GLP1R targeted radioactive probe, preparation method and application

Abstract

According to the GLP1R targeted radioactive probe, the preparation method and the application, the GLP1R targeted radioactive probe can be specifically combined with GLP1R, has the good in-vivo biological metabolism property, does not cause blood glucose reduction after being injected and is a potential glucagon-like peptide-1 receptor PET imaging probe. The GLP1R targeted radioactive probe is [68Ga] Ga-NOTA-1D32, [68Ga] Ga-NOTA-1D36, or [68Ga] Ga-NOTA-1D39, and the GLP1R targeted radioactive probe is a GLP1R targeted radioactive probe, or a GLP1R targeted radioactive probe, or a GLP1R targeted radioactive probe.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemistry, nanomaterials and biology, and particularly relates to a GLP1R-targeting radioactive probe, a preparation method of the probe, and application of the GLP1R-targeting radioactive probe in preparation of a PET imaging agent for detecting insulinoma. BACKGROUND

[0002] Insulinoma is a neuroendocrine tumor caused by excessive proliferation of islet beta cells, which is usually benign (>90%). However, the symptoms caused by uncontrolled secretion of a large amount of insulin by the tumor can be very serious, which not only affects the quality of life of the patient, but also threatens the life of the patient. Because the tumor is small in size (<1 centimeter), it is difficult to locate the lesion by using traditional imaging techniques such as abdominal ultrasound and computer tomography. Glucagon-like peptide-1 receptor (GLP1R) is a member of the B class subfamily of G protein-coupled receptors, and is also the most characteristic G protein-coupled receptor in the islet, which is widely expressed in islet beta cells, gastrointestinal cells, myocardial cells, vascular smooth muscle cells and hypothalamic nuclei. Therefore, insulinoma can be diagnosed and located by using a specific probe targeting GLP1R, thereby assisting surgical resection.

[0003] Glucagon-like peptide-1 (GLP-1) is a peptide hormone with 30 or 31 amino acids obtained by translation processing of proglucagon peptide, which is produced by amidation and cleavage of the precursor GLP-1 (1-37). However, the plasma half-life of GLP-1 is very short (about 2 minutes), which is not suitable for use as a probe or therapeutic drug. Liraglutide and semaglutide are GLP1R-targeting drugs approved for listing, which are GLP1R agonists that introduce a fatty acid chain into the structure of GLP-1-(7-37) to increase the plasma half-life. Exendin-4 is a natural GLP-1 analogue secreted by the salivary glands of Heloderma suspecturn, which has an agonistic effect on GLP1R, has a suitable plasma half-life (about 2-4 hours), and the artificial synthetic form of exendin-4, exenatide (trade name Byetta, Bai Sida), has been approved by the US FDA for the treatment of type 2 diabetes in 2005, so that the research on exendin-4-type molecular probes is the most extensive.

[0004] In 1994, a radiolabeled exendin-4 analogue [ 125 I]I-[Y 39]exendin-4 was first used to characterize GLP1R on dispersed pancreatic acinar cells and gastric chief cells of guinea pigs. In 2006, Damian Wild et al. reported that [ 111 In]In-DTPA-Lys 40 -exendin-4, biodistribution experiments showed that it could detect specific GLP1R binding sites in the stomach, pancreas, and lungs of rats and mice. Some exendin-4-based PET radioactive probes with the ability to image insulinoma have been reported, such as [ 64 Cu]Cu-(Lys 40 (DOTA)NH2)-exendin-4, [ 68 Ga]Ga / [ 64 Cu]Cu-DO3A-VS-Cys 40 -exendin-4, [ 68 Ga]Ga-NODAGA-exendin-4, etc., but all have the problems of high non-specific uptake in the kidneys, affecting the detection of pancreatic tail lesions. And exendin-4 is a sensitive GLP1R agonist, which can cause physiological reactions such as vomiting and blood sugar reduction after a small amount of injection (~ 5 μg), especially in children, which may affect the health of patients.

