Radiolabeled ligands for fibroblast activation protein-alpha imaging agents and methods of making the same

Abstract

The present application relates to a kind of fibroblast activation protein-alpha imaging agent radioactive labeled ligand and its preparation method, belong to the field of radiopharmaceutical chemistry, fibroblast activation protein-alpha imaging agent radioactive labeled ligand has the following structural formula: this kind of compound has excellent 68 Ga marking property, high stability, good FAP tumor affinity.Therefore, a kind of 68 Ga labeled FAPi radioactive labeled ligand of the present application can be used as tumor positron imaging agent.

Description

Technical Field

[0001] This invention relates to novel radiolabeled ligands (precursors) and their preparation methods, specifically a... 68 The radiolabeled ligands of Ga-labeled imaging agents targeting fibroblast activation protein-α (FAP) and their preparation methods belong to the field of radiolabeled compounds. Background Technology

[0002] Fibroblast activation protein-α (FAP) is a serine protease on the cell surface that acts on various hormones and extracellular matrix components. FAP is expressed at high levels in various cancers and is commonly used as a biomarker for tumorigenic mechanisms. In recent years, FAP has been used as a molecular target for cancer diagnosis and treatment, and numerous FAP-targeted therapies have entered the design and testing phase.

[0003] Fibroblast activation protein-α (FAP) was independently discovered in the mid-1980s and early 1990s by two groups researching different topics. Retting et al., while studying surface antigens, first described and named FAP based on its expression on fibroblasts in 1986. Another group, Aoyama et al., while studying membrane-bound proteases, discovered a gelatinase expressed on the surface of invasive melanoma cells, which they named "seprase". It wasn't until 1997 that gene sequencing results confirmed that FAP and seprase are the same protein molecule, belonging to the dipeptidyl peptidase (DPP) family. Like other DPP enzymes, FAP possesses post-proline exopeptidase activity; however, FAP has unique gelatinase activity, allowing it to be degraded, denatured, or cleaved by matrix metalloproteinases (MMPs). Structurally, FAP consists of a 6-amino acid cytoplasmic tail, a 20-amino acid transmembrane domain, and a 734-amino acid extracellular domain. The extracellular domain comprises an octetal β-propeller acting as a substrate-selective phylum and an α / β hydrolase domain. FAP monomers are inactive, but they form active homodimers and heterodimers with their closest family member, dipeptidyl peptidase IV (DPPIV). Unlike the widespread expression of DPPIV, FAP is expressed only in fetal cells, stromal fibroblasts, injured tissues, and fibroblasts in over 90% of malignant tumors, and not in benign tumors or normal adult tissues. In normal adult tissues, FAP is expressed only in bone marrow mesenchymal stem cells (BM-MSCs), while in tumors, various mesenchymal cells contain FAP, including MSCs, cancer-associated fibroblasts (CAFs), sarcoma, and melanoma cells. Cancer-associated fibroblasts (CAFs), also known as tumor-associated fibroblasts or activated fibroblasts, are a cell type in the tumor microenvironment that plays a supportive role in tumor growth and invasion. They promote extracellular matrix remodeling, enhance tumor invasiveness and angiogenesis, and induce epithelial-mesenchymal transition by secreting growth factors and cytokines. Furthermore, CAFs are involved in the immune interactions between tumors and the host. Based on the high expression of fibroblast-associated protein (FAP) in CAFs, many studies have used FAP as a biomarker for CAFs.

[0004] FAP-targeted tumor PET imaging offers significant advantages because tumors larger than 1-2 mm often exhibit supporting stroma formation, with stromal cells larger than cancer cells. Therefore, if FAP is sufficiently expressed, stroma-targeted PET imaging will be more sensitive than glucose metabolism PET imaging. FAP-targeted PET imaging also has considerable advantages in detecting tumors with low or heterogeneous glucose metabolism or tumors near highly glycolytic normal tissue. Other potential advantages include early imaging 10 minutes after injection and the elimination of the need for fasting. Last but not least, FAP-targeted PET imaging can serve as an accurate predictive biomarker for response to treatment with any FAP in most cancers. A limitation is that, due to FAP expression in many tissue remodeling processes, it is not cancer-specific. For example, this can make it difficult to distinguish between chronic pancreatitis and pancreatic ductal adenocarcinoma. Conversely, FAP-targeted PET imaging can also be used for many non-oncology imaging conditions, such as myocardial infarction, chronic inflammatory diseases, and fibrosis of the lung, liver, or kidney.

