Carbonic anhydrase ix targeting compound and nuclear medicine probe and application

Abstract

The application belongs to the field of nuclear medicine, and relates to a carbonic anhydrase IX targeting compound, a nuclear medicine probe and application. The carbonic anhydrase IX targeting compound has a structure shown in formula I. The probe obtained by further labeling the compound shown in formula I 68 Ga-T5 has obvious advantages in 1-hour gastric uptake, has a higher tumor / muscle uptake ratio, and has good in-vivo metabolic performance, and is a very potential 68 The Ga-labeled CAIX targeting molecular probe has good clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine and biopharmaceuticals, specifically relating to a carbonic anhydrase IX (CAIX) targeting compound, a nuclear medicine probe prepared from the CAIX targeting compound, and their applications. Background Technology

[0002] Hypoxia is a key characteristic of the solid tumor microenvironment, caused by insufficient oxygen supply due to rapid tumor cell proliferation and abnormal angiogenesis. Hypoxic tumor cells undergo metabolic reprogramming mediated by hypoxia-inducible factor-1α (HIF-1α), enhancing glycolysis and oxidative phosphorylation to maintain energy supply. This process is accompanied by the release of lactate and protons (HbA1c). + The accumulation of acidic metabolites such as carbon dioxide (CO2) leads to acidification of the tumor microenvironment. Tumor cells specifically activate related enzymes and transport proteins to cope with acidification stress and maintain pH homeostasis, among which carbonic anhydrase IX (CAIX) is a key regulatory enzyme.

[0003] CAIX is a transmembrane metalloenzyme that catalyzes the hydration of CO2 to produce HCO3. - and H + CAIX maintains intracellular pH stability in tumor cells and promotes intercellular matrix acidification, thereby enhancing tumor cell migration, invasion, metastasis, and resistance to radiotherapy and chemotherapy. CAIX expression is limited in healthy tissues, primarily in the stomach, gallbladder, and small intestine. Studies have shown that CAIX is highly expressed in various solid tumors, such as renal cell carcinoma (especially clear cell renal cell carcinoma), bladder cancer, and colorectal cancer. Meta-analyses have shown that tumor patients with higher CAIX expression have a higher risk of disease progression and metastasis, suggesting its significant prognostic value. Therefore, developing targeted molecular probes targeting CAIX has important theoretical basis and clinical value.

[0004] Currently reported CAIX-targeted nuclear medicine probes (with 68Represented by Ga-DPI-4452 (MASSIÈRE F, WIEDEMANN N, BORREGO I, et al. Preclinical Characterization of DPI-4452: A68 Ga / 177 Lu Theranostic Ligand for Carbonic Anhydrase IX[J / OL]. Journal of Nuclear Medicine, 2024, 65(5): 761-767. DOI:10.2967 / jnumed.123.266309.), there are still shortcomings, mainly manifested in high gastric uptake and large interference from normal tissue background, which can easily mask lesions in the stomach and adjacent areas, affecting the detection of micrometastases. If used for treatment, it may cause radiation gastritis due to gastric mucosal toxicity, affecting safety. Improving the in vivo distribution of probes, reducing non-specific uptake, and improving targeting specificity by optimizing the linker structure is of great significance for overcoming the current technical bottlenecks. Therefore, developing CAIX-targeting compounds and probes with high target affinity and excellent in vivo metabolic properties can provide more reliable imaging tools for tumor monitoring and patient screening, have potential therapeutic value, and have broad application prospects in the field of precision oncology. Summary of the Invention

[0005] The purpose of this invention is to provide a novel carbonic anhydrase IX targeting compound and a nuclear medicine probe.

[0006] To achieve the above objectives, the present invention provides a carbonic anhydrase IX targeting compound T5, which has the structure shown in Formula I: Formula I.

[0007] A second aspect of the present invention provides the application of the above-mentioned carbonic anhydrase IX targeting compound in the preparation of nuclear medicine probes.

