Targeted reversible covalent ligand, radiopharmaceutical and preparation method and application thereof

Abstract

The invention relates to the technical field of radiopharmaceuticals, and discloses a targeted reversible covalent ligand which comprises a targeted pharmacodynamic group (P), a reversible covalent connection bullet (RCL) and a combined radionuclide group (C), and the targeted reversible covalent ligand has a structural general formula: C-RCL-P, the reversible covalent linked bullet (RCL) is a thiol group-containing similar group linked bullet which can be reversibly combined with a disulfide bond in a target protein, and preferably comprises one of GSH, Lys-Cys, Cys, SeC, GCys and GSECys linkers. The target reversible covalent radiopharmaceutical has the following technical effects that the target reversible covalent radiopharmaceutical constructed by adopting the target reversible covalent ligand containing the RCL has excellent in-vivo pharmacokinetic characteristics, and can increase lesion uptake, lesion radioactivity residence time and imaging contrast, reduce liver and kidney uptake and reduce liver and kidney toxicity. The invention also discloses a targeted reversible covalent radiopharmaceutical as well as a preparation method and application thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of radiopharmaceuticals, and particularly relates to a targeted reversible covalent ligand, a radiopharmaceutical, and a preparation method and application thereof. BACKGROUND

[0002] The key to reducing the mortality rate of malignant tumors depends on early diagnosis, specific targeted molecular therapy, and precise visualized diagnosis and treatment technology for guiding treatment. Medical imaging technologies such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) have very important value in the diagnosis and treatment of malignant tumors, but their sensitivity and specificity still need to be improved. How to establish a new technology of specific targeted visualized imaging and precise treatment guidance has become a key problem in the current precise targeted diagnosis and treatment of malignant tumors. A pair of different radionuclides for imaging and a pair of different radionuclides for therapy are used to label the same or similar specific targeting ligand, to establish a nuclear medicine diagnosis and treatment integration technology, to perform molecular imaging diagnosis and guide internal radiotherapy targeted molecular therapy for the same disease, to play an important role in tumor nuclear medicine diagnosis and treatment, and to be expected to solve the key problem of precise targeted diagnosis and treatment of malignant tumors. However, the difficulty lies in developing a targeted covalent radiopharmaceutical (radiopharmaceutical is referred to as a radiopharmaceutical) with high tumor uptake and long retention time, and excellent in vivo pharmacokinetic properties for tumor specific molecular targets.

[0003] Covalent inhibitors, also known as targeted covalent inhibitors, are usually composed of a targeting pharmacophore and a covalent linker bullet, including targeted irreversible covalent inhibitors (ICIs) and targeted reversible covalent inhibitors (RCIs). The reaction of the covalent linker bullet and the target protein amino acid residue of the targeted irreversible covalent inhibitor (ICI) is permanent and irreversible, like glue that permanently bonds the affinity covalent linker bullet and the target together to form an inhibitor. The reaction of the covalent linker bullet and the target protein amino acid residue of the targeted reversible covalent inhibitor (RCI) is temporary and reversible, like a reversible “living buckle” connected between the covalent linker bullet and the target protein to form an inhibitor. The targeted reversible covalent inhibitor does not permanently bind to the target and off-target proteins of normal tissues, but can be released from the off-target proteins of normal tissues, thereby reducing the risk of undesired activation of the immune system and off-target toxicity of the targeted irreversible covalent inhibitor.

[0004] The targeting covalent drugs include targeting irreversible covalent drugs and targeting reversible covalent drugs, both of which have the biological functions and characteristics of ICI and RCI, respectively. In the prior art, the targeting FAP irreversible covalent drugs with the characteristics of ICI have been successfully developed, which significantly improves the existing targeting FAP drug tumor uptake and residence time, but may have off-target phenomena similar to ICI, and the pharmacokinetic characteristics thereof need to be improved. However, there is no literature report on the targeting reversible covalent drugs with the characteristics of RCI. If the targeting reversible covalent drugs with the characteristics of RCI can be developed, the problems existing in the existing targeting covalent drugs can be effectively solved, which has great clinical transformation significance. SUMMARY

[0005] In order to solve the problems of the prior art, the present application provides a targeting reversible covalent ligand, which adopts a reversible covalent linker bullet to couple a targeting efficacy group and a radionuclide group. The reversible covalent linker bullet can reversibly covalently react with a target protein, can increase lesion uptake and radioactive residence time, enhance imaging contrast, and effectively improve the in vivo pharmacokinetic characteristics of the targeting reversible covalent radioactive drug. The present application also provides a targeting reversible covalent radioactive drug, a preparation method and application thereof. The technical effects achieved by the present application are realized through the following technical aspects: In a first aspect, the present application provides a targeting reversible covalent ligand, which is composed of a targeting efficacy group (P) targeting a lesion tissue-specific target, a reversible covalent linker bullet (RCL), and a radionuclide group (C). The reversible covalent linker bullet (RCL) is used to couple the targeting efficacy group (P) and the radionuclide group (C). The targeting reversible covalent ligand has the following general structure: (C-RCL-P); The reversible covalent linker bullet (RCL) is a thiol-containing analog group linker bullet capable of reversibly combining with a disulfide bond in a target protein, and preferably includes one of GSH (glutathione), Lys-Cys (lysyl cysteine), Cys (cysteine), SeC (selenocysteine), GCys (glycyl cysteine), and GSeCys (glycyl selenocysteine) linkers.

[0006] Disulfide bonds (SS) are chemical bonds that link different peptide chains or two cysteine ​​residues within the same peptide chain in a protein molecule. Most target proteins contain disulfide bonds (SS). The rearrangement of disulfide bonds in proteins usually occurs through an exchange reaction between thiols and disulfide groups within the protein. The sulfides of cysteine ​​residues attack the disulfide bonds in the protein, thereby promoting the exchange reaction between the disulfide bonds and thiols. More importantly, the exchange reaction between disulfide bonds and thiols is reversible. For example, acetylcysteine, used as a mucolytic agent, contains thiol groups in its molecule that can break the disulfide bonds in glycoprotein polypeptide chains in sputum, thereby reducing the viscosity of the sputum and making it easier to cough up. It can also break DNA fibers in purulent sputum. The above-mentioned mechanism of action of acetylcysteine ​​further confirms the existence of the reversible exchange reaction between thiols and disulfide bonds. Since GSH, Lys-Cys, Cys, and GCys all contain cysteine ​​residues, while SeC and GSeCys contain selenocysteine ​​residues, and selenocysteine, as an analogue of cysteine, can also undergo reversible covalent reactions with disulfide bonds in proteins, GSH, Lys-Cys, Cys, SeC, GCys, and GSeCys are all important linkers for constructing targeted reversible covalent radiopharmaceuticals.

[0007] Preferably, the reversible covalent linker (RCL) is GSH (glutathione, a hydrophilic reversible covalent linker containing thiol and carboxyl groups). GSH, containing thiol and carboxyl groups, is an excellent hydrophilic linker that can undergo reversible covalent reactions with disulfide bonds in target proteins, possesses antioxidant properties, and improves pharmacokinetic characteristics. Hydrophilic targeted reversible covalent radiopharmaceuticals constructed using GSH-containing targeted reversible covalent ligands exhibit excellent in vivo pharmacokinetic characteristics, increasing lesion uptake, lesion radioactive retention time, and imaging contrast, while reducing hepatic and renal uptake and toxicity. This is highly beneficial for the application of targeted reversible covalent radiopharmaceuticals in the diagnosis and treatment of tumors and cardiovascular diseases.

