Nectin-4-targeting bicyclic peptide nuclide ligand, probe and preparation method and application thereof

Abstract

The invention relates to the technical field of nuclear medicines, and particularly discloses a Nectin-4-targeting bicyclic peptide nuclide ligand, a probe, a preparation method and application thereof. The invention relates to a Nectin-4-targeted bicyclic peptide nuclide ligand, which has a structure as shown in a formula I: # imgabs0 # formula I, and the Nectin-4-targeted bicyclic peptide nuclide ligand has a structure as shown in a formula II. The bicyclic peptide nuclide ligand and the probe have remarkable advantages in the aspects of radioactive stability, pharmacokinetic characteristics, tumor Nectin-4 binding specificity, tumor uptake ability, tumor and muscle uptake ratio, in-vivo metabolic performance and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of nuclear medicine, and in particular to a bicyclic peptide nuclide ligand and a probe targeting nectin-4, and a preparation method and application thereof. Background Art

[0002] Breast cancer is the most common malignant tumor in women. According to immunohistochemistry, breast cancer is mainly divided into four subtypes: luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) positive and triple-negative breast cancer (TNBC). Among them, triple-negative breast cancer is characterized by the lack of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2, accounting for about 15% to 20% of diagnosed breast cancer. Compared with other breast cancer subtypes, triple-negative breast cancer is more invasive, with higher recurrence and visceral metastasis rates. Triple-negative breast cancer is sensitive to chemotherapy, so traditional chemotherapy is still the standard treatment. However, conventional treatment has limited effect: the median overall survival of patients with triple-negative breast cancer is 10.2 months, the 5-year survival rate of patients with regional tumors is about 65%, and the 5-year survival rate of patients with tumors that spread to distant organs is 11%. Due to the lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, there is currently no effective targeted treatment for triple-negative breast cancer. Therefore, cell surface proteins that are specifically expressed in triple-negative breast cancer cells but not expressed or downregulated in normal breast tissue would become ideal biomarkers and potential therapeutic targets.

[0003] Nectin protein belongs to the adhesion receptor of the immunoglobulin superfamily (IgSF). It was originally defined as a homolog of the poliovirus receptor (PVR) and consists of four members, Nectin-1~4. Nectin-4 is a type I transmembrane cell adhesion molecule composed of three immunoglobulin (IgG)-like domains in the extracellular region. It is involved in the regulation of related signaling pathways such as cell proliferation, morphogenesis, growth and development, and immune regulation. Nectin-4 is lowly expressed in normal tissues of adults, but is overexpressed in a variety of cancers including bladder cancer, breast cancer, pancreatic cancer, oral cancer, ovarian cancer, and lung cancer. The expression rate of nectin-4 in triple-negative breast cancer and basal breast cancer is about 62%, while it is not expressed in normal breast epithelium and other normal tissues of adults. It is a cell surface biomarker for triple-negative breast cancer and can be used to develop targeted drugs.

[0004] In the field of cancer drug development, the antibody-drug conjugate Enfortumab Vedotin (PADCEV ®), which is formed by combining the humanized monoclonal antibody AGS-22M6 targeting nectin-4 with the microtubule inhibitor monomethyl auristatin E (MMAE), has been used to treat advanced or metastatic bladder cancer. Clinical studies have shown that the drug has potential efficacy in patients with locally advanced or metastatic urothelial carcinoma, providing a promising treatment option for patients whose disease has progressed after treatment with platinum-based chemotherapy, immunotherapy or the small molecule tyrosine kinase inhibitor erdafitinib.

[0005] Although antibody-drug conjugates (ADCs) targeting nectin-4 have broad clinical application prospects, issues such as how to screen patients for indications and evaluate efficacy remain unresolved. In addition, clinical studies have shown that the expression of nectin-4 in metastatic lesions is often reduced or absent, and the absence or low expression of nectin-4 may predict the occurrence of drug resistance.

[0006] Nuclear medicine molecular imaging technology can reflect the functional changes of tissue physiology, pathology and metabolism at the molecular level, and has been widely used in the study of tumor membrane receptors and immune checkpoint microenvironment. Compared with traditional imaging methods, nuclear medicine imaging has higher sensitivity. Combined with highly specific molecular probes, it can specifically, accurately and non-invasively reflect the function and metabolism of tissues and organs. Therefore, the development of nuclear medicine probes targeting Nectin-4 has important clinical significance for the diagnosis, precise positioning and efficacy evaluation of Nectin-4 positive solid tumors.

