A dimeric bicyclic peptide nuclide ligand and its application

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses a dimeric bicyclic peptide nuclide ligand and its application. The dimeric bicyclic peptide nuclide ligand of the present invention has a structure as shown in Formula I: #imgabs0# Formula I. After radionuclide labeling, the dimeric bicyclic peptide nuclide ligand of the present invention can be prepared into a nuclide probe targeting nectin-4. It has excellent radiostability and ideal pharmacokinetics. It is strongly taken up by tumors that overexpress nectin-4, and has a good tumor-to-muscle uptake ratio and in vivo metabolic performance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to a dimer bicyclic peptide nuclide ligand and application thereof. Background Art

[0002] Breast cancer is a common malignancy, and its molecular classification is crucial for clinical diagnosis and treatment. Based on immunohistochemical characteristics, breast cancer can be divided into luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-positive, and triple-negative breast cancer (TNBC). Triple-negative breast cancer lacks estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, resulting in limited clinical treatment options. Conventional chemotherapy remains the mainstay of therapy. However, this type of breast cancer exhibits high invasiveness and recurrence rates, and some patients are resistant to chemotherapy, resulting in a short median overall survival and poor long-term prognosis. Therefore, identifying specific biomarkers and novel targeted therapy strategies is crucial for improving the treatment of patients with triple-negative breast cancer.

[0003] In recent years, the role of adhesion molecules in tumor development and progression has garnered widespread attention. Nectin proteins belong to the immunoglobulin superfamily (IgSF), a family of adhesion receptors that includes four members: nectin-1, nectin-4, and nectin-4. Nectin-4 (PVRL4) is a type I transmembrane protein that is highly expressed in various malignant tumors. Studies have shown that nectin-4 expression is elevated in tumor tissues, including breast, bladder, ovarian, pancreatic, and lung cancers, and is associated with tumor cell proliferation, invasion, and drug resistance. Nectin-4 expression is particularly high in triple-negative breast cancer and basal-like breast cancer, whereas it is low or absent in normal breast tissue, making it a potential diagnostic and therapeutic target.

[0004] Currently, the research on targeted therapy for Nectin-4 is mainly focused on the field of antibody-drug conjugates (ADCs), such as Enfortumab Vedotin (PADCEV ®) has been approved for the treatment of advanced or metastatic bladder cancer. In addition, preclinical studies have also explored the use of radionuclide conjugation of nectin-4 monoclonal antibodies for molecular imaging. However, antibodies have a large molecular weight, a long circulation time in the body, and high non-specific tissue uptake, which limits their application in rapid diagnosis and treatment evaluation. In contrast, small molecule probes, such as peptide molecules, have the advantages of small molecular weight, good tissue penetration, rapid clearance, and low immunogenicity, and show good application prospects in tumor-targeted imaging and radionuclide therapy. Therefore, the development of radioactive molecular probes based on nectin-4 targeting peptides will help to achieve accurate diagnosis and efficacy evaluation of nectin-4-positive tumors, and provide an imaging basis for personalized treatment. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a nuclear ligand that can specifically target nectin-4 and has ideal pharmacokinetics.

[0006] The present invention provides a dimeric bicyclic peptide nuclide ligand having a structure as shown in Formula I:

[0007] Formula I.

[0008] The present invention constructs a dimeric bicyclic peptide nuclide ligand that can target nectin-4. Its parent nucleus, through a specific amino acid sequence and cyclization method, can achieve high affinity for the nectin-4 target. The nuclide chelating group can bind to a variety of radionuclides to achieve labeling of the bicyclic peptide targeting nectin-4. When the two bicyclic peptide nuclide ligands of the present invention are connected in a specific structure to form a dimer and then nuclide-labeled, the resulting dimer probe can increase the enrichment level of tumor tissue through a multivalent effect, while reducing nonspecific binding and improving the tumor / normal tissue ratio. In addition, the dimer probe of the present invention also exhibits higher apparent affinity and reduces the dissociation rate ( K d ), improving imaging contrast, thereby improving diagnostic accuracy. Due to the increased binding time with the target protein, the application of the dimeric bicyclic peptide nuclide ligand of the present invention can enhance tumor uptake and retention time. The dimeric bicyclic peptide molecular probe targeting Nectin-4 can be used for preclinical verification of in vivo recognition of Nectin-4 receptor-positive expression tumor models through PET whole-body imaging (which can obtain higher resolution and clearly distinguish tumor areas). It is of great significance for the early detection of triple-negative breast cancer with high Nectin-4 expression, the screening of people who benefit from Nectin-4 targeted therapeutic drugs, the monitoring and evaluation of the efficacy of anti-tumor drugs, and prognosis assessment, and has great clinical translation potential.

