A targeting ca ix compound containing a nitroimidazole group and uses thereof

Abstract

The application discloses a kind of compounds containing nitroimidazole group targeted to CA Ⅸ and preparation method and application thereof, belong to the field of radiopharmaceuticals and nuclear medicine technology.The molecular structure of the compound is mainly composed of nitroimidazole group, CA Ⅸ targeting group, chelating agent and linker, and the corresponding radioactive molecular probe can be obtained by labeling with radionuclide.It is hydrophilic material, good in vitro stability, has good targeting and specificity to CA Ⅸ.The tumor uptake in tumor-bearing mice and target and non-target ratio are high, the imaging background is clean, the image contrast is strong, has good in vivo biodistribution characteristics, can be used for the diagnosis or treatment of cancer with high expression of CA Ⅸ, and has good clinical application prospect.

Description

Technical Field

[0001] This invention relates to radiolabeled complexes and their applications, specifically to a radiopharmaceutical containing a nitroimidazole group that targets CA IX, its preparation method, and its application, belonging to the fields of radiopharmaceutical chemistry and clinical nuclear medicine. Background Technology

[0002] The tumor microenvironment (TME) refers to the local environment in which tumor cells reside. It is composed of various components, including tumor cells, the extracellular matrix, and blood vessels, and plays a crucial role in tumor development, growth, metastasis, and invasion. Due to the highly active metabolism of tumor cells and insufficient oxygen supply from blood vessels, hypoxia is one of the most important characteristics of the tumor microenvironment, and the vast majority of solid tumors exhibit hypoxic features. Under hypoxic conditions, the concentration of hydrogen ions outside cancer cells increases, forming an acidic microenvironment. To maintain pH homeostasis, cancer cells express a series of proteins to regulate the pH inside and outside the cell, thereby promoting their proliferation and metastasis. Carbonic anhydrase IX (CA IX) is one such important protein, regulating the pH inside and outside cancer cells by catalyzing the simple reaction of carbon dioxide hydration into hydrogen ions and bicarbonate ions. CA IX is a metallotransmembrane protein regulated by hypoxia-inducible factor-1α (HIF-1α). Under hypoxic conditions, HIF-1α cannot be degraded, leading to high expression of CA IX in various tumor cells, including breast cancer and head and neck cancer. In contrast, expression in normal tissues is low, with CA IX only detected in gastric and intestinal epithelial cells. Furthermore, due to gene mutations, the von Hippel-Lindau protein (VHL) pathway is dysfunctional in clear cell renal cell carcinoma (ccRCC), resulting in high expression of CA IX even under normoxic conditions. Therefore, CA IX is a biomarker strongly associated with various solid tumors and ccRCC, and is considered a promising target for cancer diagnosis and treatment.

[0003] Given the frequent coexistence of hypoxic microenvironment and CA IX in tumor tissues, introducing hypoxia-sensitive groups into molecular probes targeting CA IX may enhance tumor uptake and retention, and improve the target-to-non-target ratio. Nitroimidazole is a classic hypoxia-sensitive molecule and is widely used in designing molecular probes for detecting tissue hypoxia. In 2026, Teng Peng et al. designed a polypeptide molecular probe containing 2-nitroimidazole. 68Ga]Ga-IPM-N001 (J. Med. Chem. 2026, 69, 7474 − 7487) exhibited high tumor uptake and target-to-non-target ratio in tumor-bearing mice, demonstrating good imaging effects. Compared to peptide molecules, small molecules are generally easier to synthesize and have better stability and tissue penetration in vivo. Therefore, the development of molecular probes containing nitroimidazole groups targeting CA IX based on small molecules has important clinical value and practical significance. However, work in this area is currently lacking. Only in 2025 did Chu Taiwei et al. develop a series of radioiodine-labeled small molecule probes containing 2-nitroimidazole targeting CA IX (Eur. J. Med. Chem. 289(2025) 117443). In cell experiments, these probes showed significantly higher cellular uptake under hypoxic conditions, indicating that nitroimidazole plays an important role. However, their study only included in vitro cell experiments and lacked any in vivo evaluation, thus lacking relevant data to prove its effectiveness in vivo. Therefore, based on the above objective facts, this invention aims to develop a small molecule compound containing a nitroimidazole group that targets CA IX, and obtain a corresponding molecular probe through radiolabeling, so as to enhance tumor uptake and retention, improve the target-to-non-target ratio, and thus achieve early diagnosis, efficacy monitoring and targeted therapy of cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a small molecule compound containing a nitroimidazole group that specifically targets CA IX, which is simple to label, has high tumor uptake, strong retention, and a good target-to-non-target ratio. It exhibits high affinity and specificity for CA IX and can be used for the diagnosis and treatment of CA IX-positive tumors, achieving an integrated diagnosis and treatment effect. This invention belongs to the fields of radiopharmaceuticals and nuclear medicine.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] To achieve the above objectives, the present invention provides a compound or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof having the structure shown in formula (I):

[0007] (I),

[0008] Wherein, X and Y are diazole, triazole, or amide groups;

[0009] R is a 5-10 membered aromatic ring substituted with a sulfonic acid amino group;

[0010] NM is a nitroimidazole group, selected from any of the following:

[0011] ,

[0012] Where n is a positive integer between 1 and 6;

[0013] L1, L2, L3, and L4 are functionalized connectors, selected from any of the following:

[0014] ,

[0015] m is a positive integer between 1 and 6;

[0016] L5 is a functionalized linker, selected from any of the following:

[0017]

[0018] In some embodiments of the present invention, X is... Y is ;

[0019] In some embodiments of the present invention, L1 is a functionalized linker -(CH2)2CONH-;

[0020] In some embodiments of the present invention, L2 is a functionalized linker -(CH2)5-;

[0021] In some embodiments of the present invention, L3 is a functionalized linker -CONH(CH2CH2O)2CH2CH2NHCO-;

[0022] In some embodiments of the present invention, L4 is a functionalized linker -(CH2)2-;

[0023] In some embodiments of the present invention, NM is ;

[0024] In some embodiments of the present invention, L5 is selected from any of the following:

[0025]

[0026] In another aspect of the invention, the invention also provides a compound or a pharmaceutically acceptable salt, stereoisomer, or solvate thereof having the structure shown in formula (II):

[0027] (II)

[0028] Wherein, X and Y are diazole, triazole, or amide groups;

[0029] R is a 5-10 membered aromatic ring substituted with a sulfonic acid amino group;

[0030] NM is a nitroimidazole group, selected from any of the following:

[0031] ,

[0032] Where n is a positive integer between 1 and 6;

[0033] L1, L2, L3, and L4 are functionalized connectors, selected from any of the following:

[0034] ,

[0035] m is a positive integer between 1 and 6;

[0036] L5 is a functionalized linker, selected from any of the following:

[0037]

[0038] In some embodiments of the present invention, X is... Y is ;

[0039] In some embodiments of the present invention, L1 is a functionalized linker -(CH2)2CONH-;

[0040] In some embodiments of the present invention, L2 is a functionalized linker -(CH2)5-;

[0041] In some embodiments of the present invention, L3 is a functionalized linker -CONH(CH2CH2O)2CH2CH2NHCO-;

[0042] In some embodiments of the present invention, L4 is a functionalized linker -(CH2)2-;

[0043] In some embodiments of the present invention, NM is ;

[0044] In some embodiments of the present invention, L5 is selected from any of the following:

[0045] Specifically, the chelator in the structure shown in formula (II) of the present invention is a bifunctional chelating agent selected from any of the following:

[0046]

[0047] In some embodiments of the present invention, the compound of formula (II) is selected from any of the following:

[0048]

[0049] (CAIX-DJ01)

[0050]

[0051] (CAIX-DJ02)

[0052]

[0053] (CAIX-DJ03)

[0054]

[0055] (CAIX-DJ04)

[0056]

[0057] (CAIX-DJ05)

[0058] Or its pharmaceutically acceptable salts, stereoisomers, or solvates.

[0059] In another aspect, the present invention also provides a coordination compound formed by radiolabeling the aforementioned compound or a pharmaceutically acceptable salt, stereoisomer, or solvate.

[0060] In some embodiments of the present invention, the radionuclide is selected from... 18 F, 67 Ga、 68 Ga、 111 In、 45 Ti、 99m Tc, 94m Tc, 64 Cu、 67 Cu、 186 / 188 Re、 43 / 44 Sc、 47 Sc、 52 Mn, 86 Y、 90 Y、 89 Sr、 177 Lu、 153 Sm、 212 / 213 Bi、 212 Pb, 225 Ac、 198 Au、 161 Tb, 149 Pm, 177 Yb、 166 Ho、 131 I, 211 At、 226 / 227 Th.

[0061] In some embodiments of the present invention, the radionuclide is selected from... 68 Ga.

[0062] In some embodiments of the present invention, the coordination compound of the present invention is selected from any of the following:

[0063]

[0064] ([ 68 Ga]Ga-CAIX-DJ01)

[0065]

[0066] ([ 68 Ga]Ga-CAIX-DJ02)

[0067]

[0068] ([ 68 Ga]Ga-CAIX-DJ03)

[0069]

[0070] ([ 68 Ga]Ga-CAIX-DJ04)

[0071]

[0072] ([ 68 Ga]Ga-CAIX-DJ05)

[0073] The present invention also provides a tumor imaging agent containing a nitroimidazole group that targets CA IX, said imaging agent comprising the aforementioned compound or a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

[0074] The present invention also provides the use of the aforementioned compounds or pharmaceutically acceptable salts, stereoisomers or solvates thereof for the preparation of medicaments for the diagnosis or treatment of cancers with high expression of CA IX.

[0075] Beneficial effects:

[0076] The compounds of this invention are made from readily available and inexpensive raw materials, and their molecular skeleton mainly uses amide bonds, making their synthesis method simple.

[0077] The compound of the present invention is mainly composed of two parts: a nitroimidazole group and a target CA IX. Compared with most current probes that only contain one of these parts, it has a wider range of indications and expands the diagnostic range of the probe.

[0078] The compounds of this invention have good in vivo biodistribution properties, which effectively solves the problem of high non-target uptake of current molecular probes targeting CA IX, reduces image background interference and radiation damage to patients, and improves imaging results.

