Radiolabeled Exendin-4 polypeptide probe precursor and application thereof

Abstract

The invention relates to a radiolabeled Exendin-4 polypeptide probe precursor and application thereof, and belongs to the technical field of radiopharmaceutical chemistry and nuclear medicine diagnosis and treatment. The radioactive labeled Exendin-4 polypeptide probe precursor is R-HBED-CC-PEGn-Exendin-4, the general formula structure of the radioactive labeled Exendin-4 polypeptide probe precursor is shown in the specification, and n is equal to 3, 6 or 12; r is-OH or PEG chains with different lengths are introduced into R-HBED-CC-Exendin-4 to modify the medicine, so that the pharmacokinetics of the medicine is changed. Through a radionuclide labeling experiment, an in-vitro cell uptake experiment, an in-vitro receptor competitive binding experiment, biological distribution, model mouse imaging and other experiments, the targeting and in-vivo metabolism level of the novel probe are explored. And a novel targeted GLP-1R diagnosis and treatment drug with a clinical application prospect is screened out.

Description

Technical Field

[0001] This invention relates to a radiolabeled Exendin-4 polypeptide probe precursor and its application, belonging to the fields of radiopharmaceutical chemistry and nuclear medicine diagnostic and therapeutic technologies. Background Technology

[0002] The latest epidemiological data shows that in 2024, there were 589 million adults with diabetes worldwide, with China accounting for nearly a quarter of the total. Compared with the data in 2021, China still ranks first in the world in terms of the number of patients. Diabetes has become a major public health problem and a global chronic metabolic disease.

[0003] Diabetes mellitus is divided into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). While their causes differ, both share the typical symptoms of "polyuria, polydipsia, polyphagia, and weight loss," characterized by abnormally high blood sugar. In later stages, complications including cardiovascular disease and kidney disease can occur, necessitating early intervention. Therefore, early diagnosis and treatment of diabetes are crucial. Although the pathogenesis of T1DM and T2DM differs, both share the pathological characteristic of pancreatic β-cells showing functional loss or reduced mass (Beta Cell Mass, BCM) before the onset of obvious symptoms. Currently, routine hospital screening and diagnostic methods, primarily fasting blood glucose testing, insulin level testing, and urine glucose testing, only indirectly reflect BCM, lacking specificity, sensitivity, and reproducibility. Due to the dispersed distribution and relatively small number of pancreatic β-cells, coupled with the invasiveness and immaturity of current BCM detection technologies, early diagnosis is difficult, easily leading to missed opportunities for optimal treatment. There is an urgent need to develop specific, non-invasive monitoring and diagnostic technologies for early β-cell function in this disease. With the development of nuclear medicine radiomolecular imaging technology, specific radioactive molecular probes have provided a direction for this development.

[0004] The most common neuroendocrine tumor in the pancreas is the insulinoma. Although almost 90% of insulinoma patients are benign, the tumor produces a large amount of insulin that enters the patient's bloodstream, causing metabolic disorders. Over time, this can lead to chronic neurological damage, posing a very high risk. Early diagnosis and treatment are essential. However, there are still many problems in the diagnosis and treatment process, such as non-specific clinical manifestations, which can easily lead to difficulties in differentiation or even misdiagnosis. Current treatment methods mainly rely on surgical resection, but the resection site is very small, which is extremely demanding on the operator's skills.

[0005] Most insulinomas highly specifically express GLP-1R. Therefore, Exendin-4, as a GLP-1 (Glucagon-Like Peptide-1) analog, can be used to develop specific radioactive molecular probes for preoperative nuclear imaging localization of lesions and targeted therapy. In recent years, there have been many related studies, such as […]. 68 Ga]Ga-Exendin-4 PET / CT imaging agent exhibits extremely high GLP-1R targeting and lesion detection rate, reaching 97.7%.

[0006] GLP-1R (Glucagon-Like Peptide-1 Receptor) is often considered an important molecular target for insulinoma imaging and diabetes diagnosis. However, its endogenous ligand, glucagon-like peptide-1 (GLP-1), is inactivated by DPP4 degradation in the body, resulting in a short plasma half-life. To address this issue, researchers have developed several highly stable GLP-1R agonists. Exendin-4, a stable GLP-1 analog, is used to develop GLP-1R-targeting radioactive molecular probes due to its anti-enzymatic properties and good targeting binding ability.

[0007] In 2022, Linlin Li et al. reported Ga- 68 The labeled HBED-CC-Exendin-4, when used to scintigraphically examine insulinomas, [ 68 Ga]Ga-HBED-CC-Exendin-4 can be rapidly taken up by tumors, and its renal clearance rate in rats and humans is higher than that in […]. 68 Ga]Ga-NOTA-MAL-Cys 40 -Exendin-4 is faster. In a clinical study published in 2024 by Linlin Li et al., [ 68 Although the tumor uptake of Ga]Ga-HBED-CC-Exendin-4 was lower than [ 68 Ga]Ga-NOTA-MAL-Cys 40 -Exendin-4, but its renal uptake is reduced by about 30%, which may be more beneficial for clearly showing tumors near the left kidney in clinical practice.

[0008] The radiolabeled Exendin-4 probes reported at this stage still have the following problems: First, the problem of high renal uptake: if the renal uptake is too high in the imaging, it will affect the signal-to-noise ratio of the imaging due to the proximity of the pancreas and the kidney; Second, the lack of integration of diagnosis and treatment: most current probes are biased towards a single function and cannot meet the needs of diagnosis and treatment at the same time.

[0009] Therefore, developing a radiolabeled Exendin-4 peptide probe precursor and its application to further reduce non-specific binding of the probe to non-target tissues, making the probe more likely to accumulate in target tissues (such as insulinoma tissue), thereby further improving the contrast between tumor and background tissues and more clearly displaying the location and morphology of the tumor; further improving the stability of the probe in vivo, resisting degradation by various enzymes in the body, ensuring that it is not decomposed before reaching the target tissue, maintaining its integrity and bioactivity, and thus better exerting its imaging function; and optimizing its absorption, distribution, metabolism and excretion in vivo by adjusting the physicochemical properties such as the size, shape and charge of the probe, prolonging the half-life of the probe in the blood circulation, allowing it more time to bind to target cells, and further improving imaging efficiency, has become an urgent technical challenge to be solved in this field. Summary of the Invention

[0010] One objective of this invention is to provide a radiolabeled GLP-1R targeting peptide probe precursor with improved specificity and sensitivity. This precursor further reduces non-specific binding of the probe to non-target tissues, making the probe more likely to accumulate in target tissues (such as insulinoma tissue), thereby further improving the contrast between the tumor and background tissues and more clearly displaying the tumor location and morphology. It also further enhances the probe's stability in vivo, resisting degradation by various enzymes in the body, ensuring it is not decomposed before reaching the target tissue, maintaining its integrity and bioactivity, and thus better performing its imaging function. By adjusting the physicochemical properties of the probe, such as size, shape, and charge, its absorption, distribution, metabolism, and excretion processes in vivo are optimized, prolonging the probe's half-life in the bloodstream, allowing it more time to bind to target cells, and further improving imaging efficiency.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution:

[0012] A radiolabeled Exendin-4 peptide probe precursor R-HBED-CC-PEG n -Exendin-4, its general formula structure is as follows:

[0013]

[0014] Where n is an integer between 3 and 12; or n = 3, 6, or 12; R is -OH or

[0015] Preferably, its general formula structure is as follows:

[0016]

[0017] Where n is an integer between 3 and 12; or n = 3, 6, or 12.

[0018] Another object of the present invention is to provide the above-mentioned radiolabeled Exendin-4 polypeptide probe precursor HBED-CC-PEG. n Preparation method of -Exendin-4.

[0019] The above-mentioned objective of this invention is achieved through the following technical solution:

[0020] Radiolabeled Exendin-4 peptide probe precursor HBED-CC-PEG n The preparation of -Exendin-4 is as follows:

[0021] Step 1: Compound 1 (tert-butyl 3-(3-(((2-(tert-butoxy)-2-oxoethyl)(2-((2-(tert-butoxy)-2-oxoethyl))

[0022] Synthesis of (5-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoate)(3-[((2-(tert-butoxy)-2-oxoethyl)(2-{[2-(tert-butoxy)-2-oxoethyl][5-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl]amino}-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino)methyl]-4-hydroxyphenylpropanoate tert-butyl)

[0023] (2-((2-(tert-butoxy)-2-oxoethyl)(5-(3-(tert-butoxy)-3-oxopropyl)-2-hydroxybenzyl)

[0024] amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoic acid (3-[3-({[2-(tert-butoxy)-2-oxoethyl][2-({[5-(3-tert-butoxy-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino]ethyl}amino)methyl]-4-hydroxyphenylpropanoic acid) dissolved in anhydrous N,N-dimethylformamide. Under ice bath conditions, an anhydrous N,N-diisopropylethylamine and an anhydrous N,N-dimethylformamide solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate were added dropwise to activate the reaction. The reaction solution turned light brown. Then, 1-(2-Aminoethyl)-1H-pyrrole-2,5-diol was added dropwise. A solution of ne-2,2,2-trifluoroacetate (1-(2-aminoethyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate) in anhydrous N,N-dimethylformamide was reacted at room temperature. The reaction solution was dark brown. The N,N-dimethylformamide solvent was removed by vacuum distillation. The solution was redissolved in ethyl acetate, extracted with deionized water, and then back-extracted with ethyl acetate. The solutions were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a dark brown oily compound. The compound was purified by silica gel chromatography (mobile phase A: dichloromethane; phase B: methanol). The solvent was removed by vacuum distillation to obtain a colorless oily compound 1.

