A bnct diagnosis and treatment integrated drug based on an alpha v beta 6 targeting polypeptide and application thereof

Abstract

This invention belongs to the field of biomedical technology and discloses an integrated diagnostic and therapeutic drug for boron neutron capture therapy (BNCT) based on targeted peptides. Existing BNCT drugs lack sufficient specificity and targeting, limiting their clinical application. To address this issue, this invention designs peptide drugs with targeting and high affinity, enabling PET / CT imaging and BNCT treatment. The HKB series molecules designed in this invention show good tumor specificity and high uptake against pancreatic ductal adenocarcinoma (PDAC) with high αvβ6 expression, resulting in better BNCT treatment efficacy. Therefore, this invention has practical application value and can significantly improve the effectiveness and precision of BNCT in the clinical treatment of PDAC, providing a new solution for the treatment of tumors such as pancreatic cancer with high αvβ6 expression.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the design, synthesis and application of drugs targeting BNCT. Background Technology

[0002] The integrin receptor family consists of transmembrane heterodimers composed of α and β subunits, mediating cell-cell and cell-extracellular matrix (ECM) recognition and adhesion. Integrin receptor αvβ6 (αvβ6) is an important member of the integrin receptor family, an epithelial-specific integrin receptor that regulates various physiological activities during tumorigenesis, such as participating in intercellular and ECM adhesion reactions, influencing gene expression and intracellular signal transduction, and mediating the transformation of epithelial cells into mesenchymal cells. αvβ6 was first discovered in pancreatic ductal carcinoma (PDAC) tissue and has been persistently highly expressed in corresponding patient samples, while its expression level is significantly lower in normal pancreatic tissue. Furthermore, αvβ6 expression is significantly increased in metastatic lesions compared to primary tumors. Existing research data indicates that, in vitro, integrin αvβ6 promotes the growth, survival, migration, and invasion of PDAC cells. In addition, αvβ6 may enhance the therapeutic effect of chemotherapeutic drugs by affecting the chemosensitivity of pancreatic cancer cells.

[0003] αvβ6 is a pan-cancer target, highly expressed on the surface of various tumor cells, including pancreatic cancer. For example, in various tumors such as oral cancer, breast cancer, gastric cancer, cholangiocarcinoma, and colorectal cancer, αvβ6 expression is closely related to malignant progression. Therefore, αvβ6 has become a broad target for cancer therapy. Currently, drugs targeting αvβ6 are under development. For example, SGN-B6A developed by Seagen, as an antibody-drug conjugate targeting αvβ6, is undergoing clinical trials to evaluate its efficacy in treating non-small cell lung cancer. Currently, αvβ6 has become a potential target for the treatment of epithelial cancers, including pancreatic cancer, cholangiocarcinoma, and colorectal cancer.

[0004] Pancreatic cancer, a malignant tumor of the digestive system that is difficult to diagnose early, has a high recurrence and metastasis rate, and a high mortality rate, is known as the "king of cancers" and is the seventh leading cause of cancer-related death worldwide. Statistics show that the 5-year survival rate for pancreatic cancer is only 9%-11%, and its early diagnosis rate is less than 5%, with approximately 60% of patients already having metastasized at initial diagnosis. Due to the dense tissue, complex tumor microenvironment, and difficulty in drug penetration, the treatment of pancreatic cancer is particularly challenging. Currently, surgery and chemotherapy are commonly used to treat pancreatic cancer patients; however, these treatments have significant limitations, and further development of more treatment methods is needed to provide effective and personalized treatment plans for pancreatic cancer patients.

[0005] Therefore, developing precise and effective new methods for the early diagnosis and treatment of pancreatic cancer has become a crucial and urgent issue. This involves not only high-resolution imaging of pancreatic cancer but also the development of new treatment methods, especially targeted therapies that address the specific biological characteristics of pancreatic cancer. With a deeper understanding of the pathological process of pancreatic cancer, scientists have developed several novel targeted molecules that exhibit high specificity and therapeutic efficacy, contributing to the prolongation of survival for pancreatic cancer patients as a complement to conventional chemotherapy. For example, the FDA has approved the KRASG12C inhibitor sotorasib for the treatment of patients with KRASG12C-driven pancreatic cancer, targeting the common KRAS mutation. Furthermore, immunotherapy is also showing potential in the treatment of pancreatic cancer, particularly for patients with microsatellite instability-high (MSI-H) / mismatch repair deficiency (dMMR), where the PD-1-targeting pembrolizumab has been approved for treatment. However, the dense stroma of pancreatic cancer makes it difficult for drugs to penetrate, and the level of immune response is low. Current targeted therapies cannot effectively improve the survival rate of pancreatic cancer patients, and the development of molecularly targeted diagnostic and therapeutic drugs for pancreatic cancer still faces many difficulties.

