A bicyclic peptide ligand and probe targeting Nectin-4, its preparation method and application

Abstract

This invention relates to the field of nuclear medicine technology, specifically disclosing a bicyclic peptide isotope ligand and probe targeting Nectin-4, their preparation methods, and applications. The bicyclic peptide isotope ligand of this invention targeting Nectin-4 has the structure shown in Formula I: Formula I. The bicyclic peptide isotope ligand and probe of this invention exhibit significant advantages in terms of radiostability, pharmacokinetic properties, binding specificity to tumor Nectin-4, tumor uptake capacity, tumor-to-muscle uptake ratio, and in vivo metabolic performance.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nuclear medicine, in particular to a bicyclic peptide radionuclide ligand targeting Nectin-4, a probe and a preparation method and application thereof. BACKGROUND

[0002] Breast cancer is the most common malignant tumor in women. According to immunohistochemical methods, 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 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 subtypes of breast cancer, triple-negative breast cancer is more invasive, with higher recurrence rate and visceral metastasis rate. Triple-negative breast cancer is sensitive to chemotherapy, so traditional chemotherapy is still the standard treatment method. However, the effect of conventional treatment is limited: the median overall survival of triple-negative breast cancer patients 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 spreading to distant organs is 11%. Due to the non-expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, there is currently no effective targeted therapy for triple-negative breast cancer. Therefore, cell surface proteins that are specifically expressed in triple-negative breast cancer cells but not expressed or down-regulated in normal breast tissue will become ideal biomarkers and potential therapeutic targets.

[0003] Lectin (Nectin) protein belongs to the adhesion receptor of immunoglobulin superfamily (IgSF, Immunoglobulin superfamily), which is initially defined as the homolog of Poliomyelitis virus receptor (PVR) and consists of four members of 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 many 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%, and it is not expressed in normal breast epithelium and other normal tissues of adults, which is a cell surface biomarker of triple-negative breast cancer cells and can develop targeted drugs.

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

[0005] Although antibody drug conjugates (ADCs) targeting Nectin-4 have broad application prospects in clinical practice, issues such as how to screen patients for indications and evaluate efficacy have not been resolved. In addition, clinical studies have shown that Nectin-4 expression is often reduced or absent in metastatic lesions, and the absence or low expression of Nectin-4 may predict the occurrence of drug resistance.

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

[0007] The Nectin-4-targeted nuclear medicine diagnosis and treatment reagents currently reported are Nectin-4 monoclonal antibodies labeled with radionuclides 89 Zr、 18 F and 99m Tc. However, such imaging agents have the disadvantages of large molecular weight, long in vivo circulation time, and weak transmembrane ability, which have certain limitations when applied to clinical routine molecular imaging reagents. Therefore, there is an urgent need to develop radiolabeled molecular probes to evaluate Nectin-4 expression levels, providing visual tools and criteria for patient diagnosis, classification, treatment decisions, efficacy evaluation, and prognosis assessment. Using peptides as targeting carriers has many advantages: compared with antibody-based radiopharmaceuticals, peptides are smaller in size, can be quickly cleared from the blood and non-target tissues, are non-immunogenic, have good tissue penetration, and have natural advantages in clinical translation. Therefore, it is necessary to study the construction of Nectin-4-targeted peptides. SUMMARY

[0008] One of the purposes of the present application is to provide a Nectin-4 targeted bicyclic peptide radionuclide ligand and its application, which has better radiochemical stability, pharmacokinetic characteristics, binding specificity to tumor Nectin-4, tumor uptake ability, tumor to muscle uptake ratio and in vivo metabolic performance.

[0009] The present application provides a Nectin-4 targeted bicyclic peptide radionuclide ligand, which has a structure as shown in formula I:

[0010] Formula I.

