WL12 derivative containing functional linker modification, radioactive compound and application thereof

Abstract

The invention relates to the technical field of radiopharmaceuticals and nuclear medicine, in particular to a functional linker modified WL12 derivative, a radioactive compound and application of the functional linker modified WL12 derivative and the radioactive compound. The WL12 derivative containing the functional linker modification, disclosed by the invention, has a specific structural formula. The radioactive compound is obtained by labeling a functional linker-modified WL12 derivative with radionuclide, has relatively strong specific binding capacity to PD-L1 and good stability, can be used in a radioactive preparation, has high uptake in tumors with high expression of PD-L1, and also has the effects of reducing uptake in liver and kidney, improving the bioavailability of the radioactive preparation and improving the bioavailability of the radioactive preparation. The medicine is a novel tumor radiopharmaceutical with popularization significance.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceuticals and nuclear medicine, and in particular to WL12 derivatives containing functional linker modifications, radioactive compounds and applications thereof. Background Art

[0002] In recent years, with the advancement of immunotherapy research, programmed death protein-1 / programmed death protein-ligand-1 (PD-1 / PD-L1) immune checkpoint inhibitor therapy (ICI) has demonstrated unique advantages in cancer treatment. These inhibitors block the PD-1 / PD-L1 immune checkpoint signaling pathway, thereby stimulating the patient's own immune system to eliminate and recognize cancer cells, significantly increasing patient survival rates. However, studies have shown that PD-L1 expression levels in the patient's body significantly influence the efficacy of this type of immunotherapy. Therefore, effective monitoring of PD-L1 expression in cancer patients is crucial.

[0003] Compared to traditional clinical methods, nuclear medicine molecular imaging combined with radionuclide-labeled molecular probes targeting PD-L1 can provide noninvasive, real-time, and dynamic assessment of systemic PD-L1 expression levels in patients. This approach offers unique advantages and broad clinical application prospects. Recent studies have demonstrated that the 14-amino acid cyclic peptide WL12 exhibits high affinity and specificity for the PD-L1 protein, making WL12-based radioactive molecular probes a hot topic in both international and domestic radiopharmaceutical research. However, radionuclide-labeled WL12 currently exhibits high liver and kidney uptake. Clinically, reducing liver and kidney uptake facilitates the provision of tumor PD-L1 images with low background signal. Therefore, reducing non-target tissue uptake of WL12-based radioactive molecular probes is a pressing clinical challenge.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention provides a WL12 derivative containing a functional linker modification, a radioactive compound and its application, so as to solve the problem that the existing radionuclide-labeled WL12 still has a high liver and kidney uptake.

[0006] According to the first aspect of the present invention, the present invention provides a WL12 derivative containing a functional linker modification, the structural formula of which is shown in general formula (I): (I); where X is (PEG4) or (GYK).

[0007] Experimental studies have shown that the WL12 derivative modified with a functional linker, obtained through structural optimization, can specifically bind to PD-L1 after radiolabeling. The introduction of a hydrophilic PEG4 chain significantly increases the water solubility of the labeled compound, thereby reducing liver uptake and enhancing tumor uptake. The GYK chain is specifically recognized and cleaved by the brush border endonuclease in the renal tubules, separating the radionuclide and chelating group from the labeled compound, thereby reducing renal uptake. The radiolabeled compound exhibits high tumor-to-blood and tumor-to-muscle ratios, demonstrating excellent diagnostic efficacy for tumor PD-L1 expression levels.

[0008] In the present invention, when X is When the WL12 derivative is HYNIC-PEG4-WL12; when X is When the WL12 derivative is HYNIC-GYK-WL12.

[0009] In the present invention, the preparation method of the above-mentioned WL12 derivative is the Fmoc solid phase synthesis method, and the specific synthetic raw materials and related reagents are as follows: 1. Protected amino acids and raw materials: Fmoc-Gly-OH, Fmoc-Cys(Mtt)-OH, Fmoc-Orn(Dde)-OH, Fmoc-N-Me-Nle-OH, Fmoc-Trp(Me)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, F moc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-N-Me-Ala-OH, Fmoc-Tyr(tbu)-OH, Boc-Hynic, Fmoc-NH-PEG4-CH2CH2COOH, Boc-Lys(Fmoc)-OH; 2. Condensation reagents: HBTU, HATU, DIEA; 3. Solvents: DMF, DCM, methanol, acetonitrile; 4. Resin: Rink Amide-MBHA Resin; 5. Deprotection reagent: piperidine; 6. Cutting reagents: TFA, TIS, EDT, H2O; 7. Others: nitrogen, anhydrous ether.

[0010] Based on the above raw materials, those skilled in the art can prepare the WL12 derivatives by Fmoc solid phase synthesis.

[0011] Specifically, the preparation method of WL12 derivatives includes the following steps: 1. Resin swelling Place Rink Amide-MBHA resin in a reaction tube, add DCM (10-20 ml / g), and shake for 20-40 minutes to fully swell the resin.

[0012] 2. Connect the first amino acid Add 10-30% piperidine DMF solution (10-20 mL / g) and react for 10-20 minutes. Filter the solvent through a sand core and rinse thoroughly.

