Tumor-targeted uPAR radionuclide coupled cyclic peptide and preparation method and application thereof

Abstract

The invention discloses a radionuclide coupled cyclic peptide of a targeted tumor uPAR as well as a preparation method and application of the radionuclide coupled cyclic peptide, and belongs to the field of biological medicines. The radionuclide coupled cyclopeptide molecule can be specifically combined with tumor uPAR, tumor tissue with positive uPAR is accurately positioned through PET imaging, and the radionuclide coupled cyclopeptide molecule can be used for tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to a radionuclide-conjugated cyclic peptide targeting tumor uPAR, its preparation method and application, for radionuclide imaging and treatment of tumors with high expression of uPAR. Background Art

[0002] The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein. uPAR is a glycosylphosphatidylinositol-anchored membrane protein and belongs to the Ly-6 / uPAR (LU) protein domain family (Smith, H et al., Nat Rev Mol Cell Biol., 11: 23-36, 2018). The plasminogen system is involved in blood clot dissolution, wound healing, tissue regeneration, cancer, and many other processes affecting health and disease (Blasi, F et al., Nat Rev Mol Cell Biol, 3: 932-943, 2002). uPAR was initially thought to contribute to cell migration and directed invasion, but it is now becoming increasingly clear that this protease receptor triggers excessive cellular responses in a non-proteolytic manner, including cell adhesion, differentiation, proliferation, and migration. Cell migration is very important during normal development and tissue repair, and when it is dysregulated, it can lead to many diseases. In response to chemoattractants, cells use proteases to break unwanted attachments and anatomical barriers and clear a path. To move, cells must also modify their shape, reorganize the intracellular cytoskeleton, and continuously relax their contact with other cells and the surrounding matrix.

[0003] Many proteins are involved in cell migration, including chemoattractants and their receptors, cell adhesion molecules, proteases, cytoskeleton regulators, and several signaling enzymes. When the urokinase-type plasminogen activator receptor (uPAR) binds to the urokinase-type plasminogen activator (uPA), it simply converts the zymogen plasminogen into the serine protease plasmin 1 at the leading edge of the cell, thus promoting cell migration through its ability to digest extracellular molecules (Kathleen Dass et al., Cancer Treat Rev, 34(2): 122-136, 2008). The urokinase-type plasminogen activator receptor (uPAR) can not only promote the degradation of extracellular matrix proteins by binding to uPA, but also bind to integrins and vitronectin to mediate the activation of cell signaling pathways such as RAS / RAF / MEK / ERK and PI3K / AKT. It can not only enhance the proliferation, migration, and invasion abilities of tumor cells, but also inhibit tumor cell apoptosis and promote tumor tissue angiogenesis (Mahmood N et al., Int J Mol Sci, 22(9): 4358-4374, 2021).

[0004] Studies have shown that elevated uPAR expression levels are associated with the severity and progression of many chronic inflammatory diseases, such as most solid cancers (prostate cancer, breast cancer, colorectal cancer, etc.), kidney diseases, rheumatoid arthritis, HIV infection, novel coronavirus infection, and atherosclerosis (LeBeau AM et al., Cancer Res. 73:2070 - 2081, 2013). Clinical studies have clearly shown that overexpression of uPA, PAI-1, or uPAR is associated with a high incidence of disease recurrence and early death (Aaron M LeBeau et al., Cancer Res, 73(7):2070 - 2081, 2013). Consistent with these findings, disrupting the protein-protein interaction between uPA and its receptor has emerged as an attractive strategy to inhibit or reduce tumor growth and metastasis. To this end, scientists have been working on developing ligands that can bind to uPAR with high efficiency and specificity, including small molecules, polypeptides, and antibodies. Nevertheless, there is still a lack of drugs targeting uPAR in the clinic. On the other hand, there is also a lack of diagnostic tools targeting uPAR clinically. Imaging molecules targeting tumor uPAR can contribute to the early detection of cancer. Binding therapeutic radionuclides to molecules targeting uPAR can specifically kill cancer cells. PET, namely Positron Emission Tomography, is a commonly used non-invasive disease visualization technique in nuclear medicine, which can generate three-dimensional images of the location of various molecular processes in the body or proteins related to disease pathology, and has significant advantages in determining tumor location and cancer staging, as well as evaluating intratumoral uptake and safety of drugs. Clinically, especially from an oncology perspective, a non-invasive imaging modality for visualizing and quantifying uPAR expression in the body is necessary. Polypeptides, due to their high affinity for target proteins, low immunogenicity, and molecular weight between small molecules and antibodies, can regulate protein-protein interactions, making them a good molecular type for drug discovery. Currently, there is only one polypeptide tracer targeting uPAR, AE105, in clinical use (Morten Persson et al., J Nucl Med, 53:138 - 145, 2012). AE105 is a linear polypeptide-coupled radionuclide drug, using 64 Cu-DOTA-AE105 was used for small animal PET imaging in mice bearing uPAR-positive U87MG human glioblastoma and uPAR-negative MDA-MB-435 human breast cancer xenografts, and the results showed that the accumulation in the positive cell line was significantly higher than that in the negative cell. 64Cu-DOTA-AE105 is currently in Phase II clinical trials (Leth, JM et al., Sci Rep, 13:17248, 2023). Although AE105 showed specific binding to uPA, PET imaging in mice showed 64 Cu-DOTA-AE105 accumulates to a certain extent in normal tissues (liver, kidney, etc.), so finding new ligand molecules that can efficiently bind to uPAR and have high safety is a clinical problem that needs to be solved urgently. Compared with linear peptides, cyclic peptides usually have higher targeting affinity, longer half-life and tissue permeability, and radiopharmaceuticals based on cyclic peptides have good application prospects.