[0007] The currently reported Nectin-4 targeted nuclear medicine diagnostic and therapeutic agent is a radionuclide 89 Zr, 18 F and 99m Tc-labeled nectin-4 monoclonal antibody. However, this type of imaging agent has defects such as large molecular weight, long in vivo circulation time, and weak transmembrane ability, which have certain limitations when used as conventional clinical molecular imaging agents. Therefore, it is urgent to develop radioactive targeted molecular probes to evaluate the expression level of nectin-4, and provide visualization tools and judgment basis for patient diagnosis, typing, treatment decision-making, efficacy evaluation, and prognosis evaluation. There are many advantages to using peptides as targeting carriers: compared with radioactive drugs designed based on antibodies, peptides are smaller in size, can be quickly cleared from the blood and non-target tissues, are non-immunogenic, have good tissue permeability, and have natural advantages in clinical transformation. Therefore, it is necessary to study the construction of nectin-4 targeting peptides. Summary of the invention

[0008] One of the purposes of the present invention is to provide a bicyclic peptide nuclide ligand targeting nectin-4 and its application which has better radioactivity stability, pharmacokinetic properties, binding specificity with tumor nectin-4, tumor uptake ability, tumor to muscle uptake ratio and in vivo metabolic performance.

[0009] The present invention provides a bicyclic peptide nuclide ligand targeting nectin-4, which has a structure as shown in Formula I: Formula I.

[0010] The present invention constructs a radionuclide labeled nectin-4 targeting molecular probe (bicyclic peptide nuclide ligand targeting nectin-4) with specific covalent modification at a specific position. The mother nucleus can achieve high affinity to the nectin-4 target through a specific amino acid sequence and cyclization mode, and the nuclide chelating group can bind to a variety of radionuclides to achieve the labeling of the bicyclic peptide targeting nectin-4, and the covalent modification group (fluorosulfonyl) located at the specific binding site can be used to target tyrosine, activated serine or threonine residues, and also has reactive activity to cysteine ​​and histidine side chains, so that after binding to the nectin-4 target protein site, the amino acid residues in the binding pocket area can provide hydrogen bond donors or electrophilic groups to stabilize the removable fluoride ions, thereby awakening the activity of fluorosulfonyl, and then forming an irreversible covalent bond with the nucleophilic amino acid residues of the endogenous target protein. Due to the increase in the binding time with the target protein, the application of the specific modified targeting peptide of the present invention can enhance the uptake and retention time of the tumor. The Nectin-4 targeted molecular probe labeled with a radionuclide that has been specifically covalently modified is used for preclinical validation of the in vivo recognition of Nectin-4 receptor-positive tumor models through PET whole-body imaging (which can achieve higher resolution and clearly distinguish tumor areas). This is of great significance for the early detection of triple-negative breast cancer with high Nectin-4 expression, the screening of people who will benefit from Nectin-4 targeted therapy drugs, the monitoring and evaluation of the efficacy of anti-tumor drugs, and the prognosis assessment, and has great potential for clinical transformation.

[0011] The present invention also provides a method for preparing the above-mentioned bicyclic peptide nuclide ligand targeting nectin-4, which comprises: (1) coupling the amino acids contained in formula I by solid phase synthesis to obtain a polypeptide chain; (2) coupling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid with the polypeptide chain to obtain a DOTA-polypeptide chain; (3) After coupling 3-(4-(fluorosulfonyl)phenyl)propionic acid with the DOTA-polypeptide chain, the DOTA-polypeptide chain is reacted with 1,3,5-triacryloylhexa-1,3,5-triazine.

[0012] The present invention also provides a bicyclic peptide nuclide probe targeting nectin-4, which is the bicyclic peptide nuclide ligand targeting nectin-4 labeled with a radioactive nuclide.

[0013] In the bicyclic peptide nuclide probe targeting nectin-4 of the present invention, the radionuclide includes a diagnostic radionuclide or a therapeutic radionuclide, and the diagnostic radionuclide is 68 Ga, 64 Cu, 18 F. 86 Y. 90 Y. 89 Zr, 111 In, 99m Tc, 11 C. 123 I. 125 I and 124 I at least one; the therapeutic radionuclide comprises 177 Lu, 125 I. 131 I. 211 At 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bihe 212 At least one of Pb; preferably, the radionuclide is 68 Ga.

[0014] Compared with the imaging agents in the prior art, the small molecule tracers provided by the present invention have the advantages of reasonable half-life, stronger tumor permeability, and can provide accurate evaluation results in a shorter time, thereby obtaining more accurate results. 68 Ga-labeled bicyclic peptide nuclide ligand has the characteristics of fast labeling speed and high labeling rate, with radiochemical purity greater than 95%. The labeled compound has good hydrophilicity. 68 The Ga-labeled bicyclic peptide nuclide ligand is mainly excreted through the kidneys, and the uptake in other non-target organs is low. It has a certain uptake in tumors, showing good sensitivity and specificity.

