An inhibitor targeting carbonic anhydrase IX, a molecular probe and its application

Abstract

The present invention belongs to the field of nuclear medicine imaging agents and radionuclide radiotherapy agents, and relates to an inhibitor, molecular probe and application targeting carbonic anhydrase IX. The inhibitor is at least one of the compounds shown in formula I. The CAIX-targeted probe provided by the present invention can target the high-specific uptake of CAIX-high-expressing tumors, and has excellent in vivo metabolic performance and long tumor retention capacity. The CAIX-targeted inhibitor and its molecular probe provided by the present invention have a clear tumor-to-body ratio, can be rapidly metabolized out of the body by the kidneys, and have important application value for early accurate diagnosis and targeted radiotherapy of clear renal cells.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine imaging agents and radionuclide radiotherapy agents, and in particular, relates to a targeted carbonic anhydrase IX (CAIX inhibitor), a molecular probe and an application thereof. Background Art

[0002] Kidney cancer is one of the top ten most common malignant tumors in the world. According to cancer data, the United States is expected to have 81,800 new cases and 14,890 deaths in 2023. The main subtype of renal cell carcinoma, which accounts for 90% of kidney cancer, is clear cell renal cell carcinoma (75%). Although surgical resection can cure patients with early kidney cancer, many patients still have poor prognosis due to recurrence and metastatic disease. The commonly used detection methods, enhanced computed tomography, ultrasound and magnetic resonance imaging, are difficult to achieve accurate diagnosis of lesions, resulting in unnecessary surgery. Therefore, there is an urgent need to find new diagnostic and treatment methods to improve and increase the overall survival rate of patients with clear cell renal carcinoma.

[0003] Due to conventional MRI, CT, 18 F-FDG and other methods have serious defects in determining the subtype of early renal cancer and accurately monitoring advanced renal cancer. In this process, there is a phenomenon of overtreatment and missed resection causing secondary recurrence. Up to 20% of patients are overtreated with surgery due to benign or low-invasive lesions in the kidney (such as oncocytoma, angiomyolipomas, chromophobe renal cell carcinoma, etc.). Therefore, the precise positioning of the tumor and the integrity of surgical resection are crucial to the successful tumor resection. Nuclear medicine radionuclide probe PET-CT imaging has the advantages of real-time, dynamic, and high sensitivity. It can accurately diagnose lesions at an early stage. The use of radionuclide probes can play multiple roles in early diagnosis, efficacy evaluation, and prognosis of tumors. At the same time, radionuclide targeted therapy has certain clinical therapeutic value for metastatic lesions that cannot be surgically removed.

[0004] Carbonic anhydrase IX (CAIX) is a membrane protein that is regulated by hypoxia-inducible factor-1 (HIF-1α). When HIF-1α is upregulated in the tumor microenvironment, it induces tumor angiogenesis, cell proliferation, differentiation and metastasis. When tumor cells undergo glycolysis due to hypoxia, the interior of the tumor is in an acidic condition. At this time, CAIX, as a metalloenzyme, catalyzes the reversible generation of carbonate and protons from CO2 and water, and promotes tumor cell survival, proliferation and differentiation by regulating the pH value inside and outside the cell. CAIX is a biomarker in clear renal cell carcinoma due to the overexpression of CAIX caused by the accumulation of HIF-1α due to the mutation of its VHL gene. As a specific membrane protein of clear renal cell carcinoma, CAIX is not only a therapeutic target, but also has received attention in molecular imaging. Using CAIX as a target, a specific small molecule probe targeting CAIX is constructed. With the help of nuclear medicine PET in vivo, non-invasive, dynamic and real-time molecular imaging technology, the conventional clinical PET molecular probes are overcome. 18 The problem of insufficient specificity of F-FDG can be solved to achieve clinical problems such as early accurate diagnosis, efficacy evaluation, and prognosis evaluation of patients with clear cell renal carcinoma. At the same time, targeted radionuclide therapy can be used for patients with unresectable metastatic lesions of clear cell renal carcinoma, which is of great value in the clinical diagnosis and treatment of clear cell renal cell. Summary of the invention

[0005] The key to improving the treatment of clear renal cell carcinoma is early and accurate diagnosis, which is used to guide accurate surgical resection and avoid excessive surgery. For patients with metastatic lesions who cannot undergo surgical treatment, specific molecular targeted radiotherapy of nuclear medicine chelated radiotherapy nuclides is used to improve their treatment effect. Based on this, the present invention constructs an inhibitor and molecular probe with high affinity for CAIX by changing different core targeting groups, different linkers, and constructing dual-targeting core groups. Experiments have verified that the targeted CAIX inhibitor and molecular probe constructed by the present invention have specific high targeted uptake, long-term tumor retention ability, and good in vivo metabolic performance for clear renal cell carcinoma, and can be quickly metabolized and excreted from the body through the kidneys.