[0079] The compounds of this invention exhibit higher tumor uptake and target-to-non-target ratios, resulting in better imaging performance. They are promising radiopharmaceuticals that can be used for imaging diagnosis, efficacy monitoring, or radionuclide therapy of clear cell renal cell carcinoma or tumors that highly express CA IX. Attached Figure Description

[0080] Figure 1 Synthetic route of ligand CAIX-DJ01

[0081] Figure 2 High-resolution mass spectrometry of ligand CAIX-DJ01

[0082] Figure 3 Coordination compounds [ 68 Radioactive HPLC chromatogram of Ga]Ga-CAIX-DJ01 after C18 purification

[0083] Figure 4 Coordination compounds [ 68 HPLC analysis of the stability of Ga]Ga-CAIX-DJ01 in room temperature salt formation

[0084] Figure 5 Coordination compounds [ 68 Stability analysis of Ga]Ga-CAIX-DJ01 in mouse serum by HPLC

[0085] Figure 6 Coordination compounds [ 68 Imaging images of Ga-CAIX-DJ01 in HT-29 and OS-RC-2 tumor-bearing mice.

[0086] Figure 7 Coordination compounds [ 68 Competitive inhibition experiment of Ga-CAIX-DJ01 in HT-29 tumor-bearing rats

[0087] Figure 8 Coordination compounds [ 68 Biodistribution of Ga-CAIX-DJ01 in OS-RC-2 tumor-bearing rats

[0088] Figure 9 Synthetic route of ligand CAIX-DJ02

[0089] Figure 10 High-resolution mass spectrometry of ligand CAIX-DJ02

[0090] Figure 11 Coordination compounds [ 68 Radioactive HPLC spectrum of Ga]Ga-CAIX-DJ02 after C18 purification

[0091] Figure 12 Coordination compounds [ 68 HPLC analysis of the stability of Ga]Ga-CAIX-DJ02 in raw salt at room temperature

[0092] Figure 13 Coordination compounds [ 68 Stability analysis of Ga]Ga-CAIX-DJ02 in mouse serum by HPLC

[0093] Figure 14 Coordination compounds [ 68 Ga]Ga-CAIX-DJ02 uptake inhibition experiment in OS-RC-2 cells

[0094] Figure 15 Coordination compounds [ 68 Imaging images of Ga-CAIX-DJ02 in HT-29 and OS-RC-2 tumor-bearing mice.

[0095] Figure 16 Coordination compounds [ 68 Competitive inhibition experiment of Ga-CAIX-DJ02 in HT-29 tumor-bearing rats

[0096] Figure 17 Coordination compounds [ 68 Biodistribution of Ga-CAIX-DJ02 in OS-RC-2 tumor-bearing rats

[0097] Figure 18 Synthetic route of ligand CAIX-DJ03

[0098] Figure 19 High-resolution mass spectrometry of ligand CAIX-DJ03

[0099] Figure 20 Coordination compounds [ 68 Radioactive HPLC spectrum of Ga]Ga-CAIX-DJ03 after C18 purification

[0100] Figure 21 Coordination compounds [ 68 HPLC analysis of the stability of Ga-CAIX-DJ03 in raw salt at room temperature

[0101] Figure 22 Coordination compounds [ 68 Stability analysis of Ga]Ga-CAIX-DJ03 in mouse serum by HPLC

[0102] Figure 23 Coordination compounds [ 68 Ga]Ga-CAIX-DJ03 uptake inhibition experiment in OS-RC-2 cells

[0103] Figure 24 Coordination compounds [ 68 Imaging images of Ga-CAIX-DJ03 in HT-29 and OS-RC-2 tumor-bearing mice.

[0104] Figure 25 Coordination compounds [ 68 Competitive inhibition experiment of Ga-CAIX-DJ03 in HT-29 tumor-bearing rats

[0105] Figure 26 Coordination compounds [ 68 Biodistribution of Ga-CAIX-DJ03 in OS-RC-2 tumor-bearing rats

[0106] Figure 27 Synthetic route of ligand CAIX-DJ04

[0107] Figure 28 High-resolution mass spectrometry of ligand CAIX-DJ04

[0108] Figure 29 Coordination compounds [ 68 Radioactive HPLC chromatogram of Ga]Ga-CAIX-DJ04 after C18 purification

[0109] Figure 30 Coordination compounds [ 68 HPLC analysis of the stability of Ga]Ga-CAIX-DJ04 in room temperature salt

[0110] Figure 31 Coordination compounds [ 68 Stability analysis of Ga]Ga-CAIX-DJ04 in mouse serum by HPLC

[0111] Figure 32 Coordination compounds [ 68 Imaging images of Ga-CAIX-DJ04 in HT-29 and OS-RC-2 tumor-bearing mice.

[0112] Figure 33 Coordination compounds [ 68 Competitive inhibition experiment of Ga-CAIX-DJ04 in HT-29 tumor-bearing rats

[0113] Figure 34 Coordination compounds [ 68 Biodistribution of Ga-CAIX-DJ04 in OS-RC-2 tumor-bearing rats

[0114] Figure 35 Synthetic route of ligand CAIX-DJ05

[0115] Figure 36 High-resolution mass spectrometry of ligand CAIX-DJ05

[0116] Figure 37 Coordination compounds [ 68 Radioactive HPLC chromatogram of Ga]Ga-CAIX-DJ05 after C18 purification

[0117] Figure 38 Coordination compounds [ 68 HPLC analysis of the stability of Ga-CAIX-DJ05 in raw salt at room temperature

[0118] Figure 39 Coordination compounds [ 68 HPLC analysis of the stability of Ga]Ga-CAIX-DJ05 in mouse serum

[0119] Figure 40 Coordination compounds [ 68 Imaging images of Ga-CAIX-DJ05 in HT-29 and OS-RC-2 tumor-bearing mice.

[0120] Figure 41 Coordination compounds [ 68 Competitive inhibition experiment of Ga-CAIX-DJ05 in HT-29 tumor-bearing rats

[0121] Figure 42 Coordination compounds [ 68 Biodistribution of Ga-CAIX-DJ05 in OS-RC-2 tumor-bearing rats Detailed Implementation

[0122] The compounds of the present invention, their preparation methods, and applications are further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following examples are merely illustrative and explanatory of the present invention and do not imply any limitation on the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0123] Unless otherwise specified, all reagents, raw materials and equipment used in the following examples are commercially available products.

[0124] Example 1: Synthesis of compound CAIX-DJ01 (e.g.) Figure 1 (As shown)

[0125] This embodiment provides a synthetic scheme for a ligand CAIX-DJ01 containing a nitroimidazole group that targets CA IX, wherein the ligand CAIX-DJ01 has the following structure:

[0126]

[0127] Synthesis route:

[0128] Step 1:

[0129]

[0130] Compound 1 (2.22 g, 10.0 mmol) was dissolved in 50 mL of ethanol, and concentrated hydrochloric acid (10 mL) was added at room temperature. The mixture was refluxed at 85°C for 2 h, and the reaction was monitored by TLC (dichloromethane / methanol = 5:1, v / v). After the reaction was complete, the pH was adjusted to alkaline with saturated sodium bicarbonate solution, and then extracted with ethyl acetate (30 mL × 3). The organic phases were combined, dried over anhydrous MgSO4, filtered, and the solvent was evaporated under reduced pressure to give a white solid (compound 2, 1.57 g, 8.7 mmol).

[0131] Step Two:

[0132]

[0133] Compound 4-pentynic acid (1.75 g, 17.8 mmol) was dissolved in 20 mL of dichloromethane and cooled to 0°C in an ice-water bath. Oxaloyl chloride (2.15 g, 16.9 mmol, 1.48 mL) was then added dropwise. The mixture was stirred at room temperature for 3 h and concentrated under reduced pressure to give compound Pent-4-ynoyl chloride, which was a yellow oil.

[0134] Compound 2 (1.57 g, 8.7 mmol) and pyridine (2.37 g, 17.5 mmol, 2.42 mL) were dissolved in N,N-dimethylformamide and cooled to 0°C in an ice-water bath. Pent-4-ynoyl chloride was dissolved in dichloromethane and added dropwise to the reaction flask, followed by stirring at room temperature for 12 h. After the reaction was complete, the solvent was removed by concentration under reduced pressure, and the crude product was purified by column chromatography (eluent: ethyl acetate) to give a pale yellow solid (compound 3, 1.15 g, 4.4 mmol).

[0135] Step 3:

[0136]

[0137] Under nitrogen protection, compound 3 (0.52 g, 2.0 mmol) was dissolved in 20 mL of tetrahydrofuran. Then, 6-azidohexanoic acid (0.31 g, 2.0 mmol), sodium ascorbate (0.16 g, 0.8 mmol), and copper sulfate pentahydrate (0.1 g, 0.4 mmol) were added sequentially to the solution. The solution was stirred overnight at room temperature and monitored by TLC (dichloromethane / methanol = 20:1, v / v). After the reaction was complete, the solution was diluted with deionized water (20 mL), extracted with ethyl acetate (20 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The solution was filtered, and the filtrate was evaporated to dryness and purified by column chromatography (dichloromethane / methanol = 20:1, v / v) to give a white solid (compound 4, 0.53 g, 1.3 mmol).

[0138] Step Four:

[0139]

[0140] Compound 4 (148 mg, 0.4 mmol), Boc-NH-PEG2-C2-NH2 (104 mg, 0.4 mmol), and N,N-diisopropylethylamine (DIPEA, 100 mg, 0.8 mmol) were dissolved in DMF, followed by the addition of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 160 mg, 0.4 mmol). The reaction was stirred overnight at room temperature and monitored by TLC (dichloromethane / methanol = 10:1, v / v). After the reaction was complete, the solvent was removed under reduced pressure, and the residue was dissolved in DCM. TFA was added, and the reaction was stirred at room temperature for 4 h, monitored by TLC (dichloromethane / methanol = 5:1, v / v). After the reaction was completed, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (dichloromethane / methanol = 5:1, v / v) to give a transparent oil (compound 5, 176 mg, 0.3 mmol).

[0141] Step 5:

[0142]

[0143] Compound 6 (1.13 g, 10 mmol) and N-(2-bromoethyl)phthalimide (3.05 g, 12 mmol) were dissolved in DMF (50 mL), followed by the addition of potassium carbonate (0.98 g, 12 mmol). The solution was heated under reflux at 110°C for 3 h. After cooling to room temperature, the mixture was filtered, the filtrate was evaporated to dryness, washed with water, and dried to give a white solid (compound 7, 2.56 g, 8.9 mmol).

[0144] Step Six:

[0145]

[0146] Compound 7 (1.43 g, 5 mmol) and hydrazine hydrate (85% by mass, 0.5 mL) were dissolved in 50 mL of anhydrous ethanol. The solution was heated under reflux for 4 h, and the reaction was monitored by TLC (dichloromethane / methanol = 10:1, v / v). After the reaction was complete, the solution was cooled to room temperature and then stored at 0°C for 12 h to allow the solid to precipitate. The solid was then filtered, the filtrate was evaporated to dryness, and the residue was purified by column chromatography (dichloromethane / methanol = 10:1, v / v) to give a yellow solid (compound 8, 0.58 g, 3.7 mmol).

