PET (Polyethylene Terephthalate) tracer for targeting tumor integrin alpha5beta1 as well as labeled precursor, preparation method and application of PET tracer

Abstract

The invention relates to the technical field of biological medicines, discloses a PET (Polyethylene Terephthalate) tracer for targeting tumor integrin alpha5beta1 as well as a labeled precursor, a preparation method and application of the PET tracer, and belongs to the technical field of biological medicines. The PEG tracer agent is of an F-X-R-G structure, X is a chelating agent, R is a connecting arm, G is a polypeptide compound, the amino acid sequence of the polypeptide compound is KSSPHSN (SG) 5RGDSP, KSSPHSN (SG) 5RGDSPC or KSSPHSN (SG) 5RGDSPC-PEG4, and F is selected from 64Cu. Experimental verification shows that the prepared PET tracer has good in-vivo stability, good binding specificity with tumor alpha5beta1, long tumor residence time and easy synthesis. The PEG tracer prepared by the invention has an important value in clinical application in polypeptide targeted nuclear drug tumor diagnosis and treatment integration.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a PET tracer targeting tumor integrin α5β1 and a labeled precursor, a preparation method and an application thereof. Background Art

[0002] Integrin is a transmembrane heterodimer composed of α subunits and β subunits. At present, 18 α subunits and 8 β subunits have been discovered, and different subunits can form 24 heterodimers. As a type of cell adhesion and signaling receptor, integrin can not only bind to extracellular ligands, but also interact with the cytoskeleton through the intracellular domain. The intracellular and extracellular bidirectional signaling pathways mediated by integrin participate in a variety of important biological behaviors such as cell adhesion, migration and survival. At present, drugs targeting integrins have been widely developed in fields including cardiovascular disease, inflammatory bowel disease, multiple sclerosis and dry eye disease. Integrin α5β1 is the only known α5 integrin and is abnormally highly expressed in a variety of malignant tumors. Its overexpression is closely related to tumor cell survival, migration, invasion, angiogenesis, drug resistance, and low survival rate and poor prognosis of patients. In addition, its inhibitors can inhibit the occurrence and development of glioblastoma. Therefore, integrin α5β1 is an important target for the development of nuclear drugs for tumor targeted therapy.

[0003] Precision targeted nuclear medicine tracers refer to the process of delivering radionuclides that emit α-particles, β-particles or Auger electrons to lesions through targeted carriers (such as peptides, proteins, small molecules), and accurately imaging the lesion site through precise nuclear imaging (PET or SPECT). It can not only achieve efficient tumor diagnosis, but also guide the dosage of drugs during treatment. Among them, peptides are an important targeting carrier of nuclear medicines. Compared with antibodies and small molecules, they show unique advantages in synthetic labeling, tissue penetration, and pharmacokinetic properties. The following are currently reported targeted nuclear medicines based on integrin α5β1, all of which are used as tracers for tumor imaging. In 2013, Neubauer et al. first reported a PET tracer for integrin α5β1 imaging. 68 Ga-NODAGA-FR366, whose targeting vector is a small molecule based on a peptide analog, has a high affinity only for α5β1 in the integrin family. 68 Ga-aquibeprin has also been reported, and this tracer is also highly selective for integrin α5β1. vThe peptide isoDGR, which has a targeting effect on both α5β6 and α5β1, was modified with nitrogen methylation to obtain isoDGR(NMe)k. The modified peptide has a high affinity only for α5β1. The peptide was trimerized and labeled with nuclides through TRAP to obtain a PET tracer. 68 Ga-8 has good tumor α5β1 imaging ability. In addition, PR_b is also a polypeptide with high affinity for integrin α5β1. There are two main structural units in PR_b, the main recognition site RGDSP of α5β1 and the cooperative binding site PHSRN (19). 18 F-aluminum fluoride 18 The PET tracer is radiolabeled with F. This PET tracer exhibits α5β1 binding activity and specificity, and has rapid blood clearance and major renal excretion. However, PR_b is easily degraded in vivo and has poor stability, making it difficult to use in tumor treatment. In addition, the cell membrane penetration ability and tissue penetration of PR_b need to be further explored. Therefore, optimizing the structure of PR_b and improving its in vivo stability are expected to improve its clinical application value in the integrated diagnosis and treatment of tumors with peptide-targeted nuclear medicine. Summary of the invention

[0004] The purpose of the present invention is to provide a PET tracer targeting tumor integrin α5β1 and its labeled precursor, preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The PET tracer has good in vivo stability, good binding specificity with tumor integrin α5β1, and a long tumor retention time, and is easy to synthesize.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a labeling precursor of a PET tracer targeting tumor integrin α5β1, wherein the labeling precursor is an XRG structure, wherein X is a chelating agent, R is a connecting arm, G is a polypeptide compound, the amino side of the connecting arm is connected to the chelating agent, the carboxyl side of the connecting arm is connected to the N-terminal lysine amino group of the polypeptide compound, and the amino acid sequence of the polypeptide compound is KSSPHSRN(SG)5RGDSP or KSSPHSRN(SG)5RGDSPC.

