Carbonic anhydrase IX targeted molecular probe and application thereof

Abstract

The invention relates to a carbonic anhydrase IX targeted molecular probe and application thereof, and belongs to the technical field of nuclear medicine. The invention provides carbonic anhydrase IX targeted molecular probes 18F-SAZ and 18F-DAZ, and the two molecular probes have the following advantages: firstly, the specificity is strong, and the molecular probes can be selectively taken in a CAIX positive tumor part so as to better distinguish carbonic anhydrase IX negative and positive tumors; secondly, the compound can quickly reach a carbonic anhydrase IX positive tumor, a better imaging effect can be obtained within 30 minutes, and the compound has better retention property; and thirdly, the in-vivo metabolism is rapid, the biological half-life period is shorter, the in-vivo clearing time is obviously shorter than that of an antibody molecular probe, and the safety is higher. In conclusion, the two molecular probes can dynamically monitor the change of the level of carbonic anhydrase IX in tumor cells in real time through PET imaging, and have great application prospects in diagnosis of positive tumors of carbonic anhydrase IX.

Description

Technical Field

[0001] The present invention relates to a carbonic anhydrase IX-targeted molecular probe and its application, belonging to the technical field of nuclear medicine. Background Art

[0002] Renal cell carcinoma (referred to as kidney cancer) is a highly invasive malignant tumor, accounting for 80% - 90% of all kidney tumors, and renal clear cell carcinoma (RCCC) is the main type. Most RCCC patients are in the middle or late stage when diagnosed, and 30% of RCCC patients will experience recurrence or metastasis after the initial surgery. Clinically, CT (computed tomography) or MRI (magnetic resonance imaging) is mainly used to examine kidney cancer, but the provided images are not sufficient to determine the subtype and stage of kidney cancer, and it is difficult for spiral CT, which is commonly used for kidney cancer detection, to identify smaller kidney cancer lesions. PET imaging can quickly and non-invasively obtain high-resolution images, differentiate and diagnose the typing and staging of tumors at the molecular level, and thus effectively overcome the defects of CT or MRI in the examination of kidney cancer. 18 18F-FDG is the most commonly used broad-spectrum tumor PET imaging agent in clinical practice at present, but its sensitivity for diagnosing the primary focus of RCCC is only 62%, and it has limited specificity in differentiating between benign and malignant kidney cancers (see the literature "Lou K, Wang J, He H, et al. Value of 68 [68Ga]Ga-NYM046 PET / CT, in comparison with 18 18F-FDG PET / CT, for diagnosis of clear cell renal cell carcinoma [J]. J Nucl Med, 2024, 65(12): 1884 - 1890. DOI: 10.2967 / jnumed.124.267527.") Therefore, in order to improve the early differential diagnosis ability of kidney cancer, it is urgent to develop a PET molecular probe with stronger specificity for kidney cancer.

[0003] Carbonic anhydrase IX is a transmembrane protein that is specifically overexpressed on the surface of hypoxic tumor cells. It plays a crucial role in regulating the pH value inside and outside tumor cells and is closely related to tumor proliferation, invasion, and metastasis. Carbonic anhydrase IX is overexpressed in various hypoxic malignant tumors, including kidney cancer, breast cancer, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, colon cancer, and cervical cancer, while its expression in normal tissues is very limited. Carbonic anhydrase IX also plays a crucial role in mediating tumor progression, invasion, and metastasis. The H produced by carbonic anhydrase IX +It acidifies the extracellular matrix and induces the formation of an immunosuppressive tumor microenvironment, thereby promoting tumor invasion, metastasis, and resistance to various treatments. A large number of clinical and preclinical studies have shown that the overexpression of carbonic anhydrase IX significantly increases the likelihood of tumor metastasis and is positively correlated with chemotherapy resistance and radiotherapy tolerance, thus having an adverse impact on cancer treatment and prognosis. Therefore, there is an urgent clinical need to accurately monitor the expression of carbonic anhydrase IX in cancer patients to guide treatment.

[0004] In recent years, the development of molecular probes for carbonic anhydrase IX-targeted PET imaging has attracted much attention, but it generally faces the problems of low uptake in the tumor site (<5% ID / g) and high background. On the one hand, this is related to the metabolism of the probe in the body, and on the other hand, it is related to the fact that carbonic anhydrase IX in mice is usually expressed in the epithelial cells of the stomach and intestines. To solve this problem, researchers have conducted a lot of explorations. For example, He et al. developed a molecular probe 68 68Ga-LF-4, which showed relatively clear tumor imaging in the OS-RC-2 tumor-bearing mouse model and had a retention time of up to 4 h, but the tumor uptake was not high (<2.5% ID / g) (see the literature "He C, Liu F, Tao J, et al. A carbonic anhydrase IX dual-targeting small-molecule probe for noninvasive imaging of ccRCC[J]. Mol Pharm, 2024, 21(7): 3383-3394. DOI: 10.1021 / acs.molpharmaceut.4c00104.") 18 18F-VM4-037 is one of the few molecular probes that have entered clinical studies. It showed relatively high signal uptake (3.04 ± 1.14)% ID / g in the primary focus of renal cancer, but it was much lower than the normal renal uptake (35.4 ± 12.3)% ID / g (see the literature "Turkbey B, Lindenberg ML, Adler S, et al. PET / CT imaging of renal cell carcinoma with 18 18F-VM4-037: a phase II pilot study[J]. Abdom Radiol(NY), 2016, 41(1): 109-118. DOI: 10.1007 / s00261-015-0599-1.") In addition, Zhang et al. synthesized a molecular probe based on sulfonamide inhibitors 18F-2, PET imaging can clearly show the HT-29 tumor. The tumor uptake reached the highest 1.2% ID / g at 10 min after injection of the probe. The biodistribution results showed that the tumor uptake was (0.41 ± 0.06)% ID / g after 1 h, which was much lower than the accumulation of the probe in the liver and kidney [which were (10.7 ± 0.96) and (13.7 ± 3.96)% ID / g, respectively] (see the literature "Zhang Z, Lau J, Zhang C, et al. Design, synthesis and evaluation of 18 F-labeled cationic carbonic anhydrase IX inhibitors for PET imaging[J]. J Enzyme Inhib Med Chem, 2017, 32(1): 722-730. DOI: 10.1080 / 14756366.2017.1308928.")。Kunal et al. synthesized the molecular probe 18 F-acetazolamide based on acetazolamide. The PET imaging results showed that the uptake of the probe by the tumor was also low (<0.5% ID / g), and the probe mainly accumulated in the kidney, stomach and intestine (see the literature "More KN, Lee JY, Kim DY, et al. Acetazolamide-based 18 F]-PET tracer: in vivo validation of carbonic anhydrase IX as a sole target for imaging of CA-IX expressing hypoxic solid tumors[J]. Bioorg Med Chem Lett, 2018, 28(5): 915-921. DOI: 10.1016 / j.bmcl.2018.01.060.")。Therefore, the research and development of molecular probes for PET imaging targeting carbonic anhydrase IX still need to be continuously explored. SUMMARY OF THE INVENTION