[0025] Step 2: Compound 2(3-(3-(((carboxymethyl)(2-((carboxymethyl))(5-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoi c Synthesis of compound 1 (3-[3-({[[(carboxymethyl)(2-({[5-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl]amino}-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino]methyl})amino]methyl})-4-hydroxyphenyl]propionic acid): Compound 1 was dissolved in anhydrous trifluoroacetic acid, stirred at room temperature, diluted with dichloromethane, and the solvent was removed by vacuum distillation. The dilution and vacuum distillation were repeated 4-5 times, and the solution was dried to obtain a pale yellow oily compound. The compound was dissolved in DMSO and deionized water to a concentration of 10 mg / mL, purified by Semi pre-HPLC, and lyophilized to obtain a white powder compound 2.

[0026] Step 3: HBED-CC-PEG n Synthesis of Exedin-4: Compound 2 was dissolved in PBS solution, and Cys was added under ice bath conditions. 39 -PEG n -Exedin-4, at room temperature, stirred, overnight, purified by semi-pre-HPLC to obtain a white solid HBED-CC-PEG. n -Exendin-4, where n = an integer from 3 to 12; or n = 3, 6, or 12.

[0027] Preferably, in step 2, the HPLC separation conditions are: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0-20 min, B 5%-100%, UV=280nm, flow rate 4mL / min.

[0028] Preferably, in step 3, the Semi pre-HPLC purification is performed as follows: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0-20 min B 5%-100%, UV=280nm, flow rate 4mL / min.

[0029] Another object of the present invention is to provide the above-mentioned HBED-CC-PEG. n Applications of -Exendin-4.

[0030] The above-mentioned objective of this invention is achieved through the following technical solution:

[0031] Radiolabeled peptide probes [ 68 Ga]Ga-HBED-CC-PEG n -Exendin-4, its general structure is as follows:

[0032]

[0033] Where n is an integer between 3 and 12; or n = 3, 6, or 12.

[0034] Another object of the present invention is to provide the above-mentioned [ 68 Ga]Ga-HBED-CC-PEG n Preparation method of -Exendin-4.

[0035] The above-mentioned objective of this invention is achieved through the following technical solution:

[0036] [ 68 Ga]Ga-HBED-CC-PEG n The preparation method of -Exendin-4 is as follows:

[0037] HBED-CC-PEG n -Exendin-4 was dissolved in DMSO to prepare a DMSO solution of the precursor. In a clean vial, the precursor solution and sodium acetate buffer were added. The Ge-68 / Ga-68 generator was rinsed with high-purity hydrochloric acid to obtain […]. 68 Ga]GaCl3 hydrochloric acid solution, take the one with the highest activity [ 68 Add the above precursor solution to a GaCl3 solution (~2-4 mCi), shake to mix thoroughly, and adjust the pH of the reaction solution to 4. Place the reaction flask in a 50°C metal bath and heat the reaction. After the reaction is complete, cool the reaction flask to room temperature, take the reaction solution, dilute it with water to 500 μL, take 100 μL, and analyze it using HPLC and TLC. Calculate the radiochemical purity (RCP) based on the peak area integration of TLC and HPLC; where n = an integer from 3 to 12; or n = 3, 6, or 12.

[0038] Another object of the present invention is to provide DOTA-HBED-CC-PEG. n Preparation method of -Exendin-4.

[0039] The above-mentioned objective of this invention is achieved through the following technical solution:

[0040] A marker precursor, DOTA-HBED-CC-PEG n -Exendin-4, its general structure is as follows:

[0041]

[0042] Where n = 0, 3 or 6.

[0043] Another object of the present invention is to provide DOTA-[ 68 Ga]Ga-HBED-CC-PEG n -Exendin-4.

[0044] The above-mentioned objective of this invention is achieved through the following technical solution:

[0045] A radiolabeled compound, DOTA-[ 68 Ga]Ga-HBED-CC-PEG n -Exendin-4, its general structure is as follows:

[0046]

[0047] Where n = 0, 3 or 6.

[0048] Another object of the present invention is to provide [ 177Lu]Lu-DOTA-HBED-CC-PEG n -Exendin-4.

[0049] The above-mentioned objective of this invention is achieved through the following technical solution:

[0050] A radiolabeled compound, [ 177 Lu]Lu-DOTA-HBED-CC-PEG n -Exendin-4, its general structure is as follows:

[0051]

[0052] Where n = 0, 3 or 6.

[0053] Beneficial effects:

[0054] The radiolabeled Exendin-4 polypeptide probe precursor and its application of this invention, through the introduction of PEG, further reduce the non-specific binding of the probe to non-target tissues, making the probe more likely to accumulate in target tissues (such as insulinoma tissue), thereby further improving the contrast between the tumor and background tissues and more clearly displaying the tumor location and morphology; further improving the stability of the probe in vivo, resisting the degradation by various enzymes in the body, ensuring that it is not decomposed before reaching the target tissue, maintaining its integrity and biological activity, thereby better exerting its imaging effect; by adjusting the physicochemical properties such as the size, shape and charge of the probe, its absorption, distribution, metabolism and excretion process in vivo are optimized, prolonging the half-life of the probe in the blood circulation, allowing it more time to bind to target cells, further improving the imaging effect.

[0055] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this does not imply any limitation on the scope of protection of the present invention. Attached Figure Description

[0056] Figure 1 The radiolabeled precursor HBED-CC-PEG used in Examples 1-3 of this invention n The synthesis route of -Exendin-4 (n = 3, 6, 12);

[0057] Figure 2 HBED-CC-PEG in Application Examples 1-3 of this invention n -Ga-68 marker route for Exendin-4 (n = 0, 3, 6);

[0058] Figure 3-A In the application embodiment 1 of the present invention [ 68Ga]Ga-HBED-CC-PEG3-Exendin-4 labeled reaction solution and [ nat Ga]Ga-HBED-CC-PEG3-Exendin-4 co-injection HPLC chromatogram;

[0059] Figure 3-B In the second application embodiment of the present invention, [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 labeled reaction solution and [ nat Ga]Ga-HBED-CC-PEG6-Exendin-4 co-injection HPLC chromatogram;

[0060] Figure 3-C In the application embodiment 3 of the present invention [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 labeled reaction solution with [ nat Ga]Ga-HBED-CC-PEG 12 -Exendin-4 co-injection HPLC chromatogram;

[0061] Figure 4-A This is from application examples 1-3 of the present invention. 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG3-Exendin-4 incubated in physiological saline;

[0062] Figure 4-B This is from application examples 1-3 of the present invention. 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG6-Exendin-4 incubated in physiological saline;

[0063] Figure 4-C This is from application examples 1-3 of the present invention. 68 Ga]Ga-HBED-CC-PEG 12 HPLC chromatogram of Exendin-4 incubated in physiological saline;

[0064] Figure 5-A This is from application examples 1-3 of the present invention. 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG3-Exendin-4 incubated in rat serum;

[0065] Figure 5-B This is from application examples 1-3 of the present invention. 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG6-Exendin-4 incubated in rat serum;

[0066] Figure 5-C This is from application examples 1-3 of the present invention. 68 Ga]Ga-HBED-CC-PEG 12 HPLC chromatogram of Exendin-4 incubated in rat serum;

[0067] Figure 6-A This is from application examples 1-3 of the present invention. 68 PET / CT-MIP images of Ga-HBED-CC-PEG3-Exendin-4 in normal mice (K: kidney; B: bladder);

[0068] Figure 6-B This is from application examples 1-3 of the present invention. 68 PET / CT-MIP images of Ga-HBED-CC-PEG6-Exendin-4 in normal mice (K: kidney; B: bladder);

[0069] Figure 6-C This is from application examples 1-3 of the present invention. 68 Ga]Ga-HBED-CC-PEG 12 - PET / CT-MIP images of Exendin-4 in normal mice (K: kidney; B: bladder);

[0070] Figure 7-A This is an application example 1-application example 3 of this invention. 68 Biodistribution of Ga]Ga-HBED-CC-PEG3-Exendin-4 in normal mice;

[0071] Figure 7-B This is an application example 1-application example 3 of this invention. 68 Biodistribution of Ga]Ga-HBED-CC-PEG6-Exendin-4 in normal mice;

[0072] Figure 7-C This is an application example 1-application example 3 of this invention. 68 Ga]Ga-HBED-CC-PEG 12 -Biodistribution of Exendin-4 in normal mice;

[0073] Figure 8 This invention is applied in Examples 1-3 of the present invention. 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,

