Compound targeting fibroblast activating protein and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a compound targeting fibroblast activating protein and application of the compound. The compound forms a novel FAP-targeting skeleton molecule with phenylalanine / pyridine alanine / naphthylalanine-pyridine acyl-glycyl-prolinamide as a core, and the structure of the novel FAP-targeting skeleton molecule is as shown in a formula I in the specification. The compound designed by the invention is high in tumor uptake, low in normal tissue (such as liver and bone) uptake and high in target-to-non-target ratio, so that the properties of an original probe are greatly improved, and accurate outlining of tumors is facilitated. Meanwhile, the compound provided by the invention has strong chelating ability, and can form a stable chelate with most radioactive metals, thereby facilitating the integration of tumor diagnosis and treatment.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a compound targeting fibroblast activation protein and its application. Background Art

[0002] Fibroblast activation protein (FAP) is a type II transmembrane serine protease that belongs to the proline-specific peptidase family and has dipeptidyl peptidase and endopeptidase activities. It is highly expressed in tumor-associated fibroblasts (CAFs) of most epithelial tumors and is almost not expressed in normal tissues (only appears in some wound healing and fibrotic lesions). As a key component of the tumor microenvironment, CAFs drive tumor growth, invasion and metastasis by promoting extracellular matrix remodeling, immune escape and angiogenesis. Based on the widespread expression of FAP in the tumor stroma and its core regulatory role in the tumor microenvironment, it has become a highly promising target for tumor diagnosis and treatment ( J Biol Chem. 1999, 274, 36505-36512, Hum Pathol. 2013, 44, 2549-2557).

[0003] Currently, the development of FAP-based radiopharmaceuticals is mainly based on small molecule inhibitors (FAPIs), the core structure of which mostly contains quinoline-glycyl-prolineamide (such as FAPI-04, FAPI-46, etc. J Nucl Med. 2018, 59, 1423-1429. J Nucl Med. 2018, 59, 1415-1422), can bind to FAP with high affinity and achieve efficient radionuclide labeling. 68 Ga-labeled FAPIs have better tumor / background ratio in PET / CT imaging 18 F-FDG has unique advantages for low glucose metabolism tumors and fibrotic lesions; 64 Cu, 44 Long half-life nuclides such as Sc optimize the imaging time window and pharmacokinetic studies; 177 Lu, 225 FAPIs labeled with therapeutic radionuclides such as Ac have preliminarily demonstrated the ability of tumors to accumulate radiation doses, providing a new strategy for targeted therapy of metastatic solid tumors (Euro J Med Chem 277 (2024) 116787, Eur J Nucl Med Mol Imaging. 2022,49, 1822-1832.). However, existing FAP-targeted diagnostic and therapeutic drugs have problems such as short tumor retention time and nonspecific uptake (such as accumulation in inflammation or normal organs) (J Nucl Med 2023; 00:1–8, Eur J Nucl Med Mol Imaging(2023) 50:3050–3061), which restricts diagnostic accuracy and therapeutic effectiveness. Furthermore, molecular framework breakthroughs are urgently needed for the modification of the quinoline core of the UAMC-1110-derived probe. To address the radioactivity issues of quinoline-containing FAPs, peptide compounds such as FAP-2286 have been developed. While this compound has significantly improved tumor retention, it also suffers from the disadvantage of high renal uptake ( Eur J Nucl Med Mol Imaging (2023) 50:3050–3061); For example, the compound obtained by replacing the quinoline ring with a pyridine ring has poor affinity (at the μM level, J Med Chem. 2014, 57, 3053-3074), which has poor specificity as a radioactive probe, and has also been applied to the research of PET probes (CN202410031820.0). The compounds invented in this patent are 18 The F-labeled compounds are only suitable for diagnosis, and the tumor uptake is low (less than 1) and the tumor-to-skin ratio is around 2, which is still a big gap compared to FAPI-04 and FAPI-46, and further optimization is still needed. Similarly, nitrogen monocyclic compounds (Pharmaceuticals 16 (2023)449) have also been tried. 68 Ga-labeled, but with lower affinity (187 ± 52.0 and 17.1 ± 4.60 nM), compared to J With Chem. 2014, 57, 3053-3074, which leads to low tumor uptake and low target-to-non-target ratio, limiting further clinical application. Summary of the Invention

[0004] The purpose of this application is to address the deficiencies of the prior art. In order to address the problem of insufficient affinity and specificity of the existing FAPIs pyridine nucleus, quinoline is innovatively replaced with pyridine, and phenylalanine, pyridine alanine, and naphthylalanine are introduced next to pyridine to form a new FAP-targeting skeleton molecule with phenylalanine / pyridine alanine / naphthylalanine-pyridyl-glycyl-prolineamide as the core, which greatly enhances the affinity of the compound for FAP. By combining the introduction of different linkers with the polymerization of targeting molecules, a new single-multi-target synergistic ligand and its metal chelate FAP probe are innovatively designed and developed. This research is aimed at the dual clinical needs of solid tumor diagnosis and targeted radionuclide therapy. Through original structural optimization, a series of new FAP-targeting ligands with high resolution and high specificity and their integrated diagnosis and treatment metal radionuclide probes have been successfully developed. Based on this, the first aspect of this application provides a compound of formula I or its pharmaceutically acceptable salts, diastereomers, tautomers, hydrates, solvates, crystals,

[0005] Wherein, R1 and R2 are independently selected from H, F; R3 is selected from aromatic ring or aromatic heterocyclic ring; R py1 、R py2 、R py3 , R4 and R5 are independently selected from: hydrogen, deuterium, halogen, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl; R6 and R7, one is H, the other is CN or B (OH) 2; * is dextrorotatory, levorotatory or racemic; R a and R b Independently selected from: hydrogen, a first compound, a second compound; the first compound is a labeled precursor containing a linker and a chelating agent, a cold compound or a radioactive compound; the second compound is a labeled precursor containing a first targeting molecule, a linker and a chelating agent, a cold compound or a radioactive compound, and the first targeting molecule is a targeting molecule of non-targeted fibroblast activation protein; R a and R b Not hydrogen at the same time.