[0008] As a further description of the technical solution of the present invention, the GSH connector and the Lys-Cys connector respectively have the following structural formulas: , ; The Cys connector and the SeC connector each have the following structural formulas: , ; The GCys connector and the GSeCys connector each have the following structural formulas: , .

[0009] As a further description of the technical solution of the present invention, the targeted pharmacophore (P) preferably includes a pharmacophore (FAPI) targeting fibroblast activation proteins and a targeted integrin α. v Pharmacodynamic group (α) of β6 receptor v One of the following: β6L), a pharmacophore targeting human epidermal growth factor receptor 2 (HER2) (DH6), a pharmacophore targeting glucose-dependent insulinotropic peptide receptor (GIPR) (EG4), a pharmacophore targeting somatostatin receptor (JR11), a pharmacophore targeting myeloperoxidase (MPO) (AVP), and glutathione (GSH) targeting glutathione peroxidase.

[0010] As a further description of the technical solution of the present invention, the radioactive nuclide group (C) preferably includes one of the chelating agents 1,4,7-triazacyclononadecanyl-N',N''-diacetyl-N-acetyl (NOTA) and 1,4,7,10-tetraazacyclododecane-N',N'',N'''-triacetyl-N-acetyl (DOTA).

[0011] Preferably, the targeted reversible covalent ligand C-RCL-P includes a targeted FAP reversible covalent ligand and a targeted α-ligand. v Reversible covalent ligands targeting β6, HER2, glutathione peroxidase, SSTR2, MPO, and GIPR are among the reversible covalent ligands targeted. Representative reversible covalent ligands and their structural formulas are as follows: NOTA-GSH-FAPT and DOTA-GSH-FAPT have the following structures: , ; NOTA-NGSH-FAPT2 has the following structural formula: ; NOTA-GSH-FAPT2 and DOTA-GSH-FAPT2 have the following structural formulas: , ; NOTA-Lys-Cys-FAPT and DOTA-Lys-Cys-FAPT have the following structures: , ; NOTA-Cys-FAPT and DOTA-Cys-FAPT have the following structural formulas respectively: , ; NOTA-GCys-FAPT and DOTA-GCys-FAPT have the following structures: , ; NOTA-FAPTPS has the following structure: ; NOTA-GSH-α v β6L has the following structural formula: ; NOTA-GSH-DH6 has the following structure: ; NOTA-GSH-cH6T has the following structural formula: ; NOTA-GSH-FSK-DH6 has the following structure: ; NOTA-GSH has the following structure: ; NOTA-GSH-JR11 has the following structure: ; NOTA-GSH-BSO3-cJR11T2 has the following structural formula: ; NOTA-GSH-AVP and NOTA-GSH-cAVP have the following structures: , ; NOTA-GSH-EG4 has the following structure: ; NOTA-SeC-FAPT and NOTA-GSeCys-FAPT have the following structural formulas respectively: , ; NOTA-GSeH-FAPT has the following structural formula: .

[0012] Secondly, the present invention provides a targeted reversible covalent radiopharmaceutical, comprising a radionuclide (*R) and a targeted reversible covalent ligand (C-RCL-P) as described in any one of claims 1-4, wherein the targeted reversible covalent radiopharmaceutical has the general structural formula: *RC-RCL-P. Specifically, the targeted FAP reversible covalent radiopharmaceutical is represented as *RC-RCL-FAPI; the targeted integrin α... v β6 receptor reversible covalent radiopharmaceuticals are represented as *RC-RCL-α v β6L; reversible covalent radiopharmaceuticals targeting HER2 are represented as *RC-RCL-DH6; reversible covalent radiopharmaceuticals targeting GIPR are represented as *RC-RCL-EG4; reversible covalent radiopharmaceuticals targeting GPC3 are represented as *RC-RCL-GPC3P; reversible covalent radiopharmaceuticals targeting SSTR are represented as *RC-RCL-JR11; reversible covalent radiopharmaceuticals targeting MPO are represented as *RC-RCL-AVP; reversible covalent radiopharmaceuticals targeting glutathione peroxidase are represented as *RC-GSH.

[0013] The mechanism of action of the targeted reversible covalent radiopharmaceutical *RC-RCL-P includes two steps: First, the targeted pharmacophore (P) specifically binds reversibly and non-covalently to the target protein in the lesion, transporting the reversibly covalently linked bullet (RCL) and the radionuclide-bound group (C) containing the radionuclide (*R) to the lesion; then, the RCL forms a reversible covalent bond with the reactive amino acid residues of the adjacent target protein, forming reversible non-covalent binders and reversible covalent adducts with the target protein in the lesion, thereby enabling the targeted reversible covalent radiopharmaceutical to achieve higher radioactive uptake and longer retention time in the lesion, thus achieving highly specific and highly sensitive radionuclide diagnosis and treatment.

[0014] As a further description of the technical solution of the present invention, the radioactive nuclide (*R) includes 18 F, 68 Ga、 111 In、 99m Tc, 186 Re、 188 Re、 175 Yb、 153 Sm、 166 Ho、 88 Y、 90 Y、 177 Lu、 47 Sc、 212 Bi、 213 Bi、 123 I, 124 I, 131 I, 211 At、153 Eu、 169 Eu、 212 Pb, 64 Cu、 67 Cu、 198 Au、 223 Ra、 225 Ac and 227 One of Th, preferably 18 F, 68 Ga、 177 Lu and 64 One of the types of Cu.

[0015] Thirdly, the present invention provides a method for preparing the targeted reversible covalent ligand, comprising the following steps: The targeted pharmacophore (P) is modified by reversibly covalently linking a bullet head (RCL) to obtain the RCL-P ligand; the RCL-P ligand is linked to bind a radioactive nuclide group (C) to obtain the targeted reversible covalent ligand C-RCL-P.

[0016] Fourthly, the present invention provides a method for preparing the targeted reversible covalent radiopharmaceutical, comprising the following steps: Using the targeted reversible covalent ligand C-RCL-P as a precursor, a chelation reaction was carried out with radionuclide ions. After separation and purification by a small column, the targeted reversible covalent radiopharmaceutical *RC-RCL-P was obtained.

[0017] As a further description of the technical solution of the present invention, the radioactive nuclide ions include 18 F, 68 Ga、 111 In、 99m Tc, 186 Re、 188 Re、 175 Yb、 153 Sm、 166 Ho、 88 Y、 90 Y、 177 Lu、 47 Sc、 212 Bi、 213 Bi、 123 I, 124 I, 131 I, 211 At、 153 Eu、 169 Eu、 212 Pb, 64 Cu、 67 Cu、 198 Au、 223 Ra、 225 Ac and 227One of the ions formed in Th, preferably 18 F - or [Al] 18 F] 2+ (abbreviated as) 18 F-AlF), 68 Ga 3+ , 64 Cu 2+ and 177 Lu 3+ One of them.

[0018] Preferably, NOA-RCL-P is used as a precursor, reacting with radioactive metal nuclide ions [Al]. 18 F] 2+ , 68 Ga 3+ and 177 Lu 3+ After undergoing chelation reactions and purification by column chromatography, the products were prepared from the individual chelate groups. 18 F, 68 Ga and 177 The specific reaction process and synthetic route for the Lu-labeled targeted reversible covalent radiopharmaceutical (*R-NOTA-RCL-P) are as follows: .