[0013] Add a 3-fold molar excess of Fmoc-protected amino acid, dissolve in DMF, then add a 3-fold molar excess of HBTU, and finally add a 10-fold molar excess of DIEA, shake for 50-70 minutes, and block with pyridine and acetic anhydride.

[0014] 3. Deprotection Remove DMF, add 10-30% piperidine DMF solution (10-20 mL / g), react for 3-8 minutes, remove the solution and add 10-30% piperidine DMF solution (10-20 mL / g) again, react for 10-20 minutes.

[0015] 4. Detection Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, heat at 105℃~110℃ for 3-8 minutes, and turn dark blue for a positive reaction.

[0016] 5. Washing The mixture was washed two to three times with DMF, two to three times with methanol, and then two to three times with DMF.

[0017] 6. Condensation Add a 3-fold molar excess of Fmoc-protected amino acid, a 3-fold molar excess of HBTU, and then a 10-fold molar excess of DIEA. Finally, add DMF to dissolve and shake for 40-50 minutes.

[0018] 7. Detection Take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol solution, heat at 105-110°C for 3-8 minutes, and colorless is a negative reaction.

[0019] 8. Washing Wash with DMF, methanol, and then DMF.

[0020] 9. Repeatedly linking amino acids Repeat steps 3 to 8 to connect the desired amino acids from right to left, retaining the Fmoc protecting group of the last amino acid.

[0021] 10. Removal of specific protecting groups Deprotection of Mtt: Remove the solution, add 1-3% TFA / DMF solution, react for 1-3 hours to remove the cysteine ​​protecting group Mtt.

[0022] Alternatively, deprotection by Dde: After washing, remove the solution and add 2%-5% hydrazine hydrate / DMF solution. React for 10-30 minutes to remove the -Orn protecting group Dde.

[0023] 11. Cyclocondensation After washing, add 3-fold molar excess of HATU, 10-fold molar excess of DIEA, and 3-fold molar excess of HOBT, all dissolved in as little DMF as possible, add to the reaction tube, react for 1-3 hours, and then test with ninhydrin for colorless reaction, which is a negative reaction.

[0024] 12. Connect functional linker Repeat steps 3 to 8 to sequentially connect a functional linker (such as PEG4 or GYK) and Boc-Hynic.

[0025] 13. Washing resin Wash the resin and drain it as follows: DMF (5-15 mL / g) twice DCM (5-15 mL / g) three times Methanol (5-15 mL / g) four times Allow to drain for 5-15 minutes.

[0026] 14. Cutting Prepare cutting fluid: TFA 94-96%, water 1-3%, EDT 1-3%, TIS 0.5-1.5%. Cutting time: 150-200 minutes.

[0027] 15. Blow dry and wash The lysate was blown dry with nitrogen as much as possible, ether was precipitated, the supernatant was removed by centrifugation, the precipitate was washed with ether six times, and then evaporated to dryness at room temperature.

[0028] 16. Purification and Preparation Dissolution: Take a small amount of crude product and dissolve it in H2O / ACN.

[0029] Analysis: Take a small amount of sample and analyze it on HPLC analyzer to determine the peak time corresponding to the target peak.

[0030] Preparation: Using C18 reverse phase chromatography system: Wavelength: 220nm Flow Rate: 15mL / min Inj.Vol: 20mL Column Temp: 25℃ Buffer A: 0.1% TFA in water Buffer B: 0.1%TFA in Acetonitrile Collect the target peak solution.

[0031] Detection: Take a small amount of target peak solution in a 1.5mL centrifuge tube for mass spectrometry confirmation and purity detection.

[0032] 17. Freeze-dried The qualified target peak solution is freeze-dried to obtain the finished product.

[0033] 18. Identification A small amount of the finished peptide was taken for mass spectrometry and high performance liquid chromatography to confirm its structure and purity.

[0034] According to a second aspect of the present invention, the present invention further provides a radioactive compound, which is obtained by labeling the WL12 derivative containing a functional linker modification as described in claim 1 with a radioactive nuclide in the presence of a co-ligand; The structural formula of the radioactive compound is shown in general formula (II): , (II); where X is or , M is the radionuclide, and L is the co-ligand component.

[0035] The radioactive compound described in this invention has the advantages of good stability and simple preparation. Its application in imaging the PD-1 / PD-L1 immune checkpoint in tumors demonstrates high tumor uptake and low background signal. This has important scientific significance and broad clinical application prospects in the field of tumor immunotherapy, particularly in the development of novel tumor radiopharmaceuticals that specifically target the PD-1 / PD-L1 immune checkpoint.

