Cyclic peptide conjugate of targeted fibroblast activating protein and application of cyclic peptide conjugate

Abstract

The invention relates to the technical field of biological medicine, in particular to a cyclic peptide conjugate and application thereof. The invention provides a cyclic peptide conjugate targeting fibroblast activating protein and application of the cyclic peptide conjugate. The cyclopeptide conjugate not only improves the uptake value of the fibroblast activated protein targeting compound at the tumor site, but also reduces the uptake value at the kidney site.

Description

A cyclic peptide conjugate targeting a fibroblast activation protein and its application Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a cyclic peptide conjugate targeting a fibroblast activation protein and its application. Background Technology

[0002] Fibroblast activation protein (FAP, also known as FAPα) is significantly overexpressed in cancer-associated fibroblasts within the solid tumor microenvironment, but is usually difficult to detect in most normal tissues and benign proliferative processes. As a major component of the tumor stroma, cancer-associated fibroblasts can drive tumor growth and enhance its invasiveness, thus becoming an important target in current anti-tumor strategies. The specific enrichment of FAP on cancer-associated fibroblasts makes it a potential molecular target for tumor diagnosis and intervention targeting this cell type.

[0003] FAP is a type II transmembrane serine protease with homologous structure to dipeptidyl peptidase 4. It possesses the catalytic functions of both dipeptidyl peptidase and proline endopeptidase, participating in the regulation of dynamic extracellular matrix remodeling and tissue fibrosis. Located on the surface of cancer-associated fibroblasts, FAP promotes tumor development through multiple mechanisms: on the one hand, it mediates extracellular matrix remodeling and activates signaling pathways such as VEGF / AKT / ERK, thereby enhancing the directional migration of tumor cells along the fibrous network; on the other hand, it participates in angiogenesis, constructing a microenvironmental barrier that supports tumor growth and inhibiting the immune function of effector T cells. The expression level of FAP in the tumor stroma is directly induced by malignant tumor lesions, and its expression intensity has a clear positive correlation with adverse clinical outcomes in patients. Based on these characteristics, FAP has been regarded as a marker molecule of activated fibroblasts in malignant tumors, granulation tissue, and fibrotic lesions.

[0004] Among them, FAP-2286 is the most representative therapeutic radioligand targeting fibroblast activation proteins. It carries different nuclides (such as...) through a single molecular structure. 68 Ga is used in PET imaging. 177 Lu (used for treatment) has achieved high sensitivity and targeted radiotherapy for various solid tumors that highly express FAP, and multiple Phase I / II clinical trials are currently underway globally. Although preclinical and early clinical data show high tumor uptake and promising application prospects, some limitations have also been revealed in clinical trials: most notably, FAP-2286 exhibits high renal uptake and retention in vivo, which may lead to unnecessary radiation doses to the kidneys during treatment, potentially limiting its effectiveness. 177The safe dosage of therapeutic radionuclides such as Lu is uncertain, but there is a potential risk of nephrotoxicity. Other common drawbacks include some physiological uptake by the liver and salivary glands, and the need for further validation through larger-scale clinical trials regarding long-term safety and optimal dosing regimens.

[0005] Therefore, it is necessary to obtain better FAP-targeting molecules that, while having high tumor targeting, further reduce background uptake by normal tissues, thereby potentially improving the diagnostic contrast of such probes and reducing the toxicity of radionuclide therapy. Summary of the Invention

[0006] In view of this, the present invention aims to provide a cyclic peptide conjugate targeting a fibroblast activation protein and its application. The cyclic peptide conjugate not only increases the uptake of FAP-2286 in tumors but also reduces its uptake in the kidneys.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a cyclic peptide conjugate targeting fibroblast activation proteins, wherein the cyclic peptide conjugate has the structure shown in Formula I: Formula I; in Formula I, R1, R2, R3, R4, R5, and R6 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, , , , , , , , , , , , , , , , , , , , or In Formula I, a, b, c, d, e, f, and the -CO(CH2) are... n In NH-, n is an independent integer between 0 and 8; in Formula I, R8 is H, a halogen, or a C1-C4 alkyl group; in Formula I, X is an effector.

