longer tumor residence times.

Therefore, providing a novel inhibitor radioactive probe, which can show high affinity and high specificity to fibroblast activation protein, has better tumor uptake and longer tumor retention time, and becomes a technical problem to be solved urgently in the technical field.

Disclosure of Invention

It is an object of the present invention to provide⁶⁸Ga-labeled inhibitors of Fibroblast Activation Protein (FAP) radioactive probes show high affinity and high specificity to the FAP, have better tumor uptake and longer tumor retention time, and are more potential FAP/PET imaging agents.

The above purpose of the invention is realized by the following technical scheme:

⁶⁸the structural formula of the Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein is shown in figure 6.

It is another object of the present invention to provide the above⁶⁸A preparation method of Ga-labeled inhibitor radioactive probes of targeted fibroblast activation protein.

⁶⁸the preparation method of the Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein comprises the following steps: in the presence of alkali and condensing agent, the bifunctional linker HBED-CC and fibroblast activation protein inhibitor are subjected to condensation reaction, after the protective group is removed by acid, the obtained product is dissolved in dimethyl sulfoxide, and added⁶⁸Ga]GaCl₃Mixing the solution uniformly, and heating to obtain the solution shown in the structural formula⁶⁸Ga-labeled inhibitor radioactive probes targeting fibroblast activation proteins.

The reaction scheme is shown in FIG. 7.

Preferably, the base is N, N-diisopropylethylamine, and the addition amount is 3-5 equivalents; the condensing agent is 1-hydroxybenzotriazole and 1-ethyl- (3-dimethyl aminopropyl) carbodiimide, and the addition amount of the condensing agent is 1 equivalent; the fibroblast activation protein inhibitor is (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidine-1-yl) -2-ethoxy) -6- (3- (piperazine-1-yl) propoxy) quinoline-4-formamide, and the adding amount is 1-5 equivalents; the acid was trifluoroacetic acid and the amount added was 3 ml.

Preferably, the first and second electrodes are formed of a metal,⁶⁸the preparation method of the Ga-labeled inhibitor radioactive probe of the targeted fibroblast activation protein comprises the following specific steps:

step 1:⁶⁸synthesis of labeled precursor of Ga-labeled fibroblast activation protein-targeted inhibitor-based radioactive probe

Dissolving (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-ethoxy) -6- (3- (piperazin-1-yl) propoxy) quinoline-4-carboxamide in anhydrous dimethylformamide, adding 1-hydroxybenzotriazole, N-diisopropylethylamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 3, 3' - ((((2, 2,13, 13-tetramethyl-4, 11-dioxo-3, 12-dioxa-6, 9-diazepidecane-6, 9-diyl) bis (methylene)) bis (4-hydroxy-3 to the mixed solution, 1-phenyl)) dipropionic acid, reacting at room temperature, adding ethyl acetate and saturated saline solution into the mixed solution after overnight, washing, and collecting organic phase filtrate; drying the organic phase filtrate with anhydrous sodium sulfate, filtering, and removing solid impurities; decompressing by using a rotary evaporator, removing the solvent in the filtrate, separating by using a silica gel column by using a mixed solution (4/1/0.1, v/v/v) of dichloromethane, methanol and 25% ammonia water, collecting components, decompressing by using the rotary evaporator and an oil pump, removing the solvent to obtain a brown yellow oily substance, dissolving the obtained brown yellow oily substance in trifluoroacetic acid, stirring at room temperature, decompressing by using the rotary evaporator and the oil pump, removing the solvent, and recrystallizing by using diethyl ether to obtain a brown yellow solid; dissolving the obtained brown yellow solid in dimethyl sulfoxide, and purifying by Semi-HPLC to obtain brown yellow solid HBED-CC-04-DiF-Monomer;

step 2:⁶⁸labeling of Ga-labeled fibroblast activation protein-targeted inhibitor-based radioactive probes