[0036] Furthermore, the co-ligand component is: N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris(sulfonic acid) sodium salt (TPPTS); or, N-tris(hydroxymethyl)methylglycine (Tricine) and isonicotinic acid (ISONIC); or, N-tris(hydroxymethyl)methylglycine (Tricine) and 3,5-pyridinedicarboxylic acid (PDA); or, N-tris(hydroxymethyl)methylglycine (Tricine) and 4-pyridinesulfonic acid (4-PSA); or, N-tris(hydroxymethyl)methylglycine (Tricine) and ethylenediamine-N, N' -diacetic acid (EDDA); or, N-tris(hydroxymethyl)methylglycine (Tricine) and sodium diphenylphosphinophenyl-3-sulfonate (TPPMS); or, N-tris(hydroxymethyl)methylglycine (Tricine) and 2-(pyridin-4-yl)acetic acid (PA); or, N-tris(hydroxymethyl)methylglycine (Tricine) and nicotinic acid (NIC); or, N-tris(hydroxymethyl)methylglycine (Tricine) and 3-pyridinesulfonic acid (3-PSA); or, N-tris(hydroxymethyl)methylglycine (Tricine) and 3, 3' Disodium 2-(phenylphosphinodiyl)di(benzene-1-sulfonate) (TPPDS); or, N-Tris(hydroxymethyl)methylglycine (Tricine) and glucoheptonate; or, N-Tris(hydroxymethyl)methylglycine (Tricine) and glucosamine; or, N-Tris(hydroxymethyl)methylglycine (Tricine) and mannitol; or, N-Tris(hydroxymethyl)methylglycine (Tricine) and diphenylphosphinobenzoic acid.

[0037] In the present invention, when X is When L is N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris-sulfonate (TPPTS), the radioactive compound is 99m Tc-PEG4-HYNIC-WL12-tricine / TPPTS; when X is , when L is N-tris(hydroxymethyl)methylglycine (Tricine) and isonicotinic acid (ISONIC), the radioactive compound is 99m Tc-PEG4-HYNIC-WL12-tricine / ISONIC; when X is When L is N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris-sulfonate (TPPTS), the radioactive compound is 99m Tc-GYK-HYNIC-WL12-tricine / TPPTS; when X is , when L is N-tris(hydroxymethyl)methylglycine (Tricine) and isonicotinic acid (ISONIC), the radioactive compound is 99m Tc-GYK-HYNIC-WL12-tricine / ISONIC.

[0038] Furthermore, the co-ligand components are: N-tricine and sodium triphenylphosphine tris-metasulfonate; or N-tricine and isonicotinic acid.

[0039] Furthermore, the radioactive nuclide is a metal radionuclide; the metal radionuclide includes 99m Tc, 99 Tc, 94m Tc and 94 Tc.

[0040] Furthermore, the radioactive compound has a radiochemical purity greater than 95%, is hydrophilic, and has good in vitro stability.

[0041] According to the third aspect of the present invention, the present invention also provides a method for preparing the above-mentioned radioactive compound, wherein the radionuclide is 99m When Tc, the preparation method of the radioactive preparation comprises: WL12 derivatives, with different co-ligands, were combined with Na 99m The TcO4 eluent is reacted at 90-110°C for 20-40 minutes to obtain the radioactive preparation.

[0042] Preferably, the reaction time is 30 min.

[0043] In the specific implementation process, use 99m When other radionuclides besides Tc are used, those skilled in the art can prepare the radioactive compound by referring to the above-mentioned method for preparing the radioactive compound.

[0044] According to a fourth aspect of the present invention, the present invention further provides a radioactive preparation comprising the above-mentioned radioactive compound.

[0045] Furthermore, it also includes acceptable carriers and / or excipients in the fields of radiopharmaceuticals and nuclear medicine.

[0046] According to a fifth aspect of the present invention, the present invention also provides use of the above-mentioned radioactive compound or the above-mentioned radioactive preparation in the preparation of immunotherapy-related drugs.

[0047] Furthermore, the immunotherapy-related drug is used to detect the expression level of PD-L1; or, the immunotherapy-related drug diagnoses and / or treats the disease by specifically targeting PD-L1.

[0048] The application of the present invention has important scientific significance and broad clinical application prospects in the field of diagnosis of tumors with high PD-L1 expression and / or treatment of tumors with high PD-L1 expression.

[0049] Beneficial effects of the present invention: The present invention provides a radioactive compound derived from a WL12 derivative modified with PEG4 or GYK and labeled with a radionuclide. It exhibits strong specific binding to PD-L1 and excellent stability. When used in radioactive preparations, it exhibits high uptake in tumors that overexpress PD-L1, while exhibiting reduced uptake in the liver and kidneys. This makes it a promising new tumor radiopharmaceutical. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0051] Figure 1 This is the mass spectrometry characterization of the WL12 derivative in Example 1 of the present invention.

[0052] Figure 2 This is a high performance liquid chromatography characterization chart of the WL12 derivative in Example 1 of the present invention.

[0053] Figure 3 This is the mass spectrometry characterization diagram of the WL12 derivative in Example 2 of the present invention.

[0054] Figure 4 This is a high performance liquid chromatography characterization chart of the WL12 derivative in Example 2 of the present invention.

[0055] Figure 5 For the test examples of the present invention 99m Figure 3. SPECT / CT imaging experimental results of Tc-PEG4-WL12-TPPTS complex in tumor-bearing mice.