[0019] Preferably, DOTA-RCL-P is used as a precursor material, reacting with radioactive metal nuclide ions. 68 Ga 3+ , 64 Cu 2+ and 177 Lu 3+ After undergoing chelation reactions and purification by column chromatography, the products were prepared from the individual chelate groups. 68 Ga 3+ , 64 Cu 2+ and 177 Lu 3+ The specific reaction process and synthetic route for the labeled, targeted, reversible covalent radiopharmaceutical (*R-DOTA-RCL-P) are as follows: .

[0020] Fifthly, the present invention provides the application of the aforementioned targeted reversible covalent radiopharmaceutical, including: The application of the targeted reversible covalent radiopharmaceutical (especially the targeted reversible covalent radiopharmaceutical labeled with positron-emitting radionuclides) in the preparation of PET imaging agents for lung cancer, liver cancer, breast cancer, melanoma, lymphoma, glioma, digestive system tumors, neuroendocrine tumors, prostate cancer, thyroid cancer and other tumors and diseases (such as cardiovascular and cerebrovascular diseases); The application of the targeted reversible covalent radiopharmaceutical (especially the targeted reversible covalent radiopharmaceutical labeled with single-photon emission radionuclides) in the preparation of SPECT imaging agents for lung cancer, liver cancer, breast cancer, melanoma, lymphoma, glioma, digestive system tumors, neuroendocrine tumors, prostate cancer, thyroid cancer and other tumors and diseases (such as cardiovascular and cerebrovascular diseases). The application of the targeted reversible covalent radiopharmaceutical in the preparation of diagnostic and therapeutic agents for lung cancer, liver cancer, breast cancer, melanoma, lymphoma, glioma, digestive system tumors, neuroendocrine tumors, prostate cancer, thyroid cancer, and other tumors and diseases (such as cardiovascular and cerebrovascular diseases).

[0021] In summary, the present invention has at least the following advantages: 1. The targeted reversible covalent ligand provided by this invention employs a reversible covalently linked bullet containing a thiol-like group that can reversibly bind to disulfide bonds in target proteins to connect the targeted pharmacophore group and the radioactive nuclide group. The thiol group in the reversible covalently linked bullet can undergo a reversible covalent reaction with the disulfide bonds in the target protein, which can significantly increase lesion uptake, prolong the lesion radioactive retention time, and enhance imaging contrast. Simultaneously, it can reduce hepatic and renal uptake and reduce hepatorenal toxicity. The targeted reversible covalent radiopharmaceutical constructed using the targeted reversible covalent ligand of this invention exhibits excellent in vivo pharmacokinetic characteristics, overcoming the shortcomings of existing targeted covalent radiopharmaceuticals, and has significant application value in the diagnosis and treatment of tumors and cardiovascular diseases.

[0022] 2. The targeted reversible covalent radiopharmaceutical provided by this invention can specifically and reversibly non-covalently bind to target proteins in lesions via a targeted pharmacophore group. This transports the reversibly covalently linked bullet and the radionuclide-labeled binding radionuclide group to the lesion. Furthermore, the reversibly covalently linked bullet forms reversible covalent bonds with reactive amino acid residues of adjacent target proteins. Through this dual targeting effect of reversible non-covalent binding and reversible covalent binding, the radioactive uptake and retention time of the targeted reversible covalent radiopharmaceutical in the lesion can be effectively increased, thereby significantly improving imaging contrast. Simultaneously, it can effectively reduce hepatic and renal uptake and toxicity, achieving the goal of highly specific and sensitive radionuclide diagnosis and treatment.

[0023] 3. The method for preparing targeted reversible covalent radiopharmaceuticals provided by this invention uses the targeted reversible covalent ligand C-RCL-P as a precursor, which undergoes a chelation reaction with radioactive nuclide ions. After separation and purification by a microcolumn, the targeted reversible covalent radiopharmaceutical *RC-RCL-P can be obtained. The preparation process is simple and easy to operate, and the prepared targeted reversible covalent radiopharmaceutical has high yield and high purity, and can be automated to meet the needs of scientific research and clinical nuclear medicine diagnosis and treatment. In addition, the precursor C-RCL-P can be prepared by a combination of chelating agent modification, organic synthesis and peptide synthesis, which can effectively solve the problem of precursor source.

[0024] 4. The targeted reversible covalent radiopharmaceutical provided by this invention can be widely used in nuclear medicine imaging or radionuclide therapy for various solid tumors such as lung cancer, brain tumors, gastric cancer, colorectal cancer, prostate cancer, neuroendocrine tumors, liver cancer, pancreatic cancer, thyroid cancer, and breast cancer. It can also be further applied to the evaluation of the efficacy of nuclear medicine imaging for the above-mentioned solid tumors, and has broad application prospects. Attached Figure Description

[0025] Figure 1 The diagram shows the molecular docking of NOTA-GSH-FAPT (A), DOTA-GSH-FAPT (B), NOTA-GCys-FAPT (C), and DOTA-GCys-FAPT (D) with FAP protein (FAP protein PDB encoding: 1Z68), respectively. Figure 2 For Nota-GSH-α v β6L(A) and DOTA-GSH-α v β6L(B) and α v β6 protein (α) v A schematic diagram of molecular docking of the β6 protein PDB encoding: 4UM8; Figure 3 Schematic diagrams of the molecular docking of NOTA-GSH-DH6 (A) and DOTA-GSH-DH6 (B) with the HER2 protein, respectively; Figure 4 The diagram shows the molecular docking of Nota-GSH-EG4 (A) and DOTA-GSH-EG4 (B) with the GIPR protein (GIPR protein PDB encoding: 7FIY), respectively. Figure 5 The diagrams show the molecular docking of Nota-GSH-JR11 (A) and DOTA-GSH-JR11 (B) with the SSTR2 protein (SSTR2 protein PDB encoding: 7XAU), respectively. Figure 6The diagram shows the molecular docking of Nota-GSH-AVP (A) and DOTA-GSH-AVP (B) with MPO protein (MPO protein PDB encoding: 3F9P), respectively. Figure 7 A representative UV-HPLC chromatogram of the NOTA-GSH-FAPT precursor (A); 18 Representative radiometric HPLC chromatograms of F-GSH-FAPT product (B), radiometric HPLC chromatograms in PBS buffer at 2 h in vitro (C), and radiometric HPLC chromatograms in serum at 2 h (D); 18 The radioactive HPLC analysis chromatograms (E) of F-GSH-FAPT product in urine at 1 hour in vivo, and the radioactive HPLC analysis chromatograms (F) of F-GSH-FAPT product in serum at 1 hour in vivo; 18 F]F-GSH-FAPT product's binding rate to mouse serum albumin (G); 18 Ester-water distribution coefficient (H) of F]F-GSH-FAPT; Figure 8 For in vitro experimental results and model animals [ 18 F]AlF-GSH-α v β6L PET imaging results: [ 18 F]AlF-GSH-α v Representative radiometric HPLC chromatograms of β6L injection and radiometric HPLC chromatograms at 2 h in vitro with PBS buffer, 2 h in serum, and 1 h in mice (A); 18 F]AlF-GSH-α v Schematic diagram of β6L in vitro cell experiment results (B); 18 F]AlF-GSH-α v β6L, [ 18 F]AlF-Asp2-α v β6L and [ 18 F]AlF-Glc-α v Schematic diagram of PET imaging results of β6L in Capan-2 tumor-bearing mice (C); 18 F]AlF-GSH-α v Biodistribution of β6L in Capan-2 tumor-bearing mice (D); Figure 9 For in vitro experimental results and model animals 18 PET imaging results of F-GSH-JR11: HPLC UV chromatogram of NOTA-GSH-JR11 (A); 18 Radio-HPLC chromatogram of F-GSH-JR11 (B); 18Radio-HPLC chromatogram of F-GSH-JR11 in vitro with PBS for 2 h (C); 18 Radio-HPLC chromatogram of F-GSH-JR11 in FBS for 2 h (D); 18 Radio-HPLC chromatogram of F-GSH-JR11 in vivo blood 1 h (E). 18 Schematic diagram of the results of the F-GSH-JR11 uptake and inhibition experiment in AR42J cells (F); 18 Dynamic Micro-PET images of F-GSH-JR11 in AR42J model mice over 120 min and their corresponding time-activity curves (G). Figure 10 For in vitro experimental results and model animals 18 Schematic diagram of F-GSH-DH6 PET imaging results; Figure 11 For in vitro experimental results and model animals 18 A schematic diagram of the PET imaging results of F-GSH-EG4 (A) and a 60-minute static Micro-PET imaging image of the HT29 model mouse (B); Figure 12 A schematic diagram showing the results of the competitive inhibition experiment of Nota-GSH-FAPT and Nota-FAPI-42 on A549-FAP cells; Figure 13 This is a schematic diagram of the reversible covalent determination results of NOA-GSH-FAPT. Figure 14 For model animals [ 18 Schematic diagram of F]F-GSH-FAPT PET imaging and biodistribution results; Figure 15 for[ 18 Schematic diagram of HPLC determination results for F]F-NOTA-GSH injection (A and B); Various tumor-bearing animal models [ 18 Schematic diagram of F-NOTA-GSH PET imaging results (C and D); and a tumor-bearing model of the same organ. 68 Ga]Ga-NOTA-GSH-FAPT and [ 68 Ga]Ga-DOTA-GSH-FAPT, [ 18 F]AlF-NOTA-GSH-FAPT and [ 18 F] Schematic diagram of AlF-NOTA-Lys-Cys-FAPT imaging comparison results (E); Figure 16 For the same patient [ 18 F]F-GSH-FAPT and 18Schematic diagram of F-FDG PET imaging results. Detailed Implementation