Dissolving the HBED-CC-04-DiF-Monomer (labeled precursor) obtained in the step 1 in dimethyl sulfoxide, adding a sodium acetate solution to obtain a labeled precursor sodium acetate solution, leaching a germanium gallium generator (iThemba laboratories, 740MBq, 20mCi) by using a high-purity hydrochloric acid solution, and carrying out ion exchange to obtain the HBED-CC-04-DiF-Monomer (labeled precursor), wherein the labeled precursor sodium acetate solution is obtained by dissolving the HBED-CC-DiF-Monomer (labeled precursor) in dimethyl sulfoxide, adding a sodium acetate solution in dimethyl sulfoxide, leaching the germanium gallium generator (iThemba laboratories, 740MBq, 20mCi) by using a high-purity hydrochloric acid solution, and carrying out ion exchange to obtain the labeled precursor sodium acetate solutionDes2 [ de ]⁶⁸Ga]GaCl₃Adding hydrochloric acid solution into labeled precursor sodium acetate solution, mixing, reacting at 95 deg.C, cooling to room temperature, and detecting with radioactivity detector (Flow-Count, Eckert)&High performance liquid chromatography (radio-HPLC, Agilent 1260Infinity II system) of Ziegler) the labeling rate was determined in a mobile phase of 0.1% aqueous trifluoroacetic acid (v/v) and 0.1% acetonitrile trifluoroacetic acid (v/v) to obtain a radiochemical yield of greater than 99%⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer。

Preferably, in step 2, the test conditions of the high performance liquid chromatography with the radioactivity detector are as follows: the first mobile phase is 0.1% trifluoroacetic acid in water (v/v) and the second mobile phase is 0.1% trifluoroacetic acid in acetonitrile (v/v), and the gradient elution conditions are as follows: from 0 to 10 minutes, from 100% to 35% of the first mobile phase; 10-12 minutes, 35% -100% of the first mobile phase; 12-15 minutes, 100% of the first mobile phase; the flow rate of the mobile phase was 1 ml/min.

Dissolving (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-ethoxy) -6- (3- (piperazin-1-yl) propoxy) quinoline-4-carboxamide in anhydrous dimethylformamide, adding 1-hydroxybenzotriazole, N-diisopropylethylamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 3, 3' - ((((2, 2,13, 13-tetramethyl-4, 11-dioxo-3, 12-dioxa-6, 9-diazepidecane-6, 9-diyl) bis (methylene)) bis (4-hydroxy-3 to the mixed solution, 1-phenyl)) dipropionic acid, reacting at room temperature, adding ethyl acetate and saturated saline solution into the mixed solution after overnight, washing, and collecting organic phase filtrate; drying the organic phase filtrate with anhydrous sodium sulfate, filtering, and removing solid impurities; removing solvent from the filtrate under reduced pressure by using a rotary evaporator, separating with a silica gel column by using a mixed solution (20/1/0.1, v/v/v) of dichloromethane, methanol and 25% ammonia water, collecting components, reducing pressure by using the rotary evaporator and an oil pump, removing the solvent to obtain a brownish red oily substance, dissolving the obtained brownish red oily substance in trifluoroacetic acid, stirring at room temperature, reducing pressure by using the rotary evaporator and the oil pump, removing the solvent, and recrystallizing with diethyl ether to obtain a brownish red solid; dissolving the obtained brownish red solid in dimethyl sulfoxide, and purifying by Semi-HPLC to obtain brownish red solid HBED-CC-04-DiF-Dimer;

Dissolving HBED-CC-04-DiF-Dimer (labeled precursor) in dimethyl sulfoxide, adding sodium acetate solution to obtain labeled precursor sodium acetate solution, rinsing germanium gallium generator (iThemba laboratories, 740MBq, 20mCi) with high-purity hydrochloric acid solution, and mixing the obtained solution⁶⁸Ga]GaCl₃Adding hydrochloric acid solution into labeled precursor sodium acetate solution, mixing, reacting at 95 deg.C, cooling to room temperature, and detecting with radioactivity detector (Flow-Count, Eckert)&High performance liquid chromatography (radio-HPLC, Agilent 1260Infinity II system) of Ziegler) the labeling rate was determined in a mobile phase of 0.1% aqueous trifluoroacetic acid (v/v) and 0.1% acetonitrile trifluoroacetic acid (v/v) to obtain a radiochemical yield of greater than 99%⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer。

Has the advantages that:

according to the invention⁶⁸The Ga-labeled inhibitor radioactive probe targeting the fibroblast activation protein shows high affinity and high specificity to the fibroblast activation protein, has better tumor uptake and longer tumor retention time, and is a more potential FAP/PET imaging agent.