[0006] To achieve the above object, the first aspect of the present invention provides an inhibitor targeting carbonic anhydrase IX, wherein the inhibitor is at least one of the compounds represented by formula I:

[0007]

[0008] In Formula I, AA is an amino acid or a flexible long chain, and L is R is H or a group represented by formula II, and p is 0 or 1;

[0009]

[0010] In Formula I and Formula II, L1 is a flexible long chain or an amino acid, It is a five-membered or six-membered aromatic ring.

[0011] According to a preferred embodiment of the present invention, AA is lysine, cysteine, -(CH2)n9-, -CH2(OCH2CH2O)n 10 -, wherein n9 is an integer from 1 to 5, n 10 An integer from 1 to 5.

[0012] According to a preferred embodiment of the present invention, L1 is one of -(CH2)n1-, -CH2(OCH2CH2O)n2-, -(CH2)n3-1,2,3-triazole-(CH2)n4-, -(CH2)n5(OCH2CH2)n6NHCO(CH2)n7-, wherein n1 is an integer of 1-10, n2 is an integer of 1-5, n3 is an integer of 1-6, n4 is an integer of 1-6, n5 is an integer of 1-4, n6 is an integer of 1-4, and n7 is an integer of 1-4; or, L is M-CO-(CH2)n8-, M is a hydrophilic amino acid peptide chain, the hydrophilic amino acid peptide chain is a peptide chain of 1-5 amino acids, and n8 is an integer of 1-4.

[0013] According to a preferred embodiment of the present invention, A benzene ring or

[0014] Further preferably, n1 is an integer of 1-6, n2 is an integer of 1-4, n3 is an integer of 3-6, n4 is an integer of 2-4, n5 is an integer of 1-3, n6 is an integer of 1-2, n7 is an integer of 1-2, the hydrophilic amino acid peptide chain is a peptide chain of 2-4 amino acids, and n8 is an integer of 2-3.

[0015] Most preferably, n3 is 4 or 5, n4 is 3, n5 is 2, n6 is 2, n7 is 2, and n8 is 2; and the hydrophilic amino acid peptide chain is Gly-Gly-His.

[0016] The second aspect of the present invention provides a molecular probe, which is the above-mentioned inhibitor targeting carbonic anhydrase IX labeled with a radionuclide, wherein the radionuclide is directly labeled on the inhibitor or labeled on the inhibitor through L-Chelater, wherein L is a bridging group and Chelater is a nuclide chelating group.

[0017] The molecular probe of the present invention comprises a nuclide portion and a ligand portion. According to a preferred embodiment of the present invention, -L-Chelater is covalently linked to the inhibitor to form a ligand of the structure shown in Formula III:

[0018]

[0019] According to a more preferred embodiment of the present invention, the bridging group L is a chemical bond; the nuclide chelating group is a group formed by a bifunctional chelating agent, and the bifunctional chelating agent is selected from DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3 or isonitrile.

[0020] Specifically, the ligand is preferably selected from one of the following ligands 1-4:

[0021]

[0022]

[0023] According to the present invention, the radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide includes but is not limited to 68 Ga, 64 Cu, 18 F. 86 Y. 90 Y. 89 Zr, 111 In, 99 mTc, 11 C. 123 I. 125 I and 124 I, wherein the therapeutic radionuclide is not limited to 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.

[0024] The third aspect of the present invention provides the use of the above-mentioned molecular probe in the preparation of a radionuclide imaging diagnostic reagent or a radionuclide therapeutic reagent.

[0025] Among them, the radionuclide imaging diagnostic reagent is preferably an early detection reagent, prognosis judgment reagent or efficacy evaluation reagent for tumors with high expression of carbonic anhydrase IX, and the radionuclide therapeutic reagent is preferably a therapeutic reagent for tumors with high expression of carbonic anhydrase IX; the tumor is preferably renal cancer, more preferably clear renal cell carcinoma.

[0026] The present invention provides inhibitors and molecular probes with different targets such as benzenesulfonamide and acetazolamide that have binding force to CAIX, and the inhibitors and molecular probes with different targets all show high affinity and targeting to CAIX. In addition, the CAIX targeted inhibitor and radionuclide labeled molecular probe of the present invention have high stability in vivo and in vitro, high radionuclide labeling rate and radiochemical purity, excellent in vivo metabolic performance, and the probe has a long tumor retention ability.

