A sort of 99m Tc-labeled radioactive probes targeting fibroblast activation proteins, their preparation methods, and applications.

Abstract

This invention provides a compound 4 or a pharmaceutically acceptable salt thereof that targets fibroblast activation proteins, and also provides the use of compound 4 in the preparation of imaging agents and diagnostic formulations. The radioactive probe provided by this invention [ 99m Tc]4 has the biological characteristic of specifically targeting FAP, and can be used for early diagnosis, pathological research, disease progression, and efficacy evaluation of tumors with high FAP expression.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis technology, specifically relating to novel radionuclide-labeled ligands and their preparation methods, particularly to a method for... 99m Tc-labeled inhibitors targeting fibroblast activation protein (FAP) 4. Radionuclide-labeled ligands, their preparation methods and applications. Background Technology

[0002] Malignant tumors are among the most serious threats to human health. In 2020, my country ranked first globally in both the number of new cases and deaths from malignant tumors. Most patients are diagnosed at middle or late stages, which is a major reason for the heavy burden of cancer treatment and low survival rates in my country. Early screening, diagnosis, and treatment are crucial ways to reduce the burden of cancer. Nuclear medicine imaging techniques can provide patients and doctors with a wealth of information, including early diagnosis, differentiation between benign and malignant tumors, clinical staging, efficacy evaluation, and prognostic monitoring. It also provides molecular-level evidence for tumor research. Currently, in clinical practice, nuclear medicine receptor imaging and metabolic imaging techniques are the most commonly used for tumor screening and diagnosis, primarily through positron emission tomography / computed tomography (PET / CT) and single-photon emission computed tomography (SPECT). While PET / CT has many advantages in tumor diagnosis, its high platform construction cost and expensive single-use fees prevent its widespread adoption. Compared to PET / CT, SPECT has a lower cost, as its radionuclides are generated directly from the radionuclides, eliminating the need for expensive medical cyclotrons and making it easier to promote nationwide. Designing and developing imaging agents that can be visualized on SPECT will be beneficial for early tumor screening and treatment monitoring.

[0003] Fibroblast activation protein (FAP) is a type II transmembrane serine protease that is highly expressed in activated tumor stroma, healing wounds, or inflammation, but is virtually absent in healthy adult organs or tissues. Because FAP is expressed sparingly in normal tissues but highly in tumor stroma, radiopharmaceuticals targeting FAP are considered a promising area for development in tumor diagnosis and treatment. Currently, 68 Ga or 18F-labeled FAP inhibitor (FAPI) analogues have entered clinical use for PET diagnosis of tumors, producing images with a high target-to-background ratio. Numerous clinical studies have confirmed that FAPI-PET / CT offers higher sensitivity than FDG in certain tumors, such as liver cancer, colorectal cancer, and gastric cancer. However, due to the high cost of PET, SPECT is the primary imaging instrument used in many developing and underdeveloped regions. Therefore, it is necessary to develop radiolabeled FAPI drugs for single-photon emission computed tomography (SPECT) imaging.

[0004] Scholars have already used 99m Tc is used to label FAPI analogs for preclinical and clinical studies. Lindner et al. developed a series of... 99m Tc-labeled FAPIs, among which FAPI-34 showed better performance in animal and human imaging, and the imaging results of FAPI-34 in patients with metastatic ovarian and pancreatic cancer showed that SPECT imaging was consistent with that of FAPI-46. Diana et al. used HYNIC as... 99m A novel FAPI radiopharmaceutical was synthesized using a Tc ligand. 99m Tc-HYNIC-D-alanine-boroPro( 99m Tc-iFAP was studied and evaluated in three different solid tumors (breast cancer, lung cancer, and cervical cancer) for its effects on [ 99m Tc]Tc-iFAP uptake, and compared with [ 18 The results were compared with FDG PET / CT scans. The results showed that in both the primary tumor and metastatic lymph nodes, [ 99m Tc-iFAP exhibits good biodistribution and kinetics, enabling the acquisition of high-quality and high-contrast images. The team further investigated its application in 32 patients with different types of tumors. 99m The imaging value of Tc-iFAP. The results show that... 99m Tc-iFAPSPECT imaging has advantages in certain solid tumors, such as breast cancer and peritoneal metastases of recurrent colon cancer. Jia X et al. evaluated [ 99m The potential role of Tc-HFAPi in the clinical analysis of digestive system tumors. This prospective study recruited 40 patients for SPECT imaging and found […]. 99m Tc-HFAPi can successfully detect local recurrence lesions and has a higher diagnostic efficiency for distant metastases than enhanced CT, especially for liver metastases. Therefore, it is evident that... 99m Tc-labeled FAPI drugs have clinical application value, but currently... 99mThere are relatively few Tc-labeled FAPI drugs, so it is necessary to develop more drugs for SPECT imaging to fill the gap in early diagnosis, treatment and monitoring of tumors in economically underdeveloped areas. Summary of the Invention

[0005] This study synthesized a novel labeled prodrug for SPECT imaging, 4, with UAMC1110 as the targeting group and 6-hydrazinonicyl (HYNIC) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM) as the coordinating group, enabling it to be labeled by single-photon diagnostic radionuclides. 99m Tc labeling enables SPECT imaging.