[0015] The present invention also provides a method for preparing the above-mentioned bicyclic peptide nuclide probe targeting nectin-4, which comprises the step of labeling the above-mentioned bicyclic peptide nuclide ligand targeting nectin-4 or the bicyclic peptide nuclide ligand targeting nectin-4 prepared by the above-mentioned method with a radioactive nuclide.

[0016] The present invention also provides a medicine, which comprises the above-mentioned bicyclic peptide nuclide ligand targeting nectin-4 or bicyclic peptide nuclide probe targeting nectin-4 and pharmaceutically acceptable excipients.

[0017] "Pharmaceutically acceptable" in the present invention means that the compound or composition is chemically and / or toxicologically compatible with other ingredients constituting the formulation and with humans or mammals for the prevention, diagnosis and treatment of diseases or disorders.

[0018] In the drug of the present invention, the bicyclic peptide nuclide ligand or bicyclic peptide nuclide probe targeting nectin-4 is also coupled with a therapeutic agent that can prevent, inhibit and / or treat diseases characterized by overexpression of nectin-4.

[0019] The present invention also provides the use of the above-mentioned bicyclic peptide nuclide probe targeting nectin-4 in the preparation of nectin-4 imaging agents or PET imaging probes or reagents or kits for diagnosing, treating and / or preventing diseases; the diseases are characterized by overexpression of nectin-4.

[0020] The present invention also provides a detection reagent or a kit, which comprises the above-mentioned bicyclic peptide nuclide probe targeting nectin-4.

[0021] The beneficial effects of the present invention are at least: The present invention provides a novel bicyclic peptide nuclide ligand targeting nectin-4 and a preparation method thereof, as well as a bicyclic peptide nuclide probe targeting nectin-4 based on the ligand and a preparation method thereof, and covers the application of the ligand and the probe in the preparation of solid tumor diagnostic reagents. Compared with the existing bicyclic peptide nuclide probe targeting nectin-4, the bicyclic peptide nuclide ligand and probe of the present invention show significant advantages in radioactive stability, pharmacokinetic properties, binding specificity with tumor nectin-4, tumor uptake ability, tumor to muscle uptake ratio, and in vivo metabolic performance, etc., have significant clinical transformation potential, and provide new technical means and application prospects for the accurate diagnosis of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the mass spectrum of DOTA-HTA-SF intermediate Compd1; Figure 2 is the mass spectrum of DOTA-HTA-SF; Figure 3 The binding affinity diagram of DOTA-HTA-SF and human nectin-4 detected by SPR; Figure 4 for68 HPLC spectrum of Ga-DOTA-HTA-SF bicyclic peptide nuclide probe; Figure 5 This is a Western blot detection image of tumor cells MDA-MB-468; Figure 6 MDA-MB-468 breast cancer model mice were injected with 200 μCi of 68 Ga-DOTA-HTA-SF, 68 Ga-DOTA-HTA-SF and blocking dose of DOTA-HTA-SF (Blocked), 68 Ga-DOTA-HTA (without covalent modification) and other covalent modification groups 68 Ga-DOTA-HTA-SF2, 68 Ga-DOTA-HTA-pFS and 68 Whole-body Micro-PET / CT MIP images after Ga-DOTA-HTA-mFS probe 30 minutes and 1 hour respectively; the arrows indicate the tumor location; Figure 7 MDA-MB-468 breast cancer model mice were injected with 200 μCi of 68 Ga-DOTA-HTA-SF and 68 The ratio of the tumor / muscle uptake values ​​of Ga-DOTA-HTA-SF and blocking dose of DOTA-HTA-SF at 30 minutes, 1 hour, and 2 hours after ROI. represents P < 0.01; Figure 8 is the mass spectrum of DOTA-HTA; Fig. 9 is the structural formula of DOTA-HTA; Fig.10 It is the structural formula of Compd4 in the synthetic route of DOTA-HTA-SF2; Fig.11 is the mass spectrum of DOTA-HTA-SF2; Fig.12 is the structural formula of DOTA-HTA-SF2; Fig.13 It is the structural formula of Compd4 in the synthetic route of DOTA-HTA-mFS; Fig.14 is the mass spectrum of DOTA-HTA-mFS; Fig.15 is the structural formula of DOTA-HTA-mFS; Fig.16 It is the structural formula of Compd4 in the synthetic route of DOTA-HTA-pFS; Fig.17 is the mass spectrum of DOTA-HTA-pFS; Fig.18 This is the structural formula of DOTA-HTA-pFS. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0024] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0025] Example 1 This example discloses a method for synthesizing a bicyclic peptide DOTA-HTA-SF targeting nectin-4, specifically: Synthesis of Compd1: First, Fmoc-Lys(Dde)-OH was coupled to Rink Amide MBHA Resin, then Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-1Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH were coupled in sequence. All amino acids (1 eq) were coupled in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was protected with 20% Pip / DMF solution cleavage.