[0074] [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and Comparative Example 1 [ 68 Figure 1 shows the uptake of Ga]Ga-HBED-CC-Exendin-4 in the mouse pancreas at different time points;

[0075] Figure 9 In the second application embodiment of the present invention, [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and Comparative Example 1 [ 68 Ga]Ga-HBED-CC-Exendin-4 uptake-time curve in INS-1 cells;

[0076] Figure 10 In Example 2 of this invention, INS-1 cells were used at 60 min to […]. 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Uptake and blockade of Ga]Ga-HBED-CC-Exendin-4 (n=3, *p<0.05,**p<0.01,***p<0.001,****p<0.0001);

[0077] Figure 11-A Example 2 of the application of the present invention 68 Ga]Ga-HBED-CC-PEG6-Exendin-4( Figure 11-A PET / CT-MIP images of INS-1 tumor-bearing mice (T: tumor; K: kidney; B: bladder);

[0078] Figure 11-B For Comparative Example 1 [ 68 PET / CT-MIP images of Ga-HBED-CC-Exendin-4 in INS-1 tumor mice (T: tumor; K: kidney; B: bladder);

[0079] Figure 12 The target compound DOTA-HBED-CC-PEG in Examples 4-6 of this invention. n -Exendin-4 (n = 0, 3 or 6) synthesis route;

[0080] Figure 13 This invention applies to DOTA in Examples 4-6. 68 Ga]Ga-HBED-CC-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4 and DOTA-[68 Preparation route diagram of Ga]Ga-HBED-CC-PEG6-Exendin-4;

[0081] Figure 14-A In the application embodiment 4 of this invention, DOTA-[ 68 Radio-HPLC spectrum of Ga]Ga-HBED-CC-Exendin-4;

[0082] Figure 14-B In the application embodiment 5 of this invention, DOTA-[ 68 Radio-HPLC spectrum of Ga]Ga-HBED-CC-PEG3-Exendin-4;

[0083] Figure 14-C For the application of DOTA- in the present invention 6 68 Radio-HPLC spectrum of Ga]Ga-HBED-CC-PEG6-Exendin-4;

[0084] Figure 15-A In the application embodiment 4 of this invention, DOTA-[ 68 PET / CT-MIP image of Ga]Ga-HBED-CC-Exendin-4 in normal mice;

[0085] Figure 15-B In the application embodiment 5 of this invention, DOTA-[ 68 PET / CT-MIP image of Ga]Ga-HBED-CC-PEG3-Exendin-4 in normal mice;

[0086] Figure 15-C In the application embodiment 6 of this invention, DOTA-[ 68 PET / CT-MIP image of Ga]Ga-HBED-CC-PEG6-Exendin-4 in normal mice. Detailed Implementation

[0087] Unless otherwise specified, the reagents and raw materials used in the preparation and detection methods described in the following examples and comparative examples are all commercially available products, and the equipment used is all common equipment; all units are weight units.

[0088] 1. Experimental Materials and Instruments

[0089] Experimental materials: Cys 39 -PEG n-Exendin-4 (amino acid sequence: His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-PEG) n -Cys, n = 0, 3, 6 or 12), anhydrous DMSO and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich. All other reagents used in this part of the experiment were purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd., Beijing Innocare Technology Co., Ltd., and Beijing Chemical Plant. All solvents used were purchased from Beijing Tongguang Fine Chemical Co., Ltd. Unless otherwise specified, they can be used directly.

[0090] Table 1: Experimental Instruments

[0091]

[0092] 2. Radiolabeled precursor HBED-CC-PEG n Synthesis of -Exendin-4 (n = 3, 6, or 12)

[0093] like Figure 1 As shown, the radiolabeled precursor HBED-CC-PEG in Examples 1-3 of this invention is shown. n Synthetic routes for -Exendin-4 (n = 3, 6 or 12); HATU is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIPEA is N,N-diisopropylethylamine; TFA is trifluoroacetic acid; PBS is phosphate buffer solution.

[0094] Example 1: Preparation of HBED-CC-PEG3-Exendin-4, the steps of which are as follows:

[0095] Step 1: Preparation of compound 2, (ert-butyl-3-(3-(((2-(tert-butoxy)-2-oxoethyl)(2-((2-(tert-butoxy)-2-oxoethyl)(5-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)

[0096] Synthesis of tert-butyl propanoate (3-(3-{[(2-tert-butoxy-2-oxoethyl)(2-({[5-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl]amino}-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino)methyl]amino}methyl)-4-hydroxyphenylpropanoate):

[0097]

[0098] Dissolve 150 mg (0.214 mmol) of tert-butyl propanoic acid (3-[((2-(tert-butoxy)-2-oxoethyl)(2-((2-(tert-butoxy)-2-oxoethyl)(5-(3-(tert-butoxy)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoic acid in an ethereal medium. Anhydrous N,N-dimethylformamide solution containing N,N-diisopropylethylamine (77 mg, 105 μL, 0.6 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (106 mg, 0.28 mmol) was added dropwise to N,N-dimethylformamide under ice bath conditions. The reaction was activated for 40 minutes under ice bath conditions, and the reaction solution turned light brown. Then, 1-( A solution of 2-Aminoethyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate (1-(2-aminoethyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate) (66 mg, 0.26 mmol) in anhydrous N,N-dimethylformamide was reacted at room temperature for 4 hours. The reaction solution was dark brown. The N,N-dimethylformamide solvent was removed by vacuum distillation. The solution was redissolved in ethyl acetate, extracted four times with deionized water, and then back-extracted three times with ethyl acetate. The extracts were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a dark brown oily compound. The compound was purified by silica gel chromatography (mobile phase A: dichloromethane; phase B: methanol). The solvent was removed by vacuum distillation to obtain a colorless oily compound 2 (65 mg, 0.10 mmol), with a yield of 50%.

[0099] 1H NMR(400MHz,)δ6.99(td,J=8.7,2.2Hz,2H),6.84(dd,J=3.4,2.4Hz,2H),6.80(s,2H),6.69(dd,J=8.2,3.8Hz,2H),3.37-3.28(m,8H),3.2 3(d,J=1.6Hz,4H),2.76(dd,J=14.9,7.4Hz,4H),2.68(s,4H),2.48(t,J=7.5Hz,2H),2.35(dd,J=9.0,6.7Hz,2H),1.44(d,J=30.8Hz,27H);

[0100] HRMS theoretical value C 44 H 62 N4O 11 [M+H] + 823.4487, measured value 823.4509;

[0101] Step 2: Preparation of compound 3, the steps are as follows:

[0102] Compound 3(3-(3-(((carboxymethyl)(2-((carboxymethyl)(5-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol)

[0103] Synthesis of 3-[3-({[(carboxymethyl)(2-({[5-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl]amino}-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino]methyl})amino]methyl})-4-hydroxyphenyl]propanoic acid):

[0104]

[0105] Compound 2 (130 mg, 0.158 mmol) was dissolved in anhydrous trifluoroacetic acid and stirred at room temperature for 3 hours. It was then diluted with dichloromethane, and the solvent was removed by vacuum distillation. This dilution and vacuum distillation were repeated 4-5 times, and the solution was dried to obtain a pale yellow oily compound. The compound was dissolved in DMSO and deionized water at a 1:1 ratio to a concentration of 10 mg / mL. After purification by Semi-pre-HPLC, the solution was lyophilized to obtain a white powder compound 3 (84 mg, 0.13 mmol). The HPLC separation conditions were: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0-20 min; B: 5%-100%, UV = 280 nm, flow rate 4 mL / min. 87.6 mg of compound 3 was obtained, with a yield of 83.2%.

[0106] HRMS theoretical value C 32 H 38 N4O 11 [M+H] + 655.2609, actual value 655.2600;

[0107] Step 3: Synthesis of HBED-CC-PEG3-Exedin-4:

[0108]

[0109] Compound 3 (10 mg, 0.015 mmol) was dissolved in 3 mL of PBS (0.0067 M, pH 7.2). Cys was added under ice bath conditions. 39 -PEG3-Exedin-4 (15 mg, 3.4 μmol) was stirred overnight at room temperature and purified by Semi-pre-HPLC: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0-20 min B 5%-100%, UV=280nm, flow rate 4 mL / min, to obtain white solid compound HBED-CC-PEG3-Exedin-4 (13.7 mg, 2.7 μmol), yield: 79.6%;

[0110] HRMS theoretical value C 225 H 336 N 54 O 75 S2[M+4H] 4+ 1265.5908, measured value 1265.5918.

[0111] Example 2: Preparation of HBED-CC-PEG6-Exendin-4, the steps of which are as follows:

[0112] Step 1: Preparation of compound 2 is the same as in Example 1;

[0113] Step 2: Preparation of compound 3, same as in Example 1;

[0114] Step 3: Synthesis of HBED-CC-PEG6-Exendin-4:

[0115]

[0116] Compound 3 (11 mg, 0.017 mmol) was dissolved in 3 mL of PBS (0.0067 M, pH 7.2), and Cys was added under ice bath conditions. 39 PEG6-Exedin-4 (20 mg, 2.8 μmol) was incubated overnight at room temperature with stirring. The solution was purified by semi-pre-HPLC: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0–20 min. B 5%–100%, UV = 280 nm, flow rate 4 mL / min. The resulting white solid compound HBED-CC-PEG6-Exedin-4 (10.2 mg, 1.9 μmol) was obtained, yield: 69.8%.