[0027] The inhibitor targeting CAIX and the molecular probe thereof provided by the present invention have a clear tumor-to-target ratio and can be rapidly metabolized out of the body through the kidneys, and have important application value for early accurate diagnosis and targeted radiotherapy of clear renal cells.

[0028] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0030] Figure 1-1 to Figure 1-4 The synthetic routes of LF-1, LF-2, LF-3 and LF-4 are shown.

[0031] Figure 2-1 to Figure 2-4 The mass spectra of LF-1, LF-2, LF-3, and LF-4 are shown.

[0032] Figure 3 Shows 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 Ga-LF-4 68 Ga labeling process.

[0033] Figure 4 Shows 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 Quality control results of Ga-LF-4.

[0034] Figure 5 Shows 68Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 In vitro stability of Ga-LF-4.

[0035] Figure 6 Shows 68 Ga-LF-3, 68 In vivo stability of Ga-LF-4.

[0036] Figure 7 Shows 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 K of Ga-LF-4 and CAIX d result.

[0037] Figure 8 Shows 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 Pharmacokinetic results of Ga-LF-4.

[0038] Fig. 9 Shown is the expression of CAIX membrane protein in ccRCC tumors and PC3 tumors.

[0039] Fig.10 Shows 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 Biodistribution results of Ga-LF-4.

[0040] Fig.11 Shows 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 Micro-PET / CT imaging results of Ga-LF-4 in ccRCC tumors (normal group and blocking group) and PC3 tumors.

[0041] Fig.12 The immunohistochemical results of CAIX target proteins in ccRCC tumors and PC3 tumors and major organs are shown. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0043] If the specific conditions are not specified in the examples, all the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, all of them are conventional products that can be purchased from the market.

[0044] Example 1

[0045] Synthesis and characterization of ligands

[0046] The synthesis routes of four ligands targeting CAIX (LF-1 to LF-4, corresponding to the aforementioned ligands 1-4, respectively) are as follows: the amino acid sequence is prepared by solid phase synthesis from C-terminus to N-terminus. First, the 2-CTC resin is activated, and then the first amino acid is connected, the amino group is deprotected from Fmoc, cleaned, and the second amino acid is condensed and coupled. The above operations are repeated to couple different amino acids in the polypeptide chain in turn, and then the chelator is coupled to the polypeptide. Finally, the constructed CAIX targeting probe is cut by a lysis buffer and purified by preparative high performance liquid chromatography to obtain the target probe, and the structure is identified by mass spectrometry.

[0047] Example 1

[0048] LF-1 synthesis route Figure 1-1As shown, the synthesis steps are as follows: (1) swelling resin: 2-chlorotrityl resin (200 mg, 0.08 mmol-0.60 mmol) is placed in a solid phase synthesis tube, DCM (8 mL) is added, and the mixture is stirred and swollen for 60 min; (2) connecting the starting amino acid: the solvent is filtered, a DMF solution containing 3 times the molar amount of Fmoc-L-Lys(Dde)-OH (639 mg, 1.20 mmol) and 8 times the molar amount of DIPEA (794 μL, 4.80 mmol) is added, and the mixture is stirred for 120 min, the solvent is filtered, and the mixture is blocked with methanol (8 mL); (3) deprotection group: the methanol is filtered, and DCM (8 mL) is added. Add a solution containing 20% ​​piperidine (4 mL) into the solid phase reaction tube and stir for 40 min; (4) Deprotection detection: Filter the piperidine DMF solution, take the resin washed with ethanol, detect it with a detection reagent, heat it at 106°C for 3-5 min, and observe the color change of the resin. The reaction is dark blue and the unreacted is colorless; (5) Resin washing: Wash it three times with DMF (4 mL), three times with DCM (5 mL), and three times with DMF (4 mL); (6) Amino acid condensation: Add 1.5 times molar equivalent of Fmoc-Asp(OtBu)-OH (395 mg, 0.96 mmol), 3 times the molar equivalent of TBTU (578 mg, 1.80 mmol) and 6 times the molar equivalent of DIPEA (595 μL, 3.60 mmol) in DMF (4 mL) were stirred for 120 min; (7) Deprotection detection: the piperidine DMF solution was filtered, the resin washed with ethanol was taken, and the detection was performed with a detection reagent, and heated at 106°C for 3 min-5 min. The color change of the resin was observed. The reaction was dark blue and the unreacted was colorless; (8) Resin washing: Washed with DMF (4 mL) three times, DCM (5 mL) three times, and DMF (4 mL) three times respectively. ; (9) Repeat the third to eighth steps of the experiment, and couple the corresponding amino acids and chelating agents in the sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-NH-PEG2-CH2CH2COOH, 4-oxo-4-((4-aminosulfonylphenyl)amino)butyric acid, NOTA-BIS (tert-butyl ester); (10) Resin washing, washing with DMF (4 mL) three times, methanol (5 mL) three times, and DCM (5 mL) three times, and the resin was drained for 10 minutes; (11) Peptide product cutting: add 8 mL (TFA 95% + EDT 2% + water 1% + TIS 2%) peptide resin cutting solution was stirred for 120 minutes; (12) Analysis and purification: spin-drying, and purifying the crude product by high performance liquid chromatography; (13) Lyophilization: The target product solution was collected, concentrated, and lyophilized to obtain a white solid, namely the probe LF-1, and mass spectrometry identification: MALDI-TOFcalc.forC 53 H85 N 15 O 23 S[M+H] + 1332.5736, found 1332.600 (see Figure 2-1 ).