[0005] To solve the above problems, the present invention provides a molecular probe targeting carbonic anhydrase IX (CAIX), and the molecular probe has the following structure:

[0006]

[0007] Or, the molecular probe has the following structure:

[0008]

[0009] Wherein, R1 is R2 is The value of n1 is any integer from 3 to 5, and the values of n2 and n3 are any integers from 0 to 5.

[0010] In an embodiment of the present invention, the molecular probe has the following structure:

[0011]

[0012] Alternatively, the molecular probe has the following structure:

[0013]

[0014] In an embodiment of the present invention, the labeling precursor of the molecular probe has the following structure:

[0015]

[0016] Alternatively, the labeling precursor of the molecular probe has the following structure:

[0017]

[0018] The present invention also provides a method for preparing the above molecular probe, the method comprising: mixing azido-tetraethylene glycol-p-toluenesulfonate (N3-PEG4-OTs) with radioactive 18 F and carrying out a nucleophilic substitution reaction to obtain a compound 18 F-PEG4-N3; mixing the compound SAZ with the compound 18 F-PEG4-N3 and carrying out a cycloaddition reaction to obtain the above molecular probe;

[0019] Alternatively, the method comprises: mixing N3-(polyethylene glycol)4-p-toluenesulfonate with radioactive 18 F and carrying out a nucleophilic substitution reaction to obtain a compound 18 F-PEG4-N3; mixing the compound DAZ with the compound 18 F-PEG4-N3 and carrying out a cycloaddition reaction to obtain the above molecular probe;

[0020] The compound 18 F-PEG4-N3 has the following structure:

[0021]

[0022] The compound SAZ has the following structure:

[0023]

[0024] The compound DAZ has the structure shown below:

[0025]

[0026] In one embodiment of the present invention, the method includes: mixing azido-tetraethylene glycol-p-toluenesulfonate with radioactive 18 F and then performing a nucleophilic substitution reaction to obtain the compound 18 F-PEG4-N3; mixing the compound SAZ, the compound 18 F-PEG4-N3, sodium ascorbate, tris(3-hydroxypropyltriazylmethyl)amine (THPTA) and copper sulfate pentahydrate (CuSO4·5H2O) and then performing a cycloaddition reaction to obtain the above molecular probe;

[0027] Alternatively, the method includes: mixing azido-tetraethylene glycol-p-toluenesulfonate with radioactive 18 F and then performing a nucleophilic substitution reaction to obtain the compound 18 F-PEG4-N3; mixing the compound DAZ, the compound 18 F-PEG4-N3, sodium ascorbate, tris(3-hydroxypropyltriazylmethyl)amine and copper sulfate pentahydrate and then performing a cycloaddition reaction to obtain the above molecular probe.

[0028] In one embodiment of the present invention, the preparation method of the compound SAZ includes: deacetylating acetazolamide to obtain compound A; mixing compound A, 5-hexynoic acid, 1-hydroxybenzotriazole (HOBT), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and N,N-diisopropylethylamine (DIPEA) and then reacting to obtain the compound SAZ;

[0029] The compound A has the structure shown below:

[0030]

[0031] In one embodiment of the present invention, the preparation method of the compound DAZ includes: deacetylating acetazolamide to obtain compound A; mixing compound A, 4-azidobutyric acid, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine for a condensation reaction to obtain compound B; mixing BOC-L-glutamic acid, propargylamine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine for a condensation reaction to obtain compound 2A; mixing compound 2A, compound B, sodium ascorbate, tris(3-hydroxypropyltriazolylmethyl)amine and copper sulfate pentahydrate and then performing a click condensation reaction to obtain compound 2B; performing a deprotection reaction on compound 2B to obtain compound 2C; mixing compound 2C, 5-hexynoic acid, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine for a condensation reaction to obtain compound DAZ;

[0032] Compound B has the structure shown below:

[0033]

[0034] Compound 2A has the structure shown below:

[0035]

[0036] Compound 2B has the structure shown below:

[0037]

[0038] Compound 2C has the structure shown below:

[0039]

[0040] The present invention also provides the application of the above-mentioned molecular probe in the preparation of a carbonic anhydrase IX imaging agent or a tumor imaging agent.

[0041] In one embodiment of the present invention, the tumor is a carbonic anhydrase IX-positive tumor; the carbonic anhydrase IX-positive tumors include breast cancer, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, colon cancer, cervical cancer and / or kidney cancer.

[0042] The present invention also provides a carbonic anhydrase IX imaging agent, and the components of the imaging agent include the above-mentioned molecular probe.

[0043] The present invention also provides a tumor imaging agent, and the components of the imaging agent include the above-mentioned molecular probe.

[0044] In one embodiment of the present invention, the tumor is a carbonic anhydrase IX positive tumor; the carbonic anhydrase IX positive tumors include breast cancer, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, colon cancer, cervical cancer, and / or kidney cancer.

[0045] The technical solution of the present invention has the following advantages:

[0046] The present invention provides carbonic anhydrase IX (CAIX)-targeted molecular probes 18 F-SAZ and 18 F-DAZ, and these two molecular probes have the following advantages:

[0047] First, it has strong specificity and can selectively accumulate at the site of CAIX-positive tumors, so as to better distinguish between carbonic anhydrase IX positive and negative tumors;

[0048] Second, it can reach CAIX-positive tumors quickly, and good imaging effects can be obtained at 30 minutes, and it has good retention;

[0049] Third, its metabolism in vivo is rapid, its biological half-life is shorter, and the clearance time in vivo is significantly shorter than that of antibody-based molecular probes, so it has higher safety.