[0005] Based on existing FAP inhibitors (FAPI), Thomas Lindner et al. reported in 2018 a radioactive molecular probe targeting FAP. They synthesized a series of quinoline-based derivatives and applied them... 177 Lu-labeled compounds were used to determine their binding and internalization rates in HT-1080 cells transfected with the human or mouse FAP gene, as well as in human embryonic kidney cells transfected with CD26. All tracers showed internalization rates exceeding 90%. To confirm target specificity, binding assays were also performed using human embryonic kidney cells expressing mouse FAP and dipeptidyl peptidase 4 (CD26), which are highly homologous to human FAP. In these experiments, FAPI-02 and FAPI-04 showed strong binding to mouse FAP, with FAPI-04 showing significantly higher values, and did not bind to CD26.

[0006] Small animal PET studies were conducted using (HT-1080)FAP xenograft mice to investigate FAPI compounds that showed promising results in cell experiments. 68 Ga-FAPI-02 and 68 Ga-FAPI-04 exhibited the highest tumor uptake 1 hour post-injection (SUV max 0.88 and 1.2, respectively), with no significant decrease within 2 hours (SUV 0.71 and 1.1, respectively). This was also successfully demonstrated in the second xenograft model, SK-LMS-1. 68 The targeting of Ga-FAPI-04 was demonstrated. Furthermore, the target specificity was elucidated through blocking experiments on HT-1080FAP xenografts.

[0007] In clinical applications for breast cancer patients, intravenous injection was administered to two patients with metastatic breast cancer. 68 Diagnostic PET / CT scans were performed 10 minutes, 1 hour, and 3 hours after taking Ga-FAPI-04. In both patients, tracer accumulation in metastatic lesions was high (SUV max 7-15.5 and 15.3-29.9, respectively), while tracer uptake in normal tissues was very low.

[0008] Of all the tested derivatives, FAPI-04 was the most suitable as a therapeutic diagnostic tracer. Similar to its prodrug FAPI-02, FAPI-04 exhibits rapid internalization into FAP-positive tumors and rapid clearance from the body, resulting in very rapid accumulation at the tumor site (10 minutes after tracer administration) and a high tumor-to-organ ratio. Furthermore, effective tumor uptake of FAPI-04 was 100% higher than that of FAPI-02 after 24 hours, which is a significant advantage for the oncological application of the tracer.

[0009] Based on the structure of FAPI-04, Anastasia Loktev et al. reported a series of novel FAP-targeting radiomolecular probes in 2019, further improving drug retention time in tumors. In vitro competitive binding assays showed that all compounds exhibited high binding to FAPI, with binding values ​​equal to or higher than those of FAPI-04 after 1 and 4 hours of incubation. Except for FAPI-38, the internalization rates of all compounds were comparable to FAPI-04. Although most derivatives showed higher binding values ​​than FAPI-04 after 24 hours, compounds FAPI-38, -39, -40, and -41 were significantly cleared from FAP-expressing cells at a faster rate.

[0010] As an emerging radioactive diagnostic and therapeutic target for tumors, FAP has attracted close attention due to its high targeting accuracy, low background interference, and broad applicability to various tumors. Despite the many challenges it still faces, FAP has become the next focus of nuclear medicine.

[0011] 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid (HBED) is a non-macrocyclic bifunctional linker and was not initially used for its development. 68 Ga positron emission tomography (PET) drug research initially utilized it in conjunction with Fe... 3+ It is used as a soil fertilizer to treat iron deficiency fading in plants. Recent studies have found that HBED is an excellent source of Ga... 3+ Bifunctional binders have a thermodynamic stability constant that is much higher than that of other commonly used binders (log K).ML : HBED: 38.5; DOTA: 21.3; NOTA: 31.0; AAZTA: 22.18). HBED and Ga 3+ The energy required for coordination is relatively low compared to other bifunctional linkers, therefore, 68 Ga-HBED labeling requires lower temperatures and shorter time intervals. In the development of positron emission tomography (PET) drugs, HBED sometimes acts not only as a bifunctional linker but also provides additional targeting groups, increasing the affinity between the PET drug and the target. Eder M et al., in developing PET drugs for prostate cancer, found that compared to DOTA, HBED-containing imaging drugs showed better tumor cell uptake values. PET imaging of tumor-bearing mice demonstrated that HBED had better tumor uptake values ​​and a better target / non-target ratio.