[0006] Tumor molecular imaging is a technique for visualizing, characterizing, and measuring tumors at the molecular and cellular levels. By using specific molecular probes and highly sensitive imaging devices, key molecular targets of tumors can be imaged in vivo. Molecular imaging technology can provide non-invasive, real-time, intuitive, and accurate information on the location, volume, and activity of tumors, which is of great significance for early tumor detection, efficacy assessment, and research on drug resistance mechanisms. Positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and various optical imaging methods can all achieve high-precision tumor molecular imaging.

[0007] Based on the above background, we designed a series of molecular imaging probes targeting the integrin receptor αvβ6. Using peptides as molecular targets and linking them to imaging groups via linkers, we constructed molecular imaging probes that precisely target pancreatic cancer cells that highly express αvβ6. These probes use fluorescent groups as imaging groups, enabling imaging at both cellular and in vivo levels. Fluorescence imaging, as a commonly used early screening method, has many advantages, including low cost, ease of operation, and accurate results. Furthermore, specific diagnostic radionuclides can be used as imaging groups, and PET / CT imaging technology can be used for specific tumor imaging. PET / CT imaging technology combines PET and CT imaging techniques and is of great significance in the field of nuclear medicine. First, PET / CT provides high-sensitivity and high-resolution functional and anatomical information, accurately locating tumor lesions and assessing their biological characteristics. Second, PET / CT has systemic detection capabilities, allowing observation of tumor distribution throughout the body, providing a basis for personalized treatment. Therefore, PET / CT can not only achieve precise tumor localization but also perform biological detection, providing precise guidance for the clinical diagnosis and treatment of targeted drugs. In summary, the development of these molecular imaging probes provides scientific evidence and technical support for further preclinical research and drug development.

[0008] Boron neutron capture therapy (BNCT) is a targeted tumor radiotherapy method based on nuclear capture and fission reactions, which can selectively kill tumor cells without damaging normal tissue. Therefore, it is mainly used for advanced and recurrent cancers, such as various malignant primary brain tumors, melanoma, head and neck tumors, liver cancer, bladder cancer, locally recurrent breast cancer, lung cancer, colon cancer, and mesothelioma. However, current clinical trials for BNCT... 10 Only two types of drugs are available for B-cell delivery: BPA and BSH. Although BPA and BSH have shown some efficacy in tumor treatment in clinical trials, they still have significant shortcomings in tumor targeting and uptake rates, which is a major factor limiting the clinical application of BNCT.

[0009] Therefore, the use of highly efficient, visually targeted BNCT drugs for the precise diagnosis and treatment of αvβ6-overexpressing tumors has broad clinical application prospects. A series of HKB molecules designed based on αvβ6-targeting peptides can chelate radioactive metal ions through chelating agent structures in the molecules for tumor imaging. Simultaneously, the boron-containing carrier structure in the molecules can deliver B atoms into tumor cells for further BNCT treatment. Visually targeted BNCT drugs based on targeted peptide design are expected to overcome the limitations of BNCT drugs in clinical application, while meeting the clinical needs of integrated diagnosis and treatment of pancreatic cancer. This can provide a new approach for targeted radiotherapy of tumors, possessing significant scientific research and clinical translational value. Summary of the Invention

[0010] The purpose of this invention is to provide an integrated diagnostic and therapeutic drug based on αvβ6-targeting peptides for positron emission tomography (PET) and boron neutron capture therapy (BNCT), including the design, synthesis, labeling methods, and applications of the drug molecules.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] One of the technical solutions of the present invention is to provide a compound or a pharmaceutically acceptable salt thereof that targets tumor αvβ6, wherein the general structural formula of the compound is shown in Formula I:

[0013]

[0014] in:

[0015] R1 is a chelating group or a fluorescent group; m is 0 or 1; p is 1 or 2; q is 1, 3 or 5; a and b are equal and are 4 or 12; r and s are equal and are 0 or 1.