[0005] In recent years, with the development of cyclic peptide drugs, cyclic peptide-coupled nuclides have been widely used in clinical imaging to assist diagnosis and treatment. Compared with antibodies and small molecules, cyclic peptide radiotracers show multiple advantages: 1) Cyclic peptides have faster blood clearance than antibodies; 2) Compared with small molecules, cyclic peptide molecules have stronger tumor penetration ability, and cyclic peptide molecules have stronger target specificity and higher safety. Cyclic peptide radiopharmaceuticals are an ideal class of therapeutic and diagnostic drugs. In view of the particularity of the structure of the cyclopeptide molecule (cyclo[21,29][Cys21,29]-uPA21-30) targeting uPAR (Niko Schmiedeberg et al., J Med Chem, 45, (23): 4984-4994, 2002), the present invention constructs three uPAR-targeting cyclopeptide molecular probes CAPR-1, CAPR-2, and CAPR-3 by changing the disulfide bond configuration without affecting the binding site with the target protein, and applies them to tumor-bearing mice (MC38, B16F10) with high uPAR expression. 64 The in vivo activity study of Cu-CAPR-1 / 2 / 3 showed that 64 Cu-CAPR-1 / 2 / 3 has a certain uptake in tumor sites and can be metabolized quickly in normal tissues, showing effectiveness and safety. 64 Cu-CAPR-1 / 2 / 3 is a potential imaging agent. Despite the above achievements, continued efforts are needed to expand the imaging tool library, such as using different radionuclides, to adapt to the diversity of the tumor environment. Summary of the invention

[0006] The purpose of the present invention is to provide a radionuclide-coupled cyclic peptide targeting tumor uPAR and its preparation method and application. Although there are tracers that can target uPAR in clinical practice, they have low specificity, poor binding force, and show certain toxicity to normal tissues. Therefore, finding new ligands that can efficiently bind to uPAR and have high safety is a clinical problem that needs to be solved urgently. By modifying the side chains of the cyclic peptide molecules that can efficiently bind to uPAR reported in the literature and chelating the nuclides that can be used for PET imaging, a uPAR-targeted cyclic peptide molecule is constructed, which can achieve accurate positioning of uPAR-positive tumor tissues and achieve the purpose of early diagnosis and treatment of tumors.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a radionuclide-coupled cyclic peptide targeting tumor uPAR, wherein the cyclic peptide molecule is an XRG structure, wherein X is a chelating agent, R is a connecting arm, and G is a cyclic peptide compound, wherein the amino side of the connecting arm is connected to the chelating agent, and the carboxyl side of the connecting arm is connected to the cyclic peptide compound;

[0009] The cyclopeptide compound comprises cyclopeptides of different chirality formed by intramolecular disulfide bonds, and the amino acid sequence thereof is cyclo(1,9)[CNKYFSNIC]W-OH.

[0010] Preferably, the cyclic peptides of different chirality are:

[0011] (1) The cysteine ​​in the cyclic peptide is all L-type;

[0012] (2) The carbon-terminal cysteine ​​in the cyclic peptide is L-type or D-type, and the nitrogen-terminal cysteine ​​is D-type or L-type.