[0008] Preferably, the cyclic peptide conjugate has the structure shown in Formula II: Formula II; in Formula II, R1, R2, and R3 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, , , , , , , , , , , , , , , , , , , , or In Formula II, a, b, c, and the -CO(CH2) mentioned above... n In NH-, n is an independent integer between 0 and 8; in Equation II, X is an effector.

[0009] Preferably, the cyclic peptide conjugate has the structure shown in Formula III: Formula III; in Formula III, R1, R2, and R3 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, , , , , , , , , , , , , , , , , , , , or In formula III, a, b, c, and the -CO(CH2) mentioned above... n In NH-, n is an independent integer between 0 and 8; in Equation III, X is an effector.

[0010] Preferably, the effector includes a portion derived from a chromophore, a chelating agent, or a portion derived from a drug.

[0011] Preferably, the portion derived from the chromophore includes a phosphor or fluorophore; the chelating agent includes a radionuclide and an organic chelating ligand; and the portion derived from the drug includes a portion derived from a cytotoxic drug.

[0012] Preferably, the radionuclide includes diagnostic radionuclides or therapeutic radionuclides; the diagnostic radionuclides include 68 Ga、 18 F-Al, 64 Cu、 99m Tc, 89 Zr、 44 Sc、 111 In、 45 Ti、 52 Mn, 59 Fe、 94m Tc, 67 Ga、 82m Rb、 86 Y、 131 I and 123 One or more of I; the therapeutic radionuclide includes 177 Lu、 90 Y、 131 I, 153 Sm、 67 Cu、 89 Sr、 177 Yb、 47 Sc、 186 Re、 161 Tb, 188 Re、 149 Pm, 212 Pb, 211 At、 223 Ra、 225 Ac and 111 One or more of In; the organic chelating ligand includes , or .

[0013] Preferably, when the effector is a chelating agent, the cyclic peptide conjugate is... , , , , or .

[0014] The present invention also provides the application of the cyclic peptide conjugate described in the above technical solution in the preparation of cancer diagnostic reagents, bone metastatic solid tumor diagnostic reagents, cancer treatment drugs, or bone metastatic solid tumor diagnostic reagents.

[0015] Preferably, the cancer in the cancer diagnostic reagent or cancer treatment drug is a hypoxic cancer.

[0016] Preferably, the hypoxic cancer is prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small bowel cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumor, thyroid cancer, or intestinal cancer.

[0017] This invention provides a cyclic peptide conjugate targeting a fibroblast-activating protein and its pharmaceutically acceptable salt. The cyclic peptide conjugate and its pharmaceutically acceptable salt not only increase the uptake of FAP-2286 in tumors but also reduce its uptake in the kidneys. Attached Figure Description

[0018] Figure 1 shows the LC-MS (ESI) spectra of the cyclic peptide conjugates prepared in Examples 1-6; Figure 2 shows the HPLC spectra of the cyclic peptide conjugates prepared in Examples 1-6; Figure 3 shows... 68 PET imaging of Ga-Ga-NOTA-FAP-2286 mouse tumors; Figure 4 shows [ 68 PET imaging of Ga-Ga-NOTA-ZJUIQB-G001 mouse tumors; Figure 5 shows [ 68 PET imaging of Ga-Ga-NOTA-ZJUIQB-G002 mouse tumors; Figure 6 shows [ 68 PET imaging of Ga-Ga-NOTA-ZJUIQB-G003 mouse tumors; Figure 7 shows [ 68 PET imaging of Ga-Ga-NOTA-ZJUIQB-G004 mouse tumors; Figure 8 shows [ 68 PET imaging of tumors in Ga]-Ga-NOTA-ZJUIQB-G005 mice. Detailed Implementation

[0019] This invention provides a cyclic peptide conjugate targeting a fibroblast activation protein, the cyclic peptide conjugate having the structure shown in Formula I: Formula I; in Formula I, R1, R2, R3, R4, R5, and R6 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, , , , , , , , , , , , , , , , , , , , or In Formula I, a, b, c, d, e, f, and the -CO(CH2) are... n In NH-, n is an independent integer between 0 and 8; in Formula I, R8 is H, a halogen, or a C1-C4 alkyl group; in Formula I, X is an effector.

