68Ga labeled Exendin 4 radioactive probe and preparation method thereof

Abstract

The present invention relates to a 68 Ga-labeled exendin 4 radioactive probe [ 68 Ga]Ga‑C 12 ‑HBED‑CC‑MAL‑Cys 39 ‑Exendin‑4 and its preparation method belong to the technical field of radiopharmaceutical chemistry; its structural formula is as follows: #imgabs0# The labeling time of the radioactive probe of the present invention is 10 minutes, the labeling yield is greater than 98%, the radiochemical purity of the product determined by radio‑HPLC is greater than 98%, the product shows high affinity and high specificity for the glucagon-like peptide‑1 receptor, has a higher target organ uptake and a longer blood circulation time, and is a more potential glucagon-like peptide‑1 receptor imaging agent.

Description

Technical Field

[0001] The present invention relates to a 68 Ga-labeled exendin 4 radioactive probe [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4 and its preparation method, particularly related to a 68 A Ga-labeled exendin-4 radioactive probe targeting glucagon-like peptide-1 receptor (Glucagon-like peptide-1, GLP-1R) and a preparation method thereof belong to the technical field of radiopharmaceutical chemistry. Background Art

[0002] The International Diabetes Federation (IDF) officially released the tenth edition of the Global Diabetes Atlas on December 6, 2021. The report shows that in 2021, there are 537 million (10.5%) diabetic patients among adults aged 20-79 worldwide, and about 6 million adults die from diabetes or its complications. The total number of people with diabetes is expected to increase to 643 million (11.3%) by 2030 and to 787 million (12.2%) by 2045. In 2021, the number of people with diabetes in China was about 140 million, accounting for 1 / 4 of the total number of people with diabetes in the world. In addition, an estimated 73 million people with diabetes are undiagnosed, which means that almost one in two adult people with diabetes does not know that they have diabetes. Diabetes and its complications bring heavy social and economic burdens to people all over the world. Early diagnosis of diabetes can control the course of the disease, improve the quality of life of patients, and alleviate the economic and social burden brought by the disease.

[0003] Diabetes is mainly divided into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) according to different pathogenesis. The pathological characteristics of T1DM and T2DM are mainly manifested as the reduction of pancreatic β-cell mass (β-cell Mass, BCM) and the loss of β-cell function. The pathogenesis of T1DM is that the β-cells that secrete insulin are destroyed due to autoimmunity or other reasons, and the lack of insulin secretion causes blood sugar to rise. Once a patient is diagnosed with T1DM, he must rely on insulin treatment for life; the onset of T2DM is affected by many factors such as genetics, lifestyle and age. Patients can curb the development of the disease by controlling diet, exercising moderately and taking drugs in the early stage of the disease. About 90% of diabetic patients have T2DM, and T2DM patients often have an insidious onset and no obvious symptoms in the early stage, but at this time, the reduction of pancreatic β-cell mass or the loss of β-cell function has occurred and is irreversible. As the disease progresses, diabetic patients, in addition to increased blood sugar, are accompanied by serious complications such as diabetic nephropathy, cardiovascular disease, retinopathy, nervous system lesions, liver fibrosis, etc., which may even endanger the patient's life. Therefore, early diagnosis of diabetes and timely intervention and treatment are crucial.

[0004] Pancreatic β cells are endocrine cells that produce and secrete insulin, a hormone that regulates blood sugar homeostasis. They form islets with other pancreatic endocrine cells (α cells, δ cells, pp cells, and ε cells, etc.), and are randomly distributed in the pancreatic tissue. The reduction in the mass and dysfunction of pancreatic β cells occurs in the early stages of diabetes and plays a core role in the development of the disease. Therefore, the detection of β cell mass and function is the key to the early diagnosis of diabetes. The number of pancreatic β cells accounts for 60%-80% of the number of pancreatic endocrine cells and only 2% of the total number of pancreatic cells, which poses a huge challenge to the detection of pancreatic β cells. At present, the functional tests commonly used in clinical practice, such as blood sugar testing, insulin testing, and pancreatic C-peptide testing, cannot accurately measure the function of β cells at the level of a single pancreatic islet or β cells; although biopsy and autopsy are the "gold standard" for measuring β cell mass at this stage, the detection method has the disadvantages of invasiveness and sampling uncertainty. Patients not only have to endure the pain of biopsy, but also pancreatic slicing and fixation will affect the activity of pancreatic β cells, thereby affecting the accuracy of pancreatic β cell detection.

[0005] Nuclear medicine positron emission computed tomography (PET) is a non-invasive in vivo imaging technology with the advantages of high sensitivity and high resolution. It has been widely used in the early diagnosis, disease monitoring, efficacy evaluation and new drug development of various major diseases. Nuclear medicine imaging technology has great clinical application prospects in the detection of β-cell quality. The development of high-quality β-cell-specific radioactive probes can fundamentally make up for the shortcomings of clinical β-cell detection, realize non-invasive and accurate monitoring of BCM, and provide reliable molecular probes and technologies for the early diagnosis, disease monitoring and efficacy evaluation of diabetes.

[0006] A variety of β-cell radioactive probes based on different targets have been studied for BCM monitoring. Among them, the glucagon-like peptide 1 receptor (GLP-1R) is the most clearly studied specific receptor for BCM monitoring. GLP-1R belongs to the class B G protein-coupled receptor family, is highly expressed in pancreatic β cells, and is currently an ideal target for the treatment of T2DM. Exenatide (Exendin-4) is the first GLP-1R agonist approved by the US Food and Drug Administration (FDA) for blood sugar control in patients with T2DM. Exendin-4 has a high binding affinity to GLP-1R, can resist degradation by DPP-4, has good in vivo stability, a blood half-life of 2.4h, and high cell binding and internalization. It is the best candidate compound for the development of radioactive probes for monitoring BCM.

[0007] Radiolabeled Exendin-4's ability to monitor β-cell mass allows it to be used not only for early diagnosis of diabetes, but also for imaging and localization of insulinomas. Insulinomas are pancreatic β-cell tumors, also known as endogenous hyperinsulinemic hypoglycemia (EHH). They are a relatively rare neuroendocrine tumor (4 cases per million people per year) and the most common functional pancreatic islet cell tumor in the pancreas. They are characterized by excessive proliferation of pancreatic β-cells, uncontrolled insulin secretion, and clinical symptoms such as hypoglycemia in patients. Most insulinomas are benign tumors, and surgical resection is the main treatment. However, due to the small size of the tumor, traditional imaging techniques such as abdominal ultrasound and computed tomography are difficult to locate the lesions, and incomplete resection of the lesions may cause persistent symptoms. Therefore, the successful treatment of insulinomas depends on precise preoperative localization. The use of β-cell-specific probes can accurately locate insulinomas, guide surgical resection of insulinomas, and achieve accurate diagnosis and treatment of insulinomas.