[0013] Preferably, the linker is selected from one of the following compounds:

[0014]

[0015]

[0016] Here, n is an integer in the range of 1-10.

[0017] More preferably, the structural formula of the connecting arm is:

[0018]

[0019] Here, n is an integer in the range of 1-5.

[0020] Preferably, the chelating agent includes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N”-triacetic acid (NOTA), 4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-acetic acid (DOTAM), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutaric acid (DOTAGA), 2-(4,7-bis(2-carboxyethyl)-1,4,7-triazolin-1-yl)glutaric acid (NOTAGA), 3,6,10,13,16,19-hexaazabicyclo[6.6.6]docosane-1,8-diamine (DiAmSar), 3,6,10,13,16,19-hexaazabicyclo[6.6.6]docosane-1,8-diamine (DTPA), diethylenetriaminepentaacetic acid or deferoxamine mesylate (DFO). More preferably, the chelating agent is DOTA.

[0021] The present invention also provides a radionuclide-conjugated cyclic peptide targeting tumor uPAR, which is obtained by mixing and reacting the labeling precursor described in claim 5 with a radionuclide solution; the structure of the cyclic peptide conjugated with the radionuclide molecule is F-X-R-G, where F is selected from radionuclides 64 Cu, 67 Cu, 68 Ga, 18 F, 177 Lu, 90 Y, 89 Zr, 99m Tc, 89 Sr, 212 Pb or 225 Ac; preferably F is selected from 64 Cu.

[0022] The present invention also provides an application of the radionuclide-conjugated cyclic peptide targeting tumor uPAR in any one of the following (1)-(3):

[0023] (1) Preparing a diagnostic product labeled with a diagnostic radionuclide;

[0024] (2) Preparing a therapeutic drug labeled with a therapeutic radionuclide;

[0025] (3) Preparing a diagnostic product and / or therapeutic drug for tumors with high uPAR expression;

[0026] Wherein, the diagnostic product is a diagnostic reagent or a diagnostic drug.

[0027] Preferably, the radionuclide-conjugated cyclic peptides of the present invention are used for cancer types with high uPAR expression, including: adenoid cystic carcinoma, bladder urothelial carcinoma, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, endometrial cancer, uterine carcinosarcoma.

[0028] The present invention discloses the following technical effects:

[0029] In view of the special structure of the uPAR-targeting cyclic peptide cyclo(1,9)[CNKYFSNIC]W-OH of the present invention, without affecting its binding site with the target protein, its side chain was modified and chelated with a radionuclide for PET imaging. By changing the configuration of cysteine, three uPAR-targeting cyclic peptide molecules CARP-1, CARP-2, and CARP-3 were constructed. The three uPAR-targeting cyclic peptide molecules obtained in the present invention have good stability and are easy to synthesize. This molecule can be labeled with a radionuclide ( 64 Cu), and the resulting radioactive molecule can specifically bind to tumor uPAR. Through PET imaging, the uPAR-positive tumor tissue can be accurately located, achieving the purpose of early tumor diagnosis and treatment. The molecule in the present invention has high affinity and specificity for uPAR. This novel radionuclide-conjugated cyclic peptide can evaluate the uPA expression of primary tumors and metastatic lesions in vivo in real time. After further evaluation in clinical trials of drug use, it is expected to provide a more accurate option for early diagnosis and early treatment of cancer patients. Brief Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 HPLC chromatogram and mass spectrum of CARP-1;

[0032] Figure 2 HPLC chromatogram and mass spectrum of CARP-2;

[0033] Figure 3HPLC chromatogram and mass spectrum of CARP-3;

[0034] Figure 4 are 64 Cu-CARP-1, 64 Cu-CARP-2 and 64 PET imaging diagrams of Cu-CARP-3 in MC38 tumor-bearing mice;

[0035] Figure 5 are 64 Cu-CARP-1, 64 Cu-CARP-2 and 64 time-uptake diagrams of Cu-CARP-3 in tumor, liver and kidney;

[0036] Figure 6 are 64 PET imaging diagram of Cu-CARP-2 in B16F10 tumor-bearing mice. Detailed implementation manners

[0037] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0040] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0041] As used herein, terms such as "comprising", "including", "having", "containing", etc. are all open-ended terms, meaning including but not limited to.