[0050] In summary, these two molecular probes 18 F-SAZ can dynamically and real-time monitor the changes in the level of carbonic anhydrase IX in tumor cells through PET imaging, and has great application prospects in the diagnosis of carbonic anhydrase IX positive tumors.

[0051] In addition, the precursor synthesis steps of these two molecular probes are simple and the cost is low. Moreover, the radionuclides carried by these two molecular probes are 18 F, which belongs to a radionuclide with a short half-life, can reduce the burden on patients and improve safety, and these two aspects significantly increase the advantages of these two molecular probes in clinical application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 : Radioactive HPLC chromatograms of the reaction product of the first-step labeling 18 F-PEG4-N3 and the reaction product of the second-step labeling 18 F-SAZ.

[0053] Figure 2 : Radioactive HPLC chromatograms of the reaction product of the first-step labeling 18 F-PEG4-N3 and the reaction product of the second-step labeling 18 F-DAZ.

[0054] Figure 3 : Mass spectrum of the cold compound 19 F-SAZ.

[0055] Figure 4 : Cold compound 19 Mass spectrometry of F-DAZ

[0056] Figure 5 : 18 HPLC analysis of the stability of F-SAZ incubated in PBS for 1, 2, and 4 hours

[0057] Figure 6 : 18 HPLC analysis of the stability of F-DAZ incubated in PBS for 1, 2, and 4 hours

[0058] Figure 7 : 18 HPLC analysis of the stability of F-SAZ incubated in mouse serum for 1, 2, and 4 hours

[0059] Figure 8 : 18 HPLC analysis of the stability of F-DAZ incubated in mouse serum for 1, 2, and 4 hours

[0060] Figure 9 : 18 Results of the cellular uptake study of F-SAZ

[0061] Figure 10 : 18 Results of the cellular uptake study of F-DAZ

[0062] Figure 11 : 18 microPET imaging results of F-SAZ in tumor-bearing mice with OS-RC-2 and HCT116

[0063] Figure 12 : 18 Curves of tumor and muscle uptake of F-SAZ in tumor-bearing mice with OS-RC-2 and HCT116

[0064] Figure 13 : 18 Curves of tumor and muscle uptake of F-SAZ in tumor-bearing mice with OS-RC-2 and the blocking group

[0065] Figure 14 : 18 PET imaging experimental results of F-DAZ in tumor-bearing mice with OS-RC-2 Figure 14 In, (a) 18 microPET imaging results of F-DAZ in tumor-bearing mice with OS-RC-2; (b) 18 Curves of tumor and muscle uptake of F-DAZ in tumor-bearing mice with OS-RC-2; (c) 18Ratio of tumor to muscle uptake of F-DAZ in OS-RC-2 tumor-bearing mice.

[0066] Figure 15 : 18 Biodistribution results of F-SAZ in OS-RC-2 tumor-bearing mice.

[0067] Figure 16 : 18 Autoradiography results of F-SAZ in OS-RC-2 tumor-bearing mice.

[0068] Figure 17 : 18 Biodistribution results of F-DAZ in OS-RC-2 tumor-bearing mice.

[0069] Figure 18 : 18 Autoradiography results of F-DAZ in OS-RC-2 tumor-bearing mice.

[0070] Figure 19 : 18 Pharmacokinetic fitting graph of F-SAZ in normal mice.

[0071] Figure 20 : 18 Pharmacokinetic fitting graph of F-DAZ in normal mice. Detailed implementation mode

[0072] The following embodiments are provided to better further understand the present invention, which are not limited to the best implementation mode, do not constitute a limitation to the content and protection scope of the present invention, and any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.

[0073] For those experimental steps or conditions not specified in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0074] Example 1: A molecular probe targeting carbonic anhydrase IX (CAIX) 18 F-SAZ

[0075] This example provides a molecular probe targeting carbonic anhydrase IX (CAIX) 18 F-SAZ, and the molecular probe 18 F-SAZ has the following structure:

[0076]

[0077] Example 2: A method for preparing a carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-SAZ

[0078] This example provides a method for preparing the carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-SAZ described in Example 1, and the specific steps are as follows:

[0079] Step 1: Dissolve acetazolamide (3 g, 0.014 mol) in 3 M hydrochloric acid aqueous solution (23.4 mL), reflux at 110 °C for 3 h to obtain a deacetylated product; neutralize the deacetylated product to pH ~7 with 4 M NaOH aqueous solution first, then extract twice with ethyl acetate, and take the organic phase; wash the organic phase with saturated sodium chloride first, then dry with anhydrous sodium sulfate, and then concentrate by rotary evaporation to obtain compound A (1.06 g, yield 41.5%);

[0080] Compound A has the following structure:

[0081]

[0082] Step 2: Dissolve compound A (30.0 mg, 0.16 mmol), 5-hexynoic acid (24.4 mg, 0.22 mmol), 1-hydroxybenzotriazole (13.5 mg, 0.10 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (60.0 mg, 0.31 mmol) in DMF (N,N-dimethylformamide, 5 mL) to obtain a solution; adjust the pH of the solution with N,N-diisopropylethylamine (55.5 mg, 0.43 mmol), and react at room temperature (25 °C) for 6 h to obtain a reaction product; extract the reaction product twice with ethyl acetate, and take the organic phase; wash the organic phase with saturated sodium chloride first, then dry with anhydrous sodium sulfate, and then concentrate by rotary evaporation, and finally perform column chromatography to obtain compound SAZ (25.0 mg, yield 54.7%);

[0083] Compound SAZ has the following structure:

[0084]

[0085] Step 3: After the 18 F target water prepared by the cyclotron is loaded onto a QMA column, eluted with K2.2.2 and dried under nitrogen at 100 °C to obtain a reaction tube containing radioactive 18 F; in the reaction tube containing radioactive 18After adding azido-tetraethylene glycol-p-toluenesulfonate (2 mg, 0.0054 mmol) dissolved in DMSO (dimethyl sulfoxide, 0.7 mL) to the reaction tube of F, it was co-incubated at 100 °C for 15 min to obtain a reaction product; after the reaction product was cooled to room temperature (25 °C), the reaction product was purified by semi-preparative HPLC to obtain 18 F-PEG4-N3 (the purification conditions of semi-preparative HPLC are shown in Table 1); the product was analyzed by radio-HPLC, and the analysis results are shown in Figure 1 ;