[0008] A third aspect of the present invention provides a nuclear medicine probe, wherein the nuclear medicine probe is a carbonic anhydrase IX targeting compound labeled with a radionuclide.

[0009] According to the present invention, the radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide.

[0010] Furthermore, the diagnostic radionuclide can be... 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 I n、99m Tc, 11 C 123 I, 125 I and 124 At least one of I.

[0011] Furthermore, the therapeutic radionuclide can be... 177 Lu、 125 I, 131 I, 211 At、 111 I n、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0012] The fourth aspect of this invention provides the application of the above-mentioned nuclear medicine probe in the preparation of imaging diagnostic or therapeutic reagents targeting carbonic anhydrase IX.

[0013] The nuclear medicine probe of the present invention 68 Ga-T5 has a significant advantage in gastric uptake within 1 hour, making it particularly suitable for preparing reagents for the diagnosis or treatment of gastric diseases.

[0014] The gastric diseases mentioned therein include both gastric tumors and gastric tumor metastases.

[0015] The gastric tumor includes, but is not limited to, gastric cancer, gastric stromal tumor, or gastric lymphoma; the gastric tumor metastases include, but are not limited to, perigastric lymph node metastases or greater omentum metastases.

[0016] and 68 Compared to Ga-DPI-4452, the nuclear medicine probe of this invention 68 Ga-T5 exhibits significantly reduced gastric uptake after 1 hour, along with a high tumor-to-muscle uptake ratio, good in vivo metabolic performance, and tumor retention, making it a highly promising candidate. 68 Ga-labeled CAIX-targeting molecular probes have promising clinical application prospects, especially suitable for the diagnosis and treatment of gastric diseases.

[0017] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0018] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0019] Figure 1 This is a synthetic route diagram for the CAIX-targeted compound of this invention.

[0020] Figure 2 This is the mass spectrum of the CAIX-targeting compound of this invention.

[0021] Figure 3 For injection 68 PET / CT image of tumor-bearing mice 0.5 h after Ga-T5 treatment.

[0022] Figure 4 For injection 68 PET / CT image of tumor-bearing mice 1 hour after Ga-T5 treatment.

[0023] Figure 5 For injection 68 PET / CT image of tumor-bearing mice 1 hour after Ga-DPI-4452 treatment. Detailed Implementation

[0024] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0025] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] DPI-4452 was commercially obtained (CAS No.: 2941391-49-5) and processed according to the method reported in the literature (DOI:10.2967 / jnumed.123.266309.). 68 Ga marker, obtain 68 Ga-DPI-4452.

[0027] Example 1

[0028] Preparation of CAIX-targeted compound T5

[0029] The specific structure of T5 is shown in Equation I. The synthesis steps are as follows: Figure 1As shown. The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Reaction conditions: (a) DMF solution of 20% piperidine, DMF solution of Fmoc-11-amino-3,6,9-trioxaundecanic acid, HATU, HOBt and DIPEA; (b) DMF solution of 20% piperidine, DMF solution of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester, HATU, HOBt and DIPEA; (c) DMF solution of SnCl2·2H2O; (d) DMF solution of 1,4-sulfonylbutyric acid, HATU, HOBt and DIPEA; 2, trifluoroacetic acid, water and triisopropylsilane; (e) 1,3-di(bromomethyl)benzene, acetonitrile and aqueous solution of NH4HCO3.