[0006] In some preferred embodiments, R3 in the compound of formula I is a first group substituted by 1-3 first substituents, the first group is a benzene ring, a pyridine ring, a naphthalene ring or a quinoline ring, the first substituent is hydrogen, deuterium, halogen, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, 1-3 second substituents substituted -C 1-6 Alkyl, -OC substituted with 1-3 second substituents 1-6 Alkyl, 1-3 second substituents substituted -SC 1-6 Alkyl, alkenyl, cycloheteroalkenyl or alkynyl, the second substituent is -OH, oxygen or halogen.

[0007] Preferably, the structure of the above compound is as shown in Formula II or Formula III,

[0008] Wherein, n is 1, 2 or 3; L is a linking group selected from substituted or unsubstituted C 1-12 Alkylene, substituted or unsubstituted C 1-12 Cycloalkylene, substituted or unsubstituted C 1-12 Heteroalkylene, substituted or unsubstituted C 1-12 Heterocycloalkylene, substituted or unsubstituted C 1-12 Oxyalkylene, substituted or unsubstituted C 1-12 aminoalkylene, substituted or unsubstituted C 1-12 Alkenylene, substituted or unsubstituted C1-12 Heterocycloalkenylene, substituted or unsubstituted C 1-12 Alkenylene, substituted or unsubstituted C 1-12 Alkynylidene, substituted or unsubstituted C 1-12 Arylene, substituted or unsubstituted C 1-12 one or a combination of heteroarylene groups, polypeptide groups (one of dipeptides to hexapeptides), carbonyl groups, imine groups, imide groups, thioamide groups, phosphoramide groups, thioether groups, disulfide groups, ester groups, thioester groups, carbamate groups, carbonate groups, phosphate groups, anhydride groups, hydrazone groups, acylhydrazone groups and carbohydrates; G is 18 F. 18 F-labeled leaving group or radioactive metal chelate group; the leaving group is selected from: halogen, iodonium salt, quaternary ammonium salt, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, methanesulfonyl, trifluoromethanesulfonyl, stannyl, silyl, boronic acid, borate ester, nitrate, acetate, cyano, and thiol; the radioactive metal chelate group is a chelate group suitable for radioactive labeling, or a chelate comprising a non-radioactive isotope metal and a chelate, or a chelate comprising a radioactive isotope and a chelate.

[0009] T is a targeting molecule targeting a first protein, and the first protein is at least one of PSMA, albumin, somatostatin receptor 2, integrin, MAT2A and GPPC.

[0010] In some preferred embodiments, R1 and R2 in Formula II and Formula III are F.

[0011] When the compound is of formula II, one end of L is connected to formula I (R a and R b The amino derivative (one of which is H) is connected, and the amino derivative can further undergo 1, 2, or 3-fold coupling, and can be further coupled with a chelating group to obtain the structure of Formula II. The structure of the above compound is shown in any of the following:

[0012] Wherein, R is an amino acid residue, I is R in the structure of formula I a and R b The structure when they are not H at the same time, Q is a condensed diacid or condensed triacid of I, and when G contains a chelating agent, Q can be condensed with the chelating agent, x is selected from an integer of 0-10, and y is selected from 1, 2 or 3.

[0013] Illustratively, the structure of the above compound is shown as any one of the following:

[0014] Wherein, * represents the dextrorotatory isomer, levorotatory isomer or racemic isomer.

[0015] When the compound is of formula III, one end of L is connected to formula I (R a and R b The amino derivative (one of which is H) is coupled, and can be further coupled with another targeting molecule (T) and a chelating agent to obtain a structure of formula III. The structure of the above compound is shown in any of the following:

[0016] Wherein, R is an amino acid residue, I is R in the structure of formula I a and R b In the structure when they are not H at the same time, Z is a linker that can condense with I, and when G contains a chelating agent, Z can be coupled with the chelating agent and T at the same time, x is selected from an integer of 0-10, and y is selected from 1, 2 or 3.

[0017] Illustratively, the structure of the above compound is shown as any one of the following:

[0018] Wherein, * represents the dextrorotatory isomer, levorotatory isomer or racemic isomer.

[0019] Wherein, when G has coordination ability, the chelating agent group suitable for radiolabeling is selected from sulfur colloid, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N , N ,, N ,,, N ,,,, -tetraacetic acid (TETA), iminodiacetic acid, bis(carboxymethylimidazole)glycine, 6-nitropyridine-3-carboxylic acid (HYNIC), HBED-CC, AAZTA, DOTA, DODAGA, NOTA, NODAGA or DFO. Or selected from any of the following structures: .

[0020] Wherein, the above radioactive isotope is selected from 18 F. 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re、 188 Re、 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y.88 Y. 90 Y. 149 Pm, 165 Dy, 169 Second, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 121 Bi, 123 Bi, 72 As、 72 Se, 97 Such as 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I. 124 I. 131 I. 197 Hg, 211 At 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 227 Th, 199 Ag.

[0021] Wherein, T is selected from any of the following structures: .

[0022] In some embodiments, the structure of the above compound is shown as any one of the following:

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] .

[0039] The second aspect of the present application provides a pharmaceutical composition comprising the above-mentioned compound; in some implementations, the above-mentioned pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0040] A third aspect of the present application provides the use of the aforementioned compound or pharmaceutical composition in the preparation of a reagent for diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP). The disease characterized by overexpression of fibroblast activation protein (FAP) is selected from cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and scarring. Preferably, the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic carcinoma, glioma, glioma, astrocytoma, cervical cancer, and prostate cancer.

[0041] The fourth aspect of the present application provides a kit comprising the above-mentioned compound or the above-mentioned pharmaceutical composition.