[0006] This invention first provides a compound 4 or a pharmaceutically acceptable salt thereof for use as a target for FAP, having the structural formula shown in Formula I:

[0007]

[0008] The present invention also provides the use of the above-described compound 4 or a pharmaceutically acceptable salt thereof in the preparation of radioactive probes targeting FAP.

[0009] The present invention further provides a radioactive probe targeting FAP, comprising a radiolabeled compound 4 as described above or a pharmaceutically acceptable salt thereof; the radiolabeled compound is selected from... 99m Tc can be used for diagnosis. The radionuclide can also be selected from... 188 Re, used for treatment.

[0010] Another aspect of the present invention provides the above-described targeted FAP 99m The method for preparing Tc-labeled radioactive probes includes:

[0011] 1) Radiolabeling method for compound 4: Dissolve compound 4 as described in claim 1 in dimethyl sulfoxide to obtain a 1 mg / mL solution of compound 4. Add 50 μL of stannous chloride, 0.5 mL of ethylenediamine-N,N-diacetic acid (EDDA), 0.5 mL of tris(hydroxymethyl)methylglycine (Tricine), and 30 μL of the compound 4 solution to a reaction flask, cap the flask to keep it sealed, and add 0.5 mL of Na[ 99m [TcO4], after thorough shaking and mixing, heat at 100℃ for 25 min.

[0012] The stannous chloride solution is prepared by dissolving 1 mg of stannous chloride powder in 1 mL of 0.1 M HCl solution.

[0013] The ethylenediamine-N,N-diacetic acid (EDDA) solution was prepared by dissolving 1 mg of EDDA powder in 1 mL of 0.1 M NaOH solution.

[0014] The tris(hydroxymethyl)methylglycine (Tricine) solution was prepared by dissolving 40 mg of Tricine powder in 1 mL of 0.1 M PBS solution.

[0015] In one embodiment of the invention, it further includes obtaining [ 99m The labeling rate of Tc]4 was then determined by high-performance liquid chromatography (radio-HPLC) with a radiodetector; preferably, the high-performance liquid chromatography (radio-HPLC) with a radiodetector includes:

[0016] The first mobile phase was an aqueous solution of 0.1% trifluoroacetic acid, and the second mobile phase was an acetonitrile solution. The gradient elution conditions were: 0 min, 95% of the first mobile phase; 0-10 min, 95%-0% of the first mobile phase; 10-15 min, 100% of the second mobile phase; and the flow rate of the mobile phase was 1 mL / min.

[0017] The present invention further provides a tumor imaging agent targeting FAP, comprising compound 4 or a pharmaceutically acceptable salt thereof, or the aforementioned radioactive probe targeting FAP.

[0018] Another aspect of the present invention provides the use of the above-described compound 4 or a pharmaceutically acceptable salt thereof, or a radioactive probe targeting FAP as described above, or an imaging agent as described above, in the preparation of formulations for tumor diagnosis.

[0019] The beneficial effects of the above-described technical solution of the present invention are as follows:

[0020] The present invention provides [ 99m The Tc]4 radioactive probe has the biological characteristic of specifically targeting FAP and can be used for early diagnosis, pathological research, disease progression, and efficacy evaluation of tumors with high FAP expression. Attached Figure Description

[0021] Figure 1 The [prepared in Example 2 of this invention] 99m Radioactive HPLC chromatogram of the Tc]4 labeled reaction solution.

[0022] Figure 2 In Embodiment 3 of the present invention [ 99m Figure 1 shows the results of the Tc]4 uptake and inhibition experiments in HT-1080 and HT-1080-FAP cells.

[0023] Figure 3The biological distribution experiment in Example 4 of this invention [ 99m In vivo distribution of Tc]4 in U87MG tumor-bearing mice at 1, 2, 6, and 24 hours and in vivo distribution in the Blocking experiment.

[0024] Figure 4 In embodiment 5 of the invention [ 99m SPECT images of Tc]4 at 1 h and 3 h after injection in U87MG tumor-bearing mice. Detailed Implementation

[0025] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0026] Terms and Definitions

[0027] EDDA, ​​Ethylenediamine-N,N'-diacetic acid.