[0009] Optimizing dimeric probes involves multiple key factors, including steric hindrance, target binding efficiency, in vivo distribution, and pharmacokinetic properties. Existing studies have shown that not all probe dimerizations result in desirable performance improvements and may even lead to decreased affinity or reduced tumor uptake. For example, the article "Krasniqi A et al. Pharmacokinetics of radiolabeled dimericsdAbs constructs targeting human CD20. New biotechnology, 2018, 45: 69-79" reported that although dimerization of CD20 single-domain antibodies (sdAbs) enhanced cellular endocytosis, their apparent affinity decreased. This suggests that optimizing dimeric probes requires careful design tailored to specific targets. For example, the literature "Garousi J et al. Comparative evaluation of dimeric and monomeric forms of ADAPTscaffold protein for targeting of HER2-expressing tumours. European journal of pharmaceutics and biopharmaceutics, 2019, 134: 37-48" reported that the affinity of HER2-targeting ADAPT6 dimers was significantly improved, but their tumor uptake rate was lower than that of monomeric probes, indicating that increased molecular size may have an adverse effect on tissue permeability. Therefore, the optimization of dimeric probes does not rely solely on increasing binding sites. Different connection strategies may lead to decreased targeting ability, worsening pharmacokinetic properties, or restricted molecular conformation, thereby affecting target binding affinity. Therefore, it is necessary to comprehensively consider factors such as spatial structure, binding kinetics, and in vivo metabolism to ensure that the optimized probe exhibits good targeting performance in vivo.

[0010] The optimization of dimerization probes requires careful design based on specific targets, molecular conformations, and in vivo distribution characteristics, rather than simply achieving performance improvements through simple structural repetition. In the process of optimizing the dimeric nuclide ligand, the present invention systematically screened different connection methods and verified some failed construction schemes. Specifically, in preliminary experiments, the present invention attempted to use amide bonds to directly connect the two-end cyclic peptides, but this scheme may have changed the conformation of the cyclic peptides, thereby affecting the target binding ability. The present invention also attempted to directly couple the polyethylene glycol linker with the bicyclic peptide, but this scheme failed to succeed during the synthesis process. The possible reason is that the chemical stability of the direct coupling of polyethylene glycol and the bicyclic peptide is poor, resulting in low coupling efficiency.

[0011] To overcome the above problems, the present invention adopts a maleimide-cysteine ​​conjugation strategy, which not only improves the coupling efficiency of the dimer linker, but also ensures the spatial arrangement of the polyethylene glycol linker, thereby reducing the effect of steric hindrance on target binding. Compared with the failed scheme, the optimized dimer probe of the present invention has a higher affinity for Nectin-4 target ( K D The probe showed significant improvements in affinity, from 23.7 nM to 0.37 nM, and tumor uptake, successfully circumventing the common issues of affinity loss and poor pharmacokinetic properties associated with dimerization. Furthermore, the hydrophilicity of polyethylene glycol reduces liver uptake of the dimer probe due to its increased molecular weight, improving tumor tissue targeting and broadening its application in molecular imaging and radiotherapy.

[0012] The dimer probe of the present invention successfully overcomes the effect of steric hindrance on target binding through the optimization of specific connection mode, peptide chain length and dimerization strategy, achieves higher affinity and better pharmacokinetic properties, and overcomes the shortcomings in tumor uptake and in vivo distribution. After dimerization, the probe of the present invention not only has a significantly higher affinity than the traditional dimerization strategy ( K D The probe's affinity for the target protein was reduced from 23.7nM to 0.37nM. Furthermore, in animal experiments, tumor uptake was significantly improved compared to the control monomer probe, fully demonstrating its application value in molecular imaging and radiotherapy. This invention also optimizes the dimer connection strategy, maintaining high affinity while avoiding the problem of reduced tumor tissue permeability caused by increased dimer size.

[0013] In summary, the nectin-4-targeted dimer polypeptide probe of the present invention not only overcomes the shortcomings of existing dimerization probes but also achieves comprehensive optimization of affinity, tumor uptake, and pharmacokinetics, resolving key technical challenges in the dimerization modification process. Compared to traditional dimerization strategies, the present invention significantly improves imaging and therapeutic effects by precisely regulating the molecular structure of the probe, demonstrating greater potential for clinical application.