[0042] The fifth aspect of the present application provides an imaging agent comprising the above-mentioned compound or the above-mentioned pharmaceutical composition.

[0043] This application addresses the technical bottlenecks in the existing technology and focuses on optimizing analogs of FAPI-04 and FAPI-46 and the compounds invented in CN202410031820.0 based on the concept of rational drug design. It has the following characteristics: 1. Modification of the targeting group: By reducing the quinoline ring to a pyridine ring and simultaneously introducing phenylalanine, pyridine alanine, or naphthyl alanine at the adjacent position of the pyridine N atom, the affinity of the targeting molecule to the FAP protein was precisely increased.

[0044] 2. Improve pharmacokinetic properties: Aims to increase tumor uptake and reduce probe uptake in normal organs such as the liver and kidneys by introducing PEG chains of different lengths and polymerizing targeting molecules, thereby increasing tumor uptake and retention, and reducing liver and kidney uptake.

[0045] 3. Diversified imaging radioactivity: By introducing different chelating agents and leaving groups, 18 F. 68 Ga-labeled FAP probes are used for PET imaging, and 99m Tc-labeled FAP probes are used for SPECT imaging, providing patients with more options.

[0046] 4. Potential for integrated diagnosis and treatment: The optimized chelation system has excellent coordination ability and can be seamlessly used for therapeutic radionuclides (such as 177 Lu, 90 Y, etc.), thereby achieving accurate diagnosis and efficient targeted treatment of systemic FAP-related tumors under the same molecular carrier.

[0047] This application is expected to lead to the leapfrog development of an integrated diagnosis and treatment platform for FAP molecular imaging, providing a powerful tool for precision oncology. This application relates to cold compounds, radioactive compounds, and labeling precursors of fibroblast activation protein ligands suitable for use with positron emission tomography, single photon emission computed tomography, and radionuclide therapy, compositions containing such compounds, kits containing such compounds or compositions, and the use of such compounds, compositions, or kits for diagnostic imaging using positron emission tomography, single photon emission computed tomography, or radionuclide therapy of tumors.

[0048] The beneficial effects of this application include high tumor uptake, low normal tissue uptake (e.g., liver and bone), and a high target-to-nontarget ratio, significantly improving the properties of existing probes and facilitating precise tumor delineation. Furthermore, the compounds provided herein possess strong chelating abilities and can form stable chelates with most radioactive metals, further facilitating integrated tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Shown as [ 68 Ga] PET image of compound F1 at 1 h, the circled area is the tumor; Figure 2 Shown as [99m [Tc] SPECT image of compound F32 at 1 h, the circled area is the tumor. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments and drawings to clearly and completely describe the concept and technical effects of this application so as to fully understand the purpose, scheme and effect of this application. It should be noted that the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict.

[0051] The structures of compounds F1-F34 involved in the following content are shown below:

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] .

[0068] Example 1 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F1.

[0069] Its synthetic route is as follows:

[0070] The specific steps of synthesis are as follows: (1) Dissolve (tert-Butyloxycarbonyl)phenylalanine (0.5 g, 1.89 mmol) and HBTU (1.43 g, 3.78 mmol) in 8 mL of anhydrous DMF. Add N,N-diisopropylethylamine (1.64 mL, 9.45 mmol) dropwise at 0 °C and stir at room temperature for 15 min. Then add methyl 2-aminoisonicotinate (0.43 g, 2.84 mmol) under nitrogen protection and stir at room temperature overnight. The reaction solution was washed three times with saturated brine, and the mixture was extracted with ethyl acetate. The organic phases were combined and purified by FC to obtain compound 1 as a light yellow solid (0.35 g, 46% yield). 1 H NMR (300 MHz, CDCl3) δ 9.12 (s, 1H), 8.72 (s, 1H), 8.34 (d, J =5.0 Hz, 1H), 7.57 (d, J =4.8 Hz, 1H), 7.24–7.11 (m, 5H), 5.35 (s,1H), 4.64 (s, 1H), 3.92 (s, 3H), 3.27–3.16 (m, 1H), 3.14–3.01 (m, 1H), 1.38(s, 9H). HRMS calcd for C 21 H 26 N3O5 + , 400.1867 [M+H] + ; found,400.1860.

[0071] (2) Compound 1 (0.35 g, 0.88 mmol) was dissolved in 6 mL of methanol, and potassium carbonate (0.48 g, 3.51 mmol) was dissolved in 3 mL of water. The solution was slowly added dropwise to the above solution at 0 °C. The mixture was stirred from 0 °C to room temperature for 4 h. After stopping the reaction, the reaction solution was concentrated, and a small amount of water was added to dissolve the residue. The pH was adjusted to 4 with 2 M dilute hydrochloric acid. A white solid was precipitated and filtered to obtain compound 2 (0.18 g, 53%). HRMS calculation for C 20 H 24 N3O5 + , 386.1710 [M+H] + ; found, 386.1711.

[0072] (3) Compound 2 (0.18 g, 0.47 mmol) and HBTU (0.36 g, 0.94 mmol) were dissolved in 5 mL of anhydrous DMF, and DIPEA (0.41 mL, 2.35 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 15 min, and ( S )-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (0.09 g, 0.47 mmol) was added to the reaction mixture under nitrogen atmosphere and stirred at room temperature overnight. The reaction mixture was washed three times with saturated brine. The mixture was extracted with ethyl acetate, and the organic phases were combined and purified by FC to obtain solid compound 3 (0.17 g, 65%). 1 H NMR (300 MHz, CDCl3) δ 9.15–8.69 (m, 1H), 8.43–8.33 (m,1H), 8.26–8.14 (m, 1H), 8.02–7.79 (m, 1H), 7.37 (d, J =4.2 Hz, 1H), 7.29–7.21(m, 5H), 5.28–5.20 (m, 1H), 5.06–4.95 (m, 1H), 4.74–4.53 (m, 1H), 4.42 (dd, J =17.2, 6.0 Hz, 1H), 4.12–3.93 (m, 3H), 3.24 (td, J =13.8, 6.1 Hz, 1H), 3.13–2.98(m, 1H), 2.85–2.70 (m, 2H), 1.38 (d, J =2.4 Hz, 9H). HRMS calcd for C 27 H 31 F2N6O5 + ,557.2318 [M+H] + ; found, 557.2319.