The invention is further illustrated by the following figures and detailed description of the invention, which are not meant to limit the scope of the invention.

Drawings

FIG. 1 is the [2 ] prepared in example 1 of the present invention⁶⁸Ga]The radioactive HPLC chromatogram of the labeled reaction solution of Ga-HBED-CC-04-DiF-Monomer.

FIG. 2 is the [2 ] prepared in example 2 of the present invention⁶⁸Ga]And the radioactive HPLC spectrum of the Ga-HBED-CC-04-DiF-Dimer labeling reaction solution.

FIG. 3 is the in vitro uptake of HT1080-FAP by cells in application example 1 of the present invention⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]graph of Ga-HBED-CC-04-DiF-Dimer uptake versus time.

FIG. 4 is an assay of the in vitro HT1080-FAP cell uptake assay in application example 1 of the present invention⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]specific binding pattern of Ga-HBED-CC-04-DiF-Dimer and fibroblast activation protein.

FIG. 5 is a graph showing the binding affinity of HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer to fibroblast activator protein in an experiment for determining IC50 value in application example 2 of the present invention.

FIG. 6 shows the present invention⁶⁸Structural formula of Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein.

FIG. 7 shows the present invention⁶⁸Reaction formula of Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein.

FIG. 8 is a reaction equation for the synthesis of HBED-CC-04-DiF-Monomer in step (1) of application example 1 of the present invention.

FIG. 9 is a view showing the sequence of step (2) in application example 1 of the present invention⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer.

FIG. 10 shows the reaction equation for the synthesis of HBED-CC-04-DiF-Dimer in step (1) of application example 2.

FIG. 11 is a view showing the sequence of step (2) in application example 2 of the present invention⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer.

The specific implementation mode is as follows:

unless otherwise specified, reagents and raw materials used in the preparation methods and detection methods described in the following examples and comparative examples are commercially available, and all the equipment used are conventional equipment.

Unless otherwise indicated, the concentrations and ratios described in the following examples and comparative examples are in weight units.

Example 1([ 2 ]⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer)

The reaction equation for the synthesis of HBED-CC-04-DiF-Monomer, i.e. (S) -3- (5-methyl- ((3-carboxymethyl) (2- ((3-carboxymethyl) (5- (3- (4- (4- (4- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-oxyethyl) carbamoyl) quinolin-7-yl) -4-oxopropyl) piperazin-1-yl) -3-oxopropyl) -2-hydroxybenzyl) amino) ethyl) amino) -4-hydroxyphenyl) propanoic acid, is shown in FIG. 8.

The synthesis method comprises the following steps:

(S) -N- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-ethoxy) -6- (3- (piperazin-1-yl) propoxy) quinoline-4-carboxamide (52 mg, 0.1 mmol) was dissolved in 2 ml of anhydrous dimethylformamide, and to the mixed solution were added 1-hydroxybenzotriazole (17 mg, 0.1 mmol), N-diisopropylethylamine (68 mg, 0.5 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (25 mg, 0.1 mmol) and 3, 3' - ((2,2,13, 13-tetramethyl-4, 11-dioxo-3, 12-dioxa-6, 9-Dinitrotetradecane-6, 9-diyl) bis (methylene)) bis (4-hydroxy-3, 1-phenyl)) dipropionic acid (57 mg, 0.1 mmol), reacted at room temperature overnight, and after adding 30 ml of ethyl acetate and 10 ml of saturated saline solution to the mixed solution overnight, washing twice and collecting an organic phase filtrate; drying the organic phase filtrate with anhydrous sodium sulfate, filtering, and removing solid impurities; removing the organic solvent in the filtrate by using a rotary evaporator, separating the organic solvent by using a silica gel column (refined column chromatography silica gel, 200-mesh and 300-mesh) by using dichloromethane/methanol/25% ammonia water (4/1/0.1, v/v/v), collecting components, removing the organic solvent in the components by using the rotary evaporator and an oil pump to obtain a brown yellow oily substance, dissolving the obtained brown yellow oily substance in 3 ml of trifluoroacetic acid, stirring the mixture at room temperature for 7 hours, removing the solvent by using the rotary evaporator and the oil pump, and recrystallizing the mixture by using ether to obtain a brown yellow solid; the resulting brown yellow solid was dissolved in dimethyl sulfoxide and purified by Semi-HPLC (0.1% trifluoroacetic acid water/acetonitrile 7/3, v/v) to give HBED-CC-04-DiF-Monomer 16 mg as a brown yellow solid (yield: 20%);

confirmation of Compound HBED-CC-04-DiF-Monomer:

