A pd-1 / pd-l1 pathway targeting compound and its radionuclide label, preparation and application

Abstract

The application provides a PD-1 / PD-L1 pathway targeting compound and a nuclide label thereof, and preparation and application of the compound. The PD-1 / PD-L1 pathway targeting compound and the nuclide label thereof have excellent PD-1 / PD-L1 targeting performance. The nuclide label has the characteristics of simple labeling method and high labeling yield, and can be used for non-invasive and accurate visual detection of PD-L1 in primary tumors and metastatic tumors through PET / SPECT imaging technology.

Description

Technical Field

[0001] This invention relates to a PD-1 / PD-L1 pathway-targeting compound and its radionuclide labeling, preparation and application, belonging to the fields of radiopharmaceuticals and medical imaging technology. Background Technology

[0002] Tumor immunotherapy is revolutionizing cancer treatment. Immune checkpoint inhibitors, which directly target programmed death receptor 1 (PD-1) and its ligand (PD-L1), have shown promising clinical efficacy against various types of cancer and are considered among the most promising tumor immunotherapies today. Currently, several PD-1 / PD-L1 antibody drugs have been launched and achieved great success. PD-1 is an important immunosuppressive checkpoint, and its ligand is named PD-L1 or PD-L2. PD-L1 is a 40kDa type 1 transmembrane protein, primarily found on the surface of most tumor cells. Research has elucidated that the PD-1 / PD-L1 pathway provides inhibitory signals, suppressing the activation of antigen-specific CD8+ T cells or CD4+ helper cells, thereby mediating tumor evasion of immune surveillance. Immune checkpoint inhibitors, by blocking the PD-1 / PD-L1 signaling pathway and reactivating the immune system's recognition and killing functions, have achieved remarkable therapeutic effects in cancer treatment. Recently, several immune antibodies targeting the PD-1 / PD-L1 pathway have been used clinically, such as nivolumab, pembrolizumab, and atezolizumab. However, regrettably, not all cancer patients benefit from immunotherapy, and the therapeutic effect of immune checkpoint inhibitors is related to the individual subject's PD-L1 expression level. Therefore, accurately assessing a patient's PD-L1 expression is crucial for clinical treatment decisions.

[0003] Currently, the main methods for assessing PD-L1 expression are positron emission tomography (PET) and single-photon computed tomography (SPECT) imaging. Both PET and SPECT primarily utilize radiolabeled anti-PD-L1 antibodies, enabling real-time, quantitative, and visual assessment of PD-L1 expression in primary and metastatic tumors. Despite these significant achievements, the long clearance time of radiolabeled antibodies limits their clinical application. Compared to radiolabeled anti-PD-L1 antibodies, radioactive small molecule compounds do not have these drawbacks. Therefore, designing a radioactive small molecule compound targeting the PD-1 / PD-L1 pathway for non-invasive and precise visual detection of PD-L1 in primary and metastatic tumors using PET / SPECT imaging technology is of great significance. Summary of the Invention

[0004] This invention provides a PD-1 / PD-L1 pathway-targeting compound and its radionuclide label, preparation and application, which can effectively solve the above problems.

[0005] This invention is implemented as follows:

[0006] A PD-1 / PD-L1 pathway-targeting compound, the structural formula of which is shown in formula (I) below:

[0007]

[0008] Wherein, when R1 is a linker, it includes linker group 1 or linker group 2, wherein linker group 1 includes any one of the following structures:

[0009]

[0010] The linking group 2 includes any one of the following structures:

[0011]

[0012] Where n, n', n”, x, y, z, and w are independent integers between 0 and 10; R1 exists or does not exist;

[0013] R2 is the labeling group, which includes any one of the following structures:

[0014]

[0015] A method for preparing the above-mentioned PD-1 / PD-L1 pathway-targeting compound includes the following steps:

[0016] S1, under inert gas protection, palladium acetate and cesium carbonate are added to the first solvent, followed by 3-hydroxymethyl-2-methylbiphenyl and 6-chloro-2-methoxypyridine-3-carboxaldehyde. The mixture is heated at 75-85°C for 3.5-4.5 h. After the reaction is complete, the first solvent is removed and the mixture is purified to obtain reaction intermediate 1.

[0017] S2, reaction intermediate 1 and ethylenediamine with R1 protecting group R4 are dissolved in a second solvent, anhydrous acetic acid is added, and the reaction is carried out at room temperature for 2.5-3.5 h; then sodium borohydride acetate is added and the reaction is carried out at room temperature for 12-18 h; after the reaction is completed, water is added to quench the reaction, and then the mixture is purified to obtain reaction intermediate 2;

[0018] S3, reaction intermediate 2 is dissolved in the third solvent under ice bath conditions. After removing the ice bath, the mixture is slowly restored to room temperature and reacted for 1.5 to 2.5 hours. After the reaction is completed, the third solvent is removed and the mixture is purified to obtain reaction intermediate 3.

[0019] S4, when the R1 linker is not present, the reaction intermediate 3 is reacted with the active compound of R2 to obtain the PD-1 / PD-L1 pathway targeting compound.

[0020] When R1 linker is present and is the linking group 1, reaction intermediate 3 is first linked to R1 linker with R4 protecting group through amidation reaction to obtain reaction intermediate 4, and then deprotected to obtain reaction intermediate 5, which is then reacted with active compound R2 to obtain the PD-1 / PD-L1 pathway targeting compound.

[0021] When R1 linker is present and is the linking group 2, reaction intermediate 3 is first linked to reaction intermediate 6 through an amidation reaction to obtain reaction intermediate 7, and then reacted with reaction intermediate 8 to obtain the PD-1 / PD-L1 pathway targeting compound.

[0022] The reaction intermediates 1-8 have the following structures:

[0023]

[0024] The R2 active compound is any one of the following structures:

[0025]

[0026] The R4 protecting group is selected from one or more of the Boc protecting group, DDE protecting group, or Fmoc protecting group.