[0049] LF-2 synthesis route Figure 1-2As shown, the synthesis steps are as follows: (1) swelling resin: 2-chlorotrityl resin (200 mg, 0.08 mmol-0.60 mmol) is placed in a solid phase synthesis tube, DCM (8 mL) is added, and the mixture is stirred and swollen for 60 min; (2) connecting the starting amino acid: the solvent is filtered, a DMF solution containing 3 times the molar amount of Fmoc-L-Lys(Dde)-OH (639 mg, 1.20 mmol) and 8 times the molar amount of DIPEA (794 μL, 4.80 mmol) is added, and the mixture is stirred for 120 min, the solvent is filtered, and the mixture is blocked with methanol (8 mL); (3) deprotection group: the methanol is filtered, and a solution containing A 20% piperidine solution (4 mL) was stirred in a solid phase reaction tube for 40 min; (4) Deprotection detection: The piperidine DMF solution was filtered, and the resin washed with ethanol was tested with a detection reagent, heated at 106°C for 3-5 min, and the color change of the resin was observed. The reaction was dark blue, and the unreacted was colorless; (5) Resin washing: The resin was washed three times with DMF (4 mL), three times with DCM (5 mL), and three times with DMF (4 mL); (6) Amino acid condensation: A 1.5-fold molar equivalent of Fmoc-Asp(OtBu)-OH (395 mg, 0.96 mmol) was added to the solid phase reaction tube, A DMF (4 mL) solution of 3 times the molar equivalent of TBTU (578 mg, 1.80 mmol) and 6 times the molar equivalent of DIPEA (595 μL, 3.60 mmol) was stirred for 120 min; (7) Deprotection detection: Filter the piperidine DMF solution, take the resin washed with ethanol, detect it with a detection reagent, heat it at 106°C for 3 min-5 min, and observe the color change of the resin. The reaction is dark blue and the unreacted is colorless; (8) Resin washing: Wash it with DMF (4 mL) three times, DCM (5 mL) three times, and DMF (4 mL) three times respectively; (9) Repeat the steps of step 1 and step 2. Experimental operation from step 3 to step 8: coupling the corresponding amino acids and chelating agents in the sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-Gly-OH, N-Fmoc-N'-trityl-L-histidine, 4-oxo-4-((4-aminosulfonylphenyl)amino)butyric acid, NOTA-BIS (tert-butyl ester); (10) resin washing, washing with DMF (4 mL) three times, methanol (5 mL) three times, DCM (5 mL) three times, and the resin was drained for 10 minutes; (11) peptide product cleavage: adding 8 mL (TFA 95% + EDT 2% + water 1% + TIS 2%) peptide resin cutting solution was stirred for 120 minutes; (12) Analysis and purification: spin-drying, and purifying the crude product by high performance liquid chromatography; (13) Lyophilization: collecting, concentrating and lyophilizing the target product solution to obtain a white solid, namely the probe LF-2, mass spectrometry identification: MALDI-TOF calc.for C56 H 86 N 19 O 23 S[M+H] + 1424.5859, found 1424.592 (see Figure 2-2 ).