[0026] 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA, 1 eq) was coupled with the previous resin peptide using HATU (1.5 eq) and HOBt (1.5 eq) as coupling reagents in DIPEA (3 eq) and DMF, and the Dde protecting group was cleaved using 4% hydrazine hydrate / DMF solution.

[0027] 3-(4-(Fluorosulfonyl)phenyl)propionic acid (1eq) was coupled with the previous resin peptide in DIPEA (3eq) and DMF using HATU (1.5eq) and HOBt (1.5eq) as coupling reagents. Finally, the crude peptide Compd1 was cleaved from the resin with TFA solution at room temperature for 2h, and the crude peptide was purified by HPLC on a C18 preparative column to obtain pure Compd1 (Yield: 25.7%), which is DOTA-Cys-Pro-1Nal-DAsp-Cys-Met-HomoArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-Lys[(3-(4-(Fluorosulfonyl)phenyl)propionic acid]-NH2. The structure of Compd1 was identified by mass spectrometry, and the mass spectrum is shown in the figure. Figure 1 As shown, it is consistent with the synthesis target.

[0028] Compd1 (1 eq) was dissolved in a mixed solvent of 15 mL ACN and 15 mL H2O, and then Compd2-1,3,5-triacryloylhexa-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour. The reaction was monitored to be complete by LC-MS, and the final product DOTA-HTA-SF (Yield: 34.5%) was directly purified by reverse phase preparative liquid phase, and its structural formula is shown in Formula I below. The ligand structure of the final product was identified by mass spectrometry, and the results were as follows: Figure 2 shown.

[0029] Formula I.

[0030] The structure was confirmed by negative ion mode mass spectrometry and the purity was greater than 95% by HPLC quantitative analysis. The mass spectrometry results are shown in Table 1. Figure 2 It can be seen that 967.7081 is [M+3H] + / 3 peak, 1451.5655 is [M+2H] + / 2 peak, its molecular weight is actually 2901.29, and its structure is confirmed to be correct based on mass spectrometry.

[0031] Table 1 Mass spectrometry results of compound DOTA-HTA-SF

[0032] Example 2 This example discloses the binding affinity determination of DOTA-HTA-SF and human Nectin-4 protein, specifically: Surface plasmon resonance (SPR) affinity assays were performed on Biacore 8K to determine the affinity of DOTA-HTA-SF to human nectin-4 protein. k a (1 / Ms), k d (1 / s) and K D (M) value. First, the sensor chip was activated by freshly mixed 50mmol / L N-hydroxysuccinimide (NHS) and 200mmol / L 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 420 s. Then, 10mM sodium acetate (pH4.5) was used to dilute the nectin-4 protein to 20µg / mL. After the nectin-4 protein coupling level reached about 1200 RU, EA was passed through the chip surface to block the excess sites. The reference channel was blocked directly after activation. The diluted peptide DOTA-HTA-SF prepared in Example 1 (concentrations were 100, 50, 25, 12.5, 6.25, and 3.125 nM, respectively) was injected onto the chip at a flow rate of 30μL / min at 25°C, with a binding time of 60 seconds and a dissociation time of 60 seconds. The detection was performed using a single cycle kinetic mode. All data were processed using Biacore 8K Evaluation Software Version 4.0. Select appropriate continuous concentrations (at least 5 concentrations) for kinetic 1:1 binding and steady-state analysis. The results are shown in Figure 3 As shown in Table 2.

[0033] Table 2 Binding affinity of DOTA-HTA-SF to human nectin-4 protein

[0034] The results showed that DOTA-HTA-SF has a high binding affinity to human nectin-4 protein. K D The value is 13.8nM.

[0035] Example 3 68 Radiolabeling of Ga: The DMSO solution (concentration of 10 mg / ml) of the Nectin-4 targeted bicyclic peptide nuclide ligand prepared in Example 1 was prepared as a precursor solution, 10 μL of the precursor solution was put into a reaction bottle, 65 μL of 1 M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly washed 68 Ga eluent ( 68 Ga was eluted in 0.1M hydrochloric acid solution), and then 0.5mL deionized water was added. The pH value of the reaction solution was about 4.0. After mixing, the mixture was placed at 95℃ for 10 minutes to obtain 68 Ga-labeled complexes ( 68 Ga-DOTA-HTA-SF bicyclic peptide nuclide probe).