[0027] A pharmaceutically acceptable salt of a PD-1 / PD-L1 pathway-targeting compound, obtained by reacting the aforementioned PD-1 / PD-L1 pathway-targeting compound with an acid or base.

[0028] A PD-1 / PD-L1 pathway-targeting radionuclide label is obtained by coordinating the labeling group in the above-mentioned PD-1 / PD-L1 pathway-targeting compound or a pharmaceutically acceptable salt of the PD-1 / PD-L1 pathway-targeting compound with a labeling radionuclide.

[0029] In some embodiments, the labeled nuclide includes 18 F, 47 Sc、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 89 Zr、 86 Y、 89 Sr, 90 Y、 99m Tc, 105 Rh、 109 Pd, 111 In、119 Sb、 149 Tb, 153 Sm、 157 Gd, 161 Tb, 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

[0030] A method for preparing the above-mentioned PD-1 / PD-L1 pathway-targeting radionuclide label includes the following steps: coordinating a PD-1 / PD-L1 pathway-targeting compound or a pharmaceutically acceptable salt thereof with a labeled radionuclide to obtain a PD-1 / PD-L1 pathway-targeting radionuclide label or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, the preparation method of the above-mentioned PD-1 / PD-L1 pathway-targeting radionuclide label is to introduce the labeled radionuclide onto the above-mentioned PD-1 / PD-L1 pathway-targeting compound using a wet method or a freeze-drying method.

[0032] In some embodiments, the wet process is as follows: dissolve the PD-1 / PD-L1 pathway targeting compound in a buffer solution or deionized water or an organic solvent or a mixture of the above solvents, add a solution containing the nuclide to be labeled, and react at room temperature to 100°C for 10 to 30 minutes to obtain the product.

[0033] In some embodiments, the lyophilization process includes the following steps: dissolving the PD-1 / PD-L1 pathway targeting compound and pharmaceutically acceptable excipients in a buffer solution or deionized water, dispensing them into lyophilization containers, and sealing them after freeze-drying to form a lyophilized formulation; adding deionized water, buffer solution, organic solvent, or a mixture of the above solvents to the lyophilized formulation to dissolve it, then adding a solution containing the nuclide to be labeled, and reacting at room temperature to 100°C for 10–30 minutes to obtain the final product.

[0034] A pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising one or more of the above-mentioned PD-1 / PD-L1 pathway targeting compounds, the above-mentioned PD-1 / PD-L1 pathway targeting radionuclide markers, PD-1 / PD-L1 pathway targeting compounds and their pharmaceutically acceptable salts.

[0035] The use of the above-mentioned PD-1 / PD-L1 pathway-targeting compound, a pharmaceutically acceptable salt of the above-mentioned PD-1 / PD-L1 pathway-targeting compound, the above-mentioned PD-1 / PD-L1 pathway-targeting radionuclide label, and the above-mentioned pharmaceutical composition in the preparation of therapeutic or diagnostic drugs for PD-1 / PD-L1 pathway-related diseases.

[0036] In some embodiments, the PD-1 / PD-L1 pathway-related diseases include tumors or autoimmune diseases.

[0037] In some embodiments, the tumor includes one or more of the following: breast cancer, ovarian cancer, lung cancer, colorectal cancer, prostate cancer, lung cancer, fibrosarcoma, bone and connective tissue sarcoma, bone metastases, renal cell carcinoma, gastric cancer, pancreatic cancer, or skin melanoma.

[0038] In some embodiments, the autoimmune disease includes one or more of lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, Sjögren's syndrome, polyarteritis nodosa, and asthma.

[0039] The beneficial effects of this invention are:

[0040] 1) The PD-1 / PD-L1 pathway targeting compounds and radionuclide markers of the present invention are mainly developed based on biphenyl compounds. They not only have excellent PD-1 / PD-L1 targeting performance, but also have the characteristics of strong labeling ability, short labeling time and high labeling yield for radionuclides. They can be used for non-invasive and precise visualization detection of PD-L1 in primary tumors and metastatic tumors through PET / SPECT imaging technology.

[0041] 2) The radionuclide label of the PD-1 / PD-L1 pathway-targeting compound of the present invention introduces a small molecule chain between the targeting group and the coordination structure (i.e., the radionuclide labeling group), which can increase the distance between the targeting group and the coordination structure, avoid mutual interference, and improve targeting performance and labeling efficiency. At the same time, the R1 linker can regulate the lipid-water distribution properties of the molecule, thereby improving the pharmacokinetic properties of the radionuclide label, accelerating the clearance rate of the drug in non-target tissues, and increasing the target / non-target ratio.

[0042] 3) The PD-1 / PD-L1 pathway targeting compounds and the radionuclide labels of the PD-1 / PD-L1 pathway targeting compounds of the present invention are small molecule inhibitors compared with the peptide and antibody PD-L1 inhibitors that are currently reported. They have advantages such as clear chemical structure and no immunogenicity, and overcome the disadvantage of long clearance time of radiolabeled antibodies in traditional methods, which is more conducive to commercial application and clinical promotion. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is the mass spectrum of compound D-PMED from Example 1 of the present invention.

[0045] Figure 2 This is the mass spectrum of compound D-PEG-PMED from Example 2 of the present invention.

[0046] Figure 3 This is the mass spectrum of compound D-pep-PMED from Example 3 of the present invention.

[0047] Figure 4 This is the mass spectrum of compound N-PMED from Example 4 of the present invention.

[0048] Figure 5 The compound in Example 5 of this invention [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 HPLC spectrum of Ga]Ga-D-pep-PMED.

[0049] Figure 6 The radionuclide marker in Example 6 of this invention [ 18 HPLC spectrum of F]AlF-N-PMED.

[0050] Figure 7 The radionuclide marker in Example 7 of this invention [ 68 Stability of Ga]Ga-D-PMED in PBS and mouse serum.

[0051] Figure 8 The radionuclide marker in Example 7 of this invention [ 68 Stability of Ga]Ga-D-PEG-PMED in PBS and mouse serum.