[0011] The present application constructs a Nectin-4 targeted molecular probe (Nectin-4 targeted bicyclic peptide radionuclide ligand) which is covalently modified at a specific position. The parent nucleus thereof can realize high affinity to the Nectin-4 target through a specific amino acid sequence and cyclization mode. The radionuclide chelating group can be combined with various radionuclides to realize labeling of the Nectin-4 targeted bicyclic peptide. 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 reactivity to cysteine and histidine side chains. Therefore, after binding to the Nectin-4 target protein site, the amino acid residues in the binding pocket region can provide a hydrogen bond donor or an electrophilic group to stabilize the leaving fluorine ion, thereby awakening the activity of the fluorosulfonyl group, 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 Nectin-4 targeted peptide after the specific modification can enhance the tumor uptake and retention time. The Nectin-4 targeted molecular probe labeled with radionuclide after specific covalent modification can be used for preclinical verification of in vivo identification of Nectin-4 receptor positive tumor models through PET whole body imaging (which can obtain higher resolution and can clearly distinguish the tumor region). It has important significance for early detection of triple-negative breast cancer with high expression of Nectin-4, screening of Nectin-4 targeted therapeutic drug beneficiaries, monitoring and evaluation of the efficacy of antitumor drugs, prognosis evaluation, etc., and has great clinical transformation potential.

[0012] The present application also provides a method for preparing the Nectin-4 targeted bicyclic peptide radionuclide ligand, which comprises:

[0013] (1) coupling the amino acids contained in formula I through a solid phase synthesis method to obtain a polypeptide chain;

[0014] (2) coupling 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid with the polypeptide chain to obtain a DOTA-polypeptide chain;

[0015] (3) after coupling 3-(4-(fluorosulfonyl)phenyl)propionic acid with the DOTA-polypeptide chain, reacting with 1,3,5-triacryloylhexa-1,3,5-triazine.

[0016] The application also provides a bicyclic peptide radionuclide probe targeting Nectin-4, which is a radionuclide-labeled bicyclic peptide radionuclide ligand targeting Nectin-4.

[0017] In the bicyclic peptide radionuclide probe targeting Nectin-4 of the application, the radionuclide comprises a diagnostic radionuclide or a therapeutic radionuclide, the diagnostic radionuclide is at least one of 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; and the therapeutic radionuclide comprises at least one of 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 Bi and 212 Pb; preferably, the radionuclide is 68 Ga.

[0018] Compared with the imaging agent in the prior art, the small molecule tracer provided by the application has a reasonable half-life, stronger tumor penetration, and can give accurate evaluation results in a shorter time, and the like, thereby obtaining more accurate results. 68 The Ga-labeled bicyclic peptide radionuclide ligand has the characteristics of fast labeling speed and high labeling rate, and the radiochemical purity is greater than 95%. The labeled compound has good hydrophilicity. 68 The Ga-labeled bicyclic peptide radionuclide ligand is mainly excreted through the kidney, and the uptake of other non-target organs is low, and there is a certain uptake in tumors, showing good sensitivity and specificity.

[0019] The application also provides a method for preparing the above-mentioned Nectin-4-targeting bicyclic peptide radionuclide probe, which comprises the step of labeling the above-mentioned Nectin-4-targeting bicyclic peptide radionuclide ligand or the Nectin-4-targeting bicyclic peptide radionuclide ligand prepared by the above-mentioned method with a radionuclide.

[0020] The application also provides a medicament comprising the above-mentioned Nectin-4-targeting bicyclic peptide radionuclide ligand or Nectin-4-targeting bicyclic peptide radionuclide probe and a pharmaceutically acceptable excipient.

[0021] “Pharmaceutically acceptable” in the present application means that the compound or composition is chemically and / or toxicologically compatible with other ingredients constituting the preparation and with humans or mammals to be prevented, diagnosed and treated with the preparation.

[0022] In the medicament of the present application, the Nectin-4-targeting bicyclic peptide radionuclide ligand or Nectin-4-targeting bicyclic peptide radionuclide probe is further coupled with a therapeutic agent capable of preventing, inhibiting and / or treating a disease characterized by overexpression of Nectin-4.

[0023] The application also provides the use of the above-mentioned Nectin-4-targeting bicyclic peptide radionuclide probe in the preparation of a Nectin-4 imaging agent or a PET imaging probe or an agent or a kit for diagnosing, treating and / or preventing a disease characterized by overexpression of Nectin-4.

[0024] The application also provides a detection agent or a kit comprising the above-mentioned Nectin-4-targeting bicyclic peptide radionuclide probe.