[0042] The test methods involved in the following examples and comparative examples are as follows:

[0043] In the present invention 64 Cu was purchased from commercial channels with a radionuclide purity of 98%. Radioactive high-performance liquid chromatography (HPLC) analysis of all radioactive tracers was performed using an HPLC system (Shimadzu, Japan).

[0044] All animal experiments used in the present invention have been approved by the Animal Ethics Committee of Nanjing Medical University. All animals were cared for by humans, and the experiments were conducted according to the recommendations of the Committee on the Care and Use of Laboratory Animals. The establishment of tumor-bearing mouse models followed standard methods. Tumor-bearing C57BL / 6J mouse models were established by subcutaneous injection of MC38 or B16F10 (3×10 6 cells / mouse) cells. When the tumor volume reached approximately 100 - 300 mm 3 , the mice were used for PET / CT imaging and biodistribution studies. The mouse melanoma B16F10 and mouse colon cancer MC38 cell lines were maintained in a humidified CO 2 incubator (37 °C, 5% CO 2 ) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and passaged every three days.

[0045] The Chinese meanings of the English and abbreviations involved in the present invention are as follows:

[0046] Fmoc: 9-fluorenylmethoxycarbonyl;

[0047] Resin: resin;

[0048] DCM: dichloromethane;

[0049] DMF: N,N-dimethylformamide;

[0050] NMP: N-methylpyrrolidone;

[0051] HBTU: benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0052] HCTU: 6-chlorobenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate;

[0053] DIEA: N,N-diisopropylethylamine;

[0054] TFA: Trifluoroacetic acid;

[0055] TIS: Triisopropylsilane;

[0056] Trt: Triphenylmethyl;

[0057] tBu: Tert-butyl;

[0058] Pbf: 2,2,4,6,7-Pentamethylbenzofuran-5-sulfonyl;

[0059] DOTA: 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0060] Synthesis of CARP-1 in Example 1

[0061] The cyclic peptide cyclo(1,9)[CNKYFSNIC]W-OH is a commercially available resin peptide (Wang resin). After verifying the correct amino acid sequence, it is coupled with a linker and a chelating agent by solid-phase synthesis method. Then, the protecting groups are removed and the crude product is eluted from the resin, and the pure product CARP-1 is obtained by HPLC purification.

[0062] The specific synthesis steps are as follows:

[0063] (1) Put 200 mg of the crude peptide into a reaction tube, add 20% piperidine / DMF solution (20% piperidine: DMF = 1:4), vibrate for 5 min, remove it, then add 20% piperidine / DMF solution and vibrate for 5 min. Wash twice with DMF, twice with dichloromethane, and twice with DMF in sequence.

[0064] (2) Add the DMF solution of Fmoc-NH-PEG3-CH 2 CH 2 COOH / HCTU / DIEA, react for 30 min, wash twice with DMF, twice with dichloromethane, and twice with DMF in sequence. Evaporate the solvent, add 20% piperidine / DMF solution, for 5 min, remove it, then add 20% piperidine / DMF solution and vibrate for 5 min.

[0065] (3) Wash the resin: Wash twice with DMF, twice with dichloromethane, and twice with DMF in sequence.

[0066] (4) Add the DMF solution of DOTAtri(tBuester) / HCTU / DIEA, react for 30 min, wash twice with DMF, twice with dichloromethane, and twice with DMF in sequence. Evaporate the solvent, add 20% piperidine / DMF solution, vibrate for 5 min, remove it, then add 20% piperidine / DMF solution and vibrate for 5 min.

[0067] (5) Wash the resin: Wash it twice with DMF, twice with dichloromethane, twice with DMF, and drain the resin.

[0068] (6) Cleavage: Prepare a cleavage solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H 2 O (v:v:v = 95:2.5:2.5), cleave the polypeptide from the resin, remove the cleavage solution, dissolve the polypeptide with 50% acetonitrile / H 2 O, filter the reaction solution, and separate the clarified filtrate by high performance liquid chromatography.

[0069] (7) Purification: Filter with a 0.2 μm filter membrane and then separate and purify using an RP-HPLC system. The separation and purification conditions are as follows: The chromatographic column is a reversed-phase C18 column, mobile phase: A: H 2 O (0.1% TFA), B: acetonitrile (0.1% TFA), B: 0 - 65% (0 - 40 min). After purification, freeze-dry to obtain the white powder cyclic peptide CARP-1. Its HPLC and mass spectrometry diagrams are shown in Figure 1 , and the structural formula is as follows.