[0052] Figure 9 The radionuclide marker in Example 7 of this invention [ 68 Stability of Ga]Ga-D-pep-PMED in PBS and mouse serum.

[0053] Figure 10 In embodiment 8 of the present invention [ 177 Lu]Lu-D-PMED cell uptake and internalization at different time points.

[0054] Figure 11 In embodiment 8 of the present invention [ 18 F]AlF-N-PMED cell uptake and BMS202 (a PD-1 / PD-L1 inhibitor) inhibition at different time points.

[0055] Figure 12 In embodiment 9 of the present invention [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga]Ga-D-pep-PMED IC 50 Measurement chart.

[0056] Figure 13 In Embodiment 10 of the present invention [ 68 Ga]Ga-D-PMED 2-hour MicroPET imaging and 1-hour activity curve in mice bearing MC38 colon cancer.

[0057] Figure 14 In Embodiment 10 of the present invention [ 68 MicroPET imaging of Ga]Ga-D-PEG-PMED in mice bearing MC38 colon cancer at 2 hours and its 1-hour activity curve.

[0058] Figure 15 In Embodiment 10 of the present invention [ 68 MicroPET imaging of Ga]Ga-D-pep-PMED in mice bearing MC38 colon cancer at 2 hours and its 1-hour activity curve.

[0059] Figure 16 In Embodiment 11 of the present invention [ 68 Biodistribution data of Ga]Ga-D-PMED in mice bearing MC38 colon cancer.

[0060] Figure 17 In Embodiment 12 of the present invention [ 68 Ga]Ga-D-pep-PMED MicroPET imaging data of mice with MC38 colon cancer after being blocked with different doses of PD-L1 antibody 24 hours in advance.

[0061] Figure 18 For different quality BMS202 (a PD-1 / PD-L1 inhibitor) in the presence of [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED or [ 68PET images and target / non-target ratios in MC38 colon cancer mice loaded with Ga]Ga-D-pep-PMED. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] This invention provides a PD-1 / PD-L1 pathway-targeting compound, characterized in that its structural formula is shown in formula (I) below:

[0064]

[0065] Wherein, R1 is a linker, including linker group 1 or linker group 2;

[0066] The linking group 1 includes any one of the following structures:

[0067]

[0068] The linking group 2 includes any one of the following structures:

[0069]

[0070] Where n, n', n”, x, y, z, and w are independent integers between 0 and 10; R1 exists or does not exist;

[0071] R2 is the labeling group, which includes any one of the following structures:

[0072]

[0073] The main structure of this compound is a biphenyl-based structure, exhibiting good PD-1 / PD-L1 targeting. R2 is the labeling group to be labeled, primarily used to label radionuclides to obtain a PD-1 / PD-L1 pathway-targeting radionuclide label. This radionuclide label can be used as a probe for single-photon emission computed tomography (SPECT) and positron emission tomography (PET). The labeled radionuclides include... 18 F, 47 Sc、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 89 Zr、 86 Y、 89 Sr, 90 Y、 99m Tc, 105 Rh、 109 Pd, 111 In、 119 Sb、 149 Tb, 153 Sm、 157 Gd, 161 Tb, 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

[0074] The R1 group is a small molecule linker that increases the distance between the targeting group and the coordination structure (i.e., the radionuclide labeling group), avoiding mutual interference and thus improving targeting and labeling efficiency. Simultaneously, the small molecule chain structure provides a good basis for regulating the lipid-water distribution properties of the entire molecule, thereby improving the pharmacokinetic properties of the drug, accelerating the clearance rate of the tracer in non-target tissues, and increasing the target / non-target ratio.

[0075] This invention provides a method for preparing a PD-1 / PD-L1 pathway-targeting compound, comprising the following steps:

[0076] S1, under inert gas protection, palladium acetate and cesium carbonate are added to the first solvent, followed by 3-hydroxymethyl-2-methylbiphenyl and 6-chloro-2-methoxypyridine-3-carboxaldehyde. The mixture is heated at 75-85°C for 3.5-4.5 h. After the reaction is complete, the first solvent is removed and the mixture is purified to obtain reaction intermediate 1.

[0077] S2, reaction intermediate 1 and ethylenediamine with R1 protecting group R4 are dissolved in a second solvent, anhydrous acetic acid is added, and the reaction is carried out at room temperature for 2.5-3.5 h; then sodium borohydride acetate is added and the reaction is carried out at room temperature for 12-18 h; after the reaction is completed, water is added to quench the reaction, and then the mixture is purified to obtain reaction intermediate 2;

[0078] S3, reaction intermediate 2 is dissolved in the third solvent under ice bath conditions. After removing the ice bath, the mixture is slowly restored to room temperature and reacted for 1.5 to 2.5 hours. After the reaction is completed, the third solvent is removed and the mixture is purified to obtain reaction intermediate 3.

[0079] S4, when the R1 linker is not present, the reaction intermediate 3 is reacted with the active compound of R2 to obtain the PD-1 / PD-L1 pathway targeting compound.

[0080] When R1 linker is present and is the linking group 1, reaction intermediate 3 is first linked to the R1 structure with R4 protection group through an amidation reaction to obtain reaction intermediate 4, and then deprotected to obtain reaction intermediate 5, which is then reacted with the active compound of R2 to obtain the PD-1 / PD-L1 pathway targeting compound.

[0081] When R1 linker is present and is the linking group 2, reaction intermediate 3 is first linked to reaction intermediate 6 through an amidation reaction to obtain reaction intermediate 7, and then reacted with a peptide analog containing R2 structure with a thiol group (reaction intermediate 8) to obtain the PD-1 / PD-L1 pathway targeting compound.

[0082] The reaction intermediates 1-8 have the following structures:

[0083]

[0084] The R2 active compound is any one of the following structures:

[0085]

[0086] The R4 protecting group includes tert-butoxycarbonyl (Boc protecting group), Lys protecting group (DDE protecting group) or 9-fluorenylmethoxycarbonyl protecting group (Fmoc protecting group).