[0056] Figure 6 For the test examples of the present invention 99m Figure 3. SPECT / CT imaging experimental results of Tc-PEG4-WL12-ISONIC complex in tumor-bearing mice.

[0057] Figure 7 For the test examples of the present invention 99m Figure 3. SPECT / CT imaging experimental results of Tc-GYK-WL12-TPPTS complex in tumor-bearing mice.

[0058] Figure 8 For the test examples of the present invention 99m Figure 3. SPECT / CT imaging experimental results of Tc-GYK-WL12-ISONIC complex in tumor-bearing mice. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0060] Unless otherwise specified, the various raw materials used in the examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0061] Example 1 This example provides a WL12 derivative modified with a functional linker, HYNIC-PEG4-WL12, the preparation method of which comprises the following steps: Protected amino acids and raw materials: Fmoc-Gly-OH, Fmoc-Cys(Mtt)-OH, Fmoc-Orn(Dde)-OH, Fmoc-N-Me-Nle-OH, Fmoc-Trp(Me)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(t Bu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-N-Me-Ala-OH, Fmoc-Tyr(tbu)-OH, Boc-Hynic, Fmoc-NH-PEG4-CH2CH2COOH.

[0062] 1. Resin swelling: Place Rink Amide-MBHA Resin into a reaction tube, add DCM (15 ml / g), and shake for 30 minutes.

[0063] 2. Attach the first amino acid: Add 20% piperidine in DMF (15 mL / g) and incubate for 15 minutes. Filter the solvent through a sand core and rinse thoroughly. Add a 3-fold molar excess of the Fmoc-protected amino acid, dissolve in DMF, add a 3-fold excess of HBTU, and then add a 10-fold molar excess of DIEA. Shake for 60 minutes. Block with pyridine and acetic anhydride.

[0064] 3. Deprotection: Remove DMF, add 20% piperidine in DMF (15 mL / g) for 5 min, remove and then add 20% piperidine in DMF (15 mL / g) for 15 min.

[0065] IV. Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105°C-110°C for 5 minutes. A dark blue color indicates a positive reaction.

[0066] 5. Washing: DMF (10 mL / g) twice, methanol (10 mL / g) twice, and DMF (10 mL / g) twice.

[0067] 6. Condensation: Add a 3-fold molar excess of Fmoc-protected amino acid, a 3-fold molar excess of HBTU, and then a 10-fold molar excess of DIEA. Finally, dissolve in DMF and shake for 45 minutes.

[0068] 7. Detection: Take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol solution, and heat at 105-110°C for 5 minutes. A colorless reaction indicates a negative reaction.

[0069] 8. Washing: DMF (10 mL / g) once, methanol (10 mL / g) twice, and DMF (10 mL / g) twice.

[0070] 9. Repeat steps 3 to 8, connecting from right to left, retaining the Fmoc protecting group of the last amino acid.

[0071] 10. Deprotection of Mtt: Remove the solution and add 2% TFA / DMF solution (10 mL / g) and react for 2 h to remove the cysteine ​​protecting group Mtt.

[0072] 11. After washing, remove the solvent, add K2CO3 / DMF solution, then add iodoacetic acid, and react at room temperature under nitrogen for 4 hours.

[0073] 12. Remove the solvent and add 20% piperidine / DMF solution (15 mL / g) for 5 minutes. Remove the solvent and add 20% piperidine / DMF solution (15 mL / g) for 15 minutes to remove the Fmoc protecting group of the last amino acid. A dark blue color in the ninhydrin assay indicates a positive reaction.

[0074] 13. Cyclocondensation: After washing, add a 3-fold molar excess of HATU, a 10-fold molar excess of DIEA, and a 3-fold molar excess of HOBT, all dissolved in as little DMF as possible, to the reaction tube and react for 2 hours. A colorless reaction is considered negative by the ninhydrin test.

[0075] 14. Deprotection of Dde: After washing, remove the solution and add 2%-5% hydrazine hydrate / DMF solution (10mL / g), react for 20min, and remove -Orn Protecting group Dde, and then ninhydrin detection turns dark blue for positive reaction.

[0076] 15. Repeat steps 3 to 8 to sequentially link Fmoc-NH-PEG4-CH2CH2COOH and Boc-Hynic.

[0077] 16. Wash the resin as follows and drain: DMF (10 mL / g) twice, DCM (10 mL / g) three times, and methanol (10 mL / g) four times, draining for 10 min.

[0078] 17. Cutting: Prepare cutting solution (10 mL / g): TFA 95%, water 2%, EDT 2%, TIS 1%. Cutting time: 180 min.

[0079] 18. Drying and washing: Dry the lysate as much as possible with nitrogen gas, precipitate with ether, remove the supernatant by centrifugation, wash the precipitate with ether six times, and then evaporate to dryness at room temperature.

[0080] 19. Purification: 1. Dissolve a small amount of crude product in H2O / ACN. 2. Analyze a small amount on an HPLC analyzer to determine the elution time of the target peak. 3. Analyze using a C18 reversed-phase preparative chromatography system: Wavelength: 220 nm; Flow Rate: 15 mL / min; Inj. Vol: 20 mL; Column Temp: 25°C; Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile; collect the target peak solution. 4. Use a 1.5 mL centrifuge tube to obtain a small amount of the target peak solution for mass spectrometry confirmation and purity testing.