[0117] HRMS theoretical value C 231 H 348 N 54 O 78 S2[M+4H]4 + 1298.6164, measured value 1298.6185.

[0118] Example 3: HBED-CC-PEG 12 The preparation of -Exendin-4 is as follows:

[0119] Step 1: Preparation of compound 2 is the same as in Example 1;

[0120] Step 2: Preparation of compound 3, same as in Example 1;

[0121] Step 3: HBED-CC-Cys 39 -PEG 12 Synthesis of -Exedin-4:

[0122]

[0123] Compound 3 (15.7 mg, 0.024 mmol) was dissolved in 3 mL of PBS (0.0067 M, pH 7.2). Cys was added under ice bath conditions. 39 -PEG 12-Exedin-4 (25 mg, 5.2 μmol), at room temperature, stirred, overnight, purified by Semi-pre-HPLC: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0–20 min B 5%–100%, UV = 280 nm, flow rate 4 mL / min, to obtain a white solid compound HBED-CC-PEG. 12 -Exendin-4 (22.6 mg, 4.1 μmol), yield: 79.6%;

[0124] HRMS theoretical value C 243 H 372 N 54 O 84 S2[M+4H] 4+ 1364.6557, measured value 1364.6552.

[0125] Application Examples

[0126] The inventors designed and synthesized three novel HBED-CC-Exendin-4-derived marker precursors targeting GLP-1R, linked with PEG chains: HBED-CC-PEG3-Exendin-4, HBED-CC-PEG6-Exendin-4, and HBED-CC-PEG. 12 -Exendin-4 is used to mark Ga -68 ; obtained a new type of Ga -68 GLP-1R-targeted imaging drugs were labeled for further biological evaluation studies. All compounds were characterized by HRMS and 1H NMR or LC-MS, and the spectral results confirmed that all compounds had the correct structures.

[0127] like Figure 2 As shown, HBED-CC-PEG is used in application examples 1-3 of this invention. n Ga of -Exendin-4 (n = 3, 6, 12) -68 Mark routes; including [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 Preparation of -Exendin-4;

[0128] Application Example 1

[0129] [ 68 The preparation steps of Ga]Ga-HBED-CC-PEG3-Exendin-4 are as follows:

[0130] The compound HBED-CC-PEG3-Exedin-4 was dissolved in DMSO to prepare a DMSO solution of 2 mg / mL precursor. In a clean 10 mL vial, 13 μL (~25 μg, 5 nmol) of the precursor solution and 17 μL of sodium acetate buffer (NaOAc / HAc, 3 M, pH 5.2) were added. The solution was then rinsed with 5 mL of 0.1 M high-purity hydrochloric acid. -68 / Ga -68 Generator (Newrite Medical), yielding 5mL [ 68 Ga]GaCl3 hydrochloric acid solution, take 300 μL of the solution with the highest activity [ 68 Add the above precursor solution to Ga]GaCl3 solution (~2-4 mCi), shake and mix thoroughly to make the pH of the reaction solution 4. Place the reaction flask in a 50℃ metal bath and heat for 5 min. After the reaction is complete, cool the reaction flask to room temperature, take 50 μL of the reaction solution, dilute with water to 500 μL, take 100 μL, and analyze using HPLC and TLC. Calculate the radiochemical purity (RCP) based on the peak area integration of TLC and HPLC.

[0131] HPLC analysis conditions: Analytical column: 5μm C18(2)LC Column 150×4.6mm, flow rate 1mL / min, mobile phase A is 0.1% TFA aqueous solution, mobile phase B is 0.1% TFA acetonitrile solution (0-10min, 5%-100%, mobile phase B; 10-14min, 100% mobile phase B; 14-15min, 100%-5% mobile phase B).

[0132] Application Example 2

[0133] [ 68 Preparation of Ga]Ga-HBED-CC-PEG6-Exendin-4

[0134] The rest is the same as in Application Example 1, except that: the compound HBED-CC-PEG6-Exendin-4 was dissolved in DMSO to prepare a DMSO solution of 2 mg / mL precursor, and 13 μL (~26 μg, 5 nmol) of precursor solution was added to a clean 10 mL vial.

[0135] Application Example 3

[0136] [ 68 Ga]Ga-HBED-CC-PEG 12 Preparation of -Exendin-4

[0137] Everything else is the same as in Application Example 1, except that: the compound HBED-CC-PEG is used. 12 -Exendin-4 was dissolved in DMSO to prepare a DMSO solution of 2 mg / mL precursor. 14 μL (~27 μg, 5 nmol) of the precursor solution was added to a clean 10 mL vial.

[0138] [ 68 Ga]Ga-HBED-CC-PEG n -Exendin-4 identification

[0139] To further identify the structure of the labeled products, three types of labeled precursors and stable gallium ions were prepared respectively. nat Ga 3+ Complex products of ) nat Ga]Ga-HBED-CC-PEG3-Exendin-4,[ nat Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ nat Ga]Ga-HBED-CC-PEG 12 -Exendin-4, and the purity of the "cold compound" was determined by LC-MS to ensure that the purity was greater than 95%, and then compared with the corresponding "hot compound" [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 co-injection;

[0140] Comparative Example 1

[0141] [ nat The preparation of Ga]Ga-HBED-CC-PEG3-Exendin-4 involves the following steps: [Ga]Ga-HBED-CC-PEG3-Exendin-4 nat Ga]GaCl3 was dissolved in 0.05M high-purity hydrochloric acid to prepare the following solution: nat Ga 3+ A 0.0114 M solution was prepared; 104 μL (200 μg, ~40 nmol) was added to a clean 10 mL vial.

[0142] HBED-CC-PEG3-Exendin-4 solution, add 100 μL of water, and add 6 μL of […]. natGaCl3 hydrochloric acid solution (0.0114M, 68.4 nmol), 8 μL sodium acetate buffer solution (NaOAc / HAc, 3M, pH 5.2), adjusted to pH 4 of the reaction solution, shaken, and mixed thoroughly; after placing the reaction flask at room temperature for 4 h, it was heated at 50 °C for 10 min; after the reaction was completed, it was cooled to room temperature, and the reaction solution was analyzed by LC-MS and high-resolution mass spectrometry (analytical column: Agilent Eclipse XDB-C18, 5 μm, 4.6 × 250 mm, UV = 280 nm; mobile phase conditions: mobile phase A is 0.1% TFA aqueous solution, mobile phase B is 0.1% TFA acetonitrile solution, 0-20 min 5%-100% mobile phase B);

[0143] LC-MS confirmed that the purified stable gallium ion structure was correct and the purity was greater than 95%. 100 μL (100 μg, 20 nmol) of the cold compound ([ nat A solution of Ga[Ga-HBED-CC-PEG3-Exendin-4] was mixed with 10 μL of labeled reaction solution, and analyzed by HPLC to obtain the compound. nat Ga 3+ Complexes and [ 68 Ga]Ga 3+ HPLC spectra of the complex under the same conditions.

[0144] Comparative Example 2

[0145] [ nat The preparation of Ga]Ga-HBED-CC-PEG6-Exendin-4 is as follows: The rest is the same as in Comparative Example 1, except that: [ nat Ga]GaCl3 was dissolved in 0.05M high-purity hydrochloric acid to prepare the following solution: nat Ga 3+ A 0.0114 M solution was prepared; 104 μL (208 μg, ~40 nmol) of the compound HBED-CC-PEG6-Exendin-4 was added to a clean 10 mL vial.

[0146] Comparative Example 3

[0147] [ nat Ga]Ga-HBED-CC-PEG 12 The preparation of -Exendin-4 is as follows: Everything else is the same as in Comparative Example 1, except that: [ nat Ga]GaCl3 was dissolved in 0.05M high-purity hydrochloric acid to prepare the following solution: nat Ga 3+A 0.0114 M solution was prepared; 112 μL (216 μg, ~40 nmol) of compound HBED-CC-PEG was added to a clean 10 mL vial. 12 -Exendin-4 solution.

[0148] As can be seen from the co-injection results of the compounds (Figures 3A-3C), under the same HPLC conditions, [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 has the same retention time as its corresponding cold compound.

[0149] In summary, suitable labeling conditions for the three compounds can be obtained: a precursor amount of approximately 25 μg (5 nmol, concentration of 10 μM), heating at 50 °C for 5 min, yields a labeled product with a radiochemical purity greater than 95%. Subsequent in vitro and in vivo evaluation experiments can be conducted using these labeling conditions.