[0016] Furthermore, when R1 is a chelating group, it is selected from one of the following structures:

[0017]

[0018]

[0019] Preferably, R1 is DOTA:

[0020]

[0021] When R1 is a fluorescent group selected from one of the following structures:

[0022]

[0023]

[0024] Specifically, the compounds of the present invention are selected from the following compounds:

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] The second technical solution of the present invention provides a method for preparing a compound or a pharmaceutically acceptable salt thereof that targets tumor αvβ6, the preparation method comprising the following steps [compounds or pharmaceutically acceptable salts numbered O and C are used as boron-free controls, and the preparation process does not include step (2)]:

[0031] (1) Synthesis of the polypeptide, linker and chelate or fluorescent group conjugate product of Formula II by solid-phase polypeptide synthesis (SPPS):

[0032]

[0033] Where R1 is DOTA or sulfo-Cy5, p = 1 or 2; r = 0 or 1, s = 0 or 1; a = b = 4 or 12.

[0034] The synthesis method is as follows: amino acid condensation is completed on MBHA resin according to the polypeptide sequence using solid phase polypeptide synthesis (SPPS). Then, the Fmoc protecting group at the N-terminus of the polypeptide chain is removed, and the chelating agent DOTA is coupled to the exposed amino group of the polypeptide chain using an amide condensation reaction.

[0035] (2) The compound shown in Formula I or its pharmaceutically acceptable salt is obtained by linking the compound shown in Formula II with the boron-containing support shown in Formula III via an amide condensation reaction:

[0036]

[0037] The value of q is limited to that of claim 1, where q = 1, 3 or 5.

[0038] The third technical solution of the present invention: provides a method for labeling a compound that targets tumor αvβ6 or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0039] The radionuclide eluent was mixed with the compound or its pharmaceutically acceptable salt and incubated. The pH was adjusted to 4–10 with a buffer solution, and the reaction was carried out at 20–110°C for 10 minutes to overnight to obtain the PET tracer targeting tumor integrin αvβ6.

[0040] The buffer solution is a sodium acetate, water, ethanol, phosphate, salt buffer solution, or dimethyl sulfoxide. Preferably, before mixing the labeled precursor with the radionuclide solution, the pH is adjusted to 4.5-5.5 with a buffer solution, wherein the buffer solution is a sodium acetate and water buffer solution, and the incubation conditions are 99°C for 5-15 min.

[0041] The radionuclide is a radionuclide used for diagnosis or treatment, and the radionuclide is selected from... 43 Sc、 44 Sc、 51 Mn, 52 Mn,64 Cu、 67 Ga、 68 Ga、 90 Y、 89 Zr、 99m Tc, 111 ln、 152 Tb, 155 Tb, 201 Tl、 203 Pb, 212 Pb, 213 Bi、 212 Bi、 225 Ac、 211 At、 186 Re、 67 Cu、 188 Re、 177 Lu、 18 F, 76 Br、 77 Br、 123 I, 124 I, 125 I, 89 Zr or 11 At least one of C.

[0042] Fourth technical solution of the present invention: The present invention provides the use of a compound targeting αvβ6 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of one or more αvβ6-mediated diseases;

[0043] Preferably, the disease is selected from tumors, autoimmune diseases, kidney diseases, cardiovascular diseases, inflammation, metabolic / endocrine dysfunction, or neurological diseases; the tumor is one or more tumors expressing αvβ6 or benign tumors, preferably selected from lung cancer, breast cancer, pancreatic cancer, bladder cancer, thyroid cancer, liver cancer, pleural cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, testicular cancer, colon cancer, colorectal cancer, anal cancer, bile duct cancer, gastrointestinal carcinoids, esophageal cancer, gallbladder cancer, rectal cancer, and appendix cancer. Cancer, small intestine cancer, stomach cancer, kidney cancer (i.e., renal cell carcinoma), central nervous system cancer, skin cancer, oral squamous cell carcinoma, cutaneous squamous cell carcinoma, choriocarcinoma, head and neck tumors, bone cancer, osteosarcoma, prosarcoma, neuroblastoma, melanoma, leukemia (such as acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or hairy cell leukemia), lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, B-cell lymphoma, or Burkitt lymphoma), and multiple myeloma.

[0044] Preferably, the treatment is boron neutron capture therapy.

[0045] In other words, the present invention also provides one or more methods for preventing and / or treating αvβ6-mediated diseases, the method comprising administering to a subject in need a preventive and / or therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof.