[0081] 20. Freeze-dry the qualified target peak solution to obtain the finished product.

[0082] 21. Identification: Take a small amount of the finished peptide and perform mass spectrometry and high performance liquid chromatography. The characterization diagram is shown in Figure 1 and Figure 2 .

[0083] This example further provides a radioactive preparation obtained using HYNIC-PEG4-WL12 99m Tc-PEG4-HYNIC-WL12-tricine / TPPPTS and 99m Tc-PEG4-HYNIC-WL12-tricine / ISONIC, the preparation method thereof comprises the following steps: Take 5 μg of ligand HYNIC-PEG4-WL12, 3 mg of triphenylphosphine tris(-)sulfonate (TPPTS) or 2 mg of isonicotinic acid (ISONIC), 3 mg of N-tris(hydroxymethyl)methylglycine (Tricine), 10 μg of SnCl2·2H2O (30 μg of SnCl2·2H2O is used for ISONIC labeling), add 0.4 mL of normal saline to dissolve 0.5 mL of succinate buffer (0.2 mol / L, pH = 4.6), and then add 0.1 mL of Na 99m The total volume of TcO4 eluent was controlled within 1 mL, and the reaction was carried out in a boiling water bath for 30 min to obtain the target complex. 99m Tc-PEG4-WL12-TPPTS and 99m Tc-PEG4-HYNIC-WL12-tricine / ISONIC. The radiochemical purity was >95% as determined by iTLC and HPLC.

[0084] Example 2 This example provides a WL12 derivative modified with a functional linker, HYNIC-GYK-WL12, the preparation method of which comprises the following steps: Protected amino acid raw materials include: Fmoc-Gly-OH, Fmoc-Cys(Mtt)-OH, Fmoc-Orn(Dde)-OH, Fmoc-N-Me-Nle-OH, Fmoc-Trp(Me)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu )-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-N-Me-Ala-OH, Fmoc-Tyr(tbu)-OH, Fmoc-Gly-OH, Boc-Lys(Fmoc)-OH, Boc-Hynic.

[0085] 1. Resin swelling: Place Rink Amide-MBHA Resin into a reaction tube, add DCM (15 mL / g), and shake for 30 minutes.

[0086] 2. Attach the first amino acid: Add 20% piperidine in DMF (15 mL / g) and incubate for 15 minutes. Filter the solvent through a sand core and rinse thoroughly. Add a 3-fold molar excess of the Fmoc-protected amino acid, dissolve in DMF, add a 3-fold excess of HBTU, and then add a 10-fold molar excess of DIEA. Shake for 60 minutes. Block with pyridine and acetic anhydride.

[0087] 3. Deprotection: Remove DMF, add 20% piperidine in DMF (15 mL / g) for 5 min, remove and then add 20% piperidine in DMF (15 mL / g) for 15 min.

[0088] IV. Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105-110°C for 5 minutes. A dark blue color indicates a positive reaction.

[0089] 5. Washing: DMF (10 mL / g) twice, methanol (10 mL / g) twice, and DMF (10 mL / g) twice.

[0090] 6. Condensation: Add a 3-fold molar excess of Fmoc-protected amino acid, a 3-fold molar excess of HBTU, and then a 10-fold molar excess of DIEA. Finally, dissolve in DMF and shake for 45 minutes.

[0091] 7. Detection: Take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol solution, and heat at 105-110°C for 5 minutes. A colorless reaction indicates a negative reaction.

[0092] 8. Washing: DMF (10 mL / g) once, methanol (10 mL / g) twice, and DMF (10 mL / g) twice.

[0093] 9. Repeat steps 3 to 8, connecting from right to left, retaining the Fmoc protecting group of the last amino acid.

[0094] 10. Deprotection of Mtt: Remove the solution and add 2% TFA / DMF solution (10 mL / g) and react for 2 h to remove the cysteine ​​protecting group Mtt.

[0095] 11. After washing, remove the solvent, add K2CO3 / DMF solution, then add iodoacetic acid, and react at room temperature under nitrogen for 4 hours.

[0096] 12. Remove the solvent and add 20% piperidine / DMF solution (15 mL / g) for 5 minutes. Remove the solvent and add 20% piperidine / DMF solution (15 mL / g) for 15 minutes to remove the Fmoc protecting group of the last amino acid. A dark blue color in the ninhydrin assay indicates a positive reaction.

[0097] 13. Cyclocondensation: After washing, add a 3-fold molar excess of HATU, a 10-fold molar excess of DIEA, and a 3-fold molar excess of HOBT, all dissolved in as little DMF as possible, to the reaction tube and react for 2 hours. A colorless reaction is considered negative by the ninhydrin test.

[0098] 14. Deprotection of Dde: After washing, remove the solution and add 2%-5% hydrazine hydrate / DMF solution (10mL / g), react for 20min, and remove -Orn Protecting group Dde, and then ninhydrin detection turns dark blue for positive reaction.