[0005] Avexitide [exendin(9-39)], which is obtained by removing the first 8 amino acids from the N terminus of exendin-4, is a GLP-1R antagonist, and some reports have used it for radioactive probe design. [ 111 In]In-BnDTPA-exendin(9-39) SPECT / CT study in mice obtained clear tumor images at 30 minutes after injection. These results suggest that 111In-BnDTPA-exendin(9-39) may be a useful SPECT molecular imaging probe for insulinoma localization. [ 125 I]I-BH-exendin(9-39) can recognize all human and rat tumors expressing GLP-1R with high affinity. [ 18 F]F-FB40-exendin(9-39) has moderate affinity for GLP-1R and accumulates less in the liver and kidneys. However, compared with exendin-4-based probes, exendin(9-39)-based probes generally have insufficient uptake and faster in vivo washout. Studies have shown that the deletion of only one amino acid from the N terminus of the exendin-4 peptide chain can result in a 100-fold decrease in binding affinity, which may be the reason for the undesirable properties of exendin(9-39)-based probes.

[0006] In 2020, Fan Wu et al. analyzed the crystal structure of GLP1R by electron microscopy, and studied the dynamic process of GLP-1 binding to GLP1R by molecular dynamics simulation. The study found that the extracellular region (ECD) of GLP-1R is more flexible in conformation than the transmembrane region (TMD): when there is no GLP-1 binding, its extracellular domain (ECD) usually presents a unique closed conformation, and occasionally forms an open conformation. The binding of GLP-1 to GLP1R presents a dual-domain activation pathway: when GLP1R is in a closed conformation, GLP-1 first binds to the ECD through the C-terminal, causing the receptor to change to an open conformation, and the N-terminal of GLP-1 penetrates into the core region of TMD and binds to the key sites of TMD to play an agonistic role; the partially open GLP1R can directly accommodate GLP-1 to play a role. Therefore, the C-terminal of GLP-1 is crucial for polypeptide-receptor binding, and the hydrophobicity of the C-terminal affects the initial binding to the receptor, while the structure of the N-terminal mainly affects the agonistic effect of the polypeptide on GLP1R.

[0007] Therefore, under the premise of retaining the key binding sites of exendin-4 to GLP1R as much as possible, changing the N-terminal sequence of exendin-4 to weaken the agonistic effect of the polypeptide on GLP1R, designing a new type of radioactive probe that can specifically bind to GLP1R, has good in vivo biological metabolism, and does not cause blood glucose reduction after injection, has become a technical problem urgently to be solved in the technical field. SUMMARY

[0008] To overcome the defects of the prior art, the technical problem to be solved by the present application is to provide a GLP1R-targeted radioactive probe which can specifically bind to GLP1R, has good in vivo biological metabolism, and does not cause blood glucose reduction after injection, and is a more potential glucagon-like peptide-1 receptor PET imaging probe.

[0009] The technical scheme of the present application is that the GLP1R-targeted radioactive probe is [ 68 Ga]Ga-NOTA-1D32, [ 68 Ga]Ga-NOTA-1D36, or [ 68 Ga]Ga-NOTA-1D39, and the structural formula is as follows: .

[0010] A preparation method of the GLP1R-targeted radioactive probe is also provided, in which a chelating agent NOTA and a maleimide are subjected to condensation reaction in the presence of a base and a condensing agent, a protecting group is removed by using an acid, and then 1D32, 1D36, or 1D39 is connected, the obtained product is dissolved in a sodium acetate buffer, and [ 68Ga]GaCl3 solution, mixed well, heated to obtain GLP1R-targeting radioactive probe.