¹H NMR(600MHz,DMSO)δ8.91-8.85(m,1H),8.05(dd,J＝9.2,2.0Hz,1H), 7.89(s,1H),7.66-7.59(m,1H),7.55-7.51(m,1H),7.07(t,J＝7.7Hz,4H),6.80(d,J ＝8.2Hz,2H),5.11(d,J＝7.4Hz,1H),4.45-4.35(m,2H),4.34-4.16(m,8H),4.16- 3.96(m,10H),3.33-3.28(m,2H),3.23-3.21(m,4H),2.95-2.80(m,2H),2.69(t,J＝ 7.3Hz,4H),2.60(d,J＝8.3Hz,2H),2.26-2.17(m,2H).

HRMS (ESI) theoretical molecular weight C₅₀H₅₈F₂N₈O₁₂[M+H]⁺1001.4221 molecular weight 1001.4239[ M + H ] found]⁺；

[⁶⁸Ga]The labeling reaction equation of Ga-HBED-CC-04-DiF-Monomer is shown in FIG. 9.

The marking method comprises the following steps:

dissolving 8 micrograms of labeled precursor HBED-CC-04-DiF-Monomer in 80 microliters of dimethyl sulfoxide, and adding 135 microliters of 3 mol/L sodium acetate buffer solution to obtain a labeled precursor sodium acetate buffer solution; leaching the germanium-gallium generator with 6 ml of high-purity 0.6 mol/L hydrochloric acid solution (iThemba laboratories, 740MBq, 20mCi), to obtain [ product ], [ product ] of the present invention⁶⁸Ga]GaCl₃Adding 0.3 ml of hydrochloric acid solution into labeled precursor sodium acetate buffer solution, mixing, reacting at 95 deg.C for 5 min, cooling to room temperature, and detecting with radioactive detector (Flow-Count, Eckert)&Ziegler) high performance liquid chromatography (radio-HPLC, Agilent 1260Infinity II system) the labeling rate was determined in a mobile phase of 0.1% trifluoroacetic acid in water (v/v) and 0.1% trifluoroacetic acid in acetonitrile (v/v) to obtain radiochemicalThe yield is 99.56%⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer；

As shown in FIG. 1, is the product of the present invention prepared in example 1 of the present invention⁶⁸Ga]A radioactive HPLC chromatogram of the labeled reaction solution of Ga-HBED-CC-04-DiF-Monomer, which is shown in the chromatogram⁶⁸Ga]The radiochemical purity of Ga-HBED-CC-04-DiF-Monomer is more than 99 percent;

in the radio-HPLC assay in the above step, the first mobile phase is 0.1% trifluoroacetic acid in water (v/v), the second mobile phase is 0.1% trifluoroacetic acid in acetonitrile (v/v), and the gradient elution conditions are as follows: from 0 to 10 minutes, from 100% to 35% of the first mobile phase; 10-12 minutes, 35% -100% of the first mobile phase; 12-15 minutes, 100% of the first mobile phase; the flow rate of the mobile phase was 1 ml/min.

Example 2([ 2 ])⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer)

The reaction equation for the synthesis of HBED-CC-04-DiF-Dimer, i.e., 2' - (ethane-1, 2-diylbis (5- (3- (4- (4- (2- (2- ((S) -2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-oxoethyl) carbamoyl) quinolin-7-yl) -4-oxopropyl) piperazin-1-yl) -3-oxopropyl) -2-hydroxybenzyl)) azacyclo-diacetic acid is shown in FIG. 10.