[0147] Step Seven:

[0148]

[0149] Compound 8 (94 mg, 0.6 mmol), fluorenemethoxycarbonyl-L-glutamic acid 1-tert-butyl ester (213 mg, 0.5 mmol), and DIPEA (142 mg, 1.1 mmol) were weighed and dissolved in DMF (5 mL). HATU (228 mg, 0.6 mmol) was added with stirring, and the reaction was carried out overnight at room temperature, monitored by TLC (dichloromethane / methanol = 30:1, v / v). After the reaction was complete, the reaction solution was diluted with 15 mL of deionized water, and then extracted with ethyl acetate (20 mL × 3). The solution was dried over anhydrous sodium sulfate. After filtration, the filtrate was evaporated to dryness, and the residue was purified by column chromatography (dichloromethane / methanol = 30:1, v / v) to give a pale yellow solid (compound 9, 245 mg, 0.4 mmol).

[0150] Step 8:

[0151]

[0152] Compound 9 (74 mg, 0.1 mmol) was dissolved in 1.5 mL of trifluoroacetic acid, and the mixture was stirred at room temperature for 0.5 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in DMF (5 mL). Subsequently, compound 5 (71 mg, 0.1 mmol), DIPEA (38 mg, 0.2 mmol), and HATU (61 mg, 0.2 mmol) were added sequentially, and the reaction mixture was stirred overnight at room temperature. The reaction was monitored by TLC (dichloromethane / methanol = 10:1, v / v). After the reaction was complete, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (dichloromethane / methanol = 10:1, v / v) to give a pale yellow solid (compound 10, 96 mg, 0.1 mmol).

[0153] Step Nine:

[0154]

[0155] Compound 10 (52 mg, 0.05 mmol) was dissolved in DMF (2 mL), and DEA (1 mL) was added. The solution was stirred at room temperature for 2 h. After the reaction was complete, the solvent was removed by rotary evaporation to obtain compound 11.

[0156] Step 10:

[0157]

[0158] Compound 11 was dissolved in DMF (3 mL), followed by the addition of DIPEA (65 mg, 0.5 mmol) and DOTA-NHS-ester (30 mg, 0.06 mmol, dissolved in 2 mL DMF). The mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was evaporated, and the residue was purified by HPLC to obtain compound CAIX-DJ01 (29 mg, 0.03 mmol).

[0159] The product CAIX-DJ01 was characterized by mass spectrometry. The theoretical value was 1200.4764, and the measured value was [M+K]. + : 1239.4402, such as Figure 2 As shown.

[0160] Example 2: Molecular probe [ 68 Preparation and quality control analysis of Ga]Ga-CAIX-DJ01

[0161]

[0162] [ 68 The marking process of Ga]Ga-CAIX-DJ01

[0163] This embodiment provides a molecular probe. 68 The preparation and quality control analysis of Ga-CAIX-DJ01 were performed as follows: An appropriate amount of ligand CAIX-DJ01 was weighed and prepared into a 1 mg / mL ligand solution using DMSO. The germanium-gallium generator was rinsed with 5 mL of high-purity 0.1 M hydrochloric acid solution to obtain [Ga-CAIX-DJ01]. 68 Ga]GaCl3 solution. Take 1 mL of the solution obtained by rinsing. 68 A solution of Ga]GaCl3 was prepared, and 30 μL of ligand solution was added. The pH of the solution was then adjusted to 4.0-4.5 using high-purity sodium acetate solution (1 M). After thorough mixing, the resulting solution was capped and sealed, and reacted at 95°C for 10 min. After the reaction was complete, the solution was cooled to room temperature and then purified using a C18 column to obtain [ 68 Ga]Ga-CAIX-DJ01 labeled solution.

[0164] In the above steps, during the C18 column purification, 10 mL of ethanol and 10 mL of water were used to pre-activate the C18 column. The target product was loaded using a 1 mL syringe. 68 Ga]Ga-CAIX-DJ01 was adsorbed onto a C18 column, then the column was washed with 0.8 mL of anhydrous ethanol, dried under nitrogen at 50°C, and finally 1 mL of physiological saline for injection was added to obtain the purified [Ga-CAIX-DJ01]. 68 Ga]Ga-CAIX-DJ01 solution;

[0165] The quality control of the above products was performed using thin-layer chromatography (TLC) and radio-HPLC. For TLC, rapid chromatography paper was used as the support, and 1 M citric acid solution and 15% HCl methanol solution were used as the developing solvents, respectively. After capillary spotting, the sample was developed in a developing bar. The paper was removed and dried when the solution reached approximately 1 cm from the top of the paper strip. The portion that the solution had reached was divided into 10 equal parts, and the radioactivity count of each part was measured using a γ-counter to calculate its radiochemical purity. The Rf values ​​of each radioactive component in the two systems are shown in the table below.

[0166] Deployment System <![CDATA[[ 68 Ga]GaCl3]]> <![CDATA[[ 68 Ga]Ga(OH)3·xH2O]]> markers 1 M citric acid solution 0.8-1.0 0-0.1 0-0.1 15% HCl methanol solution 0.8-1.0 0-0.1 0.6-1.0

[0167] For radio-HPLC, a C18 column was used. Phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and phase B was acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution conditions were: 0-2 min, 10% B; 2-5 min, 10%-90% B; 5-15 min, 90% B; 15-25 min, 90%-10% B. The flow rate of the mobile phase was 1 mL / min.

[0168] like Figure 3 As shown, this is the [prepared] in Example 2 of the present invention. 68 The radio-HPLC chromatogram of Ga]Ga-CAIX-DJ01 solution showed that... 68 The radiochemical purity of Ga]Ga-CAIX-DJ01 is 99%.

[0169] Example 3: [ 68 In vitro stability test of Ga]Ga-CAIX-DJ01

[0170] This embodiment provides [ 68 The in vitro stability experiment of Ga-CAIX-DJ01 was conducted as follows:

[0171] Experiment 1: The [[] prepared in Example 2] 68 The Ga]Ga-CAIX-DJ01 solution was mixed with physiological saline and allowed to stand at room temperature. The solution was then analyzed by radio-HPLC at 1 h and 2 h. The results are as follows: Figure 4 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ01 exhibits good stability in raw salt at room temperature.

[0172] Experiment 2: The [[] prepared in Example 2] 68 Ga]Ga-CAIX-DJ01 solution was mixed with mouse serum to obtain a mixture, which was then incubated at 37°C. At 1 h and 2 h, the incubation solution was transferred to centrifuge tubes, a small amount of acetonitrile was added to precipitate the protein, centrifuged, and the supernatant was collected. The organic solvent was removed by nitrogen blowing, and then filtered through a 0.22 μm filter membrane. The filtrate was analyzed using radio-HPLC, and the results are shown below. Figure 5 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ01 showed good stability in mouse serum.

[0173] Example 4: [ 68 Experiment on the determination of the lipid-water partition coefficient of Ga]Ga-CAIX-DJ01

[0174] This embodiment provides [ 68 The specific steps for determining the lipid-water partition coefficient of Ga]Ga-CAIX-DJ01 are as follows:

[0175] Take three 5 mL centrifuge tubes, add 0.9 mL of PBS (pH = 7.4) and 1 mL of n-octanol solution to each tube, and then add 0.1 mL of the solution prepared in Example 2. 68Ga]Ga-CAIX-DJ01 solution. Vortex mix thoroughly, then centrifuge (3000 rpm / min) to separate and equilibrate the two phases. Subsequently, take 0.1 mL of each phase solution into plastic tubes, measure the radioactivity counts of both phases using a γ-counter, and calculate the lipid-water partition coefficient log D. 7.4 (log D) 7.4 = C o / C w ), where C o C represents the radioactivity count in the n-octanol phase. w This represents the radioactivity count in the aqueous phase. The average of the three sets of data is taken as the lipid-water partition coefficient, and the result is expressed as mean ± standard deviation.

[0176] Experimental results show that, 68 Ga]Ga-CAIX-DJ01's lipid distribution coefficient log D 7.4 The value is -3.81 ± 0.06, indicating that it is a hydrophilic substance.

[0177] Example 5: [ 68 Imaging experiments of Ga-CAIX-DJ01 on HT-29 and OS-RC-2 tumor-bearing mouse models

[0178] The animal models were HT-29 and OS-RC-2 tumor subcutaneous heterotopic transplantation models established using BABL / c nude mice, which were mouse models constructed from human colorectal cancer and human renal cancer cells, respectively. One animal model of each was randomly selected, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ01 (3.7 MBq, 100 μL) injection solution was injected into mice via the tail vein. Sixty minutes after injection, mice were anesthetized with isoflurane and subjected to PET / CT imaging in a prone position. The imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 6 As shown (the white arrow points to the tumor), [ 68 Ga-CAIX-DJ01 exhibits significant uptake at the tumor site, demonstrating good CA IX targeting. Furthermore, the images show that... 68 Ga]Ga-CAIX-DJ01 is primarily excreted via the kidneys and bladder.

[0179] Example 6: [ 68 Competitive inhibition experiment of Ga-CAIX-DJ01 in HT-29 tumor-bearing rat model

[0180] The animal model is a subcutaneous heterotopic HT-29 tumor transplantation model established in BABL / c nude mice, and a mouse model constructed from human colorectal cancer cells. The same HT-29 tumor-bearing mouse used in Example 5 for the imaging experiment was injected with an inhibitor (compound 1, 200 μg) 1 h prior to the procedure, and then given the same activity and volume of […]. 68 Ga]Ga-CAIX-DJ01 injection. Mice were anesthetized with isoflurane 60 min after injection and subjected to PET / CT imaging in a prone position. The imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 7 As shown (the white arrow points to the tumor), after administration of the inhibitor, the uptake at the tumor site was significantly reduced, indicating that the tumor's response to […]. 68 The uptake of Ga]Ga-CAIX-DJ01 is specific.

[0181] Example 7: [ 68 Biodistribution of Ga]Ga-CAIX-DJ01 in OS-RC-2 tumor-bearing rat model

[0182] The animal model is an OS-RC-2 tumor subcutaneous heterotopic transplantation model established in BABL / c nude mice, which is a mouse model constructed from human renal cell carcinoma cells. Four tumor-bearing mice were randomly selected as a group, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ01 (0.74 MBq, 100 μL) injection solution was injected into mice via the tail vein. Mice were anesthetized and euthanized 60 min after injection. Tumors and other organs or tissues of interest (heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestine, blood) were collected, weighed, and their radioactivity counts were measured using a gamma counter. The uptake was expressed as a percentage of the injected dose per gram of tissue (%ID / g). Results are as follows: Figure 8 As shown, the results indicate that [ 68 Ga]Ga-CAIX-DJ01 is mainly distributed in the kidneys, lungs, stomach, and tumors, with low uptake in other non-target organs, showing good in vivo biodistribution characteristics.