[0025] The application has at least the following beneficial effects:

[0026] The application provides a new Nectin-4-targeting bicyclic peptide radionuclide ligand and a preparation method thereof, as well as a Nectin-4-targeting bicyclic peptide radionuclide probe based on the ligand and a preparation method thereof, and covers the use of the ligand and the probe in the preparation of a solid tumor diagnostic agent. Compared with the existing Nectin-4-targeting bicyclic peptide radionuclide probe, the Nectin-4-targeting bicyclic peptide radionuclide ligand and probe of the present application have significant advantages in radioactivity stability, pharmacokinetic properties, binding specificity to tumor Nectin-4, tumor uptake capacity, tumor-to-muscle uptake ratio and in vivo metabolic performance, and have significant clinical transformation potential, providing a new technical means and application prospect for the precise diagnosis of solid tumors. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Mass spectrum of DOTA-HTA-SF intermediate product Compd1;

[0028] Figure 2 Mass spectrum of DOTA-HTA-SF;

[0029] Figure 3 Graph of binding affinity of DOTA-HTA-SF to human Nectin-4 detected by SPR;

[0030] Figure 4 68 HPLC profile of Ga-DOTA-HTA-SF bicyclic peptide radionuclide probe;

[0031] Figure 5 Western blot detection of tumor cell MDA-MB-468;

[0032] Figure 6 Whole body Micro-PET / CT MIP imaging of breast cancer MDA-MB-468 tumor model mice injected with 200 μCi of 68 Ga-DOTA-HTA-SF, 68 Ga-DOTA-HTA-SF with blocking dose of DOTA-HTA-SF (Blocked), 68 Ga-DOTA-HTA (without covalent modification) and other covalent modification group schemes of 68 Ga-DOTA-HTA-SF2, 68 Ga-DOTA-HTA-pFS and 68 Ga-DOTA-HTA-mFS probes at 30 min and 1 h post injection, respectively; the arrow indicates the tumor location;

[0033] Figure 7 Whole body Micro-PET / CT MIP imaging of breast cancer MDA-MB-468 tumor model mice injected with 200 μCi of 68 Ga-DOTA-HTA-SF and 68 Ga-DOTA-HTA-SF with blocking dose of DOTA-HTA-SF at 30 min, 1 h and 2 h post injection, respectively. P < 0.01;

[0034] Figure 8 Mass spectrum of DOTA-HTA;

[0035] Figure 9 Structural formula of DOTA-HTA;

[0036] Figure 10 Structural formula of Compd 4 in the synthesis route of DOTA-HTA-SF2;

[0037] ​Figure 11 Mass spectrum of DOTA-HTA-SF2;

[0038] Figure 12 Structural formula of DOTA-HTA-SF2;

[0039] Figure 13 Structural formula of Compd4 in the synthetic route of DOTA-HTA-mFS;

[0040] Figure 14 Mass spectrum of DOTA-HTA-mFS;

[0041] Figure 15 Structural formula of DOTA-HTA-mFS;

[0042] Figure 16 Structural formula of Compd4 in the synthetic route of DOTA-HTA-pFS;

[0043] Figure 17 Mass spectrum of DOTA-HTA-pFS;

[0044] Figure 18 Structural formula of DOTA-HTA-pFS. DETAILED DESCRIPTION

[0045] The preferred embodiments of the present application will be described in detail below with reference to the examples. It should be understood that the following examples are given only to illustrate the present application and are not intended to limit the scope of the present application. Those skilled in the art can make various modifications and substitutions to the present application without departing from the spirit and principles of the present application.

[0046] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially or prepared according to conventional methods in the art unless otherwise specified.

[0047] Example 1

[0048] This example discloses a method for synthesizing the Nectin-4 double-loop peptide DOTA-HTA-SF2, specifically:

[0049] Synthesis of Compd 1 : Fmoc-Lys(Dde)-OH was first 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 sequentially. All amino acid (1 eq) couplings were performed using DIC (3 eq), HOBt (3 eq) as coupling reagents in DMF. Fmoc protecting groups were cleaved using 20% Pip / DMF solution.

[0050] 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA, 1 eq) was coupled to the previous resin peptide using HATU (1.5 eq), HOBt (1.5 eq) as coupling reagents in DIPEA (3 eq), DMF. The Dde protecting group was cleaved using 4% hydrazine hydrate / DMF solution.