[0026] To facilitate understanding of the present invention, a more comprehensive description will be provided below with reference to specific embodiments and accompanying drawings. Preferred embodiments of the invention are shown in the embodiments; however, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0027] The use of terms such as "one embodiment," "implementation," and "exemplary embodiment" in the specification means a described implementation, but not every implementation may include specific features, structures, or characteristics, including specific features, structures, or characteristics. Furthermore, such phrases do not necessarily refer to the same implementation. Additionally, when a specific feature, structure, or characteristic is described in connection with certain implementations, whether explicitly described or not, the effect of applying that feature, structure, or characteristic to other implementations is within the knowledge of those skilled in the art.

[0028] In the following embodiments, the structural description of the targeted reversible covalent ligand may omit the binding radionuclide group to avoid making the structural description too lengthy. For example, the targeted FAP reversible covalent ligand 18 F-NOTA-LP, also written as 18 FLP. Additionally, due to space limitations, it is not provided in the examples. 89 Zr、 99m Tc, 111 In、 90 Y or 188 Specific examples of targeted reversible covalent radiopharmaceuticals labeled with radionuclides such as Re can be found in the specific embodiments provided in this application for preparation by those skilled in the art. 89 Zr、 99m Tc, 111 In、 90 Y or 188 Targeted reversible covalent radiopharmaceuticals labeled with radionuclides such as Re.

[0029] Example 1: Molecular Docking Experiment Using Autocock Vina software, Nota-GSH-FAPT, DOTA-GSH-FAPT, Nota-GCys-FAPT, and DOTA-GCys-FAPT were docked with the FAP protein (PDB number: 1Z68), and the docking was visualized using PyMol software (e.g., ...). Figure 1 (As shown in AD). Compared to NOTA-GCys-FAPT, NOTA-GSH-FAPT exhibits a superior binding energy of -10.5 kcal / mol, and the ligand is linked to the protein by 7 hydrogen bonds; compared to DOTA-GCys-FAPT, DOTA-GSH-FAPT exhibits a superior binding energy of -11.2 kcal / mol, and the ligand is linked to the protein by 7 hydrogen bonds.

[0030] Use Autocock Vina software to modify Nota-GSH-α v β6L and DOTA-GSH-α v β6L and α v Docking was performed on β6 protein (PDB ID: 4UM8) and the docking was visualized using PyMol software (e.g., ...). Figure 2 (As shown in AB). NOTA-GSH-α v β6L and DOTA-GSH-α v β6L and α v The minimum binding energies of the β6 protein are -8.7 kcal / mol and -8.1 kcal / mol, respectively, and the ligands and proteins are connected by 14 and 12 hydrogen bonds, respectively, exhibiting high affinity.

[0031] Using Autocock Vina software, Nota-GSH-DH6 and DOTA-GSH-DH6 were docked with the HER2 protein (PDB number: 5TDN), and the docking was visualized using PyMol software (e.g., ...). Figure 3 (As shown in AB). The minimum binding energies of NOTA-GSH-DH6 and DOTA-GSH-DH6 to HER2 protein are -8.3 kcal / mol and -7.4 kcal / mol, respectively, and the ligands and proteins are connected by 9 and 6 hydrogen bonds, respectively, exhibiting high affinity.

[0032] Molecular docking results of NOTA-GSH-EG4 with GIPR protein show (e.g.) Figure 4 As shown in Figure A), the binding energy is -11.8 kcal / mol, and it forms 8 hydrogen bonds with the GIPR protein, indicating a high affinity for GIPR. The molecular docking results of DOTA-GSH-EG4 with the GIPR protein show (e.g., ...). Figure 4(As shown in B), the binding energy is -11.3 kcal / mol, and it forms 2 hydrogen bonds with the GIPR protein, indicating that it has general GIPR affinity.

[0033] Molecular docking results of NOTA-GSH-JR11 with SSTR2 protein showed (e.g.) Figure 5 As shown in Figure A), the binding energy is -8.6 kcal / mol, and it forms 10 hydrogen bonds with the SSTR2 protein, indicating a high affinity for SSTR2. The molecular docking results of DOTA-GSH-JR11 with the SSTR2 protein show (e.g., ...). Figure 5 (As shown in B), the binding energy is -9.5 kcal / mol, and it forms 10 hydrogen bonds with the SSTR2 protein, indicating a high affinity for SSTR2.

[0034] Using Autocock Vina software, Nota-GSH-AVP and DOTA-GSH-AVP were docked with the MPO protein (PDB number: 3F9P), and the docking was visualized using PyMol software (e.g., ...). Figure 6 (As shown in AB). The minimum binding energies of NOTA-GSH-AVP and DOTA-GSH-AVP to MPO protein are -8.9 kcal / mol and -8.2 kcal / mol, respectively, and the ligands and proteins are connected by 14 and 11 hydrogen bonds, respectively, exhibiting high affinity.

[0035] The minimum binding energies of the representative targeted reversible covalent ligands C-RCL-P to the target protein molecules in this embodiment are shown in Table 1 below.

[0036] Table 1

[0037] Example 2 Preparation of precursor material for the targeted reversible covalent ligand NOTA-RCL-P Preparation of NOTA-GSH-FAPT: FAPI is used to target and modify GSH to form FAPT-GSH. FAPT-GSH is then modified with -NOTA to generate NOTA-GSH-FAPT. The product peak is then separated and purified using preparative HPLC to obtain the purified precursor product NOTA-GSH-FAPT. NOTA-GSH-FAPT has a high chemical yield and a chemical purity greater than 95% (e.g., ...). Figure 7 (As shown in A). The molecular weight (Mr.) of NOTA-GSH-FAPT was determined by mass spectrometry (MS) (m / z) to be 1061.13.