[0030] Take a certain mass of resin 1 (0.05 mmol) into a 10 mL solid-phase synthesis tube, add 2 mL of dichloromethane (DCM) to swell, repeat three times, 5 minutes each time, then use... N,NWash three times with dimethylformamide (DMF) for 5 minutes each time. Remove the amino protecting group Fmoc using a DMF solution (v / v) containing 20% ​​piperidine. Specifically, react 2 mL of 20% piperidine DMF solution for 2 minutes, 10 minutes, and 10 minutes, followed by washing 3-5 times with 2 mL DMF for 2 minutes each time. Add 5 times the stoichiometric amount of Fmoc-NH-PEG3-CH2COOH (relative to resin 0.05 mmol) activated with 5 times the stoichiometric amount of HATU in the presence of 10 times the stoichiometric amount of DIPEA to the synthesis tube, and react for 1 hour with electromagnetic stirring. Remove the amino protecting group Fmoc using a DMF solution (v / v) containing 20% ​​piperidine, followed by washing 3-5 times with 2 mL DMF for 2 minutes each time. Relative to the resin (0.05 mmol), 5 times the stoichiometric amount of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester, activated with 5 times the stoichiometric amount of HATU in the presence of 10 times the stoichiometric amount of DIPEA, was added to the synthesis tube and reacted under electromagnetic stirring for 1 hour. The mixture was then washed 3-5 times with 2 mL of DMF, 2 minutes each time. The nitro group attached to the phenylalanine residue was reduced to an amino group using SnCl2·2H2O. Specifically, 1 MS of DMF solution of SnCl2·2H2O (340 mg SnCl2·2H2O in 1.5 mL of DMF) was added to the synthesis tube, and the mixture was reacted overnight under electromagnetic stirring. The mixture was then washed 3-5 times with 2 mL of DMF, 2 minutes each time. Five times the stoichiometric amount of 4-sulfonylbutyric acid (4-sulfonylbutyric acid) relative to the resin (0.05 mmol) was added to the synthesis tube after activation with five times the stoichiometric amount of HATU in the presence of 10 times the stoichiometric amount of DIPEA. The reaction was carried out overnight under electromagnetic stirring for acylation, followed by washing with 2 mL DMF 3–5 times, 2 minutes each time. Ligand dissociation from the resin and removal of Trt were achieved by stirring with 5 mL trifluoroacetic acid / triisopropylsilane / water (95:2.5:2.5, v / v / v) for 2 hours. The resin was washed with 2 mL trifluoroacetic acid, and all filtrates were collected. After removing trifluoroacetic acid under reduced pressure, the crude product was prepared by reverse HPLC. After lyophilization, intermediate 2 was obtained, which was then diluted to 60 mL with acetonitrile / water (1:1, v / v). 1,3-Di(bromomethyl)benzene and ammonium bicarbonate were added, and the mixture was stirred overnight. The crude product was prepared by reverse HPLC and lyophilized to obtain the target compound T5. The ligand structure was identified by mass spectrometry, as shown in the figure. Figure 2 As shown.

[0031] Example 2

[0032] Marking and Quality Control

[0033] mark: Accurately weigh a certain mass of the ligand (compound T5) into the sample, add DMSO (dimethyl sulfoxide) to dissolve it, and dilute the ligand concentration to 2 nmol / μL. Pipette 10 μL of the ligand solution and 130 μL of 1 mol / L NaOAc solution into a vial, and add 2 mL of freshly rinsed solution. 68 Ga 3+ The ionic solution (0.05 mol / L hydrochloric acid solution with a radioactivity of 7-10 mCi) was shaken well, sealed, and reacted at 95°C for 10 minutes. After cooling to room temperature, the reaction solution was purified using a Sep-Pak C18 solid-phase extraction column, and the quality was controlled by HPLC analysis.

[0034] Quality control: 68 The radiochemical purity of Ga-labeled complexes was determined by HPLC (high performance liquid chromatography) with an aqueous solution containing 20% ​​acetonitrile (containing 0.1% TFA) as the mobile phase. The radiochemical purity of all complexes was greater than 95%, and they were used for further study without purification.