[0183] Example 8: Synthesis of compound CAIX-DJ02 (e.g.) Figure 9 (As shown)

[0184] This embodiment provides a synthetic scheme for a ligand CAIX-DJ02 containing a nitroimidazole group that targets CA IX, wherein the ligand CAIX-DJ02 has the following structure:

[0185]

[0186] Synthesis route:

[0187] Step 1:

[0188]

[0189] Compound 12 (514 mg, 2.0 mmol) and N-hydroxysuccinimide (230 mg, 2.0 mmol) were dissolved in dichloromethane, followed by the addition of dicyclohexylcarbodiimide (DCC, 413 mg, 2.0 mmol). The solution was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered, the filtrate was washed with deionized water (10 mL × 3), dried over anhydrous MgSO4, and then filtered again. The solvent was removed from the filtrate under reduced pressure to give a white solid (compound 13, 606 mg, 1.7 mmol).

[0190] Step Two:

[0191]

[0192] Weigh 129 mg (0.6 mmol) of 3-(1-naphthyl)-alanine and 106 mg (1.0 mmol) of sodium carbonate and dissolve them in 20 mL of a H₂O / dioxane mixed solvent (volume ratio 1:2). Then, add a 1,4-dioxane solution of compound 13 (177 mg, 0.5 mmol) and stir the solution overnight at room temperature. After the reaction is complete, adjust the pH of the system to 3 with 1 M hydrochloric acid solution, then extract with ethyl acetate (20 mL × 3) and dry with anhydrous sodium sulfate. Filter, evaporate the filtrate to dryness, and purify the residue by column chromatography (dichloromethane / methanol = 30:1, v / v) to give a white solid (compound 14, 100 mg, 0.2 mmol).

[0193] Step 3:

[0194]

[0195] Compound 14 (23 mg, 0.05 mmol) and compound 11 (41 mg, 0.05 mmol) were dissolved in DMF (3 mL), followed by the addition of DIPEA (14 mg, 0.11 mmol) and HATU (23 mg, 0.06 mmol) with stirring. The reaction was carried out overnight at room temperature. The reaction was monitored by TLC (dichloromethane / methanol = 10:1, v / v). After the reaction was complete, the solvent was evaporated to dryness, and the residue was purified by column chromatography (dichloromethane / methanol = 10:1, v / v) to give a pale yellow solid (compound 15, 41 mg, 0.03 mmol).

[0196] Step Four:

[0197]

[0198] Compound 15 (41 mg, 0.03 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of trifluoroacetic acid (1 mL), and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in DMF (3 mL). Subsequently, DIPEA (39 mg, 0.3 mmol) and DOTA-NHS-ester (20 mg, 0.04 mmol) were added sequentially, and the solution was reacted overnight at room temperature. After the reaction was complete, the solvent was evaporated, and the residue was purified by HPLC to give a white solid (CAIX-DJ02, 24 mg, 0.02 mmol).

[0199] The product CAIX-DJ02 was characterized by mass spectrometry. The theoretical value was 1536.6602, and the measured value was [M+2H]. 2+ 788.3146, for example Figure 10 As shown.

[0200] Example 9: Molecular probe [ 68 Preparation and quality control analysis of Ga]Ga-CAIX-DJ02

[0201]

[0202] [ 68 The marking process of Ga]Ga-CAIX-DJ02

[0203] This embodiment provides a molecular probe. 68 The preparation and quality control analysis of Ga-CAIX-DJ02 were performed as follows: An appropriate amount of ligand CAIX-DJ02 was weighed and prepared into a 1 mg / mL ligand solution using DMSO. The germanium-gallium generator was rinsed with 5 mL of high-purity 0.1 M hydrochloric acid solution to obtain [Ga-CAIX-DJ02]. 68 Ga]GaCl3 solution. Take 1 mL of the solution obtained by rinsing. 68 A solution of Ga]GaCl3 was prepared, and 30 μL of ligand solution was added. The pH of the solution was then adjusted to 4.0-4.5 using high-purity sodium acetate solution (1 M). After thorough mixing, the resulting solution was capped and sealed, and reacted at 95°C for 10 min. After the reaction was complete, the solution was cooled to room temperature and then purified using a C18 column to obtain [ 68 Ga]Ga-CAIX-DJ02 labeled solution.

[0204] In the above steps, during the C18 column purification, 10 mL of ethanol and 10 mL of water were used to pre-activate the C18 column. The target product was loaded using a 1 mL syringe. 68Ga]Ga-CAIX-DJ02 was adsorbed onto a C18 column, then the column was washed with 0.8 mL of anhydrous ethanol, dried under nitrogen at 50°C, and finally 1 mL of physiological saline for injection was added to obtain the purified [Ga]Ga-CAIX-DJ02. 68 Ga]Ga-CAIX-DJ02 labeled solution;

[0205] The quality control of the above products was performed using thin-layer chromatography (TLC) and radio-HPLC. For TLC, rapid chromatography paper was used as the support, and 1 M citric acid solution and 15% HCl methanol solution were used as the developing solvents, respectively. After capillary spotting, the sample was developed in a developing bar. The paper was removed and dried when the solution reached approximately 1 cm from the top of the paper strip. The portion that the solution had reached was divided into 10 equal parts, and the radioactivity count of each part was measured using a γ-counter to calculate its radiochemical purity. The Rf values ​​of each radioactive component in the two systems are shown in the table below.

[0206] Deployment System <![CDATA[[ 68 Ga]GaCl3]]> <![CDATA[[ 68 Ga]Ga(OH)3·xH2O]]> markers 1 M citric acid solution 0.8-1.0 0-0.1 0-0.1 15% HCl methanol solution 0.8-1.0 0-0.1 0.6-1.0

[0207] For radio-HPLC, a C18 column was used. Phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and phase B was acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution conditions were: 0-2 min, 10% B; 2-5 min, 10%-90% B; 5-15 min, 90% B; 15-25 min, 90%-10% B. The flow rate of the mobile phase was 1 mL / min.

[0208] like Figure 11 As shown, this is the [prepared] in Example 9 of the present invention. 68 The radio-HPLC chromatogram of Ga]Ga-CAIX-DJ02 solution showed that... 68 The radiochemical purity of Ga]Ga-CAIX-DJ02 is 99%.

[0209] Example 10: [ 68 In vitro stability test of Ga]Ga-CAIX-DJ02

[0210] This embodiment provides [ 68 The in vitro stability experiment of Ga-CAIX-DJ02 was conducted as follows:

[0211] Experiment 1: The [[] prepared in Example 9 68 The Ga]Ga-CAIX-DJ02 solution was mixed with physiological saline and allowed to stand at room temperature. The solution was then analyzed by radio-HPLC at 1 h and 2 h. The results are as follows: Figure 12 As shown, the graph displays [ 68Ga]Ga-CAIX-DJ02 exhibits good stability in raw salt at room temperature.

[0212] Experiment 2: The [[] prepared in Example 9 68 Ga]Ga-CAIX-DJ02 solution was mixed with mouse serum to obtain a mixture, which was then incubated at 37°C. At 1 h and 2 h, the incubation solution was transferred to centrifuge tubes, a small amount of acetonitrile was added to precipitate the protein, centrifuged, and the supernatant was collected. The organic solvent was removed by nitrogen blowing, and then filtered through a 0.22 μm filter membrane. The filtrate was analyzed using radio-HPLC, and the results are shown below. Figure 13 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ02 showed good stability in mouse serum.

[0213] Example 11: [ 68 Experiment on the determination of the lipid-water partition coefficient of Ga]Ga-CAIX-DJ02

[0214] This embodiment provides [ 68 The specific steps for determining the lipid-water partition coefficient of Ga]Ga-CAIX-DJ02 are as follows:

[0215] Take three 5 mL centrifuge tubes, add 0.9 mL of PBS (pH = 7.4) and 1 mL of n-octanol solution to each tube, and then add 0.1 mL of the solution prepared in Example 9. 68 Ga]Ga-CAIX-DJ02 solution. Vortex mix thoroughly, then centrifuge (3000 rpm / min) to separate and equilibrate the two phases. Subsequently, take 0.1 mL of each phase solution into plastic tubes, measure the radioactivity counts of both phases using a γ-counter, and calculate the lipid-water partition coefficient log D. 7.4 (log D) 7.4 = C o / C w ), where C o C represents the radioactivity count in the n-octanol phase. w This represents the radioactivity count in the aqueous phase. The average of the three sets of data is taken as the lipid-water partition coefficient, and the result is expressed as mean ± standard deviation.

[0216] Experimental results show that, 68 Ga]Ga-CAIX-DJ02's fat-water partition coefficient log D 7.4 The value is -3.08 ± 0.06, indicating that it is a water-soluble substance.

[0217] Example 12: [ 68 Ga]Ga-CAIX-DJ02 uptake inhibition assay in HT-29 cells

[0218] This embodiment provides [ 68 The uptake and inhibition experiments of Ga-CAIX-DJ02 on CA IX-positive HT-29 cells were conducted to verify its targeting specificity. The specific steps were as follows: HT-29 cells were cultured in DMEM / F12 (1:1) medium until they reached a sufficient density for cell experiments. Cells were digested with 0.25% trypsin, collected in centrifuge tubes, and diluted with medium to 1 × 10⁻⁶ cells / mL. 6 Cells. Add 0.5 mL of the above cell suspension to a cell plate and incubate overnight at 37°C with 5% CO2 to allow adhesion. After aspirating and washing, add 0.1 mL of [ 68 Ga]Ga-CAIX-DJ02 labeled solution (10 μCi / well) and 0.4 mL of culture medium (n = 5) were added, and then incubated at 37°C for 1 h. For the inhibition group, 5 μg of inhibitor (compound 1) was added 1 h in advance for incubation, and the remaining treatments were the same as the normal group. After incubation, the culture medium was aspirated, and the cells were washed twice with ice-cold PBS (containing 0.5% BSA). Finally, 1 M sodium hydroxide solution was added to lyse the cells, and the lysate was collected. The radioactivity count was measured using a γ-counter. The final result is expressed as the percentage of cell radioactivity count relative to the total added radioactive dose (%AD). The experimental results are as follows: Figure 14 As shown, the results indicate that [ 68 Cellular uptake of Ga]Ga-CAIX-DJ02 can be significantly inhibited, demonstrating good targeting specificity.