[0087] In some embodiments, the first solvent is anhydrous toluene; the second solvent is anhydrous dichloromethane; and the third solvent includes one or more of hydrochloric acid-ethyl acetate, hydrazine hydrate, and piperidine-DMF.

[0088] This invention provides a radionuclide label for a PD-1 / PD-L1 pathway-targeting compound, obtained by introducing a radionuclide onto the aforementioned PD-1 / PD-L1 pathway-targeting compound. The radionuclide label is diluted with physiological saline or water for injection and filtered through a sterile membrane to generate a radionuclide-labeled complex injection solution.

[0089] This invention provides a method for preparing radionuclide markers of the above-mentioned PD-1 / PD-L1 pathway targeting compounds, wherein the radionuclide is introduced onto the above-mentioned PD-1 / PD-L1 pathway targeting compounds by wet or lyophilization methods.

[0090] In some embodiments, the wet process involves dissolving the PD-1 / PD-L1 pathway targeting compound in a buffer solution or deionized water, then adding a solution containing a radionuclide, and reacting at room temperature to 100°C for 10 to 30 minutes to obtain the final product.

[0091] In some embodiments, the lyophilization process includes the following steps: dissolving the PD-1 / PD-L1 pathway targeting compound in a buffer solution or deionized water, aliquoting it into lyophilization containers, and sealing it after freeze-drying to form a lyophilized formulation; adding deionized water or buffer solution to the lyophilized formulation to dissolve it, then adding a solution containing a radionuclide, and reacting at room temperature to 100°C for 10–30 minutes to obtain the final product. Compared with wet labeling, lyophilized formulations have advantages such as better uniformity, convenient transportation, and simpler labeling.

[0092] This invention provides an application of the radionuclide markers of the aforementioned PD-1 / PD-L1 pathway-targeting compounds in the preparation of therapeutic or diagnostic drugs for PD-1 / PD-L1-related diseases. The radionuclide markers of the PD-1 / PD-L1 pathway-targeting compounds can achieve highly specific and sensitive imaging in animal tumor models (MC38 colon cancer), and hold promise for non-invasive in vivo imaging of PD-L1 expression levels in patient tumor sites. This is expected to provide a new imaging method for early non-invasive screening of PDL1 patients and monitoring PD-L1 expression levels during immunotherapy. The radionuclide markers of the PD-1 / PD-L1 pathway-targeting compounds can be prepared as injectable preparations for intravenous administration.

[0093] In some embodiments, the PD-1 / PD-L1-related disease is a tumor or an autoimmune disease.

[0094] In some embodiments, the tumor includes, but is not limited to, one or more of the following: breast cancer, ovarian cancer, lung cancer, colorectal cancer, prostate cancer, lung cancer, fibrosarcoma, bone and connective tissue sarcoma, bone metastases, renal cell carcinoma, gastric cancer, pancreatic cancer, or skin melanoma.

[0095] In some embodiments, the autoimmune diseases include, but are not limited to, one or more of lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, Sjögren's syndrome, polyarteritis nodosa, and asthma.

[0096] In some embodiments, the treatment includes, but is not limited to, radionuclide-targeted therapy, immunotherapy, or a combination of therapies. The detection includes single-photon emission computed tomography (SPECT) and positron emission tomography (PET).

[0097] Example 1: A method for preparing a PD-1 / PD-L1 pathway-targeting compound is as follows:

[0098]

[0099] 1) Synthesis of compound 2

[0100] Under argon protection, palladium acetate (80 mg, 0.36 mmol) and cesium carbonate (2.23 g, 6.83 mmol) were added to 30 mL of anhydrous toluene, followed by the addition of 3-hydroxymethyl-2-methylbiphenyl (0.88 g, 4.4 mmol) and 6-chloro-2-methoxypyridine-3-carboxaldehyde (0.56 g, 3.4 mmol). The mixture was heated at 80 °C for 4 hours. After the reaction was complete, the mixture was filtered through diatomaceous earth to remove the solvent. The crude product was purified by silica gel column chromatography, and the product was recrystallized from diethyl ether to give compound 2.

[0101] 2) Synthesis of compound 3

[0102] Compound 2 (66 mg, 0.2 mmol) and BOC-ethylenediamine (44 mg, 0.4 mmol) were dissolved in 10 mL of anhydrous dichloromethane, and one drop of anhydrous acetic acid was added. The mixture was reacted at room temperature for 3 h. Then, sodium borohydride acetate (66 mg, 0.3 mmol) was added, and the mixture was reacted at room temperature for 15 h. After the reaction was complete, the reaction was quenched with water, and then extracted with an equal volume of dichloromethane. The organic phase was removed from the solvent, and the crude product was purified by silica gel column chromatography to obtain compound 3.

[0103] 3) Synthesis of compound 4

[0104] Compound 3 (47 mg, 0.1 mmol) was dissolved in 5 mL of hydrochloric acid-ethyl acetate (4 M) under ice bath conditions. After removing the ice bath, the mixture was slowly allowed to return to room temperature and reacted for 2 h. After the reaction was complete, the solvent was removed, and compound 4 was obtained by HPLC separation.

[0105] 4) Synthesis of compound D-PMED

[0106] Compound 4 (2 mg, 5 μmol) and DOTA-NHS (4 mg, 5 μmol) were dissolved in 200 μL of DMSO, and then 15 μL of triethylamine was added and the mixture was reacted overnight. The crude product was separated by HPLC. The mass spectrum of D-PMED is shown below. Figure 1 As shown.

[0107] MS(ESI) calculation C 39 H 54 N7O9([M+H)) + ): It should be 763.4, but we get 764.6.

[0108] MS(ESI) calculation C 39 H 53 KN7O9([M+K)) + ): It should be 802.4, but we get 802.6.

[0109] Example 2: A method for preparing a PD-1 / PD-L1 pathway-targeting compound is as follows:

[0110]

[0111] 1) Synthesis of Compound 5

[0112] Compound 4 (38 mg) obtained in Example 1 was mixed with BOC-NH-PEG6-COOH (40 mg) and 10 ml of anhydrous DMF was added. Then 76 mg of HATU was added and the mixture was reacted at room temperature for 2 h. Compound 5 was obtained after separation by HPLC.