[0014] In addition, the optimization process of the present invention involves the regulation of multiple key parameters, which poses high technical challenges. This technical solution is not only applicable to Nectin-4 targeted probes, but can also be extended to the design of other bicyclic peptide targeted probes, providing important technical support for molecular imaging and radiotherapy of similar targets.

[0015] The present invention also provides a method for preparing the above-mentioned dimer bicyclic peptide nuclide ligand, which comprises:

[0016] (1) coupling the amino acids contained in formula I by solid phase synthesis to obtain compound 1;

[0017] (2) Compound 1 and compound 2 were subjected to a cyclization reaction in a mixed solvent of ammonium carbonate buffer and ACN / H2O (volume ratio 1:1) to obtain compound 3;

[0018] Preferably, the volume ratio of the ACN / H2O mixed solvent to the ammonium carbonate buffer is 3:1, and the concentration of NH4HCO3 in the ammonium carbonate buffer is 1M;

[0019] The molar ratio of compound 1 to compound 2 is 1:1.5;

[0020] (3) Compound 4 was dissolved in DMF, activated in the presence of DCC, HOSu and DIPEA, and then subjected to acid-amine condensation with compound 3; after the reaction, DMF was removed, and liquid E was added for reaction, followed by addition of ether and drying to obtain compound 5; the liquid E refers to a mixture of 90% TFA + 5% thioanisole + 2.5% phenol + 2.5% 1,2-ethanedithiol;

[0021] Preferably, the molar ratio of compound 3 to compound 4 is 1:2;

[0022] The molar ratio of DCC, HOSu, and DIPEA was 2:2:5; the molar ratio of compound 3 to DIPEA was 1:5;

[0023] (4) Compound 7 was dissolved in DMF, activated in the presence of DCC, HOSu, and DIPEA, and then subjected to acid-amine condensation with compound 6. After the reaction, DMF was removed to obtain compound 8;

[0024] Preferably, the molar ratio of compound 6 to compound 7 is 1:1.2;

[0025] The molar ratio of DCC, HOSu, and DIPEA was 1.2:1.2:5; the molar ratio of compound 6 to DIPEA was 1:5;

[0026] (5) Compound 8 was dissolved in DMF and subjected to acid-amine condensation with compound 9 in the presence of EDC·HCl, HOBt, and NMM. After the reaction was complete, DMF was removed, the tert-butyl protecting group was removed with TFA solution, ether was added, and the mixture was dried to obtain compound 10.

[0027] Preferably, the molar ratio of compound 8 to compound 9 is 1:5;

[0028] The molar ratio of EDC·HCl, HOBt, and NMM was 1:1:1; the molar ratio of compound 8 to NMM was 1:5;

[0029] (6) Compound 10 and compound 5 were dissolved in ACN / H2O (1:1), and then phosphate buffer (pH = 7.20.2M) was added for co-reaction to obtain the dimer bicyclic peptide nuclide ligand;

[0030] Preferably, the molar ratio of compound 5 to compound 10 is 2.5:1;

[0031] The structural formula of the compound 1 is as follows:

[0032] ;

[0033] The compound 2 is 1,3,5-triacryloyl-1,3,5-triazine;

[0034] The structural formula of the compound 3 is as follows:

[0035] ;

[0036] The structural formula of the compound 4 is as follows:

[0037] ;

[0038] The structural formula of the compound 5 is as follows:

[0039] ;

[0040] The structural formula of the compound 6 is as follows:

[0041] ;

[0042] The structural formula of the compound 7 is as follows:

[0043] ;

[0044] The structural formula of the compound 8 is as follows:

[0045] ;

[0046] The structural formula of the compound 9 is as follows:

[0047] ;

[0048] The structural formula of the compound 10 is as follows:

[0049] .

[0050] The present invention also provides a nuclide probe targeting nectin-4, which is the above-mentioned dimeric bicyclic peptide nuclide ligand labeled with radioactive nuclide or the dimeric bicyclic peptide nuclide ligand prepared by the above-mentioned method.

[0051] In the radionuclide 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 includes 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.