[0051] 3-(4-(fluorosulfonyl)phenyl)propanoic acid (1 eq) was coupled to the previous resin peptide using HATU (1.5 eq), HOBt (1.5 eq) as coupling reagents in DIPEA (3 eq), DMF. Finally, the peptide was cleaved from the resin using TFA solution at room temperature for 2 h to give the crude peptide. The crude peptide was purified by HPLC on a C18 preparative column to give pure Compd 1 (Yield: 25.7%) as DOTA-Cys-Pro-1Nal-DAsp-Cys-Met-HomoArg-Asp-Trp-Ser-Thr-Pro-Hyp-Trp-Cys-Lys[(3-(4-(fluorosulfonyl)phenyl)propanoic acid]-NH2. The structure of Compd 1 was identified by mass spectrometry, and the mass spectrum is shown in Figure 1 which is consistent with the synthesis target.

[0052] Compd1 (1 eq) was dissolved in 15 mL ACN and 15 mL H2O mixed solvent, then Compd2-1, 3, 5-triacryloylhexa-1, 3, 5-triazine (1.5 eq), 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour, and LC-MS was used to monitor the reaction. The final product DOTA-HTA-SF (Yield: 34.5%) was obtained by direct reversed-phase preparative liquid chromatography purification, and its structural formula is shown in Formula I below. The structure of the final product ligand was identified by mass spectrometry, and the results are shown in Figure 2 .

[0053] Formula I.

[0054] 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. As can be seen in the mass spectrum Figure 2 , the 967.7081 is [M+3H] + / 3 peak, and the 1451.5655 is [M+2H] + / 2 peak. The molecular weight is actually 2901.29, and the structure is correct according to the mass spectrometry structure confirmation.

[0055] Table 1 Mass spectrometry results of compound DOTA-HTA-SF

[0056]

[0057] Example 2

[0058] This example discloses the binding affinity determination of DOTA-HTA-SF to human Nectin-4 protein, specifically:

[0059] Surface plasmon resonance affinity determination (SPR) experiments were performed on Biacore 8K to determine the k a (1 / Ms)、 k d (1 / s) and K D(M) values. The sensor chip 420 was first activated by freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) for 420 s. Subsequently, the Nectin-4 protein was diluted to 20 µg / mL using 10 mM sodium acetate (pH 4.5), and after the Nectin-4 protein coupling level reached about 1200 RU, EA was flowed over the chip surface to block the excess sites. The reference channel was activated and then directly blocked. The diluted polypeptide DOTA-HTA-SF prepared in Example 1 (concentrations were 100, 50, 25, 12.5, 6.25, 3.125 nM, respectively) was injected into the chip at a flow rate of 30 µL / 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 run. All data were processed using Biacore 8K evaluation software version 4.0. The appropriate serial concentrations (at least 5 concentrations) were selected for kinetic 1:1 binding and steady-state analysis, and the results are shown in Table 2 and FIG. 2. Figure 3 and Table 2.

[0060] Table 2 Binding affinity of DOTA-HTA-SF to human Nectin-4 protein

[0061]

[0062] The results show that DOTA-HTA-SF has a very high binding affinity to human Nectin-4 protein, and the K D The value is 13.8 nM.

[0063] Example 3

[0064] 68 Radiolabeling of Ga:

[0065] A DMSO solution (concentration of 10 mg / ml) of the Nectin-4 targeting bicyclic peptide radionuclide ligand prepared in Example 1 was configured as a precursor solution, 10 μL of the precursor solution was taken into a reaction bottle, 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly eluted 68 Ga eluent 68 Ga eluent in 0.1M hydrochloric acid solution) were added, and 0.5 mL of deionized water was added. The pH value of the reaction solution was about 4.0, and after mixing, the mixture was placed in a 95°C reaction for 10 min to obtain 68 Ga-labeled complex 68Ga-DOTA-HTA-SF bicyclic peptide radionuclide probe.

[0066] Quality control of Nectin-4 targeting probes:

[0067] 68 The radiochemical purity of Ga-DOTA-HTA-SF bicyclic peptide radionuclide probe was determined by radio-high performance liquid chromatography (Radio-HPLC). Chromatographic method: mobile phase: 0.1% TFA in water and 0.1% TFA in acetonitrile solution, 0-20 min: 30-50% acetonitrile phase. The radiochemical purity of the product was greater than 95% by Radio-HPLC detection, and the detection results are shown in Figure 4 .