[0046] The disease is selected from: tumors originating from the central nervous system, preferably gliomas, such as glioblastoma, glial sarcoma, anaplastic astrocytoma, low-grade astrocytoma, pilocytic astrocytoma, oligodendroglioma or brainstem glioma, meningioma, peripheral neuroepithelial tumor, primitive neuroectodermal tumor, neuroblastoma, germ cell tumor, and brain metastases, wherein the tumor is a malignant tumor or a metastatic tumor.

[0047] In some implementations, the subject has pancreatic cancer, such as locally advanced or metastatic pancreatic cancer, locally advanced, unresectable or metastatic pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma (PDAC).

[0048] Fifth technical solution of the present invention: The present invention also provides a diagnostic radionuclide marker targeting αvβ6, wherein the preferred diagnostic method is radionuclide imaging, and more preferably includes PET imaging and SPECT imaging; the preferred treatment is radionuclide therapy, and more preferably includes beta radionuclide therapy and alpha radionuclide therapy.

[0049] The beneficial technical effects of the present invention are as follows:

[0050] This invention links a linear polypeptide, a chelating agent, and a boron-containing carrier together via a linker arm. The synthesized molecule can simultaneously serve as a tracer precursor for PET imaging and a drug molecule for BNCT therapy. Furthermore, the molecule is... 68 Ga labeling exhibits good in vitro and in vivo stability and demonstrates good tumor diagnosis and BNCT treatment effects in mice with high αvβ6 expression, enabling the development of integrated drugs for precision diagnosis and treatment of pancreatic cancer. Attached Figure Description

[0051] Figure 1 This is a schematic diagram illustrating the synthesis of a therapeutic drug based on targeted peptides for boron neutron capture therapy (BNCT).

[0052] Figure 2 Mass spectra of ortho-carborane analogs B1, B2, and B3

[0053] Figure 3 Mass spectra of HKB1-8 and its corresponding CY5 coupling products

[0054] Figure 4 To analyze the affinity of CY5-conjugated HKB-C cells for HKB-9-16 cells using flow cytometry

[0055] Figure 5 Fluorescence imaging images of HKB-10 and 16 subcutaneous tumor-bearing mice.

[0056] Figure 6 for[ 68 Ga]Ga-HKB-O and [ 68 Stability study of Ga]Ga-HKB-1-4,7-8 in PBS, FBS, mouse serum and urine

[0057] Figure 7 for[ 68 Ga]Ga-HKB-O and [ 68 The lipid-water partition coefficient of Ga]Ga-HKB-1-4,7-8

[0058] Figure 8 for[ 68 Ga]Ga-HKB-O and [ 68 PET image of Ga]Ga-HKB-1-8

[0059] Figure 9 The graph shows the BNCT treatment results in HKB-8BxPC-3 subcutaneous tumor-bearing mice, where (A) is the tumor relative volume curve and (B) is the mouse body weight change curve. Detailed Implementation

[0060] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0061] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0062] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0063] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0064] In this invention, "boron neutron capture therapy" (BNCT) refers to a tumor treatment method that includes administering a boron-containing compound to the subject requiring treatment and irradiating the subject with thermal neutrons.

[0065] The treatments mentioned in this invention can be used as standalone treatments or as other conventional surgical or chemotherapy treatments besides boron neutron capture therapy.

[0066] In this invention 68 Ga from 68 Ge / 68 Ga generator (ITM, Germany). Radiometric high-performance liquid chromatography (HPLC) analysis of all radiotracers was performed using an HPLC system (Shimadzu, Japan).

[0067] All animal experiments used in this invention were approved by the Animal Ethics Committee of Nanjing Medical University. All animals were cared for by humans, and experiments were conducted according to the recommendations of the Laboratory Animal Care and Use Committee. The establishment of the tumor-bearing mouse model followed standard procedures. This was achieved through subcutaneous injection of BxPC-3 (1×10⁻⁶). 6 A tumor-bearing Nude-Bulb / c mouse model was established using cells / mice. The tumor volume reached approximately 100-300 mm². 3 Mice were used for PET / CT imaging and biodistribution studies. The BxPC-3 cell line was maintained in a humidified CO2 incubator (37°C, 5% CO2) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Subcultures were performed every two days.

[0068] Example 1

[0069] The overall synthetic route for the αvβ6 targeting molecule is shown below. Figure 1 As shown, the specific synthesis steps are as follows:

[0070] Step 1: Solid-phase synthesis and mass spectrometry identification of peptides

[0071] 1. Resin swelling: Weigh 200mg of MBHA resin, place it in a synthesis tube, add 3-4mL of dichloromethane (DCM), and shake at 200rpm for 30min to allow the resin to swell fully.