[0007] Preferably, the linker arm has one of the compounds represented by the following structural formula:

[0008]

[0009] Here, n is an integer in the range of 1-10.

[0010] Preferably, the structural formula of the connecting arm is: Here, n is an integer in the range of 1-5.

[0011] Preferably, the chelating agent comprises 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or 1,4,7-triazacyclononane-N,N',N"-triacetic acid.

[0012] Preferably, the chelating agent is 1,4,7-triazacyclononane-N,N',N"-triacetic acid.

[0013] The present invention also provides a method for preparing the labeling precursor, comprising the following steps:

[0014] synthesizing the polypeptide compound;

[0015] Carrying out coupling reaction between the polypeptide compound, the connecting arm and the chelating agent to obtain a crude product of the labeling precursor;

[0016] The crude product of the labeling precursor is purified to obtain the labeling precursor.

[0017] Preferably, the molar ratio of the polypeptide compound, the linker arm and the chelating agent is 1:(1-5):(1-5);

[0018] The purification reagent is a mixture of diethyl ether and n-hexane, and the volume ratio of the diethyl ether to the n-hexane is 1:1.

[0019] The present invention also provides a PET tracer targeting tumor integrin α5β1, wherein the PET tracer is obtained by chelating a nuclide in the labeling precursor, and the structure of the PET tracer is FXRG, wherein F is selected from a nuclide 64 Cu.

[0020] The present invention also provides a method for preparing the PET tracer targeting tumor integrin α5β1, comprising the following steps:

[0021] The labeled precursor is mixed with a radionuclide solution and incubated to obtain the PET tracer targeting tumor integrin α5β1; wherein the radionuclide solution is 64 CuCl2 solution.

[0022] Preferably, before the labeling precursor is mixed with the nuclide solution, the pH value is adjusted to 4.5-5.5 with a buffer solution, and the buffer solution is sodium acetate, water, ethanol, phosphoric acid, saline buffer solution or dimethyl sulfoxide;

[0023] The incubation condition is 70-90° C. for 5-15 min.

[0024] The present invention also provides a pegylated PET tracer targeting tumor integrin α5β1, wherein the polypeptide compound in the PET tracer is connected by four-arm polyethylene glycol to form a tetrameric polypeptide, namely, the pegylated PET tracer targeting tumor integrin α5β1.

[0025] The present invention also provides a method for preparing the PEGylated PET tracer targeting tumor integrin α5β1, comprising the following steps:

[0026] The labeled precursor, the four-arm polyethylene glycol and the radionuclide solution are mixed and incubated to obtain a PEGylated PET tracer targeting tumor integrin α5β1; wherein the radionuclide solution is 64 CuCl2 solution,

[0027] Preferably, before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is sodium acetate, water, ethanol, phosphate buffer solution or dimethyl sulfoxide, and the pH value is 4.5-5.5;

[0028] The incubation condition is 70-90° C. for 5-15 min.

[0029] More preferred is polyethylene glycol (Creative PEGWorks, PSB-453), the structure of which is shown below:

[0030]

[0031] The present invention also provides the use of the labeled precursor in preparing a PET tracer targeting tumor integrin α5β1.

[0032] The present invention also provides the labeled precursor, the PET tracer targeting tumor integrin α5β1, or the PEGylated PET tracer targeting tumor integrin α5β1.

[0033] Application in any of the following (1)-(3):

[0034] (1) Preparation of diagnostic products and / or therapeutic drugs labeled with diagnostic nuclides;

[0035] (2) preparing therapeutic drugs labeled with therapeutic nuclides;

[0036] (3) preparing diagnostic products and / or therapeutic drugs for tumors with high integrin α5β1 expression;

[0037] Wherein, the diagnostic product is a diagnostic reagent or a diagnostic drug.