[0020] In this invention, the halogen is preferably F, Cl, Br or I.

[0021] In this invention, the C1-C4 alkyl group is preferably a substituted or unsubstituted C1-C4 alkyl group; when the C1-C4 alkyl group is unsubstituted, it is preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, primary butyl, or tert-butyl; when the C1-C4 alkyl group is substituted, the substituent is preferably C1-C4 alkyl, -O-(C1-C4)alkyl, -aryl, -CO-R', -O-CO-R', -COOR', -CONH2, -CONHR', -CONR'2, -NH-CO-R', -SO2-R', -SO-R', -OH, -halogen, -NH2, -NHR', -NR'2, and -CN; wherein each R' is independently selected from -(C1-C4)alkyl or halogen. The substituted C1-C4 alkyl group is more preferably -CF3 or -CH2CF3.

[0022] In this invention, the cyclic peptide conjugate preferably has the structure shown in Formula II: Formula II; in Formula II, R1, R2, and R3 are preferably -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, or -CO(CH2). n NH-, , , , , , , , , , , , , , , , , , , , or In Formula II, a, b, c, and the -CO(CH2) mentioned above... n In NH-, n is preferably an integer between 0 and 8; in Formula II, X is preferably an effector.

[0023] In this invention, the cyclic peptide conjugate preferably has the structure shown in Formula III: Formula III; in Formula III, R1, R2, and R3 are preferably -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, or -CO(CH2). n NH-, , , , , , , , , , , , , , , , , , , , or In formula III, a, b, c, and the -CO(CH2) mentioned above... n In NH-, n is preferably an integer between 0 and 8; in Formula III, X is preferably an effector.

[0024] In this invention, a, b, c in Formulas I, II, and III, and the -CO(CH2) n The n in NH- is preferably 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0025] In this invention, the effector preferably comprises a portion derived from a chromophore, a chelating agent, or a portion derived from a drug. In this invention, the portion derived from a chromophore preferably comprises a phosphor or a fluorophore, more preferably a fluorescein or rhodamine. In this invention, the chelating agent preferably comprises a radionuclide and an organic chelating ligand; the radionuclide preferably comprises a diagnostic radionuclide or a therapeutic radionuclide; the diagnostic radionuclide preferably comprises… 68 Ga、 18 F-Al, 64 Cu、99m Tc, 89 Zr、 44 Sc、 111 In、 45 Ti、 52 Mn, 59 Fe、 94m Tc, 67 Ga、 82m Rb、 86 Y、 131 I and 123 One or more of I; the therapeutic radionuclide preferably includes 177 Lu、 90 Y、 131 I, 153 Sm、 67 Cu、 89 Sr、 177 Yb、 47 Sc、 186 Re、 161 Tb, 188 Re、 149 Pm, 212 Pb, 211 At、 223 Ra、 225 Ac and 111 One or more of In; when the radionuclides are two or more of the specific selections mentioned above, the present invention does not impose any special limitation on the ratio of the specific substances, and they can be mixed in any ratio. In the present invention, the organic chelating ligand preferably includes , or .

[0026] In this invention, the portion derived from the drug preferably includes a portion derived from a cytotoxic drug.

[0027] In this invention, when the effector is a chelating agent, the cyclic peptide conjugate is preferably... (NOTA-ZJUIQB-G001) (NOTA-ZJUIQB-G002) (NOTA-ZJUIQB-G003) (NOTA-ZJUIQB-G004) (NOTA-ZJUIQB-G005) or (NOTA-ZJUIQB-G006).

[0028] In this invention, when the peptide structure disclosed herein has a chiral center, its stereostructure is independently selected from the R-configuration, S-configuration, or a mixture of R- and S-configurations.

[0029] In this invention, the preferred method for preparing the cyclic peptide conjugate includes the following steps: preparing Fmoc-Phe-2-nitroimidazole or Fmoc-Glu-2-nitroimidazole in solution, then preparing a linear polypeptide on CTC resin using a solid-phase synthesis method, cleaving the obtained linear polypeptide from the solid phase, cyclizing it in solution to obtain a cyclic peptide, then conjugating it with a nitroimidazole derivative, and finally conjugating it with an effector X group, followed by preparative HPLC purification to obtain the cyclic peptide conjugate; wherein the Fmoc-Phe-2-nitroimidazole is... The Fmoc-Glu-2-nitroimidazole is .