[0068] 68 Lipid-water partition coefficient of Ga-DOTA-HTA-SF bicyclic peptide radionuclide probe:

[0069] The same volume (0.5 mL:0.5 mL) of n-octanol (organic phase) and phosphate buffer solution (aqueous phase, pH=7.4) were used to dissolve 68 Ga-DOTA-HTA-SF bicyclic peptide radionuclide probe in an EP tube (about 3.7 MBq). Shake well for 5 min, and centrifuge to separate the layers for 5 min in a centrifuge. The speed was 2000 r / min. Take 100 μL of the organic phase and the aqueous phase respectively in a 1.5 mL EP tube, and measure the radioactivity count rate in a well-type gamma detector. The lipid-water partition coefficient (P) was calculated from the ratio of the radioactivity count rates of the organic phase and the aqueous phase.

[0070] P=log(N0 / N aq );

[0071] Where: N0and N aq are the radioactivity count rates of the organic phase and the aqueous phase samples, respectively, s -1 . Repeat the experiment 3 times, and take the average value as the lipid-water partition coefficient of the marker. 68 The lipid-water partition coefficient of Ga-DOTA-HTA-SF bicyclic peptide radionuclide probe was logP=-0.28±0.03 (n=3), indicating 68 Ga-DOTA-HTA-SF has certain hydrophilicity.

[0072] Example 4

[0073] Western blot detection of Nectin-4 expression in human breast cancer MDA-MB-468 cell line:

[0074] 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 then take it out and stand on ice for 30 min, shaking every 10 min. Centrifuge at 12,000 rpm at 4°C for 30 min, and collect the supernatant, which is the total protein solution. Protein concentration determination: Detect the protein concentration using a BCA protein concentration determination kit. Protein sample preparation: Mix the treated protein solution, RAPI lysis solution, and SDS-PAGE protein loading buffer according to the proportions, denature in a metal bath at 95°C for 5 min, and store in a -20°C refrigerator for standby. SDS-PAGE electrophoresis: Add the protein sample and protein marker in turn according to the sample layout, and apply a constant voltage of 80V for 15 min and 120V for 1 hr. Membrane transfer: Place the membrane transfer tank in an ice water bath and transfer at a constant current of 300 mA for 1 hr. Blocking: Incubate with 5% skimmed milk on a shaker at room temperature for 1 hr. Primary antibody incubation: Incubate the PVDF membrane with the primary antibody on a constant temperature shaker at 4°C overnight. Secondary antibody incubation: Incubate the PVDF membrane with the secondary antibody on a shaker at room temperature for 1 hr. ECL color development: Add the color developing solution and use a chemiluminescence imaging system for automatic exposure. The Western blot detection results are shown in Figure 5 Figure 2. The molecular weight of Nectin-4 protein is about 60-70 kDa, with GAPDH protein as the internal standard. The relative expression of Nectin-4 and GAPDH in MDA-MB-468 tumor tissue is relatively high, while no expression of Nectin-4 is detected in the control MDA-MB-231 tumor tissue. This 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, serving as a positive control to prove the effectiveness of the antibody and the reliability of the Western blot experiment operation.

[0075] Example 5

[0076] Human breast cancer MDA-MB-468 model mouse Micro-PET / CT imaging:

[0077] This example discloses the in vivo imaging test of the Nectin-4 targeted bicyclic peptide radionuclide ligand prepared in Example 1. The MDA-MB-468 xenograft tumor model nude mice were given tail vein injection (200 μCi each) when the tumor tissue reached 100-300 mm 3 After the scan, the scan data were iteratively reconstructed (OSEM 3D), and the two tumors and other tissues were outlined as regions of interest (ROIs) to calculate the radioactivity uptake (%ID / g) of each ROI using QD software. Among them, the breast cancer MDA-MB-468 tumor model mice were injected with 200 μCi of the Nectin-4 targeted bicyclic peptide radionuclide ligand prepared in Example 3, respectively.68 Ga-DOTA-HTA-SF, 68 Ga-DOTA-HTA-SF with a blocking dose (1000-fold excess) of DOTA-HTA-SF, 68 Ga-DOTA-HTA (without covalent modification) and other covalent modification group alternatives 68 Ga-DOTA-HTA-SF2, 68 Ga-DOTA-HTA-pFS and 68 Ga-DOTA-HTA-mFS probes, respectively, at 30 min and 1 h post-injection, whole body Micro-PET / CT MIPs are shown in Figure 6 Breast cancer MDA-MB-468 tumor model mice were injected with 200 μCi of 68 Ga-DOTA-HTA-SF and 68 Ga-DOTA-HTA-SF with a blocking dose of DOTA-HTA-SF, respectively, at 30 min, 1 h, 2 h post-injection, the tumor / muscle uptake ratios of the ROI-delineated tumors are shown in Figure 7 (data are presented as mean ± standard deviation (n = 3).