[0113] 2) Synthesis of Compound 6

[0114] 20 mg of compound 5 was dissolved in 2 ml of ethyl acetate, and then 2 ml of hydrochloric acid-ethyl acetate (4 M) was added. The mixture was reacted at room temperature for 5 h, and compound 6 was obtained after separation by HPLC.

[0115] 3) Synthesis of compound D-PEG-PMED

[0116] Compound 6 (7 mg, 10 μmol) and DOTA-NHS (8 mg, 10 μmol) were dissolved in 400 μL of DMSO, and then 15 μL of triethylamine was added and the mixture was reacted overnight. The crude product was separated by HPLC. The mass spectrum of D-PEG-PMED is shown below. Figure 2 As shown.

[0117] MS(ESI) calculation C 54 H 83 N8O 16 ([M+H)) + ): It should be 1099.6, but we get 1099.3.

[0118] MS(ESI) calculation C 54 H 84 N8O 16 ([M+2H)) 2+ / 2): It should be 550.3, but we get 550.1.

[0119] Example 3: A method for preparing a PD-1 / PD-L1 pathway-targeting compound is as follows:

[0120]

[0121] Synthesis of Compound 7

[0122] 1 mg of compound 4 obtained in Example 1 and 1.05 mg of 3-(maleimide)propionic acid N-hydroxysuccinimide ester were dissolved in 200 μL of DMSO, and then 15 μL of triethylamine was added and reacted overnight. The crude product was separated by HPLC.

[0123] Synthesis of compound D-pep-PMED

[0124] 1 mg of compound 7 was dissolved in 100 μL of acetonitrile, and 2 mg of the peptide was dissolved in 200 μL of pure water and reacted overnight. The crude product was separated by HPLC. The mass spectrum of D-pep-PMED is shown below. Figure 3 As shown.

[0125] MS(ESI) calculation C 65 H 92 N 13 O 21 S([M+H)) + ): It should be 1422.6, but we get 1422.9.

[0126] MS(ESI) calculation C 65 H 92 kN 13 O 21 S([M+H+k)) 2+ / 2): It should be 730.8, but we get 730.9.

[0127] Example 4

[0128]

[0129] Synthesis of compound N-PMED

[0130] The compound 4 (2 mg, 5 μmol) and NOTA-NHS (4 mg, 6 μmol) obtained in Example 1 were dissolved in 200 μL of DMSO, and then 15 μL of triethylamine was added and the reaction was carried out overnight. The crude product was separated by HPLC to obtain N-PMED. The mass spectrum of N-PMED is shown below. Figure 4 As shown.

[0131] MS(ESI) calculation C 35 H 47 N6O7([M+H)) + The value should be 663.34, but we get 663.45.

[0132] Example 5 68 The Ga nuclide labeling process is as follows:

[0133] Wet method: approximately 1100 MBq 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) was added to centrifuge tubes containing 0.5 mL of acetate-acetate solution of D-PMED, D-PEG-PMED and D-pep-PMED (50 μg) prepared in Examples 1, 2 and 3, respectively. The mixture was reacted at room temperature to 100°C for 20 minutes, then cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain the labeled compound injection solution.

[0134] Lyophilization method: A certain amount of buffer solution and approximately 1100 MBq of... 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) was added to a lyophilized kit containing D-PMED, D-PEG-PMED and D-pep-PMED (3) (50 μg) prepared in Examples 1, 2 and 3. After mixing and dissolving, the mixture was placed at room temperature to 100°C for 20 minutes and then cooled to room temperature. The mixture was diluted with physiological saline or water for injection and then sterile filtered to obtain the labeled compound injection solution.

[0135] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeling solution with 10 mL of water and load it onto the C18 separation column. Rinse the separation column with water to remove unreacted components. 68 Ga ions were obtained by rinsing with ethanol solution. 68 Ga-labeled complexes were prepared. The organic solvent was removed by nitrogen blowing, and the solution was diluted with physiological saline and then sterile filtered to obtain the labeled compound injection solution.

[0136] like Figure 5 As shown, for the labeled compound [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [68 The Ga-D-pep-PMED sample was analyzed and identified by HPLC. No other radioactive impurity peaks were found, and its radiochemical purity was greater than 95%.

[0137] Example 6 18 F nuclide labeling:

[0138] Preparation of N-PMED lyophilized medicine boxes: Take an appropriate amount of N-PMED obtained in Example 4 and dissolve it in 0.5 mol / L acetate-sodium acetate buffer solution (pH = 4.2) to prepare a 0.5 mg / mL solution. Then, weigh an appropriate amount of aluminum chloride (AlCl3) and dissolve it in the acetate-sodium acetate solution (pH = 4.2) to obtain a final concentration of 0.008 mg / mL. Mix the two solutions in equal volumes, filter aseptically, and dispense into 1.5 mL Laxygen non-sticking cryovials. Place them in a freeze dryer and freeze-dry for 24 hours. Seal the freeze-dried medicine boxes to obtain the lyophilized medicine boxes. Depending on the production volume of the medicine boxes and the required component content in each box, the amount of N-PMED and aluminum chloride can be adjusted so that their weight ratio falls within the range of (20–100):1.

[0139] [ 18 Labeling of F]AlF-N-PMED: Take the lyophilized kit prepared above, first add 0.5 mL of 0.5 mol / L acetate-acetate buffer (pH 4.2), and after complete dissolution, add approximately 740 MBq of [ 18 F]F - Target water (obtained directly from the accelerator) 18 (O water), react at 95℃ for 15 minutes in a sealed container, then cool.

[0140] Purity was verified by Radio-TLC. If the radiochemical purity was below 95%, purification was required. The purification steps were as follows: Take a Sep-Pak C18 separation column and activate it by rinsing it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted reagents. 18 F- was obtained by rinsing with an ethanol solution. 18 F-labeled complexes. Organic solvents were removed by nitrogen evaporation, and the resulting product was diluted with physiological saline and sterile filtered to obtain the labeled compound. 18 F]AlF-N-PMED injection.