[0030] The present invention also provides the application of the cyclic peptide conjugate described in the above technical solution in the preparation of cancer diagnostic reagents, bone metastatic solid tumor diagnostic reagents, cancer treatment drugs, or bone metastatic solid tumor diagnostic reagents.

[0031] In this invention, the cancer in the cancer diagnostic reagent or cancer treatment drug is preferably a hypoxic cancer; the hypoxic cancer is preferably prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small bowel cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumor, thyroid cancer, or intestinal cancer.

[0032] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0033] Synthesis of key intermediate compound: Fmoc-Phe-2-nitroimidazole Step 1: Compound (S)-BOC-4-(dihydroxyboryl)phenylalanine (1.00 g, 3.2 mmol, 1.0 eq), 2-nitroimidazole (0.42 g, 3.8 mmol, 1.2 eq), phenoxyline (0.33 g, 1.6 mmol, 0.5 eq), and copper acetate (0.32 g, 1.6 mmol, 0.5 eq) were dissolved in methanol (20 mL). DIPEA (diisopropylethylamine) was added to pH 8. The mixture was stirred in a 40°C water bath for 48 h. The reaction progress was monitored by TLC until the reaction was completed. Insoluble impurities were removed by centrifugation, and the crude product in the supernatant was obtained. The crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain compound A (pale blue solid, 0.62 g, yield 50.1%). Step 2: Compound A was dissolved in 50 mL of methanol. In a 1:1 volume ratio of TFA (trifluoroacetic acid) and DCM, the mixture was reacted at room temperature for 2 h. DCM and TFA were removed by rotary evaporation to obtain the crude product of compound B. Step 3: The crude product of compound B (~1.6 mmol) was dissolved in THF (tetrahydrofuran, 20 mL), and DIPEA was added until the pH reached 8. Fmoc-Osu (0.65 g, 1.9 mmol, 1.2 eq) and sodium bicarbonate (0.27 g, 3.2 mmol, 2.0 eq) were added, and the mixture was stirred at room temperature for 2 h. The reaction progress was monitored by TLC until completion. The solvent was removed by rotary evaporation to obtain the crude product of Fmoc-Phe-2-nitroimidazole. The crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain Fmoc-Phe-2-nitroimidazole (pale yellow solid, 0.42 g, yield 52.7%).

[0034] Fmoc-Glu-2-nitroimidazole Step 1: 2-Nitroimidazole (1.00 g, 8.8 mmol, 1.0 eq), N-Boc-3-aminopropyl bromide (3.14 g, 13.2 mmol, 1.5 eq), and potassium carbonate (6.07 g, 44.0 mmol, 5.0 eq) were dissolved in N,N-dimethylformamide (DMF, 50 mL). The mixture was stirred at room temperature for 2 h. The reaction progress was monitored by LC-MS until the reaction was complete. 1 M hydrochloric acid aqueous solution was added to the reaction solution, and the product was extracted with DCM. The resulting organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain compound C (pale yellow solid, 1.71 g, yield 72.0%). Step 2: The above compound C was dissolved in 50 mL of DCM. In a 1:1 volume ratio mixture of TFA (trifluoroacetic acid) and DCM, the mixture was reacted at room temperature for 2 hours. DCM and TFA were removed using a rotary evaporator to obtain the crude product of compound D. Step 3: The crude product of compound D (~6.3 mmol) was dissolved in DCM (dichloromethane, 50 mL), and DIPEA was added until the pH reached 8. Then, Fmoc-L-Glu 1-OtBu (3.76 g, 8.8 mmol, 1.4 eq) and HATU (3.64 g, 8.8 mmol, 1.4 eq) were added. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate), reacted at room temperature for 2 h, then 1 mol / L hydrochloric acid aqueous solution was added to the reaction solution to adjust the pH to 6), the product was extracted with DCM, the organic phase was washed with saturated brine, dried with anhydrous sodium sulfate, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography with silica gel (DCM / MeOH system) to obtain compound E (pale yellow powder solid, 2.60 g, yield 71.4%); Step 4: Dissolve the above compound E in 50 The mixture of TFA (trifluoroacetic acid) and DCM in a 1:1 volume ratio was reacted at room temperature for 2 h. DCM and TFA were removed by rotary evaporation to obtain crude Fmoc-Glu-2-nitroimidazole. The crude product was purified by column chromatography using silica gel (DCM / MeOH system) to obtain Fmoc-Glu-2-nitroimidazole (pale yellow powder solid, 2.08 g, yield 88.9%).