[0073] (4) Compound 3 (0.2 g, 0.36 mmol) was dissolved in 5 mL of anhydrous DCM. 1 mL of trifluoroacetic acid was slowly added dropwise at 0 °C. The mixture was stirred from 0 °C to room temperature for 6 h. LC-MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain a foamy solid compound 4 (0.14 g, 86%). HRMS calculation for C 22 H 23 F2N6O3 + , 457.1794 [M+H] + ; found, 457.1795.

[0074] (5) HBTU (0.27 g, 0.70 mmol) and 14-(Boc-amino)-3,6,9,12-tetraoxatetradecane-1-carboxylic acid (0.18 g, 0.53 mmol) were dissolved in 5 mL of anhydrous DMF. DIPEA (0.3 mL, 1.75 mmol) was added dropwise at 0 °C and stirred at room temperature for 15 min. Compound 4 (0.18 g, 0.35 mmol) was then added. The mixture was stirred at 50 °C overnight under nitrogen protection. After stopping the reaction, the reaction solution was washed three times with saturated brine. The mixture was extracted with ethyl acetate, the organic phases were combined, and purified by FC to obtain compound 5 as a yellow oil (0.18 g, 0.23 mmol, 66%). 1 H NMR (300 MHz, CDCl3) δ 9.72 (d, J =16.7 Hz, 1H), 8.39–8.20 (m, 2H), 8.19–8.09 (m, 1H), 7.59 (d, J =6.6 Hz, 1H),7.35–7.27 (m, 1H), 7.26–7.11 (m, 5H), 5.23 (s, 1H), 5.10–4.90 (m, 2H), 4.54–4.34 (m, 1H), 4.10–4.00 (m, 3H), HRMS calcdfor C 35 H 46 F2N7O9 + , 790.3581 [M+H] + ; found, 790.3584.

[0075] (6) Compound 5 (0.95 g, 0.12 mmol) was dissolved in 3 mL of anhydrous DCM, and 0.6 mL of trifluoroacetic acid was added dropwise at 0 °C. The mixture was stirred at room temperature for 3 h. LC-MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 6 (0.07 g, 85%) as a yellow foamy solid. HRMS calculation for C 32 H 42 F2N7O8 + , 690.3057 [M+H] + ; found, 690.3059.

[0076] (7) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (21.5 mg, 0.035 mmol) was dissolved in DMSO, TEA (16 μL, 0.12 mmol) was added at 0 °C and stirred, followed by the addition of compound 6 (16 mg, 0.02 mmol). The mixture was stirred at room temperature under nitrogen protection and purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 78 / 22) gave a retention time of 26.3 min to obtain compound F1 (6.5 mg, 28%) as a white solid. HRMS calcd for C 51 H 72 F2N 11 O 17 + , 574.7571 [M+2H] 2+ ; found,574.7569.

[0077] Example 2 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F2.

[0078] Its synthetic route is as follows:

[0079] The specific steps of synthesis are as follows: Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (20 mg, 0.039 mmol) and compound 1 (17.91 mg, 0.026 mmol) in 1.5 mL of anhydrous acetonitrile. Add DIPEA (23.2 μL, 0.13 mmol) dropwise to the solution, and stir at room temperature overnight under nitrogen. Purify by HPLC. RP-HPLC (0.1% aqueous formic acid / acetonitrile = 72 / 28) yielded Compound F2 (4.3 mg, 15%) as a white solid with a retention time of 21.4 min. HRMS calculation for C 48 H 68 F2N 11 O 15 + , 1076.4859 [M+H] + ; found, 1076.4856.

[0080] Example 3 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F3.

[0081] Its synthetic route is as follows:

[0082] The specific steps of synthesis are as follows: (1) HBTU (0.24 g, 0.62 mmol) and 1-tert-butyl 5,8,11-trioxa-2-azatridecanedioate (0.14 g, 0.47 mmol) were dissolved in 5 mL of anhydrous DMF. DIPEA (0.27 mL, 1.55 mmol) was added dropwise at 0 °C and stirred at room temperature for 15 min. Compound 1 (0.14 g, 0.31 mmol) was then added. The mixture was stirred at 50 °C overnight under nitrogen protection. After stopping the reaction, the reaction solution was washed three times with saturated brine. The mixture was extracted with ethyl acetate, the organic phases were combined, and purified by FC to obtain compound 2 (0.14 g, 61%) as a yellow oil. 1 H NMR (300 MHz, CDCl3) δ 9.34 (d, J =28.2 Hz,1H), 8.35 (s, 1H), 8.20 (dd, J =10.6, 5.1 Hz, 1H), 7.58 (d, J =7.0 Hz, 1H), 7.35–7.30 (m, 1H), 7.24 (m, 5H), 5.14 (s, 1H), 5.08–5.00 (m, 1H), 4.97–4.85 (m,1H), 4.45 (ddd, J =22.8, 17.3, 6.1 Hz, 1H), 4.16–3.90 (m, 6H), 3.73–3.58 (m,8H), 3.54 (dd, J =9.2, 5.1 Hz, 2H), 3.35–3.24 (m, 3H), 3.17–3.04 (m, 1H), 2.84–2.71 (m, 2H), 1.45–1.39 (m, 9H). HRMS calcd for C 35 H 46 F2N7O9 + , 746.3319 [M+H] + ;found, 746.3321.