[0052] Compared to monoclonal antibodies, the radionuclide probe of the present invention has a reasonable half-life, can more effectively penetrate tumor tissue, and can provide accurate assessment results in a shorter time, thereby achieving more precise diagnosis. Furthermore, the radionuclide probe targeting nectin-4 of the present invention features rapid labeling and a high labeling rate. Its purity exceeds 95%, and it is primarily excreted through the kidneys. Uptake in other non-target organs is low, while it exhibits ideal uptake in tumor tissue, demonstrating high sensitivity and specificity.

[0053] The present invention also provides a method for preparing the above-mentioned nuclide probe targeting nectin-4, which comprises the step of labeling the above-mentioned dimeric bicyclic peptide nuclide ligand or the dimeric bicyclic peptide nuclide ligand prepared by the above-mentioned method with a radioactive nuclide.

[0054] The present invention also provides the use of a nuclide probe targeting nectin-4 in the preparation of a nectin-4 imaging agent or a PET imaging probe.

[0055] The present invention also provides the use of a nuclear probe targeting Nectin-4 in preparing a reagent or kit for diagnosing, treating and / or preventing a disease; the disease is characterized by overexpression of Nectin-4.

[0056] The present invention also provides a detection reagent or a kit, which comprises the above-mentioned nuclide probe targeting Nectin-4.

[0057] The present invention also provides a medicine, which comprises the above-mentioned dimer bicyclic peptide nuclide ligand or nuclide probe targeting nectin-4 and pharmaceutically acceptable excipients.

[0058] In the present invention, "pharmaceutically acceptable" 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 conditions.

[0059] In the drug of the present invention, the dimer bicyclic peptide nuclide ligand or nuclide probe targeting nectin-4 is further coupled with a therapeutic agent that can prevent, inhibit and / or treat diseases characterized by overexpression of nectin-4.

[0060] The dimer bicyclic peptide nuclide ligand of the present invention has an excellent targeting effect and can be combined with a substance having pharmaceutical activity to precisely deliver the therapeutic agent to the lesion site, thereby improving the targeting of the drug, reducing side effects, and enhancing the therapeutic effect.

[0061] The beneficial effects of the present invention are at least:

[0062] The present invention provides a new dimeric bicyclic peptide nuclide ligand that can target nectin-4. Compared with existing bicyclic peptide nuclide probes targeting nectin-4, the dimeric bicyclic peptide nuclide ligand and probe of the present invention have excellent stability, ideal pharmacokinetics, strong uptake by tumors that overexpress nectin-4, good tumor-to-muscle uptake ratio and in vivo metabolic performance, and have significant clinical translation potential, providing a new technical means for the accurate diagnosis of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is one of the schematic diagrams of the synthesis route of the bicyclic peptide nuclide ligand DOTA-HTA-DM of the present invention;

[0064] Figure 2 This is the second schematic diagram of the synthesis route of the bicyclic peptide nuclide ligand DOTA-HTA-DM of the present invention;

[0065] Figure 3 This is the third schematic diagram of the synthesis route of the bicyclic peptide nuclide ligand DOTA-HTA-DM of the present invention;

[0066] Figure 4 This is the fourth schematic diagram of the synthesis route of the bicyclic peptide nuclide ligand DOTA-HTA-DM of the present invention;

[0067] Figure 5 is the mass spectrum of DOTA-HTA-DM;

[0068] Figure 6 The binding affinity diagram of DOTA-HTA-DM and human nectin-4 was detected by SPR;

[0069] Figure 7 for 68 HPLC spectrum of Ga-DOTA-HTA-DM bicyclic peptide nuclide probe;

[0070] Figure 8 This is a Western blot analysis of tumor cells MDA-MB-468;

[0071] Figure 9 200 μCi of 68 Ga-DOTA-HTA-DM, 68 Ga-DOTA-HTA-DM and blocking doses of DOTA-HTA-DM (blocking) and 68 Whole-body Micro-PET / CT MIP images 30 minutes, 1 hour, and 2 hours after Ga-DOTA-HTA (monomer); arrows indicate the tumor location;

[0072] Figure 10 200 μCi of 68 Ga-DOTA-HTA-DM and 68 The ratio of the tumor / muscle uptake values ​​of Ga-DOTA-HTA-DM and blocking dose of DOTA-HTA-DM at 30 min, 1 h, and 2 h after ROI delineation; represents P < 0.05, represents P < 0.01, ns represents no significant difference;

[0073] Figure 11 50 μCi of 68 Uptake values ​​of Ga-DOTA-HTA-DM in tumors and other tissues and organs at 30 min, 1 h, and 2 h (%ID / g) (mean ± SD, n = 3);