[0036] Quality control of Nectin-4 targeted probe: 68 The radiochemical purity of the Ga-DOTA-HTA-SF bicyclic peptide nuclide probe was determined using radio-HPLC. Chromatographic method: Mobile phase: 0.1% TFA in water and 0.1% TFA in acetonitrile, 0-20min: 30-50% acetonitrile phase. The radiochemical purity of the product was greater than 95% as determined by Radio-HPLC. Figure 4 shown.

[0037] 68 Lipid-water partition coefficient of Ga-DOTA-HTA-SF bicyclic peptide nuclide probe: Use equal volumes (0.5mL:0.5mL) of n-octanol (organic phase) and phosphate buffer solution (aqueous phase, pH=7.4) to 68 The Ga-DOTA-HTA-SF bicyclic peptide nuclide probe was dissolved in an EP tube (about 3.7 MBq). The mixture was fully shaken for 5 minutes and centrifuged in a centrifuge for 5 minutes. The speed was 2000r / min. 100μL of the organic phase and the aqueous phase were taken in 1.5mL EP tubes respectively, and their radioactivity counting rates were measured in a well-type γ detector. The lipid-water partition coefficient (P) was calculated from the ratio of the radioactivity counting rates of the organic phase and the aqueous phase.

[0038] P=log(N0 / N aq ); Where: N0 and N aq are the radioactivity counting rates of the organic and aqueous phase samples, s -1 The experiment was repeated 3 times and the average value was taken as the lipid-water partition coefficient of the marker. 68 The lipid-water partition coefficient of Ga-DOTA-HTA-SF bicyclic peptide nuclide probe is logP=-0.28±0.03 (n=3), indicating that 68Ga-DOTA-HTA-SF has certain hydrophilicity.

[0039] Example 4 Western blot detection of Nectin-4 expression in human breast cancer MDA-MB-468 cell line: Sample lysis: Add an appropriate amount of lysis solution (containing protease inhibitors) to the tissue, grind it with a tissue grinding low-temperature homogenizer, and let it stand on ice for 30 minutes after taking it out, shaking it once every 10 minutes. Centrifuge at 12000rpm, 4℃ for 30 minutes, collect the supernatant, which is the total protein solution. Protein concentration determination: Use the BCA protein concentration determination kit to detect the protein concentration. Protein sample preparation: Mix the treated protein solution, RAPI lysis solution and SDS-PAGE protein loading buffer in proportion, denature at 95℃ for 5 minutes in a metal bath, and store in a -20℃ refrigerator for standby use. SDS-PAGE electrophoresis: Add protein samples and protein markers in sequence according to the sample layout, constant voltage 80V / 15min, 120V / 1hr. Transfer: Place the transfer tank in an ice water bath and transfer at 300mA for 1hr. Blocking: Incubate in a shaker at room temperature with 5% skim milk for 1hr. Primary antibody incubation: Incubate the PVDF membrane with the primary antibody in a constant temperature shaker at 4℃ overnight. Secondary antibody incubation: Incubate the PVDF membrane with the secondary antibody on a shaker at room temperature for 1 hour. ECL color development: Add color development solution and use the chemiluminescence imaging system for automatic exposure. Western blot detection results are shown in Figure 5 As shown. With GAPDH protein as the internal standard, the molecular weight of Nectin-4 protein is about 60-70kDa. The relative expression of Nectin-4 and GAPDH in MDA-MB-468 tumor tissue is relatively high, while the expression of Nectin-4 was not detected in the control MDA-MB-231 tumor tissue. It proves that MDA-MB-468 is a high expression model of Nectin-4, while MDA-MB-231 is a low expression model of Nectin-4. T-47D has been reported to express Nectin-4, and it is used as a positive control to prove the effectiveness of the antibody and the reliability of Western blot experimental operation.

[0040] Example 5 Micro-PET / CT imaging of human breast cancer MDA-MB-468 model mice: This example discloses an in vivo imaging experiment of the bicyclic peptide nuclide ligand targeting nectin-4 prepared in Example 1. Nude mice with MDA-MB-468 xenograft tumors were transplanted with tumor tissues to 100-300 mm 3The mice were injected with 200 μCi / mouse via the tail vein at different time points (30, 60 and 120 min) after injection, and the tumors were clearly visualized. After the scan, the scan data was iteratively reconstructed (OSEM 3D), and the two tumors and other tissues were delineated as regions of interest (ROI) using QD software to calculate the radioactivity uptake (%ID / g) of each ROI. 68 Ga-DOTA-HTA-SF, 68 Ga-DOTA-HTA-SF and blocking dose (1000 times excess) of DOTA-HTA-SF, 68 Ga-DOTA-HTA (without covalent modification) and other covalent modification groups 68 Ga-DOTA-HTA-SF2, 68 Ga-DOTA-HTA-pFS and 68 The whole body was subjected to Micro-PET / CT MIP 30 minutes and 1 hour after the Ga-DOTA-HTA-mFS probe was inserted. The images were as follows Figure 6 MDA-MB-468 breast cancer model mice were injected with 200 μCi of 68 Ga-DOTA-HTA-SF and 68 Comparison of the uptake values ​​of the tumor / muscle outlined by ROI at 30 min, 1 h, and 2 h after Ga-DOTA-HTA-SF and blocking dose of DOTA-HTA-SF Figure 7 Data are presented as mean ± SD (n = 3).