[0219] Example 13: [ 68 Imaging experiments of Ga-CAIX-DJ02 on HT-29 and OS-RC-2 tumor-bearing mouse models

[0220] The animal models were HT-29 and OS-RC-2 tumor subcutaneous heterotopic transplantation models established using BABL / c nude mice, which were mouse models constructed from human colorectal cancer and human renal cancer cells, respectively. One animal model of each was randomly selected, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ02 (3.7 MBq, 100 μL) injection solution was administered to mice via the tail vein. Sixty minutes post-injection, the mice were anesthetized with isoflurane and subjected to PET / CT imaging in a prone position. The imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 15 As shown (the white arrow points to the tumor), [ 68Ga-CAIX-DJ02 showed significant uptake at mouse tumor sites, with even higher uptake and better imaging contrast in the OS-RC-2 model, which had higher CA IX expression. This indicates that tumor uptake is correlated with CA IX expression levels, demonstrating good CA IX targeting specificity. 68 Compared to Ga-CAIX-DJ01, it has higher tumor uptake and target-to-non-target ratio, higher image contrast, and better imaging quality.

[0221] Example 14: [ 68 Competitive inhibition experiment of Ga]Ga-CAIX-DJ02 in HT-29 tumor-bearing mouse model

[0222] The animal model is a subcutaneous heterotopic transplantation model of HT-29 tumors established in BABL / c nude mice, and a mouse model constructed from human colorectal cancer cells. The same mouse used in Example 6 for the imaging experiment was injected with the inhibitor (compound 1, 200 μg) 1 h in advance, and then given the same activity and volume of […]. 68 Ga]Ga-CAIX-DJ02 injection. Mice were anesthetized with isoflurane 60 min after injection and subjected to PET / CT imaging in a prone position. Imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 16 As shown (the white arrow points to the tumor), after administration of the inhibitor, tumor uptake was significantly reduced, indicating that the tumor's uptake of […]. 68 The uptake of Ga]Ga-CAIX-DJ02 is specific.

[0223] Example 15: [ 68 Biodistribution of Ga]Ga-CAIX-DJ02 in OS-RC-2 tumor-bearing rat model

[0224] The animal model is an OS-RC-2 tumor subcutaneous heterotopic transplantation model established in BABL / c nude mice, which is a mouse model constructed from human renal cell carcinoma cells. Four tumor-bearing mice were randomly selected as a group, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ02 (0.74 MBq, 100 μL) injection solution was injected into mice via the tail vein. Mice were anesthetized and euthanized 60 min after injection. Tumors and other organs or tissues of interest (heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestine, blood) were collected, weighed, and then the radioactivity count of each organ or tissue was measured using a gamma counter. Tissue uptake was expressed as a percentage of the injected dose per gram of tissue (%ID / g). Results are as follows: Figure 17 As shown, the results indicate that [ 68 Ga-CAIX-DJ02 is mainly distributed in the kidneys, lungs, stomach, and tumors, with tumor uptake being significantly higher than [68 Ga]Ga-CAIX-DJ01, while having lower uptake in other non-target organs, shows good in vivo biodistribution characteristics.

[0225] Example 16: Synthesis of compound CAIX-DJ03 (e.g.) Figure 18 (As shown)

[0226] This embodiment provides a synthetic scheme for a ligand CAIX-DJ03 containing a nitroimidazole group that targets CA IX, wherein the ligand CAIX-DJ03 has the following structure:

[0227]

[0228] Synthesis route:

[0229] Step 1:

[0230]

[0231] Compound 16 (502 mg, 2.0 mmol) was weighed and dissolved in tetrahydrofuran (10 mL), followed by the addition of N-hydroxysuccinimide (230 mg, 2.0 mmol) and a solution of dichloromethane (5 mL) containing DCC (413 mg, 2.0 mmol). The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 3:2, v / v) to give a white solid (compound 17, 620 mg, 1.8 mmol).

[0232] Step Two:

[0233]

[0234] Weigh 129 mg (0.6 mmol) of 3-(1-naphthyl)-alanine and 106 mg (1.0 mmol) of sodium carbonate and dissolve them in 20 mL of H₂O / dioxane mixed solvent (volume ratio 1:2). Then, add a 1,4-dioxane solution of compound 17 (174 mg, 0.5 mmol) and stir the solution overnight at room temperature. After the reaction is complete, adjust the pH of the system to 3 with 1 M hydrochloric acid solution, then extract with ethyl acetate (20 mL × 3) and dry with anhydrous sodium sulfate. Filter, evaporate the filtrate to dryness, and purify the residue by column chromatography (petroleum ether / ethyl acetate = 1:1, v / v) to give a white solid (compound 18, 162 mg, 0.4 mmol).

[0235] Step 3:

[0236]

[0237] Compound 18 (23 mg, 0.05 mmol) and compound 11 (41 mg, 0.05 mmol) were dissolved in DMF (3 mL), followed by the addition of DIPEA (14 mg, 0.11 mmol) and HATU (23 mg, 0.06 mmol) with stirring. The reaction was carried out overnight at room temperature. The reaction was monitored by TLC (dichloromethane / methanol = 10:1, v / v). After the reaction was complete, the solvent was evaporated, and the residue was purified by column chromatography (dichloromethane / methanol = 10:1, v / v) to give a pale yellow solid (compound 19, 44 mg, 0.04 mmol).

[0238] Step Four:

[0239]

[0240] Compound 19 (42 mg, 0.03 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of trifluoroacetic acid (1 mL), and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in DMF (3 mL). Subsequently, DIPEA (39 mg, 0.3 mmol) and DOTA-NHS-ester (20 mg, 0.04 mmol) were added sequentially, and the solution was reacted overnight at room temperature. After the reaction was complete, the solvent was evaporated, and the residue was purified by HPLC to give a white solid (CAIX-DJ03, 10 mg, 0.01 mmol).

[0241] The product CAIX-DJ03 was characterized by mass spectrometry. The theoretical value was 1530.6133, and the measured value was [M+H]. + : 1531.6211, such as Figure 19 As shown.

[0242] Example 17: Molecular probe [ 68 Preparation and quality control analysis of Ga-CAIX-DJ03

[0243]

[0244] [ 68 The marking process of Ga]Ga-CAIX-DJ03

[0245] This embodiment provides a molecular probe. 68 The preparation and quality control analysis of Ga-CAIX-DJ03 were performed as follows: An appropriate amount of ligand CAIX-DJ03 was weighed and prepared into a 1 mg / mL ligand solution using DMSO. The germanium-gallium generator was rinsed with 5 mL of high-purity 0.1 M hydrochloric acid solution to obtain [Ga-CAIX-DJ03].68 Ga]GaCl3 solution. Take 1 mL of the solution obtained by rinsing. 68 A solution of Ga]GaCl3 was prepared, and 30 μL of ligand solution was added. The pH of the solution was then adjusted to 4.0-4.5 using high-purity sodium acetate solution (1 M). After thorough mixing, the resulting solution was capped and sealed, and reacted at 95°C for 10 min. After the reaction was complete, the solution was cooled to room temperature and then purified using a C18 column to obtain [ 68 Ga]Ga-CAIX-DJ03 labeled solution.

[0246] In the above steps, during the C18 column purification, 10 mL of ethanol and 10 mL of water were used to pre-activate the C18 column. The target product was loaded using a 1 mL syringe. 68 Ga]Ga-CAIX-DJ03 was adsorbed onto a C18 column, then the column was washed with 0.8 mL of anhydrous ethanol, dried under nitrogen at 50°C, and finally 1 mL of physiological saline for injection was added to obtain the purified [Ga]Ga-CAIX-DJ03. 68 Ga]Ga-CAIX-DJ03 labeled solution;

[0247] The quality control of the above products was performed using thin-layer chromatography (TLC) and radio-HPLC. For TLC, rapid chromatography paper was used as the support, and 1 M citric acid solution and 15% HCl methanol solution were used as the developing solvents, respectively. After capillary spotting, the sample was developed in a developing bar. The paper was removed and dried when the solution reached approximately 1 cm from the top of the paper strip. The portion that the solution had reached was divided into 10 equal parts, and the radioactivity count of each part was measured using a γ-counter to calculate its radiochemical purity. The Rf values ​​of each radioactive component in the two systems are shown in the table below.

[0248] Deployment System <![CDATA[[ 68 Ga]GaCl3]]> <![CDATA[[ 68 Ga]Ga(OH)3·xH2O]]> markers 1 M citric acid solution 0.8-1.0 0-0.1 0-0.1 15% HCl methanol solution 0.8-1.0 0-0.1 0.6-1.0

[0249] For radio-HPLC, a C18 column was used. Phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and phase B was acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution conditions were: 0-2 min, 10% B; 2-5 min, 10%-90% B; 5-15 min, 90% B; 15-25 min, 90%-10% B. The flow rate of the mobile phase was 1 mL / min.

[0250] like Figure 20 As shown, this is the [prepared] in Example 16 of the present invention. 68 The radio-HPLC chromatogram of Ga]Ga-CAIX-DJ03 solution showed that... 68 The radiochemical purity of Ga]Ga-CAIX-DJ03 is 100%.

[0251] Example 18: [ 68 In vitro stability test of Ga]Ga-CAIX-DJ03

[0252] This embodiment provides [ 68 The in vitro stability experiment of Ga-CAIX-DJ03 was conducted as follows:

[0253] Experiment 1: The [[] prepared in Example 17 68 The Ga]Ga-CAIX-DJ03 solution was mixed with physiological saline and allowed to stand at room temperature. The solution was then analyzed by radio-HPLC at 1 h and 2 h. The results are as follows: Figure 21 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ03 exhibits good stability in raw salt at room temperature.

[0254] Experiment 2: The [[] prepared in Example 17 68 The Ga-CAIX-DJ03 solution was mixed with mouse serum to obtain a mixture, which was then incubated at 37°C. At 1 h and 2 h, the incubation solution was transferred to centrifuge tubes, a small amount of acetonitrile was added to precipitate the protein, and the mixture was centrifuged. The supernatant was collected, and the organic solvent was removed by nitrogen blowing, followed by filtration through a 0.22 μm filter membrane. The filtrate was analyzed using radio-HPLC, and the results are shown below. Figure 22 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ03 showed good stability in mouse serum.

[0255] Example 19: [ 68 Experiment on the determination of the lipid-water partition coefficient of Ga]Ga-CAIX-DJ03

[0256] This embodiment provides [ 68 The specific steps for determining the lipid-water partition coefficient of Ga]Ga-CAIX-DJ03 are as follows:

[0257] Take three 5 mL centrifuge tubes, add 0.9 mL of PBS (pH = 7.4) and 1 mL of n-octanol solution to each tube, and then add 0.1 mL of the solution prepared in Example 17. 68 Ga]Ga-CAIX-DJ03 solution. Vortex mix thoroughly, then centrifuge (3000 rpm / min) to separate and equilibrate the two phases. Subsequently, take 0.1 mL of each phase solution into plastic tubes, measure the radioactivity count of both phases using a γ-counter, and calculate the lipid-water partition coefficient log D. 7.4 (log D) 7.4 = C o / C w), where C o C represents the radioactivity count in the n-octanol phase. w This represents the radioactivity count in the aqueous phase. The average of the three sets of data is taken as the lipid-water partition coefficient, and the result is expressed as mean ± standard deviation.