[0008] At present, several Exendin4-type PET radioactive probes with imaging capabilities for β cells or insulinomas have been reported, such as Ram K. Selvaraju et al. 68 Ga-DO3A-Exendin-4 was used to quantify GLP-1R receptors in the pancreas, and its optimal dose was explored by dose escalation for imaging in rodents, non-human primates, and humans. 68 Ga-DO3A-Exendin-4 human study results, injection 68 5 minutes after Ga-DO3A-Exendin-4, the pancreas, kidneys and liver began to take up Ga-DO3A-Exendin-4. In the following 60 minutes, the uptake in the pancreas and kidneys continued to increase, while other non-target organs were rapidly cleared.

[0009] The Chinese invention patent with patent application number 202110305961.3 and invention name "Exendin-4 radioactive probe targeting glucagon-like peptide-1 receptor and preparation method thereof" discloses an exendin-4 radioactive probe targeting glucagon-like peptide-1 receptor and a preparation method thereof. The labeling time of the radioactive probe is 8 minutes, the labeling yield is >99%, and the radiochemical purity of the product determined by radio-HPLC is >99%. The product shows high affinity and high specificity for the glucagon-like peptide-1 receptor, and has good in vivo biological metabolic properties. It is a more potential glucagon-like peptide-1 receptor PET imaging agent.

[0010] In summary, the radioactive probe with Exendin4 as the targeting group and GLP-1R as the target can be used for BCM monitoring to achieve early diagnosis of diabetes, and can also be used for insulinoma imaging and localization to guide insulinoma treatment, and has received close attention from everyone.

[0011] However, the probes reported so far all have problems such as low specific uptake in pancreatic β cells, excessive nonspecific uptake in the kidneys, and rapid clearance from the body. The pancreas is close to the kidneys, and the high radioactive uptake in the kidneys will seriously interfere with the imaging results, which has become a technical problem that needs to be urgently solved in this field of technology.

[0012] Therefore, there is a lot of room for improvement in radioactive probes with Exendin4 as the targeting group and GLP-1R as the target; 68 The Ga-labeled Exendin 4 radioactive probe has mild labeling reaction conditions and high efficiency, and has higher pancreatic β-cell-specific uptake, insulinoma uptake and target / non-target ratio. It shows high affinity and high specificity for GLP-1R, and has good in vivo biological metabolic properties and high pancreatic radioactive imaging contrast. It has a high target organ uptake and a long blood circulation time. It is an excellent β-cell-specific imaging probe with the potential to be used in the early diagnosis of diabetes and the imaging of insulinomas, which has become a technical problem that urgently needs to be solved in this technical field. Summary of the invention

[0013] One of the purposes of the present invention is to provide a 68 The Ga-labeled Exendin 4 radioactive probe has mild labeling reaction conditions and high efficiency, and has higher pancreatic β-cell specific uptake, insulinoma uptake and target / non-target ratio. It shows high affinity and high specificity for GLP-1R, and has good in vivo biological metabolic properties and high pancreatic radioactive imaging contrast. It has a high target organ uptake and a long blood circulation time. It is an excellent β-cell specific imaging probe with the potential to be used in the early diagnosis of diabetes and the imaging of insulinoma.

[0014] The above object of the present invention is achieved through the following technical solutions:

[0015] A sort of 68 Ga-labeled exendin 4 radioactive probes, respectively [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4, whose structural formulas are as follows:

[0016]

[0017] Another object of the present invention is to provide the above 68 Preparation method of Ga-labeled Exendin 4 radioactive probe.

[0018] The above object of the present invention is achieved through the following technical solutions:

[0019] A sort of 68 The preparation method of Ga-labeled Exendin 4 radioactive probe comprises the following steps:

[0020] (1) In the presence of a base and a condensing agent, the bifunctional linker HBED-CC is subjected to a condensation reaction with the regulating group and maleimide respectively; after removing the protecting group with an acid, the linker reacts with Cys 39 -Exendin-4 connection;

[0021] (2) The obtained product was dissolved in sodium acetate buffer and [ 68 Ga]GaCl3 solution, mix well, heat, and obtain 68 Ga-labeled exendin 4 radioactive probe.

[0022] The reaction formula is as follows:

[0023]

[0024] Preferably, in step (1), the base is N,N-diisopropylethylamine, and the amount added is 3-5 equivalents; the condensing agent is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and the amount added is 1.2 equivalents; and the regulating group is dodecylamine, 4-(p-iodophenyl)butyric acid or 4-(p-methylphenyl)butyric acid.

[0025] Preferably, in step (1), the acid is trifluoroacetic acid, and the amount added is 2 ml; the Cys 39 The polypeptide sequence of -Exendin-4 is: Cysteine-Proline-Proline-Proline-Alanine-Glycine-Serine-Serine-Proline-Glycine-Glycine-Asparagine-Lysine-Leucine-Tryptophan-Glutamic Acid-Isoleucine-Phenylalanine-Leucine-Arginine-Valine-Alanine-Glutamic Acid-Glutamic Acid-Glutamic Acid-Methionine-Glutamine-Lysine-Serine-Leucine-Aspartic Acid-Serine-Threonine-Phenylalanine-Threonine-Glycine-Glutamic Acid-Glycine-Histidine.