The synthesis method comprises the following steps:

(S) -N- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-ethoxy) -6- (3- (piperazin-1-yl) propoxy) quinoline-4-carboxamide (100 mg, 0.2 mmol) was dissolved in 2 ml of anhydrous dimethylformamide, and to the mixed solution were added 1-hydroxybenzotriazole (23 mg, 0.2 mmol), N-diisopropylethylamine (84 mg, 0.6 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (32 mg, 0.2 mmol), and 3, 3' - ((2,2,13, 13-tetramethyl-4, 11-dioxo-3, 12-dioxa-6, 9-Dinitrotetradecane-6, 9-diyl) bis (methylene)) bis (4-hydroxy-3, 1-phenyl)) dipropionic acid (23 mg, 0.04 mmol) was reacted at room temperature overnight, and then 30 ml of ethyl acetate and 10 ml of saturated saline were added to the mixed solution, and washed twice to collect an organic phase filtrate; drying the organic phase filtrate with anhydrous sodium sulfate, filtering, and removing solid impurities; removing the solvent in the filtrate by using a rotary evaporator under reduced pressure, separating the filtrate by using a silica gel column (refined column chromatography silica gel, 200-mesh and 300-mesh) with dichloromethane/methanol/25% ammonia water (20/1/0.1, v/v/v), collecting components, removing the organic solvent in the components by using the rotary evaporator and an oil pump to obtain a brownish red oily substance, dissolving the obtained brownish red oily substance in 3 ml of trifluoroacetic acid, stirring the mixture at room temperature for 7 hours, removing the solvent by using the rotary evaporator and the oil pump, and recrystallizing the mixture by using ether to obtain a brownish red solid; the resulting brownish red solid was dissolved in dimethyl sulfoxide and purified by Semi-HPLC (0.1% trifluoroacetic acid water/acetonitrile 7/3, v/v) to give HBED-CC-04-DiF-Monomer 20 mg (yield: 40%) as a brownish red solid;

confirmation of Compound HBED-CC-04-DiF-Dimer:

¹H NMR(600MHz,DMSO)δ8.84(d,J＝4.3Hz,2H),8.03(d,J＝9.2Hz,2H), 7.88(d,J＝2.2Hz,2H),7.56(d,J＝4.2Hz,2H),7.48(dd,J＝9.2,1.5Hz,2H),7.13- 7.06(m,4H),6.80(d,J＝8.0Hz,2H),5.13(dd,J＝9.2,2.3Hz,2H),4.28-4.18(m, 12H),4.02-3.99(m,4H),3.66-3.63(m,8H),3.34-3.31(m,10H),3.21-3.19(m,6H), 2.98-2.76(m,6H),2.72-2.69(m,4H),2.62-2.59(m,4H),2.25-2.20(m,4H).

HRMS (ESI) theoretical molecular weight C₇₄H₈₄F₄N₁₄O₁₄[M+H]⁺1469.6306 molecular weight 1469.6306[ M + H ] found]⁺；

[⁶⁸Ga]The labeling reaction equation of Ga-HBED-CC-04-DiF-Dimer is shown in FIG. 11.

The marking method comprises the following steps:

dissolving 12 micrograms of labeled precursor HBED-CC-04-DiF-Dimer in 120 microliters of dimethyl sulfoxide, and adding 135 microliters of 3 mol/liter sodium acetate buffer solution to obtain a precursor solution; washing germanium gallium generator (iThemba laboratories, 740MBq, 20mCi) with 6 ml of high purity 0.6 mol/L hydrochloric acid solution, and subjecting the obtained product to the reaction of⁶⁸Ga]GaCl₃0.3 ml of hydrochloric acid solution is taken and added into the precursorMixing the solution, reacting at 95 deg.C for 5 min, cooling to room temperature, and detecting with radioactivity detector (Flow-Count, Eckert)&High performance liquid chromatography (radio-HPLC, Agilent 1260Infinity II system) of Ziegler) the labeling rate was measured in a mobile phase of 0.1% trifluoroacetic acid aqueous solution (v/v) and 0.1% trifluoroacetic acid acetonitrile solution (v/v), to obtain a radiochemical yield of 99.89%⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer；

As shown in FIG. 2, is the product of the present invention prepared in example 2⁶⁸Ga]A radioactive HPLC chromatogram of the labeling reaction solution of Ga-HBED-CC-04-DiF-Dimer, which shows⁶⁸Ga]The radiochemical purity of Ga-HBED-CC-04-DiF-Dimer is more than 99 percent;