[0083] (2) Compound 2 (0.16 g, 0.21 mmol) was dissolved in 3 mL of anhydrous DCM, and 0.6 mL of trifluoroacetic acid was added dropwise at 0 °C. The mixture was stirred at room temperature for 3 h. MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 3 (0.11 g, 81%). HRMS calculation for C 35 H 46 F2N7O9 + , 746.3319 [M+H] + ; found, 746.3322.

[0084] (3) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (63 mg, 0.11 mmol) was dissolved in DMSO, TEA (0.05 mL, 0.37 mmol) was added at 0 °C, and the mixture was stirred at room temperature. Compound 3 (50 mg, 0.076 mmol) was added, and the mixture was stirred at room temperature under nitrogen protection. HPLC purification was performed. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 78 / 22) showed a retention time of 21.1 min to obtain compound F3 (22 mg, 26%) as a white solid. HRMS calcd for C 49 H 68 F2N 11 O 16 + , 1104.4808 [M+H] + ; found,1104.4805.

[0085] Example 4 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F4.

[0086] Its synthetic route is as follows:

[0087] The specific steps of synthesis are as follows: (1) HBTU (0.203 g, 0.54 mmol) and 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azadocosa-22-oic acid (0.16 g, 0.402 mmol) were dissolved in 5 mL of anhydrous DMF. DIPEA (0.23 mL, 1.34 mmol) was added dropwise at 0 °C and stirred at room temperature for 15 min. Compound 1 (0.132 g, 0.268 mmol) was then added. The mixture was stirred at 50 °C overnight under nitrogen protection. After stopping the reaction, the reaction solution was washed three times with saturated brine. The mixture was extracted with ethyl acetate, the organic phases were combined, and purified by FC to obtain compound 2 (0.15 g, 67%) as a yellow oil. 1 H NMR (300 MHz, CDCl3) δ 9.58 (d, J =16.7 Hz, 1H), 8.42–8.11 (m, 3H), 7.57 (d, J =6.3 Hz, 1H),7.39–7.13 (m, 6H), 5.25–4.88 (m, 3H), 4.61–4.37 (m, 1H), 4.10 (dd, J =15.2, 8.5Hz, 4H), 3.73–3.56 (m, 17H), 3.54–3.48 (m, 2H), 3.29 (s, 3H), 3.15–3.05 (m,1H), 2.93–2.69 (m, 2H), 1.44 (s, 9H). HRMS calcd for C 39 H 54 F2N7O 11 + , 834.3843 [M+H] + ; found, 834.3847.

[0088] (2) Compound 2 (75 mg, 0.09 mmol) was dissolved in 2 mL of anhydrous DCM. 0.5 mL of trifluoroacetic acid was added dropwise at 0 °C and stirred at room temperature for 3 h. MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 3 (58 mg, 88%) as a yellow foamy solid. HRMS calculation for C 34 H 46 F2N7O9 + , 734.3319 [M+H] + ; found, 734.3321.

[0089] (3) Compound 3 (96 mg, 0.153 mmol) was dissolved in DMSO, TEA (71 μL, 0.51 mmol) was added at 0 °C, stirred at room temperature, and 2-(( S )-20-amino-2-benzyl-4-oxo-6,9,12,15,18-pentaoxo-3-azaazepineamide)-N-(2-(( S )-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide (75 mg, 0.102 mmol) was stirred at room temperature under nitrogen protection and purified by HPLC. RP-HPLC (0.1% aqueous formic acid / acetonitrile = 77 / 23) yielded Compound F4 (27 mg, 22%) as a white solid at a retention time of 21.7 min. HRMS calculation for C 53 H 76 F2N 11 O 18 + ,1192.5332 [M+H] + ; found, 1192.5335.

[0090] Example 5 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F5.

[0091] Its synthetic route is as follows:

[0092] The specific steps of synthesis are as follows: (1) HBTU (0.21 g, 0.56 mmol) and 14-(Boc-amino)-3,6,9,12-tetraoxatetradecane-1-carboxylic acid (0.15 g, 0.42 mmol) were dissolved in 5 mL of anhydrous DMF. DIPEA (0.24 mL, 1.4 mmol) was added dropwise at 0 °C and stirred at room temperature for 15 min. Compound 4 (0.13 g, 0.28 mmol) was then added. The mixture was stirred at 45 °C for 8 h under nitrogen protection. After stopping the reaction, the reaction solution was washed three times with saturated brine. The mixture was extracted with ethyl acetate, the organic phases were combined, and purified by FC to obtain compound 5 (0.14 g, 63%) as a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 9.70 (d, J=9.3 Hz, 1H),8.60 (s, 1H), 8.46 (s, 1H), 8.35–8.04 (m, 3H), 7.86–7.66 (m, 2H), 7.35–7.19(m, 2H), 5.31–5.20 (m, 1H), 5.14–4.96 (m, 2H), 4.50 (dd, J =35.8, 11.5 Hz, 1H),4.16–3.96 (m, 5H), 3.70–3.54 (m, 12H), 3.50–3.44 (m, 2H), 3.38–3.07 (m, 4H),2.90–2.67 (m, 2H), 1.40 (s, 9H). HRMS calcd for C 36 H 49 F2N8O 10 + , 791.3534 [M+H] + ;found, 791.3537.

[0093] (2) Compound 5 (0.14 g, 0.18 mmol) was dissolved in 3 mL of anhydrous DCM, and 0.5 mL of trifluoroacetic acid was added dropwise at 0 °C. The mixture was stirred at room temperature for 3 h. MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 6 (0.1 g, 78%) as a yellow foamy solid. HRMS calculation for C 31 H 41 F2N8O8 + , 691.3009 [M+H] + ; found, 691.3006.