[0150] In vitro stability test

[0151] 1. Experimental Methods

[0152] a) Prepared according to the labeling methods in Application Examples 1-3, respectively, to obtain [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4; Ga is performed on HBED-CC-Exendin-4 using the same labeling method. -68 Mark, get [ 68 Ga]Ga-HBED-CC-Exendin-4, the specific activity of all product solutions was 4 μCi / μL; the products were analyzed by radio-HPLC, and RCP>95%, which met the requirements for subsequent experiments;

[0153] b) Stability determination of the product in physiological saline: Take a sample of the product solution ([ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68Ga]Ga-HBED-CC-PEG 12 Add 200 μL of Exendin-4 to 800 μL of physiological saline, mix well to obtain raw salt incubation solution, and incubate at room temperature (37℃). Samples are taken at 30 min, 60 min, 90 min and 120 min of incubation. The samples are analyzed by radio-HPLC. The radiochemical purity at each time point is calculated based on the peak area integration of the HPLC spectrum.

[0154] C) Stability determination of the product solution in rat serum: 4-week-old male SD-1 rats were anesthetized, and blood was collected from their hearts. The blood samples were centrifuged at 10,000 rpm for 5 min, and the supernatant was collected for later use. 50 μL of the product solution was taken and mixed with 200 μL of rat serum. After vortexing, the mixture was incubated at 37℃ for 30 min, 60 min, 90 min, and 120 min. At each time point, 50 μL was taken and added to 100 μL of acetonitrile. After vortexing to fully precipitate the protein, the mixture was centrifuged at 12,000 rpm for 5 min. An appropriate amount of supernatant was collected, diluted, and analyzed by radio-HPLC. The radiochemical purity at each time point was calculated based on the peak area integral of the HPLC spectrum.

[0155] D) Compare the calculated radiochemical purity of samples at each time point to confirm the stability of the labeled product in physiological saline or mouse serum.

[0156] 2. HPLC analysis conditions

[0157] The analysis column is 5μm C18(2) Column 150×4.6mm, flow rate 1mL / min, mobile phase A is 0.1% TFA aqueous solution, mobile phase B is 0.1% TFA acetonitrile solution (0-10min, 5%-100%, mobile phase B; 10-14min, 100% mobile phase B; 14-15min, 100%-5% mobile phase B) UV detection wavelength λ=254nm;

[0158] like Figure 4-A As shown, [ 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG3-Exendin-4 incubated in physiological saline; as shown Figure 4-B As shown,

[0159] [ 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG6-Exendin-4 incubated in physiological saline; as shown Figure 4-C As shown,

[0160] [68 Ga]Ga-HBED-CC-PEG 12 HPLC chromatogram of Exendin-4 incubated in physiological saline; as shown Figure 5-A As shown,

[0161] [ 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG3-Exendin-4 incubated in rat serum; as shown Figure 5-B As shown,

[0162] [ 68 HPLC chromatogram of Ga]Ga-HBED-CC-PEG6-Exendin-4 incubated in rat serum; as shown Figure 5-C As shown,

[0163] [ 68 Ga]Ga-HBED-CC-PEG 12 HPLC chromatogram of Exendin-4 incubated in rat serum;

[0164] As shown in Figures 4A-4C and 5A-5C, [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 remained stable after incubation in physiological saline and rat serum for 120 min (radiochemical purity RCP of the labeled product > 95%), which met the requirements of subsequent evaluation experiments.

[0165] Normal animal evaluation experiment

[0166] PET imaging and biodistribution experiments were performed on normal mice to analyze the uptake and in vivo metabolic level of the new GLP-1R targeting probe linked with PEG in various tissues and organs, and to obtain the results of drug metabolism analysis in macroscopic in vivo.

[0167] 1. PET Imaging Experiment Method for Normal Mice

[0168] a) Prepare [the following] according to the labeling methods in Application Examples 1-3. 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12-Exendin-4; Ga-label HBED-CC-Exendin-4 using the same labeling method. -68 Mark, get [ 68 Ga]Ga-HBED-CC-Exendin-4, the specific activity of all product solutions was 4 μCi / μL, and the products were analyzed by radio-HPLC. The RCP was >95%, which met the requirements for subsequent experiments.

[0169] b) Take an appropriate amount of [based on the required activity] 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4 was prepared as an isotonic solution containing the labeled product (2.5 nmol / mL) in 1500 μL of physiological saline. The isotonic solution (0.5 nmol, ~150 μCi / 200 μL) containing the radioactive probe to be tested was injected into random 4-week-old (~25 g) normal CD-1 male mice via the tail vein.

[0170] c) Anesthetize the mice after injection and perform PET / CT imaging (8min PET / 40s CT) at 15min, 30min and 60min after injection;

[0171] d) Data reconstruction was performed using OsiriX software and processed by P-MOD software to obtain PET / CT maximum intensity projection (MIP) images. By delineating the region of interest, the standard uptake value (SUV) of that region was obtained. SUV is a commonly used semi-quantitative index in PET imaging. SUVmean is used to assess the degree of radiopharmaceutical uptake by a specific tissue or organ.

[0172] 2. Biodistribution assay method in normal mice

[0173] a) Prepare [the following] according to the labeling methods in Application Examples 1-3. 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12-Exendin-4; Using the same labeling method, perform Ga... -68 Mark, get [ 68 Ga]Ga-HBED-CC-Exendin-4, the specific activity of all product solutions was 4 μCi / μL, and the products were analyzed by radio-HPLC. The RCP was >95%, which met the requirements for subsequent experiments.

[0174] b) Take an appropriate amount of [based on the required activity] 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4 was prepared as an isotonic solution containing the labeled product (2.5 nmol / mL) using physiological saline.

[0175] c) The experimental subjects were normal CD-1 male mice aged 4 weeks (~25g). The mice were randomly divided into groups of three, and 12 groups (including the control group) were set up with three time phases for each different drug. The mice were injected with an isotonic solution (0.5nmol, ~150μCi / 200μL) containing the radioactive probe to be tested into the mice via the tail vein.

[0176] d) At 15 min, 30 min, and 60 min after injection, the mice were sacrificed and dissected to obtain the organs of interest: blood, brain, heart, liver, spleen, lung, kidney, pancreas, muscle, bone, stomach wall, large intestine, and small intestine. After being placed in tubes, the organs were weighed to obtain their actual weight, and radioactivity was counted using a γ-counter.

[0177] e) Calculation method for drug uptake by various organs and tissues (unit: %ID / g):

[0178] 3. Experimental Results and Discussion

[0179] The novel GLP1-R targeting probe of the present invention [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 and positive control[ 68The PET imaging results of Ga-HBED-CC-Exendin-4 in normal mice are shown in Figures 6A-6C. 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 PET / CT-MIP images of Exendin-4 in normal mice (K: kidney; B: bladder); biodistribution in vivo is shown in Figures 7A-7C;

[0180] This invention, through cross-validation of PET imaging and biodistribution experiments, discovered that three novel PEG-linked probes all exhibited rapid organ accumulation characteristics within 15 minutes post-injection. The kidneys showed significant dose uptake (>150% ID / g), and quantitative analysis showed…

[0181] [ 68 Ga]Ga-HBED-CC-PEG3-Exendin-4,[ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG 12 -The renal uptake values ​​of Exendin-4 at 15 min were 171.09±24.82, 182.98±3.07, and 446.39±53.98%ID / g, respectively, indicating a positive correlation between the increase in PEG chain length and the degree of renal accumulation. Notably, pharmacokinetic characteristics showed that the first two probes exhibited a trend of renal radiometabolism from 30 to 60 min, while […]. 68 Ga]Ga-HBED-CC-PEG 12 -Exendin-4 continued to accumulate during this period (30 min: 434.24±16.31% ID / g; 60 min: 529.34±24.22% ID / g), and its metabolism was somewhat delayed compared to the other two compounds. This may be because the addition of the PEG chain prolongs the half-life of the protein drug, resulting in a longer residence time of the drug in the blood and body. At the same time, due to the increase in drug molecular weight, the glomerular filtration rate is reduced, and the renal clearance rate also shows a decreasing trend.

[0182] Based on the targeting and distribution characteristics of GLP-1R in pancreatic β cells, this invention focuses on analyzing the pancreatic uptake kinetics of the probe. 68Ga]Ga-HBED-CC-PEG3-Exendin-4 reached its peak uptake at 15 min (4.96±1.87% ID / g), compared to the control group. 68 Ga]Ga-HBED-CC-Exendin-4 (3.89±0.63% ID / g) increased by 27.5%, and its metabolic rate was significantly accelerated within 60 min (44.35% vs 14.65% decrease within 60 min). Although its pancreas / kidney ratio was the same as the control group (0.03 vs 0.03), its rapid metabolic characteristics meet clinical needs; it is worth noting that [ 68 Ga-HBED-CC-PEG6-Exendin-4 exhibited superior targeting performance, with pancreatic uptake exceeding that of the control drug throughout the entire timeframe. 68 Ga]Ga-HBED-CC-Exendin-4, with an uptake value of 1.72 times that of the control group at 30 min (5.94±0.82% ID / g vs. 3.46±1.29% ID / g), and a pancreas / kidney ratio of 0.025 at the same time point (0.020 in the control group), has the potential to improve imaging contrast and meets clinical imaging needs.