[0072] 2. Resin deprotection: Rinse the resin with 5 mL of N,N-dimethylformamide (DMF), then add 4 mL of 20% piperidine solution (piperidine to DMF volume ratio of 1:5), shake at 200 rpm for 30 min, then completely extract the piperidine solution. Repeat this operation once to ensure that the Fmoc protecting group is completely removed.

[0073] 3. Washing: Wash the resin alternately with DMF and DCM, adding 5 mL of washing solution each time, shaking for 30 seconds, repeating 5 times, and then completely extracting the washing solution.

[0074] 4. Amino Acid Condensation: The reactant amounts are precisely calculated based on the MBHA resin loading molar amount (0.39 mmol / g). For conventional amino acid linkage, add the equivalent of 4 equivalents of Fmoc-amino acid, 4 equivalents of O-(7-aminophenyl)-N,N,N',N'-tetramethylchlorourea (HCTU), and 3 equivalents of N,N-diisopropylethylamine (DIPEA). For molecules requiring the linkage of polyethylene glycol (PEG) chains, add 4 equivalents of Fmoc-PEG. a -COOH (where a = 4 or 12), 4 equivalents of HCTU, and 3 equivalents of DIPEA. Dissolve the above reagents in 4 mL of DMF, add to the peptide synthesis tube, and react with shaking at 200 rpm for 2 h. Then, completely withdraw the reaction solution. Repeat the above deprotection, washing, and amino acid ligation steps until the synthesis of the entire peptide or peptide-PEG chain is completed.

[0075] Step Two: Verification of DOTA's Connections and Structure

[0076] 1. Deprotection of peptides: Add 4 mL of 20% piperidine solution to the tube, shake at 200 rpm for 30 min, then completely remove the piperidine solution. Repeat once to ensure that the Fmoc protecting group at the end of the peptide chain is completely removed.

[0077] 2. Washing: Wash the resin with DMF and DCM alternately, adding 5 mL of washing solution each time, shaking for 30 seconds, repeating 5 times, and then completely extracting the washing solution.

[0078] 3. DOTA Condensation: The resin was rinsed with dimethyl sulfoxide (DMSO). Four equivalents of DOTA-3tBu, four equivalents of HCTU, and three equivalents of DIPEA were added to 4 mL of DMSO and then added to the peptide synthesis tube. The mixture was shaken at 200 rpm for 4 hours, after which the reaction solution was completely removed. The resin was washed three times with 5 mL of DCM each time, followed by the addition of 4 mL of methanol and shaking for 1 minute. The resin was then dried under vacuum until completely dehydrated.

[0079] Step 3: Shearing and purification of the peptide-DOTA conjugate product

[0080] 1. Peptide shearing: Add 5-6 mL of lysis buffer (TFA:H2O:TIS volume ratio of 95:2.5:2.5) to the EP tube, seal the tube, and place it on a shaker. React at 200 rpm for 2 h. Filter the shearing system, rinse the resin with an appropriate amount of TFA, collect the filtrate in the EP tube, and distill under reduced pressure to obtain the solid.

[0081] 2. Crude separation of peptides: Add an appropriate amount of methyl tert-butyl ether to the solid. After a white precipitate is formed, sonicate for 30 seconds, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant.

[0082] 3. Peptide structure confirmation: Dissolve a small amount of the precipitate in 1 mL of 20% acetonitrile solution, and use LC-MS to confirm the structure of the peptide-DOTA conjugate product.

[0083] Step 4: Conjugation of BSH derivatives and purification and structural confirmation of HKB series molecules

[0084] 1. Synthesis and structural confirmation of BSH derivatives: 1.0 g of o-borane was weighed and reacted at -78℃ for 1 h in 2 mL of 1.6 M n-butyllithium to introduce a mercapto group, generating a triethylamine salt. 2.0 g of methyl 2-bromoacetate was weighed, dissolved in ethanol, and reacted at 75℃ for 16 h. After adding 15 mL of glacial acetic acid and reacting for 15 min, the reaction was carried out in a 1:1 sulfuric acid aqueous solution for 3 h. The reaction solution was poured into cold water, extracted with n-hexane, and distilled under reduced pressure to obtain the crude product. The crude product was dissolved in water, extracted with petroleum ether, distilled under reduced pressure, washed three times with n-hexane, and filtered to obtain molecule B1 (in the synthetic routes of molecules B2 and B3, methyl 2-bromoacetate was replaced with methyl 4-bromobutyrate and methyl 6-bromohexanoate, respectively, while the rest of the synthetic methods were the same). The structures of molecules B1, B2, and B3 were confirmed by LC-MS.