[0028] Hynic, Hydrazinonicotinic acid

[0029] PET / CT, Positron Emission Tomography / Computed Tomography

[0030] SPECT, Single-Photon Emission Computed Tomography.

[0031] FAP, Fibroblast activation protein

[0032] FAPI, Fibroblast activation protein inhibitor

[0033] Example 1: Synthesis of radiolabeled ligand 4

[0034] Synthesis reaction equation:

[0035]

[0036] Synthesis method:

[0037] Compounds 1 and 2 were synthesized according to the methods described in the references (Synthesis of radiolabeled cytarabine conjugates; Development of Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein).

[0038] Compound 3: Compound 1 (87.5 mg, 0.25 mmol) was dissolved in 5 mL of anhydrous dichloromethane. Compound 2 (100 mg, 0.25 mmol) and triethylamine (37.9 mg, 0.375 mmol) were added sequentially to the mixed solution. After reacting overnight at room temperature, the organic phase in the filtrate was removed under reduced pressure using a rotary evaporator. The filtrate was separated by silica gel column chromatography with ethyl acetate / petroleum ether (v / v, 8 / 2). The target fraction was collected, and the solvent was removed under reduced pressure to give 109 mg of a pale yellow oil.

[0039] Compound 4: Compound 3 (90 mg, 0.138 mmol) was dissolved in 5 mL of trifluoroacetic acid and reacted at room temperature for 3 hours. The organic phase in the filtrate was removed by rotary evaporation under reduced pressure. The filtrate was separated by silica gel column chromatography with dichloromethane / methanol / ammonia (v / v / v, 90 / 9 / 1). The target component was collected and the solvent was removed under reduced pressure to obtain 40.4 mg of a pale yellow oil.

[0040] Example 2 Radiolabeled ligand 4 99m Tc radiolabeling

[0041] (1) Dissolve 1 gram of compound 4 in DMSO to prepare a compound 4 solution with a concentration of 1 μg / μL;

[0042] (2) Add 50 μL of stannous chloride, 0.5 mL of ethylenediamine-N,N-diacetic acid (EDDA), 0.5 mL of tris(hydroxymethyl)methylglycine (Tricine), and 30 μL of Compound 4 solution to the reaction flask. Cap the flask to maintain a sealed state. Add 0.5 mL of Na[[] to the flask using a syringe. 99m [TcO4], after thorough shaking and mixing, heat at 100℃ for 25 min. Analyze the product using high-performance liquid chromatography (HPLC). Figure 1 As shown, radiochemical purity > 99% can be obtained. 99m Tc]4.

[0043]

[0044] Example 3 Cell Uptake and Inhibition Experiment

[0045] (1) HT1080-FAP (FAP positive) and HT-1080 (FAP negative) cells (~1×10⁻⁶) 5 (Cells were seeded in each well of a 24-well plate and incubated for 48 hours. Once cell coverage reached approximately 90%, the culture medium was aspirated, and the cells were washed twice with phosphate-buffered saline (PBS). 2 μC / well of [a specific ingredient / component] was then added to each well. 99m Tc]4 was incubated at 37°C for 10, 30, 60, 120 and 360 minutes. In the inhibition experiment, 100 μM FAP inhibitor UAMC1110 was used to block cell uptake. After incubation, the solution was aspirated and the cells were washed three times with ice-cold phosphate buffered saline to stop cell uptake. All cells were lysed with sodium hydroxide and the lysate was collected for radioactivity counting.

[0046] (2) Figure 2 As shown in the results of the in vitro cell uptake experiment, [ 99m Tc]4 uptake in HT1080-FAP (FAP-positive) cells was higher than in HT1080 (FAP-negative) cells in vitro; the uptake in HT1080-FAP (FAP-positive) cells was blocked after the addition of a fibroblast activating protein inhibitor, indicating that [ 99m Tc]4 specifically binds to fibroblast activation protein.

[0047] Example 4: Biodistribution in tumor-bearing mice

[0048] 1. [ 99m Biodistribution of Tc]4 in tumor-bearing mice

[0049] (1) Establishment of U87-MG xenograft nude mouse model: After one week of acclimatization feeding of 4-5 week old BALB / C-nu / nu nude mice weighing 16-22g, U87-MG cells in the logarithmic growth phase were digested, centrifuged, and resuspended in PBS to prepare 5×10⁻⁶ cells. 7 A cell suspension of 100 μL / mL was prepared by inoculating the U87-MG cell suspension into the right axilla of nude mice.