[0099] 15. Repeat steps 3 to 8 to sequentially link Fmoc-Gly-OH, Fmoc-Tyr(tbu)-OH, Boc-Lys(Fmoc)-OH, and Boc-Hynic.

[0100] 16. Wash the resin as follows and drain: DMF (10 mL / g) twice, DCM (10 mL / g) three times, and methanol (10 mL / g) four times, draining for 10 min.

[0101] 17. Cutting: Prepare cutting solution (10 mL / g): TFA 95%, water 2%, EDT 2%, TIS 1%. Cutting time: 180 min.

[0102] 18. Drying and washing: Dry the lysate as much as possible with nitrogen gas, precipitate with ether, remove the supernatant by centrifugation, wash the precipitate with ether six times, and then evaporate to dryness at room temperature.

[0103] 19. Purification: 1. Dissolve a small amount of crude product in H2O / ACN. 2. Analyze a small amount on an HPLC analyzer to determine the elution time of the target peak. 3. Analyze using a C18 reversed-phase preparative chromatography system: Wavelength: 220 nm; Flow Rate: 15 mL / min; Inj. Vol: 20 mL; Column Temp: 25°C; Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in acetonitrile; collect the target peak solution. 4. Use a 1.5 mL centrifuge tube to obtain a small amount of the target peak solution for mass spectrometry confirmation and purity testing.

[0104] 20. Freeze-dry the qualified target peak solution to obtain the finished product.

[0105] 21. Identification: Take a small amount of the finished peptide and perform mass spectrometry and high performance liquid chromatography. The characterization diagram is shown in Figure 3 and Figure 4 .

[0106] This example further provides a radioactive preparation obtained using HYNIC-GYK-WL12 99m Tc-GYK-HYNIC-WL12-tricine / TPPPTS and 99mTc-GYK-HYNIC-WL12-tricine / ISONIC, the preparation method thereof comprises the following steps: Take 5 μg of ligand HYNIC-GYK-WL12, 3 mg of triphenylphosphine tris(-)sulfonate (TPPTS) or 2 mg of isonicotinic acid (ISONIC), 3 mg of N-tris(hydroxymethyl)methylglycine (Tricine), 10 μg of SnCl2·2H2O (30 μg of SnCl2·2H2O is used for ISONIC labeling), add 0.4 mL of normal saline to dissolve 0.5 mL of succinate buffer (0.2 mol / L, pH = 4.6), and then add 0.1 mL of Na 99m The total volume of TcO4 eluent was controlled within 1 mL, and the reaction was carried out in a boiling water bath for 30 min to obtain the target complex. 99m Tc-GYK-WL12-TPPTS and 99m Tc-GYK-HYNIC-WL12-tricine / ISONIC. The radiochemical purity was >95% as determined by iTLC and HPLC.

[0107] Test example This test example provides the 99m Tc-PEG4-HYNIC-WL12-tricine / TPPPTS、 99m Tc-PEG4-HYNIC-WL12-tricine / ISONIC, 99m Tc-GYK-HYNIC-WL12-tricine / TPPPTS and 99m The performance of the Tc-GYK-HYNIC-WL12-tricine / ISONIC complex was tested as follows: (1) Identification of complexes Instant Thin Layer Chromatography (iTLC) Identification: The development system is: instant thin layer chromatography as the support, ACD buffer (pH = 5) as the developing agent. Under this system, the R f The values ​​are shown in Table 1.

[0108] Table 1 Chromatographic results of each component of the complex (R f value)

[0109] The radiochemical purity of the labeled substance determined by the above chromatographic identification was >95%.

[0110] (2) Determination of lipid-water partition coefficient of complex Place 0.85 mL of n-octanol and 0.80 mL of pH 7.4 (0.01 M) phosphate buffer in a 2 mL centrifuge tube. Add 0.05 mL of the complex solution to the tube, cover with a stopper, vortex thoroughly to mix, and centrifuge for 5 minutes (3000 rpm). Take 0.2 mL from each of the organic and aqueous phases, measure the radioactivity counts in both phases, and calculate the log D 7.4 value( D = Radioactivity of the organic phase / Radioactivity of the aqueous phase). The lipid-water partition coefficient of the complex is shown in Table 2: Table 2 Results of lipid-water partition coefficients of complexes

[0111] The results of lipid-water partition coefficients showed that the complexes were all water-soluble substances.

[0112] (3) In vitro stability determination of the complex The radiochemical purity of the labeled complex was measured after being placed in mouse serum at room temperature and at 37°C for 4 hours. The experimental results showed that the radiochemical purity of the complex was greater than 95% after being placed in mouse serum at room temperature and at 37°C for 4 hours, indicating good in vitro stability.