[0012] Therefore, combining HBED and FAP inhibitors in radiopharmaceutical research may lead to... 68 Ga-labeling offers milder reaction conditions, higher efficiency, and more suitable in vivo pharmacokinetic properties, potentially meeting future clinical needs in tumor diagnosis. Furthermore, HBED-CC can also be combined with […]. 18 F]AlF coordination marker F-18 is used for PET imaging, and with carbonyl technetium 99m Tc(CO)3 and rhenium carbonyl 188 / 186 Re(CO)3 coordination labeling yields radioactive agents for SPECT imaging and treatment. Summary of the Invention

[0013] One of the objectives of this invention is to provide a 68 This is a Ga-labeled radiolabeled ligand for fibroblast activation protein-α (FAP) imaging agent. The radiolabeled ligand is easy and efficient to prepare and has good in vivo bio-metabolic properties, making it a promising tumor imaging drug.

[0014] Another object of the present invention is to provide the above-mentioned 68 A method for preparing radiolabeled ligands for Ga-labeled fibroblast activating protein-α (FAP) imaging agents.

[0015] Another object of the present invention is to provide the above. 68 The use of Ga-labeled radiolabeled ligands as tumor imaging agents for fibroblast activation protein-α (FAP) imaging agents.

[0016] According to one aspect of the present invention, the present invention provides a radiolabeled ligand for a fibroblast activation protein-α (FAP) imaging agent with the following structural formula:

[0017]

[0018] Where R is -OH or

[0019]

[0020] According to another aspect of the present invention, the present invention provides a method for preparing the radiolabeled ligand of the above-mentioned fibroblast activation protein-α (FAP) imaging agent, the steps of which are as follows: In the presence of a base and a condensing agent, a bifunctional linker HBED-CC and a FAP inhibitor are subjected to a condensation reaction, and then the protecting group is removed using an acid to obtain the fibroblast activation protein-α (FAP) imaging agent, the reaction formula of which is as follows:

[0021]

[0022] Where R is -OH or

[0023]

[0024] According to another aspect of the present invention, the radiolabeled ligand of the fibroblast activation protein-α (FAP) imaging agent of the present invention is used for 68 Ga positron-emitting drugs, the imaging agent's chemical structure contains elements that are more readily associated with... 68 The Ga-bound bifunctional linker HBED makes radioactive preparation more convenient and efficient.

[0025] Beneficial effects:

[0026] The radiolabeled ligand of the fibroblast activation protein-α (FAP) imaging agent of the present invention, with its biological characteristic of specifically targeting FAP, has important clinical potential value in the early diagnosis of tumors, preoperative staging, treatment guidance, and detection of recurrence and metastasis, and is expected to lead to the development of tumor imaging drugs with good biological properties.

[0027] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings, but this does not mean that the scope of protection of the present invention is limited. Attached Figure Description

[0028] Figure 1 The [prepared in step (3) of Example 3 of this invention] 68 HPLC chromatogram of Ga]Ga-A1 labeled reaction solution.

[0029] Figure 2 The [prepared in step (3) of Example 4 of this invention] 68 HPLC chromatogram of Ga]Ga-A2 labeled reaction solution.

[0030] Figure 3 This is a binding affinity diagram of A1 and A2 to FAP in Embodiment 5 of the present invention.

[0031] Figure 4 In embodiment 6 of the present invention [ 68 Ga]Ga-A1 and [ 68 PET / CT image of mice with U87MG tumor (FAP+) of Ga]Ga-A2. Detailed Implementation

[0032] Unless otherwise specified, the raw materials and reagents mentioned in the embodiments of the present invention are all commercially available conventional raw materials and reagents, the testing methods used are all conventional methods used in the art, and the equipment and apparatus used are all conventional equipment and apparatus used in the art.