Application example 1

[⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]in vitro cellular uptake of Ga-HBED-CC-04-DiF-Dimer

(1) HT1080-FAP (FAP-positive) cells (. about.5X 10)⁵One hole) is inoculated in a 6-hole plate and cultured in an incubator for 60 hours, and the cell coverage rate is 90-100 percent; after 60 hours, the culture solution was aspirated, the cells were washed twice with a phosphate buffered saline solution, and 12. mu. Ci of⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer or [2 ]⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer or [2 ]⁶⁸Ga]Ga-FAPI-04, incubating at 37 ℃ for 10, 30, 60, 90 and 120 minutes, blocking cell uptake at 60 minutes by using 10 mu M UAMC-1110(FAP inhibitor), sucking out the solution after the incubation is finished, and rapidly washing the cells with phosphate buffered saline solution for three times to stop cell uptake; using sodium hydroxide to crack all cells, collecting a cracking solution and carrying out radioactive counting;

(2) the results of in vitro cellular uptake experiments show that: [⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]the Ga-HBED-CC-04-DiF-Dimer has high uptake in HT1080-FAP (FAP positive) cells in vitro, and the uptake is increased along with the increase of time, and the uptake in 10 minutes is higher than that of the Ga-HBED-CC-04-DiF-Dimer⁶⁸Ga]Maximum uptake of Ga-FAPI-04, 120 min uptake: (>60 percent of the total content of the components is obviously higher than the total content of the components⁶⁸Ga]Ga-FAPI-04; when the fibroblast activation protein inhibitor UAMC-1110 was added, the uptake of both was blocked, indicating that⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer specifically binds to fibroblast activation protein.

As shown in FIG. 3, in vitro uptake of HT1080-FAP in cells is shown in example 1 of use of the present invention⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]graph of Ga-HBED-CC-04-DiF-Dimer uptake versus time, with n being 3.

As shown in FIG. 4, the in vitro HT1080-FAP cell uptake assay of the present invention as described in example 1⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]specific binding pattern of Ga-HBED-CC-04-DiF-Dimer to fibroblast activation protein, n is 3, and significant difference in cellular uptake between the group without UAMC1110 and the group with UAMC1110 showed: [⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer binds specifically to fibroblast activation protein.

Application example 2

Determination of IC50 values of HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer

(1) HT1080-FAP (FAP-positive) cells (. about.5X 10)⁵Perwell) was inoculated in a 6-well plate, cultured in an incubator for 60 hours with a cell coverage of 90 to 100%, after 60 hours, the culture solution was aspirated, the cells were washed twice with a phosphate buffered saline solution, and 12. mu. Ci of⁶⁸Ga]Ga-FAPI-04 and HBED-CC-04-DiF-Monomer or HBED-CC-04-DiF-Dimer or FAPI-04, so that the final concentration of HBED-CC-04-DiF-Monomer or HBED-CC-04-DiF-Dimer or FAPI-04 is 10^-9.5、10^-9、10^-8.5、10^-8、10^-7And 10^-6M/l, incubation at 37 ℃ for 60 min, aspiration of the solution, rapid washing of the cells three times with phosphate buffered saline, termination of cellular uptake, hydrogen additionSodium oxide lyses all cells, and lysate is collected for radioactive counting;

as shown in fig. 5, for the analysis of binding affinity of HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer to fibroblast activation protein in the experiment for determining IC50 values in application example 2 of the present invention, n is 3, and IC50 values in nM scale show: HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer have high binding affinity to fibroblast activation protein;

(2) the results of the experiment were determined from the IC50 values: the binding affinity of HBED-CC-04-DiF-Monomer and HBED-CC-04-DiF-Dimer to fibroblast activator protein is high, and the IC50 values are respectively 5.06 +/-1.09 nM and 5.10 +/-1.41 nM and are basically consistent with FAPI-04(5.16 +/-0.75 nM).