[0011] Another preparation method of the GLP1R-targeting radioactive probe is also provided, which comprises the following steps: (1) Synthesis of labeling precursor of GLP1R-targeting radioactive probe: carboxyl-protected NOTA is dissolved in anhydrous dimethylformamide, and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 1-(2-aminoethyl)-1 H -pyrrole-2,5-dione; after reacting at room temperature for 12 hours, ethyl acetate is added to the mixed solution, which is washed with water and saturated brine; the organic phase is dried with anhydrous magnesium sulfate, filtered, and after removing solid impurities, separated by silica gel column with a mixture of dichloromethane, methanol and ammonia water, and the components are collected, the solvent is removed under reduced pressure, the obtained colorless oil is dissolved in trifluoroacetic acid, stirred at room temperature, and the solvent is removed, and recrystallized with ethanol or diethyl ether; the crude product is dissolved in DMSO dimethyl sulfoxide DMSO, and purified by semi-preparative high performance liquid chromatography HPLC to obtain colorless oil NOTA-MAL; after dissolving NOTA-MAL in DMSO, adding a phosphate buffered saline solution PBS containing 1D32, 1D36 or 1D39, and reacting overnight at room temperature, the reaction solution is purified by semi-preparative HPLC to obtain white flocculent solid NOTA-1D32, NOTA-1D36 or NOTA-1D39; (2) Labeling of GLP1R-targeting radioactive probe: the labeling precursor NOTA-1D32, NOTA-1D36 or NOTA-1D39 is dissolved in sodium acetate buffer to obtain a sodium acetate solution of the labeling precursor, and the obtained 68 Ga]GaCl3 eluate is added to the sodium acetate solution of the labeling precursor, the pH of the system is adjusted to 4.5, and after reacting at 50°C, the solution is diluted with water, cooled, purified by C18 column, and the labeled product is diluted with physiological saline and determined by high performance liquid chromatography with a radioactivity detector to obtain [Ga]Ga-NOTA-1D32, [Ga]Ga-NOTA-1D36 or [Ga]Ga-NOTA-1D39 with radiochemical yield > 95%. 68 Ga]Ga-NOTA-1D32, [Ga]Ga-NOTA-1D36 or [Ga]Ga-NOTA-1D39. 68 Ga]Ga-NOTA-1D32, [Ga]Ga-NOTA-1D36 or [Ga]Ga-NOTA-1D39. 68 Ga]Ga-NOTA-1D32, [Ga]Ga-NOTA-1D36 or [Ga]Ga-NOTA-1D39.

[0012] The GLP1R-targeting radioactive probe is also provided for use in preparing a PET imaging agent for detecting insulinoma.

[0013] The GLP1R targeting radioactive probe of the application shows high specificity to the glucagon-like peptide-1 receptor, has good in-vivo biological metabolism, and does not cause blood sugar reduction after injection, and is a potential glucagon-like peptide-1 receptor PET imaging agent. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 The radio-HPLC spectrum of the labeling reaction solution of [ 68 Ga]Ga-NOTA-1D32 prepared in Example 1 of the application.

[0015] Figure 2 The radio-HPLC spectrum of the labeling reaction solution of [ 68 Ga]Ga-NOTA-1D36 prepared in Example 2 of the application.

[0016] Figure 3 The radio-HPLC spectrum of the labeling reaction solution of [ 68 Ga]Ga-NOTA-1D39 prepared in Example 3 of the application.

[0017] Figure 4 The blood sugar condition chart of normal mice after injection of the NOTA-1D32 or NOTA-1D36 or NOTA-1D39 of the application.

[0018] Figure 5 The kidney uptake comparison chart of the [ 68 Ga]Ga-NOTA-1D32 or the [ 68 Ga]Ga-NOTA-1D36 or the [ 68 Ga]Ga-NOTA-1D39 in insulinoma model mice.

[0019] Figure 6 The key organ distribution comparison chart of the [ 68 Ga]Ga-NOTA-1D32 or the [ 68 Ga]Ga-NOTA-1D36 or the [ 68 Ga]Ga-NOTA-1D39 in insulinoma model mice.

[0020] Figure 7 The in-vivo small animal PET / CT imaging result chart of the [ 68 Ga]Ga-NOTA-1D32 or the [ 68 Ga]Ga-NOTA-1D36 or the [ 68 Ga]Ga-NOTA-1D39 in insulinoma model mice. DETAILED DESCRIPTION

[0021] The GLP1R-targeting radioactive probe is 68 Ga]Ga-NOTA-1D32, 68 Ga]Ga-NOTA-1D36, or 68 Ga]Ga-NOTA-1D39, and has the following structural formula: .