[0113] (4) Biodistribution experiment of the complex in tumor-bearing mice To verify that the complex can achieve low background uptake while retaining PD-L1 targeting, uptake experiments were conducted in MC38-B7H1 or MC38 tumor-bearing mice, and inhibition experiments were performed using the peptide inhibitor WL12 that was not modified by HYNIC. The experiments included control, low-expression, and inhibition groups. In the control and low-expression groups, 0.1 mL of the complex solution (0.74 MBq) was injected into the tail vein of each female C57BL / 6N mouse bearing MC38-B7H1 or MC38 tumor, respectively; in the inhibition group, 50 μg of WL12 and 0.1 mL of HYNIC were injected into the tail vein of each female C57BL / 6N mouse bearing MC38-B7H1 tumor. 99m Tc-WL12-M solution (0.74 MBq). Two hours after administration, mice were sacrificed, and tissues and organs, including kidneys, heart, lungs, blood, and muscle, were removed, cleaned, weighed, and radioactivity counted using a γ-counter. The percent injected dose per gram (% ID / g) in the kidney was calculated. Biodistribution results are shown in Tables 3-6.

[0114] Table 3 99mBiodistribution of Tc-PEG4-HYNIC-WL12-tricine / TPPTS in MC38-B7H1 and MC38 tumor-bearing mice over the entire time period (%ID / g, mean ± SD, n = 5, t = 2 h)

[0115] Table 4 99m Biodistribution of Tc-PEG4-HYNIC-WL12-tricine / ISONIC over the entire time period in MC38-B7H1 and MC38 tumor-bearing mice (%ID / g, mean ± SD, n = 5, t = 2 h)

[0116] Table 5 99m Biodistribution of Tc-GYK-HYNIC-WL12-tricine / TPPTS in MC38-B7H1 and MC38 tumor-bearing mice over the entire time period (%ID / g, mean ± SD, n = 5, t = 2 h)

[0117] Table 6 99m Biodistribution of Tc-GYK-HYNIC-WL12-tricine / ISONIC over the entire time period in MC38-B7H1 and MC38 tumor-bearing mice (%ID / g, mean ± SD, n = 5, t = 2 h)

[0118] As can be seen from Table 3-6, in the control group, MC38-B7H1 tumors, as tumors with high expression of PD-L1, 99m Tc-PEG4-HYNIC-WL12-tricine / TPPPTS、 99m Tc-PEG4-HYNIC-WL12-tricine / ISONIC, 99m Tc-GYK-HYNIC-WL12-tricine / TPPPTS and 99m Tc-GYK-HYNIC-WL12-tricine / ISONIC showed high tumor uptake 2 hours after administration, while uptake in other non-target tissues and organs was low and blood clearance was fast. 99m Compared with Tc-WL12-TPPTS, the introduction of PEG4 chain significantly reduced 99mThe liver uptake of Tc-PEG4-HYNIC-WL12-tricine / TPPTS was significantly improved (2 h after administration: 12.00 ± 1.11 vs. 7.52 ± 1.68 %ID / g), which improved the tumor / liver ratio. 99m Compared with Tc-WL12-ISONIC, the introduction of PEG4 chain 99m Tc-PEG4-HYNIC-WL12-tricine / ISONIC demonstrated a 2.63-fold increase in tumor uptake (2 hours after administration: 6.63 ± 0.80 vs. 24.10 ± 2.37 %ID / g). The introduction of the GYK chain reduced renal uptake of the probe molecule to varying degrees. Co-injection of the inhibitor WL12 significantly reduced MC38-B7H1 tumor uptake, demonstrating a robust inhibitory effect, demonstrating specific binding of the four complexes to PD-L1.

[0119] (5) SPECT / CT imaging experiments of the complex in tumor-bearing mice To further verify that the complex is a tumor imaging agent that specifically targets PD-L1, SPECT / CT imaging experiments were performed using the unmodified peptide inhibitor WL12 and the radiolabeled complex. The experiment included a control group and an inhibition group. In the control group, 0.1 mL of the complex solution (37 MBq) was injected into the tail vein of each tumor-bearing female C57BL / 6N mouse; in the inhibition group, 50 μg of WL12 and 0.1 mL of the complex solution (37 MBq) were injected into the tail vein of each tumor-bearing female C57BL / 6N mouse. SPECT / CT imaging was performed 2 hours after administration. The results are shown in the figure below. Figure 5-8 shown.

[0120] MC38-B7H1 tumor is a tumor model with high expression of PD-L1. SPECT / CT imaging results in tumor-bearing mice showed that 99m The Tc-PEG4-HYNIC-WL12-tricine / ISONIC complex enabled clear tumor delineation. Co-injection of the inhibitor WL12 resulted in no visible tumors, demonstrating a significant inhibitory effect, demonstrating specific targeting of PD-L1. Furthermore, no clear tumor delineation was observed in mice bearing MC38 tumors that underexpress PD-L1. Besides the tumor, no significant radioactive signal was detected in non-target tissues.