[0050] (2) Take 20 model mice and randomly divide them into 5 groups of 4 mice each. After marking, […]. 99m After diluting the Tc-4 solution with physiological saline, each model mouse was injected with approximately 40 μCi (1-6 h) and 300 μCi (24 h) via the tail vein. The mice were euthanized by cervical dislocation at 1, 2, 6, and 24 h, and the tissue of interest was collected for radioactivity counting. Blocking experiment: Excess DOTA-FAPI04 (45 μg / mouse) was mixed with […]. 99mAfter mixing the Tc]4 drug solution (approximately 40 μCi), it was injected via the tail vein. Two hours later, the victim was euthanized by cervical dislocation, and the tissue of interest was harvested to measure the radioactivity count.

[0051] (3) The results of the biological distribution experiment show that, for example Figure 3 As shown, [ 99m Tc]4 showed high uptake in U87-MG tumors in nude mice; uptake in U87-MG tumors was significantly reduced in the blocking group.

[0052] Example 5 Imaging of tumor-bearing mice

[0053] 1. SPECT imaging of U87-MG in tumor-bearing mice

[0054] The steps for establishing the U87-MG xenograft nude mouse model are the same as before. Imaging is performed when the tumor in the tumor-bearing nude mouse is approximately 8 mm in diameter. Mice are anesthetized, and 0.1 mL of […] is injected via the tail vein. 99m After 1 hour and 3 hours, the mice were fixed on a scanning plate for SPECT scanning. The SPECT scanning parameters were as follows: low-energy collimator, peak energy of 140 keV, matrix of 256×256, and scanning time of 10 min.

[0055] As can be seen from the U87-MG imaging results, such as Figure 4 As shown, [ 99m Tc]4 showed significant uptake in U87-MG tumors.

[0056] The present invention 99m Tc-labeled inhibitors of fibroblast activating protein (FAP) have excellent... 99m Tc labeling properties, simple and efficient radioactive preparation, and high stability of the resulting formulation. This molecular probe contains the UAMC1110 group, which exhibits good FAP affinity. 99m Tc]4 has a high affinity for FAP and exhibits significant specific uptake by tumors. Therefore, the present invention 99m Molecular probes made from Tc-labeled radioligands that target fibroblast activating protein (FAP) inhibitors can be used as SPECT molecular probes for imaging FAP-overexpressing lesions.

[0057] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound 4 targeting fibroblast activation protein, or a pharmaceutically acceptable salt thereof, characterized in that, Compound 4 has the structural formula shown in Formula I: 。 2. The use of compound 4 as described in claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a radioactive probe targeting FAP.

3. A radioprobe targeting FAP, characterized in that, A compound of claim 1 or a pharmaceutically acceptable salt thereof, comprising a radionuclide selected from the group consisting of 99m Tc.

4. The method for preparing a radioactive probe targeting FAP as described in claim 3, characterized in that, Includes the following steps: 1) Dissolve compound 4 according to claim 1 in dimethyl sulfoxide to obtain a 1 mg / ml solution of compound 4; 2) Add 50 μL of stannous chloride solution, 0.5 mL of ethylenediamine-N,N-diacetic acid (EDDA) solution, 0.5 mL of tris(hydroxymethyl)methylglycine (Tricine) solution, and 30 μL of compound 4 solution to the reaction flask. Cap the flask to ensure a tight seal. Then, use a syringe to add 0.5 mL of Na[ 99m The TcO4 solution was thoroughly shaken and mixed, then heated at 100°C for 25 min. The stannous chloride solution is prepared by dissolving 1 mg of stannous chloride powder in 1 mL of 0.1 M HCl solution. The ethylenediamine-N,N-diacetic acid (EDDA) solution was prepared by dissolving 1 mg of EDDA powder in 1 mL of 0.1 M NaOH solution. The tris(hydroxymethyl)methylglycine (Tricine) solution was prepared by dissolving 40 mg of Tricine powder in 1 mL of 0.1 M PBS solution.

5. The production method according to claim 4, wherein It also includes determining the labeling rate of the radioactive probe using high-performance liquid chromatography (radio-HPLC) with a radiodetector after obtaining the radioactive probe.

6. The production method according to claim 5, wherein Radio-HPLC with a radiodetector includes: The first mobile phase was an aqueous solution of 0.1% trifluoroacetic acid, and the second mobile phase was an acetonitrile solution. The gradient elution conditions were: 0 min, 95% of the first mobile phase; 0-10 min, 95%-0% of the first mobile phase; 10-15 min, 100% of the second mobile phase; and the flow rate of the mobile phase was 1 mL / min.

7. A tumor imaging agent targeting FAP, characterized by, It comprises compound 4 as described in claim 1 or a pharmaceutically acceptable salt thereof, or a radioactive probe targeting FAP as described in claim 3.

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