[0026] Preferably, 68 The specific preparation steps of Ga-labeled Exendin 4 radioactive probe are as follows:

[0027] Step 1: 68 Synthesis of Ga-labeled Exendin 4 radioactive probe labeling precursor:

[0028] The compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid was dissolved in ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added to the mixed solution in sequence under ice bath condition to obtain a mixed solution; dodecylamine was dissolved in ultra-dry N'N-dimethylformamide, and added dropwise to the mixed solution to react at room temperature (2-6 hours); ethyl acetate was added to the mixture, and the organic phase was washed with water (twice), and then with saturated brine, dried over anhydrous sodium sulfate, filtered to remove solid impurities; the organic solution was removed by vacuum rotary evaporation, and the obtained yellow oily liquid was purified by silica gel column chromatography The crude product was purified by HPLC to obtain a crude product, which was redissolved in ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added in sequence to obtain a mixed solution; 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate was dissolved in ultra-dry N'N-dimethylformamide and added dropwise to the above mixed solution; the mixture was reacted at room temperature (2-6 hours), ethyl acetate was added to the mixture, the organic phase was washed with water (twice), and then with saturated brine, dried with anhydrous sodium sulfate, filtered, and solid impurities were removed; the organic solution was removed by vacuum rotary evaporation, and purified by silica gel column chromatography to obtain a colorless oily compound; the obtained colorless oily compound was dissolved in trifluoroacetic acid, stirred at room temperature (1 hour), a dichloromethane solution was added, diluted, vacuum distilled, the organic solvent was removed, and the mixture was purified by SEMI Pre-HPLC purification was performed to obtain a white solid compound, which was dissolved in phosphate buffer solution and added with Cys 39 -Exendin-4, stirred at room temperature overnight, purified by Semi pre-HPLC to obtain white solid compound C 12 -HBED-CC-MAL-Cys 39 -Exendin-4;

[0029] Step 2: 68 Labeling of Ga-labeled Exendin 4 radioactive probe

[0030] The labeled precursor C prepared in step (1) 12 -HBED-CC-MAL-Cys 39 -Exendin-4 was dissolved in sodium acetate buffer to obtain a sodium acetate solution of the labeled precursor; a germanium gallium generator (iThemba Laboratories, 740MBq, 20mCi) was eluted with a high-purity hydrochloric acid solution, and the obtained [ 68 The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the labeling precursor, mixed evenly, reacted at 50°C (10 min), cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4.

[0031] Preferably, in step 2, the test conditions of the high performance liquid chromatography with a radioactive detector are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v / v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v / v), and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 5%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0032] Preferably, 68 The specific preparation steps of Ga-labeled Exendin 4 radioactive probe are as follows:

[0033] Step 1: 68 Synthesis of Ga-labeled Exendin 4 radioactive probe labeling precursor:

[0034] The compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid was dissolved in ultra-dry N'N-dimethylformamide. Under ice bath conditions, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added to the mixed solution in sequence to obtain a mixed solution. 4-aminobutyl-4-iodophenylbutyramide was dissolved in ultra-dry N'N-dimethylformamide and added dropwise to the mixed solution to react at room temperature (4h). Ethyl acetate was added to the mixture, and the organic phase was washed with water (twice), then with saturated brine, dried over anhydrous sodium sulfate, filtered to remove solid impurities, and the organic solution was removed by vacuum rotary evaporation to obtain a yellow oily liquid. , purified by silica gel column chromatography to obtain a crude product, which was redissolved in ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added in sequence to obtain a mixed solution; 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate was dissolved in ultra-dry N'N-dimethylformamide, and added dropwise to the above mixed solution. After reacting at room temperature (4h), ethyl acetate was added to the mixture, and the organic phase was washed with water (twice), and then washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and solid impurities were removed; the organic solution was removed by vacuum rotary evaporation, and purified by silica gel column chromatography to obtain a colorless oily compound; the obtained colorless oily compound was dissolved in trifluoroacetic acid, stirred at room temperature (1h), diluted with dichloromethane solution, and vacuum distilled to remove the organic solvent, and used Semi Pre-HPLC purification was performed to obtain a white solid compound, which was dissolved in phosphate buffer solution and added with Cys 39 -Exendin-4, stirred at room temperature overnight, and purified by Semi pre-HPLC to obtain a white solid compound pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4;

[0035] Step 2: 68 Labeling of Ga-labeled Exendin 4 radioactive probe

[0036] The labeled precursor pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 was dissolved in sodium acetate buffer to obtain a sodium acetate solution of the labeled precursor, and a germanium gallium generator (iThemba Laboratories, 740MBq, 20mCi) was eluted with a high-purity hydrochloric acid solution to obtain the obtained [ 68The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the labeling precursor, mixed evenly, reacted at 50°C (10 min), cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain a radiochemical yield of >98% [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4.

[0037] Preferably, in step 2, the test conditions of the high performance liquid chromatography with a radioactive detector are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v / v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v / v), and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 5%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0038] Preferably, 68 The specific preparation steps of Ga-labeled Exendin 4 radioactive probe are as follows:

[0039] Step 1: 68 Synthesis of Ga-labeled Exendin 4 radioactive probe labeling precursor:

[0040] The compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid was dissolved in ultra-dry N'N-dimethylformamide, and a mixed solution was obtained under ice bath conditions. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexachloride was added to the mixed solution in sequence. Fluorophosphate, N,N-diisopropylethylamine, to obtain a mixed solution; N-(4-aminobutyl)-4-p-tolylbutanamide was dissolved in ultra-dry N'N-dimethylformamide, and added dropwise to the above mixed solution, and reacted at room temperature (4h) to obtain a mixture; ethyl acetate was added to the mixture, and the organic phase was washed with water (twice), and then with saturated brine, dried over anhydrous sodium sulfate, filtered, and solid impurities were removed; the organic solution was removed by vacuum rotary evaporation to obtain to a yellow oily liquid, which was purified by silica gel column chromatography to obtain a crude product, which was redissolved in ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added in sequence to obtain a mixed solution, 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate was dissolved in ultra-dry N'N-dimethylformamide, and added dropwise to the above mixed solution; after reaction at room temperature (4h), ethyl acetate was added to the mixture, the organic phase was washed with water (twice), and then with saturated brine, dried over anhydrous sodium sulfate, filtered, and solid impurities were removed; the organic solution was removed by vacuum rotary evaporation, and purified by silica gel column chromatography to obtain a colorless oily compound; the obtained colorless oily compound was dissolved in trifluoroacetic acid, stirred at room temperature (1h), diluted with dichloromethane solution, and vacuum distilled to remove the organic solvent, and used Semi Pre-HPLC purification was performed to obtain a white solid compound, which was dissolved in phosphate buffer solution and added with Cys 39 -Exendin-4, stirred at room temperature overnight, purified by Semi pre-HPLC to obtain a white solid compound p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4;

[0041] Step 2: 68 Labeling of Ga-labeled Exendin 4 radioactive probe

[0042] The labeling precursor p-Me-ABM-HBED-CC-MAL-Cys prepared in step 1 39 -Exendin-4 was dissolved in sodium acetate buffer to obtain a sodium acetate solution of the labeled precursor, and a germanium gallium generator (iThemba Laboratories, 740MBq, 20mCi) was eluted with a high-purity hydrochloric acid solution to obtain the obtained [68 The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the labeling precursor, mixed evenly, reacted at 50°C (10 min), cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain a radiochemical yield of >99% [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4.