[0050] LF-3 synthesis route Figure 1-3As shown, the synthesis steps are as follows: (1) swelling resin: 2-chlorotrityl resin (200 mg, 0.08 mmol-0.60 mmol) is placed in a solid phase synthesis tube, DCM (8 mL) is added, and the mixture is stirred and swollen for 60 min; (2) connecting the starting amino acid: the solvent is filtered, a DMF solution containing 3 times the molar amount of Fmoc-L-Lys(Dde)-OH (639 mg, 1.20 mmol) and 8 times the molar amount of DIPEA (794 μL, 4.80 mmol) is added, and the mixture is stirred for 120 min, the solvent is filtered, and the mixture is blocked with methanol (8 mL); (3) deprotection group: the methanol is filtered, and a solution containing 2 0% piperidine solution (4 mL) was stirred in a solid phase reaction tube for 40 min; (4) Deprotection detection: the piperidine DMF solution was filtered, and the resin washed with ethanol was tested with a detection reagent, heated at 106°C for 3-5 min, and the color change of the resin was observed. The reaction was dark blue and the unreacted was colorless; (5) Resin washing: the resin was washed three times with DMF (4 mL), three times with DCM (5 mL), and three times with DMF (4 mL); (6) Amino acid condensation: 1.5 times molar equivalent of Fmoc-Asp(OtBu)-OH (395 mg, 0.96 mmol), 3 times molar equivalent of A DMF (4 mL) solution of TBTU (578 mg, 1.80 mmol) and 6 times the molar equivalent of DIPEA (595 μL, 3.60 mmol) was stirred for 120 min; (7) Deprotection detection: Filter the piperidine DMF solution, take the resin washed with ethanol, detect it with a detection reagent, heat it at 106°C for 3 min-5 min, and observe the color change of the resin. The reaction is dark blue and the unreacted is colorless; (8) Resin washing: Wash it with DMF (4 mL) three times, DCM (5 mL) three times, and DMF (4 mL) three times respectively; (9) Repeat the third to eighth steps in sequence. Step 1 of the experimental operation was to couple the corresponding amino acids and chelating agents in the sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, 5-azidohexanoic acid; (10) Click reaction: Cuprous iodide (11.42 mg, 0.06 mmol), TBTA (31.4 mg, 0.06 mmol)-N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)hex-5-ynamide (329 mg, 1.2 mol) were added to the resin in a mixed solution of DMF (1 mL) and tetrahydrofuran (1 mL) and reacted at room temperature for 2 h.(11) Dde deprotection: 2% hydrazine hydrate DMF solution (4 mL) was added to the solid phase reaction tube and stirred for 60 min; the cleaning experimental process and coupling with NOTA-BIS (tert-butyl ester) were repeated; (12) Resin cleaning: three times with DMF (4 mL), three times with methanol (5 mL), and three times with DCM (5 mL), and the resin was drained for 10 min; (13) Peptide product cleavage: 8 mL (TFA 95% + EDT 2% + water 1% + TIS 2%) of peptide resin cleavage solution was added and stirred for 120 min; (14) Analysis and purification: spin-drying, and the crude product was purified by high performance liquid chromatography; (15) Lyophilization: The target product solution was collected, concentrated, and lyophilized to obtain a white solid, namely, the probe LF-3, mass spectrometry identification: MALDI-TOF calc.forC. 49 H 79 N 19 O 20 S2[M+H] + 1318.5263, found 1318.484 (see Figure 2-3 ).

[0051] LF-4 synthesis route Figure 1-4As shown, the synthesis steps are as follows: (1) swelling resin: 2-chlorotrityl resin (200 mg, 0.08 mmol-0.60 mmol) is placed in a solid phase synthesis tube, DCM (8 mL) is added and stirred for swelling for 60 min; (2) connecting the starting amino acid: the solvent is filtered, a DMF solution containing 3 times the molar amount of Fmoc-L-Lys(Dde)-OH (639 mg, 1.20 mmol) and 8 times the molar amount of DIPEA (794 μL, 4.80 mmol) is added and stirred for 120 min, the solvent is filtered, and methanol (8 mL) is used for blocking; (3) deprotection group: methanol is filtered, and a solution containing 20% ​​piperidine (4 mL) is added. Stir in a solid phase reaction tube for 40 min; (4) Deprotection detection: Filter the piperidine DMF solution, take the resin washed with ethanol, detect it with a detection reagent, heat it at 106°C for 3-5 min, and observe the color change of the resin. The reaction is dark blue and the unreacted is colorless; (5) Resin washing: Wash it three times with DMF (4 mL), three times with DCM (5 mL), and three times with DMF (4 mL); (6) Amino acid condensation: Add 1.5 times molar equivalent of Fmoc-Asp(OtBu)-OH (395 mg, 0.96 mmol), 3 times molar equivalent of TBTU (578 mg, 1.80 mmol) to the solid phase reaction tube. ) and 6 times the molar equivalent of DIPEA (595 μL, 3.60 mmol) in DMF (4 mL) were stirred for 120 min; (7) Deprotection detection: the piperidine DMF solution was filtered, the resin washed with ethanol was taken, and the detection reagent was used for detection, and the resin was heated at 106°C for 3 min-5 min to observe the color change of the resin. The reaction was dark blue and the unreacted was colorless; (8) Resin washing: the resin was washed three times with DMF (4 mL), three times with DCM (5 mL), and three times with DMF (4 mL); (9) The experimental operations from the third to the eighth steps were repeated in sequence to couple the corresponding amino acids and chelating agents in the sequence, and Fmoc-Arg(Pb f)-OH, Fmoc-Asp(OtBu)-OH, (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-azidopentanoic acid, 6-azidohexanoic acid, (10) Click reaction: Click reaction: Cuprous iodide (11.42 mg, 0.06 mmol), TBTA (31.84 mg, 0.06 mmol)-N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)hex-5-ynamide (493 mg, 1.80 mmol) were added to the resin in a mixed solution of DMF (1 mL) and tetrahydrofuran (1 mL) and reacted at room temperature for 2 h.(11) Dde deprotection: 2% hydrazine hydrate DMF solution (4 mL) was added to the solid phase reaction tube and stirred for 60 min; the cleaning process and coupling with NOTA-BIS (tert-butyl ester) were repeated; (12) Resin cleaning: three times with DMF (4 mL), three times with methanol (5 mL), and three times with DCM (5 mL), and the resin was drained for 10 min; (13) Peptide product cleavage: 8 mL (TFA 95% + EDT 2% + water 1% + TIS 2%) of peptide resin cleavage solution was added and stirred for 120 min; (14) Analysis and purification: spin-drying, and the crude product was purified by high performance liquid chromatography; (15) Lyophilization: The target product solution was collected, concentrated, and lyophilized to obtain a white solid, namely, the probe LF-4, mass spectrometry identification: MALDI-TOF calc.forC. 64 H 101 N 27 O 24 S4[M+H] + 1760.6468, found 1760.613 (see Figure 2-4 ).