[0035] Example 3

[0036] Imaging of labeled products

[0037] Take 0.1 mL of freshly prepared 68 Ga-labeled complexes (5.6 MBq-7.4 MBq) were injected via the tail vein into female mice carrying the CAIX-transfected mouse renal cell carcinoma line Renca (CAIX) (tumor diameter approximately 1 cm). After 0.5 h and 1 h, the mice were anesthetized with isoflurane and subjected to small animal PET / CT (SUPER-NOVA, Ping Sheng Technology, China) imaging. The regions of interest were delineated using standard uptake values ​​(SUVs).

[0038] like Figure 3 As shown in Table 1, after 0.5 hours 68 Ga-T5 complexes showed significant concentrations in the tumor region. The SUVmax ratio of tumor to kidney was 5.971±3.532, the SUVmax ratio of tumor to muscle was 85.472±36.216, and the SUVmax ratio of tumor to liver was 20.058±12.999. Figure 4 As shown in Table 1, after 1 hour 68 Ga-T5 complexes were significantly concentrated in the tumor area. The SUVmax of the stomach was 0.201±0.042, the ratio of the SUVmax of the tumor to that of the kidney was 6.699±4.275, the ratio of the SUVmax of the tumor to that of the muscle was 142.583±132.044, and the ratio of the SUVmax of the tumor to that of the liver was 25.379±17.337.

[0039] like Figure 5 As shown in Table 1, after 1 hour 68 Ga-DPI-4452 was significantly concentrated in the tumor area. The SUVmax of the stomach was 0.737±0.158, the ratio of the SUVmax of the tumor to that of the kidney was 7.706±1.447, the ratio of the SUVmax of the tumor to that of the muscle was 260.884±173.675, and the ratio of the SUVmax of the tumor to that of the liver was 32.050±9.349.

[0040] and 68 Compared to the Ga-DPI-4452, at the 1-hour imaging time point, 68 The radioactive uptake of Ga-T5 in the stomach was only 0.201±0.042, significantly lower than that in the control group. 68 The Ga-DPI-4452 has a value of 0.737 ± 0.158, and, 68 Ga-T5 exhibits high absolute tumor uptake, a high tumor-to-muscle uptake ratio, and good in vivo metabolic performance. Furthermore... 68 The tumor uptake value of Ga-T5 slightly increased from 1.846 from 0.5h to 1h and then stabilized at 1.863, indicating that the probe has good tumor retention properties, providing a wider and more stable imaging time window and higher tolerance for errors in clinical PET scans. Therefore, 68 The Ga-T5 is a very promising... 68 Ga-labeled CAIX-targeting molecular probes have promising clinical application prospects.

[0041] Table 1

[0042] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A carbonic anhydrase IX-targeting compound, characterized in that, The carbonic anhydrase IX targeting compound has the structure shown in Formula I: Equation I.

2. The application of the carbonic anhydrase IX targeting compound according to claim 1 in the preparation of nuclear medicine probes.

3. A nuclear medicine probe, characterized in that, The nuclear medicine probe is a carbonic anhydrase IX-targeting compound of claim 1 labeled with a radionuclide.

4. The nuclear medicine probe according to claim 3, wherein, The radionuclide mentioned is a diagnostic radionuclide or a therapeutic radionuclide.

5. The nuclear medicine probe according to claim 4, wherein, The diagnostic radionuclide is 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I.

6. The nuclear medicine probe according to claim 4, wherein, The therapeutic radionuclide is 177 Lu、 125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

7. The use of the nuclear medicine probe according to any one of claims 3-6 in the preparation of imaging diagnostic reagents or therapeutic reagents targeting carbonic anhydrase IX.

8. The application according to claim 7, wherein, The imaging diagnostic reagent or therapeutic reagent is a reagent used for diagnosing or treating gastric diseases.

9. The application according to claim 8, wherein, The gastric disease referred to is a gastric tumor or a metastatic lesion of a gastric tumor.

10. The application according to claim 9, wherein, The gastric tumor is gastric cancer, gastric stromal tumor, or gastric lymphoma; the gastric tumor metastases are perigastric lymph node metastases or greater omentum metastases.

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