[0041] 68 The preparation method of Ga-DOTA-HTA is: Synthesis of Compd1: First, Fmoc-Cys(Trt)-OH was coupled to Rink Amide MBHA Resin, then Fmoc-Trp(Boc)-OH, Fmoc-Hyp-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-2Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH were coupled in sequence. All amino acids (1 eq) were coupled in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved using 20% ​​Pip / DMF solution. DOTA was coupled with the resin peptide in DIPEA (3eq) and DMF using HATU (1.5eq) and HOBt (1.5eq) as coupling reagents. Finally, the resin was cleaved with TFA solution at room temperature for 2h to obtain crude peptide Compd1, which was purified by HPLC on a C18 preparative column to obtain pure Compd1 (Yield: 26.8%).

[0042] Synthesis of DOTA-HTA: Compd1 (1 eq) was dissolved in a mixed solvent of 15 mL ACN and 15 mL H2O, and then Compd2-1,3,5-triacryloylhexa-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour. The reaction was monitored by LC-MS to be complete. DOTA-HTA (Yield: 34.5%) was directly purified by reverse phase preparative liquid chromatography. The structure of the final product ligand was identified by mass spectrometry. The results are as follows: Figure 8 As shown, the structural formula is shown in Fig. 9 .

[0043] 68 Radioactive labeling of Ga: Prepare the prepared DOTA-HTA DMSO solution (concentration of 10 mg / ml) as the precursor solution, take 10 μL of the precursor solution and put it into the reaction bottle, add 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly washed 68 Ga eluent ( 68 Ga was eluted in 0.1M hydrochloric acid solution), and then 0.5mL deionized water was added. The pH value of the reaction solution was about 4.0. After mixing, the mixture was placed at 95℃ for 10 minutes to obtain 68 Ga-labeled complexes68 Ga-DOTA-HTA bicyclic peptide radionuclide probe.

[0044] 68 The preparation method of Ga-DOTA-HTA-SF2 is: Synthesis of Compd1: First, Fmoc-Lys(Boc)-OH was coupled to Rink Amide MBHA Resin, then Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-2Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH were coupled in sequence. All amino acids (1 eq) were coupled in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved using 20% ​​Pip / DMF solution. DOTA (1eq) was coupled with the resin peptide in DIPEA (3eq) and DMF using HATU (1.5eq) and HOBt (1.5eq) as coupling reagents. Finally, the resin was cleaved with TFA solution at room temperature for 2h to obtain the crude peptide Compd1, which was purified by HPLC on a C18 preparative column to obtain pure Compd1 (Yield: 27.3%).

[0045] Compd1 (1 eq) was dissolved in a mixed solvent of 10 mL ACN and 10 mL H2O, and then Compd2-1,3,5-triacryloylhexa-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour, and the reaction was complete after LC-MS monitoring. Compd3 (Yield: 34.5%) was directly purified by reverse phase preparative liquid chromatography.

[0046] Compd 4 (1eq) (Structural formula see Fig.10) was dissolved in 10 ml DMF, and then DCC (1.2 eq) and HOSu (1.2 eq) were added, and the reaction was carried out at room temperature for 6 hours. Then Compd 3 (1 eq) and DIEA (3 eq) were added, and the reaction was carried out at room temperature for 1 hour. The reaction was monitored by LC-MS. DMF was removed by rotation, and DOTA-HTA-SF2 (Yield: 33.7%) was obtained after purification by reverse phase preparative liquid phase. The structure of the final product ligand was identified by mass spectrometry. The results are as follows Fig.11 As shown, the structural formula is shown in Fig.12 .

[0047] 68 Radioactive labeling of Ga: Prepare the prepared DOTA-HTA-SF2 DMSO solution (concentration of 10 mg / ml) as the precursor solution, take 10 μL of the precursor solution into the reaction bottle, add 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly washed 68 Ga eluent ( 68 Ga was eluted in 0.1M hydrochloric acid solution), and then 0.5mL deionized water was added. The pH value of the reaction solution was about 4.0. After mixing, the mixture was placed at 95℃ for 10 minutes to obtain 68 Ga-labeled complexes 68 Ga-DOTA-HTA-SF2 bicyclic peptide radionuclide probe.