[0035] Example 1: Synthesis of compound Nota-ZJUIQB-G001: Take 120 mg of CTC resin (loading capacity of 1.319 mmol / g) and place it in a peptide synthesis tube. Add 4 mL of DMF to swell the resin for 30 min and wash. 3.0 molar equivalents of Fmoc-Cys(Trt)-OH and 6.0 molar equivalents of DIPEA were dissolved in 4 mL of DCM. The resulting solution was added to the above-mentioned peptide synthesis tube and reacted for 2 h to obtain intermediate compound F. Step a: 4 mL of a 1:4 mixture of piperidine and DMF was added, and after reacting for 10 min, the resin was washed three times each with alternating DCM and DMF. Step b: 3.0 molar equivalents of Fmoc-Ala-OH, 3.0 molar equivalents of HOBt (1-hydroxybenzotriazole), and 3.0 molar equivalents of DIC (N,N'-diisopropylcarbodiimide) were dissolved in 4 mL of DCM. After shaking for 10 min to activate the amino acids, the resulting reaction solution was added to the above-mentioned peptide synthesis tube and reacted for 2 h to obtain intermediate compound G. The reaction progress was detected using the ninhydrin colorimetric method. By repeatedly performing steps a and b until all the required amino acids were coupled to the resin, compound H was obtained. Finally, 5 The peptide was lysed from the resin using a mixture of TFA, TIS, and H2O in a volume ratio of 95:2.5:2.5. After 2 h, the lysate was collected and TFA and DCM were removed using a rotary evaporator. The lysate was then added dropwise to ice-cold diethyl ether, centrifuged, and the crude peptide product (compound I) was collected. The crude product of compound I was dried to powder at room temperature and dissolved in acetonitrile (ACN) and ethanol in a volume ratio of 1:1. 6.0 equivalents of DIPEA and 1.5 equivalents of 1,3,5-tribromomethylbenzene were added, and the mixture was stirred at room temperature for 2 h to obtain a crude product solution of compound J. Insoluble substances were removed using a filter, and the filtrate was purified by HPLC and lyophilized to obtain compound J. Separately, 100 mg of MBHA resin (loading capacity of 0.631 mmol / g) was placed in a peptide synthesis tube, and 4 mL of DMF was added to swell the resin for 30 min, followed by washing.Step b: Dissolve 3.0 molar equivalents of Fmoc-Cys(Trt)-OH, 3.0 molar equivalents of HOBt (1-hydroxybenzotriazole), and 3.0 molar equivalents of DIC (N,N'-diisopropylcarbodiimide) in 4 mL of DMF, shake for 10 min to activate the amino acids, and then add the resulting reaction solution to the above peptide synthesis tube. React for 2 h to obtain intermediate compound K. The reaction progress is detected using the ninhydrin colorimetric method. Step a: Add 4 mL of a 1:4 mixture of piperidine and DMF, react for 10 min, and then wash the resin three times each with alternating DCM and DMF. Dissolve 3.0 molar equivalents of Fmoc-Glu-2-nitroimidazole, 3.0 molar equivalents of HOBt (1-hydroxybenzotriazole), and 3.0 molar equivalents of DIC (N,N'-diisopropylcarbodiimide) in 4 mL of DMF, shake for 10 min. After activating the amino acids, the resulting reaction solution was added to the peptide synthesis tube and reacted for 2 h to obtain intermediate compound K. The reaction progress was detected using the ninhydrin colorimetric method. In step a, 4 mL of a 1:4 mixture of piperidine and DMF was added, and after reacting for 10 min, the resin was washed three times each with alternating DCM and DMF to obtain compound M. Finally, the peptide was lysed from the resin using 5 mL of a 95:2.5:2.5 TFA / TIS / H2O mixture. After 2 h, the lysate was collected and TFA and DCM were removed using a rotary evaporator. The lysate was then added dropwise to ice-cold ether, centrifuged, and the crude peptide product (compound N) was collected. The crude product of compound N was dried at room temperature to a powder (approximately 150 mg). Compound J and 2 equivalents of compound N were dissolved in a mixture of 1 mL ACN and 1 mL ethanol, and 6.0 equivalents of DIPEA were added. The mixture was stirred at room temperature for 1.5 h. h, a crude product solution containing compound O was obtained. Insoluble substances were removed using a filter. The filtrate was purified by HPLC and lyophilized to obtain compound O. Compound O was dissolved in 300 μL DMSO, and 6.0 equivalents of DIPEA and 2.0 equivalents of NOA-NHS were added. The mixture was stirred at room temperature for 2 h to obtain a crude product solution. The solution was filtered, and the filtrate was purified by HPLC and lyophilized to obtain compound NOA-ZJUIQB-G001. The detection result of compound NOA-ZJUIQB-G001 is: LC-MS (ESI) calculated for C. 75 H 108 N 18 O 21 S31693.97 [M+2H] 2+ , found 848.06.