[0038] The targeted reversible covalent ligands, namely, NOTA-GSH, DOTA-GSH-FAPT, NOTA-GCys-FAPT, DOTA-GCys-FAPT, NOTA-GSeCys-FAPT, and DOTA-GSeCys-FAPT, were prepared using the same preparation method as NOTA-GSH-FAPT. The chemical yields and chemical purities of all the finally prepared targeted reversible covalent ligands were high, and all were greater than 95%. The molecular weights (Mr.) of NOTA-GSH, DOTA-GSH-FAPT, NOTA-GCys-FAPT, DOTA-GCys-FAPT, NOTA-GSeCys-FAPT, and DOTA-GSeCys-FAPT were determined by mass spectrometry (MS / z) to be 739.8, 1162.23, 1004.08, 1105.18, 1050.99, and 1152.09, respectively.

[0039] Nota-GSH-α was prepared using the same method as Nota-GSH-FAPT. v β6L and DOTA-GSH-α v β6L, prepared as NOTA-GSH-α v β6L and DOTA-GSH-α v The β6L yields were all high, and the chemical purity was greater than 95%. Mass spectrometry (MS / z) was used to determine the NOTA-GSH-α... v The molecular weight (Mr.) of β6L is 1653.60, and the DOTA-GSH-α... v The molecular weight (Mr.) of β6L is 1752.81.

[0040] Nota-GSH-DH6 and DOTA-GSH-DH6 were prepared using the same method as Nota-GSH-FAPT. Both Nota-GSH-DH6 and DOTA-GSH-DH6 showed high chemical yields and chemical purities greater than 95%. The molecular weights (Mr.) of Nota-GSH-DH6 and DOTA-GSH-DH6 were determined by mass spectrometry (MS / z) to be 1694.93 and 1796.03, respectively.

[0041] Furthermore, the molecular weights (Mr.) of NOTA-GSH-EG4 and DOTA-GSH-EG4 were determined by mass spectrometry (m / z) to be 4202.71 and 4303.82, respectively; the molecular weights (Mr.) of NOTA-GSH-JR11 and DOTA-GSH-JR11 were determined by mass spectrometry (m / z) to be 1877.48 and 1978.59, respectively; and the molecular weights (Mr.) of NOTA-GSH-AVP and DOTA-GSH-AVP were determined by mass spectrometry (m / z) to be 1746.71 and 1847.82, respectively. Other targeted reversible covalent ligands were prepared using similar methods.

[0042] Example 3 18 Radiosynthesis of F-AlF-labeled targeted reversible covalent radiopharmaceuticals In a reaction flask containing the precursor NOTA-GSH-P (50 µg / µL, 50 µL), 6 μL of 2 mmol / L AlCl3 solution, 5 μL of glacial acetic acid, and 300 µL of acetonitrile were added sequentially and mixed thoroughly. The mixture was then passed through a cyclotron. 18 O(p, n) 18 F nuclear reaction produces 18 F - The ions were captured in a Sep-Pak QMA anion exchange column. The ions were then removed from the QMA anion exchange column using 0.3–0.4 mL of physiological saline or sodium acetate buffer. 18 F - The eluent was transferred to a vial, and 50 µL of this eluent was added to the reaction flask. After stirring and mixing, the mixture was heated at 100°C for 10–15 min. After cooling, 6–8 mL of water was added to the reaction flask, mixed, and transferred to a SEP-PAK C18 column. After all the solution in the reaction flask had been transferred, the column was rinsed with 10 mL × 3 mL of water for injection and dried. Finally, the eluent was collected in a receiving bottle after passing through a sterile filter membrane with 1.5 mL of ethanol, and diluted with physiological saline to a product solution containing 5% ethanol to obtain the desired targeted reversible covalent radiopharmaceutical. 18 F]AlF-GSH-P injection. Wherein, P includes FAPT, GSH, and α. v One of β6L, DH6, EG4, JR11, and AVP.

[0043] 18 The radiochemical yield after attenuation correction by F-NOTA-GSH-FAPT was 27.23 ± 7.03% (n = 5), and the total radiosynthesis time was approximately 30 min. 18 The radiochemical yield after F-NOTA-GSH attenuation correction was 11.37 ± 8.32% (n = 3), and the total radiosynthesis time was approximately 30 min; 18F]AlF-GSH-α v After β6L attenuation correction, the radiochemical yield was 31.32 ± 10.42% (n = 4), and the total radiosynthesis time was approximately 30 min. 18 The radiochemical yield after attenuation correction of F-GSH-DH6 was 14.6 ± 0.07%, and the total radiosynthesis time was 30 min. 18 The attenuation-corrected radiochemical yield of F-GSH-EG4 was 10.2 ± 3.6%, and the total radiosynthesis time was 25 min. 18 After attenuation correction, the radiochemical yield of F-GSH-JR11 was 21.2 ± 5.3%, and the total radiosynthesis time was 30 min. 18 The radiochemical yield after F-GSH-AVP attenuation correction was 14.1±3.6%, and the total radiosynthesis time was 30 min.

[0044] Example 4 68 Radiosynthesis of GaCl3-labeled targeted reversible covalent radiopharmaceuticals Made of metal 68 Ga 3+ Chelation reaction preparation 68 Ga-NOTA-GSH-FAPT (or 68 Ga-DOTA-GSH-FAPT), mainly includes the following steps: (1) 68 Rinsing of the Ga generator: Draw 4 ml of hydrochloric acid (0.05 M) into a syringe, connect it to the inlet tube of the generator through a filter membrane, and slowly push the hydrochloric acid into the generator. 68 Ga eluent was collected in a vacuum bottle; (2) Chelation reaction: Ga eluent was collected in a vacuum bottle. 68 Ga eluent was added to a solution containing Nota-GSH-FAPT (or 68 (2) Mix the reaction mixture with 50 μg of Ga-DOTA-GSH-FAPT (50 μg, 50 μl of water) and sodium acetate buffer (pH = 4; 0.5 ml; 0.25 M), and heat the reaction mixture at 100°C for 10 min. (3) Column trapping: Add 6 ml of water to the reaction mixture, cool to room temperature, and then transfer to a Sep Pak plus C18 column. (4) Separation and purification: Rinse the Sep Pak plus C18 column once with 10 ml of water, then rinse the Sep Pak plus C18 column with 1.5 ml of 50% ethanol. Transfer the product to a transfer bottle, and finally collect the product in a sterile product bottle after sterile filtration. 68 Ga-NOTA-GSH-FAPT (or 68 Ga-DOTA-GSH-FAPT).

[0045] 68The radiochemical yield after Ga-NOTA-GSH-FAPT attenuation correction was 63.50±1.49% (n = 3), and the total radiosynthesis time was approximately 25 min. 68 The radiochemical yield after Ga-DOTA-GSH-FAPT attenuation correction was 71.14 ± 1.12% (n = 3), and the total radiosynthesis time was approximately 25 min. Other... 68 Ga-NOTA-GSH-P or 68 Ga-DOTA-GSH-P targeted reversible covalent radiopharmaceuticals can be prepared and synthesized using the methods described above.

[0046] Example 5 177 Radiosynthesis of LuCl3-labeled targeted reversible covalent radiopharmaceuticals In a reaction tube, add 50µg of the precursor NOA-GSH-P (or DOTA-GSH-P), 2mg of gentianic acid, 160µl of sodium acetate, and free [unclear - possibly a specific ingredient or compound] in sequence. 177 0.100–1.000 mL of Lu solution was added and heated in a water bath at 106 °C for 15 minutes. The reaction was monitored by HPLC. After passing through a C18 column, the product was rinsed with 30 mL of water and then washed with 1 mL of 50% ethanol. The product was collected. Finally, it was diluted with physiological saline and filtered through a sterile membrane into a receiving flask to obtain the required product. 177 Lu-NOTA-GSH-P (or 177 Lu-DOTA-GSH-P injection.