[0038] The present invention discloses the following technical effects:

[0039] The present invention first connects the chelating agent to the polypeptide compound targeting α5β1 through a connecting arm to synthesize a PET tracer labeled precursor QM-2301 with a sequence of KSSPHSRN(SG)5RGDSP, and synthesizes a PET tracer labeled precursor QM-2302 with a sequence of KSSPHSRN(SG)5RGDSPC through cysteine ​​modification at the carbon end. 64 Cu labeling to obtain high purity PET tracer [ 64 Cu]QM-2301 and [ 64 Cu]QM-2302. At the same time, through the modification method of PEGylation and 64 Cu-labeled, synthetic PEGylated PET tracer [ 64 Cu] QM-2303, the experiment found that: the PET tracer prepared by the present invention has good in vivo stability, good binding specificity with tumor α5β1, long tumor retention time, and is easy to synthesize. The PEG tracer prepared by the present invention has important value for the clinical application of polypeptide targeted nuclear medicine in the integrated diagnosis and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 HPLC and mass spectrometry analysis results of PET tracer labeled precursors QM-2301 (AB) and QM-2302 (CD);

[0042] Figure 2 for[ 64 Cu]QM-2301 and [ 64 Radioactive high performance liquid chromatography (HPLC) analysis results of Cu]QM-2302;

[0043] Figure 3 for[ 64 Radioactive high performance liquid chromatography (HPLC) analysis results of Cu]QM-2303;

[0044] Figure 4 for[ 64 Cu]QM-2301, 64 Cu]QM-2302 and [ 64 Study on the stability (A), time and cell binding (B), and concentration and cell binding (C) of Cu]QM-2303 in vitro;

[0045] Figure 5 for[ 64 Cu]QM-2302 and [ 64 In vivo PET imaging study of QM-2303 in normal mice; A: [ 64 Cu]QM-2302 in vivo PET imaging; B: [ 64 Cu]QM-2303 in vivo PET imaging; C:[ 64 Cu]QM-2302 aggregation in different tissues; D: [ 64 Cu]Statistical diagram of QM-2303 aggregation in different tissues;

[0046] Figure 6 for[ 64 Cu]QM-2301 and [ 64 In vivo PET imaging study of Cu]QM-2302 in tumor-bearing mice; A: [ 64 PET imaging scan results of QM-2301; B: [ 64 Cu]QM-2302 PET imaging scan results; C: analysis of tracer uptake in tumors; D: in vivo metabolism analysis of tracers in tumors;

[0047] Figure 7 for[ 64 In vivo PET imaging study of Cu]QM-2303 in tumor-bearing mice; A: [ 64 Cu]QM-2303 PET imaging results; B: Analysis of tracer uptake in tumors; C: In vivo metabolism analysis of tracers in tumors; D: [ 64 Cu]QM-2301, 64 Cu]QM-2302 and [ 64 Cu]QM-2303 retention time in tumors;

[0048] Figure 8 for[ 64 Cu]isoDGR,[ 64 Cu]QM-2303 and [ 64 In vivo PET imaging study of Cu]QM-2302_PEG mixture in tumor-bearing mice; A: [ 64 Cu]isoDGR;B:[ 64 Cu]QM-2303;C:[ 64 Cu]QM-2302_PEG mixture; D: Statistical graph of the uptake of different tracers in tumors. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] In the present invention 64 Cu was purchased from commercial sources with a radionuclide purity of 98%. Radioactive high performance liquid chromatography (HPLC) analysis of all radiotracers was performed using an HPLC system (Shimadzu, Japan).

[0055] The present invention uses mouse melanoma B16F10 cell line as a cell model, and its culture medium is: DMEM high-glucose culture medium + 10% fetal bovine serum + 1% penicillin / streptomycin, and the incubator is set at: 37°C, 5% CO2. The cell culture is performed once every three days, and the medium is changed once every two days.

[0056] All animal experiments used in this invention were approved by the Animal Ethics Committee of the National Institute for Quantum Science, Japan. All animal experiments followed the 3R principle and were conducted under the guidance of the Experimental Animal Care and Ethics Committee. The tumor-bearing C57BL / 6J mouse model was established by subcutaneous injection of B16F10 cells. Each mouse was injected unilaterally in the axilla with 1×10 6 cells, and one tumor was established in each mouse. After 2 weeks, when the tumor tissue grew to about 100-300mm 3 At 4 pm, mice were dosed and subjected to PET / CT imaging studies.