[0036] Example 2: Synthesis of compound NOTA-ZJUIQB-G002: NOTA-ZJUIQB-G002; the preparation method is the same as in Example 1, except that Fmoc-5-amino-3-oxovaleric acid is introduced into compound N in Example 1, and the condensation conditions are the same as in step b; the detection result of compound NOTA-ZJUIQB-G002 is: LC-MS (ESI) calculated for C 79 H 115 N 19 O 23 S31795.08 [M+2H] 2+ , found 898.21.

[0037] Example 3: Synthesis of compound Nota-ZJUIQB-G003: NOTA-ZJUIQB-G003; the preparation method is the same as in Example 1, except that Fmoc-6-aminohexanoic acid is introduced into compound N in Example 1, and the condensation conditions are the same as in step b; the detection result of compound NOTA-ZJUIQB-G00G003 is: LC-MS (ESI) calculated for C 81 H 119 N 19 O 22 S31805.79 [M+2H] 2+ , found 904.25.

[0038] Example 5: Synthesis of compound Nota-ZJUIQB-G004: NOTA-ZJUIQB-G004; the preparation method is the same as in Example 1, except that Fmoc-Glu-2-nitroimidazole is replaced with Fmoc-Phe-2-nitroimidazole, while the condensation conditions remain unchanged; the detection result of the compound NOTA-ZJUIQB-G004 is as follows: LC-MS (ESI) calculated for C 76 H 103 N 17 O 20 S31669.67 [M+2H] 2+ , found 836.24.

[0039] Example 5: Synthesis of compound Nota-ZJUIQB-G005: NOTA-ZJUIQB-G005; the preparation method is the same as in Example 1, except that Fmoc-Glu-2-nitroimidazole is replaced with Fmoc-Phe-2-nitroimidazole, the condensation conditions remain the same, and Fmoc-5-amino-3-oxovaleric acid is introduced into compound N in Example 1, with the condensation conditions being the same as in step b; the detection result of compound NOTA-ZJUIQB-G005 is: LC-MS (ESI) calculated for C 80 H 110 N 18 O 22 S31770.72 [M+2H] 2+ , found 886.92.

[0040] Example 6: Synthesis of compound Nota-ZJUIQB-G006: NOTA-ZJUIQB-G006; the preparation method is the same as in Example 1, except that Fmoc-Glu-2-nitroimidazole is replaced with Fmoc-Phe-2-nitroimidazole, the condensation conditions remain the same, and Fmoc-6-aminohexanoic acid is introduced into compound N in Example 1, with the condensation conditions being the same as in step b; the detection result of compound NOTA-ZJUIQB-G006 is: LC-MS (ESI) calculated for C 82 H 114 N 18 O 21 S31782.76 [M+2H] 2+ , found 892.93.