[0043] Preferably, in step 2, the test conditions of the high performance liquid chromatography with a radioactive detector are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v / v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v / v), and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 5%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0044] Beneficial effects:

[0045] The present invention 68 The Ga-labeled Exendin 4 radioactive probe exhibits high affinity and specificity for the glucagon-like peptide-1 receptor protein, has a higher target organ uptake and a longer blood circulation time, and is a potential glucagon-like peptide-1 receptor imaging agent.

[0046] The present invention is further described below through the accompanying drawings and specific implementation modes, but it is not intended to limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Radio-HPLC spectrum of the labeling reaction solution of Exendin-4.

[0048] Figure 2 [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Radio-HPLC spectrum of the labeling reaction solution of Exendin-4.

[0049] Figure 3 [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39-Radio-HPLC spectrum of the labeling reaction solution of Exendin-4.

[0050] Figure 4 The in vitro INS-1 cell uptake in Example 1 of the present invention [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 uptake-time curve.

[0051] Figure 5 The in vitro INS-1 cell uptake experiment analysis in Example 1 of the present invention [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 specifically binds to the glucagon-like peptide 1 receptor.

[0052] Figure 6 The pancreas, kidney and blood of rats 15 minutes after injection in Application Example 2 of the present invention [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 radioactivity concentration graph.

[0053] Figure 7 In the application example 2 of the present invention, respectively inject [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Ex vivo PET images of some organs of rats 15 minutes after exendin-4. DETAILED DESCRIPTION

[0054] Unless otherwise specified, the reagents and raw materials used in the preparation methods and detection methods described in the following examples and comparative examples are all commercially available products, and the equipment used are all commonly used equipment.

[0055] Unless otherwise specified, the concentrations and ratios described in the following examples and comparative examples are all in mass units.

[0056] Example 1: 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 Preparation of -Exendin-4

[0057] Step 1: 68 Synthesis of Ga-labeled Exendin-4 radioactive probe targeting the glucagon-like peptide-1 receptor

[0058] C 12 -HBED-CC-MAL-Cys 39- Exendin-4 i.e., N,N'-bis[2-hydroxy-5-(acetyldodecylamino)-benzyl]ethylenediamine-N,N'-diacetic acid-N-ethylsuccinimide-(cysteine-proline-proline-proline-alanine-glycine-serine-serine-proline-glycine-glycine-asparagine-lysine-leucine-tryptophan-glutamate-isoleucine-phenylalanine-leucine-arginine-valine-alanine-glutamate-glutamate-glutamate-methionine-glutamine-lysine-serine-leucine-aspartic acid-serine-threonine-phenylalanine-threonine-glycine-glutamate-glycine-histidine);

[0059] Synthesis reaction equation:

[0060]

[0061] Synthesis method:

[0062] Compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid (199 mg, 0.309 mmol) was dissolved in 6 ml of ultra-dry N'N-dimethylformamide. Under ice bath conditions, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (129 mg, 0.340 mmol) was added to the solution. mol), N,N-diisopropylethylamine (80.1 mg, 0.619 mmol), dodecylamine (57.3 mg, 0.309 mmol) was dissolved in 8 ml of ultra-dry N'N-dimethylformamide and added dropwise to the above mixed solution; reacted at room temperature overnight, vacuum rotary evaporation was performed to remove most of it, 40 ml of ethyl acetate was added to the mixture, the organic phase was washed twice with 15 ml of saturated brine, and then washed with 15 ml of water, the organic phase was dried over anhydrous sodium sulfate, filtered, and solid impurities were removed; vacuum rotary evaporation was used to remove The yellow oily liquid obtained by organic solution was purified by silica gel column chromatography to obtain a crude product, which was redissolved in 10 ml of ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (45.6 mg, 0.120 mmol), N,N-diisopropylethylamine (25.9 mg, 0.199 mmol), 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate (23.8 mg, 0.0936 mmol) was dissolved in 8 ml of ultra-dry N'N-dimethylformamide and added dropwise to the above mixed solution; reacted at room temperature overnight, vacuum rotary evaporation was performed to remove most of it, 40 ml of ethyl acetate was added to the mixture, the organic phase was washed twice with 15 ml of saturated brine, and then with 15 ml of water, the organic phase was dried over anhydrous sodium sulfate, filtered to remove solid impurities; vacuum rotary evaporation was performed to remove the organic solution, and the compound C was purified by silica gel column chromatography (dichloromethane / methanol / ammonia water, v / v / v=90 / 10 / 1) to obtain a colorless oily compound C 12 -HBED-CC(t-Bu)2-MAL (32.2 mg, 0.0345 mmol), yield 11.2%;

[0063] Compound C 12 -Confirmation of HBED-CC(t-Bu)2-MAL:

[0064] HRMS theoretical molecular weight C 52 H 80 N5O 10 [M+H] + 934.5900. Measured molecular weight 934.5905

[0065] 1H NMR (600MHz CD3OD): δ7.08-6.47(m,5H),3.56(d,6H,J=120.9Hz),3.21(s,7H),2.99(s,3H),2.66(d,7H J=28.9Hz),2.50-2.17(m,4H),1.35(s,18H),1.31-1.05(m,20H),0.78(d,4H,J=6.5Hz);