[0022] A preparation method of the GLP1R-targeting radioactive probe is also provided, in which a chelating agent NOTA and a maleimide are subjected to condensation reaction in the presence of a base and a condensing agent, a protecting group is removed by using an acid, and then the GLP1R-targeting radioactive probe is obtained by connecting 1D32, 1D36, or 1D39, dissolving the obtained product in a sodium acetate buffer, adding a [ 68 Ga]GaCl3 solution, uniformly mixing, and heating.

[0023] Another preparation method of the GLP1R-targeting radioactive probe is also provided, which comprises the following steps: (1) Synthesis of a labeling precursor of the GLP1R-targeting radioactive probe: carboxyl-protected NOTA is dissolved in anhydrous dimethylformamide, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and 1-(2-aminoethyl)-1 H pyrrole-2,5-dione are sequentially added to the mixed solution; after reaction at room temperature for 12 hours, ethyl acetate is added to the mixed solution, which is washed with water and saturated brine; the organic phase is dried with anhydrous magnesium sulfate, filtered, and then separated by a silica gel column with a mixture of dichloromethane, methanol, and ammonia water; the components are collected, and the solvent is removed under reduced pressure; the obtained colorless oil is dissolved in trifluoroacetic acid, stirred at room temperature, and then the solvent is removed; recrystallization is performed with ethanol or diethyl ether; the crude product is dissolved in dimethyl sulfoxide (DMSO), and then purified by semi-preparative high performance liquid chromatography (HPLC) to obtain colorless oil NOTA-MAL; after the NOTA-MAL is dissolved in DMSO, a phosphate buffered saline (PBS) solution containing 1D32, 1D36, or 1D39 is added, and the reaction is performed at room temperature overnight; the reaction solution is purified by semi-preparative HPLC to obtain white flocculent solid NOTA-1D32, NOTA-1D36, or NOTA-1D39; (2) Labeling of the GLP1R-targeting radioactive probe: the labeling precursor NOTA-1D32, NOTA-1D36, or NOTA-1D39 is dissolved in a sodium acetate buffer to obtain a sodium acetate solution of the labeling precursor, and then eluted by a germanium-gallium generator to obtain a [ 68The labeling precursor Ga]GaCl3 elution solution is added into sodium acetate solution, the pH of the system is adjusted to 4.5, and the system is reacted at 50°C. After cooling, the system is diluted with water, purified by a C18 column, and the labeled product is diluted with normal saline and determined by high performance liquid chromatography with a radioactivity detector. The radiochemical yield of the product is greater than 95%, 68 Ga]Ga-NOTA-1D32, 68 Ga]Ga-NOTA-1D36, or 68 Ga]Ga-NOTA-1D39.

[0024] The application also provides the use of the GLP1R-targeted radioactive probe in the preparation of a PET imaging agent for detecting insulinoma.

[0025] The GLP1R-targeted radioactive probe of the application has high specificity for the glucagon-like peptide-1 receptor, good in-vivo metabolic properties, and does not cause blood glucose reduction after injection, and is a potential PET imaging agent for the glucagon-like peptide-1 receptor.

[0026] The embodiments of the application will be described in detail below with reference to the accompanying drawings.

[0027] 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 commercially available products, and the equipment used is common equipment.