[0183] Combining pharmacokinetic properties with organ-specific distribution characteristics, 68 Ga]Ga-HBED-CC-PEG3-Exendin-4 and [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 has demonstrated clinical application value in balancing targeted uptake and metabolic clearance; especially [ 68 [Ga]Ga-HBED-CC-PEG6-Exendin-4, by optimizing the PEG chain length, significantly enhances pancreatic targeted uptake while maintaining reasonable renal clearance. Its superior target / non-target ratio at 30 min provides experimental evidence for further research on its deep targeting mechanism. In comparison, [ 68 Ga]Ga-HBED-CC-PEG 12 The imaging background interference caused by excessive renal retention of Exendin-4 confirmed the nonlinear relationship between the degree of PEG modification and imaging quality, providing an important reference for molecular design.

[0184] In vitro cell uptake and inhibition experiment

[0185] The GLP-1R targeting specificity and in vitro targeted uptake of a series of radioactive probes with PEG chains were evaluated and compared with existing standards. 68 Comparison of Ga]Ga-HBED-CC-Exendin-4)

[0186] 1. Experimental Methods

[0187] a) Cell culture method: INS-1 cells (rat islet cell tumor cells) specifically expressing GLP-1R were selected. The INS-1 cell culture medium was prepared as follows: RPMI 1640, 10% FBS, 1% penicillin-streptomycin, and 50 μM 2-mercaptoethanol. Using this medium, INS-1 cells were cultured in a carbon dioxide incubator (5% CO2, 37℃) for 4-5 weeks to obtain an appropriate number of cells.

[0188] b) Cell aliquoting: When the cells are in good condition, digest them into 15 mL centrifuge tubes using 0.25% trypsin. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 5 mL of PBS, gently pipette to mix, centrifuge again at 1000 rpm for 5 min, discard the supernatant, add serum-free basal culture medium to prepare 2 × 10⁶ cells / tubes. 6 500 μL (1×10⁶) of cell suspension per mL were taken separately. 6 (cells) into a 2.5 mL round-bottom sterile EP tube;

[0189] c) Using the present invention 68 Ga]Ga-HBED-CC-PEG6-Exendin-4; HBED-CC-Exendin-4 was labeled with Ga using the same method. -68 Mark, get [ 68 Ga]Ga-HBED-CC-Exendin-4, the specific activity of all product solutions was 4 μCi / μL, and the products were analyzed by radio-HPLC. The RCP was >95%, which met the requirements for subsequent experiments.

[0190] d) Preparation of incubation solution for the intake group: Take 10 μL [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 was added to 9990 μL of serum-free RPMI-1640 basal culture medium and diluted to a concentration of 4 μCi / 10 pmol / mL for use as an incubation solution for the uptake group.

[0191] e) Preparation of inhibition group incubation solution: Take 10 μL of the labeled product solution of the radioactive probe, add 10,000 equivalents of GLP-1R agonist Exendin-4 to it, and dilute it with 9,990 μL of serum-free RPMI-1640 basal culture medium to obtain the inhibition group incubation solution containing the inhibitor Exendin-4 (100 nmol / mL) (the concentration of the radioactive probe to be tested is 4 μCi / 10 pmol / mL).

[0192] f) Add the incubation solution (500 μL / tube) of the test sample to the EP tube containing cells and incubate them in a 37°C incubator. For the same test sample at the same time, set up parallel groups n=3. After incubation for 10 min, 30 min and 60 min, remove the centrifuge tubes from the incubator and centrifuge at 1000 rpm / min for 5 min. After discarding the supernatant, add 1 mL of ice-cold PBS solution to stop the uptake. Shake to mix for 1-2 min, centrifuge at 1000 rpm / min for 5 min, discard the supernatant, and put the centrifuge tubes into a plastic tube for radioactivity counting using a γ-counter. For the inhibition group, after incubation for 60 min, perform radioactivity counting in the same way. Calculate the average number of cells in each group to obtain the average radioactivity count.

[0193] g) Determination of the average radioactivity count of the drug added to each well: Take 1 mL of the incubation solution of the uptake group and the incubation solution of the inhibition group for each probe, in three parallel groups, and use γ-counter to determine the radioactivity count for subsequent calculations;

[0194] h) Calculation method for cellular drug uptake (unit: %ID / cell number):

[0195]

[0196] 2. Experimental Results and Discussion

[0197] like Figure 8 As shown, [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4 uptake-time curve in INS-1 cells; cell uptake experiments showed that, [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and positive control compound[ 68 Ga]Ga-HBED-CC-Exendin-4 can be rapidly taken up by INS-1 cells overexpressing GLP-1R. With prolonged incubation time,

[0198] [ 68 Cellular uptake of Ga-HBED-CC-Exendin-4 fluctuated, showing a slight overall increase, while [ 68 The cell uptake of Ga-HBED-CC-PEG6-Exendin-4 showed a significant increasing trend, with the cell uptake value after 30 min of incubation exceeding [ 68Cellular uptake of Ga-HBED-CC-Exendin-4 (2.16±0.09 vs. 2.53±0.23% % ID / 1×10⁻⁶) 6 The cells maintained a relative advantage after 60 minutes of incubation, demonstrating good targeting affinity.

[0199] like Figure 9 As shown, at 60 min, INS-1 cells showed [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Uptake and blockade of Ga-HBED-CC-Exendin-4 (n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001); In the cellular uptake blockade experiment,

[0200] [ 68 The total intake of Ga]Ga-HBED-CC-PEG6-Exendin-4 was higher than [ 68 Ga]Ga-HBED-CC-Exendin-4 (1.67±0.13vs.1.44±0.04%, ID / 1×10 6 Cells), and when blocked by excessive GLP-1R agonist Exendin-4, their nonspecific uptake was simultaneously lower than [ 68 Ga]Ga-HBED-CC-Exendin-4(0.22±0.01vs.0.81±0.12% ID / 1×10 6 Cells) were significantly inhibited (P < 0.0001), demonstrating excellent targeting specificity. Figure 10 ).

[0201] In vivo PET imaging experiment of INS-1 tumor-bearing mice

[0202] By performing PET imaging on INS-1 tumor-bearing (GLP-1R positive) mice, the ability of radiopharmaceuticals to visualize GLP-1R positive tumors was evaluated at the in vivo animal level, and the distribution and metabolism of the drug in tumor-bearing mice were further assessed.

[0203] 1. Experimental Methods

[0204] a) Prepare [the following according to the labeling method in Application Example 2] 68 Ga]Ga-HBED-CC-PEG6-Exendin-4; HBED-CC-Exendin-4 was labeled with Ga using the same method. -68 Mark, get [ 68Ga]Ga-HBED-CC-Exendin-4, the specific activity of all product solutions was 4 μCi / μL, and the products were analyzed by radio-HPLC. The RCP was >95%, which met the requirements for subsequent experiments.

[0205] b) Take an appropriate amount of [based on the required activity] 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4 was prepared as an isotonic solution containing the labeled product (2.5 nmol / mL) in 1500 μL of physiological saline. The isotonic solution (0.5 nmol, ~150 μCi / 200 μL) containing the radioactive probe to be tested was injected into INS-1 tumor-bearing mice via the tail vein.

[0206] c) Anesthetize the mice after injection and perform PET / CT imaging (8 min PET / 40 s CT) at 15 min, 30 min, 60 min and 120 min after injection;

[0207] d) In the uptake blocking experiment, Exendin-4 was selected as a GLP-1R inhibitor. It was dissolved and diluted with the radioactive probe labeling solution to be tested at a concentration of 50 equivalents and then diluted to an isotonic solution (concentration of test compound: 0.5 nmol, ~150 μCi / 200 μL; concentration of inhibitor: 25 nmol / 200 μL). It was injected into INS-1 tumor-bearing mice via the tail vein. The mice were anesthetized in advance, and PET / CT imaging was performed 60 min after injection (8 min PET / 40 s CT).

[0208] e) Data reconstruction was performed using OsiriX software and processed by P-MOD software to obtain PET / CT maximum intensity projection (MIP) images. By delineating the region of interest, the standard uptake value (SUV) of that region was obtained. SUV is a commonly used semi-quantitative index in PET imaging. SUVmean is used to assess the degree of radiopharmaceutical uptake by a specific tissue or organ.

[0209] 2. Experimental Results and Discussion

[0210] [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and positive control compound[ 68The PET / CT results of Ga]Ga-HBED-CC-Exendin-4 in mice with GLP-1R-positive INS-1 tumors are shown in Figures 11A-11B.

[0211] As can be seen from the imaging image and SUVmean, [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4 exhibits specific uptake in GLP-1R-positive INS-1 tumors, and this uptake can be competitively inhibited by Exendin-4. 68 The blocking rate of Ga-HBED-CC-PEG6-Exendin-4 was 96.90%, further illustrating that... 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 exhibits GLP-1R targeting specificity, and dynamic uptake analysis shows that, 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 showed significant pharmacokinetic advantages 15 min after administration: at 60 min, the tumor SUVmean reached 7.02±0.43, compared with [ 68 Ga]Ga-HBED-CC-Exendin-4 (6.20±0.37) increased by 13.2%; and [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 has a more advantageous tumor retention effect (120 min [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 The tumor uptake retention rates of Ga-HBED-CC-Exendin-4 were 97.89% vs. 81.68%, which was 19.8% higher than that of the control group; meanwhile, the PET / CT images showed that... 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4 has similar renal uptake, but lower uptake in non-target organs such as the liver and lungs, which is beneficial for tumor imaging.