[0033] Example 1

[0034] Synthesis of radiolabeled ligand A1 in fibroblast activation protein-α (FAP) imaging agent

[0035] (S)-3-(3-((carboxymethyl)(2-((carboxymethyl)(5-(3-(4-((4-((2-(2-(2-cyanopyrrolid-1-yl)-2-oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazin-1-yl)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propionic acid

[0036] Synthesis reaction equation:

[0037]

[0038] Synthesis method:

[0039] Compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid (64 mg, 0.1 mmol) was dissolved in 2 mL of anhydrous dimethylformamide. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 28.5 mg, 0.15 mmol), 1-hydroxybenzotriazole (HOBt, 25.3 mg, 0.15 mmol), and N,N-diisopropylethylamine (DIPEA, 41.3 mg, 0.32 mmol) were added sequentially to the mixture. The mixture contained (S)-N-(2-(2-cyanopyrrolo-1-yl)-2-ethoxy)-6-(3-(-1-piperazin-1-yl)propoxy)quinoline-4-carboxamide (45 mg, 0.1 mmol); after reacting overnight at room temperature, 30 mL of ethyl acetate was added to the mixture, and the solution was washed with water (10 mL × 2) and saturated brine (10 mL); the organic phase was dried over anhydrous magnesium sulfate and filtered to remove solid impurities; the organic phase in the filtrate was removed under reduced pressure using a rotary evaporator, and the mixture was separated by silica gel column chromatography with dichloromethane / ethanol / ammonia (v / v / v, 90 / 10 / 1), the fraction was collected, and the solvent was removed under reduced pressure to obtain 45 mg of a pale yellow oil.

[0040] The obtained pale yellow oily substance was dissolved in 5 mL of trifluoroacetic acid and stirred at room temperature for 3 hours. The solvent was removed under reduced pressure using a rotary evaporator. The residue was separated using a semi-preparative high-performance liquid chromatography (HPLC) column to obtain 15.2 mg of product A1 (yield: 10.5%). The product was identified as the target product by LC / MS. HRMS (ESI) theoretical molecular weight C 50 H 61 N8O 12 (M+H) + , 964.4331; measured molecular weight, 964.4376. In the above semi-preparative high performance liquid chromatography (HPLC) column, the first mobile phase was 0.1% trifluoroacetic acid aqueous solution, the second mobile phase was acetonitrile, and the gradient elution conditions were: 0 min, 100% of the first mobile phase; 0–10 min, 100%–0% of the first mobile phase; the flow rate of the mobile phase was 4 ml / min.

[0041] Example 2

[0042] Synthesis of radiolabeled ligand A2 of fibroblast activation protein-α (FAP) imaging agent

[0043] 2,2'-(ethane-1,2-diylbis((5-(3-(4-((2-((S)-2-cyanopyrrolidone-1-yl)-2-oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazin-1-yl)-3-oxopropyl)-2-hydroxybenzyl)azacyclic diacetic acid

[0044] Synthesis reaction equation:

[0045]

[0046] Synthesis method:

[0047] Compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid (128 mg, 0.2 mmol) was dissolved in 5 mL of anhydrous dimethylformamide. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 57 mg, 0.3 mmol), 1-hydroxybenzotriazole (HOBt, 51.3 mg, 0.3 mmol), and N,N-diisopropylethylamine (DIPEA, 82.6 mg, 0.64 mmol) were added sequentially to the mixed solution. and (S)-N-(2-(2-cyanopyrrolo-1-yl)-2-ethoxy)-6-(3-(-1-piperazin-1-yl)propoxy)quinoline-4-carboxamide (45 mg, 0.1 mmol); after reacting overnight at room temperature, 30 mL of ethyl acetate was added to the mixed solution, and the mixture was washed with water (10 mL × 2) and saturated brine (10 mL); the organic phase was dried over anhydrous magnesium sulfate, filtered, and solid impurities were removed; the organic phase in the filtrate was removed under reduced pressure using a rotary evaporator, and the mixture was separated by silica gel column chromatography with dichloromethane / ethanol / ammonia (v / v / v, 90 / 10 / 1), the fraction was collected, and the solvent was removed under reduced pressure to obtain 80 mg of a pale yellow oil.

[0048] The resulting pale yellow oily substance was dissolved in 10 mL of trifluoroacetic acid and stirred at room temperature for 3 hours. The solvent was removed by rotary evaporation under reduced pressure. The residue was separated by semi-preparative high-performance liquid chromatography (HPLC) to obtain 25.9 mg of product A2 (yield: 41%). The product was identified as the target product by LC / MS. HRMS (ESI) theoretical molecular weight C 74 H 89 N 14 O 14 (M+H) +, 1397.6683; Measured molecular weight, 1397.6746. In the above semi-preparative high performance liquid chromatography (HPLC) column, the first mobile phase was 0.1% trifluoroacetic acid aqueous solution, the second mobile phase was acetonitrile, and the gradient elution conditions were: 0 min, 100% of the first mobile phase; 0–10 min, 100%–0% of the first mobile phase; the flow rate of the mobile phase was 4 ml / min.