[0028] Example 1 ([ 68 Ga]Ga-NOTA-1D32) Step 1: Synthesis of the labeling precursor of the radioactive probe NOTA-1D32, namely 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid-N-ethyl succinimide-(cysteine-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-aspartic acid) synthesis; Synthesis reaction equation: ; Synthesis method: The compound NOTA chelator with carboxyl protected (35 mg, 0.1 mmol) was dissolved in anhydrous dimethylformamide, and then benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 57 mg, 0.15 mmol), 1-hydroxybenzotriazole (HOBt, 25.3 mg, 0.15 mmol), N,N-diisopropylethylamine (DIPEA, 41.3 mg, 0.32 mmol) and l-(2-aminoethyl)-lH-pyrrole-2,5-dione (14 mg, 0.1 mmol) were added successively to the mixed solution. After the reaction at room temperature overnight, 30 mL of ethyl acetate was added to the mixed solution, which was washed with water and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, and the solid impurities were removed by filtration. The crude product was separated by silica gel column with dichloromethane / methanol / ammonia water (v / v / v, 90 / 9 / 1), and the obtained colorless oil was dissolved in 5 mL of trifluoroacetic acid, stirred at room temperature for 4 hours. After removing the solvent, the white solid was recrystallized with ethanol / ether. The obtained white solid was dissolved in DMSO, and purified by Semi-HPLC (0.1% TFA aqueous solution / acetonitrile = 9 / 1) to obtain colorless oil NOTA-MAL 62 mg. After dissolving NOTA-MAL with DMSO, 10 μL (1 mg, 1.5 μmol) was added to 3 mL of PBS solution with 1D32 (3 mg, 0.9 μmol), and the reaction was carried out at room temperature overnight. The reaction solution was purified by Semi-HPLC (0.1% TFA aqueous solution / 0.1% TFA acetonitrile = 8 / 2), and freeze-dried to obtain white flocculent solid NOTA-1D32.

[0029] Step 2: Labeling of the radioprobes targeting glucagon-like peptide-1 receptor Labeling reaction equation: ; Forty micrograms of the labeling precursor NOTA-1D32 was dissolved in 93 μL of 1.25 N sodium acetate buffer, and 5 mL of high-purity 0.1 N hydrochloric acid solution was used to elute the germanium gallium generator. One mL of the obtained hydrochloric acid solution of [ 68 Ga]GaCl3was added to the precursor sodium acetate solution, and uniformly mixed. After the reaction at 90°C for 10 min, the solution was purified by C18 column, eluted with 75% ethanol, and the labeling rate was determined by radio-HPLC. The radiochemical yield of [ 68 Ga]Ga-NOTA-1D32 was >95%.

[0030] Example 2 ([ 68 Ga]Ga-NOTA-1D36) Step 1: Synthesis of the labeling precursor of the radioprobes Synthesis of NOTA-1D36, i.e., 2,2',2''-(1,4,7-triazonane-1,4,7-triyl)triacetic acid-N- ethylsuccinimidyl-(cysteine-serine-proline-proline-proline-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-aspartic acid); Synthesis reaction equation: ; Synthesis method: The compound NOTA chelator with carboxyl protected (35 mg, 0.1 mmol) was dissolved in anhydrous dimethylformamide, and benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 57 mg, 0.15 mmol), 1-hydroxybenzotriazole (HOBt, 25.3 mg, 0.15 mmol), N,N-diisopropylethylamine (DIPEA, 41.3 mg, 0.32 mmol) and 1-(2-aminoethyl)-1H-pyrrole-2,5-dione (14 mg, 0.1 mmol) were sequentially added to the mixed solution; after reaction at room temperature overnight, 30 mL of ethyl acetate was added to the mixed solution, which was washed with water and saturated brine; the organic phase was dried with anhydrous magnesium sulfate, and the solid impurities were removed by filtration; the crude product was separated by silica gel column with dichloromethane / methanol / ammonia water (v / v / v, 90 / 9 / 1), and the obtained colorless oil was dissolved in 5 mL of trifluoroacetic acid, stirred at room temperature for 4 hours, and then recrystallized with ethanol / ether after removal of the solvent; the obtained white solid was dissolved in DMSO, and purified by Semi-HPLC (0.1% TFA aqueous solution / acetonitrile = 9 / 1) to obtain colorless oil NOTA-MAL 62 mg; After NOTA-MAL was dissolved in DMSO, 10 μL (1 mg, 1.5 μmol) was taken and added to 3 mL of PBS containing 1D36 (3 mg, 0.9 μmol), and reacted at room temperature overnight; the reaction solution was purified by Semi-HPLC (0.1% TFA aqueous solution / 0.1% TFA acetonitrile = 8 / 2), and freeze-dried to obtain white flocculent solid NOTA-1D36.