[0074] Figure 12 is the mass spectrum of DOTA-HTA;

[0075] Figure 13 for 68 The structural formula of Ga-DOTA-HTA; Figure 14is the mass spectrum of DOTA-HTA-DM intermediate compound 1;

[0076] Figure 15 is the mass spectrum of DOTA-HTA-DM intermediate compound 3;

[0077] Figure 16 is the mass spectrum of DOTA-HTA-DM intermediate compound 5;

[0078] Figure 17 is the mass spectrum of DOTA-HTA-DM intermediate compound 8;

[0079] Figure 18 This is the mass spectrum of DOTA-HTA-DM intermediate compound 10. DETAILED DESCRIPTION

[0080] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only 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.

[0081] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available or prepared according to conventional methods in the art.

[0082] Example 1

[0083] This example discloses a method for synthesizing a bicyclic peptide DOTA-HTA-DM targeting Nectin-4 dimer (see the schematic diagram of the synthesis route). Figures 1 to 4 ), specifically:

[0084] Synthesis of compound 1: First, Fmoc-Cys(Trt)-OH was coupled to Rink Amide MBHA Resin, then 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 cleaved with 20% Pip / DMF solution. Finally, the crude peptide compound 1 was cleaved from the resin with TFA solution at room temperature for 2 h. The crude peptide was purified by HPLC on a C18 preparative column to obtain pure compound 1 (Yield: 23%). Its structure was identified by mass spectrometry. The mass spectrum is shown in FIG. Figure 14 As shown, it is consistent with the synthesis target.

[0085] Synthesis of Compound 3: Compound 1 (1 eq) was dissolved in a mixed solvent of 75 mL ACN and 75 mL H2O, followed by the addition of Compound 2—1,3,5-triacryloyl-1,3,5-triazine (1.5 eq) and 50 mL 1M NH4HCO3. The reaction was allowed to proceed at room temperature for 1 hour. LC-MS monitored the reaction to completion, and the product was directly purified by reverse-phase preparative liquid chromatography to obtain Compound 3 (Yield: 28%). The product was identified by mass spectrometry, as shown in the mass spectrum. Figure 15 shown.

[0086] Synthesis of compound 5: Compound 4 (2eq) was dissolved in 10ml DMF, and then DCC (2eq) and HOSu (2eq) were added. The reaction was allowed to proceed at room temperature for 6 hours. Compound 3 (1eq) and DIPEA (5eq) were then added. The reaction was allowed to proceed at room temperature for 1 hour. The reaction was monitored by LC-MS until completion. The DMF was removed by vortexing, and 10ml of E solution (90% TFA + 5% thioanisole + 2.5% phenol + 2.5% 1,2-ethanedithiol) was added. The reaction was allowed to proceed at room temperature for 2 hours. 100ml of ether was added, and a large amount of solid precipitated. The solid was centrifuged and dried. After purification by reverse phase preparative liquid chromatography, compound 5 (Yield: 28.8%) was obtained. Compound 5 was identified by mass spectrometry. The mass spectrum is shown in FIG. Figure 16 shown.

[0087] Synthesis of Compound 8: Compound 7 (1.2 eq) was dissolved in 10 ml of DMF, and then DCC (1.2 eq) and HOSu (1.2 eq) were added. The mixture was reacted at room temperature for 6 hours. Compound 6 (1 eq) and DIPEA (5 eq) were then added. The mixture was reacted at room temperature for 1 hour. The reaction was monitored by LC-MS. The DMF was removed by spin-drying and the mixture was purified by reverse phase preparative liquid chromatography to obtain Compound 8 (Yield: 63%). Compound 8 was identified by mass spectrometry. The mass spectrum is shown in FIG. Figure 17 shown.

[0088] Synthesis of compound 10: Compound 8 (1 eq) was dissolved in 10 ml DMF, HOBt (5 eq) and EDC·HCl (5 eq) were added, and the mixture was reacted at 0°C for 10 minutes. Compound 9 (5 eq) and NMM (5 eq) were then added, and the mixture was transferred to room temperature for 12 hours. The reaction was monitored by LC-MS to be complete. The DMF was dried and 10 mL TFA solution was added to react at room temperature for 2.5 hours. 100 ml of ether was added, and a large amount of solid precipitated. The mixture was centrifuged and dried. Compound 10 (yield: 16%) was obtained after purification by reverse phase preparative liquid chromatography. Compound 10 was identified by mass spectrometry, and the mass spectrum is shown in FIG. Figure 18 shown.