[0049] Example 3

[0050] Radiolabeled ligand A1 68 Ga radiolabeling

[0051]

[0052] (1) Dissolve 1 mg of compound A1 ((S)-3-(3-((carboxymethyl)(2-((carboxymethyl)(5-(3-(4-((4-((2-(2-(2-cyanopyrrolid-1-yl)-2-oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazin-1-yl)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propionic acid) in 1 mL of 0.05 N sodium acetate buffer solution, and then add sodium hydroxide solution to adjust the pH to 5 to obtain a compound A1 solution with a concentration of 1 mg / mL;

[0053] (2) Rinse the germanium-gallium generator with 4 mL of high-purity 0.05N hydrochloric acid solution to obtain [ ] with an activity of 8-10 mCi. 68 Hydrochloric acid solution of Ga]GaCl3;

[0054] (3) Add 50 μL of 0.5 M sodium acetate buffer to the reaction vessel, then add 30 μL of the compound A1 solution prepared in step (1), mix thoroughly, and then add 500 μL of the solution prepared in step (2). 68 A hydrochloric acid solution of Ga]GaCl3 was prepared, and after shaking, the mixture was reacted at room temperature for 10–30 min. After cooling to room temperature, the labeling rate was determined using a semi-preparative high-performance liquid chromatography (HPLC) column. 68 Ga]Ga-A1;

[0055] In the semi-preparative high performance liquid chromatography column (HPLC) described in step (3), the first mobile phase is 0.1% trifluoroacetic acid aqueous solution, the second mobile phase is acetonitrile, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0-10 min, 100%-0% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0056] like Figure 1 As shown, this is the product prepared in step (3) of embodiment 3 of the present invention. 68HPLC chromatogram of Ga]Ga-A1 labeled reaction solution.

[0057] Example 4

[0058] Radiolabeled ligand A2 68 Ga radiolabeling

[0059]

[0060] (1) Dissolve 1 mg of compound A2 (2,2'-(ethane-1,2-diylbis((5-(3-(4-((2-(((S)-2-cyanopyrrolidone-1-yl)-2-oxoethyl)carbamoyl)quinoline-6-yl)oxy)propyl)piperazin-1-yl)-3-oxopropyl)-2-hydroxybenzyl)azacyclodiacetic acid) in 1 mL of 0.05 N sodium acetate buffer solution, then add sodium hydroxide solution to adjust the pH to 5, to obtain a compound A2 solution with a concentration of 1 mg / mL;

[0061] (2) Rinse the germanium-gallium generator with 4 mL of high-purity 0.05N hydrochloric acid solution to obtain [ ] with an activity of 8-10 mCi. 68 Hydrochloric acid solution of Ga]GaCl3;

[0062] (3) Add 50 μL of 0.5 M sodium acetate buffer to the reaction vessel, then add 30 μL of the compound A2 solution prepared in step (1), mix thoroughly, and then add 500 μL of the solution prepared in step (2). 68 A hydrochloric acid solution of Ga]GaCl3 was prepared, and after shaking, the mixture was reacted at room temperature for 10–30 min. After cooling to room temperature, the labeling rate was determined by HPLC. 68 Ga]Ga-A2;

[0063] In the semi-preparative high performance liquid chromatography column (HPLC) described in step (3), the first mobile phase is 0.1% trifluoroacetic acid aqueous solution, the second mobile phase is acetonitrile, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0-10 min, 100%-0% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0064] like Figure 2 As shown, this is the product prepared in step (3) of Example 4 of the present invention. 68 HPLC chromatogram of Ga]Ga-A2 labeled reaction solution.

[0065] Example 5

[0066] ICs A1 and A2 50 Measurement

[0067] Experimental steps:

[0068] (1) Cell treatment: HT1080-FAP cells (FAP+) were digested from the culture flask using 0.25% trypsin and then added to DMED high-glucose complete culture medium to prepare 5×10⁶ cells / year. 5 5 × 10⁶ cells / 3 mL of single-cell suspension were seeded into each well of a 6-well plate. 5 After culturing the cells in an incubator (5% CO2, 37℃) for 60 hours, the cell coverage rate was 90-100%, which can be used for cell uptake experiments;

[0069] (2) Remove the culture medium from the well plate and wash the cells twice with PBS solution;

[0070] (3) Add 8 μCi / 32 pmol / 2 mL / well of [ ] to the well plate. 68 Ga]Ga-FAPI-04 and Al (or A2), such that the final concentrations of Al (or A2) are 10 -9.5 10 -9 10 -8.5 10 -8 10 -7 and 10 -6 M, incubate at 37°C for 60 min;

[0071] (4) For uptake blocking (non-specific uptake) experiments, add 8 μCi / 32 pmol / 2 mL / well of [ ] to the well plate. 68 Ga]Ga-FAPI-04 (containing 10 μM UAMC-1110) was incubated at 37°C for 60 min;

[0072] (5) After incubation, remove the drug solution and wash the cells three times with ice-cold PBS solution to stop cell uptake.