[0086] The compound 18 F-PEG4-N3 has the following structure:

[0087]

[0088] Step four: Hang 18 F-PEG4-N3 on a C18 column, and then elute it with 1 mL of N,N-dimethylformamide into the reaction flask to obtain 18 F-PEG4-N3 eluate (~1.1 MBq); in 18 After adding compound SAZ (1.5 mg, 0.0055 mmol), sodium ascorbate (0.84 mg, 0.0044 mmol), tris(3-hydroxypropyltriazylmethyl)amine (1.91 mg, 0.0044 mmol) and copper sulfate pentahydrate (0.55 mg, 0.0022 mmol) previously dissolved in 400 μL of water to the F-PEG4-N3 eluate, first bubble with nitrogen, and then react at 45 °C for 45 min to obtain a reaction product; the reaction product was purified by semi-preparative HPLC to obtain a molecular probe targeting carbonic anhydrase IX (CAIX) 18 F-SAZ (the purification conditions of semi-preparative HPLC are shown in Table 1); the product was analyzed by radio-HPLC, and the analysis results are shown in Figure 1 。By calculation, it can be known that 18 The radiochemical purity of F-SAZ is greater than 99%, the yield is about 5.6% after decay correction, and the specific activity is about 7.90 MBq / mmol.

[0089] Table 1 Purification conditions of semi-preparative HPLC

[0090]

[0091] Example 3: A molecular probe targeting carbonic anhydrase IX (CAIX) 18 F-DAZ

[0092] This example provides a molecular probe targeting carbonic anhydrase IX (CAIX) 18F-DAZ, the molecular probe 18 F-DAZ has the structure shown below:

[0093]

[0094] Example 4: A method for preparing a carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-DAZ

[0095] This example provides a method for preparing the carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-DAZ as described in Example 1, and the specific steps are as follows:

[0096] Step 1: First, place 4-azidobutyric acid (400 mg, 2.89 nmol) in a 50 mL round-bottom flask, set up a condenser reflux device, then under N2 protection, inject thionyl chloride (15 mL) into the condenser tube with a syringe, and then under N2 protection, reflux at 70 °C for 1 h (phenomenon: bubbles are generated) to obtain a reflux product; first concentrate the reflux product by rotary evaporation, then redissolve it in dichloromethane to the original volume, and then remove the excess thionyl chloride by rotary evaporation to obtain an oily liquid; under ice bath conditions, add compound A (175 mg, 0.93 nmol) dissolved in 3 mL of N,N-dimethylformamide dropwise to the oily liquid, and then react at room temperature (25 °C) for 2.5 h to obtain a reaction product; after the reaction is monitored by TLC to be completed (the developing agent used for TLC monitoring is a mixed liquid of chloroform and methanol, where the volume ratio of chloroform to methanol is 4:1), extract the reaction product twice with ethyl acetate, and take the organic phase; wash the organic phase with saturated sodium chloride first, then dry it with anhydrous sodium sulfate, then concentrate it by rotary evaporation, and finally perform column chromatography to obtain compound B (180 mg, with a yield of 67%).

[0097] Step 2: Dissolve BOC-L-glutamic acid (300 mg, 1.22 nmol) in 5 mL of N,N-dimethylformamide to obtain a solution. Sequentially add propargylamine (233 μL, 3.66 nmol), 1-hydroxybenzotriazole (98.82 mg, 0.732 nmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (467.75 mg, 2.44 nmol), and N,N-diisopropylethylamine (630.69 mg, 4.88 nmol) to the solution, and then react overnight (16 h) at room temperature to obtain a reaction product. After monitoring the completion of the reaction by TLC (the developing agent used for TLC monitoring is a mixed liquid of chloroform and methanol, where the volume ratio of chloroform to methanol is 10:1), extract the reaction product twice with ethyl acetate, and take the organic phase. Wash the organic phase with saturated sodium chloride first, then dry it with anhydrous sodium sulfate, concentrate it by rotary evaporation, and finally perform column chromatography to obtain compound 2A (240 mg, yield 62%).

[0098] Step 3: Dissolve compound B (20 mg, 0.09 nmol) and compound 2A (12.2 mg, 0.04 nmol) in 1.6 mL of N,N-dimethylformamide to obtain a solution. Add sodium ascorbate (11.3 mg, 0.053 nmol) dissolved in 400 μL of water, tris(3-hydroxypropyltriazylmethyl)amine (8.28 mg, 0.019 nmol), and copper sulfate pentahydrate (13.3 mg, 0.053 nmol) to the solution, and then react at a constant temperature of 45 °C for 1.5 h under N2 protection to obtain a reaction product containing compound 2B. Add a mixed solution of trifluoroacetic acid and dichloromethane (TFA:DCM = 1:1, 4 mL) to the reaction product containing compound 2B, and then perform a deprotection reaction at room temperature for 1 h to obtain a reaction product. Purify the reaction product by semi-preparative HPLC (the purification conditions of semi-preparative HPLC are shown in Table 1) to obtain compound 2C (9.1 mg, yield 30%);

[0099] Step 4: Mix compound 2C (8.0 mg, 0.01 nmol), 5-hexynoic acid (1.34 mg, 0.012 nmol), 1-hydroxybenzotriazole (0.81 mg, 0.059 nmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (5.88 mg, 0.297 nmol), and N,N-diisopropylethylamine (10.28 mg, 0.792 nmol), and then react at 25 °C for 6 h to obtain a reaction product. Purify the reaction product by semi-preparative HPLC (the purification conditions of semi-preparative HPLC are shown in Table 1) to obtain compound DAZ (6.34 mg, yield 71%);

[0100] Compound DAZ has the following structure:

[0101]

[0102] Step 5: Hang 18 F-PEG4-N3 on the C18 column, and then elute it with 1 mL of DMF into the reaction flask to obtain 18 F-PEG4-N3 eluent (~1.1 MBq); Add compound DAZ (1.5 mg, 0.0017 mmol), sodium ascorbate (0.26 mg, 0.0013 mmol), tris(3-hydroxypropyltriazylmethyl)amine (0.56 mg, 0.0013 mmol), and copper sulfate pentahydrate (0.17 mg, 0.0006 mmol) which were previously dissolved in 400 μL of water into the 18 F-PEG4-N3 eluent. First, bubble with nitrogen, and then react at 45 °C for 45 min to obtain the reaction product; Purify the reaction product by semi-preparative HPLC to obtain the carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-DAZ (the purification conditions of semi-preparative HPLC are shown in Table 1); Analyze the product by radio-HPLC, and the analysis results are shown in Figure 2 . It can be calculated that 18 The radiochemical purity of F-DAZ is greater than 99%, the yield is about 7.2% after decay correction, and the specific activity is about 8.50 MBq / mmol.