[0089] The structural formula of the compound 1 is as follows:

[0090] ;

[0091] The compound 2 is 1,3,5-triacryloyl-1,3,5-triazine;

[0092] The structural formula of the compound 3 is as follows:

[0093] ;

[0094] The structural formula of the compound 4 is as follows:

[0095] ;

[0096] The structural formula of the compound 5 is as follows:

[0097] ;

[0098] The structural formula of the compound 6 is as follows:

[0099] ;

[0100] The structural formula of the compound 7 is as follows:

[0101] ;

[0102] The structural formula of the compound 8 is as follows:

[0103] ;

[0104] The structural formula of the compound 9 is as follows:

[0105] ;

[0106] The structural formula of the compound 10 is as follows:

[0107] .

[0108] Synthesis of DOTA-HTA-DM: Compound 10 (1 eq) and compound 5 (2.5 eq) were dissolved in 10 mL of ACN / H2O (1:1), and then 10 mL of pH = 7.2 0.2 M phosphate buffer was added. The reaction was allowed to react at room temperature for 1 hour. The reaction was monitored to be complete by LC-MS. DOTA-HTA-DM (Yield: 18.6%) was directly purified by reverse phase preparative liquid chromatography, and its structural formula is shown in Formula I.

[0109] Formula I.

[0110] 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 5 As can be seen, 1186.6854 is [M+5H] + / 5 peak, [M+4H] at 1483.1050 + / 4 peak, its molecular weight is actually 5928.82, and its structure is confirmed to be correct based on mass spectrometry.

[0111] Table 1 Mass spectrometry results of compound DOTA-HTA-DM

[0112]

[0113] Example 2

[0114] This example discloses the determination of the binding affinity between DOTA-HTA-DM and human Nectin-4 protein, specifically:

[0115] Surface plasmon resonance affinity assay (SPR) experiments were performed on Biacore 8K to determine the k affinity of DOTA-HTA-DM to human nectin-4 protein. a (1 / Ms), k d (1 / s) and K D(M) value. The sensor chip was first activated for 420 s with a fresh mixture of 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Nectin-4 protein was then diluted to 20 µg / mL using 10 mM sodium acetate (pH 4.5). Once the nectin-4 protein binding level reached approximately 800 RU, EA was passed over the chip surface to block excess sites. The reference channel was blocked directly after activation. Diluted peptide DOTA-HTA-DM prepared in Example 1 (concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 nM, respectively) was injected onto the chip at a flow rate of 30 µL / min at 25°C. The association time was 60 s and the dissociation time was 600 s. Detection was performed using single-cycle kinetic mode. All data were processed using Biacore 8K Evaluation Software Version 4.0. Appropriate continuous concentrations (at least 5 concentrations) were selected for kinetic 1:1 binding and steady-state analysis. The results are shown in the table below. Figure 6 As shown in Table 2.

[0116] Table 2 Binding affinity of DOTA-HTA-DM to human nectin-4 protein

[0117]

[0118] The results showed that DOTA-HTA-DM has a high binding affinity to human Nectin-4 protein. K D The value is 0.37nM.

[0119] Example 3

[0120] 68 Radiolabeling with Ga:

[0121] The DMSO solution of the Nectin-4 targeted dimer bicyclic peptide nuclide ligand prepared in Example 1 (concentration of 10 mg / ml) was prepared as a precursor solution. 20 μL of the precursor solution was placed in a reaction flask. 65 μL of 1 M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly washed 68 Ga eluent ( 68 Ga was washed 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 ( 68Ga-DOTA-HTA-DM bicyclic peptide radionuclide probe).

[0122] Quality control of Nectin-4 targeted probe:

[0123] 68 The radiochemical purity of the Ga-DOTA-HTA-DM bicyclic peptide nuclide probe was determined using radio-HPLC. Chromatographic method: Mobile phase: 0.1% TFA in water and 0.1% TFA in acetonitrile, 0-20 min: 30-50% acetonitrile phase. The radiochemical purity of the product was greater than 95% as determined by Radio-HPLC. Figure 7 shown.