[0073] (6) Lyse the cells in each well with 0.8 mL of 1 M NaOH for 10-15 min;

[0074] (7) Use filter paper to absorb the lysed liquid, put the filter paper into a finger cot, insert the finger cot into the tubing, and use a gamma counter to measure the radioactivity count in the tubing.

[0075] (8) The calculation of drug uptake by cells is: radioactivity count of the tube / radioactivity count of 2 mL of drug per well × 100, in %ID / 10. 6 cells;

[0076] (9) Use GraphPad Prism 6.0 software to analyze the data and calculate [ 68The concentration of the analyte precursor compound when the binding of Ga-FAPI-O4 to FAP is inhibited by 50%, i.e., the half-inhibition constants (IC50) of A1 and A2. 50 ).

[0077] Experimental results

[0078] like Figure 3 As shown, this is an example of embodiment 5 of the present invention. 68 The graph shows the uptake of Ga-FAPI-04 in FAP+ cells as a function of A1 or A2 concentrations, n=3, IC50 values ​​for A1 and A2. 50 The values ​​were 6.62±1.73 nM and 4.99±0.73 nM, respectively, in the nM range of IC50. 50 The values ​​indicate that A1 and A2 have high binding affinity for FAP.

[0079] Example 6

[0080] [ 68 Ga]Ga-A1 and [ 68 Ga]Ga-A2 Small Animal PET / CT Imaging

[0081] Experimental steps

[0082] (1) Intake group: [ 68 Ga]Ga-A1 or [ 68 Ga]Ga-A2 or [ 68 Ga-FAPI-04 was diluted to an isotonic solution and injected (0.6 nmol, 150-250 μL, 50-100 μCi) into U87MG tumor (FAP+) mice via the tail vein. Mice were anesthetized with 2% isoflurane and fixed in a prone position on a PET scanner (IRIS, Inviscan). PET imaging was performed at 15, 30, 60, 120, and 180 min after injection. At each time point, a 10-minute static PET scan was performed first, followed by a 2-minute CT localization scan. PET / CT images were obtained by reconstructing data using OsiriX software, and MIP images were obtained by analyzing the images using P-MOD (version 3.908) software.

[0083] (2) Intake blocking group: [ 68 Ga]Ga-A1 or [ 68 Ga]Ga-A2 or [ 68Ga-FAPI-04 was diluted to an isotonic solution and injected via the tail vein (0.6 nmol, 150-250 μL, 50-100 μCi, containing 80 nmol FAPI-04) into U87MG tumor (FAP+) mice. PET imaging was performed 60 min later. For each different drug, the same tumor mouse was used in both the uptake group and the uptake blockade group.

[0084] Experimental results

[0085] like Figure 4 As shown, this is an example of embodiment 6 of the present invention. 68 Ga]Ga-A1 and [ 68 PET / CT image of a mouse with a Ga]Ga-A2 U87MG tumor (FAP+). 68 Ga]Ga-A1 and [ 68 Ga]Ga-A2 was rapidly and strongly taken up by U87MG tumors (FAP+), with both showing superior tumor uptake and retention compared to [ 68 Ga]Ga-FAPI-04. After adding the FAP inhibitor, [ 68 Ga]Ga-A1 and [ 68 Tumor uptake of Ga-A2 was blocked, indicating that [ 68 Ga]Ga-A1 and [ 68 The uptake of Ga-A2 in tumors is due to its binding with FAP, i.e. 68 Ga]Ga-A1 and [ 68 Ga]Ga-A2 specifically binds to tumors that highly express FAP.