[0070]

[0071] Example 2 Synthesis of CARP-2

[0072] The cyclic peptide cyclo(1,9)[cNKYFSNIC]W-OH is a commercially available resin peptide (Wang resin). Then it is coupled with a linker and a chelating agent by solid-phase synthesis method, and then the protecting groups are removed and the crude product is eluted from the resin, and the pure product CARP-2 is obtained by HPLC purification.

[0073] The specific synthesis steps are as follows:

[0074] (1) Put 200 mg of the crude peptide into a reaction tube, add a 20% piperidine / DMF solution (20% piperidine:DMF = 1:4), vibrate for 5 min, remove it, add another 20% piperidine / DMF solution and vibrate for 5 min. Wash twice with DMF, twice with dichloromethane, and twice with DMF.

[0075] (2) Add a DMF solution of Fmoc-NH-PEG3-CH 2 CH 2 COOH / HCTU / DIEA, react for 30 min, wash twice with DMF, twice with dichloromethane, twice with DMF, remove the solvent, add a 20% piperidine / DMF solution, for 5 min, remove it, add another 20% piperidine / DMF solution and vibrate for 5 min.

[0076] (3) Wash the resin: Wash it twice with DMF, twice with dichloromethane, and twice with DMF successively.

[0077] (4) Add a DMF solution of DOTAtri(tBuester) / HCTU / DIEA, react for 30 min, wash twice with DMF, twice with dichloromethane, and twice with DMF successively, remove the solvent, add a 20% piperidine / DMF solution, vibrate for 5 min, remove it, and then add a 20% piperidine / DMF solution again and vibrate for 5 min.

[0078] (5) Wash the resin: Wash it twice with DMF, twice with dichloromethane, twice with DMF successively, and dry the resin by suction.

[0079] (6) Cleavage: Prepare a cleavage solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H 2 O (v:v:v = 95:2.5:2.5), cleave the polypeptide from the resin, remove the cleavage solution, dissolve the polypeptide with 50% acetonitrile / H 2 O, filter the reaction solution, and separate the clarified filtrate by high performance liquid chromatography.

[0080] (7) Purification: After filtering with a 0.2 μm filter membrane, perform separation and purification using an RP-HPLC system. The separation and purification conditions are as follows: The chromatographic column is a reversed-phase C18 column, mobile phase: A: H 2 O (0.1% TFA), B: acetonitrile (0.1% TFA), B: 0 - 65% (0 - 40 min). After purification, lyophilize to obtain the white powdery cyclic peptide CARP-2, and its HPLC and mass spectrometry diagrams are shown in Figure 2 , and the structural formula is as follows.

[0081]

[0082] Example 3 Synthesis of CARP-3

[0083] The cyclic peptide cyclo(1,9)[CNKYFSNIc]W-OH is a commercially available resin peptide (Wang resin). Then, it is coupled with a linker and a chelating agent by solid-phase synthesis method, and then the protecting groups are removed and the crude product is eluted from the resin, and the pure product CARP-3 is obtained by HPLC purification.

[0084] The specific synthesis steps are as follows:

[0085] (1) Put 200 mg of the crude peptide into a reaction tube, add a 20% piperidine / DMF solution (20% piperidine: DMF = 1:4), vibrate for 5 min, remove it, then add a 20% piperidine / DMF solution again and vibrate for 5 min, wash twice with DMF, twice with dichloromethane, and twice with DMF successively.

[0086] (2) Add the DMF solution of Fmoc-NH-PEG3-CH 2 CH 2 COOH / HCTU / DIEA, react for 30 min, wash twice with DMF, twice with dichloromethane, twice with DMF successively, remove the solvent, add the 20% piperidine / DMF solution for 5 min, remove it, and then add the 20% piperidine / DMF solution and vibrate for 5 min.

[0087] (3) Wash the resin: Wash twice with DMF, twice with dichloromethane, and twice with DMF successively.

[0088] (4) Add the DMF solution of DOTAtri(tBuester) / HCTU / DIEA, react for 30 min, wash twice with DMF, twice with dichloromethane, and twice with DMF successively, remove the solvent, add the 20% piperidine / DMF solution and vibrate for 5 min, remove it, and then add the 20% piperidine / DMF solution and vibrate for 5 min.