[0057] The Chinese meanings of the English and abbreviations involved in the present invention are as follows:

[0058] Fmoc: 9-methoxycarbonyl;

[0059] RinkAmide-MBHAResin: 4-Methylbenzhydrylamine resin hydrochloride;

[0060] DMF: N,N,-dimethylformamide;

[0061] DCM: dichloromethane;

[0062] HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0063] HCTU: 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate;

[0064] NMP: N-methylpyrrolidone;

[0065] TFA: trifluoroacetic acid;

[0066] DIEA: N,N-diisopropylethylamine;

[0067] TIS: triisopropylsilane;

[0068] Trt: trityl;

[0069] tBu: tert-butyl;

[0070] Pbf: 2,2,4,6,7-pentamethylbenzofuran-5-sulfonyl;

[0071] NOTA: 1,4,7-triazacyclononane-1,4,7-triacetic acid;

[0072] DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.

[0073] Example 1 Labeled precursor QM-2301 of PET tracer targeting tumor α5β1 and PET tracer [64 Synthesis of Cu]QM-2301

[0074] Using the Fmoc-based solid phase peptide synthesis method, a peptide with the sequence of KSSPHSRN(SG)5RGDSP was synthesized, and then coupled with a linker and a chelating agent, followed by deprotection and shearing of the crude product, and purified by HPLC to obtain pure QM-2301. The synthetic route is as follows:

[0076] The specific synthesis steps are:

[0077] (1) Resin swelling: 100 mg of Rink Amide-MBHA Resin with a degree of substitution of 0.32 mmol / g was added to a reaction tube, followed by 3 mL of dichloromethane, and the mixture was reacted for 30 minutes under nitrogen blowing.

[0078] (2) Removal of Fmoc protecting group from resin: discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 5 minutes, discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 15 minutes.

[0079] (3) Resin cleaning: Wash with DMF, dichloromethane, and DMF in sequence, twice each.

[0080] (4) First amino acid connection: The solvent was filtered off using a sand core, and Fmoc-Pro-OH (4 eq, 43.18 mg) was first added to the reaction tube, followed by DIPEA (8 eq, 130 mg) and HCTU (4 eq, 120 mg), and finally DMF was added to dissolve, and the reaction was allowed to proceed for 60 minutes under nitrogen.

[0081] (5) Resin washing: Wash with DMF, dichloromethane, and DMF in sequence, twice each.

[0082] (6) Removal of Fmoc protecting group from resin: discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 5 minutes, discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 15 minutes.

[0083] (7) Resin washing: Wash with DMF, dichloromethane, and DMF in sequence, twice each.

[0084] (8) Second amino acid connection: Fmoc-Ser(tBu)-OH (4 eq, 49.08 mg) was added to the reaction tube, followed by DIPEA (8 eq, 130 mg) and HCTU (4 eq, 120 mg), and finally DMF was added to dissolve, and the mixture was reacted for 30 minutes under nitrogen blowing.

[0085] Repeat the above steps and connect in sequence: Fmoc-Asp(otBu)-OH(52.67mg), Fmoc-Gly-OH(38.05mg), Fmoc-Arg(Pbf)-OH(83.04mg), Fmoc-Gly-OH(38.05mg), Fmoc-Ser(tBu)-OH(49.08mg), Fmoc-Gly-OH(38.05mg), Fmoc-Ser(tBu)-OH(49.08mg), Fmoc-Gly-OH(38.05mg), Fmoc-Ser(tBu)-OH(49.08mg), Fmoc-Gly-OH(38.05mg), Fmoc-Ser(tBu)-OH(49.08mg), Fmoc-Gly-OH(38.05mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Asn(Trt)-OH (76.37 mg), Fmoc-Arg(Pbf)-OH (83.04 mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-His(Trt)-OH (79.32 mg), Fmoc-Pro-OH (43.18 mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Lys(Boc)-OH (59.97 mg), Fmoc-NH-PEG3-CH2CH2COOH (66.5 mg), NOTAtri(tBuester) (86 mg), the amount of the above substances used is 4 eq.

[0086] (9) Drain the solution and wash the resin: wash with DMF, methanol, and DMF three times each. Drain the resin.

[0087] (10) Peptide shearing: Add shearing solution (trifluoroacetic acid (TFA): triisopropylsilane (TIPS): H2O = 95:2.5:2.5, v:v:v) to the reaction tube, place on a shaker for 2 hours, and remove the shearing solution;

[0088] (11) Filtration: Plug cotton into a 1 mL pipette tip, pour the shear system into the filter, rinse the resin with an appropriate amount of TFA after filtration, collect the filtrate in a reaction tube, and then blow dry the filtrate with nitrogen to obtain a crude peptide solid;

[0089] (12) Peptide purification: A cold ether + hexane mixture (ether: hexane = 1:1, v:v) was added to the crude peptide solid to generate a white precipitate, which was then sonicated for 30 seconds and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the precipitate was blown dry with nitrogen. After purification, the peptide precursor QM-2301 was obtained by freeze drying in the form of a white powder.