[0141] like Figure 6 As shown, for the labeled compound [ 18 The F]AlF-N-PMED sample was analyzed and identified by HPLC, and its radiochemical purity was greater than 95%.

[0142] Example 7

[0143] 1. Determination of lipid-water distribution coefficient (log P)

[0144] 100 μL of the labeled compound prepared in Example 5 [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga-D-pep-PMED was added to a centrifuge tube containing a mixture of 2.9 mL PBS and 3 mL n-octanol. The mixture was vortexed for 3 minutes, then centrifuged at 6000 rpm for 5 minutes. 100 μL of liquid was taken from both the aqueous and n-octanol phases and counted using a γ-counter. The experiment was repeated four times, and the average value was taken. The formula for calculating P is: P = I 有机相 / I 水相

[0145] Among them I 有机相 Represents the radioactivity count measured in the organic phase, I 水相 This represents the radioactivity count measured in the aqueous phase. The lipid-water distribution coefficient of each radiolabeled target probe is then determined through calculation.

[0146] Table 1. Labeled compounds [ 68Ga Ga-D-PMED, [ 68Ga Ga-D-PEG-PMED and [ 68Ga Radioactivity count of Ga-D-pep-PMED

[0147]

[0148] The labeled compound tested [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 The log P values ​​of Ga]Ga-D-pep-PMED were -0.19±0.02, -0.56±0.11 and -2.31±0.09, respectively, all of which showed water solubility.

[0149] The experimental results above show that the water solubility of the compound was greatly improved after modification with small molecule chains, and logP decreased from -0.19±0.02 to -2.31±0.09. This is more conducive to reducing the uptake of non-target sites and improving pharmacokinetic properties.

[0150] 2. In vitro stability test

[0151] The labeled compound dissolved in PBS [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68Ga-D-pep-PMED was left at room temperature for different times, and samples were analyzed by HPLC. At the tested time points, the probe still maintained a radiochemical purity of >95%, indicating that it is stable in the specified solution and does not easily decompose. 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 The stability of Ga]Ga-D-pep-PMEDPBS was determined by HPLC as follows: Figure 7-9 As shown.

[0152] Similarly, the labeled compound [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga]Ga-D-pep-PMED was placed in serum at room temperature for different times, and samples were analyzed by HPLC to determine its serum stability. Figure 7-9 It can be seen that in this system, [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga]Ga-D-pep-PMED maintained high stability (>95%), and no significant decomposition was observed after 2 hours.

[0153] Example 8

[0154] 1.[ 177 Lu]Lu-D-PMED Cell Uptake Experiment

[0155] Collect MC38 cells separately and dilute them with culture medium to a concentration of 2 × 10⁻⁶. 5 Cells / mL, seeded into 24-well plates, add 0.5 mL of cell suspension to each well, aspirate the culture medium from the wells, add 0.5 mL of pre-cooled (4°C) binding buffer along the plate wall, wash twice and set aside. Add [...] to each well of the experimental group. 177 Lu]Lu-D-PMED(1μci)([ 177 The preparation method of Lu-D-PMED is the same as in Example 5. An excess of PD-1 / PD-L1 inhibitor BMS202 was added to the culture medium of the inhibition group, and the incubation was carried out at 37°C at the corresponding time points. Each incubation time point was repeated 3 times.

[0156] After incubation, remove the original solution from the wells, add 0.5 mL of stripping buffer, incubate at room temperature for 5 minutes, and then collect the stripping buffer into sample tubes for analysis (membrane binding). Next, add 0.5 mL of 1 M NaOH, incubate at room temperature for 5 minutes, and then collect the sample tubes for analysis (internalization). Finally, use a WIZARE 24802 automated gamma counter to count the radioactivity in the sample tubes, and use GraphPadPrism 7.0 for data processing and plotting.

[0157] The result is as follows Figure 10 As shown, [ 177 Lu-D-PMED achieved the highest cellular uptake within 24 hours, and it could be specifically inhibited by the standard control BMS202, demonstrating that [ 177 Lu]Lu-D-PMED and the standard control BMS202 have the same binding site, both being the PD-L1 receptor.

[0158] 2.[ 18 F]AlF-N-PMED cell uptake experiment

[0159] Collect MC38 cells separately and dilute them with culture medium to a concentration of 2 × 10⁻⁶. 5 Cells / mL, seeded into 24-well plates, 0.5 mL of cell suspension added to each well, culture medium removed from the plate, 0.5 mL of pre-cooled (4°C) binding buffer added along the wall, washed twice. For the experimental group, add the [prepared in Example 6] to each well. 18 [F]AlF-N-PMED (1 μCi) was added to the culture medium of the inhibition group, with an excess of the PD-1 / PD-L1 inhibitor BMS202 added. The samples were incubated at 37°C for the corresponding time points, with each incubation time point repeated three times. Finally, the radioactivity of the sample tubes was counted using a WIZARE 24802 automated gamma counter, and the data was processed and plotted using GraphPadPrism 7.0.

[0160] The result is as follows Figure 11 As shown, [ 18 F]N-PMED achieved the highest cellular uptake at 2 hours, and it could be specifically inhibited by the standard control BMS202, demonstrating that [ 18 F]AlF-N-PMED and the standard control BMS202 have the same binding site, both being the PD-L1 receptor.

[0161] Example 9

[0162] Collect MC38 cells separately and dilute them with culture medium to a concentration of 2 × 10⁻⁶. 5Cells / mL, seeded into 24-well plates, 0.5 mL of cell suspension added to each well, culture medium removed from the plate, 0.5 mL of pre-cooled (4°C) binding buffer added along the wall, washed twice. For the experimental group, add the [prepared in Example 5] to each well. 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga]Ga-D-pep-PMED (1 μCi) and the non-radioactive PD-1 / PD-L1 inhibitor BMS202 (1 × 10⁻⁶) at increasing concentrations. -11 M-1×10 - 4 Each concentration was repeated three times, incubated at 37°C for 1 hour, and then washed three times with binding buffer. After incubation, the original solution was aspirated from the wells, and the plates were washed twice with 0.5 mL of PBS. Finally, 1 mL of 1M NaOH was added, and the plates were incubated at room temperature for 5-10 minutes. The samples were then collected into sample tubes, and the radioactivity was counted using an automated gamma counter. Data processing was performed using GraphPadPrism 7.0 to calculate the IC50. 50 .