[0047] 177 After attenuation correction using Lu-NOTA-GSH-FAPT, the radiochemical yield is greater than 95%, and the radiochemical purity is greater than 95%. Other... 177 Lu-NOTA-RCL-P or 177 The Lu-DOTA-RCL-P targeted reversible covalent radiopharmaceutical can be prepared and synthesized using the method described above.

[0048] Example 6: Determination of radiochemical purity and stability of the product 18 The radioactive HPLC analysis results of F-NOTA-GSH-FAPT injection are as follows: Figure 7 As shown in B, the retention time is 8.25 min, and the radiochemical purity is greater than 95%. 18 The F-NOTA-GSH injection solution underwent radiochemical HPLC analysis, showing a radiochemical purity greater than 95%. 18 F]AlF-GSH-α v The HPLC analysis results of β6L injection showed a retention time of 8.43 min and a radiochemical purity greater than 95%. 18The HPLC results for F-GSH-JR11 showed a radiochemical purity greater than 95% and a retention time of 7.21 min.

[0049] 18 The in vitro and in vivo stability of F-NOTA-GSH-FAPT was determined by HPLC, and the results showed (e.g.) Figure 7 (As shown in CF): Within 2 hours, 18 F-NOTA-GSH-FAPT showed radiochemical purity greater than 95% in PBS buffer in vitro, with no defluorination or decomposition, but decomposition was observed in fetal bovine serum in vitro; within 1 hour, 18 F-NOTA-GSH-FAPT decomposes in serum and urine in vivo, and no defluorination was observed.

[0050] [ 18 F]AlF-GSH-α v The in vitro and in vivo stability of β6L was determined by HPLC. The results showed that within 2 hours, [ 18 F]AlF-GSH-α v β6L showed radiochemical purity greater than 95% in both PBS buffer and fetal bovine serum in vitro, with no defluorination or decomposition observed; within 1 hour, [ 18 F]AlF-GSH-α v β6L also exhibits good stability in serum and urine in vivo, with a radiochemical purity greater than 95%, and no defluorination or decomposition was observed.

[0051] 18 HPLC analysis showed that F-GSH-JR11 had a radiochemical purity greater than 95% and a retention time of 7.21 min. It exhibited good stability in PBS buffer at 37°C and in serum for 1 h and 2 h, respectively. It also showed good stability in Kunming rat blood for 1 h.

[0052] [ 18 The HPLC results of F]AlF-NOTA-GSH-DH6 showed a radiochemical purity greater than 95% and a retention time of 10.696 min. It exhibited certain stability in PBS buffer at 37°C and in vitro serum for 1 h. It also showed good stability in Kunming rat blood for 1 h.

[0053] 18 The HPLC results of F-GSH-EG4 showed a radiochemical purity greater than 95%, a retention time of 12.11 min, and good stability in PBS buffer at 37°C and in vitro serum for 2 h; it also showed good stability in Kunming rat blood for 1 h.

[0054] Example 7: Experiment for Determination of Ester-Water Partition Coefficient The ester-water partition coefficient was determined using the conventional method. 18 The lipid-water partition coefficient of F-NOTA-GSH-FAPT injection was -2.51 ± 0.12, as shown in the results. Figure 7 As shown in H, [the following is an explanation] 18 F]AlF-NOTA-GSH exhibits significant hydrophilicity; as determined, [ 18 F]AlF-GSH-α v The lipid-water partition coefficient of β6L injection was -2.73 ± 0.06 (n = 5). The results showed that... 18 F]AlF-GSH-α v β6L exhibits significant hydrophilicity; 18 The lipid-water partition coefficient of F-GSH-JR11 is LogD = -2.58 ± 0.04, indicating good hydrophilicity; 18 The lipid-water partition coefficient of F]AlF-NOTA-GSH-DH6 is LogD=-2.24±0.23, indicating significant hydrophilicity; 18 The lipid-water partition coefficient of F-GSH-JR11 is LogD = -1.24 ± 0.03, indicating good hydrophilicity.

[0055] Example 8 Albumin Binding Experiment According to the measurement, 18 The binding rate of F-NOTA-GSH-FAPT to serum albumin was 24.09±4.06, as shown in the results. Figure 7 As shown in G, [ 18 F]F-NOTA-GSH-FAPT has a small amount of binding.

[0056] Example 9 In vitro cell uptake, inhibition and IC50 assay Results of cell uptake and inhibition experiments (e.g.) Figure 8 As shown): [ 18 F]AlF-GSH-α v The uptake of β6L in 293T-β6 cells gradually increased over time, reaching 1.27 ± 0.18 %AD / million cells at 60 minutes and 1.44 ± 0.23 %AD / million cells at 120 minutes. In the cell inhibition assay, [ 18 F]AlF-GSH-α v The uptake of β6L in 293T-β6 cells was significantly reduced after 60 minutes, indicating that [ 18 F]AlF-GSH-α v β6L exhibits relatively specific uptake in 293T-β6 cells.

[0057] 18 The results of the F-GSH-JR11 inhibition experiment on AR42J uptake in SSTR-positive cells showed (e.g.) Figure 9 (as shown) 18 F-GSH-JR11 showed high uptake in SSTR-positive cells, reaching a peak rapidly within 15 minutes (8.62 ± 1.10% AD / million cells), followed by a slow decline.

[0058] The inhibition experiment used Nota-JR11 inhibition, 60 min. 18 The uptake of F-GSH-JR11 was significantly inhibited (5.46 ± 0.22% AD / million cells vs. 0.89 ± 0.04% AD / million cells, P < 0.05), indicating that it acts on the SSTR2 target and has good specificity.

[0059] [ 18 The results of the experiment on the inhibition of SKOV-3 uptake in HER2-positive cells by F]AlF-NOTA-GSH-DH6 showed (e.g.) Figure 10 As shown): [ 18 F]AlF-NOTA-GSH-DH6 exhibits high uptake in SKOV-3 cells, with uptake increasing over time to reach a peak (3.63 ± 0.39% AD / million cells).

[0060] 18 The results of the F-GSH-EG4 uptake and inhibition experiment in GIPR-positive HT29 cells showed (e.g.) Figure 11 (as shown) 18 F-GSH-EG4 showed high uptake in GIPR-positive cells, with an uptake value of 28.74 ± 1.28% AD / million cells at 60 min.

[0061] The inhibition experiment used EG4 inhibition for 60 min. 18 The uptake of F-GSH-EG4 was significantly inhibited (9.14±0.41%AD / million cells, P<0.05), indicating that it acts on the same GIPR target and has good specificity.

[0062] NOTA-GSH-FAPT and NOTA-FAPI-42 are respectively with 18 The competitive binding assay of F-FAPI-42 in A549-FAP cells showed (e.g.) Figure 12 As shown in the figure): the IC50 values ​​of NOTA-GSH-FAPT and NOTA-FAPI-42 are 0.81 nM and 0.97 nM, respectively, indicating that the two drugs have similar affinity.