[0085] 2. Condensation of BSH derivatives with peptides and confirmation of the final product structure: Accurately weigh 5.0 mg of the peptide-DOTA conjugate, 4.8 mg of BSH-COOH, 4.9 mg of N-hydroxysuccinimide (NHS), and 5.4 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), place them in a 1.5 mL EP tube, add 300 μL of DMF to dissolve, and activate the reaction at 200 rpm for 30 min. Add 1 μL of DIPEA to the activated mixture, mix thoroughly, then add the peptide-DOTA conjugate, and react at 200 rpm for 2 h. Purify the reaction system using high-performance liquid chromatography (HPLC), and lyophilize to obtain the final product. Confirm the structure of the product using LC-MS.

[0086] Step 5: Synthesis, purification, and structural identification of CY5-conjugated HKB fluorescent probe molecules

[0087] 1. Synthesis of polypeptide molecules: The synthesis, cleavage, and BSH coupling steps of the polypeptide chain portion of the fluorescent probe molecule are the same as those of the HKB series molecules, and will not be repeated here.

[0088] 2. Deprotection of peptides: Take 150 mg of resin, add it to the peptide synthesis tube, add 4 mL of 20% piperidine solution, shake at 200 rpm for 30 min, and then extract the piperidine solution.

[0089] 3. Washing: Wash the resin with DMF and DCM alternately, adding 5 mL of washing solution each time, shaking for 30 seconds, repeating 5 times, and then completely extracting the washing solution.

[0090] 4. Cy5 condensation: Add 30 mg of NHS-CY5 and 30 μL of DIPEA, dissolve in 4 mL of DMF, and then add the entire solution to the peptide synthesis tube. Incubate in the dark with shaking for 6 hours, then completely remove the reaction mixture. Add 4 mL of methanol, shake for 1 minute, and then dry the resin until completely dehydrated, continuing the dehydration process in the dark. Confirm the structure of the product using LC-MS.

[0091] Example 2

[0092] The molecules prepared in Example 1 (numbered B1, B2, and B3) were analyzed by MS under the following conditions: ion source: ES; nebulizer gas (NEB): 12.00; nitrogen (CUR): 6.00; ion spray voltage (IS): ±4500; temperature (TEM): 0.00; run time: 5 minutes. The mass spectra are shown below. Figure 2 The results showed that the synthesized structure was correct.

[0093] Example 3

[0094] The molecules prepared in Example 1 (numbered B1, B2, B3, and 1-16) were analyzed by MS under the following conditions: ion source: ES; nebulizer gas (NEB): 12.00; nitrogen (CUR): 6.00; ion spray voltage (IS): ±4500; temperature (TEM): 0.00; run time: 10 minutes. The mass spectra are shown below. Figure 3 The results showed that the synthesized structure was correct.

[0095] Example 4

[0096] Flow cytometry was used to analyze the affinity of CY5-coupled molecules (numbered 9-16) for BxPC-3 cells. The specific experimental procedures are as follows:

[0097] 1. Co-incubation of cells and drugs:

[0098] (1) Drug quantification: Prepare a PBS:DMSO buffer solution of 6:1 to dissolve the sample. Take a small amount of CY5-HKB conjugate product and dissolve it in the buffer solution. Use NanoDrop to measure its absorbance at 650 nm and calculate its precise concentration according to Lambert-Beer law.

[0099] (2) Drug incubation: Take two sterile 12-well plates and add 300 μL of 1640 medium to each well; digest one dish of cells from the culture dish and dissolve them in 400 μL of 1640 medium, then add 20 μL of cell suspension to each well and incubate overnight. Before the measurements at 4h, 2h, and 1h, add 10 μL of drug solution to each well and incubate in the dark.

[0100] 2. Prepare samples and perform flow cytometry analysis:

[0101] (1) Cell sample preparation: Aspirate the culture medium containing drug molecules from the 12-well plate, wash three times with PBS, then detach the cells from the plate, centrifuge at 1200 rpm for 5 min, and discard the supernatant. Use 400 μL of serum-free 1640 medium.

[0102] (2) Flow cytometry analysis: Thermo Fisher Scientific flow cytometer was used for analysis. First, the blank control group samples were analyzed to adjust the appropriate voltage and gate. Then, the cell suspensions of each group were analyzed sequentially. The analysis results were processed using FlowJo software. The experimental results are shown below. Figure 4 .