[0066] Compound C 12 -HBED-CC(t-Bu)2-MAL (7.20 mg, 7.71 μmol) was dissolved in 2 ml of trifluoroacetic acid, stirred at room temperature for 1 h, and then 2 ml of dichloromethane solution was added, diluted, and distilled under reduced pressure to remove the organic solvent. The compound was purified by semi-pre-HPLC (first mobile phase: 0.1% trifluoroacetic acid aqueous solution, second mobile phase: 0.1% trifluoroacetic acid acetonitrile solution, 0-20 min second mobile phase 5%-100%, UV=280 nm, flow rate 4 ml / min) to obtain a white solid compound. The obtained white solid compound (3.02 mg, 3.67 μmol) was dissolved in 3 ml of PBS (0.0067 M, pH=7.2) solution, and Cys was added under ice bath conditions. 39 -Exedin-4 (5.00 mg, 1.19 μmol), stirred at room temperature overnight, purified by Semi pre-HPLC (first mobile phase: 0.1% trifluoroacetic acid aqueous solution, second mobile phase: 0.1% trifluoroacetic acid acetonitrile solution, 0-15 min second mobile phase 5%-100%, UV = 280 nm, flow rate 4 ml / min) to obtain white solid compound C 12 -HBED-CC-MAL-Cys 39 -Exendin-4 (4.8 mg, 0.955 μmol);

[0067] HRMS theoretical molecular weight C 288 H 348 N 55 O 69 S2[M+3H] 3+ 1674.8279, measured molecular weight 1674.8278; HRMS theoretical molecular weight C 288 H 347 N 55 O 69 NaS2[M+2H+Na] 3+ 1682.1552, measured molecular weight 1682.1488;

[0068] HRMS theoretical molecular weight C 288 H 349 N55 O 69 S2[M+4H] 4+ 1256.3727, measured molecular weight 1256.3691; HRMS theoretical molecular weight C 288 H 348 N 55 O 69 NaS2[M+3H+Na] 4+ 1261.8268, measured molecular weight 1261.8637;

[0069] HRMS theoretical molecular weight C 288 H 350 N 55 O 69 S2[M+5H] 5+ 1005.2996, measured molecular weight 1005.3023; HRMS theoretical molecular weight C 288 H 349 N 55 O 69 NaS2[M+4H+Na] 5+ 1009.6960, measured molecular weight 1009.6891;

[0070] Step 2: 68 Labeling of Ga-labeled Exendin-4 targeted to the glucagon-like peptide-1 receptor

[0071] Label the reaction equation:

[0072]

[0073] Marking method:

[0074] 40 μg of labeled precursor C 12 -HBED-CC-MAL-Cys 39 -Exendin-4 was dissolved in 150 μl of 3N sodium acetate buffer, and a germanium gallium generator (iThemba Laboratories, 740 MBq, 20 mCi) was eluted with 6 ml of high-purity 0.6N hydrochloric acid solution. 300 μl of the obtained [ 68 The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the precursor, mixed evenly, reacted at 50°C for 10 minutes, cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain a radiochemical yield of >98% [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4;

[0075] like Figure 1 As shown, it is [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Radioactive HPLC spectrum of the labeling reaction solution of Exendin-4, the spectrum shows that [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 - The radiochemical purity of Exendin-4 is greater than 98%;

[0076] The test conditions of the high performance liquid chromatography with a radioactive detector described in the above steps are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v / v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v / v), and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 0%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0077] Example 2: 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 preparation, the steps are as follows:

[0078] Step 1: 68 Synthesis of Ga-labeled Exendin-4 radioactive probe targeting the glucagon-like peptide-1 receptor

[0079] The synthesis reaction equation is as follows:

[0080]

[0081] Compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid (140 mg, 0.217 mmol) was dissolved in 6 ml of ultra-dry N'N-dimethylformamide. Under ice bath conditions, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (88.4 mg, 0.232 mmol) and N,N-diisopropylethylamine (55.3 mmol) were added to the solution. g, 0.425 mmol) to obtain a mixed solution; 4-aminobutyl-4-iodophenylbutyramide (77.1 mg, 0.214 mmol) was dissolved in 8 ml of ultra-dry N'N-dimethylformamide and added dropwise to the mixed solution; after overnight reaction at room temperature, most of the N'N-dimethylformamide was removed by vacuum rotary evaporation, 40 ml of ethyl acetate was added to the mixture, the organic phase was washed twice with 15 ml of saturated brine, and then with 15 ml of water, the organic phase was dried over anhydrous sodium sulfate, and solid impurities were removed by filtration; the organic solution was removed by vacuum rotary evaporation, and the obtained yellow oily liquid was purified by simple The crude product obtained by single silica gel chromatography was redissolved in 10 ml of ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (29.9 mg, 0.0787 mmol) and N,N-diisopropylethylamine (15.6 mg, 0.120 mmol) were added in sequence. 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate (15.6 mg, 0.0619 mmol) was dissolved in 8 ml of ultra-dry N'N-dimethylformamide and added dropwise to the above mixed solution; the reaction was allowed to react at room temperature overnight. Afterwards, most of the N'N-dimethylformamide was removed by rotary evaporation under reduced pressure, 40 ml of ethyl acetate was added to the mixture, the organic phase was washed twice with 15 ml of saturated brine and then with 15 ml of water, the organic phase was dried over anhydrous sodium sulfate, and solid impurities were removed by filtration; the organic solution was removed by rotary evaporation under reduced pressure, and the product was purified by silica gel column chromatography (dichloromethane / methanol / ammonia water, v / v / v=90 / 10 / 1) to obtain a colorless oily compound pI-ABM-HBED-CC(t-Bu)2-MAL (35.4 mg, 0.0319 mmol) with a yield of 14.7%.

[0082] Confirmation of compound I-ABM-HBED-CC(t-Bu)2-MAL:

[0083] HRMS theoretical molecular weight C 54 H 75 IN6 11 [M+H] + 1109.4458. Measured molecular weight 1109.4454

[0084] 1 H NMR (600MHz CD3OD): δ7.55(s,1H),7.24-6.51(m,5H),3.58(d,4H) J=50.2Hz),3.20(dd,8H,J=84.2,32.3Hz),2.94-2.45(m,7H),2.33(dd,3H, J=33.4,6.6Hz),2.14(d,2H,J=6.9Hz),1.84(d,2H,J=6.6Hz),1.42(s,18H).