[0103] Example 5: 18 Cold compound of F-SAZ 19 F-SAZ

[0104] This example provides 18 Cold compound of F-SAZ 19 F-SAZ, and the cold compound 19 F-SAZ has the following structure:

[0105]

[0106] Example 6: A method for preparing 18 Cold compound of F-SAZ 19 F-SAZ

[0107] This example provides a method for preparing the 18 Cold compound of F-SAZ 19 F-SAZ described in Example 1, and the specific steps are as follows:

[0108] Step 1: N3-PEG4-OTS (50.0 mg, 0.18 mmol) was mixed with tetrabutylammonium fluoride (94.0 mg, 0.36 mmol) and co-incubated at 100 °C for 15 min to obtain a reaction product. The reaction product was extracted with ethyl acetate twice for the aqueous phase, and the organic phase was then washed with saturated sodium chloride. The organic phase was taken, dried over anhydrous sodium sulfate first, and then concentrated by rotary evaporation to obtain 19 F-PEG4-N3 (32.2 mg, yield 81.0%).

[0109] Step 2: 19 F-PEG4-N3 (5.0 mg, 0.023 mmol) and compound SAZ (7.0 mg, 0.0253 mmol) were dissolved in 2 mL of N,N-dimethylformamide to obtain a solution. Sodium ascorbate (6.8 mg, 0.0345 mmol), tris(3-hydroxypropyltriazylmethyl)amine (5.0 mg, 0.0115 mmol), and copper sulfate pentahydrate (2.9 mg, 0.0115 mmol) previously dissolved in 500 μL of water were added to the solution, and the reaction was carried out at 45 °C for 45 min to obtain a reaction product. The reaction product was extracted twice with ethyl acetate, and the organic phase was taken. The organic phase was washed with saturated sodium chloride first, then dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and finally purified by semi-preparative HPLC to obtain 18 The cold compound of F-SAZ 19 F-SAZ (3.8 mg, yield 60.0%) (the purification conditions of semi-preparative HPLC are shown in Table 1); the product was analyzed by mass spectrometry, and the analysis results are shown in Figure 3 , 496.40 is the molecular ion peak of [M+Na] + of.

[0110] Example 7: 18 The cold compound of F-DAZ 19 F-DAZ

[0111] This example provides 18 The cold compound of F-DAZ 19 F-DAZ, and the cold compound 19 F-DAZ has the following structure:

[0112]

[0113] Example 8: A method for preparing 18 The cold compound of F-DAZ 19 F-DAZ

[0114] This example provides a preparation of the 18 The cold compound of F-DAZ 19The method of F-DAZ is as follows:

[0115] Dissolve 19 F-PEG4-N3 (4.4 mg, 0.023 mmol) and compound DAZ (15.0 mg, 0.0253 mmol) in 2 mL of N,N-dimethylformamide to obtain a solution; add sodium ascorbate (2.6 mg, 0.0345 mmol), tris(3-hydroxypropyltriazylmethyl)amine (2.9 mg, 0.0115 mmol), and copper(II) sulfate pentahydrate (3.34 mg, 0.0115 mmol) that have been previously dissolved in 500 μL of water to the solution, and react at 45 °C for 45 min to obtain a reaction product; extract the reaction product twice with ethyl acetate and take the organic phase; wash the organic phase with saturated sodium chloride first, then dry it with anhydrous sodium sulfate, concentrate it by rotary evaporation, and finally purify it using semi-preparative HPLC to obtain 18 the cold compound of F-DAZ 19 F-DAZ (7.48 mg, yield 40.1%) (the purification conditions of semi-preparative HPLC are shown in Table 1); analyze the product by mass spectrometry, and the analysis results are shown in Figure 4 , 1119.60 is the molecular ion peak of [M+H] + , and 560.40 is the molecular ion peak of [(M + 2H) / 2] + of the molecular ion peak.

[0116] Experimental Example 1: In vitro stability experiment of a molecular probe targeting carbonic anhydrase IX (CAIX)

[0117] This experimental example provides an in vitro stability experiment of a molecular probe targeting carbonic anhydrase IX (CAIX), and the specific process is as follows:

[0118] Experiment 1: Mix the molecular probe 18 F-SAZ or 18 F-DAZ with PBS buffer (pH = 7.4, 0.01 M) at a volume ratio of 1:9 to obtain a mixture; incubate the mixture at 37 °C for 1, 2, or 4 h; after the incubation ends, take the incubation solution and perform radioactive HPLC analysis using a GabiNova radioactive detector, and the analysis results are shown in Figures 5 - 6 .

[0119] Experiment 2: Mix the molecular probe 18 F-SAZ or 18F-DAZ was mixed with mouse serum (purchased from Nanjing Senbeijia Biotechnology Co., Ltd.) at a volume ratio of 1:9 to obtain a mixed solution; the mixed solution was incubated at 37 °C for 1, 2, 3, or 4 h; after the incubation ended, 20 μL of the incubation solution was taken, an equal volume of acetonitrile was added, and the mixture was centrifuged at 12,000 r / min for 5 min to separate the serum from the protein; after centrifugation, the supernatant was aspirated and analyzed by radioactive HPLC using a Gabi Nova radioactive detector. The analysis results are shown in Figures 7 - 8 .

[0120] As can be seen from Figures 5 - 8 , the molecular probe targeting carbonic anhydrase IX 18 F-SAZ or 18 F-DAZ had a main peak ratio greater than 95% in the HPLC chromatogram after incubation in mouse serum and PBS at 37 °C for 1-4 h, indicating good in vitro stability.