[0052] Example 2

[0053] CAIX targeting probe 68 Ga labeling

[0054] LF series probes 68 Ga labeling process Figure 3 As shown, the specific method is as follows: the probe precursor is dissolved in ultrapure water (1 mg / mL), and stored at -20°C after being divided into aliquots. The probe precursor is then rinsed with 4 mL of 0.05 M HCl. 68 Ge- 68 Ga generator, collect 3mL of eluent and place it in a 4mL reaction tube, add sodium acetate (180μL, pH 4.0) and 20μL of probe precursor, and heat the reaction at 95℃ for 15min. After the reaction, add 3mL of pure water to the system and purify it through a pre-activated Sep-Pak LightC18 solid phase extraction column, and rinse the column with 5mL of pure water. Elute the C18 solid phase extraction column with 0.5mL of 80% ethanol, add 5mL of physiological saline to the eluent, and filter it through a 0.2μm microporous filter membrane to obtain a radioactive probe that can be used in the experiment.

[0055] Example 3

[0056] 68 Quality control of Ga-labeled probes:

[0057] 68The radiochemical purity of the Ga-labeled molecular probe targeting CAIX was verified using Radio-HPLC analysis. Figure 4 As shown, the results showed that before purification 68 Ga-LF-1, 68 Ga-LF-2, 68 Ga-LF-3, 68 The labeling efficiency of all Ga-LF-4 probes was >90%, and the radiochemical purity of all radiopharmaceutical products was >95% after purification by C18 solid phase extraction cartridges.

[0058] Example 4

[0059] 68 Stability of Ga-labeled probe in vivo and in vitro

[0060] Will 68 The Ga-labeled CAIX targeting molecular probe was incubated with physiological saline and 5% HSA (human serum albumin) in vitro, and the in vitro stability of the probe was detected by Radio-HPLC at 0, 30, 60, 120, and 240 minutes. Five healthy KM mice were used for the in vivo stability experiment, and each mouse was injected with 37 MBq 68 Ga-labeled CAIX-targeting molecular probe, blood and urine were collected at 0, 30, 60, 120, and 240 minutes, and the radiochemical purity was analyzed by Radio-HPLC to detect the in vivo stability of the probe. Figure 5 As shown, the present invention 68 After incubation in physiological saline and 5% HSA (human serum albumin), the radiochemical purity of the Ga-labeled CAIX targeting molecular probe reached more than 90% within 1 h. Figure 6 As shown, 68 Ga-LF-3 and 68 The radiochemical purity of Ga-LF-4 1h in urea is greater than 80%, and the radiochemical purity in blood is greater than 60%. The results show that the probe constructed by the present invention has good in vitro and in vivo stability.