[0086] Example 7

[0087] [ 68 Ga]Ga-A1 and [ 68 Biodistribution experiment of Ga]Ga-A2

[0088] Experimental steps

[0089] (1) Intake group: [ 68 Ga]Ga-A1 and [ 68 Ga]Ga-A2 was diluted to an isotonic solution, and the drug (0.3 nmol, 150-250 μL, 50-100 μCi) was injected into U87MG tumor (FAP+) mice via the tail vein. Each group of mice was anesthetized and sacrificed at 30, 60 and 120 min, respectively. The mice were dissected, and blood, brain, heart, liver, spleen, lung, kidney, pancreas, flesh, bone, stomach, large intestine, small intestine, gallbladder and tumor were weighed and counted for radioactivity.

[0090] (2) Intake blocking group: [ 68Ga]Ga-A1 and [ 68 Ga]Ga-A2 was diluted to an isotonic solution, and the drug (0.3 nmol, 150-250 μL, 50-100 μCi, containing 80 nmol FAPI-04) was injected into mice via the tail vein. Each group of mice was anesthetized and sacrificed after 60 minutes. The mice were dissected, and blood, brain, heart, liver, spleen, lung, kidney, pancreas, flesh, bone, stomach, large intestine, small intestine, gallbladder, and tumors were weighed and counted for radioactivity.

[0091] The drug uptake by each organ and tissue is calculated as: (radioactivity count of the sampled tissue / 1% of the radioactivity count of the drug injected into each mouse) / weight of the sampled tissue, in %ID / g.

[0092] Experimental results

[0093] As shown in Tables 1 and 2, respectively, in Embodiment 7 of the present invention, […] 68 Ga]Ga-A1 and [ 68 Biodistribution data of Ga-A2 in U87MG tumor (FAP+) mice. 68 Ga]Ga-A1 and [ 68 The biodistribution data of Ga]Ga-A2 matched the PET / CT images, and both specifically bound to FAP+ tumors, with high tumor uptake.

[0094] Table 1

[0095]

[0096] Table 2

[0097]

[0098] The radiolabeled ligand of the fibroblast activation protein-α (FAP) imaging agent of the present invention possesses excellent... 68 Ga labeling properties allow for rapid and efficient radioactive preparation, resulting in highly stable formulations. 68 The Ga molecular probe contains a (S)-N-(2-(2-cyanopyrrolo-1-yl)-2-ethoxy)-6-(3-(-1-piperazin-1-yl)propoxy)quinoline-4-carboxamide group, which exhibits good FAP affinity. 68 Ga]Ga-A1 and [ 68 Ga]Ga-A2 specifically targets FAP, exhibiting high binding affinity for FAP and high uptake and long retention in FAP+ tumors; therefore, the present invention 68 Molecular probes made from radiolabeled ligands of Ga-labeled fibroblast activation protein-α (FAP) targeting molecular probes can serve as positron-emitting molecular probes for tumors.

[0099] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A radiolabeled ligand for a fibroblast activation protein-α imaging agent, comprising compounds A1 and A2, with the following structural formulas: ； 。 2. The method for preparing the radiolabeled ligand of the fibroblast activation protein-α imaging agent as described in claim 1, comprising the following steps: a condensation reaction is performed between the bifunctional linker HBED-CC and the FAP inhibitor FAPi in the presence of a base and a condensing agent, followed by deprotection of the protecting group using an acid to obtain the fibroblast activation protein-α imaging agent; the structures of the bifunctional linker HBED-CC and the FAP inhibitor FAPi are as follows: 。 3. The method of preparing a radiolabeled ligand for fibroblast activation protein-α imaging agent according to claim 1, wherein: ###0001### is added to the reaction mixture. The radiolabeled ligand of the fibroblast activation protein-α imaging agent is compound A1, and its preparation steps are as follows: Compound 1 is dissolved in anhydrous dimethylformamide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, and compound 2 are added sequentially to the mixed solution; after reacting overnight at room temperature, ethyl acetate is added to the mixed solution, and the solution is washed with water and saturated brine; the organic phase is dried with anhydrous magnesium sulfate, filtered, and solid impurities are removed; the organic phase in the filtrate is removed under reduced pressure using a rotary evaporator, and the solution is separated by silica gel column chromatography with dichloromethane / ethanol / ammonia water, the fraction is collected, and the solvent is removed under reduced pressure to obtain a pale yellow oily compound A1; the structural formulas of compound 1 and compound 2 are as follows: Compound 1: ; Compound 2: .