[0258] Experimental results show that, 68 Ga]Ga-CAIX-DJ03's lipid-water partition coefficient log D 7.4 The value of -3.00 ± 0.03 indicates that it is a water-soluble substance.

[0259] Example 20: [ 68 Ga]Ga-CAIX-DJ03 uptake inhibition assay in HT-29 cells

[0260] This embodiment provides [ 68 The uptake and inhibition experiments of Ga-CAIX-DJ03 on CA IX-positive HT-29 cells were conducted to verify its targeting specificity. The specific steps were as follows: HT-29 cells were cultured in DMEM / F12 (1:1) medium until they reached a sufficient density for cell experiments. Cells were digested with 0.25% trypsin, collected in centrifuge tubes, and diluted with medium to 1 × 10⁻⁶ cells / mL. 6 Cells. Add 0.5 mL of the above cell suspension to a cell plate and incubate overnight at 37°C with 5% CO2 to allow adhesion. After aspirating and washing, add 0.1 mL of […]. 68 Ga]Ga-CAIX-DJ03 labeled solution (10 μCi / well) and 0.4 mL of culture medium (n = 5) were added, and then incubated at 37°C for 1 h. For the inhibition group, 5 μg of inhibitor (compound 1) was added 1 h in advance for incubation, and the remaining treatments were the same as the normal group. After incubation, the culture medium was aspirated, and the cells were washed twice with ice-cold PBS (containing 0.5% BSA). Finally, 1 M sodium hydroxide solution was added to lyse the cells, and the lysate was collected. The radioactivity count was measured using a γ-counter. The final result is expressed as the percentage of cell radioactivity count relative to the total added radioactive dose (%AD). The experimental results are as follows: Figure 23 As shown, the results indicate that [ 68 Ga]Ga-CAIX-DJ03 cellular uptake is relatively [ 68 Ga]Ga-CAIX-DJ02 is higher and can also be significantly inhibited, indicating that it has better affinity and target specificity.

[0261] Example 21: [ 68 Imaging experiments of Ga-CAIX-DJ03 on HT-29 and OS-RC-2 tumor-bearing mouse models

[0262] The animal models were HT-29 and OS-RC-2 tumor subcutaneous heterotopic transplantation models established using BABL / c nude mice, which were mouse models constructed from human colorectal cancer and human renal cancer cells, respectively. One animal model of each was randomly selected, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ03 (3.7 MBq, 100 μL) injection solution was injected into mice via the tail vein. Sixty minutes after injection, mice were anesthetized with isoflurane and subjected to PET / CT imaging in a prone position. The imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 24 As shown (the white arrow points to the tumor), [ 68 Ga-CAIX-DJ03 exhibits high tumor uptake and significantly reduced uptake of non-target organs in tumor-bearing mice, resulting in strong image contrast and good imaging performance. 68 Ga]Ga-CAIX-DJ01 and [ 68 Ga]Ga-CAIX-DJ02 is better and has greater potential for clinical application.

[0263] Example 22: [ 68 Competitive inhibition experiment of Ga-CAIX-DJ03 in HT-29 tumor-bearing mouse model

[0264] The animal model is a subcutaneous heterotopic transplantation model of HT-29 tumors established in BABL / c nude mice, and a mouse model constructed from human colorectal cancer cells. The same mouse used in Example 21 for the imaging experiment was injected with the inhibitor (compound 1, 200 μg) 1 h in advance, and then given the same activity and volume of […]. 68 Ga]Ga-CAIX-DJ03 injection. Mice were anesthetized with isoflurane 60 min after injection and subjected to PET / CT imaging in a prone position. Imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 25 As shown (the white arrow points to the tumor), after administration of the inhibitor, tumor uptake was significantly reduced, indicating that the tumor's uptake of […]. 68 The uptake of Ga]Ga-CAIX-DJ03 is specific.

[0265] Example 23: [ 68 Biodistribution of Ga]Ga-CAIX-DJ03 in OS-RC-2 tumor-bearing rat model

[0266] The animal model is an OS-RC-2 tumor subcutaneous heterotopic transplantation model established in BABL / c nude mice, which is a mouse model constructed from human renal cell carcinoma cells. Four tumor-bearing mice were randomly selected as a group, and the labeled and purified […]. 68Ga-CAIX-DJ03 (0.74 MBq, 100 μL) injection solution was injected into mice via the tail vein. Mice were anesthetized and euthanized 60 min after injection. Tumors and other organs or tissues of interest (heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestine, blood) were collected, weighed, and then the radioactivity count of each organ or tissue was measured using a gamma counter. Tissue uptake was expressed as a percentage of the injected dose per gram of tissue (%ID / g). Results are as follows: Figure 26 As shown, the results indicate that [ 68 Ga]Ga-CAIX-DJ03 is mainly distributed in the kidneys, lungs, stomach, and tumors. It has high uptake in tumors and low uptake in non-target organs or tissues, resulting in a good tumor / blood and tumor / meat ratio, showing excellent in vivo biodistribution characteristics.

[0267] Example 24: Synthesis of compound CAIX-DJ04 (e.g.) Figure 27 (As shown)

[0268] This embodiment provides a synthetic scheme for a ligand CAIX-DJ04 containing a nitroimidazole group that targets CA IX, wherein the ligand CAIX-DJ04 has the following structure:

[0269]

[0270] Synthesis route:

[0271] Step 1:

[0272]

[0273] Weigh 517 mg (2.0 mmol) of 3-(1-naphthyl)-alanine and 424 mg (4.0 mmol) of sodium carbonate and dissolve them in 40 mL of a H₂O / dioxane mixed solvent (volume ratio 1:2). Then, add a 1,4-dioxane solution of compound 20 (622 mg, 2 mmol) and stir the solution overnight at room temperature. After the reaction is complete, adjust the pH of the system to 3 with 1 M hydrochloric acid solution, then extract with ethyl acetate (20 mL × 3) and dry with anhydrous sodium sulfate. Filter, evaporate the filtrate to dryness, and purify the residue by column chromatography (petroleum ether / ethyl acetate = 3:2, v / v) to give a pale yellow solid (compound 21, 720 mg, 1.7 mmol).

[0274] Step Two:

[0275]

[0276] Compound 21 (37 mg, 0.09 mmol) and compound 11 (57 mg, 0.07 mmol) were dissolved in DMF (3 mL), followed by the addition of DIPEA (20 mg, 0.15 mmol) and HATU (34 mg, 0.09 mmol) with stirring. The reaction was carried out overnight at room temperature. The reaction was monitored by TLC (dichloromethane / methanol = 10:1, v / v). After the reaction was complete, the solvent was evaporated, and the residue was purified by column chromatography (dichloromethane / methanol = 10:1, v / v) to give a pale yellow solid (compound 22, 50 mg, 0.04 mmol).

[0277] Step 3:

[0278]

[0279] Compound 22 (50 mg, 0.04 mmol) was dissolved in dichloromethane (2 mL), followed by the addition of trifluoroacetic acid (1 mL), and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in DMF (3 mL). Subsequently, DIPEA (52 mg, 0.4 mmol) and DOTA-NHS-ester (25 mg, 0.05 mmol) were added sequentially, and the solution was reacted overnight at room temperature. After the reaction was complete, the solvent was evaporated, and the residue was purified by HPLC to give a white solid (CAIX-DJ04, 13 mg, 0.01 mmol).

[0280] The product CAIX-DJ04 was characterized by mass spectrometry. The theoretical value was 1494.6133, and the measured value was [M+H]. + : 1495.6126, such as Figure 28 As shown.

[0281] Example 25: Molecular probe [ 68 Preparation and quality control analysis of Ga-CAIX-DJ04

[0282]

[0283] [ 68 The marking process of Ga]Ga-CAIX-DJ04

[0284] This embodiment provides a molecular probe. 68 The preparation and quality control analysis of Ga-CAIX-DJ04 were performed as follows: An appropriate amount of ligand CAIX-DJ04 was weighed and prepared into a 1 mg / mL ligand solution using DMSO. The germanium-gallium generator was rinsed with 5 mL of high-purity 0.1 M hydrochloric acid solution to obtain [Ga-CAIX-DJ04]. 68Ga]GaCl3 solution. Take 1 mL of the solution obtained by rinsing. 68 A solution of Ga]GaCl3 was prepared, and 30 μL of ligand solution was added. The pH of the solution was then adjusted to 4.0-4.5 using high-purity sodium acetate solution (1 M). After thorough mixing, the resulting solution was capped and sealed, and reacted at 95°C for 10 min. After the reaction was complete, the solution was cooled to room temperature and then purified using a C18 column to obtain [ 68 Ga]Ga-CAIX-DJ04 labeled solution.

[0285] In the above steps, during the C18 column purification, 10 mL of ethanol and 10 mL of water were used to pre-activate the C18 column. The target product was loaded using a 1 mL syringe. 68 Ga]Ga-CAIX-DJ04 was adsorbed onto a C18 column, then the column was washed with 0.8 mL of anhydrous ethanol, dried under nitrogen at 50 °C, and finally 1 mL of physiological saline for injection was added to obtain the purified [Ga]Ga-CAIX-DJ04. 68 Ga]Ga-CAIX-DJ04 labeled solution;

[0286] The quality control of the above products was performed using thin-layer chromatography (TLC) and radio-HPLC. For TLC, rapid chromatography paper was used as the support, and 1 M citric acid solution and 15% HCl methanol solution were used as the developing solvents, respectively. After capillary spotting, the sample was developed in a developing bar. The paper was removed and dried when the solution reached approximately 1 cm from the top of the paper strip. The portion that the solution had reached was divided into 10 equal parts, and the radioactivity count of each part was measured using a γ-counter to calculate its radiochemical purity. The Rf values ​​of each radioactive component in the two systems are shown in the table below.

[0287] Deployment System <![CDATA[[ 68 Ga]GaCl3]]> <![CDATA[[ 68 Ga]Ga(OH)3·xH2O]]> markers 1 M citric acid solution 0.8-1.0 0-0.1 0-0.1 15% HCl methanol solution 0.8-1.0 0-0.1 0.6-1.0

[0288] For radio-HPLC, a C18 column was used. Phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and phase B was acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution conditions were: 0-2 min, 10% B; 2-5 min, 10%-90% B; 5-15 min, 90% B; 15-25 min, 90%-10% B. The flow rate of the mobile phase was 1 mL / min.

[0289] like Figure 29 As shown, this is the [material] prepared in Example 25 of the present invention. 68 The radio-HPLC chromatogram of Ga]Ga-CAIX-DJ04 solution showed that... 68 The radiochemical purity of Ga]Ga-CAIX-DJ04 is 98%.