[0094] (3) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (62 mg, 0.1 mmol) was dissolved in DMSO, TEA (47 μL, 0.34 mmol) was added at 0 °C, and the mixture was stirred at room temperature for 15 min. Compound 6 (46 mg, 0.067 mmol) was added, and the mixture was stirred at room temperature under nitrogen protection. The mixture was purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 89 / 11) with a retention time of 18.7 min gave Compound F5 (22 mg, 26%) as a white solid. HRMS calcd for C 50 H 71 F2N 12 O 17 +, 1149.5023 [M+H] + ; found,1194.5022.

[0095] Example 6 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F6.

[0096] Its synthetic route is as follows:

[0097] The specific steps of synthesis are as follows: Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolidinyl) ester (16 mg, 0.032 mmol) and compound 1 (14.7 mg, 0.021 mmol) in 1 mL of anhydrous acetonitrile. Add DIPEA (18.8 μL, 0.105 mmol) dropwise to the solution, and stir at room temperature overnight under nitrogen. Purify by HPLC. RP-HPLC (0.1% aqueous formic acid / acetonitrile = 85 / 15) yielded Compound F6 (2.6 mg, 11.5%) as a white solid with a retention time of 19.4 min. HRMS calculation for C 47 H 67 F2N 12 O 15 + , 1077.4811 [M+H] + ; found, 1077.4814.

[0098] Example 7 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F7.

[0099] Its synthetic route is as follows:

[0100] The specific steps of synthesis are as follows: (1) Dissolve N-tert-butyloxycarbonyl-L-glutamic acid (13 mg, 0.052 mmol) in 3 mL DMF, add HBTU (68 mg, 0.18 mmol), add DIPEA (46 μL, 0.26 mmol) dropwise under ice bath, then add compound R (95 mg, 0.13 mmol), and stir at room temperature under nitrogen for 6 h. After the reaction, dilute with ethyl acetate, wash with saturated brine 3 times, wash with saturated sodium bicarbonate water once, extract with ethyl acetate twice, dry and concentrate the organic phase, and purify by FC to obtain orange compound 1. HRMScalcd for C 74H 96 F4N 15 O 20 + , 1590.6887 [M+H] + ; found, 1590.6885.

[0101] (2) Compound 1 (58 mg, 0.037 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 0.2 mL of trifluoroacetic acid was added dropwise under ice bath. The mixture was stirred at room temperature for 2 h. MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 2 (50 mg, 92%) as a yellow oil. HRMS calculation for C 69 H 88 F4N 15 O 18 + , 1490.6362 [M+H] + ; found, 1490.6365.

[0102] (3) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (32 mg, 0.051 mmol) was dissolved in DMSO, TEA (24 μL, 0.17 mmol) was added at 0 °C, and the mixture was stirred at room temperature. Compound 2 (50 mg, 0.034 mmol) was added, and the mixture was stirred at room temperature under nitrogen protection. HPLC purification was performed. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 73 / 27) had a retention time of 19.9 min to obtain compound F7 (11 mg, 16%) as a white solid. HRMS calcd for C 88 H 117 F4N 19 O 27 + , 1949.8409 [M+H] + ; found,1949.8406.

[0103] Example 8 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F8.

[0104] Referring to the synthesis method of Example 7, compound F8 was obtained; HRMS calculated for, C 86 H 116 F4N 21 O 27 + ,1950.8280 [M+H]+ ; found, 1950.8284.

[0105] Example 9 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F9.

[0106] Its synthetic route is as follows:

[0107] The specific steps of synthesis are as follows: (1) Dissolve amino tert-butyl ester-tris-(carbonylethoxymethyl)-methane (16 mg, 0.037 mmol) in 2 mL of DMF, add HBTU (68 mg, 0.18 mmol), add DIPEA (65 μL, 0.46 mmol) dropwise under ice bath, and then add compound R (167 mg, 0.19 mmol). Stir at room temperature under nitrogen for 6 h. After the reaction, HPLC purification was performed. RP-HPLC (water / acetonitrile = 72 / 28) gave compound 1 (55 mg, 59%) as a colorless oil. HRMS calculated for C 114 H 150 F6N 22 O 32 2+ , 1227.0357 [M+2H] 2+ ; found, 1227.0355.

[0108] (2) Compound 1 (55 mg, 0.022 mmol) was dissolved in 1 mL of anhydrous dichloromethane, and 0.2 mL of trifluoroacetic acid was added dropwise under ice bath. The mixture was stirred at room temperature for 2 h. MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 2 (48 mg, 91%) as a yellow oil. HRMS calculation for C 109 H 142 F6N 22 O 30 2+ , 1176.5078 [M+2H] 2+ ; found, 1176.5075.

[0109] (3) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (18.7 mg, 0.03 mmol) was dissolved in DMSO, TEA (0.014 mL, 0.1 mmol) was added at 0 °C, and the mixture was stirred at room temperature. Compound 2 (48 mg, 0.02 mmol) was added, and the mixture was stirred at room temperature overnight under nitrogen protection. The reaction was stopped and purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 71 / 29) showed a peak elution time of 19.3 min to obtain a white solid compound F9 (11 mg, 16%). HRMS calcd for C 128 H 172 F6N 26 O 39 2+ , 1405.6084[M+2H] 2+ ; found, 1405.6485.

[0110] Example 10 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F10.

[0111] Its synthetic route is as follows:

[0112] The specific steps of synthesis are as follows: (1) Dissolve N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid tert-butyl ester (150 mg, 0.233 mmol) and 2,3,5,6-tetrafluorophenol (38.7 mg, 0.233 mmol) in 10 mL of anhydrous dichloromethane. Slowly add a 2 mL dichloromethane solution of DCC (72 mg, 0.35 mmol) dropwise under ice bath. Stir at room temperature for 4 h. LC-MS monitoring shows no further increase in product, and the reaction is stopped. The reaction solution is concentrated, redissolved in ethyl acetate, and DCU is filtered. The filtrate is concentrated and loaded onto silica gel. Purification by FC gives compound 1 (113 mg, 61%). HRMS calcd for C 40 H 49 F4N2O 10 + , 793.3318 [M+H] + ; found,793.3319.