[0078] 68 Ga-DOTA-HTA was prepared by:

[0079] Synthesis of Compd1: First, Fmoc-Cys(Trt)-OH is coupled to Rink Amide MBHA. Resin was then performed, followed by sequential coupling of 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, and Fmoc-Cys(Trt)-OH. All amino acids (1 eq) were coupled in DMF using DIC (3 eq) and HOBt (3 eq) as coupling agents. The Fmoc protecting group was cleaved using a 20% Pip / DMF solution. DOTA was coupled to the preceding resin peptide in DIPEA (3 eq) and DMF using HATU (1.5 eq) and HOBt (1.5 eq) as coupling reagents. Finally, the peptide was lysed from the resin in TFA solution at room temperature for 2 h to obtain crude peptide Compd1. The crude peptide was purified by HPLC on a C18 preparative column to obtain pure Compd1 (Yield: 26.8%).

[0080] Synthesis of DOTA-HTA: Compd1 (1 eq) was dissolved in a mixed solvent of 15 mL ACN and 15 mL H2O, followed by the addition of Compd2-1,3,5-triacryloylhexa-1,3,5-triazine (1.5 eq) and 10 mL 1 M NH4HCO3. The reaction was carried out at room temperature for 1 hour, and the reaction was monitored by LC-MS until complete. DOTA-HTA (Yield: 34.5%) was obtained directly by reverse-phase preparative HPLC purification. The ligand structure of the final product was identified by mass spectrometry, and the results are as follows. Figure 8 As shown, the structural formula is... Figure 9 .

[0081] 68 Radiolabeling of Ga: A DMSO solution of DOTA-HTA (concentration 10 mg / ml) was prepared as a precursor solution. 10 μL of the precursor solution was placed in a reaction flask, and 65 μL of 1 M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly rinsed solution were added to the flask. 68 Ga rinsing solution ( 68 Ga was rinsed in 0.1M hydrochloric acid solution, and then 0.5 mL of deionized water was added. The pH of the reaction solution was approximately 4.0. After mixing, the mixture was placed at 95℃ for 10 min to obtain... 68 Ga-labeled complexes68 Ga-DOTA-HTA bicyclic peptide radionuclide probe.

[0082] 68 The preparation method of Ga-DOTA-HTA-SF2 is as follows:

[0083] Synthesis of Compd 1: Fmoc-Lys(Boc)-OH was coupled to Rink Amide MBHA Resin first, 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 turn, all the coupling of amino acids (1 eq) were carried out in DMF using DIC (3 eq), HOBt (3 eq) as coupling reagents, and the Fmoc protecting group was cleaved using 20% Pip / DMF solution. DOTA (1 eq) was coupled with the previous resin peptide using HATU (1.5 eq), HOBt (1.5 eq) as coupling reagents in DIPEA (3 eq), DMF. Finally, the crude peptide Compd 1 was obtained by cleaving from the resin using TFA solution at room temperature for 2 h, and the crude peptide was purified by HPLC on a C18 preparative column to obtain pure Compd 1 (Yield: 27.3%).

[0084] Compd 1 (1 eq) was dissolved in 10 mL ACN and 10 mL H2O mixed solvent, then Compd 2-1, 3, 5-triacryloyl hexa-1, 3, 5-triazine (1.5 eq), 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 h, and LC-MS was used to monitor the completion of the reaction. Compd 3 was obtained by direct reversed-phase preparative liquid chromatography purification (Yield: 34.5%).

[0085] Compd 4 (1 eq) (structural formula is shown in Figure 10The ligand was dissolved in 10 ml of DMF, 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 until completion. The DMF was removed, and the product was purified by reverse-phase preparative HPLC to obtain DOTA-HTA-SF2 (Yield: 33.7%). The final product ligand structure was identified by mass spectrometry, and the results are as follows. Figure 11 As shown, the structural formula is... Figure 12 .