[0041] Test Example 1: The cyclic peptide conjugates prepared in Examples 1-6 were characterized by HPLC and LC-MS (ESI). The characterization results are shown in Table 1 and Figures 1-2 (Figure 1 is the LC-MS (ESI) spectrum of the cyclic peptide conjugates prepared in Examples 1-6, and Figure 2 is the HPLC spectrum of the cyclic peptide conjugates prepared in Examples 1-6): Table 1 Characterization data of cyclic peptide conjugates prepared in Examples 1-6

[0042] 2. The cyclic peptide conjugates prepared in Examples 1-2, 4, and 5 are used for... 68 Ga-labeled PET imaging of mouse tumors: 200 μg of the cyclic peptide conjugates prepared in Examples 1-6 and FAP-2286 were dissolved in 1 mL of ultrapure water. The pH was adjusted to 4-5 with 4 mL of 0.1 mol / L acetate buffer. 4 mL of radioactive... 68GaCl3 was reacted at room temperature for 30 min to obtain a crude product labeled with a radionuclide. This product was purified by a C-18 reversed-phase column and washed with 2 mL of 50% (v / v) ethanol-water solution to obtain a reflectance-pure product (referred to as […]). 68 Ga]-Ga-NOTA-ZJUIQB-G001、[ 68 Ga]-Ga-NOTA-ZJUIQB-G002、[ 68 Ga]-Ga-NOTA-ZJUIQB-G003、[ 68 Ga]-Ga-NOTA-ZJUIQB-G004、[ 68 Ga]-Ga-NOTA-ZJUIQB-G005 and control compound [ 68 [Ga]-Ga-FAP-2286), its radiochemical purity was determined to be greater than 95% by radio-HPLC; FAP-HEK293T cells transfected with FAP were cultured, and 1-2 million FAP-HEK293T cells were injected subcutaneously into nude mice (athymic nude mice). When the tumors grew to 1-2 cm in size, the radioactive amount of the above-mentioned positron-emitting nuclide-68Ga-labeled reflectance-pure product was injected into the tail vein at a dose of 3.7-7.4 MBq. Then, the tumor-bearing mice were anesthetized, and the animals were fixed on the PET scanning bed to ensure stable body position during the scanning process; then the PET equipment was turned on for scanning, and data was acquired in 15-120 minutes; after the scanning, the acquired data was reconstructed using Siemens Inveon PET / CT image reconstruction software to generate visualized images. The imaging results are shown in Figures 3-7; Figure 3 shows the control compound [ 68 PET imaging of tumors in Ga-Ga-FAP-2286 mice. As shown in Figure 3, the tumor imaging was positive, but the uptake decreased slightly over 30-120 minutes. Uptake was high in non-target tissues such as the kidneys, gallbladder, and intestines. Figure 4 shows... 68 PET imaging of tumors in Ga-Ga-NOTA-ZJUIQB-G001 mice. As shown in Figure 4, the tumors were positive and maintained high retention for 30-120 minutes. Uptake was low in the kidneys (non-target tissue), and no significant uptake was observed in other non-target tissues. Figure 5 shows... 68 PET imaging of tumors in Ga-Ga-NOTA-ZJUIQB-G002 mice. As shown in Figure 5, the tumors were positive, with higher uptake and high retention throughout the 30-120 min period. Uptake was low in the non-target tissue, specifically the kidneys, with particularly low uptake at 120 min. No significant uptake was observed in other non-target tissues. Figure 6 shows... 68PET imaging of tumors in Ga-Ga-NOTA-ZJUIQB-G003 mice, as shown in Figure 6, revealed a positive tumor scintigraphy and maintained high retention rates from 30 to 120 minutes. Uptake in the kidneys significantly decreased at 120 minutes, while no significant uptake was observed in other non-target tissues. Figure 7 shows... 68 PET imaging of tumors in Ga-Ga-NOTA-ZJUIQB-G004 mice, as shown in Figure 7, revealed a positive tumor scintigraphy and maintained high retention rates from 30 to 120 minutes. Uptake in the kidneys gradually decreased from 30 to 120 minutes, while no significant uptake was observed in other non-target tissues. Figure 8 shows... 68 PET imaging of tumors in Ga-Ga-NOTA-ZJUIQB-G005 mice; As shown in Figure 8, the tumor imaging was strongly positive and maintained high retention for 30-120 min. The uptake in the kidneys gradually decreased from 30 to 120 min, and there was no significant uptake in other non-target tissues.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cyclic peptide conjugate targeting a fibroblast activation protein, characterized in that, The cyclic peptide conjugate has the structure shown in Formula I: Formula I; in Formula I, R1, R2, R3, R4, R5, and R6 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or In Formula I, a, b, c, d, e, f, and the -CO(CH2) are... n In NH-, n is an independent integer between 0 and 8; in Formula I, R8 is H, a halogen, or a C1-C4 alkyl group; in Formula I, X is an effector.