[0103] Cellular affinity assays showed that molecules numbered 9-16 had higher affinity than the control group, with molecules 11 and 16 having the highest affinity.

[0104] Example 5

[0105] The drug was weighed at a dose of 2 mg / kg, 200 μL / mouse, and dissolved in PBS buffer. The drug was administered via tail vein injection, with each drug administered to three mice repeatedly, and the injection time was recorded. Fluorescence imaging of tumor-bearing mice was performed at 1, 2, 4, 8, and 12 hours post-injection. Mice were anesthetized with isoflurane, and after losing mobility, they were placed ventrally upwards in a small animal fluorescence imaging system for fluorescence imaging. Image acquisition, processing, and fluorescence intensity quantification were performed using OI-IMAGEIVIS software. All images were acquired under the same lighting settings and software parameters. Experimental results are as follows: Figure 5 .

[0106] Example 6

[0107] Will 68 Ga-labeled HKB molecules were incubated for 4 hours on a shaker in PBS, FBS, mouse serum, and mouse urine at 37°C. 20 μL samples were collected at different time intervals and analyzed by RP-HPLC. 68Radiochemical purity of Ga-HKB series molecules was determined under the following conditions: column: YMC-tria-C18; mobile phase gradient: mobile phase A, 0.1% aqueous trifluoroacetic acid (TFA); mobile phase B, acetonitrile (0.1% TFA); flow time: 15 min; mobile phase B from 20% to 90%; flow rate: 1.0 mL / min. Results are shown below. Figure 4 From this, we can calculate [ 68 The Ga-HKB series molecules exhibit good stability in PBS, FBS, mouse serum, and mouse urine, with a radioactivity purity greater than 99%. Experimental results are as follows: Figure 6 .

[0108] Example 7

[0109] 68 The specific method for determining the lipid-water partition coefficient of Ga-labeled HKB series molecules is as follows:

[0110] Take 3.7MBq 68 Ga-labeled molecules 1-8 were added to an equal volume mixture of 2 mL of n-octanol and water. After thorough mixing, the mixture was allowed to stand at room temperature for 30 minutes. Once complete separation of the two phases was observed, the n-octanol phase and the aqueous phase were separately aspirated, and their radioactivity was measured using a gamma counter. The lipid-water partition coefficients were then calculated. The results are as follows: Figure 7

[0111] Example 8

[0112] 68 In vivo PET imaging study of Ga-labeled HKB series molecules in tumor-bearing mice

[0113] PET experiments were performed using Inveon Micro-PET / CT (Siemens Medical Solutions, Knoxville, Munich, Germany). Each mouse was injected intravenously via tail vein with 3.7 MBq dissolved in 100 μL of saline. 68 Ga]Ga-HKB. Static PET images were acquired at 0.5 hours, 1 hour, 2 hours, and 4 hours post-injection. Results are shown in […]. Figure 8 .

[0114] Example 9

[0115] HKB-8BxPC-3 subcutaneous tumor-bearing mouse BNCT treatment experiment

[0116] Mice were randomly divided into a blank control group, a neutron irradiation group, and a treatment group, with 6 mice in each group. The blank control group and the neutron irradiation group received a tail vein injection of 100 μL of physiological saline, while the treatment group received a tail vein injection of 100 μL of 10.0 mg / mL HKB-8 physiological saline solution. Forty-eight hours later, the neutron irradiation group and the treatment group underwent neutron irradiation, which continued for 7 days before being discontinued. Tumor size and body weight were measured daily from the irradiation date. Results showed that tumor growth in the treatment group was significantly inhibited compared to the blank control group and the neutron irradiation group. Simultaneously, no significant difference in body weight was observed among the groups. Figure 9 .

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, in: R1 is a chelating group or a fluorescent group; m is 0 or 1; p is 1 or 2; q is 1, 3 or 5; a and b are equal and are 4 or 12; r and s are equal and are 0 or 1.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, R1 is a chelating group selected from one of the following structures; 3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, R1 is a fluorescent group selected from one of the following structures; 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, In Equation I, m = 0, p = 1, r = s = 0; m = 1, p = 1, q = 1 or 3 or 5, r = s = 0; m = 1, p = 1, q = 1, r = s = 0 or 1, a = b = 4 or 12; Or m = 1, p = 2, q = 1, r = s = 0.