[0086] 68 Radiolabeling of Ga: A DMSO solution of DOTA-HTA-SF2 (concentration 10 mg / ml) was prepared as a precursor solution. 10 μL of the precursor solution was placed in a reaction flask, and 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly rinsed solution were added to the flask. 68 Ga rinsing solution ( 68 Ga was rinsed in 0.1M hydrochloric acid solution, and then 0.5 mL of deionized water was added. The pH of the reaction solution was approximately 4.0. After mixing, the mixture was placed at 95℃ for 10 min to obtain... 68 Ga-labeled complexes 68 Ga-DOTA-HTA-SF2 bicyclic peptide nuclide probe.

[0087] 68 The preparation method of Ga-DOTA-HTA-mFS is as follows:

[0088] Synthesis of Compd 1: Fmoc-Lys(Boc)-OH was first 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 sequentially coupled. All the coupling of amino acids (1 eq) were carried out in DMF using DIC (3 eq), HOBt (3 eq) as coupling reagents. Fmoc protecting group was cleaved using 20% Pip / DMF solution. DOTA (1 eq) was coupled to the previous resin peptide using HATU (1.5 eq), HOBt (1.5 eq) as coupling reagents in DIPEA (3 eq), DMF. Finally, the crude peptide Compd 1 was cleaved from the resin using TFA solution at room temperature for 2 h. The crude peptide was purified by HPLC on C18 preparative column to give pure Compd 1 (Yield: 27.3%).

[0089] Compd 1 (1 eq) was dissolved in 10 mL ACN and 10 mL H2O mixed solvent, then Compd 2-1, 3, 5-triacryloylhexa-1, 3, 5-triazine (1.5 eq), 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 h. LC-MS was used to monitor the reaction completion. The product was directly purified by reverse phase preparative liquid chromatography to give Compd 3 (Yield: 34.5%).

[0090] Compd 4 (1 eq) (structure see Figure 13 ) was dissolved in 10 mL DMF, then DCC (1.2 eq) and HOSu (1.2 eq) were added. The reaction was carried out at room temperature for 6 h. Then Compd 3 (1 eq) and DIEA (3 eq) were added. The reaction was carried out at room temperature for 1 h. LC-MS was used to monitor the reaction completion. DMF was removed. The product was purified by reverse phase preparative liquid chromatography to give DOTA-HTA-mFS (Yield: 30.2%). The structure of the final product ligand was identified by mass spectrometry. The results are shown in Figure 14 Figure 15

[0091] 68 ​​Radiolabeling of Ga: A DMSO solution (10 mg / ml) of prepared DOTA-HTA-mFS was configured as precursor solution, 10 μL of the precursor solution was put into a reaction bottle, 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly eluted 68 Ga elution solution (0.1M HCl) 68 Ga elution in 0.1M HCl), and 0.5 mL of deionized water were added. The pH of the reaction solution was about 4.0, and after mixing, the mixture was placed in a 95°C reaction for 10 min to obtain 68 Ga-labeled complex 68 Ga-DOTA-HTA-mFS bicyclic peptide radionuclide probe.

[0092] 68 The preparation method of Ga-DOTA-HTA-pFS is as follows:

[0093] Synthesis of Compd1: Fmoc-Lys(Boc)-OH was first coupled to Rink Amide MBHA Resin, and 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 sequentially coupled, and the coupling of all amino acids (1 eq) was carried out in DMF using DIC (3 eq) and HOBt (3 eq) as coupling reagents. The Fmoc protecting group was cleaved using a 20% Pip / DMF solution. 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. Finally, the crude peptide Compd1 was obtained by cleaving from the resin using a TFA solution at room temperature for 2 h, and the crude peptide was purified by HPLC on a C18 preparative column to obtain the pure Compd1 (Yield: 27.3%).

[0094] Compd 1 (1 eq) was dissolved in 10 mL ACN and 10 mL H2O mixed solvent, then Compd 2-1, 3, 5-triacryloylhexa-1, 3, 5-triazine (1.5 eq), 10 mL 1M NH4HCO3 were added. The reaction was carried out at room temperature for 1 hour, LC-MS was used to monitor the reaction completion, and then the product Compd 3 was obtained by direct reversed-phase preparative liquid chromatography purification (Yield: 34.5%).

[0095] Compd 4 (1 eq) (see structural formula Figure 16 ) was dissolved in 10 mL DMF, 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, and then the DMF was removed. After reversed-phase preparative liquid chromatography purification, DOTA-HTA-pFS was obtained (Yield: 30.2%). The structure of the final product ligand was identified by mass spectrometry, and the results are shown in Figure 17 , and the structural formula is shown in Figure 18 .