2. The cyclic peptide conjugate as described in claim 1, characterized in that, The cyclic peptide conjugate has the structure shown in Formula II: Formula II; in Formula II, R1, R2, and R3 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or In Formula II, a, b, c, and the -CO(CH2) mentioned above... n In NH-, n is an independent integer between 0 and 8; in Equation II, X is an effector.

3. The cyclic peptide conjugate as described in claim 1, characterized in that, The cyclic peptide conjugate has the structure shown in Formula III: Formula III; in Formula III, R1, R2, and R3 are independently -CH2-, -CONH-, -O-, -S-, -CO-NCH3-, -OCH2-, and -CO(CH2). n NH-, 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or In formula III, a, b, c, and the -CO(CH2) mentioned above... n In NH-, n is an independent integer between 0 and 8; in Equation III, X is an effector.

4. The cyclic peptide conjugate according to any one of claims 1 to 3, characterized in that, The effector includes a portion derived from a chromophore, a chelating agent, or a portion derived from a drug.

5. The cyclic peptide conjugate as described in claim 4, characterized in that, The portion derived from the chromophore includes a phosphor or fluorophore; the chelating agent includes a radionuclide and an organic chelating ligand; the portion derived from the drug includes a portion derived from a cytotoxic drug.

6. The cyclic peptide conjugate as described in claim 5, characterized in that, The radionuclides include diagnostic radionuclides or therapeutic radionuclides; the diagnostic radionuclides include 68 Ga、 18 F-Al, 64 Cu、 99m Tc, 89 Zr、 44 Sc、 111 In、 45 Ti、 52 Mn, 59 Fe、 94m Tc, 67 Ga、 82m Rb、 86 Y、 131 I and 123 One or more of I; the therapeutic radionuclide includes 177 Lu、 90 Y、 131 I, 153 Sm、 67 Cu、 89 Sr、 177 Yb、 47 Sc、 186 Re、 161 Tb, 188 Re、 149 Pm, 212 Pb, 211 At、 223 Ra、 225 Ac and 111 One or more of In; the organic chelating ligand includes 、 or 。 7. The cyclic peptide conjugate as described in claim 6, characterized in that, When the effector is a chelating agent, the cyclic peptide conjugate is 、 、 、 、 or 。 8. The use of the cyclic peptide conjugate according to any one of claims 1 to 7 in the preparation of cancer diagnostic reagents, bone metastatic solid tumor diagnostic reagents, cancer therapeutic drugs, or bone metastatic solid tumor diagnostic reagents.

9. The application as described in claim 8, characterized in that, The cancers mentioned in the cancer diagnostic reagents or cancer treatment drugs are specifically hypoxic cancers.

10. The application as described in claim 9, characterized in that, The hypoxic cancers mentioned are prostate cancer, breast cancer, pancreatic cancer, liver cancer, lung cancer, sarcoma, colorectal cancer, cholangiocarcinoma, chordoma, small bowel cancer, pheochromocytoma, gastric cancer, kidney cancer, ovarian cancer, bladder cancer, esophageal cancer, head and neck cancer, thymic cancer, cervical cancer, endometrial cancer, neuroendocrine tumors, thyroid cancer, or intestinal cancer.

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