[0090] QM-2301 mass spectrometry and HPLC detection diagrams are shown in Figure 1 As shown in A. The structure of the labeled precursor (QM-2301) of the synthesized PET tracer is shown below:

[0091]

[0092] (13) Radionuclide labeling: 20 μg of QM-2301 was dissolved in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.1) and then added 64 CuCl2 (10 mCi) solution, incubate at 80°C for 10 minutes. Dilute with saline and sterilize by filtration using a 0.22 μm filter. Radio-HPLC analysis using a YMC-tria-C18 filter column 64 The radiochemical purity of QM-2301 was determined by the following analysis conditions:

[0093] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% trifluoroacetic acid (TFA)); flow time: 20 min, acetonitrile from 10% to 90%; flow rate was 1.0 mL / min.

[0094] The PET tracer prepared in this example [ 64 Cu]QM-2301 was tested by Radio-HPLC, and the results were: 64 The radiochemical yield of Cu]QM-2301 was greater than 99% (Table 1) and the retention time was approximately 6.54 min (e.g. Figure 2 As shown in Table 1, the molar activity of the tracer was greater than 74 GBq / μmol.

[0095] Table 1. 64 Cu]QM-2301, 64 Cu]QM-2302 and [ 64 Quality Control Results of Cu]QM-2303

[0096]

[0097] Example 2 Labeled precursor QM-2302 of PET tracer targeting tumor α5β1 and PET tracer [ 64Synthesis of Cu]QM-2302

[0098] Using the Fmoc-based solid phase peptide synthesis method, a peptide with the sequence of KSSPHSRN(SG)5RGDSPC was synthesized, and then coupled with a linker and a chelating agent, followed by deprotection and shearing of the crude product, and purified by HPLC to obtain pure QM-2302. The synthetic route is as follows:

[0099]

[0100] The specific synthesis steps are:

[0101] (1) Resin swelling: 100 mg of Rink Amide-MBHA Resin with a degree of substitution of 0.32 mmol / g was added to a reaction tube, followed by 3 mL of DCM, and the mixture was reacted for 30 minutes under nitrogen blowing.

[0102] (2) Removal of Fmoc protecting group from resin: discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 5 minutes, discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 15 minutes.

[0103] (3) Resin cleaning: Wash with DMF, dichloromethane, and DMF in sequence, twice each.

[0104] (4) First amino acid connection: Use a sand core to filter out the solvent, first add Fmoc-Cys(Trt)-OH (4 eq, 43.18 mg) to the reaction tube, then add DIPEA (8 eq, 130 mg) and HCTU (4 eq, 120 mg), and finally add DMF to dissolve, blow nitrogen gas and react for 60 minutes.

[0105] (5) Resin washing: Wash with DMF, dichloromethane, and DMF in sequence, twice each.

[0106] (6) Removal of Fmoc protecting group from resin: discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 5 minutes, discard the solvent, add 3 mL of 20% piperidine / DMF solution (20% piperidine:DMF=1:4, v:v), react for 15 minutes.

[0107] (7) Resin washing: Wash with DMF, dichloromethane, and DMF in sequence, twice each.

[0108] (8) Second amino acid connection: Fmoc-Pro-OH (4 eq, 49.08 mg) was added to the reaction tube, followed by DIPEA (8 eq, 130 mg) and HCTU (4 eq, 120 mg), and finally DMF was added to dissolve, and the mixture was reacted for 30 minutes under nitrogen blowing.

[0109] Repeat the above steps and connect in sequence: Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Asp(otBu)-OH (52.67 mg), Fmoc-Gly-OH (38.05 mg), Fmoc-Arg(Pbf)-OH (83.04 mg), Fmoc-Gly-OH (38.05 mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Gly-OH (38.05 mg), Fmoc-Ser(tBu)-OH (49.08 mg), Fmoc-Gly-OH (38.05 mg), Fmoc-Ser(tBu)-OH (49.08 mg). mg), Fmoc-Gly-OH (38.05mg), Fmoc-Ser(tBu)-OH (49.08mg), Fmoc-Asn(Trt)-OH (76.37m g), Fmoc-Arg(Pbf)-OH(83.04mg), Fmoc-Ser(tBu)-OH(49.08mg), Fmoc-His(Trt)-OH(79 .32mg), Fmoc-Pro-OH (43.18mg), Fmoc-Ser(tBu)-OH (49.08mg), Fmoc-Ser(tBu)-OH (49.08mg), Fmoc-Lys(Boc)-OH (59.97mg), Fmoc-NH-PEG3-CH2CH2COOH (66.5mg), NOTAtri(tBu) ester) (86 mg), the amount of the above substance used is 4 eq.