[0096] 68 Radiolabeling of Ga: The prepared DMSO solution (concentration of 10 mg / ml) of DOTA-HTA-pFS was used as a precursor solution. 10 μL of the precursor solution was taken into a reaction bottle, 65 μL of 1M sodium acetate buffer (pH 4.5) and 0.5 mL of freshly eluted 68 Ga eluent 68 Ga eluent in 0.1M hydrochloric acid solution) were added to the reaction bottle, and then 0.5 mL of deionized water was added. The pH value of the reaction solution was about 4.0. After mixing, the mixture was placed in a 95°C reaction for 10 min to obtain 68 Ga-labeled complex 68 Ga-DOTA-HTA-pFS bicyclic peptide radionuclide probe.

[0097] As shown in Figure 6 and Figure 7 , 68 Ga-DOTA-HTA-SF was obviously concentrated in the tumor area, had good sensitivity, and was mainly excreted out of the body through the kidneys. With the extension of time, the uptake ratio of the complex in the tumor to muscle increased. At 30 min, 1 h and 2 h 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 ratio of the tumor to muscle was 6.27±0.51, 8.55±1.82 and 7.47±1.48, respectively. There was a certain uptake in the heart position, which was due to the high blood drug concentration and a certain enrichment in the heart position. In the blocking imaging experiment 68In Ga-DOTA-HTA-SF+block group, it can be seen that there is still high uptake in kidney and bladder, less uptake in other organs, and no significant uptake in tumor site. The tumor uptake of probe in block group is significantly lower than that in positive group, with 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 group schemes 68 Ga-DOTA-HTA-SF2, 68 Ga-DOTA-HTA-pFS and 68 Ga-DOTA-HTA-mFS probe in MDA-MB-468 tumor model are significantly lower than 68 Ga-DOTA-HTA-SF. The above studies show that, 68 Ga-DOTA-HTA-SF has good prospects for clinical research.

[0098] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A bicyclic peptide radionuclide ligand targeting Nectin-4, characterized in that, having a structure as shown in Formula I: 。 2. A method of making the bicyclic peptide radionuclide ligand targeted to Nectin-4 of claim 1, characterized in that, comprising: (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) coupling 3-(4-(fluorosulfonyl)phenyl)propionic acid with the DOTA-polypeptide chain, and then reacting with 1,3,5-trisacryloylhexa-1,3,5-triazine.

3. A bicyclic peptide radionuclide probe targeting Nectin-4, characterized in that, The Nectin-4 targeting bicyclic peptide radionuclide ligand of claim 1 labeled with a radionuclide; The radionuclide includes a diagnostic radionuclide or a therapeutic radionuclide, the diagnostic radionuclide is at least one of 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; the therapeutic radionuclide includes at least one of 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 Bi and 212 Pb.

4. A method of preparing the bicyclic peptide radionuclide probe targeted to Nectin-4 of claim 3, characterized in that, comprising the step of labeling the Nectin-4 targeting bicyclic peptide radionuclide ligand of claim 1 with a radionuclide or the Nectin-4 targeting bicyclic peptide radionuclide ligand prepared by the method of claim 2.

5. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. comprising the Nectin-4 targeting bicyclic peptide radionuclide ligand of claim 1 or the Nectin-4 targeting bicyclic peptide radionuclide probe of claim 3 and a pharmaceutically acceptable excipient.

6. The medicament according to claim 5, characterized in that, The Nectin-4 targeting bicyclic peptide radionuclide ligand or the Nectin-4 targeting bicyclic peptide radionuclide probe is further coupled with a therapeutic agent that can prevent, inhibit and / or treat a disorder characterized by overexpression of Nectin-4.

7. Use of the Nectin-4 targeting bicyclic peptide radionuclide probe of claim 3 in the preparation of a Nectin-4 imaging agent or a PET imaging probe.

8. Use of the Nectin-4 targeting bicyclic peptide radionuclide probe of claim 3 in the preparation of a reagent or a kit for diagnosis, treatment and / or prevention of a disease; the disease is characterized by overexpression of Nectin-4.

9. An assay reagent or kit characterized in that, comprising the Nectin-4 targeting bicyclic peptide radionuclide probe of claim 3.

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