[0061] Example 5

[0062] 68 Lipid-water partition coefficient of Ga-labeled probe

[0063] 68 Determination of the octanol-water partition coefficient of Ga-labeled CAIX targeting molecular probe: n-octanol, water and 68The mixture of Ga-labeled probes was vortexed for 3 minutes and centrifuged at 3000×rpm for 5 minutes, then 3 samples (50 μL) were taken from each phase and the radioactivity was counted using a gamma counter (Wizard II, Perkin Elmer Inc., Germany). 8 The lipid-water partition coefficient of Ga-LF-4 is logP = -1.78 ± 0.14 (n = 5), indicating that 68 Ga-LF-4 has excellent hydrophilicity.

[0064] Example 6

[0065] (Western Blot, WB) Verification of protein expression of the model

[0066] The expression of CAIX in PC3 and ccRCC tumor tissues was detected by WB. The protein concentration in the tumor tissue lysate was determined by the BCA method. After SDS-PAGE, the samples were separated and transferred to a PVDF membrane. Then 5% skim milk was added, shaken at room temperature, and incubated for 1 hour. Anti-CAIX antibody ab15086 and anti-GAPDH antibody 60004-1-lg (dilution 1:5000) were incubated at 4°C overnight. The next day, the samples were washed several times with TBST, and sheep anti-rabbit antibody GB23303 and anti-rat antibody GB23302 (dilution 1:3000) were added and incubated at room temperature for 2 hours. After washing several times with TBST, imaging was performed using an enhanced chemiluminescence kit. The results are shown in the figure. Fig. 9 As shown, with GAPDH protein as the internal standard, the molecular weight of CAIX protein is about 55kDa. The relative expression ratio of CAIX to GAPDH in ccRCC tumor tissue is very high, while the expression of CAIX was not detected in the control PC3 tumor tissue. This proves that ccRCC is a model with high CAIX expression, while PC3 is a model with low CAIX expression.

[0067] Example 7

[0068] 68 Protein binding assay using Ga-labeled probes

[0069] Before the experiment, CAIX protein at a concentration of 1 mg / mL was added to a 96-well plate at a volume of 100 μL per well. The plate was then incubated overnight at 4°C. The next day, the lysate was discarded and then washed several times with PBST. 200 μL of 5% skim milk was added to each well and incubated at 37°C for 2 hours. After washing several times with PBST, 68Different CAIX targeting molecular probes labeled with Ga were added to 96-well plates at different concentrations (0.032-1.59 nmol / L, n=5) and incubated at 37°C for 2 h. After washing with PBST, the radioactivity of each well was measured using a γ counter. Figure 7 As shown, 68 The Kd value of Ga-LF-1 is 18.66nM. 68 The Kd value of Ga-LF-2 is 11.01nM. 68 The Kd value of Ga-LF-3 is 15.14nM. 68 The Kd value of Ga-LF-4 was 6.619 nM, showing strong affinity for CAIX.

[0070] Example 8

[0071] 68 Pharmacokinetics of Ga-labeled probe

[0072] Five-week-old healthy KM mice were injected with 3.7 MBq 68 Ga-labeled different CAIX-targeting molecular probes were then used to collect blood samples from the eye sockets at 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 120 minutes, and 240 minutes (n=5). After measuring the blood weight using an electronic balance, the radioactivity was detected using a γ counter. The experimental results are shown in Figure 8 Shown, displayed 68 The half-life of the distribution phase and elimination phase of Ga-LF-1 in the body is 1.06 and 18.43 minutes respectively. 68 The half-life of the distribution phase and elimination phase of Ga-LF-2 in the body is 1.91 and 33.4 minutes respectively. 68 The half-life of the distribution phase and elimination phase of Ga-LF-3 in the body is 1.08 and 43.66 minutes respectively. 68 The half-lives of the distribution phase and elimination phase of Ga-LF-4 in the body are 0.37 and 21 minutes, respectively, and both can be rapidly cleared from the body.

[0073] Example 9

[0074] 68 Study on the biodistribution of Ga-labeled probes

[0075] Healthy KM mice, ccRCC (CAIX high expression) tumor model mice, and PC3 (CAIX low expression) tumor model mice were injected with 20 μCi 68 Healthy KM mice aged 5 weeks were euthanized at 30, 60, 120, and 240 minutes after injection of different CAIX-targeting molecular probes labeled with Ga. 68ccRCC tumor model mice (with or without injection of corresponding labeled precursors) and PC3 tumor model mice with different CAIX-targeting molecular probes labeled with Ga were sacrificed at 1 h. Organs (heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, brain, and tumor; n = 5) were weighed and measured using a γ-counter. Fig.10 As shown, the biodistribution results showed that each probe had a certain uptake in organs where CAIX was physiologically expressed, such as lung, kidney, and small intestine, and the uptake decreased with time, which was consistent with the results of Western blot. The uptake in other organs was low, and the probe was excreted from the body through kidney metabolism.