[0063] Example 10 Reversible Covalent Bonding Experiment In vitro binding experiments of NOTA-GSH-FAPT with cystine showed (e.g.) Figure 13 (As shown in A): After reacting with cysteine ​​in vitro for 1 h, two new peaks appeared in the HPLC UV spectrum of Nota-GSH-FAPT. The peak areas of the new peaks gradually increased at 2 h and 12 h, while the original peaks of Nota-GSH-FAPT and cysteine ​​decreased slightly over time. This indicates that Nota-GSH-FAPT can break the existing disulfide bonds within cysteine ​​and form new disulfide bonds that bind with cysteine.

[0064] Furthermore, utilizing the ability of the free thiol group (-SH) of GSH to form disulfide bonds with cysteine ​​residues (Cys) in the target protein sequence, the GSH ligand and the target protein were incubated under physiological conditions. The free thiol group (-SH) of GSH in the GSH ligand underwent an exchange reaction with the disulfide bond (SS) of the target protein, forming a ligand GSS-target protein containing a new disulfide bond. Subsequently, by combining a bottom-up proteomics strategy, the covalently modified protein was digested by enzymes and analyzed by LC-MS / MS to obtain the specific modification sites and molecular modification characteristics.

[0065] The results showed a modification signal of 592.25 Da detected in the peptide VCLQWLK at the Cys305 site. This signal is consistent with the characteristic of a partial cleavage of 468.17 Da following the formation of a disulfide bond between the ligand and Cys305 (e.g., Figure 13 (As shown in B). Meanwhile, in the control group, only IAA modification was detected at this site, indicating that it was in a free state and capable of specific binding to the ligand. Furthermore, small fragment modifications of +89.0061 Da were detected at Cys305, Cys438, and Cys448 (e.g., Figure 13 (As shown in B), this is the smallest skeleton remaining after the ligand is broken by HCD.

[0066] Example 11 PET Imaging Experiment of Model Animals 18 PET imaging results of F-NOTA-GSH-FAPT in nude mice bearing A549-FAP tumors (e.g.) Figure 14 (as shown) 18 F-NOTA-GSH-FAPT is primarily eliminated from the body rapidly through the kidneys; 18 F-NOTA-GSH-FAPT showed high uptake in the A549-FAP tumor-bearing model with high FAP expression, and the tumor uptake gradually increased over time; prolonged contrast imaging at 1-5 h in the same nude mouse bearing A549-FAP tumor revealed... 18The tumor uptake and tumor retention capacity of F-NOTA-GSH-FAPT (1h: 9.3±0.03 %ID / g, 3h: 9.55±0.03 %ID / g, 5h: 10.1±0.06 %ID / g) were significantly superior to existing tumor-specific uptake and retention capacity. 18 F-FAPI-42.

[0067] 18 In vivo PET imaging results of F-NOTA-GSH in mice bearing HCT116 colorectal cancer, AR42J pancreatic cancer, SKOV3 ovarian cancer, A549-FAP lung adenocarcinoma, and U87MG glioma showed (e.g.) Figure 15 (as shown) 18 F-NOTA-GSH showed some uptake in larger HCT116 (0.83 ± 0.15 %ID / g) and AR42J (0.65 ± 0.14 %ID / g) tumors, while it was less active in smaller SKOV3 (0.30 ± 0.10 %ID / g), A549-FAP (0.34 ± 0.08 %ID / g), and U87MG (0.46 ± 0.15 %ID / g) tumors. 18 Lower uptake of F-NOTA-GSH indicates higher levels of oxidative stress in larger HCT116 and AR42J tumors, while slightly lower levels of oxidative stress are observed in smaller SKOV3, A549-FAP, and U87MG tumors. Furthermore, [ 18 F]F-NOTA-GSH is mainly cleared from the body through the kidneys, and partially excreted through the liver and gallbladder. In addition to the kidneys, the gallbladder and intestines have high radioactive uptake, while other normal tissues and organs have low uptake.

[0068] [ 18 F]AlF-GSH-α v PET imaging results of β6L in nude mice bearing Capan-2 tumors showed (e.g. Figure 8 As shown): [ 18 F]AlF-GSH-α v β6L is primarily cleared from the body via rapid renal elimination, with no significant radioactive uptake observed in other normal tissues and organs; 18 F]AlF-GSH-α v β6L in α v Capan-2 showed high uptake in a pancreatic cancer model with high β6 expression, and this uptake could be specifically inhibited; prolonged contrast imaging at 1-4 h in the same nude mouse bearing Capan-2 tumor showed that, 18 F]AlF-GSH-α v The tumor uptake value of β6L is significantly higher than that of existing [ 18F]AlF-Asp2-α v β6L and [ 18 F]AlF-Glc-α v β6L. Furthermore, [ 18 F]AlF-GSH-α v β6L has a relatively high tumor retention capacity. 18 F]AlF-Asp2-α v β6L and [ 18 F]AlF-Glc-α v β6L was significantly increased. 1-4 hours after injection, [ 18 F]AlF-GSH-α v The tumor uptake of β6L decreased slightly (1 h: 1.06 ± 0.05 %ID / g, 4 h: 0.92 ± 0.08 %ID / g), while the tumor-background contrast gradually increased over time. 18 F]AlF-Asp2-α v β6L and [ 18 F]AlF-Glc-α v The tumor uptake and tumor-background contrast of β6L drugs decreased significantly over time, indicating that [ 18 F]AlF-GSH-α v β6L has better tumor retention ability than [ 18 F]AlF-Asp2-α v β6L and [ 18 F]AlF-Glc-α v β6L. Furthermore, [ 18 F]AlF-GSH-α v The renal uptake value of β6L is between [ 18 F]AlF-Asp2-α v β6L and [ 18 F]AlF-Glc-α v β6L levels were between the two and gradually decreased over time, while the tumor-to-kidney ratio gradually increased over time, showing […]. 18 F]AlF-GSH-α v β6L has rapid renal clearance efficiency.

[0069] 18 Dynamic imaging of F-GSH-JR11 in AR42J mice shows (e.g.) Figure 9 (as shown) 18F-GSH-JR11 is primarily cleared through the renal urinary system. It exhibits high uptake in AR42J tumors, reaching peak uptake at 60 min and maintaining high uptake in tumors until 120 min (2.73±0.74%ID / g, 60 min; 2.55±0.08%ID / g, 120 min). Uptake in non-target organs (heart, lungs, liver, etc.) is low, resulting in a high target-to-substance ratio.

[0070] [ 18 Static imaging of F]AlF-NOTA-GSH-DH6 in SKOV3 nude mice shows (e.g.) Figure 10 As shown): [ 18 F]AlF-NOTA-GSH-DH6 is mainly cleared through the renal urinary system. At 30 min, SKOV-3 tumor uptake was high at 2.9±0.20%ID / g, and at 60 min, uptake decreased to 1.4±0.08%ID / g.

[0071] 18 1-hour static imaging of F-GSH-EG4 in HT29 model mice showed (e.g.) Figure 11 (as shown) 18 F-GSH-EG4 is mainly cleared through the renal urinary system. It is taken up in HT29 tumors, with an uptake value of 0.60±0.11%ID / g at 60 min. The uptake in non-target organs (heart, lungs, liver, etc.) is low, except for the kidneys, and it has a high target-to-supplement ratio.

[0072] Example 12 In vivo biodistribution experiment 18 The results of the 1-h biodistribution experiment of F-NOTA-GSH-FAPT in nude mice bearing A549-FAP tumors (e.g.) Figure 14 (as shown) 18 F-NOTA-GSH-FAPT exhibits the highest radioactive uptake in tumors, while normal tissues and organs such as the liver and gallbladder show lower uptake, indicating that the probe is primarily excreted through the kidneys; 18 F]F-NOTA-GSH-FAPT exhibits certain radioactive uptake in A549-FAP tumors and can be inhibited by inhibitors, indicating that [ 18 F]F-NOTA-GSH-FAPT exhibits specific uptake in tumors; joints and spine show moderate uptake, while other organs such as the stomach, spleen, heart, brain, muscles, and pancreas show lower uptake.