[0212] As shown in Figure 11A, [ 68 PET / CT-MIP images of Ga-HBED-CC-PEG6-Exendin-4 in INS-1 tumor mice (T: tumor; K: kidney; B: bladder); as shown in Figure 11B. 68PET / CT-MIP images of Ga-HBED-CC-Exendin-4 in INS-1 tumor mice (T: tumor; L: liver; K: kidney; B: bladder); injection [ 68 Ga]Ga-HBED-CC-PEG6-Exendin-4 and [ 68 Ga]Ga-HBED-CC-Exendin-4, after 15 min, 30 min, 60 min and 120 min.

[0213] Example 4 (e.g.) Figure 13 (As shown): Preparation of DOTA-HBED-CC-MAL-PEG3-Exendin-4

[0214]

[0215] Step 1: Synthesis of Compound 5: Compound 4 (100 mg, 0.067 mmol) was placed in 2 ml of dry DMF and stirred until dissolved. DIPEA (41.36 mg, 0.32 mmol, 4 eq.) was added dropwise to the system at 0 °C. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 36.5 mg, 0.096 mmol, 1.2 eq.) was weighed, dissolved in 2 ml of anhydrous DMF, and slowly added dropwise to the mixed solution under ice bath conditions. The mixture was first placed in an ice bath for 10 min, then stirred at room temperature for 20 min to complete the activation step. 1-(2-aminoethyl)-1H-pyrrole-2, 5-Diketone (40.64 mg, 0.16 mmol, 2 eq.) was dissolved in 2 mL of anhydrous DMF and added dropwise to a flask. The mixture was stirred continuously at room temperature (25 °C) for 12 h to carry out the condensation reaction. After the reaction was completed, 20 mL of ethyl acetate was weighed and extracted twice with deionized water and 20 mL of saturated NaCl solution. The aqueous phase was washed four times with ethyl acetate. The organic phases were combined and then extracted three times with saturated brine. Anhydrous Na2SO4 was added to the combined solution to remove residual water. The mixture was filtered to remove any clumps of solid. The solution was concentrated under reduced pressure at 39 °C using a rotary evaporator to remove the ethyl acetate organic phase from the filtrate. After drying with an oil pump, 132 mg of a pale yellow solid, namely compound 5, was obtained.

[0216] ESI-MS theoretical value: C 70 H 110 N 10 O 17 Na[M+Na] + 1385.8050, Measured value: [M+Na] + 1385.7531;

[0217]

[0218] Step 2: Synthesis of Compound 6: 132 mg of the pale yellow solid compound 5 obtained by vacuum drying was dissolved in (8 mL DCM + 2 mL MeOH). 2 mL of the mixture was taken out, dried, and then dissolved in 2 mL trifluoroacetic acid. The mixture was stirred at room temperature for 2.5 h until all protecting groups (O-tBu) were removed. A larger amount of DCM was added to quench the reaction. The solvent was removed by rotary evaporation in small batches under reduced pressure until no acidic odor remained. The mixture was then dried using an oil pump. The sample to be purified was dissolved in DMSO, and an appropriate amount of solution was transferred to a sterile vial. Group analysis was performed using liquid chromatography-mass spectrometry (LC-MS) to optimize the gradient. After determining a suitable mobile phase gradient, the elution parameter system was prepared using a high-performance liquid chromatography (HPLC) system with a binary mobile phase consisting of "mobile phase A: an ultrapure aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B: a chromatographically pure acetonitrile solution containing 0.1% trifluoroacetic acid". The gradient program was set to linearly increase the proportion of phase B from 10% to 35% within 0 to 10 minutes, the detection wavelength was 280 nm, the column temperature was maintained at 25 °C, and the flow rate was set to 4 mL / min. After purification, the solution was lyophilized to obtain 4.53 mg of white solid, compound 6.

[0219] ESI-MS theoretical value: C 50 H 71 N 10 O 17 [M+H] + 1083.4920, Measured value: [M+H] + 1083.5026; Theoretical value: Fragment 1: C 28 H 43 N6O9 + 607.3086, Measured value: 607.3090, Theoretical value: Fragment 2: C 34 H 55 N8O 13 + 783.3883, measured value: 783.3841;

[0220]

[0221] Step 3: Synthesis of DOTA-HBED-CC-MAL-PEG3-Exendin-4: Compound 6: DOTA-HBED-CC-MAL (2.24 mg, 2.07 μmol, 2 eq.) was dissolved in 2 mL Dissolve in PBS, stir in an ice-water bath, weigh PEG3-Exendin-4 (4.54 mg, 1.03 μmol, 1 eq.), add to the above reaction flask, stir at room temperature for 7 h, and detect the reaction by LCMS. On a high performance liquid chromatography system, prepare a binary mobile phase according to the following: "Mobile phase A: ultrapure aqueous solution containing 0.1% trifluoroacetic acid, mobile phase B: chromatographic grade acetonitrile solution containing 0.1% trifluoroacetic acid". The gradient program is set to linearly increase the proportion of phase B from 15% to 50% within 0 to 15 min. The detection wavelength is 280 nm, the column temperature is maintained at 25 °C, and the flow rate is set to 4 mL / min. 2.77 mg of white solid (DOTA-HBED-CC-MAL-PEG3-Exendin-4) is obtained.

[0222] ESI-MS theoretical value: C 243 H 372 N 60 O 81 S2[M+4H] 4+ 1372.6490, Measured value: [M+4H] 4+ 1372.6659;

[0223] The purity was determined to be greater than 95% by LC-MS.

[0224] Example 5: Synthesis and preparation of compound DOTA-HBED-CC-MAL-PEG6-Exendin-4:

[0225]

[0226] Compound 6 (2.29 mg, 2.1 μmol, 2 eq.) was dissolved in 2 mL of PBS and stirred in an ice-water bath. PEG6-Exendin-4 (4.81 mg, 1.06 μmol, 1 eq.) was weighed and added to the same reaction flask. After stirring at room temperature for 7 h, the reaction was detected by LCMS. A binary mobile phase was prepared using a high-performance liquid chromatography system according to the following formula: mobile phase A: ultrapure aqueous solution containing 0.1% trifluoroacetic acid, mobile phase B: chromatographic grade acetonitrile solution containing 0.1% trifluoroacetic acid. The gradient program was set to linearly increase the proportion of phase B from 15% to 50% within 0 to 15 min. The detection wavelength was 280 nm, the column temperature was maintained at 25 °C, and the flow rate was set to 4 mL / min. 3.2 mg of white solid (DOTA-HBED-CC-MAL-PEG6-Exendin-4) was obtained.

[0227] ESI-MS theoretical value: C 249 H 384 N 60 O 84 S2[M+4H] 4+ 1405.6687, Measured value: [M+4H] 4+ 1405.6857; purity determined by LC-MS is greater than 95%.

[0228] Application Example 4

[0229] DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG6-Exendin-4 and DOTA-[ 68 The preparation route of Ga]Ga-HBED-CC-Exendin-4 is as follows: Figure 13 As shown;

[0230] First, the prepared compound DOTA-HBED-CC-MAL-PEG n -Exendin-4 (n=3, 6) and DOTA-[ 68 [Ga]Ga-HBED-CC-Exendin-4, mixed at a ratio of 2 μg / μL with DMSO as the solvent, yielded a precursor solution. 13.72 μL, 14.05 μL, and 13.21 μL of the preparation solution, along with 17 μL of 3M sodium acetate buffer, were added to a 10 mL sterile vial. Then, 170 μL of pure water was added, and the volume was adjusted to 200 μL. The pH was adjusted to 4-5, and the solution was rinsed with 0.1M high-purity hydrochloric acid at a rate of 2-3 mL / min to obtain […]. 68 After the GaCl3 eluent was measured by a radioactivity meter (2-4 mCi), 300 μL of the eluent with the highest activity was added to the pretreated vials. The mixture was vortexed and transferred to a metal bath. All reaction flasks were tested at 60°C for 5 min. After the reaction was terminated and the temperature was cooled for a period of time, 50 μL of each flask was taken and diluted with 450 μL of pure water. 100 μL of each flask was taken by pipette for subsequent chromatographic analysis and thin-layer chromatography. The analysis report was obtained by integrating the results to evaluate the radiochemical purity.

[0231] As can be seen from Figures 14A-14C, [ 68 Ga]Ga 3+ The retention time under these HPLC conditions was 2.95 min.

[0232] DOTA-[ 68Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Under these HPLC conditions, the retention times of Ga-HBED-CC-MAL-PEG6-Exendin-4 and DOTA-HBED-CC-Exendin-4 were 10.41 min, 10.42 min, and 10.535 min, respectively, indicating that they could bind with free […]. 68 Ga]Ga 3+ Separate, DOTA-[ 68 The Ga-HBED-CC-MAL-PEG6-Exendin-4 and DOTA-HBED-CC-Exendin-4 labeled products have a radiochemical purity greater than 95%, requiring no further purification and meeting the requirements for subsequent evaluation.