[0110] (9) Drain the solution and wash the resin: wash with DMF, methanol, and DMF three times each. Drain the resin.

[0111] (10) Peptide shearing: Add shearing solution (trifluoroacetic acid (TFA): triisopropylsilane (TIPS): H2O = 95:2.5:2.5, v:v:v) to the reaction tube, place on a shaker for 2 hours, and remove the shearing solution;

[0112] (11) Filtration: Plug cotton into a 1 mL pipette tip, pour the shear system into the filter, rinse the resin with an appropriate amount of TFA after filtration, collect the filtrate in a reaction tube, and then blow dry the filtrate with nitrogen to obtain a crude peptide solid;

[0113] (12) Peptide purification: A low-temperature ether / hexane mixture (ether:hexane=1:1, v:v) was added to the crude peptide solid to generate a white precipitate, which was then sonicated for 30 seconds and centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded and the precipitate was blown dry with nitrogen. After purification, the peptide precursor QM-2302 was obtained by freeze drying in the form of a white powder.

[0114] QM-2302 mass spectrometry and HPLC detection diagrams are shown in Figure 1 As shown in B. The structure of the labeled precursor (QM-2302) of the synthesized PET tracer is shown below:

[0115]

[0116] (13) Dissolve 20 μg of QM-2302 in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.1) and then add 64 CuCl2 (10 mCi) solution, incubate at 80°C for 10 minutes. Dilute with saline and sterilize by filtration using a 0.22 μm filter. Radio-HPLC analysis using a YMC-tria-C18 filter column 64 The radiochemical purity of QM-2302 was determined by the following analysis conditions:

[0117] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% trifluoroacetic acid (TFA)); flow time: 20 min, acetonitrile from 10% to 90%; flow rate was 1.0 mL / min.

[0118] The PET tracer prepared in this example [ 64 The results of Radio-HPLC test of QM-2302 were as follows: 64 The radiochemical yield of Cu]QM-2302 was greater than 99% (Table 1) and the retention time was approximately 6.54 minutes ( Figure 2 As shown in Table 1, the molar activity of the tracer was greater than 74 GBq / μmol.

[0119] Example 3 PET tracer [ 64 The synthesis of Cu]QM-2303 is as follows:

[0120] 20 μg of QM-2302 was dissolved in sodium acetate solution (0.1 mol / L, 100 mL, pH 4.1), and then PEG molecules, 64CuCl2 (10 mCi) solution, incubated at 80°C for 6 hours. Diluted with physiological saline and sterilized by filtration using a 0.22 μm filter. Radio-HPLC analysis using a YMC-tria-C18 filter column 64 The radiochemical purity of QM-2303 was determined by the following analysis conditions:

[0121] Solvent gradient: solvent A, deionized water; solvent B, acetonitrile (0.1% trifluoroacetic acid (TFA)); flow time: 20 min, acetonitrile from 10% to 90%; flow rate was 1.0 mL / min.

[0122] The PET tracer prepared in this example [ 64 The results of Radio-HPLC test of QM-2303 were as follows: 64 The radiochemical yield of Cu]QM-2303 was about 57% (Table 1) and the retention time was about 8.69 minutes ( Figure 3 The molar activity of the tracer was greater than 30 GBq / μmol (Table 1).

[0123] Example 4

[0124] This embodiment discloses the compound [ 64 Cu]QM-2301, 64 Cu]QM-2302, 64 In vitro stability study of Cu]QM-2303. Specifically, the three prepared PET tracers were incubated in mouse serum on a shaker for 0 to 1 hour at a reaction temperature of 37°C. At different time intervals, 20 μL samples were collected and analyzed using a Shimadzu high-performance liquid chromatography (HPLC) system and a C-18 column. The results showed that [ 64 Cu]QM-2301, 64 Cu]QM-2302 and [ 64 Cu]QM-2303 mice have a certain stability, and [ 64 Cu]QM-2303 has higher stability ( Figure 4 (A).