[0031] Step 2: Labeling of the radioprobes targeting the glucagon-like peptide-1 receptor Labeling reaction equation: ; 40 μg of the labeled precursor NOTA-1D36 was dissolved in 93 μL of 1.25 N sodium acetate buffer. The germanium-gallium generator was rinsed with 5 mL of high-purity 0.1 N hydrochloric acid solution. The resulting 1 mL [ 68 A hydrochloric acid solution of Ga]GaCl3 was added to a sodium acetate solution of the precursor, mixed thoroughly, and reacted at 90°C for 10 min. The mixture was then purified using a C18 column, eluted with 75% ethanol, and the labeling rate was determined by radio-HPLC, yielding a radiochemical yield >95% for [Ga]GaCl3. 68 Ga]Ga- NOTA-1D36.

[0032] Example 3 ([ 68 Ga]Ga-NOTA-1D39) Step 1: Synthesis of labeled precursors for radioactive probes The synthesis of NOTA-1D39, namely 2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid-N-ethylsuccinimide-(cysteine-serine-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-methionine-glutamine-lysine-serine-leucine-aspartic acid-serine-threonine-phenylalanine-threonine-glycine-glutamic acid-glycine-aspartic acid); Synthesis reaction equation: ; Synthesis method: The compound NOTA chelator with carboxyl protected (35 mg, 0.1 mmol) was dissolved in anhydrous dimethylformamide, and then benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU, 57 mg, 0.15 mmol), 1-hydroxybenzotriazole (HOBt, 25.3 mg, 0.15 mmol), N, N-diisopropyl ethylamine (DIPEA, 41.3 mg, 0.32 mmol) and 1-(2-aminoethyl)-1H-pyrrole-2,5-dione (14 mg, 0.1 mmol) were added successively into the mixed solution. After the reaction at room temperature overnight, 30 mL of ethyl acetate was added into the mixed solution, which was washed with water and saturated brine. The organic phase was dried over anhydrous magnesium sulfate, and the solid impurities were removed by filtration. The crude product was separated by silica gel column with dichloromethane / methanol / ammonia water (v / v / v, 90 / 9 / 1), and the obtained colorless oil was dissolved in 5 mL of trifluoroacetic acid, stirred at room temperature for 4 hours. After removing the solvent, the white solid was recrystallized with ethanol / ether, and then dissolved in DMSO, and purified by Semi-HPLC (0.1% TFA aqueous solution / acetonitrile = 9 / 1) to obtain colorless oil NOTA-MAL 62 mg. After dissolving NOTA-MAL with DMSO, 10 μL (1 mg, 1.5 μmol) was added into 3 mL of PBS containing 1D39 (3 mg, 0.9 μmol), and the reaction was carried out at room temperature overnight. The reaction solution was purified by Semi-HPLC (0.1% TFA aqueous solution / 0.1% TFA acetonitrile = 8 / 2), and freeze-dried to obtain white flocculent solid NOTA-1D39.

[0033] Step 2: Labeling of the radioprobes targeting glucagon-like peptide-1 receptor Labeling reaction equation: ; 45 μg of the labeling precursor NOTA-1D39 was dissolved in 93 μL of 1.25 N sodium acetate buffer, and the germanium-gallium generator was eluted with 5 mL of high-purity 0.1 N hydrochloric acid solution. 1 mL of the obtained hydrochloric acid solution of [ 68 Ga]GaCl3was added into the precursor sodium acetate solution, and uniformly mixed. After the reaction at 90°C for 10 min, the solution was purified by C18 column, eluted with 75% ethanol, and determined for the labeling rate by radio-HPLC to obtain [ 68 Ga]Ga-NOTA-1D39 with radiochemical yield >95%.