[0073] [ 18 F]AlF-GSH-α v The results of the 1-hour biodistribution experiment of β6L in nude mice bearing Capan-2 tumors (e.g.) Figure 8 As shown): [18 F]AlF-GSH-α v β6L exhibits the highest radioactive uptake in the kidneys, while normal tissues and organs such as the liver and gallbladder show lower uptake, indicating that the probe is primarily excreted through the kidneys; 18 F]AlF-GSH-α v β6L exhibits some radioactive uptake in Capan-2 tumors and can be converted by excess α v β6-targeting peptide inhibition indicates that [ 18 F]AlF-GSH-α v β6L exhibits specific uptake in tumors; moderate uptake is observed in blood and lungs, while lower uptake is observed in other organs such as the stomach, spleen, heart, brain, muscles, and pancreas.

[0074] Example 13: Clinical study of targeted reversible covalent radiopharmaceuticals Six enrolled patients were injected with... 18 F-GSH-FAPT and 18 A PET / CT scan was performed 60 minutes after F-FDG, and the results showed (e.g.) Figure 16 (as shown) 18 F-GSH-FAPT showed significant uptake of lesions at 60 minutes, compared to 18 F-FDG can reveal more tiny lesions. 18 F-GSH-FAPT is mainly excreted through the urinary system in the human body. Its uptake by the gallbladder, bile ducts, and intestines is relatively low, and its renal excretion rate is similar to that of other organs. 18 F-FDG are similar. Furthermore, 18 F-GSH-FAPT uptake in the pancreas is higher than existing methods. 18 F-FAPT and 18 Drugs such as F-SP2A-FAPT have lower levels, which may help solve the problem. 18 The challenge of high pancreatic uptake of F-labeled targeted FAP radiopharmaceuticals.

[0075] The above description is merely an example and illustration of the structure of the present invention, and while the description is relatively specific and detailed, it should not be construed as limiting the scope of the present invention. It should be noted that, due to space limitations, no embodiments are provided. 89 Zr、 99m Tc, 111 In、 90 Y、 223 Ra、 225 Ac or 188Specific examples of Re-labeled targeted reversible covalent radiopharmaceuticals are provided, but those skilled in the art can make various modifications and improvements to these radiopharmaceuticals without departing from the concept of the present invention, and these obvious substitutions are all within the scope of protection of the present invention.

Claims

1. A targeted reversible covalent ligand, characterized in that, It is composed of a targeting pharmacophore group (P), a reversibly covalently linked bullet head (RCL), and a radioactive nuclide binding group (C), and the targeting reversible covalent ligand has the general structural formula: C-RCL-P; The reversible covalent linker (RCL) is a linker containing a thiol-like group that can reversibly bind to disulfide bonds in the target protein, including one of GSH, Lys-Cys, Cys, SeC, GCys, and GSeCys linkers.

2. The targeted reversible covalent ligand according to claim 1, characterized in that, The GSH connector has the following structural formula: ； The Lys-Cys connector has the following structural formula: ； The Cys connector has the following structural formula: ； The SeC connector has the following structural formula: ； The GCys connector has the following structural formula: ； The GSeCys connector has the following structural formula: 。 3. The targeted reversible covalent ligand according to claim 1, characterized in that, The targeted pharmacophore (P) includes a pharmacophore targeting fibroblast activation proteins (FAPI) and a targeted integrin α. v Pharmacodynamic group (α) of β6 receptor v One of the following: β6L), a pharmacophore targeting human epidermal growth factor receptor 2 (HER2) (DH6), a pharmacophore targeting glucose-dependent insulinotropic peptide receptor (GIPR) (EG4), a pharmacophore targeting somatostatin receptor (JR11), a pharmacophore targeting myeloperoxidase (MPO) (AVP), and glutathione (GSH) targeting glutathione peroxidase.

4. The targeted reversible covalent ligand according to claim 1, characterized in that, The radioactive nuclide group (C) includes one of the following chelating agents: 1,4,7-triazacyclononadecanyl-N',N''-diacetyl-N-acetyl (NOTA) and 1,4,7,10-tetraazacyclododecane-N',N'',N'''-triacetyl-N-acetyl (DOTA).

5. A targeted reversible covalent radiopharmaceutical, characterized in that, Composed of a radionuclide (*R) and the targeted reversible covalent ligand (C-RCL-P) as described in any one of claims 1-4, the targeted reversible covalent radiopharmaceutical having the general structural formula: *RC-RCL-P.

6. The targeted reversible covalent radiopharmaceutical according to claim 5, characterized in that, The radionuclides (*R) include 18 F, 68 Ga、 111 In、 99m Tc, 186 Re、 188 Re、 175 Yb、 153 Sm、 166 Ho、 88 Y、 90 Y、 177 Lu、 47 Sc、 212 Bi、 213 Bi、 123 I, 124 I, 131 I, 211 At、 153 Eu、 169 Eu、 212 Pb, 64 Cu、 67 Cu、 198 Au、 223 Ra、 225 Ac and 227 One of the Th types.

7. A method for preparing the targeted reversible covalent ligand according to any one of claims 1-4, characterized in that, Includes the following steps: The targeted pharmacophore (P) is modified by reversibly covalently linking a bullet head (RCL) to obtain the RCL-P ligand; the RCL-P ligand is linked to bind a radioactive nuclide group (C) to obtain the targeted reversible covalent ligand C-RCL-P.

8. A method for preparing a targeted reversible covalent radiopharmaceutical according to any one of claims 5-6, characterized in that, Includes the following steps: Using the targeted reversible covalent ligand C-RCL-P as a precursor, a chelation reaction was carried out with radionuclide ions. After separation and purification by a small column, the targeted reversible covalent radiopharmaceutical *RC-RCL-P was obtained.

9. The method for preparing a targeted reversible covalent radiopharmaceutical according to claim 8, characterized in that, The radioactive nuclide ions include 18 F, 68 Ga、 111 In、 99m Tc, 186 Re、 188 Re、 175 Yb、 153 Sm、 166 Ho、 88 Y、 90 Y、 177 Lu、 47 Sc、 212 Bi、 213 Bi、 123 I, 124 I, 131 I, 211 At、 153 Eu、 169 Eu、 212 Pb, 64 Cu、 67 Cu、 198 Au、 223 Ra、 225 Ac and 227 One of the ions formed in Th.

10. The application of the targeted reversible covalent radiopharmaceutical according to any one of claims 5-6, characterized in that, include: The application of the targeted reversible covalent radiopharmaceutical in the preparation of PET imaging agents for lung cancer, liver cancer, breast cancer, melanoma, lymphoma, glioma, digestive system tumors, neuroendocrine tumors, prostate cancer, and thyroid cancer; The application of the targeted reversible covalent radiopharmaceutical in the preparation of SPECT imaging agents for lung cancer, liver cancer, breast cancer, melanoma, lymphoma, glioma, digestive system tumors, neuroendocrine tumors, prostate cancer, and thyroid cancer; The application of the targeted reversible covalent radiopharmaceutical in the preparation of diagnostic and therapeutic agents for lung cancer, liver cancer, breast cancer, melanoma, lymphoma, glioma, digestive system tumors, neuroendocrine tumors, prostate cancer, and thyroid cancer.

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