[0121] Experimental Example 2: Experiment on the lipophilic-hydrophilic partition coefficient of the molecular probe targeting carbonic anhydrase IX (CAIX)

[0122] This experimental example provided an experiment on the lipophilic-hydrophilic partition coefficient of the molecular probe targeting carbonic anhydrase IX (CAIX). The specific procedure was as follows:

[0123] Take a centrifuge tube, first add 1 mL of deionized water and 1 mL of n-octanol, and then add the molecular probe targeting carbonic anhydrase IX (CAIX) prepared in Example 2 18 F-SAZ (30 μCi) to obtain a mixed solution; after shaking the mixed solution for 5 min, it was centrifuged at 6000 r / min for 5 min to break the emulsion and separate the two phases; 500 μL of the n-octanol phase and the aqueous phase were taken into a radioimmunoassay tube, and then the radioactivity of the n-octanol phase and the aqueous phase was detected using a γ counter and LogP was calculated (Log P = LogC o / C w ), where C o represents the radioactive dose of 18 F-SAZ in the n-octanol phase, and C w represents the radioactive dose of 18 F-SAZ in the aqueous phase. After the first test, 500 μL of n-octanol and 500 μL of water were added back to the original centrifuge tube, and it was shaken, centrifuged, sampled, and Log P was measured again; the experiment was repeated multiple times until three consecutive LogP values were close, and the average value of the three groups of data was taken as the lipophilic-hydrophilic partition coefficient value, and the result was expressed as the average value ± standard deviation. The lipophilic-hydrophilic partition coefficient of the molecular probe targeting carbonic anhydrase IX prepared in Example 4 18 F-DAZ was detected using the same method.

[0124] The experimentally measured molecular probe targeting CAIX18 F-SAZ and 18 The lipophilic-hydrophilic partition coefficients of F-SAZ and F-DAZ are -0.38±0.08 and -1.83±0.03 respectively, indicating that both are water-soluble and have the advantages of excretion from the kidneys and reduced liver metabolism in terms of metabolism.

[0125] Experimental Example 3: Cellular Uptake Experiment of a Carbonic Anhydrase IX (CAIX)-Targeted Molecular Probe

[0126] This experimental example provides a cellular uptake experiment of a carbonic anhydrase IX (CAIX)-targeted molecular probe, and the specific process is as follows:

[0127] The human renal cancer cell line OS-RC-2 (positive expression of carbonic anhydrase IX) and the human colon cancer cell line HCT116 (negative expression of carbonic anhydrase IX) were both purchased from the Cell Bank of the Chinese Academy of Sciences. OS-RC-2 cells were cultured in RPMI-1640 medium containing 1% (v / v) penicillin-streptomycin in a cell culture incubator at 37°C with 5% (v / v) CO2, and HCT116 cells were cultured in DMEM medium containing 1% (v / v) penicillin-streptomycin in a cell culture incubator at 37°C with 5% (v / v) CO2.

[0128] Set up the OS-RC-2 group, the OS-RC-2 blocking group, and the HCT116 group, with one radioimmunoassay tube in each group. The corresponding cells were added to the three groups of radioimmunoassay tubes at an addition amount of 1.0×10 6 cells per tube. After the cell addition, the cells in the OS-RC-2 blocking group were pre-incubated with cold compound 19 F-SAZ (2 μg) in a water bath at 37°C for 30 min, and then 37 kBq of the carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-SAZ prepared in Example 2 was added to each tube, and they were incubated in a water bath at 37°C for 30, 60, and 120 min respectively. After the incubation, 500 μL of cold (4°C) PBS buffer (pH = 7.4, 0.01 M) was added to each tube, and centrifuged at 4000 r / min for 5 min. After centrifugation, the above operation was repeated once, and the CPM value of the sample was measured using a gamma counter, and the percentage of cellular radioactivity (percentage of radioactivity, %AD) was calculated. The calculation results are shown in Figure 9 , where the result of the percentage of cellular radioactivity is expressed as the ratio of the CPM in the cells to the CPM of the total dose. The cellular uptake of the carbonic anhydrase IX-targeted molecular probe 18 F-DAZ prepared in Example 4 was detected using the same method, and the calculation results are shown in Figure 10 .

[0129] As Figure 9Shown is a molecular probe targeting carbonic anhydrase IX 18 F-SAZ can be rapidly taken up by carbonic anhydrase IX-positive cells OS-RC-2. The cellular uptake at 30 min was (1.47 ± 0.24)% AD, reached the maximum of (1.78 ± 0.03)% AD at 60 min, and decreased to (1.39 ± 0.15)% AD at 120 min. While the uptake of the probe by carbonic anhydrase IX-negative cells HCT116 was relatively low, with the cellular uptake only being (0.50 ± 0.05)% AD at 30 min, showing a statistically significant difference from OS-RC-2 cells. When pre-blocked with cold compound 19 F-SAZ, the uptake of the probe by OS-RC-2 cells was significantly inhibited, being only (0.60 ± 0.07)% AD at 30 min. The above results indicate that the target probe 18 F-SAZ can specifically target cells with high expression of carbonic anhydrase IX.

[0130] As Figure 10 Shown is a molecular probe targeting carbonic anhydrase IX 18 F-DAZ can be rapidly taken up by carbonic anhydrase IX-positive cells OS-RC-2. The cellular uptake at 30 min was (2.24 ± 0.07)% AD, reached the maximum of (2.52 ± 0.20)% AD at 60 min, and decreased to (1.77 ± 0.145)% AD at 120 min. While the uptake of the probe by carbonic anhydrase IX-negative cells HCT116 was relatively low, with the cellular uptake only being (0.67 ± 0.15)% AD at 30 min, showing a statistically significant difference from OS-RC-2 cells. When pre-blocked with cold compound 19 F-DAZ, the uptake of the probe by OS-RC-2 cells was significantly inhibited, being only (0.94 ± 0.12)% AD at 30 min. The above results indicate that the target probe 18 F-DAZ can specifically target cells with high expression of carbonic anhydrase IX.

[0131] Experimental Example 4: Mouse PET imaging experiment of a molecular probe targeting carbonic anhydrase IX (CAIX)

[0132] This experimental example provides a mouse PET imaging experiment of a molecular probe targeting carbonic anhydrase IX (CAIX), and the specific process is as follows:

[0133] Female BALB / c nude mice, 4 - 6 weeks old (~20 mg), were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. Fifteen nude mice were raised in a specific pathogen-free environment for 1 week and then inoculated with OS-RC-2 or HCT116 cells (4×10 6 cells per mouse) under the right axilla of the nude mice. Wait until the tumor grows to about 200 mm 3Perform imaging experiments.