[0113] (2) Compound 1 (57 mg, 0.072 mmol) was dissolved in 1.5 mL of trifluoroacetic acid and stirred at room temperature overnight. LC-MS monitoring showed no residual starting material. The reaction was stopped and trifluoroacetic acid was removed to obtain an oily compound 2 (41 mg, 88%) without further purification. HRMS calculation for C 32 H 33 F4N2O 10 + , 681.2066 [M+H] + ; found, 681.2067.

[0114] (3) Compound 2 (43 mg, 0.063 mmol) was dissolved in 1 mL of anhydrous DMSO, and TEA (29 μL, 0.21 mmol) was added dropwise under ice bath. Finally, compound 3 (29 mg, 0.042 mmol) was added and stirred at room temperature overnight under nitrogen. The reaction solution was filtered and the filtrate was purified by HPLC using acetonitrile / 0.1% formic acid aqueous solution at 2 mL / min (0-16 min, 10-80% acetonitrile; 16-25 min, 80% acetonitrile) with a retention time of 18.9 min. Compound F10 (5.3 mg, 10%) was obtained. HRMS calculated for C 58 H 72 F2N9O 17 + , 1204.5009 [M+H] + ; found, 1204.5006.

[0115] Example 11 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F11.

[0116] Its synthetic route is as follows:

[0117] The specific steps of synthesis are as follows: (1) S)-2-((tert-Butyloxycarbonyl)amino)-3-(naphthalen-1-yl)propanoic acid (100 mg, 0.32 mmol), PyBOP (211 mg, 0.41 mmol), and HOBt (4 mg, 0.029 mmol) were dissolved in 5 mL of anhydrous DCM and stirred at room temperature for 30 min. Methyl 2-aminoisonicotinate (44 mg, 0.29 mmol) was then added and stirred at room temperature under nitrogen for 6 h. After stopping the reaction, the DCM was spin-dried, redissolved in ethyl acetate, washed three times with saturated brine and once with saturated sodium bicarbonate. The organic phase was collected, dried, concentrated, and purified by FC on silica gel to obtain compound 1 as a white solid (60 mg, 47%). HRMS calculation for C 25 H 28 N3O5 + ,450.2023 [M+H] + ; found, 451.0962.

[0118] (2) Compound 1 (52 mg, 0.12 mmol) was dissolved in 1.5 mL of methanol. An aqueous solution of potassium carbonate (64 mg, 0.46 mmol) was added to the reaction mixture under ice-cooling. The mixture was stirred at room temperature for 4 h. When no starting material remained after MS monitoring, the reaction was stopped. The reaction mixture was concentrated, and a small amount of water was added to the residue. The pH was adjusted to 4 with 2 M dilute hydrochloric acid. A white solid precipitated and was filtered to obtain compound 2 (20 mg, 38%) as a white solid. HRMS calculation for C 24 H 26 N3O5 + , 436.1867 [M+H] + ; found,437.1156.

[0119] (3) Compound 2 (20 mg, 0.05 mmol) and HBTU (23 mg, 0.06 mmol) were dissolved in 0.5 mL DMF, and DIPEA (44 μL, 0.25 mmol) was added dropwise under ice bath, followed by addition of ( S )-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (9.5 mg, 0.05 mmol) was stirred at room temperature for 6 h under protective conditions. The reaction was stopped after MS monitoring revealed no residual starting material. The reaction solution was diluted with dichloromethane, washed three times with saturated brine and once with tap water. The organic phase was dried, concentrated, and purified by FC on silica gel to obtain compound 3 (13 mg, 43%) as a light yellow solid. HRMS calculation for C 31 H 33 F2N6O5 + , 607.2475 [M+H]+ ; found, 607.1223.

[0120] (4) Compound 3 (75 mg, 0.13 mmol) was dissolved in 0.5 mL of anhydrous DCM, 1 mL of TFA was added under ice-cooling, and the mixture was stirred at room temperature for 1 h. When no starting material remained after MS monitoring, the reaction was stopped. The reaction solution was concentrated to obtain compound 4 (58 mg, 88%) as a yellow solid. HRMS calculation for C 26 H 25 F2N6O3 + , 507.1951 [M+H] + ; found, 507.1942.

[0121] (5) 2,2-Dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azadocosa-22-oic acid (60 mg, 0.15 mmol) and HBTU (57 mg, 0.15 mmol) were dissolved in 2 mL DMF, and DIPEA (0.11 mL, 0.62 mmol) was added under ice bath, followed by compound 4 (63 mg, 0.13 mmol). Under nitrogen protection, the mixture was stirred at room temperature for 4 h. When no amino group remained after MS monitoring, the reaction was stopped. The reaction solution was diluted with ethyl acetate, washed with saturated brine three times, washed with tap water once, and the organic phase was concentrated and dried. The product was purified by FC on silica gel to obtain compound 5 (79 mg, 69%) as a white foamy solid. HRMS calcd for C 43 H 56 F2N7O 11 + , 884.4000 [M+H] + ; found, 884.3868.

[0122] (6) Compound 5 (79 mg, 0.09 mmol) was dissolved in 0.5 mL of anhydrous DCM, 1 mL of TFA was added under ice-cooling, and the mixture was stirred at room temperature for 1 h. After MS monitoring, no starting material remained, and the reaction was stopped. The reaction solution was concentrated to obtain compound 6 (61 mg, 87%) as a yellow oil. HRMS calculation for C 38 H 48 F2N7O9 + , 784.3476 [M+H] + ; found, 784.3477.