[0163] like Figure 12 As shown, via IC 50 Measurement 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga]Ga-D-pep-PMED IC 50 The values ​​are 90.7 nM, 160.8 nM, and 51.6 nM, respectively.

[0164] Example 10

[0165] MicroPET imaging of a mouse tumor model. Radiochemically pure [material] with a purity greater than 95% was prepared according to Example 5. 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 Ga]Ga-D-pep-PMED labeled complex solution, 0.2 mL (about 11 MBq) was injected into the tail vein of Mc38 colon cancer mice, and MicroPET imaging was performed 120 minutes after injection.

[0166] Depend on Figure 13-15 As shown, the area indicated by the white circle is the location of the tumor, as described in Embodiments 1, 2, and 3 of this invention. 68 Ga-labeled probes [ 68 Ga]Ga-D-PMED, [68 Ga]Ga-D-PEG-PMED and [ 68 Ga-D-pep-PMED showed significant enrichment and retention in mouse tumors, and significant uptake in the liver and kidneys, indicating that it is primarily metabolized by the liver and kidneys. At 120 minutes, [ 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED and [ 68 The tumor-to-sarcoma ratios of Ga-D-pep-PMED were 2.3±0.2, 4.40±1.2, and 6.2±1.2, respectively. Based on imaging, we found that the […] obtained by modifying short peptide chains… 68 Ga-D-pep-PMED exhibits superior metabolic properties in vivo, significantly reducing uptake at non-target sites and resulting in a marked improvement in the tumor-to-liver and tumor-to-kidney ratios. Compared to existing radiolabeled PD-L1 small molecule inhibitor probes, [ 68 Ga]Ga-D-pep-PMED has a higher tumor-to-meat ratio and lower gastrointestinal uptake, which is beneficial for clinical application.

[0167] Example 11

[0168] Select male C57 mice (approximately 8 weeks old and weighing about 20g) and inject them with 1×10 oz. 5 MC38 cells, when the subcutaneous tumor diameter is 0.5-1.0 cm, were injected with approximately 0.74 MBq of the labeling compound prepared in Example 5 via the tail vein of mice. 68 In Ga]Ga-D-PMED, mice were decapitated and euthanized at different time points after injection (3 mice per group). Blood, brain, heart, liver, lungs, kidneys, intestines, spleen, muscles, and bones of interest were collected, weighed, and their radioactivity counts were measured. The results were expressed as the percentage uptake dose per gram of tissue or organ (%ID / g).

[0169] like Figure 16 of[ 68 The Ga-D-PMED biodistribution results showed that the signal value of tumor tissue reached its maximum at 120 minutes, with %ID / g reaching 7, which was significantly higher than that of muscle tissue. Over time, the %ID / g values ​​of blood and non-target organs gradually decreased, while the uptake at the tumor site remained at a certain level, demonstrating good specificity.

[0170] Example 12

[0171] According to Example 5, a radiochemically pure [[] was prepared with a purity greater than 95% 68Ga]Ga-D-pep-PMED labeled complex solution, 0.2 mL (approximately 11 MBq) was injected via tail vein into MC38 colon cancer-bearing mice pretreated with different doses (0, 25, 50, 100, 200 μg) of PD-L1 antibody 24 hours prior. 68 MicroPET imaging was performed 120 minutes after Ga]Ga-D-pep-PMED injection.

[0172] like Figure 17 As shown in the imaging, we found that with increasing doses of PD-L1 antibody pretreatment, [ 68 The tumor uptake of Ga-D-pep-PMED was significantly reduced, demonstrating that [Ga]Ga-D-pep-PMED significantly reduced tumor uptake, demonstrating that 68 Ga]Ga-D-pep-PMED and PD-L1 both target the same target, PD-L1.

[0173] Example 13

[0174] The [contains] different quality levels of BMS202 (a PD-1 / PD-L1 inhibitor) 68 Ga]Ga-D-PMED, [ 68 Ga]Ga-D-PEG-PMED or [ 68 Ga]Ga-D-pep-PMED solution was injected into MC38 colon cancer mice via tail vein, and MicroPET imaging was performed 120 minutes after injection.

[0175] Figure 18 PET images and target / non-target ratios of different groups of radionuclide markers are shown. PET imaging experiments revealed that the PET imaging effect of PD-1 / PD-L1 pathway-targeted radionuclide markers did not significantly change in the presence of different masses of BMS202. This indicates that BMS202 does not affect the imaging effect of the radionuclide markers described in this invention.

[0176] In summary, the radionuclide label of the PD-1 / PD-L1 pathway-targeting compound of the present invention introduces a small molecule chain between the targeting group and the coordination structure, which has the following technical effects: improves its pharmacokinetic properties, accelerates the clearance rate of the tracer in non-target tissues; has a higher tumor-to-cytoplasm ratio, increasing the target / non-target ratio; increases its stability in serum; and increases the affinity of the targeting group for PD-1 / PD-L1, etc.