[0085] pI-ABM-HBED-CC(t-Bu)2-MAL (15.3 mg, 13.8 μmol) was dissolved in 2 ml of trifluoroacetic acid, stirred at room temperature for 1 h, and then diluted with 2 ml of dichloromethane solution. The organic solvent was removed by distillation under reduced pressure, and purified by Semi pre-HPLC (first mobile phase: 0.1% trifluoroacetic acid aqueous solution, second mobile phase: 0.1% trifluoroacetic acid acetonitrile solution, 0-20 min second mobile phase 5%-100%, UV=280 nm, flow rate: 4 ml / min) to obtain a white solid compound. The white solid compound (2.88 mg, 2.88 μmol) was dissolved in 3 ml of PBS (0.0067 M, pH=7.2) solution, and Cys was added under ice bath conditions. 39 -Exedin-4 (5.00 mg, 1.19 μmol) was stirred at room temperature overnight and purified by Semi pre-HPLC (first mobile phase: 0.1% trifluoroacetic acid aqueous solution, second mobile phase: 0.1% trifluoroacetic acid acetonitrile solution, 0-15 min second mobile phase 5%-100%, UV = 280 nm, flow rate 4 ml / min) to obtain a white solid compound pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4

[0086] Compound pI-ABM-HBED-CC-MAL-Cys 39 -Confirmation of Exendin-4

[0087] HRMS theoretical molecular weight C 230 H 342 N 56 O 70 S2I[M+3H] 3+ 1733.1131 Measured molecular weight 1733.1126;

[0088] HRMS theoretical molecular weight C 230 H 341 N 55 O 69 NaS2I[M+2H+Na]3+ 1740.4410 Measured molecular weight 1740.4483;

[0089] HRMS theoretical molecular weight C 230 H 343 N 56 O 70 S2I[M+4H] 4+ 1300.0866 Measured molecular weight 1300.0843;

[0090] HRMS theoretical molecular weight C 230 H 342 N 55 O 69 NaS2I[M+3H+Na] 4+ 1305.5821 Measured molecular weight 1305.5819;

[0091] HRMS theoretical molecular weight C 230 H 344 N 56 O 70 S2I[M+5H] 5+ 1040.2707 Measured molecular weight 1040.2662;

[0092] HRMS theoretical molecular weight C 230 H 343 N 55 O 69 NaS2I[M+4H+Na] 5+ 1044.6671 Measured molecular weight 1044.6694;

[0093] Step 2: 68 Labeling of Ga-labeled Exendin-4 targeted to the glucagon-like peptide-1 receptor

[0094] 40 μg of the labeled precursor pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 was dissolved in 150 μl of 3N sodium acetate buffer, and a germanium gallium generator (iThemba Laboratories, 740 MBq, 20 mCi) was eluted with 6 ml of high-purity 0.6N hydrochloric acid solution. 300 μl of the obtained [ 68 The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the precursor, mixed evenly, reacted at 50°C for 10 minutes, cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain a radiochemical yield of >98% [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39-Exendin-4

[0095] like Figure 2 As shown, it is [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Radioactive HPLC spectrum of the labeling reaction solution of Exendin-4, the spectrum shows that [ 68 Ga]pI-ABM-HBED-CC-MAL-Cys 39 - The radiochemical purity of Exendin-4 is greater than 98%;

[0096] The test conditions of the high performance liquid chromatography with a radioactive detector described in the above steps are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v / v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v / v), and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 0%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0097] Example 3: 68 Ga]Ga-p-Me-ABM--HBED-CC-MAL-Cys 39 -Exendin-4 preparation, the steps are as follows:

[0098] Step 1: 68 Synthesis of Ga-labeled Exendin-4 radioactive probe targeting the glucagon-like peptide-1 receptor

[0099] The synthesis reaction equation is as follows:

[0100]

[0101] Compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid (181 mg, 0.281 mmol) was dissolved in 6 ml of ultra-dry N'N-dimethylformamide. Under ice-bath conditions, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (119.4 mg, 0.314 mmol) and N,N-diisopropylethylamine (7 3.3 mg, 0.568 mmol) to obtain a mixed solution, N-(4-aminobutyl)-4-p-tolylbutanamide (70.4 mg, 0.283 mmol) was dissolved in 8 ml of ultra-dry N'N-dimethylformamide and added dropwise to the above mixed solution; the reaction was allowed to react at room temperature overnight, and most of it was removed by rotary evaporation under reduced pressure. 40 ml of ethyl acetate was added to the mixture, and the organic phase was washed twice with 15 ml of saturated brine and then with 15 ml of water. The organic phase was dried over anhydrous sodium sulfate and filtered to remove solid impurities; the organic solution was removed by rotary evaporation under reduced pressure, and the obtained yellow oil was obtained. The crude product was purified by silica gel column chromatography to obtain a crude product, which was redissolved in 10 ml of ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (98.8 mg, 0.260 mmol) and N,N-diisopropylethylamine (55.9 mg, 0.430 mmol) were added in sequence to obtain a mixed solution. 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate (56.1 mg, 0.221 mmol) was dissolved in 8 ml of ultra-dry N'N-dimethylformamide and added dropwise to the mixture. The above mixed solution was reacted at room temperature overnight, and most of it was removed by rotary evaporation under reduced pressure. 40 ml of ethyl acetate was added to the mixture, and the organic phase was washed twice with 15 ml of saturated brine, and then washed with 15 ml of water. The organic phase was dried over anhydrous sodium sulfate and filtered to remove solid impurities. The organic solution was removed by rotary evaporation under reduced pressure, and purified by silica gel column chromatography (dichloromethane / methanol / ammonia water, v / v / v=90 / 10 / 1) to obtain p-Me-ABM--HBED-CC-MAL (37.1 mg, 0.0372 mmol) as a colorless oil with a yield of 13.2%;

[0102] Confirmation of compound p-Me-ABM--HBED-CC(t-Bu)2-MAL:

[0103] HRMS theoretical molecular weight C 55 H 77 N6O 11 [M+H] + 997.5645, measured molecular weight 997.5638;

[0104] 1 H NMR(400MHz CD3OD): δ6.93(s,3H),6.86(d,2H,J=1.6Hz),6.71-6.62(m,2H),6.55(dd,2H,J= 8.2,3.4Hz),3.53(s,2H),3.48-3.40(m,3H),3.25-3.15(m,2H),3.09(s,4H),3.0 1(t,4H,J=6.1Hz),2.69-2.56(m,4H),2.54(s,3H),2.49-2.38(m,2H),2.30-2.1 6(m,3H),2.15(s,2H),2.10-1.99(m,2H),1.83-1.67(m,2H),1.41-1.21(m,18H);