5. The compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from...

6. A method for preparing the compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, for the preparation of the compound of formula I, comprising the following steps [compounds numbered O and C are used as boron-free controls, and the preparation process does not include step (2)]: (1) Synthesis of the polypeptide, linker and chelate or fluorescent group conjugate product of Formula II by solid-phase polypeptide synthesis (SPPS): in, R1 is DOTA or sulfo-Cy5, p = 1 or 2; r = 0 or 1, s = 0 or 1; a = b = 4 or 12; (2) The compound shown in Formula I or its pharmaceutically acceptable salt is obtained by linking the compound shown in Formula II with the boron-containing support shown in Formula III via an amide coupling reaction: The value of q is limited to that of claim 1, where q = 1, 3 or 5.

7. A diagnostic radionuclide marker, comprising a radionuclide-labeled compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein, The radionuclide is a diagnostic or therapeutic radionuclide selected from... 43 Sc、 44 Sc、 51 Mn, 52 Mn, 64 Cu、 67 Ga、 68 Ga、 90 Y、 89 Zr、 99m Tc, 111 ln、 152 Tb, 155 Tb, 201 Tl、 203 Pb, 212 Pb, 213 Bi、 212 Bi、 225 Ac、 211 At、 186 Re、 67 Cu、 188 Re、 177 Lu、 18 F, 76 Br、 77 Br、 123 I, 124 I, 125 I, 89 Zr or 11 At least one of C.

8. The diagnostic radionuclide marker according to claim 7, wherein, The radionuclide labeling method involves eluting the radionuclide eluent, mixing it with the compound or a pharmaceutically acceptable salt of any one of claims 1-5, adjusting the pH to 4-10 using a buffer solution, wherein the buffer solution is sodium acetate, water, ethanol, phosphate, salt buffer solution or dimethyl sulfoxide; and reacting at 20-110°C for 10 minutes to overnight.

9. Use of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a boron neutron capture therapeutic agent for treating tumors.

10. The application according to claim 9, wherein the tumor is selected from: tumors originating from the central nervous system, preferably gliomas, such as glioblastoma, glial sarcoma, anaplastic astrocytoma, low-grade astrocytoma, pilocytic astrocytoma, oligodendroglioma or brainstem glioma, meningioma, peripheral neuroepithelial tumor, primitive neuroectodermal tumor, neuroblastoma, germ cell tumor, brain metastasis, wherein the tumor is a malignant tumor or a metastatic tumor.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable diluent, carrier, and / or excipient.

12. The use of the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof or at least one diagnostic radionuclide marker of claim 7 or the pharmaceutical composition of claim 11 in the preparation of a medicament for the prevention, diagnosis and / or treatment of αvβ6-mediated diseases.

13. The application according to claim 12, characterized in that: The αvβ6-mediated diseases include: one or more tumors with high αvβ6 expression, autoimmune diseases, kidney diseases, cardiovascular diseases, inflammation, metabolic / endocrine disorders, or neurological diseases; the one or more tumors with high αvβ6 expression are selected from lung cancer, breast cancer, pancreatic cancer, bladder cancer, thyroid cancer, liver cancer, pleural cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, testicular cancer, colon cancer, colorectal cancer, anal cancer, bile duct cancer, gastrointestinal carcinoids, esophageal cancer, gallbladder cancer, rectal cancer, appendix cancer, small intestine cancer, gastric cancer, kidney cancer (i.e., renal cell carcinoma), central nervous system cancers, and skin cancers. Cancer, oral squamous cell carcinoma, cutaneous squamous cell carcinoma, choriocarcinoma, head and neck tumors, bone cancer, osteosarcoma, anterior sarcoma, neuroblastoma, melanoma, leukemia (such as acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or hairy cell leukemia), lymphoma (such as non-Hodgkin lymphoma, Hodgkin lymphoma, B-cell lymphoma, or Burkitt lymphoma), and multiple myeloma; in some implementations, the subject has pancreatic cancer, such as locally advanced or metastatic pancreatic cancer, locally advanced, unresectable or metastatic pancreatic adenocarcinoma, or pancreatic ductal adenocarcinoma (PDAC).

14. The application according to claim 12, characterized in that, The diagnostic method is radionuclide imaging, and more preferably includes PET imaging and SPECT imaging; The treatment is selected from radionuclide therapy or boron neutron capture therapy, wherein radionuclide therapy further preferably includes beta-nucleoside therapy and alpha-nucleoside therapy.

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