[0124] Example 4

[0125] Western blot detection of Nectin-4 expression in human breast cancer MDA-MB-468 cell line:

[0126] Sample Lysis: Add an appropriate amount of lysis buffer (containing protease inhibitors) to the tissue and grind using a cryogenic tissue grinder. Remove and place on ice for 30 minutes, shaking every 10 minutes. Centrifuge at 12,000 rpm at 4°C for 30 minutes, and collect the supernatant to obtain the total protein solution. Protein Concentration: Measure protein concentration using a BCA protein assay kit. Protein Sample Preparation: Mix the treated protein solution, RAPI lysis buffer, and SDS-PAGE protein loading buffer in the appropriate proportions, denature in a metal bath at 95°C for 5 minutes, and store in a refrigerator at -20°C until use. SDS-PAGE Electrophoresis: Add protein sample and protein marker according to the sample layout, maintaining a constant voltage of 80 V for 15 minutes and 120 V for 1 hour. Transfer: Place the transfer chamber in an ice-water bath and transfer at a constant current of 300 mA for 1 hour. Blocking: Incubate with 5% skim milk at room temperature on a shaker for 1 hour. Primary Antibody Incubation: Incubate the PVDF membrane with the primary antibody overnight at 4°C on a shaker. 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 as follows Figure 8As shown. Using GAPDH as the internal standard, the molecular weight of nectin-4 protein is approximately 60-70 kDa. Nectin-4 and GAPDH expression are relatively high in MDA-MB-468 tumor tissue, while no nectin-4 expression is detected in the control MDA-MB-231 tumor tissue. This demonstrates that MDA-MB-468 is a model with high nectin-4 expression, while MDA-MB-231 is a model with low nectin-4 expression. T-47D, which has been reported to express nectin-4, serves as a positive control to demonstrate the validity of the antibody and the reliability of the Western blot experimental procedure.

[0127] Example 5

[0128] Micro-PET / CT imaging of human breast cancer MDA-MB-468 model mice:

[0129] This example discloses the in vivo imaging experiment of the dimeric bicyclic peptide nuclide ligand targeting nectin-4 prepared in Example 1. Nude mice with MDA-MB-468 xenograft tumors were implanted with the tumor tissue at a depth of 100-300 mm. 3 The mice were injected with 200 μCi of the drug (prepared in Example 3) into the tail vein at different time points (30, 60, and 120 minutes) after injection. Static scans were performed for 10 minutes, and the tumors were clearly visualized. After the scans, the scan data was iteratively reconstructed (OSEM 3D). The two tumors and other tissues were delineated as regions of interest (ROIs) using QD software, and the radioactivity uptake %ID / g in each ROI was calculated. 68 Ga-DOTA-HTA-DM, 68 Ga-DOTA-HTA-DM and DOTA-HTA-DM at a blocking dose (1000-fold excess) and 68 After Ga-DOTA-HTA (monomer), whole-body Micro-PET / CTMIP was performed 30 minutes, 1 hour, and 2 hours later. The images are shown in the figure below. Figure 9 As shown. MDA-MB-468 breast cancer tumor model mice were injected with 200 μCi of 68 Ga-DOTA-HTA-DM and 68 Comparison of tumor / muscle uptake values ​​of ROI outlined after 30 min, 1 h, and 2 h after Ga-DOTA-HTA-DM and blocking dose of DOTA-HTA-DM Figure 10 Data are presented as mean ± SD (n = 3).

[0130] 68 The preparation method of Ga-DOTA-HTA (monomer) is:

[0131] (1) Preparation of DOTA-HTA (monomer):

[0132] Compound 1 (1 eq) prepared in Example 1 was dissolved in a mixed solvent of 15 mL ACN and 15 mL H2O, followed by the addition of compound 2-1,3,5-triacryloyl-1,3,5-triazine (1.5 eq) and 10 mL 1M NH4HCO3. The reaction was allowed to proceed at room temperature for 1 hour. LC-MS monitored the reaction to be complete, and DOTA-HTA (yield: 34.5%) was directly purified by reverse phase preparative liquid chromatography. The mass spectrum is shown in FIG. Figure 12 。 See the structural formula Figure 13 .

[0133] (2) 68 Radioactive labeling of Ga: See Example 3 for preparation. 68 Ga-DOTA-HTA.