[0290] Example 25: [68 In vitro stability test of Ga-CAIX-DJ04

[0291] This embodiment provides [ 68 The in vitro stability experiment of Ga-CAIX-DJ04 was conducted as follows:

[0292] Experiment 1: The [[] prepared in Example 25] 68 The Ga]Ga-CAIX-DJ04 solution was mixed with physiological saline and allowed to stand at room temperature. The solution was then analyzed by radio-HPLC at 1 h and 2 h. The results are as follows: Figure 30 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ04 exhibits good stability in raw salt at room temperature.

[0293] Experiment 2: The [[] prepared in Example 25] 68 Ga]Ga-CAIX-DJ04 solution was mixed with mouse serum to obtain a mixture, which was then incubated at 37°C. At 1 h and 2 h, the incubation solution was transferred to centrifuge tubes, a small amount of acetonitrile was added to precipitate the protein, centrifuged, and the supernatant was collected. The organic solvent was removed by nitrogen blowing, and then filtered through a 0.22 μm filter membrane. The filtrate was analyzed using radio-HPLC, and the results are shown below. Figure 31 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ04 showed good stability in mouse serum.

[0294] Example 26: [ 68 Experiment on the determination of the lipid-water partition coefficient of Ga]Ga-CAIX-DJ04

[0295] This embodiment provides [ 68 The specific steps for determining the lipid-water partition coefficient of Ga]Ga-CAIX-DJ04 are as follows:

[0296] Take three 5 mL centrifuge tubes, add 0.9 mL of PBS (pH = 7.4) and 1 mL of n-octanol solution to each tube, and then add 0.1 mL of the solution prepared in Example 25. 68 Ga]Ga-CAIX-DJ04 solution. Vortex mix thoroughly, then centrifuge (3000 rpm / min) to separate and equilibrate the two phases. Subsequently, take 0.1 mL of each phase solution into plastic tubes, measure the radioactivity counts of both phases using a γ-counter, and calculate the lipid-water partition coefficient log D. 7.4 (log D7.4 = C) o / C w ), where C oC represents the radioactivity count in the n-octanol phase. w This represents the radioactivity count in the aqueous phase. The average of the three sets of data is taken as the lipid-water partition coefficient, and the result is expressed as mean ± standard deviation.

[0297] Experimental results show that, 68 The fat-water partition coefficient of Ga]Ga-CAIX-DJ04, log D 7.4 The value is -3.24 ± 0.06, indicating that it is a water-soluble substance.

[0298] Example 27: [ 68 Imaging experiments of Ga-CAIX-DJ04 on HT-29 and OS-RC-2 tumor-bearing mouse models

[0299] The animal models were HT-29 and OS-RC-2 tumor subcutaneous heterotopic transplantation models established using BABL / c nude mice, which were mouse models constructed from human colorectal cancer and human renal cancer cells, respectively. One animal model of each was randomly selected, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ04 (3.7 MBq, 100 μL) injection solution was administered to mice via the tail vein. Sixty minutes post-injection, mice were anesthetized with isoflurane and subjected to PET / CT imaging in a prone position. Imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 32 As shown, [ 68 Ga-CAIX-DJ04 showed significant uptake at mouse tumor sites, indicating good CA IX targeting. Compared to [ 68 Ga]Ga-CAIX-DJ03, [ 68 Ga]Ga-CAIX-DJ04 has lower uptake in non-target organs and good biodistribution characteristics in vivo.

[0300] Example 28: [ 68 Competitive inhibition experiment of Ga-CAIX-DJ04 in HT-29 tumor-bearing rat model

[0301] The animal model is a subcutaneous heterotopic transplantation model of HT-29 tumors established in BABL / c nude mice, and a mouse model of human colorectal cancer cells. The same mouse used in Example 27 for the imaging experiment was injected with the inhibitor (compound 1, 200 μg) 1 h in advance, and then given the same activity and volume of […]. 68 Ga]Ga-CAIX-DJ04 injection. Mice were anesthetized with isoflurane 60 min after injection and subjected to PET / CT imaging in a prone position. Imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 33As shown (the white arrow points to the tumor), after administration of the inhibitor, tumor uptake was significantly reduced, indicating that the tumor's uptake of […]. 68 The uptake of Ga]Ga-CAIX-DJ04 is specific.

[0302] Example 29: [ 68 Biodistribution of Ga]Ga-CAIX-DJ04 in OS-RC-2 tumor-bearing rat model

[0303] The animal model is an OS-RC-2 tumor subcutaneous heterotopic transplantation model established in BABL / c nude mice, which is a mouse model constructed from human renal cell carcinoma cells. Four tumor-bearing mice were randomly selected as a group, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ04 (0.74 MBq, 100 μL) injection solution was injected into mice via the tail vein. Mice were anesthetized and euthanized 60 min after injection. Tumors and other organs or tissues of interest (heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestine, blood) were collected, weighed, and their radioactivity counts were measured using a gamma counter. The uptake was expressed as a percentage of the injected dose per gram of tissue (%ID / g). Results are as follows: Figure 34 As shown, the results indicate that [ 68 Ga]Ga-CAIX-DJ04 is mainly distributed in the kidneys, lungs, stomach, and tumors, while its uptake in other non-target organs is low, and it exhibits moderate tumor uptake, demonstrating good in vivo biodistribution characteristics.

[0304] Example 29: Synthesis of compound CAIX-DJ05 (e.g.) Figure 35 (As shown)

[0305] This embodiment provides a synthetic scheme for a ligand CAIX-DJ05 containing a nitroimidazole group that targets CA IX, wherein the ligand CAIX-DJ05 has the following structure:

[0306]

[0307] Synthesis route:

[0308] Step 1:

[0309]

[0310] Anhydrous SnCl2 (1.123 g, 5.9 mmol) was dissolved in concentrated hydrochloric acid, and a methanol suspension containing compound 23 (240 mg, 0.99 mmol) was added at room temperature. The mixture was heated under reflux for 2 h. After the reaction was complete, the mixture was cooled to room temperature, and the reaction was terminated by adding 10% Na2CO3 aqueous solution. The mixture was diluted with 40 mL of ethyl acetate, filtered, and the filtrate was washed with deionized water (30 mL × 3) and dried over anhydrous sodium sulfate. The filtrate was filtered, evaporated to dryness, and the residue was purified by column chromatography (petroleum ether / ethyl acetate = 4:1, v / v) to give a reddish-brown oil (compound 24, 100 mg, 0.5 mmol).

[0311] Step Two:

[0312]

[0313] Compounds 12 (49 mg, 0.19 mmol) and 24 (50 mg, 0.25 mmol) were dissolved in acetonitrile, and N-methylimidazole (NMI, 55 mg, 0.67 mmol) and N,N,N′,N′-tetramethylchloroformamide hexafluorophosphate (TCFH, 62 mg, 0.22 mmol) were added sequentially. The mixture was stirred overnight at room temperature. TLC monitoring was performed (petroleum ether / ethyl acetate = 1:1, v / v). After the reaction was complete, the mixture was diluted with deionized water (10 mL) and extracted with ethyl acetate (15 mL × 3). The organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was purified by column chromatography (petroleum ether / ethyl acetate = 1:1, v / v) to give a white solid (compound 25, 47 mg, 0.10 mmol).

[0314] Step 3:

[0315]

[0316] Compound 25 (18 mg, 0.04 mmol) was dissolved in a MeOH / H₂O mixed solvent (v / v = 5:1), and LiOH (5 mg, 0.2 mmol) was added. The mixture was stirred at room temperature for 5 h. After the reaction was complete, the pH of the system was adjusted to 3 with 1 M hydrochloric acid solution, and then extracted with ethyl acetate (10 mL × 3). The organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent was evaporated, and the residue was dissolved in DMF. EDCI (10 mg, 0.05 mmol), HOBt (7 mg, 0.05 mmol), and triethylamine (10 μL, 0.06 mmol) were added sequentially under an ice-water bath, and the mixture was stirred for 0.5 h. Then, compound 11 was added, the mixture was allowed to rise naturally to room temperature, and the reaction was allowed to proceed overnight. The reaction was monitored by TLC (dichloromethane / methanol = 85:15, v / v). After the reaction was completed, the solvent was evaporated, and the residue was purified by column chromatography (dichloromethane / methanol = 85:15, v / v) to give a pale yellow solid (26, 34 mg, 0.03 mmol of compound).

[0317] Step Four:

[0318]

[0319] Compound 26 (32 mg, 0.03 mmol) was dissolved in dichloromethane, and TFA (1 mL) was added. The mixture was stirred at room temperature for 3 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in DMF (3 mL). Then, DIPEA (39 mg, 0.3 mmol) and DOTA-NHS ester (20 mg, 0.04 mmol) were added sequentially, and the solution was stirred at room temperature overnight. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was purified by HPLC to give a pale yellow solid (CAIX-DJ05, 12 mg, 0.01 mmol).

[0320] The product CAIX-DJ05 was characterized by mass spectrometry. The theoretical value was 1508.6289, and the measured value was [M+H]. + : 1509.6369, such as Figure 36 As shown.

[0321] Example 30: Molecular probe [ 68 Preparation and quality control analysis of Ga-CAIX-DJ05

[0322]

[0323] [ 68 The marking process of Ga]Ga-CAIX-DJ05

[0324] This embodiment provides a molecular probe. 68 The preparation and quality control analysis of Ga-CAIX-DJ05 were performed as follows: An appropriate amount of ligand CAIX-DJ05 was weighed and prepared into a 1 mg / mL ligand solution using DMSO. The germanium-gallium generator was rinsed with 5 mL of high-purity 0.1 M hydrochloric acid solution to obtain [Ga-CAIX-DJ05]. 68 Ga]GaCl3 solution. Take 1 mL of the solution obtained from rinsing. 68 A solution of Ga]GaCl3 was prepared, and 30 μL of ligand solution was added. The pH of the solution was then adjusted to 4.0-4.5 using high-purity sodium acetate solution (1 M). After thorough mixing, the resulting solution was capped and sealed, and reacted at 95°C for 10 min. After the reaction was complete, the solution was cooled to room temperature and then purified using a C18 column to obtain [ 68 Ga]Ga-CAIX-DJ05 labeled solution.