[0089] (5) Wash the resin: Wash twice with DMF, twice with dichloromethane, twice with DMF successively, and dry the resin.

[0090] (6) Cleavage: Prepare the cleavage solution of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and H 2 O (v:v:v = 95:2.5:2.5), cleave the polypeptide from the resin, remove the cleavage solution, dissolve the polypeptide with 50% acetonitrile / H 2 O, filter the reaction solution, and separate the clarified filtrate by high performance liquid chromatography.

[0091] (7) Purification: Filter with a 0.2 μm filter membrane and then separate and purify using an RP-HPLC system. The separation and purification conditions are as follows: The chromatographic column is a reverse phase C18 column, mobile phase: A: H 2 O (0.1% TFA), B: acetonitrile (0.1% TFA), B: 0 - 65% (0 - 40 min). After purification, freeze-dry to obtain the cyclic peptide CARP-3 in white powder form. Its HPLC and mass spectrometry diagrams are shown in Figure 3 , and the structural formula is as follows.

[0092]

[0093] Example 4

[0094] This example discloses the synthesis of the compound 64 Cu-CARP-1, specifically as follows:

[0095] Add the cyclic peptide ligand CARP-1 (20 μg) and 64 CuCl successively into the reaction tube2 (10 mCi) 1.0 mL of the solution was adjusted to pH 5.0 with 1.0 mL of sodium acetate solution (0.1 mol / L) and reacted at 80 °C for 10 min. Finally, it was diluted with normal saline and filtered through a 0.22 μm needle-type sterile filter membrane and collected in a receiving bottle. Analyzed by RP-HPLC 64 The radiochemical purity of Cu-CAP-1, conditions were as follows:

[0096] Column: YMC-tria-C18;

[0097] Solvent gradient: Solvent A, deionized water; Solvent B, acetonitrile (0.1% trifluoroacetic acid (TFA));

[0098] Elution time: 20 min, acetonitrile from 10% to 90%;

[0099] Flow rate was 1.0 mL / min.

[0100] The prepared 64 Cu-CARP-1 was detected by Radio-HPLC, and the results were as follows: 64 The radiochemical yield of Cu-CARP-1 was greater than 99%, the radiochemical purity was greater than 97% (see Table 1), the retention time was about 8.33 min, and the molar activity of the tracer was greater than 70 GBq / μmol (see Table 1).

[0101] Table 1 Data of the labeling results of three tracers

[0102] Name Radiochemical yield (%) Radiochemical purity (%) <![CDATA[Molar activity (GBq·μmol -1 )]]> <![CDATA 64 Cu]CARP-1]]> ﹥99 ﹥97 70±10 <![CDATA 64 Cu]CARP-2]]> ﹥99 ﹥99 70±10 <![CDATA 64 Cu]CARP-3]]> ﹥99 ﹥99 70±10

[0103] The prepared 64 Cu-CARP-1 was subjected to an in vitro stability experiment, incubated and gently stirred in PBS buffer solution and mouse serum at 37 °C for 0 to 3 hours, and the samples (20 μL) were analyzed using a Shimadzu high performance liquid chromatography (HPLC) system and a C-18 column respectively. The results showed 64 Cu-CARP-1 had good stability in mice at 30 min, 65.552% of the parent remained at 140 min, while it had better stability in PBS buffer solution, and the content of the parent was greater than 95% after 5 h (see Table 2).

[0104] Table 2 Physicochemical properties of three tracers

[0105] Name Stability in salt solution (5 h) % Stability in serum (140 min) % <![CDATA 64 Cu]CARP-1]]> ﹥95 65.552 <![CDATA 64 Cu]CARP-2]]> ﹥95 38.458 <![CDATA 64 Cu]CARP-3]]> ﹥95 43.549

[0106] Example 5

[0107] This example discloses the synthesis of the compound 64 Cu-CARP-2, specifically as follows:

[0108] Add cyclo - peptide ligand CARP - 2 (20 μg) and 64 CuCl 2 (10 mCi) solution (1.0 mL) into the reaction tube in sequence, adjust the pH to 5.0 with sodium acetate solution (0.1 mol / L, 1.0 mL), and react at 80 °C for 10 min. Finally, dilute with normal saline and filter through a 0.22 - μm needle - type sterile filter membrane, then collect into a receiving bottle. Analyze the radiochemical purity of 64 Cu - CAP - 2 under the following conditions:

[0109] Column: YMC - tria - C18;

[0110] Solvent gradient: Solvent A, deionized water; Solvent B, acetonitrile (0.1% trifluoroacetic acid (TFA));

[0111] Elution time: 20 min, acetonitrile from 10% to 90%;

[0112] Flow rate: 1.0 mL / min.