[0105] The compound p-Me-ABM--HBED-CC(t-Bu)2-MAL (18.7 mg, 18.7 μmol) was dissolved in 2 ml of trifluoroacetic acid, stirred at room temperature for 1 h, and then 2 ml of dichloromethane solution was added, diluted, and distilled under reduced pressure to remove the organic solvent. The compound was purified by Semipre-HPLC (first mobile phase: 0.1% trifluoroacetic acid aqueous solution, second mobile phase: 0.1% trifluoroacetic acid acetonitrile solution, 0-15 min second mobile phase 5%-100%, UV=280 nm, flow rate 4 ml / min) to obtain a white solid compound p-Me-ABM--HBED-CC-MAL (2.65 mg, 2.99 μmol), which was dissolved in 3 ml of PBS (0.0067 M, pH=7.2) solution, and Cys was added under ice bath conditions. 39 -Exedin-4 (5.00 mg, 1.19 μmol) was stirred at room temperature overnight and purified by Semi pre-HPLC (first mobile phase: 0.1% trifluoroacetic acid aqueous solution, second mobile phase: 0.1% trifluoroacetic acid acetonitrile solution, 0-15 min second mobile phase 5%-100%, UV = 280 nm, flow rate 4 ml / min) to obtain a white solid compound p-Me-ABM--HBED-CC-MAL-Cys 39 -Exendin-4;

[0106] Compound p-Me-ABM--HBED-CC-MAL-Cys 39 -Confirmation of Exendin-4:

[0107] HRMS theoretical molecular weight C 231 H 345 N 56 O 70 S2[M+3H] 3+1695.8194, measured molecular weight 1695.8191;

[0108] HRMS theoretical molecular weight C 231 H 344 N 55 O 69 NaS2[M+2H+Na] 3+ 1703.1367, measured molecular weight 1703.1387;

[0109] HRMS theoretical molecular weight C 231 H 346 N 56 O 70 S2[M+4H] 4+ 1272.1163, measured molecular weight 1272.1133;

[0110] HRMS theoretical molecular weight C 231 H 345 N 55 O 69 NaS2[M+3H+Na] 4+ 1277.6118, measured molecular weight 1277.6024;

[0111] HRMS theoretical molecular weight C 231 H 347 N 56 O 70 S2[M+5H] 5+ 1017.8945, measured molecular weight 1017.8886;

[0112] HRMS theoretical molecular weight C 231 H 346 N 55 O 69 NaS2[M+4H+Na] 5+ 1022.2909, measured molecular weight 1022.2938;

[0113] Step 2: 68 Labeling of Ga-labeled Exendin-4 targeted to the glucagon-like peptide-1 receptor

[0114] 40 μg of the labeling precursor p-Me-ABM-HBED-CC-MAL-Cys prepared in step 1 was added 39 -Exendin-4 was dissolved in 150 μl of 3N sodium acetate buffer, and a germanium gallium generator (iThemba Laboratories, 740 MBq, 20 mCi) was eluted with 6 ml of high-purity 0.6N hydrochloric acid solution. 300 μl of the obtained [ 68The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the precursor, mixed evenly, reacted at 50°C for 10 minutes, cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography (radio-HPLC) with a radioactive detector to obtain a radiochemical yield of >98% [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4;

[0115] like Figure 3 As shown, it is [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Radioactive HPLC spectrum of the labeling reaction solution of Exendin-4, the spectrum shows that [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 - The radiochemical purity of Exendin-4 is greater than 98%;

[0116] The test conditions of the high performance liquid chromatography with a radioactive detector described in the above steps are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution (v / v), the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution (v / v), and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 0%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

[0117] Application Example 1

[0118] [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 uptake into INS-1 cells in vitro

[0119] INS-1 cells were prepared to 2×10 6 500 μL of the cell suspension was taken into 45 sterile EP tubes, and the 45 EP tubes were divided into three groups, with 15 tubes in each group. 37 KBq of [ 68 Ga]Ga-C 12-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 or [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 solution, at 37°C, at 30, 60, 90 and 120 minutes of incubation in each group, 3 EP tubes containing cells were taken out from each group, and PBS solution was used to terminate their uptake, shaken and mixed, centrifuged for 5 minutes, and the centrifuge tubes were inserted into plastic test tubes to measure their radioactivity counts; Cys 39 -Exendin-4, at 60 minutes, blocked the cell uptake. After the incubation, PBS solution was used to terminate its uptake, and the mixture was shaken and centrifuged for 5 minutes. The centrifuge tube was inserted into a plastic test tube and its radioactivity count was determined.

[0120] like Figure 4 The results are shown in Example 1 of the present invention, in vitro INS-1 cell uptake [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 uptake-time curve (n=3);

[0121] like Figure 5 The figure shows the in vitro INS-1 cell uptake experimental analysis in Application Example 1 of the present invention. 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39- Specific binding diagram of Exendin-4 to glucagon-like peptide 1 receptor (n=3).

[0122] The results of in vitro cell uptake experiments showed that INS-1 cells and [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39

[0123] -Exendin-4, with the extension of incubation time, the uptake gradually increased, among which [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 The cellular uptake of -Exendin-4 at each time point was significantly higher than that of the other two radioactive probes. 39 In the presence of -Exendin-4 peptide, the uptake of the radioactive probe is significantly reduced, indicating that the uptake of the probe by INS-1 cells is specific, and the binding of GLP-1R to the radioactive probe of the present invention is specific.

[0124] Application Example 2

[0125] [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 in vitro imaging:

[0126] Male SD rats aged 4-5 weeks weighing 130 to 150 g were fasted for 12 hours and 3.7 MBq [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39-Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 solution was injected into the tail vein of SD rats. After 15 minutes, the rats were decapitated and the organs of interest were removed by dissection and placed on polyamide thin layer plates. After 10 minutes of PET scanning, the organs of interest were placed in plastic test tubes, weighed and their radioactivity counts were measured;

[0127] like Figure 6 As shown, the injection in Application Example 2 of the present invention [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 radioactivity concentrations in the pancreas, kidneys and blood of rats 15 minutes after injection; 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4, the radioactivity concentrations in the pancreas, kidneys and blood were measured 15 minutes later. Figure 6 It can be seen that: 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4,

[0128] [ 68Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4, the pancreatic uptake and blood retention at 15 minutes after injection were higher than [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, renal uptake was significantly lower than [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4.