[0048] 68 The preparation method of Ga-DOTA-HTA-mFS is: Synthesis of Compd1: First, Fmoc-Lys(Boc)-OH was coupled to Rink Amide MBHA Resin, then Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-2Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH were coupled in sequence. All amino acids (1 eq) were coupled in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved using 20% ​​Pip / DMF solution. DOTA (1eq) was coupled with the resin peptide in DIPEA (3eq) and DMF using HATU (1.5eq) and HOBt (1.5eq) as coupling reagents. Finally, the resin was cleaved with TFA solution at room temperature for 2h to obtain the crude peptide Compd1, which was purified by HPLC on a C18 preparative column to obtain pure Compd1 (Yield: 27.3%).

[0049] Compd1 (1 eq) was dissolved in a mixed solvent of 10 mL ACN and 10 mL H2O, and then Compd2-1,3,5-triacryloylhexa-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour, and the reaction was complete after LC-MS monitoring. Compd3 (Yield: 34.5%) was directly purified by reverse phase preparative liquid chromatography.

[0050] Compd 4(1eq) (Structural formula see Fig.13 ) was dissolved in 10 ml DMF, and then DCC (1.2 eq) and HOSu (1.2 eq) were added, and the reaction was carried out at room temperature for 6 hours. Then Compd 3 (1 eq) and DIEA (3 eq) were added, and the reaction was carried out at room temperature for 1 hour. The reaction was monitored by LC-MS. DMF was removed by rotation, and DOTA-HTA-mFS (Yield: 30.2%) was obtained after purification by reverse phase preparative liquid phase. The structure of the final product ligand was identified by mass spectrometry. The results are as follows Fig.14 As shown, the structural formula is shown in Fig.15 .

[0051] 68Radioactive labeling of Ga: Prepare the prepared DOTA-HTA-mFS DMSO solution (concentration of 10 mg / ml) as the precursor solution, take 10 μL of the precursor solution into the reaction bottle, add 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly washed 68 Ga eluent ( 68 Ga was eluted in 0.1M hydrochloric acid solution), and then 0.5mL deionized water was added. The pH value of the reaction solution was about 4.0. After mixing, the mixture was placed at 95℃ for 10 minutes to obtain 68 Ga-labeled complexes 68 Ga-DOTA-HTA-mFS bicyclic peptide radionuclide probe.

[0052] 68 The preparation method of Ga-DOTA-HTA-pFS is: Synthesis of Compd1: First, Fmoc-Lys(Boc)-OH was coupled to Rink Amide MBHA Resin, then Fmoc-Cys(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HomoArg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Cys(Trt)-OH, Fmoc-DAsp(OtBu)-OH, Fmoc-2Nal-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH were coupled in sequence. All amino acids (1 eq) were coupled in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved using 20% ​​Pip / DMF solution. DOTA (1eq) was coupled with the resin peptide in DIPEA (3eq) and DMF using HATU (1.5eq) and HOBt (1.5eq) as coupling reagents. Finally, the resin was cleaved with TFA solution at room temperature for 2h to obtain the crude peptide Compd1, which was purified by HPLC on a C18 preparative column to obtain pure Compd1 (Yield: 27.3%).

[0053] Compd1 (1 eq) was dissolved in a mixed solvent of 10 mL ACN and 10 mL H2O, and then Compd2-1,3,5-triacryloylhexa-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour, and the reaction was complete after LC-MS monitoring. Compd3 (Yield: 34.5%) was directly purified by reverse phase preparative liquid chromatography.

[0054] Compd 4(1eq) (Structural formula see Fig.16 ) was dissolved in 10 ml DMF, and then DCC (1.2 eq) and HOSu (1.2 eq) were added, and the reaction was carried out at room temperature for 6 hours. Then Compd 3 (1 eq) and DIEA (3 eq) were added, and the reaction was carried out at room temperature for 1 hour. The reaction was monitored by LC-MS. DMF was removed by rotation, and DOTA-HTA-pFS (Yield: 30.2%) was obtained after purification by reverse phase preparative liquid phase. The structure of the final product ligand was identified by mass spectrometry. The results are as follows Fig.17 As shown, the structural formula is shown in Fig.18 .