According to the invention⁶⁸Ga-labeled inhibitor radioactive probes of targeted fibroblast activation protein (FGPA) have excellent properties⁶⁸Ga-labelling properties, benefited from N, N' -bis [ 2-hydroxy-5- (carboxyethyl) -benzyl]ethylenediamine-N, N' -diacetic acid (HBED-CC) as excellent Ga³⁺Bifunctional linkers, HBED-CC and Ga³⁺Has a high thermodynamic stability constant (logK)_ML: 38.5), the energy required for coordination is low, therefore, the [ alpha ], [ beta ] is a ] or a ] is a⁶⁸Ga]The marking of Ga-HBED-CC is rapid and efficient. Comparison [2 ]⁶⁸Ga]Ga-FAPI-04 needs to be heated for 20 minutes at 100 ℃ to prepare a product with the radiochemical purity of more than 90 percent, the method⁶⁸The Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein can be quickly prepared by heating at 95 ℃ for 5 minutes, and the product has high radiochemical yield and radiochemical purity (both)>99 percent) and high stability.

According to the invention⁶⁸The Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein contains (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidine-1-yl) -2-ethoxy) -6- (3- (piperazine-1-yl) propoxy) quinoline-4-formamide groups, and the groups have good fibroblast activation protein affinity; in addition, the introduction of HBED-CC may increase tumor uptake. We expect⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer and⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer has good affinity to tumors expressing fibroblast activation protein and tumor uptakeHigh value and long tumor retention time. Accordingly, the invention⁶⁸The Ga-labeled inhibitor radioactive probe of the target fibroblast activation protein can be used as a tumor positron molecular probe for tumor imaging.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Claims

1.⁶⁸Ga-labeled inhibitors radioactive probes targeting fibroblast activation protein are respectively: [⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer or [2 ]⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer, the structural formula is as follows:

2.⁶⁸the preparation method of the Ga-labeled inhibitor radioactive probe targeting fibroblast activation protein comprises the following steps: in the presence of alkali and condensing agent, the bifunctional linker HBED-CC and fibroblast activation protein inhibitor are subjected to condensation reaction, after the protective group is removed by acid, the obtained product is dissolved in dimethyl sulfoxide, and added⁶⁸Ga]GaCl₃Mixing the solution, and heating to obtain⁶⁸Ga-labeled inhibitor radioactive probes targeting fibroblast activation proteins.

3. The method for preparing the fibroblast activation protein-targeted inhibitor-based radioactive probe according to claim 2, wherein the method comprises the following steps: the alkali is N, N-diisopropylethylamine, and the addition amount is 3-5 equivalents; the condensing agent is 1-hydroxybenzotriazole and 1-ethyl- (3-dimethyl aminopropyl) carbodiimide, and the addition amount of the condensing agent is 1 equivalent; the fibroblast activation protein inhibitor is (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidine-1-yl) -2-ethoxy) -6- (3- (piperazine-1-yl) propoxy) quinoline-4-formamide, and the adding amount is 1-5 equivalents; the acid was trifluoroacetic acid and the amount added was 3 ml.

4. The method for preparing the fibroblast activation protein-targeted inhibitor-based radioactive probe according to claim 2, wherein the method comprises the following steps: the method comprises the following specific steps:

step 1: synthesis of labeled precursor of inhibitor-based radioactive probe targeting fibroblast activation protein

Dissolving (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-ethoxy) -6- (3- (piperazin-1-yl) propoxy) quinoline-4-carboxamide in anhydrous dimethylformamide, adding 1-hydroxybenzotriazole, N-diisopropylethylamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 3, 3' - ((((2, 2,13, 13-tetramethyl-4, 11-dioxo-3, 12-dioxa-6, 9-diazepidecane-6, 9-diyl) bis (methylene)) bis (4-hydroxy-3 to the mixed solution, 1-phenyl)) dipropionic acid, reacting at room temperature, adding ethyl acetate and saturated saline solution into the mixed solution after overnight, washing, and collecting organic phase filtrate; drying the organic phase filtrate with anhydrous sodium sulfate, filtering, and removing solid impurities; removing the solvent in the filtrate under reduced pressure by using a rotary evaporator, separating the filtrate by using a silica gel column through a mixed solution (4:1:0.1, v/v/v) of dichloromethane, methanol and 25% ammonia water, collecting components, removing the organic solvent in the components by using the rotary evaporator and an oil pump to obtain a brown yellow oily substance, dissolving the obtained brown yellow oily substance in trifluoroacetic acid, stirring the mixture at room temperature, removing the solvent by using the rotary evaporator and the oil pump, and recrystallizing the mixture by using diethyl ether to obtain a brown yellow solid; dissolving the obtained brown yellow solid in dimethyl sulfoxide, and purifying by Semi-HPLC to obtain brown yellow solid HBED-CC-04-DiF-Monomer;

step 2: labelling of inhibitor-based radioactive probes targeting fibroblast activation proteins