4. The method of preparing a radiolabeled ligand for fibroblast activation protein-α imaging agent according to claim 1, wherein: ###00002### is added to the reaction mixture. The radiolabeled ligand of the fibroblast activation protein-α imaging agent is compound A2, and its preparation steps are as follows: Compound 1 is dissolved in anhydrous dimethylformamide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-hydroxybenzotriazole, and compound 2 are added sequentially to the mixed solution; after reacting overnight at room temperature, ethyl acetate is added to the mixed solution, and the solution is washed with water and saturated brine; the organic phase is dried with anhydrous magnesium sulfate, filtered, and solid impurities are removed; the organic phase in the filtrate is removed under reduced pressure using a rotary evaporator, and the solution is separated by silica gel column chromatography with dichloromethane / ethanol / ammonia water, the fraction is collected, and the solvent is removed under reduced pressure to obtain a pale yellow oily compound A2; the structural formulas of compounds 1 and 2 are as follows: Compound 1: ; Compound 2: .

5. The use of the radiolabeled ligand of fibroblast activation protein-α imaging agent according to claim 1 for the preparation of a Ga-labeled positron drug, said Ga-labeled positron drug having the following structural formulae, respectively: 68 Ga-labeled positron drug, said Ga-labeled positron drug having the following structural formulae, respectively: 68 Ga-labeled positron drug, said Ga-labeled positron drug having the following structural formulae, respectively: 。 6. The use of a radiolabeled ligand for fibroblast activation protein-α imaging agent according to claim 5, wherein the fibroblast activation protein-α is a fibroblast activation protein-α. The 68 Ga-labeled positron emitting drug 68 The labeling method of GaA1 is as follows: (1) Dissolve 1 mg of compound A1 in 1 mL of 0.05 N sodium acetate buffer, then add sodium hydroxide solution to adjust the pH to 5, and obtain a compound A1 solution with a concentration of 1 mg / mL; (2) The germanium gallium generator is eluted with 4 mL of high purity 0.05 N hydrochloric acid solution to obtain a hydrochloric acid solution of [Ga]GaCl3having an activity of 8-10 mCi; 68 Ga]GaCl3. (3) Add 50 μL of 0.5 M sodium acetate buffer to the reaction vessel, then add 30 μL of the compound A1 solution prepared in step (1), mix thoroughly, and then add 500 μL of the solution prepared in step (2). 68 A hydrochloric acid solution of Ga]GaCl3 was prepared, and after shaking, the mixture was reacted at room temperature for 10-30 minutes. After cooling to room temperature, the labeling rate was determined using a semi-preparative high-performance liquid chromatography column. 68 GaA1; The structural formula of compound A1 is as follows: 。 7. The use of a radiolabeled ligand for fibroblast activation protein-α imaging agent according to claim 6, wherein the fibroblast activation protein-α is a fibroblast activation protein-α. In the semi-preparative high performance liquid chromatography column described in step (3), the first mobile phase is 0.1% trifluoroacetic acid aqueous solution, the second mobile phase is acetonitrile, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0~10 min, 100%~0% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

8. The use of a radiolabeled ligand for fibroblast activation protein-α imaging agent according to claim 5, wherein: ###00003### The 68 Ga-labeled positron emitting drug 68 The labeling method of GaA2 is as follows: (1) Dissolve 1 mg of compound A2 in 1 mL of 0.05 N sodium acetate buffer, then add sodium hydroxide solution to adjust the pH to 5, and obtain a compound A2 solution with a concentration of 1 mg / mL; (2) The germanium gallium generator is eluted with 4 mL of high purity 0.05 N hydrochloric acid solution to obtain a hydrochloric acid solution of [Ga]GaCl3having an activity of 8-10 mCi; 68 Ga]GaCl3. (3) Add 50 μL of 0.5 M sodium acetate buffer to the reaction vessel, then add 30 μL of the compound A2 solution prepared in step (1), mix thoroughly, and then add 500 μL of the solution prepared in step (2). 68 A hydrochloric acid solution of Ga]GaCl3 was prepared, and after shaking, the mixture was reacted at room temperature for 10-30 minutes. After cooling to room temperature, the labeling rate was determined by HPLC. 68 GaA2; The structural formula of compound A2 is as follows: 。 9. The use of a radiolabeled ligand for fibroblast activation protein-α imaging agent according to claim 8, characterized in that: In the semi-preparative high performance liquid chromatography column described in step (3), the first mobile phase is 0.1% trifluoroacetic acid aqueous solution, the second mobile phase is acetonitrile, and the gradient elution conditions are: 0 min, 100% of the first mobile phase; 0~10 min, 100%~0% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

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