[0134] like Figure 9 and Figure 10 As shown, 68 Ga-DOTA-HTA-DM was significantly concentrated in the tumor area, with good sensitivity, and was mainly excreted from the body through the kidneys. As time went on, the uptake ratio of the complex in the tumor to the muscle increased. At 30 minutes, 1 hour, and 2 hours after injection, the tumor uptake values ​​were 2.18±0.34, 2.01±0.33, and 1.98±0.22%ID / g, respectively, and the tumor to muscle uptake ratios were 4.52±1.55, 9.41±1.41, and 6.83±1.75, respectively. In the blocking imaging experiment ( 68 In the Ga-DOTA-HTA-DM+blocking group, it was found that the uptake in the kidney and bladder was still high, the uptake in other organs was low, and there was no significant uptake in the tumor. The uptake of the probe in the blocking group was significantly lower than that in the positive group, with a significant statistical difference. 68 Ga-DOTA-HTA-DM has good clinical research prospects.

[0135] Example 6

[0136] Tissue distribution in human breast cancer MDA-MB-468 model mice:

[0137] MDA-MB-468 xenograft tumor model nude mice were transplanted with the tumor tissue to 100-300 mm 3 Tail vein injection 68Ga-DOTA-HTA-DM (50 μCi / mouse) was injected, and blood was collected at 30, 60, and 120 minutes after injection. Tumor tissue, as well as heart, liver, spleen, lung, stomach, pancreas, small intestine, large intestine, kidney, muscle, and bone tissues and organs were removed from each group of mice, weighed, and their radioactivity (CPM) was measured. The results were converted to ID% / g. Figure 11 .

[0138] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A dimeric bicyclic peptide nuclide ligand, characterized in that: Having the structure shown in Formula I: 。 2. A method for preparing the dimeric bicyclic peptide nuclide ligand according to claim 1, characterized in that: include: (1) coupling the amino acids contained in formula I by solid phase synthesis to obtain compound 1; (2) Compound 1 and compound 2 were subjected to a cyclization reaction in ammonium carbonate buffer and ACN / H2O mixed solvent to obtain compound 3; (3) Compound 4 was dissolved in DMF, activated in the presence of DCC, HOSu and DIPEA, and then subjected to acid-amine condensation with compound 3; after the reaction, DMF was removed, and liquid E was added for reaction, followed by addition of ether and drying to obtain compound 5; the liquid E refers to a mixture of 90% TFA + 5% thioanisole + 2.5% phenol + 2.5% 1,2-ethanedithiol; (4) Compound 7 was dissolved in DMF, activated in the presence of DCC, HOSu, and DIPEA, and then subjected to acid-amine condensation with compound 6. After the reaction, DMF was removed to obtain compound 8; (5) Compound 8 was dissolved in DMF and subjected to acid-amine condensation with compound 9 in the presence of EDC·HCl, HOBt, and NMM. After the reaction was complete, DMF was removed, the tert-butyl protecting group was removed with TFA solution, ether was added, and the mixture was dried to obtain compound 10. (6) Compound 10 and compound 5 were dissolved in ACN / H2O, and phosphate buffer was added for co-reaction to obtain the dimer bicyclic peptide nuclide ligand; The structural formula of the compound 1 is as follows: ； The compound 2 is 1,3,5-triacryloyl-1,3,5-triazine; The structural formula of the compound 3 is as follows: ； The structural formula of the compound 4 is as follows: ； The structural formula of the compound 5 is as follows: ； The structural formula of the compound 6 is as follows: ； The structural formula of the compound 7 is as follows: ； The structural formula of the compound 8 is as follows: ； The structural formula of the compound 9 is as follows: ； The structural formula of the compound 10 is as follows: 。 3. A radionuclide probe targeting Nectin-4, characterized in that: The dimeric bicyclic peptide nuclide ligand of claim 1 or the dimeric bicyclic peptide nuclide ligand prepared by the method of claim 2 is labeled with a radionuclide.

4. The nuclear probe targeting Nectin-4 according to claim 3, characterized in that: The radionuclide includes a diagnostic radionuclide or a therapeutic radionuclide. 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 includes 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 nuclide probe targeting nectin-4 according to claim 3 or 4, characterized in that: The method comprises the step of labeling the dimeric bicyclic peptide nuclide ligand according to claim 1 or the dimeric bicyclic peptide nuclide ligand prepared by the method of claim 2 with a radioactive nuclide.

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

7. Use of the nuclide probe targeting nectin-4 according to claim 3 or 4 in the preparation of a reagent or kit for diagnosing a disease; the disease is breast cancer characterized by overexpression of nectin-4.

8. A detection reagent or kit, characterized in that: The invention comprises the nuclear probe targeting Nectin-4 according to claim 3 or 4.

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