[0055] 68 Radioactive labeling of Ga: Prepare the prepared DOTA-HTA-pFS DMSO solution (concentration of 10 mg / ml) as the precursor solution, take 10 μL of the precursor solution into the reaction bottle, add 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly washed 68 Ga eluent ( 68 Ga was eluted in 0.1M hydrochloric acid solution), and then 0.5mL deionized water was added. The pH value of the reaction solution was about 4.0. After mixing, the mixture was placed at 95℃ for 10 minutes to obtain 68 Ga-labeled complexes 68 Ga-DOTA-HTA-pFS bicyclic peptide radionuclide probe.

[0056] like Figure 6 and Figure 7 As shown, 68 Ga-DOTA-HTA-SF is significantly concentrated in the tumor area, with good sensitivity, and is mainly excreted from the body through the kidneys. As time goes on, the uptake ratio of the complex in the tumor and muscle increases. At 30 minutes, 1 hour and 2 hours after injection, the tumor uptake values ​​were 5.09±0.60, 4.77±0.68 and 4.28±0.59%ID / g, respectively, and the uptake ratios of the tumor and muscle were 6.27±0.51, 8.55±1.82 and 7.47±1.48, respectively. There is a certain uptake in the heart, which is due to the high blood drug concentration and a certain enrichment in the heart. In the blocking imaging experiment ( 68In the Ga-DOTA-HTA-SF+blocking group, it can be seen that there is still a high uptake in the kidney and bladder, less uptake in other organs, and no significant uptake in the tumor. The probe uptake in the blocking group was significantly lower than that in the positive group, with a significant statistical difference. 68 Ga-DOTA-HTA-SF drug has good specificity and targeting. 68 Ga-DOTA-HTA (without covalent modification) and other covalent modification groups 68 Ga-DOTA-HTA-SF2, 68 Ga-DOTA-HTA-pFS and 68 The uptake of Ga-DOTA-HTA-mFS probe in MDA-MB-468 tumor model was significantly lower than 68 Ga-DOTA-HTA-SF. The above studies show that 68 Ga-DOTA-HTA-SF has good clinical research prospects.

[0057] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A bicyclic peptide nuclide ligand targeting nectin-4, characterized in that: Having the structure shown in Formula I: Formula I.

2. A method for preparing the bicyclic peptide nuclide ligand targeting nectin-4 according to claim 1, characterized in that: include: (1) coupling the amino acids contained in formula I by solid phase synthesis to obtain a polypeptide chain; (2) coupling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid with the polypeptide chain to obtain a DOTA-polypeptide chain; (3) After coupling 3-(4-(fluorosulfonyl)phenyl)propionic acid with the DOTA-polypeptide chain, the DOTA-polypeptide chain is reacted with 1,3,5-triacryloylhexa-1,3,5-triazine.

3. A bicyclic peptide nuclide probe targeting nectin-4, characterized in that: The bicyclic peptide nuclide ligand targeting nectin-4 according to claim 1 is labeled with a radionuclide.

4. The bicyclic peptide nuclide probe targeting nectin-4 according to claim 3, characterized in that: The radionuclides include diagnostic radionuclides or therapeutic radionuclides. The diagnostic radionuclides are 68 Ga, 64 Cu, 18 F. 86 Y. 90 Y. 89 Zr, 111 In, 99m Tc, 11 C. 123 I. 125 I and 124 I at least one; the therapeutic radionuclide comprises 177 Lu, 125 I. 131 I. 211 At 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bihe 212 At least one of Pb.

5. A method for preparing the bicyclic peptide nuclide probe targeting nectin-4 according to claim 3 or 4, characterized in that: The method comprises the step of labeling the bicyclic peptide nuclide ligand targeting nectin-4 as claimed in claim 1 or the bicyclic peptide nuclide ligand targeting nectin-4 prepared by the method of claim 2 with a radioactive nuclide.

6. A drug, characterized in that The invention comprises the bicyclic peptide nuclide ligand targeting nectin-4 as claimed in claim 1 or the bicyclic peptide nuclide probe targeting nectin-4 as claimed in claim 3 or 4 and pharmaceutically acceptable excipients.

7. The drug according to claim 6, characterized in that The bicyclic peptide nuclide ligand or bicyclic peptide nuclide probe targeting nectin-4 is also coupled with a therapeutic agent that can prevent, inhibit and / or treat a disease characterized by overexpression of nectin-4.

8. Use of the bicyclic peptide nuclide probe targeting nectin-4 according to claim 3 or 4 in the preparation of nectin-4 imaging agent or PET imaging probe.

9. Use of the bicyclic peptide nuclide probe targeting nectin-4 according to claim 3 or 4 in the preparation of a reagent or kit for diagnosing, treating and / or preventing a disease characterized by overexpression of nectin-4.

10. A detection reagent or kit, characterized in that: It comprises the bicyclic peptide nuclide probe targeting nectin-4 as described in claim 3 or 4.

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