[0121] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention include, in addition to the WL12 polypeptide modified with a PEG4 chain or a GYK chain involved in the present invention, HYNIC-WL12 polypeptides containing other different linker modifications and the corresponding co-ligands M are N-tris(hydroxymethyl)methylglycine (Tricine) and ethylenediamine-N, N'-diacetic acid (EDDA), N-tris(hydroxymethyl)methylglycine (Tricine) and sodium diphenylphosphinophenyl-3-sulfonate (TPPMS), N-tris(hydroxymethyl)methylglycine (Tricine) and 2-(pyridin-4-yl)acetic acid (PA), N-tris(hydroxymethyl)methylglycine (Tricine) and nicotinic acid (NIC), N-tris(hydroxymethyl)methylglycine (Tricine) and 3-pyridinesulfonic acid (3-PSA), N-tris(hydroxymethyl)methylglycine (Tricine) and 3, The radioactive preparations obtained by radionuclide labeling of HYNIC-containing polypeptides or HYNIC-WL12 polypeptides with different linker modifications and co-ligand M with 3'-(phenylphosphinodiyl)di(benzene-1-sulfonic acid) disodium (TPPDS), N-tris(hydroxymethyl)methylglycine (Tricine) and glucoheptonate, N-tris(hydroxymethyl)methylglycine (Tricine) and glucosamine, N-tris(hydroxymethyl)methylglycine (Tricine) and mannitol, and N-tris(hydroxymethyl)methylglycine (Tricine) and diphenylphosphinobenzoic acid all fall within the scope of protection claimed in the present invention.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A WL12 derivative modified with a functional linker, characterized in that: Its structural formula is shown in general formula (I): (I); where X is or .

2. The WL12 derivative modified with a functional linker according to claim 1, characterized in that: The WL12 derivative was prepared by Fmoc solid phase synthesis.

3. The WL12 derivative modified with a functional linker according to claim 2, characterized in that The Fmoc protected amino acid used in the Fmoc solid phase synthesis method is selected from one or more of Fmoc-Gly-OH, Fmoc-Cys(Mtt)-OH, Fmoc-Orn(Dde)-OH, Fmoc-N-Me-Nle-OH, Fmoc-Trp(Me)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-N-Me-Ala-OH, Fmoc-Tyr(tbu)-OH, Boc-Hynic, Fmoc-NH-PEG4-CH2CH2COOH and Boc-Lys(Fmoc)-OH; And / or, the condensation reagents used in the Fmoc solid phase synthesis method include HBTU, HATU and DIEA; And / or, the solvent used in the Fmoc solid phase synthesis method is selected from one or more of DMF, DCM, methanol and acetonitrile; And / or, the resin used in the Fmoc solid phase synthesis method is Rink Amide-MBHA resin; And / or, the deprotection reagent used in the Fmoc solid phase synthesis method is piperidine; And / or, the cleavage reagent used in the Fmoc solid phase synthesis method consists of TFA, TIS, EDT and H2O.

4. A radioactive compound, characterized in that The radioactive compound is obtained by labeling the WL12 derivative containing a functional linker modification as described in any one of claims 1 to 3 with a radionuclide in the presence of a co-ligand; The structural formula of the radioactive compound is shown in general formula (II): , (II); where X is or , M is the radionuclide, and L is the co-ligand component.

5. The radioactive compound according to claim 4, characterized in that The co-ligand components are: N-tris(hydroxymethyl)methylglycine and sodium triphenylphosphine tris-metasulfonate; or, N-tris(hydroxymethyl)methylglycine and isonicotinic acid; or, N-tris(hydroxymethyl)methylglycine and 3,5-pyridinedicarboxylic acid; or, N-tris(hydroxymethyl)methylglycine and 4-pyridinesulfonic acid; or, N-tris(hydroxymethyl)methylglycine and ethylenediamine-N,N'-diacetic acid; or, N-tris(hydroxymethyl)methylglycine and sodium diphenylphosphine benzene-3-sulfonate; or, N-tris(hydroxymethyl)methylglycine and 2-(pyridin-4-yl)acetic acid; or, N-tris(hydroxymethyl)methylglycine and nicotinic acid; or, N-tris(hydroxymethyl)methylglycine and 3-pyridinesulfonic acid; or, N-tris(hydroxymethyl)methylglycine and 3,3' Disodium 1-(phenylphosphinodiyl)di(benzene-1-sulfonate); or, N-tricine and glucoheptonic acid; or, N-tricine and glucosamine; or, N-tricine and mannitol; or, N-tricine and diphenylphosphinobenzoic acid.

6. The radioactive compound according to claim 5, characterized in that The co-ligand components are: N-tricine and sodium triphenylphosphine tris-metasulfonate; or N-tricine and isonicotinic acid; And / or, the radionuclide is a metal radionuclide; the metal radionuclide includes 99m Tc, 99 Tc, 94m Tc and 94 One or more of Tc.

7. A radioactive preparation, characterized in that The invention comprises the radioactive compound according to any one of claims 4 to 6.

8. The radioactive preparation according to claim 7, characterized in that Also included are acceptable carriers and / or excipients in the fields of radiopharmaceuticals and nuclear medicine.

9. Use of the radioactive compound according to any one of claims 4 to 6, or the radioactive preparation according to claim 7 or 8, in the preparation of immunotherapy-related drugs.

10. The use according to claim 9, characterized in that The immunotherapy-related drug is used to detect the expression level of PD-L1; alternatively, the immunotherapy-related drug diagnoses and / or treats the disease by specifically targeting PD-L1.

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