[0034] Application Example 1 Effects of 1D32, 1D36 and 1D39 on blood glucose in mice (1) Take normal feeding and drinking water mice, after measuring the initial blood glucose, intraperitoneal injection of 5 ug 1D32 / 1D36 / 1D39 and 0.5 mg glucose (n = 3), control group injection of equal amount of saline and glucose, positive control group injection of 5 ug exendin-4 peptide, negative control group injection of 5 ug peptide, 5, 10, 15, 20, 30, 60, 90, 120 min after injection respectively measure the change of blood glucose of mice.

[0035] (2) From the experimental results can be seen: after injection of exendin-4, the blood glucose of mice decreased rapidly, while after injection of 1D32 / 1D36 / 1D39 and exendin(9-39), the blood glucose of mice changed little compared with the control group, and decreased slowly with the metabolism of the drug 30 minutes after injection. It is proved that 1D32 / 1D36 / 1D39 has similar effect with exendin(9-39), and will not cause acute hypoglycemia after injection.

[0036] As Figure 4 shown, it is the blood glucose chart of normal mice after injection of NOTA-1D32 or NOTA-1D36 or NOTA-1D39 of the application in application example 1.

[0037] Application example 2 [ 68 Ga]Ga-NOTA-1D32 or[ 68 Ga]Ga-NOTA-1D36 or[ 68 Ga]Ga-NOTA-1D39 in tumor-bearing mice (1) The prepared [ 68 Ga]Ga-NOTA-1D32 or[ 68 Ga]Ga-NOTA-1D36 or[ 68 Ga]Ga-NOTA-1D39 solution is diluted with normal saline, and the INS-1 insulin tumor-bearing mice (n = 3) are injected with 60 μCi of the solution through the tail vein, and the positive control group is injected with equal amount of [ 68 Ga]Ga-NOTA-exendin-4, and killed after 15, 30 min, and the interested tissues are taken to measure the radioactivity count; From the results of biodistribution experiment, it can be seen that [ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 has higher uptake in INS-1 insulinoma, and is mainly metabolized through the kidney, with higher kidney uptake; and [ 68Ga]Ga-NOTA-exendin-4 showed reduced uptake compared to tumors, but uptake by non-target organs throughout the body, such as the lungs, pancreas, and kidneys, was also significantly reduced.

[0038] like Figure 5 As shown, this is an experimental analysis of biological distribution in Example 1 of the present invention: [ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Image showing renal uptake of Ga-NOTA-1D39 in mice bearing INS-1 tumors (insulinomas).

[0039] like Figure 6 As shown, this is an experimental analysis of biological distribution in Example 1 of the present invention: [ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 uptake in the tumor, lung, and pancreas of mice bearing A549 tumors (insulinomas).

[0040] Application Example 3 [ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga-NOTA-1D39 in tumor-bearing mice: Small animal PET / CT imaging (1) After the labeling was completed, the solution was diluted with physiological saline, and 100 μCi of drug solution was injected into the tail vein with a syringe. After 15 minutes, the small animal was PET / CT imaged.

[0041] (2) Blocking experiment: Cys 39 -exendin-4 peptide (10mg / kg body weight) and [ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 After mixing Ga]Ga-NOTA-1D39 solution (100μCi), it was injected into the tail vein of a tumor model mouse. After 15 minutes, the small animal was PET / CT imaged.

[0042] (3) The results of the small animal PET / CT imaging experiment show that: [ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 exhibits high specific uptake in insulinomas.

[0043] As Figure 7 shown, the application of the present application in Example 3[ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 in tumor-bearing mice in vivo small animal PET / CT imaging results figure; tumor-bearing mice and blocking tumor uptake of significant difference show[ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 and glucagon-like peptide-1 receptor in insulinoma cells specific binding;[ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 suitable for insulinoma imaging.