[0134] Set up the OS-RC-2 group, the OS-RC-2 blocking group, and the HCT116 group, with three tumor-bearing nude mice in each group. After grouping, inject the cold compound 19 F-SAZ (injection dose 20 mg / kg, solvent is 100 μL of normal saline) into the tail vein of the tumor-bearing nude mice in the blocking group 30 min in advance, and then inject the carbonic anhydrase IX (CAIX)-targeted molecular probe prepared in Example 2 18 F-SAZ (injection dose ~4.5 MBq, solvent is 100 μL of normal saline) into the tail vein of the tumor-bearing nude mice in each group; perform static PET scans for 10 min at 30, 60, 90, and 120 min after injection, collect images and perform quantitative analysis. The PET imaging results are shown in Figure 11 ; use the region of interest (ROI) technique in the ASIPRO software to outline and analyze the distribution of the probe in the tumor site and other organ tissues. The analysis results are shown in Figures 12 - 13 , where the uptake values of the molecular probe in various tissues in vivo are expressed as %ID / g (percentage of injection dose per gram). Use the same method to perform the mouse PET imaging experiment of the carbonic anhydrase IX-targeted molecular probe 18 F-DAZ prepared in Example 4. The experimental results are shown in Figure 14 .

[0135] As Figures 11 - 13 shown, from 30 min to 120 min after injection, it can be observed that 18 F-SAZ has obvious tumor uptake in OS-RC-2 tumor-bearing mice. The tumor uptake reaches the maximum value of (2.92 ± 0.07)%ID / g at 30 min, and then slowly decreases, dropping to (1.99 ± 0.06)%ID / g at 120 min; while 18 the maximum uptake of F-SAZ in the tumors of HCT116 tumor-bearing mice with negative carbonic anhydrase IX expression is only (1.12 ± 0.07)%ID / g; after 19 F-SAZ blockade, the uptake of the probe by the tumors of OS-RC-2 tumor-bearing mice is significantly inhibited, with a maximum of only (1.36 ± 0.02)%ID / g; 18 the uptake of F-SAZ in the muscles of OS-RC-2 tumor-bearing mice remains low, with a maximum uptake of only (1.34 ± 0.11)%ID / g; the tumor / muscle uptake ratio reaches the maximum of (2.79 ± 0.08) at 30 min, which is significantly higher than that of the HCT116 group (1.57 ± 0.23) and the blocking group (1.37 ± 0.29) at the same time point. Figures 11 - 13 The results of 18F-SAZ has excellent specificity and can selectively accumulate at the sites of carbonic anhydrase IX-positive tumors, thus better distinguishing carbonic anhydrase IX-positive and -negative tumors. At the same time, 18 F-SAZ can reach carbonic anhydrase IX-positive tumors relatively quickly and achieve good imaging effects at 30 min, and it has good retention. In addition, the probe 18 F-SAZ did not show obvious uptake in the bone marrow of tumor-bearing nude mice, indicating that the probe 18 F-SAZ did not undergo in vivo defluorination and had excellent in vivo stability.

[0136] As Figure 14 shown, from 30 min to 120 min after injection, obvious tumor uptake of 18 F-DAZ was observed in OS-RC-2 tumor-bearing mice. The tumor uptake reached the maximum value of (3.29 ± 0.16)% ID / g at 30 min, and then decreased slowly, dropping to (1.82 ± 0.01)% ID / g at 120 min. And, 18 the uptake of F-DAZ in the muscle of OS-RC-2 tumor-bearing mice remained low continuously, with the maximum uptake being only (0.57 ± 0.05)% ID / g. In addition, 18 the tumor / muscle uptake ratio of F-DAZ in OS-RC-2 tumor-bearing mice reached the maximum of (5.75 ± 0.36) at 30 min.

[0137] Experimental Example 5: Mouse biodistribution experiment of a carbonic anhydrase IX (CAIX)-targeted molecular probe

[0138] This experimental example provides a mouse biodistribution experiment of a carbonic anhydrase IX (CAIX)-targeted molecular probe. The specific process is as follows:

[0139] Refer to the method in Experimental Example 4 to prepare OS-RC-2 tumor-bearing nude mice. Take OS-RC-2 tumor-bearing nude mice (3 mice) and inject the carbonic anhydrase IX (CAIX)-targeted molecular probe 18 F-SAZ prepared in Example 2 via the tail vein (injection dose ~7.4 MBq, solvent is 100 μL normal saline); 1 h after injection, sacrifice the tumor-bearing nude mice, take an equal volume of solution as a reference, collect each tissue of the tumor-bearing nude mice respectively and weigh them, perform γ counting and calculate the percentage of injected dose per gram of tissue (%ID / g) to obtain the biodistribution of the probe in OS-RC-2 tumor-bearing mice at 1 h. Make frozen sections with a thickness of 30 μm from the tumor and muscle tissues, and perform quantitative analysis after exposure by a phosphor imaging system. Use the same method to conduct the mouse biodistribution experiment of the carbonic anhydrase IX-targeted molecular probe 18 F-DAZ prepared in Example 4.

[0140] 18 The results of F-SAZ are as follows Figures 15 - 16 shown. Overall, the biodistribution results are consistent with the PET imaging results. In OS-RC-2 tumor-bearing nude mice, the uptake of the probe 18 F-SAZ by the tumor was 5.59 ± 0.65% ID / g, which was significantly higher than that of muscle (1.60 ± 0.41% ID / g). Figure 12 The results also showed that the radioactivity in the OS-RC-2 tumor tissue (14.54 ± 0.20×10 4 DLU / mm2) was significantly higher than that of muscle (7.67 ± 0.34×10 4 DLU / mm 2 ). Figures 15 - 16 The results further confirmed that 18 F-SAZ has excellent specificity and can selectively accumulate in carbonic anhydrase IX-positive tumor sites, enabling better discrimination between carbonic anhydrase IX-positive and -negative tumors.