[0113] Perform Radio - HPLC detection on the prepared 64 Cu - CARP - 2, and the results are as follows: 64 The radiochemical yield of Cu - CARP - 2 is greater than 99% (see Table 1), the retention time is about 8.43 min, and the molar activity of the tracer is greater than 70 GBq / μmol (see Table 2).

[0114] Perform in vitro stability experiments on the prepared 64 Cu - CARP - 2. Incubate in mouse serum at 37 °C with gentle stirring for 0 to 3 hours, sample at 30 min and 140 min respectively, and analyze the samples (20 μL) using a Shimadzu high - performance liquid chromatography (HPLC) system and a C - 18 column. The results show that 64 Cu - CARP - 2 has good stability in mice at 30 min. At 140 min, 38.458% of the parent compound remains, while it has better stability in PBS buffer solution, and the content of the parent compound is greater than 95% after 5 h (see Table 2).

[0115] Example 6

[0116] This example discloses the synthesis of compound 64 Cu - CARP - 3, specifically as follows:

[0117] Add CARP - 3 (20 μg) and 64 CuCl 21.0 mL of the (10 mCi) solution was adjusted to pH 5.0 with 1.0 mL of sodium acetate solution (0.1 mol / L), and reacted at 80 °C for 10 min. Finally, it was diluted with normal saline and filtered through a 0.22 μm needle-type sterile filter membrane and collected into a receiving bottle. Analyzed by RP-HPLC 64 The radiochemical purity of Cu-CARP-3, conditions:

[0118] Column: YMC-tria-C18;

[0119] Solvent gradient: Solvent A, deionized water; Solvent B, acetonitrile (0.1% trifluoroacetic acid (TFA));

[0120] Elution time: 20 min, acetonitrile from 10% to 90%;

[0121] Flow rate was 1.0 mL / min.

[0122] The prepared 64 Cu-CARP-3 was detected by Radio-HPLC, and the results were: 64 The radiochemical yield of Cu-CARP-3 was greater than 99% (see Table 1), the retention time was about 8.18 min, and the molar activity of the tracer was greater than 70 GBq / μmol (see Table 2).

[0123] The prepared 64 Cu-CARP-3 was subjected to an in vitro stability experiment, incubated in mouse serum at 37 °C with gentle stirring for 0 to 3 hours, sampled at 30 min and 140 min respectively, and the samples (20 μL) were analyzed using a Shimadzu high performance liquid chromatography (HPLC) system and a C-18 column. The results showed 64 Cu-CARP-3 had good stability in mice at 30 min, with 43.549% of the parent remaining at 140 min, and had better stability in PBS buffer solution, with the parent content greater than 95% after 5 h (see Table 2).

[0124] Example 7

[0125] This example was for the in vivo PET imaging study of compounds 64 Cu-CARP-1, 64 Cu-CARP-2 and 64 Cu-CARP-3 in MC38 tumor-bearing C57BL / 6J mice. The specific implementation was as follows:

[0126] MC38 tumor-bearing mice (n = 3) were intravenously injected with 3.70 MBq dissolved in 0.1 mL of saline 64 Cu-CARP-1, 64Cu-CARP-2 and 64 Cu-CARP-3. Mice were sacrificed by cervical dislocation after dynamic scanning within 60 min of injection. Tumors, livers, hearts, and kidneys were dissected and weighed wet. Subsequently, radioactivity was measured using an automatic gamma counter (PerkinElmer, ma, USA) and expressed as a percentage of the injected radioactivity dose per gram (%ID / g).

[0127] PET-CT serial images of the mice showed that within 0 - 5 min 64 Cu-CARP-1,[[]] 64 Cu-CARP-2 and 64 Cu-CARP-3 accumulated significantly in tumor tissues (see Figure 4 ). Tumor uptake gradually increased, and the radioactivity content showed a gradually decreasing trend after 5 min; within 60 min of injection, 64 Cu-CARP-2,[[]] 64 Cu-CARP-3 uptake in the liver and kidneys gradually decreased (see Figure 5 ).