[0044] The radioprobes targeting glucagon-like peptide-1 receptor of the present application, in the weak alkaline pH environment of the human body, the 1st amino acid of the antagonist Avexitide [exendin (9-39)] is negatively charged, while the 1st amino acid of the agonist GLP-1 (7-37) and the liraglutide, semaglutide and exendin-4 designed on this basis are positively charged. The charge of the 1st amino acid may affect the agonistic effect of the polypeptide on GLP-1R, and all the polypeptide structures designed in the present application replace the 1st amino acid of exendin-4 with negatively charged amino acid Asp. The hydrophobicity of the C-terminal affects the initial binding with the receptor, and the length of the polypeptide may affect the stable binding of the polypeptide with the receptor, and 1D36 in the present application is connected with hydrophobic residue PPPS at the C-terminal based on 1D32. The present inventors found that the modified[ 68 Ga]Ga-NOTA-1D32 / [ 68 Ga]Ga-NOTA-1D36 / [ 68 Ga]Ga-NOTA-1D39 show high specificity for glucagon-like peptide-1 receptor, and have good in vivo metabolic properties, and do not cause blood glucose reduction after injection, and are potential glucagon-like peptide-1 receptor targeting insulinoma PET imaging agents, which are expected to meet the needs of future clinical insulinoma diagnosis, reduce side effects and reduce kidney uptake.

[0045] The above is only the preferred embodiment of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiment according to the technical essence of the present application are still within the protection scope of the technical solution of the present application.

Claims

1. A GLP1R targeted radioactive probe, characterized in that: It is [ 68 Ga]Ga-NOTA-1D32,[ 68 Ga]Ga-NOTA-1D36, or [ 68 Ga]Ga-NOTA-1D39, the structural formula is as follows: 。 2. The method for preparing the GLP1R targeted radioactive probe according to claim 1, characterized in that: In the presence of a base and a condensing agent, the chelating agent NOA and maleimide undergo a condensation reaction. After deprotecting the group with acid, the product is linked to 1D32, 1D36, or 1D39. The resulting product is dissolved in sodium acetate buffer and […]. 68 The Ga]GaCl3 solution was mixed thoroughly and heated to obtain the GLP1R targeted radioactive probe.

3. The method for preparing the GLP1R targeted radioactive probe according to claim 1, characterized in that: It includes the following steps: (1) Synthesis of the labeled precursor of the GLP1R-targeted radioactive probe: Carboxyl-band-protected NOTA was dissolved in anhydrous dimethylformamide, and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and 1-(2-aminoethyl)-1 H -pyrrole-2,5-dione; after reacting at room temperature for 12 hours, ethyl acetate was added to the mixed solution, and the mixture was washed with water and saturated brine; the organic phase was dried with anhydrous magnesium sulfate, filtered, and after removing solid impurities, it was separated by silica gel column chromatography with a mixture of dichloromethane, methanol and ammonia, the fractions were collected, the solvent was removed under reduced pressure, the resulting colorless oil was dissolved in trifluoroacetic acid, stirred at room temperature and the solvent was removed, and the product was recrystallized with ethanol or ether; the crude product was dissolved in dimethyl sulfoxide (DMSO) and purified by semi-preparative high-performance liquid chromatography (HPLC) to obtain a colorless oil, NOA-MAL; NOA-MAL was dissolved in DMSO and added to PBS containing 1D32, 1D36, or 1D39, and reacted overnight at room temperature. The reaction solution was purified by semi-preparative HPLC to obtain white flocculent solids NOA-1D32, NOA-1D36, or NOA-1D39; (2) Labeling of GLP1R-targeted radioactive probes: Dissolve the labeled precursors Nota-1D32, Nota-1D36, or Nota-1D39 in sodium acetate buffer to obtain a sodium acetate solution of the labeled precursors. Rinse the germanium-gallium generator with the resulting [ 68 [Ga]GaCl3 eluent was added to the sodium acetate solution of the labeled precursor, the pH of the system was adjusted to 4.5, and the reaction was carried out at 50℃. After dilution and cooling with water, the product was purified by C18 column chromatography. The labeled product was diluted with physiological saline and the labeling rate was determined by high performance liquid chromatography with a radiochemical detector. The radiochemical yield was >95%. 68 Ga]Ga-NOTA-1D32,[ 68 Ga]Ga-NOTA-1D36, or [ 68 Ga]Ga-NOTA-1D39.

4. The application of the GLP1R targeted radioactive probe according to claim 1, characterized in that: It is used in the preparation of PET imaging agents for detecting insulinomas.

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