[0141] 18 The results of F-DAZ are as follows Figures 17 - 18 shown. Overall, the biodistribution results are consistent with the PET imaging results. In OS-RC-2 tumor-bearing nude mice, the uptake of the probe 18 F-DAZ by the tumor was 7.32 ± 0.88% ID / g, which was significantly higher than that of muscle (1.31 ± 0.38% ID / g). Figure 12 The results also showed that the radioactivity in the OS-RC-2 tumor tissue (16.57 ± 0.25×10 4 DLU / mm2) was significantly higher than that of muscle (7.16 ± 0.26×10 4 DLU / mm 2 ). Figures 17 - 18 The results further confirmed that 18 [[ID=3,9]]F-DAZ has excellent specificity and can selectively accumulate in carbonic anhydrase IX-positive tumor sites, enabling better discrimination between carbonic anhydrase IX-positive and -negative tumors.

[0142] Experimental Example 6: Pharmacokinetic experiment of carbonic anhydrase IX-targeted molecular probe

[0143] This experimental example provides a pharmacokinetic experiment of a carbonic anhydrase IX-targeted molecular probe, and the specific process is as follows:

[0144] Take white rats (n = 3, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) and inject the carbonic anhydrase IX (CAIX)-targeted molecular probe prepared in Example 2 through the tail vein 18F-SAZ (injection dose ~ 6 MBq, solvent is 100 μL of normal saline), and blood samples were taken from the tail vein and weighed at 1, 3, 5, 7, 10, 15, 20, 30, 45, 60, 90, and 120 min respectively. The radioactivity content in the blood was measured using a γ counter, and decay correction was performed. The experimental results were expressed as the percentage of radioactivity dose per gram of blood sample (%ID / g), and pharmacokinetic fitting was performed on the data using DAS 2.1 software. The same method was used for the carbonic anhydrase IX-targeted molecular probe prepared in Example 4 18 Pharmacokinetic experiment of F-DAZ.

[0145] Pharmacokinetics mainly studies the law of the dynamic change of drug blood concentration in vivo over time. 18 The results of F-SAZ are as Figure 19 shown, 18 The metabolic process of F-SAZ in vivo conforms to the two-compartment model, and the elimination half-life is 44.98 min. 18 The results of F-DAZ are as Figure 20 shown, 18 The metabolic process of F-DAZ in vivo conforms to the two-compartment model, and the elimination half-life is 40.05 min.

[0146] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A carbonic anhydrase IX-targeted molecular probe, characterized in that, The molecular probe has the structure shown below: Alternatively, the molecular probe has the structure shown below: In the formula, R1 is R2 is The value of n1 is any integer from 3 to 5, and the values of n2 and n3 are any integers from 0 to 5.

2. The molecular probe according to claim 1, wherein The molecular probe has the structure shown below: Alternatively, the molecular probe has the structure shown below:

3. The molecular probe according to claim 1 or 2, characterized in that, The labeling precursor of the molecular probe has the structure shown below: Alternatively, the labeling precursor of the molecular probe has the structure shown below:

4. A method for preparing the molecular probe according to any one of claims 1 to 3, characterized in that, The method includes: After mixing azido-tetraethylene glycol-p-toluenesulfonate with radioactive 18 F, a nucleophilic substitution reaction is carried out to obtain compound 18 F-PEG4-N3; After mixing compound SAZ with compound 18 F-PEG4-N3, a cycloaddition reaction is carried out to obtain the molecular probe described in claim 1; Alternatively, the method includes: mixing N3-(polyethylene glycol)4-p-toluenesulfonate with radioactive 18 F and carrying out a nucleophilic substitution reaction to obtain compound 18 F-PEG4-N3; mixing compound DAZ with compound 18 F-PEG4-N3 and carrying out a cycloaddition reaction to obtain the molecular probe described in claim 1; The compound 18 F-PEG4-N3 has the structure shown below: The compound SAZ has the structure shown below: The compound DAZ has the structure shown below:

5. The method according to claim 4, characterized in that, The method includes: mixing azido-tetraethylene glycol-p-toluenesulfonate with radioactive 18 F and carrying out a nucleophilic substitution reaction to obtain compound 18 F-PEG4-N3; mixing compound SAZ, compound 18 F-PEG4-N3, sodium ascorbate, tris(3-hydroxypropyltriazylmethyl)amine and copper sulfate pentahydrate and carrying out a cycloaddition reaction to obtain the molecular probe described in claim 1; Alternatively, the method includes: mixing azido-tetraethylene glycol-p-toluenesulfonate with radioactive 18 F and performing a nucleophilic substitution reaction to obtain a compound 18 F-PEG4-N3; mixing compound DAZ, compound 18 F-PEG4-N3, sodium ascorbate, tris(3-hydroxypropyltriazylmethyl)amine, and copper sulfate pentahydrate and performing a cycloaddition reaction to obtain the molecular probe described in claim 1.

6. The method according to claim 4 or 5, characterized in that The preparation method of the compound SAZ includes: deacetylating acetazolamide to obtain compound A; mixing compound A, 5-hexynoic acid, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine and reacting them to obtain compound SAZ; The compound A has the structure shown below:

7. The method according to claim 4 or 5, characterized in that, The preparation method of the compound DAZ includes: deacetylating acetazolamide to obtain compound A; mixing compound A, 4-azidobutyric acid, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine and performing a condensation reaction to obtain compound B; mixing BOC-L-glutamic acid, propargylamine, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine and performing a condensation reaction to obtain compound 2A; mixing compound 2A, compound B, sodium ascorbate, tris(3-hydroxypropyltriazolylmethyl)amine, and copper sulfate pentahydrate and performing a click condensation reaction to obtain compound 2B; performing a deprotection reaction on compound 2B to obtain compound 2C; mixing compound 2C, 5-hexynoic acid, 1-hydroxybenzotriazole, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine and performing a condensation reaction to obtain compound DAZ; The compound B has the structure shown below: The compound 2A has the structure shown below: The compound 2B has the structure shown below: The compound 2C has the structure shown below:

8. Use of the molecular probe according to any one of claims 1 to 3 in the preparation of a carbonic anhydrase IX imaging agent or a tumor imaging agent.

9. A carbonic anhydrase IX imaging agent, characterized in that, The imaging agent contains the molecular probe according to any one of claims 1 to 3.

10. A tumor imaging agent, characterized in that, The imaging agent contains the molecular probe according to any one of claims 1 to 3.

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