[0233] DOTA-[ 68 The Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4 labeled product has a radiochemical purity greater than 85%, and further purification can meet the requirements of subsequent evaluation.

[0234] Application Example 5

[0235] PET / CT Imaging of Novel Therapeutic Targeted GLP-1 Receptor Radiopharmaceuticals

[0236] This section only performs in vivo imaging experiments on normal mice, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG6-Exendin-4, DOTA-HBED-CC-Exendin-4 and [ 68 The study compared Ga-HBED-CC-Exendin-4 to preliminarily assess drug uptake in the kidneys and non-target organs (muscle) and metabolic pathways; four healthy experimental mice were selected and subjected to fasting pretreatment before the formal experiment, and DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG6-Exendin-4, DOTA-HBED-CC-Exendin-4 and [ 68Ga]Ga-HBED-CC-Exendin-4, diluted to isotonic solution with 1500 μL of physiological saline, was administered in 200 μL doses (~0.5 nmol, 300 μCi) via the tail vein to four normal mice. Subsequently, tomographic images were acquired at three time points (10 min, 30 min, and 60 min) using a small animal PET / CT imaging device (IRIS, Inviscan). The images were processed using P-MOD software to obtain 12 PET / CT-MIP images; as shown in Figures 15A-15C, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG6-Exendin-4 and DOTA-[ 68 PET / CT-MIP images of Ga-HBED-CC-Exendin-4 in normal mice (K: kidney; B: bladder); the experimental results indicate that DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG6-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-Exendin-4 and [ 68 The radioactivity of four drugs, Ga]Ga-HBED-CC-Exendin-4, was mainly concentrated in the kidney tissue, with renal excretion being the primary metabolic pathway. In contrast, hepatic uptake was reduced, and background clearance was accelerated, preliminarily demonstrating that the model compound DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG3-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG6-Exendin-4, DOTA-[ 68 Ga]Ga-HBED-CC-Exendin-4 has the potential to become a bifunctional chelating agent and a radiotherapeutic agent.

[0237] The radioactive probe of the present invention has the following characteristics: 1. High GLP-1R targeting affinity and specificity; 2. Long retention time in the target tissue; 3. Low uptake in non-target tissues, such as the kidney; 4. Fast clearance rate in vivo.

[0238] The PEG introduced in the radioactive probe of this invention has advantages such as being non-toxic, non-immunogenic, non-antigenic, and highly water-soluble. At the same time, PEGylation changes the conformation, electrostatic binding ability, hydrophilicity and hydrophobicity of the drug, increases the drug's in vivo retention time, increases the plasma half-life, prolongs the absorption time, improves the affinity of the drug for cell receptors, and enhances the drug's tumor targeting.

[0239] This invention introduces PEG chains of different lengths into HBED-CC-Exendin-4 to modify the drug and alter its pharmacokinetics. The targeting properties and in vivo metabolic levels of the new probes were investigated through experiments including radionuclide labeling, in vitro cellular uptake, in vitro receptor competitive binding, biodistribution, and mouse model imaging. Novel probes targeting GLP-1R with clinical application potential were screened.

[0240] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A radiolabeled Exendin-4 polypeptide probe precursor R-HBED-CC-PEG n -Exendin-4, its general formula structure is as follows: in, n = 3, 6, or 12; R is -OH or 2. The radiolabeled Exendin-4 polypeptide probe precursor R-HBED-CC-PEG as described in claim 1 n The preparation method of -Exendin-4, wherein, When R is -OH, the steps are as follows: Step 1: Compound 1 (tert-butyl 3-(3-(((2-(tert-butoxy)-2-oxoethyl)(2-((2-(tert-butoxy)-2-oxoethyl)) Synthesis of (5-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoate)(3-[((2-(tert-butoxy)-2-oxoethyl)(2-{[2-(tert-butoxy)-2-oxoethyl][5-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl]amino}-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino)methyl]-4-hydroxyphenylpropanoate tert-butyl) (2-((2-(tert-butoxy)-2-oxoethyl)(5-(3-(tert-butoxy)-3-oxopropyl)-2-hydroxybenzyl) amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoic acid (3-[3-({[2-(tert-butoxy)-2-oxoethyl][2-({[5-(3-tert-butoxy-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino]ethyl}amino)methyl]-4-hydroxyphenylpropanoic acid) dissolved in anhydrous N,N-dimethylformamide. Under ice bath conditions, an anhydrous N,N-diisopropylethylamine and an anhydrous N,N-dimethylformamide solution of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate were added dropwise to activate the reaction. The reaction solution turned light brown. Then, 1-(2-Aminoethyl)-1H-pyrrole- A solution of 2,5-dione-2,2,2-trifluoroacetate (1-(2-aminoethyl)-1H-pyrrole-2,5-dione 2,2,2-trifluoroacetate) in anhydrous N,N-dimethylformamide was reacted at room temperature. The reaction solution was dark brown. The N,N-dimethylformamide solvent was removed by vacuum distillation. The solution was redissolved in ethyl acetate, extracted with deionized water, and then back-extracted with ethyl acetate. The solutions were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain a dark brown oily compound. The compound was purified by silica gel chromatography (mobile phase A: dichloromethane; phase B: methanol). The solvent was removed by vacuum distillation to obtain a colorless oily compound 1. Step 2: Compound 2(3-(3-(((carboxymethyl)(2-((carboxymethyl))(5-(3-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-3-oxopropyl)-2-hydroxybenzyl)amino)ethyl)amino)methyl)-4-hydroxyphenyl)propanoi c Synthesis of compound 1 (3-[3-({[[(carboxymethyl)(2-({[5-(3-{[2-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)ethyl]amino}-3-oxopropyl)-2-hydroxybenzyl]amino}ethyl)amino]methyl})amino]methyl})-4-hydroxyphenyl]propionic acid): Compound 1 was dissolved in anhydrous trifluoroacetic acid, stirred at room temperature, diluted with dichloromethane, and the solvent was removed by vacuum distillation. The dilution and vacuum distillation were repeated 4-5 times, and the solution was dried to obtain a pale yellow oily compound. The compound was dissolved in DMSO and deionized water to a concentration of 10 mg / mL, purified by Semi pre-HPLC, and lyophilized to obtain a white powder compound 2. Step 3: HBED-CC-Cys 39 -PEG n Synthesis of Exedin-4: Compound 2 was dissolved in PBS solution, and Cys was added under ice bath conditions. 39 -PEG n -Exedin-4, at room temperature, stirred, overnight, purified by semi-pre-HPLC to obtain a white solid HBED-CC-PEG. n -Exendin-4, where n is an integer of 3, 6, or 12.

3. The preparation method according to claim 2, characterized in that: In step 2, the HPLC separation conditions are: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0-20 min B 5%-100%, UV=280nm, flow rate 4mL / min.

4. The preparation method according to claim 3, characterized in that: In step 3, Semi pre-HPLC purification: A: 0.1% TFA aqueous solution, B: 0.1% TFA acetonitrile solution, 0-20 min B 5%-100%, UV=280nm, flow rate 4mL / min.

5. Radiolabeled Exendin-4 peptide probe [ 68 Ga]Ga-HBED-CC-PEG n -Exendin-4; its general structure is shown below: in, n = 3, 6 or 12.

6. The radiolabeled Exendin-4 polypeptide probe of claim 5 [ 68 Ga]Ga-HBED-CC-PEG n The preparation method of -Exendin-4 is as follows: Compound HBED-CC-PEG3-Exedin-4, compound HBED-CC-PEG6-Exedin-4, or compound HBED-CC-PEG... 12 -Exedin-4 was dissolved in DMSO to prepare a DMSO solution of the precursor. In a clean vial, the precursor solution and sodium acetate buffer were added. The Ge-68 / Ga-68 generator was rinsed with high-purity hydrochloric acid to obtain […]. 68 Ga]GaCl3 hydrochloric acid solution, take the one with the highest activity [ 68 Add the above precursor solution to the Ga]GaCl3 solution, shake to mix thoroughly, and adjust the pH of the reaction solution to 4. Place the reaction flask in a 50°C metal bath and heat the reaction. After the reaction is complete, cool the reaction flask to room temperature, take the reaction solution, dilute it with water to 500 μL, take 100 μL, and analyze it using HPLC and TLC. Calculate the radiochemical purity based on the peak area integration of TLC and HPLC.

7. The application of the radiolabeled Exendin-4 polypeptide probe of claim 5 in insulinoma imaging.

8. A labeled precursor, DOTA-HBED-CC-MAL-PEG n -Exendin-4, its general structure is as follows: in, n = 0, 3 or 6.

9. A radiolabeled compound, DOTA-[ 68 Ga]Ga-HBED-CC-MAL-PEG n -Exendin-4, its general structure is as follows: in, n = 0, 3 or 6.

10. A radiolabeled compound, [ 177 Lu]Lu-DOTA-HBED-CC-MAL-PEG n -Exendin-4, its general structure is as follows: in, n = 0, 3 or 6.

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