[0125] Example 5

[0126] In this example, the compound [ 64 Cu]QM-2301, 64 Cu]QM-2302 and [ 64 The specific binding ability of Cu]QM-2303 to tumor cell α5β1 in vitro was compared. Figure 4 Specifically, B16F10 cells were cultured at 1×10 5The cells were seeded at a density of 100 cells / well in a 12-well plate and grown to a confluence of 70-80%. For tumor cell uptake experiments, the radioactive labeled tracers in the culture medium (all three were 740 KBq / mL) were added to the cells and incubated at 37°C for 5, 20, 40, 60, and 80 minutes. At each time point, the culture medium was discarded and the cells were washed three times with ice-cold DPBS; 300 μL of NaOH solution (concentration: 0.2 M) was added to collect the cell lysate. The radioactivity was detected using an autogamma counter (PerkinElmer WIZARD22480, ma, USA). For binding affinity (IC 50 ) measurement, 64 Cu]QM-2301, 64 Cu]QM-2302, 64 Cu]QM-2303 (20 μCi / mL) was added to B16F10 cells, and different concentrations of α5β1 antibody were added at the same time, and co-cultured at 37°C for 120 min. The IC 50 They are 1.94μM, 1.55μM and 1.30μM respectively.

[0127] Example 6

[0128] This example shows that the tracer [ 64 Cu]QM-2301, 64 Cu]QM-2302, 64 Cu]QM-2303 was studied in PET imaging of normal mice, specifically:

[0129] PET scanning was performed using Inveon Micro-PET / CT (Siemens Medical Solutions, Knoxville, Munich, Germany). Each mouse was injected via the tail vein with 3.7 MBq (100 μL) of [ 64 Cu]QM-2302, dynamic PET scanning was started immediately after injection and continued for 60 minutes (n = 3). Each mouse was injected with 3.7 MBq (100 μL) [ 64 Cu]QM-2303, static PET scans were performed at 50 minutes, 9 hours, and 33 hours after injection (n=3). 64 Cu]QM-2302 and [ 64 Cu]QM-2303 is mainly metabolized through the renal pathway and less accumulated in the liver, indicating that the tracer has low in vivo toxicity ( Figure 5 ).

[0130] Example 7

[0131] The compounds of the present invention [ 64 Cu]QM-2301 and [ 64 In vivo PET imaging study of Cu]QM-2302 in B16F10 tumor-bearing mice, specifically:

[0132] Each tumor-bearing mouse was injected with 3.7 MBq (100 μL) [ 64 Cu]QM-2301 and [ 64 Cu]QM-2302, dynamic PET scanning was started immediately after injection and continued for 60 minutes (n=3) to analyze the uptake of the tracer in the tumor and its metabolism in vivo.

[0133] PET-CT series images show [ 64 Cu]QM-2301 and [ 64 Cu]QM-2302 showed significant uptake in the tumor site 30 minutes after injection. However, 1 hour after injection, the tumor uptake of both was significantly reduced. Similarly, in tumor-bearing mice, the main metabolic pathway of the drug was also renal metabolism ( Figure 6 ).

[0134] Example 8

[0135] The compounds of the present invention [ 64 In vivo PET imaging study of Cu]QM-2303 in B16F10 tumor-bearing mice, specifically:

[0136] Each tumor-bearing mouse was injected with 3.7 MBq (100 μL) [ 64 Cu]QM-2303, PET scans were performed 30 minutes, 1 hour, 5 hours, 10 hours, and 24 hours after injection (n=3) to analyze the uptake of the tracer in the tumor and its metabolism in vivo.

[0137] PET-CT serial images showed that 30 minutes to 24 hours after injection, [ 64 Cu]QM-2303 has significant uptake in tumor sites. It is worth noting that compared with [ 64 Cu]QM-2301 and [ 64 Cu]QM-2302,[ 64 Cu]QM-2303 significantly prolonged its retention time in the tumor, which was closely related to its improved stability. 64 Cu]QM-2303 retention in the heart was also significantly improved, further indicating that the tracer had improved stability in the blood ( Figure 7 ).

[0138] Example 9

[0139] The compounds of the present invention [64 Cu]QM-2303 and [ 64 Comparison of in vivo PET imaging studies of Cu]isoDGR in B16F10 tumor-bearing mice, specifically:

[0140] Each tumor-bearing mouse was injected with 3.7 MBq (100 μL) [ 64 Cu]QM-2303 and [ 64 Cu]isoDGR, dynamic PET scanning was started immediately after injection and lasted for 60 minutes (n=3) to analyze the uptake of the tracer in the tumor and its metabolism in vivo. PET-CT serial images showed that 30 minutes to 1 hour after injection, [ 64 The uptake of QM-2303 in tumors was significantly higher than that in 64 Cu]isoDGR( Figure 7 ). In addition, this embodiment also discloses [ 64 Cu]QM-2303 and non-covalently linked [ 64 Comparison of PET imaging of Cu]QM-2302 and PEG molecule mixture in tumor-bearing mice. 64 Cu]QM-2303 also showed high tumor uptake, which indicates that [ 64 Importance of covalent bonding in the synthesis of Cu]QM-2303 ( Figure 8 ).