[0076] Example 10

[0077] 68 Micro-PET / CT Imaging Study of Ga-labeled Probe Model Mouse

[0078] Micro-PET / CT imaging studies were performed in ccRCC tumor models with high CAIX expression and PC3 models with low CAIX expression. 68 After different CAIX-targeting molecular probes labeled with Ga were injected into the tail vein of each mouse, images were collected using Micro-PET / CT at 30, 60, 120, and 240 minutes. For blocking studies, 300 μg of the corresponding unlabeled probe precursor was pre-injected 1 hour in advance. Then, 100 μCi of the corresponding 68 After Ga-labeled probes, mice were anesthetized with isoflurane for image acquisition. After CT-AC reconstruction of the acquired data, the maximum uptake value SUVmax of the region of interest was depicted, and each organ was depicted at least 5 times. Fig.11 As shown, the Micro-PET / CT imaging results show that the probe constructed by the present invention can be specifically and highly taken up in ccRCC tumors with high CAIX expression, but basically no uptake in PC3 tumor model mice with low CAIX expression. In the blocking group, the tumor probe uptake is significantly reduced, and the probe is quickly excreted from the body through kidney metabolism, with a clear imaging background. 68 The SUVmax of Ga-LF-1 was 0.94±0.01 at 60 min. 68 The SUVmax of Ga-LF-2 was 0.57±0.01 at 60 min. 68 The SUVmax of Ga-LF-3 at 60 min was 1.88 ± 0.01, 120 min: 1.59 ± 0.03, 240 min: 1.48 ± 0.01; 68The SUVmax of Ga-LF-4 was 1.75±0.01 at 60min, 1.85±0.01 at 120min, and 1.74±0.02 at 240min. The tumor uptake of each probe in the blocking group and PC3 latent group was significantly lower than that in the positive group, with significant statistical differences.

[0079] Embodiment 11

[0080] Immunohistochemistry of tumor model mice

[0081] ccRCC and PC3 tumor tissues and various organ tissues were fixed and embedded with 5% tissue fixative, and the slice thickness was 5 μm. Before the experiment began, the slices were dewaxed and then rehydrated with gradient xylene and alcohol. In order to block the activity of endogenous peroxidase, the tumor slices were treated with 3% H2O2 for 10 minutes. Then add the antigen retrieval solution and heat it in a microwave for 10 minutes. Wash several times with PBS and then block with goat serum at room temperature for 40 minutes. After sealing, add the primary antibody ab108351 at 1:200 and incubate at 4°C overnight. The next day, take out the slices and wash several times with PBS. Add the secondary antibody and incubate at room temperature for 30 minutes. Then use the DAB matrix kit and hematoxylin staining for hydrochloric acid identification. After gradient alcohol dehydration, seal with neutral gum to observe the staining results. As Fig.12 As shown, immunohistochemistry results showed that CAIX was highly expressed in ccRCC tumors, with some expression in the lungs and gastrointestinal tract, but was lowly expressed in PC3 tumors and other organs.

[0082] The above experimental results indicate that the CAIX-targeted probe provided by the present invention can specifically target tumors with high CAIX expression for uptake.

[0083] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An inhibitor targeting carbonic anhydrase IX, characterized in that: The inhibitor is the following compound: 。 2. A molecular probe, characterized in that The molecular probe is the inhibitor targeting carbonic anhydrase IX according to claim 1 which is labeled with a radionuclide, wherein the radionuclide is directly labeled on the inhibitor or is labeled on the inhibitor via an L-Chelater, wherein L is a bridging group and Chelater is a nuclide chelating group.

3. The molecular probe according to claim 2, wherein -L-Chelater is covalently attached to the inhibitor to form ligand 4: 。 4. The molecular probe according to claim 2, wherein The radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide is 68 Ga, 64 Cu, 18 F. 86 Y. 90 Y. 89 Zr, 111 In, 99 mTc, 11 C. 123 I. 125 I and 124 I, 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.

5. Use of the molecular probe according to any one of claims 2 to 4 in the preparation of a radionuclide imaging diagnostic reagent or a radionuclide therapeutic reagent; The radionuclide imaging diagnostic reagent is an early detection reagent, a prognosis judgment reagent or a therapeutic efficacy evaluation reagent for tumors with high carbonic anhydrase IX expression; The radionuclide therapeutic agent is a therapeutic agent for tumors with high expression of carbonic anhydrase IX; The tumor is renal cancer.

6. The use according to claim 5, wherein: The tumor was clear cell renal carcinoma.

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