Dissolving the HBED-CC-04-DiF-Monomer obtained in the step 1 in dimethyl sulfoxide, adding a sodium acetate solution to obtain a labeled precursor sodium acetate solution, leaching the germanium and gallium generator with a high-purity hydrochloric acid solution, and filtering the solution to obtain the product⁶⁸Ga]GaCl₃Adding marked precursor vinegar into hydrochloric acid solutionMixing with sodium solution, reacting at 95 deg.C, cooling to room temperature, and measuring its labeling rate by high performance liquid chromatography (radio-HPLC) with radioactivity detector to obtain sodium sulfate solution with radiochemical yield of greater than 99%⁶⁸Ga]Ga-HBED-CC-04-DiF-Monomer。

5. The method for preparing the fibroblast activation protein-targeted inhibitor-based radioactive probe according to claim 2, wherein the method comprises the following steps: the method comprises the following specific steps:

Dissolving (S) -N- (2- (2-cyano-4, 4-difluoropyrrolidin-1-yl) -2-ethoxy) -6- (3- (piperazin-1-yl) propoxy) quinoline-4-carboxamide in anhydrous dimethylformamide, adding 1-hydroxybenzotriazole, N-diisopropylethylamine, 1-ethyl- (3-dimethylaminopropyl) carbodiimide and 3, 3' - ((((2, 2,13, 13-tetramethyl-4, 11-dioxo-3, 12-dioxa-6, 9-diazepidecane-6, 9-diyl) bis (methylene)) bis (4-hydroxy-3 to the mixed solution, 1-phenyl)) dipropionic acid, reacting at room temperature, adding ethyl acetate and saturated saline solution into the mixed solution after overnight, washing, and collecting organic phase filtrate; drying the organic phase filtrate with anhydrous sodium sulfate, filtering, and removing solid impurities; removing the solvent in the filtrate under reduced pressure by using a rotary evaporator, separating the filtrate by using a silica gel column through a mixed solution (20:1:0.1, v/v/v) of dichloromethane, methanol and 25% ammonia water, wherein the volume ratio of the mixed solution is 20:1:0.1, collecting components, removing the organic solvent in the components by using the rotary evaporator and an oil pump to obtain a brownish red oily substance, dissolving the obtained brownish red oily substance in trifluoroacetic acid, stirring at room temperature, removing the solvent by using the rotary evaporator and the oil pump, and recrystallizing by using diethyl ether to obtain a brownish red solid; dissolving the obtained brownish red solid in dimethyl sulfoxide, and purifying by Semi-HPLC to obtain brownish red solid HBED-CC-04-DiF-Dimer;

Dissolving the HBED-CC-04-DiF-Dimer obtained in the step 2 in dimethyl sulfoxide, adding a sodium acetate solution to obtain a labeled precursor sodium acetate solution,leaching the germanium-gallium generator with high-purity hydrochloric acid solution to obtain the product⁶⁸Ga]GaCl₃Adding hydrochloric acid solution into labeled precursor sodium acetate solution, mixing, reacting at 95 deg.C, cooling to room temperature, and measuring its labeling rate by high performance liquid chromatography with radioactivity detector to obtain the final product with radiochemical yield greater than 99%⁶⁸Ga]Ga-HBED-CC-04-DiF-Dimer。

6. The method for preparing the fibroblast activation protein-targeted inhibitor-based radioactive probe according to claim 4 or 5, wherein the method comprises the following steps: in step 2, the test conditions of the high performance liquid chromatography with the radioactivity detector are as follows: the first mobile phase is 0.1% trifluoroacetic acid water solution, the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution, and the gradient elution conditions are as follows: from 0 to 10 minutes, from 100% to 35% of the first mobile phase; 10-12 minutes, 35% -100% of the first mobile phase; 12-15 minutes, 100% of the first mobile phase; the flow rate of the mobile phase was 1 ml/min.