[0129] like Figure 7 As shown, it is the in vitro PET imaging of some organs in Example 2 of the present invention. From the in vitro PET imaging, it can be seen that the reported GLP-1R targeted radioactive probe [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4 is metabolized rapidly and mainly through the kidneys. 15 minutes after injection, most of the radioactive tracer is rapidly metabolized to the kidneys. Under this chromatic contrast condition, pancreatic uptake is not obvious; [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4 is mainly metabolized by the liver, with significantly reduced uptake by the kidneys and a certain uptake by the pancreas; 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 is mainly metabolized by the kidneys, and the pancreatic uptake is significant and higher than that reported previously [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4.

[0130] The present invention 68 Ga-labeled Exendin 4 radioactive probe ( 68 Ga-labeled radioactive probe targeting glucagon-like peptide 1 receptor) with excellent 68 The Ga labeling properties are due to the excellent Ga 3+Bifunctional linker, HBED-CC with Ga 3+ The thermodynamic stability constant is high (logK ML :38.5), the coordination energy is low, therefore, [ 68 The labeling of Ga]Ga-HBED-CC is fast and efficient. 68 Ga-labeled Exendin 4 radioactive probe ( 68 The Ga-labeled radioactive probe targeting the glucagon-like peptide 1 receptor can be quickly prepared by heating at 50°C for 5 minutes. The product has high radiochemical yield and radiochemical purity (both >98%) and high stability.

[0131] The present invention 68 Ga-labeled Exendin 4 radioactive probe ( 68 Ga-labeled radioactive probe targeting glucagon-like peptide 1 receptor) contains Exendin-4 structure, which can ensure that the present invention has good glucagon-like peptide 1 receptor affinity; compared with the reported [ 68 Ga]Ga-HBED-CC-MAL-Cys 39 -Exendin-4, [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4 also has a lipophilic group, which allows it to be metabolized by the liver, preventing excessive kidney uptake and affecting pancreatic imaging; [ 68 Ga]Ga-p-Me-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 has a plasma albumin binding group, which ensures that the present invention has a higher blood drug concentration and a longer blood retention time, and improves the problem of low target organ uptake caused by too fast blood clearance. 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 and [ 68 Ga]Ga-pI-ABM-HBED-CC-MAL-Cys 39 -Exendin-4 pancreatic uptake was significant and higher than that reported previously [ 68 Ga]Ga-HBED-CC-MAL-Cys 39-Exendin-4. Therefore, the Exendin-4 radioactive probe targeting the glucagon-like peptide-1 receptor of the present invention can be used for β cell mass measurement and insulinoma imaging.

[0132] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. 68 Ga-labeled Exendin 4 radioactive probe [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4, whose structural formula is as follows:

2. According to claim 1 68 Ga-labeled exendin 4 radioactive probe [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -The preparation method of Exendin-4, the specific steps are as follows: Step 1: 68 Synthesis of Ga-labeled Exendin 4 radioactive probe labeling precursor: The compound 3,3'-(((2,2,13,13-tetramethyl-4,11-dioxo-3,12-dioxa-6,9-diazatetradecane-6,9-diyl)bis(methylene))bis(4-hydroxy-3,1-phenylene))dipropionic acid was dissolved in ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added to the mixed solution in sequence under ice bath condition to obtain a mixed solution; dodecylamine was dissolved in ultra-dry N'N-dimethylformamide, and added dropwise to the mixed solution to react at room temperature; ethyl acetate was added to the mixture, and the organic phase was washed with water and then with saturated brine, dried over anhydrous sodium sulfate, filtered to remove solid impurities; the organic solution was removed by vacuum rotary evaporation, and the obtained yellow oily liquid was purified by silica gel Column chromatography was performed to obtain a crude product, which was redissolved in ultra-dry N'N-dimethylformamide, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added in sequence to obtain a mixed solution; 1-(2-aminoethyl)-1H-pyrrole-2,5-dione trifluoroacetate was dissolved in ultra-dry N'N-dimethylformamide and added dropwise to the above mixed solution; the mixture was reacted at room temperature, ethyl acetate was added to the mixture, the organic phase was washed with water, and then with saturated brine, dried with anhydrous sodium sulfate, filtered, and solid impurities were removed; the organic solution was removed by vacuum rotary evaporation, and the mixture was purified by silica gel column chromatography to obtain a colorless oily compound; the obtained colorless oily compound was dissolved in trifluoroacetic acid, stirred at room temperature, a dichloromethane solution was added, diluted, vacuum distilled, the organic solvent was removed, and the mixture was purified by Semi pre-HPLC to obtain a white solid compound, which was dissolved in a phosphate buffer solution, and Cys was added under ice bath conditions. 39 -Exendin-4, stirred at room temperature overnight, purified by Semi pre-HPLC to obtain white solid compound C 12 -HBED-CC-MAL-Cys 39 -Exendin-4; Step 2: 68 Labeling of Ga-labeled Exendin 4 radioactive probe The labeled precursor C prepared in step 1 12 -HBED-CC-MAL-Cys 39 -Exendin-4 was dissolved in sodium acetate buffer to obtain a sodium acetate solution of the labeled precursor; the germanium gallium generator was eluted with a high-purity hydrochloric acid solution, and the obtained [ 68 The hydrochloric acid solution of Ga]GaCl3 was added to the sodium acetate solution of the labeling precursor, mixed evenly, reacted at 50°C, cooled to room temperature, and the labeling rate was determined by high performance liquid chromatography with a radioactive detector to obtain [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -Exendin-4.

3. According to claim 2 68 Ga-labeled exendin 4 radioactive probe [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -The preparation method of Exendin-4 is characterized by: The reaction time at room temperature in step 1 is 2-6 hours.

4. According to claim 3 68 Ga-labeled Exendin 4 radioactive probe [ 68 Ga]Ga-C 12 -HBED-CC-MAL-Cys 39 -The preparation method of Exendin-4 is characterized by: In step 2, the test conditions of the high performance liquid chromatography with a radioactive detector are: the first mobile phase is 0.1% trifluoroacetic acid aqueous solution, the second mobile phase is 0.1% trifluoroacetic acid acetonitrile solution, and the gradient elution conditions are: 0-10 minutes, 95%-0% of the first mobile phase; 10-11 minutes, 5%-95% of the first mobile phase; 11-15 minutes, 5% of the first mobile phase; the flow rate of the mobile phase is 1 ml / min.

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