[0177] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A PD-1 / PD-L1 pathway targeting compound, characterized in that, Its chemical structural formula is shown in formula (I) below: Equation (I), R1 is a linker, selected from linker group 1 or linker group 2; The linking group 1 is selected from any of the following chemical structures: ； The linking group 2 has the following structure: ； Where n, n', n'', x, y, z, and w are independent integers between 0 and 10; R1 exists or does not exist; R2 is the labeling group, which is selected from any of the following structures: 。 2. A method for preparing the PD-1 / PD-L1 pathway-targeting compound as described in claim 1, characterized in that, The preparation method includes the following steps: S1, under inert gas protection, palladium acetate and cesium carbonate are added to the first solvent, followed by 3-hydroxymethyl-2-methylbiphenyl and 6-chloro-2-methoxypyridine-3-carboxaldehyde. The mixture is heated for 3.5-4.5 hours. After the reaction is complete, the first solvent is removed and the mixture is purified to obtain reaction intermediate 1. S2, reaction intermediate 1 and ethylenediamine with protecting group R4 are dissolved in the second solvent, anhydrous acetic acid is added, and the reaction is carried out at room temperature for 2.5-3.5 h; then sodium borohydride acetate is added and the reaction is carried out at room temperature for 12-18 h; after the reaction is completed, water is added to quench the reaction, and then the mixture is purified to obtain reaction intermediate 2; S3, reaction intermediate 2 was dissolved in hydrochloric acid-ethyl acetate under ice bath conditions. After removing the ice bath, the mixture was slowly brought back to room temperature and reacted for 1.5-2.5 h. After the reaction was completed, the solvent was removed and the mixture was purified to obtain reaction intermediate 3. S4, when the R1 linker is not present, the reaction intermediate 3 is reacted with the active compound of R2 to obtain the PD-1 / PD-L1 pathway targeting compound. When R1 linker is present and is the linking group 1, reaction intermediate 3 is first linked to R1 linker with R4 protecting group through amidation reaction to obtain reaction intermediate 4, and then deprotected to obtain reaction intermediate 5, which is then reacted with active compound R2 to obtain the PD-1 / PD-L1 pathway targeting compound. When R1 linker is present and is the linking group 2, reaction intermediate 3 is first linked to reaction intermediate 6 through an amidation reaction to obtain reaction intermediate 7, and then reacted with reaction intermediate 8 to obtain the PD-1 / PD-L1 pathway targeting compound. The chemical structures of reaction intermediates 1-8 are as follows: The chemical structure of the R2 active compound is any one of the following: ； The R4 protecting group is selected from one or more of the Boc protecting group, DDE protecting group, or Fmoc protecting group.

3. A pharmaceutically acceptable salt of a PD-1 / PD-L1 pathway-targeting compound, characterized in that, It is obtained by reacting a PD-1 / PD-L1 pathway targeting compound with an acid or base; the PD-1 / PD-L1 pathway targeting compound is the PD-1 / PD-L1 pathway targeting compound as described in claim 1.

4. A PD-1 / PD-L1 pathway-targeting radionuclide marker, characterized in that, It is obtained by coordinating the labeling group R2 in a pharmaceutically acceptable salt of the PD-1 / PD-L1 pathway targeting compound of claim 1 or the PD-1 / PD-L1 pathway targeting compound of claim 3 with a labeled nuclide.

5. A PD-1 / PD-L1 pathway-targeting radionuclide marker according to claim 4, characterized in that, The labeled nuclide is selected from 18 F, 47 Sc、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 89 Zr、 86 Y、 89 Sr, 90 Y、 99m Tc, 105 Rh、 109 Pd, 111 In、 119 Sb、 149 Tb, 153 Sm、 157 Gd, 161 Tb, 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 At least one of Ac.

6. A method for preparing the PD-1 / PD-L1 pathway-targeting radionuclide marker as described in claim 4 or 5, characterized in that, Includes the following steps: By coordinating a PD-1 / PD-L1 pathway-targeting compound or its pharmaceutically acceptable salt with a labeled nuclide, a PD-1 / PD-L1 pathway-targeting nuclide label is obtained.

7. A method for preparing the PD-1 / PD-L1 pathway-targeting radionuclide marker as described in claim 6, characterized in that, The labeled nuclide is introduced into the PD-1 / PD-L1 pathway targeting compound of claim 1 or a pharmaceutically acceptable salt of the PD-1 / PD-L1 pathway targeting compound of claim 3 using either a wet or lyophilization method. The wet method involves dissolving the PD-1 / PD-L1 pathway targeting compound in a buffer solution, deionized water, an organic solvent, or a mixture thereof, adding a solution containing the nuclide to be labeled, and reacting at room temperature to 100 °C for 10-30 minutes. The lyophilization method involves dissolving the PD-1 / PD-L1 pathway targeting compound and a pharmaceutically acceptable excipient in a buffer solution, deionized water, an organic solvent, or a mixture thereof, dispensing the solution into lyophilization containers, freezing and drying the mixture, sealing it to form a lyophilized formulation, adding deionized water or a buffer solution to the lyophilized formulation to dissolve it, then adding a solution containing the nuclide to be labeled, and reacting at room temperature to 100 °C for 10-30 minutes.

8. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient is selected from one or more of the PD-1 / PD-L1 pathway targeting compound of claim 1, the PD-1 / PD-L1 pathway targeting radionuclide label of claim 4, and a pharmaceutically acceptable salt of the PD-1 / PD-L1 pathway targeting compound of claim 3.

9. The use of a PD-1 / PD-L1 pathway targeting compound of claim 1, a targeting compound prepared by the method of claim 2, a pharmaceutically acceptable salt of the PD-1 / PD-L1 pathway targeting compound of claim 3, a PD-1 / PD-L1 pathway targeting radionuclide marker of any one of claims 4-5, a radionuclide marker prepared by the method of any one of claims 6-7, and a pharmaceutical composition of claim 8 in the preparation of therapeutic or diagnostic drugs for PD-1 / PD-L1 pathway-related diseases.

10. The application according to claim 9, characterized in that, The PD-1 / PD-L1 pathway-related diseases are selected from tumors or autoimmune diseases; the tumors are selected from one or more of the following: breast cancer, ovarian cancer, lung cancer, colorectal cancer, prostate cancer, fibrosarcoma, bone and connective tissue sarcoma, bone metastases, renal cell carcinoma, gastric cancer, pancreatic cancer, or skin melanoma; the autoimmune diseases are selected from one or more of the following: lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, Sjögren's syndrome, polyarteritis nodosa, and asthma.

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