[0128] Example 8

[0129] This example was an in vivo PET imaging study of the compound 64 Cu-CAP-2 in B16F10 tumor-bearing C57BL / 6J mice, specifically:

[0130] B16F10 tumor-bearing mice (n = 3) were intravenously injected with 3.70 MBq of 64 Cu-CARP-2 dissolved in 0.1 mL of saline. Dynamic scanning was performed within 60 min of injection. Mice were sacrificed by cervical dislocation. Tumors were dissected, and then radioactivity was measured using an automatic gamma counter (PerkinElmer, ma, USA) and expressed as a percentage of the injected radioactivity dose per gram (%ID / g). PET-CT serial images of the mice showed that within 0 - 5 min 64 Cu-CARP-2 accumulated significantly in tumor tissues (see Figure 6 ). After 5 min of injection, the radioactivity content showed a gradually decreasing trend.

[0131] The above-described examples are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A radionuclide-conjugated cyclic peptide targeting tumor uPAR, characterized in that, the cyclic peptide molecule has an X-R-G structure, where X is a chelating agent, R is a linker arm, and G is a cyclic peptide compound. The amino side of the linker arm is connected to the chelating agent, and the carboxyl side of the linker arm is connected to the cyclic peptide compound; the cyclic peptide compound includes cyclic peptides with different chiralities formed by intramolecular disulfide bonds, and its amino acid sequence is cyclo(1,9)[CNKYFSNIC]W-OH.

2. The radionuclide-conjugated cyclic peptide targeting tumor uPAR according to claim 1, characterized in that, the cyclic peptides with different chiralities are: (1) All cysteines in the cyclic peptide are of the L type; (2) The cysteine at the C-terminus of the cyclic peptide is of the L type or D type, and the cysteine at the N-terminus is of the D type or L type.

3. The radionuclide-conjugated cyclic peptide targeting tumor uPAR according to claim 1, characterized in that, the linker arm is selected from one of the following compounds: wherein, n is an integer in the range of 1-10.

4. The radionuclide-conjugated cyclic peptide targeting tumor uPAR according to claim 1, characterized in that, the chelating agent includes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclononane-N,N',N”-triacetic acid, 4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-acetic acid, 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)glutaric acid, 2-(4,7-bis(2-carboxyethyl)-1,4,7-triazolin-1-yl)glutaric acid, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]docosane-1,8-diamine, diethylenetriaminepentaacetic acid or deferoxamine mesylate.

5. A radionuclide-conjugated cyclic peptide targeting tumor uPAR, characterized in that, The cyclic peptide molecule can be used as a labeling precursor, chelated with a radionuclide to obtain a cyclic peptide conjugated with a radionuclide for targeting uPAR; the molecular structure of the cyclic peptide conjugated with the radionuclide is F-X-R-G, where F is selected from radionuclides 64 Cu, 67 Cu, 68 Ga, 18 F, 177 Lu, 90 Y, 89 Zr, 99m Tc, 89 Sr, 212 Pb or 225 Ac.

6. A preparation method of the radionuclide-conjugated cyclic peptide targeting tumor uPAR according to claim 5, characterized in that, Mix the cyclic peptide with a radionuclide solution and incubate it, then purify to obtain the cyclic peptide conjugated with a radionuclide targeting tumor uPAR; wherein, the radionuclide solution is 64 CuCl 2 、 67 CuCl 2 、 68 GaCl 3 、Al 18 F、 177 LuCl 3 、 90 YCl 3 、 89 ZrCl 4 、Na 99m TcO 4 、 89 SrCl 2 、 212 PbCl 2 or 225 AcCl 3 solution.

7. An application of the radionuclide-conjugated cyclic peptide targeting tumor uPAR according to claim 5 in any one of the following (1)-(3): (1) Preparing a diagnostic product labeled with a diagnostic radionuclide; (2) Preparing a therapeutic drug labeled with a therapeutic radionuclide; (3) Preparing a diagnostic product and / or a therapeutic drug for tumors with high uPAR expression; wherein, the diagnostic product is a diagnostic reagent or a diagnostic drug.

8. According to the application described in claim 7, the radionuclide-conjugated cyclic peptide targeting tumor uPAR of the present invention is used for cancer types with high uPAR expression, including: adenoid cystic carcinoma, bladder urothelial carcinoma, invasive breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, acute myeloid leukemia, low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, melanoma, gastric adenocarcinoma, testicular germ cell tumor, thyroid cancer, thymoma, endometrial cancer, uterine carcinosarcoma.

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