[0325] In the above steps, during the C18 column purification, 10 mL of ethanol and 10 mL of water were used to pre-activate the C18 column. The target product was loaded using a 1 mL syringe. 68 Ga]Ga-CAIX-DJ05 was adsorbed onto a C18 column, then the column was washed with 0.8 mL of anhydrous ethanol, dried under nitrogen at 50°C, and finally 1 mL of physiological saline for injection was added to obtain the purified [Ga]Ga-CAIX-DJ05. 68 Ga]Ga-CAIX-DJ05 labeled solution;

[0326] The quality control of the above products was performed using thin-layer chromatography (TLC) and radio-HPLC. For TLC, rapid chromatography paper was used as the support, and 1 M citric acid solution and 15% HCl methanol solution were used as the developing solvents, respectively. After capillary spotting, the sample was developed in a developing bar. The paper was removed and dried when the solution reached approximately 1 cm from the top of the paper strip. The portion that the solution had reached was divided into 10 equal parts, and the radioactivity count of each part was measured using a γ-counter to calculate its radiochemical purity. The Rf values ​​of each radioactive component in the two systems are shown in the table below.

[0327] Deployment System <![CDATA[[ 68 Ga]GaCl3]]> <![CDATA[[ 68 Ga]Ga(OH)3·xH2O]]> markers 1 M citric acid solution 0.8-1.0 0-0.1 0-0.1 15% HCl methanol solution 0.8-1.0 0-0.1 0.6-1.0

[0328] For radio-HPLC, a C18 column was used. Phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and phase B was acetonitrile containing 0.1% trifluoroacetic acid. The gradient elution conditions were: 0-2 min, 10% B; 2-5 min, 10%-90% B; 5-15 min, 90% B; 15-25 min, 90%-10% B. The flow rate of the mobile phase was 1 mL / min.

[0329] like Figure 37 As shown, this is the [prepared] in Example 30 of the present invention. 68 The radio-HPLC chromatogram of Ga]Ga-CAIX-DJ05 solution showed that... 68 The radiochemical purity of Ga]Ga-CAIX-DJ05 is 98%.

[0330] Example 31: [ 68 In vitro stability test of Ga-CAIX-DJ05

[0331] This embodiment provides [ 68 The in vitro stability experiment of Ga-CAIX-DJ05 was conducted as follows:

[0332] Experiment 1: The [[] prepared in Example 30] 68 The Ga]Ga-CAIX-DJ05 solution was mixed with physiological saline and allowed to stand at room temperature. The solution was then analyzed by radio-HPLC at 1 h and 2 h. The results are as follows: Figure 38 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ05 exhibits good stability in raw salt at room temperature.

[0333] Experiment 2: The [[] prepared in Example 30] 68 The Ga-CAIX-DJ05 solution was mixed with mouse serum to obtain a mixture, which was then incubated at 37°C. At 1 h and 2 h, the incubation solution was transferred to centrifuge tubes, a small amount of acetonitrile was added to precipitate the protein, and the mixture was centrifuged. The supernatant was collected, and the organic solvent was removed by nitrogen blowing, followed by filtration through a 0.22 μm filter membrane. The filtrate was analyzed using radio-HPLC, and the results are shown below. Figure 39 As shown, the graph displays [ 68 Ga]Ga-CAIX-DJ05 showed good stability in mouse serum.

[0334] Example 32: [ 68 Experiment on the determination of the lipid-water partition coefficient of Ga]Ga-CAIX-DJ05

[0335] This embodiment provides [ 68 The specific steps for determining the lipid-water partition coefficient of Ga]Ga-CAIX-DJ05 are as follows:

[0336] Take three 5 mL centrifuge tubes, add 0.9 mL of PBS (pH = 7.4) and 1 mL of n-octanol solution to each tube, and then add 0.1 mL of the solution prepared in Example 34. 68Ga]Ga-CAIX-DJ05 solution. Vortex mix thoroughly, then centrifuge (3000 rpm / min) to separate and equilibrate the two phases. Subsequently, take 0.1 mL of each phase solution into plastic tubes, measure the radioactivity counts of both phases using a γ-counter, and calculate the lipid-water partition coefficient log D. 7.4 (log D7.4 = C) o / C w ), where C o C represents the radioactivity count in the n-octanol phase. w This represents the radioactivity count in the aqueous phase. The average of the three sets of data is taken as the lipid-water partition coefficient, and the result is expressed as mean ± standard deviation.

[0337] Experimental results show that, 68 Ga]Ga-CAIX-DJ05's lipid-water partition coefficient log D 7.4 The value is -2.69 ± 0.02, indicating that it is a water-soluble substance.

[0338] Example 33: [ 68 Imaging experiments of Ga-CAIX-DJ05 on HT-29 and OS-RC-2 tumor-bearing mouse models

[0339] The animal models were HT-29 and OS-RC-2 tumor subcutaneous heterotopic transplantation models established using BABL / c nude mice, which were mouse models constructed from human colorectal cancer and human renal cancer cells, respectively. One animal model of each was randomly selected, and the labeled and purified […]. 68 Ga]Ga-CAIX-DJ05 (3.7 MBq, 100 μL) injection solution was administered to mice via the tail vein. Sixty minutes post-injection, mice were anesthetized with isoflurane and subjected to PET / CT imaging in a prone position. Imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 40 As shown, [ 68 Ga]Ga-CAIX-DJ05 showed significant uptake at mouse tumor sites, indicating that it has good CA IX targeting.

[0340] Example 34: [ 68 Competitive inhibition experiment of Ga-CAIX-DJ05 in HT-29 tumor-bearing mouse model

[0341] The animal model is a subcutaneous heterotopic transplantation model of HT-29 tumors established in BABL / c nude mice, and a mouse model of human colorectal cancer cells. The same mouse used in Example 33 for the imaging experiment was injected with the inhibitor (compound 1, 200 μg) 1 h in advance, and then given the same activity and volume of […]. 68Ga]Ga-CAIX-DJ05 injection. Mice were anesthetized with isoflurane 60 min after injection and subjected to PET / CT imaging in a prone position. Imaging data were reconstructed using software to obtain images, which were then analyzed using PMOD software. Results are as follows: Figure 41 As shown (the white arrow points to the tumor), after administration of the inhibitor, tumor uptake was significantly reduced, indicating that the tumor's uptake of […]. 68 The uptake of Ga]Ga-CAIX-DJ05 is specific.

[0342] Example 35: [ 68 Biodistribution of Ga]Ga-CAIX-DJ05 in OS-RC-2 tumor-bearing rat model

[0343] The animal model is an OS-RC-2 tumor subcutaneous heterotopic transplantation model established in BABL / c nude mice, which is a mouse model constructed from human renal cell carcinoma cells. Four tumor-bearing mice were randomly selected as a group, and the labeled and purified […]. 68 Ga-CAIX-DJ05 (0.74 MBq, 100 μL) injection solution was injected into mice via the tail vein. Mice were anesthetized and euthanized 60 min after injection. Tumors and other organs or tissues of interest (heart, liver, lung, kidney, spleen, stomach, bone, muscle, intestine, blood) were collected, weighed, and their radioactivity counts were measured using a gamma counter. The uptake was expressed as a percentage of the injected dose per gram of tissue (%ID / g). Results are as follows: Figure 42 As shown, the results indicate that [ 68 Ga]Ga-CAIX-DJ05 exhibits moderate tumor uptake, with significantly reduced uptake in other non-target organs such as the lungs, kidneys, and stomach.

[0344] The molecular probe obtained by radiolabeling the nitroimidazole group-containing CA IX-targeting compound of the present invention exhibits high tumor uptake and low uptake in non-target organs or tissues in tumor-bearing mice, high image contrast, good imaging effect, and good in vivo biodistribution properties. It is expected to be used for early cancer diagnosis, efficacy monitoring, and radionuclide targeted therapy.

[0345] Obviously, the above-described embodiments are merely illustrative examples and not intended to limit the implementation. Therefore, any modifications, equivalent substitutions, or improvements made without departing from the spirit of this invention are within the scope of protection claimed by this invention.

Claims

1. A structure having the form shown in formula (Ⅰ): （Ⅰ）， in, X and Y are diazole, triazole, or amide groups; R is a 5-10 membered aromatic ring substituted with a sulfonic acid amino group; NM is a nitroimidazole group, selected from any of the following: ， L1, L2, L3, and L4 are functionalized connectors, selected from any of the following: ， n is a positive integer between 1 and 6; L5 is a functionalized linker, selected from any of the following: 。 2. A compound having the structure shown in formula (II): （Ⅱ）， in, X and Y are diazole, triazole, or amide groups; R is a 5-10 membered aromatic ring substituted with a sulfonic acid amino group; NM is a nitroimidazole group, selected from any of the following: ， L1, L2, L3, and L4 are functionalized connectors, selected from any of the following: ， n is a positive integer between 1 and 6; L5 is a functionalized linker, selected from any of the following: 。 3. The compound of formula (II) according to claim 2, wherein the chelator in the structure is a bifunctional chelating agent selected from any of the following: 。 4. The compound of formula (II) according to claim 2, wherein the compound is selected from: (CAIX-DJ01) (CAIX-DJ02) (CAIX-DJ03) (CAIX-DJ04) (CAIX-DJ05) Or its pharmaceutically acceptable salts, stereoisomers, or solvates.

5. A radiolabeled complex containing a nitroimidazole group targeting CA IX, obtained by radiolabeling the compound of formula (II) according to claim 2.

6. The nitroimidazole group containing, CA IX targeting complex according to claim 5, said radionuclide is selected from 18 F, 67 Ga, 68 Ga, 111 In, 45 Ti, 99m Tc, 94m Tc, 64 Cu, 67 Cu, 186 / 188 Re, 43 / 44 Sc, 47 Sc, 52 Mn, 86 Y, 90 Y, 89 Sr, 177 Lu, 153 Sm, 212 / 213 Bi, 212 Pb, 225 Ac, 198 Au, 161 Tb, 149 Pm, 177 Yb, 166 Ho, 131 I, 211 At, 226 / 227 Th.

7. The coordination compound according to claim 5, wherein the compound is selected from: in, M 68 Ga or 177 Lu, [ 68 Ga / 177 Lu]Ga / Lu-CAIX-DJ01, or Where M= 68 Ga or 177 Lu, [ 68 Ga / 177 Lu]Ga / Lu-CAIX-DJ02, or Where M= 68 Ga or 177 Lu, [ 68 Ga / 177 Lu]Ga / Lu-CAIX-DJ03, or Where M= 68 Ga or 177 Lu, [ 68 Ga / 177 Lu]Ga / Lu-CAIX-DJ04, or Among them, M= 68 Ga or 177 Lu, [ 68 Ga / 177 Lu]Ga / Lu-CAIX-DJ05 Or its pharmaceutically acceptable salts, stereoisomers, or solvates.

8. A tumor imaging agent, characterized in that... The developing agent comprises any one of the compounds of claims 1 to 7 that contain a nitroimidazole group and target CA IX.

9. The use of the compound containing a nitroimidazole group targeting CA IX as described in any one of claims 1 to 7 in the preparation of targeted tumor radiodiagnostic / therapeutic drugs.

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