[0141] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A labeled precursor of a PET tracer targeting tumor integrin α5β1, characterized in that: The labeling precursor is an XRG structure, wherein X is a chelating agent, R is a connecting arm, and G is a polypeptide compound. 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 N-terminal lysine amino group of the polypeptide compound. The amino acid sequence of the polypeptide compound is KSSPHSRN(SG)5RGDSP or KSSPHSRN(SG)5RGDSPC.

2. The labeled precursor of the PET tracer targeting tumor integrin α5β1 according to claim 1, characterized in that: The connecting arm is selected from one of the compounds with the following structural formula: Here, n is an integer in the range of 1-10.

3. The labeled precursor of the PET tracer targeting tumor integrin α5β1 according to claim 2, characterized in that: The structural formula of the connecting arm is: Here, n is an integer in the range of 1-5.

4. The labeled precursor of the PET tracer targeting tumor integrin α5β1 according to claim 1, characterized in that: The chelating agent includes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or 1,4,7-triazacyclononane-N,N',N"-triacetic acid.

5. The labeled precursor of the PET tracer targeting tumor integrin α5β1 according to claim 4, characterized in that: The chelating agent is 1,4,7-triazacyclononane-N,N',N"-triacetic acid.

6. A method for preparing a labeling precursor according to any one of claims 1 to 5, characterized in that: The following steps are involved: synthesizing the polypeptide compound; Carrying out coupling reaction between the polypeptide compound, the connecting arm and the chelating agent to obtain a crude product of the labeling precursor; The crude product of the labeling precursor is purified to obtain the labeling precursor.

7. The preparation method according to claim 6, characterized in that: The molar ratio of the polypeptide compound, the linker and the chelating agent is 1:(1-5):(1-5); The purification reagent is a mixture of diethyl ether and n-hexane, and the volume ratio of the diethyl ether to the n-hexane is 1:

1.

8. A PET tracer targeting tumor integrin α5β1, characterized in that: The PET tracer is obtained by chelating a nuclide in a labeling precursor according to any one of claims 1 to 5, and the structure of the PET tracer is FXRG, where F is selected from the nuclide 64 Cu.

9. A method for preparing a PET tracer targeting tumor integrin α5β1 as claimed in claim 8, characterized in that: The following steps are involved: The labeled precursor is mixed with a radionuclide solution and incubated to obtain the PET tracer targeting tumor integrin α5β1; wherein the radionuclide solution is 64 CuCl2 solution.

10. The preparation method according to claim 9, characterized in that: Before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is sodium acetate, water, ethanol, phosphate buffer solution or dimethyl sulfoxide, and the pH value is 4.5-5.5; The incubation condition is 70-90° C. for 5-15 min.

11. A pegylated PET tracer targeting tumor integrin α5β1, characterized in that: The polypeptide compound in the PET tracer according to claim 8 is connected by four-arm polyethylene glycol to form a tetrameric polypeptide, which is a PEGylated PET tracer targeting tumor integrin α5β1.

12. A method for preparing a PEGylated PET tracer targeting tumor integrin α5β1 as claimed in claim 11, characterized in that: The following steps are involved: The labeled precursor, the four-arm polyethylene glycol and the radionuclide solution are mixed and incubated to obtain a PEGylated PET tracer targeting tumor integrin α5β1; wherein the radionuclide solution is 64 CuCl2 solution.

13. The preparation method according to claim 12, characterized in that: Before the labeling precursor is mixed with the nuclide solution, the labeling precursor is first dissolved in a buffer solution, wherein the buffer solution is sodium acetate, water, ethanol, phosphoric acid, saline buffer solution or dimethyl sulfoxide, and the pH value is 4.5-5.5; The incubation condition is 70-90° C. for 5-15 min.

14. Use of the labeled precursor according to any one of claims 1 to 5 in the preparation of a PET tracer targeting tumor integrin α5β1.

15. Use of the labeled precursor according to any one of claims 1 to 5, the PET tracer targeting tumor integrin α5β1 according to claim 8, or the PEGylated PET tracer targeting tumor integrin α5β1 according to claim 11 in any one of the following (1) to (3): (1) Preparation of diagnostic products and / or therapeutic drugs labeled with diagnostic nuclides; (2) preparing therapeutic drugs labeled with therapeutic nuclides; (3) preparing diagnostic products and / or therapeutic drugs for tumors with high integrin α5β1 expression; in, The diagnostic product is a diagnostic reagent or a diagnostic drug.

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