[0123] (7) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (67 mg, 0.11 mmol) was dissolved in 0.3 mL DMSO. TEA (150 μL, 0.89 mmol) was added at 0 °C, followed by compound 6 (70 mg, 0.09 mmol). The mixture was stirred at room temperature for 10 h under nitrogen protection. HPLC purification was performed. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 81 / 19) showed a retention time of 21.3 min to obtain compound F11 (19 mg, 17%) as a white solid. HRMS calcd for C 57 H 78 F2N 11 O 18 + , 1242.5489 [M+H] + ; found,1242.1074.

[0124] Example 12 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F12.

[0125] Its synthetic route is as follows:

[0126] The specific steps of synthesis are as follows: (1) Dissolve N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid tert-butyl ester (300 mg, 0.47 mmol) and 2,3,5,6-tetrafluorophenol (154.7 mg, 0.932 mmol) in 25 mL of anhydrous dichloromethane. Slowly add a 5 mL dichloromethane solution of DCC (144 mg, 0.7 mmol) dropwise under ice bath. Stir at room temperature for 6 h. LC-MS monitoring shows no further increase in product, and the reaction is stopped. The reaction solution is concentrated, redissolved in ethyl acetate, and DCU is filtered. The filtrate is concentrated and loaded onto silica gel. Purification by FC gives compound 1 (321 mg, 73%). HRMS calcd for C 46 H 49 F8N2O 10 + , 941.3254 [M+H] + ; found,941.3255.

[0127] (2) Compound 1 (60 mg, 0.064 mmol) was dissolved in 1.5 mL of trifluoroacetic acid and stirred at room temperature overnight. LC-MS monitoring showed no residual starting material. The reaction was stopped and trifluoroacetic acid was removed to obtain an oily compound 2 (44 mg, 83%) without further purification. HRMS calculation for C 38 H 33 F8N2O 10 + , 829.2002 [M+H] + ; found, 829.2006.

[0128] (3) Compound 2 (44 mg, 0.053 mmol) was dissolved in 1 mL of anhydrous DMSO, and TEA (37 μL, 0.27 mmol) was added dropwise under ice bath. Finally, 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S )-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide (29 mg, 0.042 mmol) was stirred at room temperature overnight under nitrogen. The reaction mixture was filtered, and the filtrate was purified by HPLC using acetonitrile / 0.1% formic acid in water at 2 mL / min (0-16 min, 10-60% acetonitrile; 16-20 min, 60% acetonitrile), with a retention time of 17.5 min. Compound F12 (13 mg, 17%) was obtained. HRMS calculation for C 90 H 111 F4N 16 O 24 + , 1875.7888 [M+H] + ; found,1875.7885.

[0129] Example 13 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F13.

[0130] Its synthetic route is as follows:

[0131] Compound F13 (6.3 mg, 16%) was obtained by referring to the method of Example 4. Its characterization results were as follows: HRMS calcd for C 46 H 63 F2N 10 O 17 + , 1065.4335 [M+H]+ ; found, 1065.4333.

[0132] Example 14 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F14.

[0133] Its synthetic route is as follows:

[0134] The specific steps of synthesis are as follows: Triphosgene (10 mg, 0.034 mmol) was dissolved in 3 mL of anhydrous DCM, and a solution of deferoxamine mesylate (48 mg, 0.073 mmol) and TEA (22 μL, 0.158 mmol) in 3 mL of anhydrous DCM was slowly added dropwise under ice-bath. After stirring at room temperature for 0.5 h, 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S A solution of 2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide (50 mg, 0.073 mmol) and TEA (22 μL, 0.158 mmol) in 3 mL of anhydrous DCM was stirred at room temperature for 2 h, concentrated to remove the solvent, and the residue was re-dissolved in DMSO / water (V / V = 1 / 1). The filtrate obtained after membrane filtration was purified by RP-HPLC to obtain compound F14 (15 mg, 16.1%). HRMS calculation for C 58 H 88 F2N 13 O 17 + , 1276.6384 [M+H] + ; found, 1276.6382.

[0135] Example 15 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F15.

[0136] Its synthetic route is as follows:

[0137] The specific steps of synthesis are as follows: (1) 3,4-diethoxycyclobut-3-ene-1,2-dione (2 g, 11.76 mmol) was dissolved in 40 mL of anhydrous THF, and tert-butyl (2-aminoethyl)carbamate (1.88 g, 11.76 mmol) was added. The mixture was stirred at room temperature overnight. After stopping the reaction, the reaction solution was concentrated, redissolved in ethyl acetate, washed once with saturated ammonium chloride and water, and the organic phase was dried and concentrated. The organic phase was separated by FC on silica gel to obtain tert-butyl (2-((2-ethoxy-3,4-dioxocyclobutane-1-en-1-yl)amino)ethyl)carbamate (3.14 g, 94%). 1 H NMR (300 MHz, DMSO-d6) δ 8.60 (d, J =57.0 Hz, 1H), 6.88 (s,1H), 4.64 (q, J =7.0 Hz, 2H), 3.47 (d, J =5.3 Hz, 1H), 3.30–3.23 (m, 1H), 3.09(dd, J =11.5, 5.7 Hz, 2H), 1.47–1.30 (m, 12H). HRMS calcd for C 13 H 20 N2NaO5 + ,307.1269 [M+Na] + ; found, 307.1266.

[0138] (2) 2,2-Dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-oic acid (55 mg, 0.203 mmol) was dissolved in 2 mL DMF, HBTU (77 mg, 0.202 mmol) was added, DIPEA (0.09 mL, 0.51 mmol) was added dropwise under ice bath, and then compound 1 (50 mg, 0.101 mmol) was added. The mixture was stirred at 50 °C for 6 h under nitrogen protection. After the reaction was stopped, the mixture was diluted with ethyl acetate, washed with saturated brine three times, washed with saturated sodium bicarbonate water once, extracted with ethyl acetate twice, dried and concentrated, and purified by FC to obtain compound 2 (55 mg, 77%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 9.97–9.38 (m, 1H), 8.49–7.95 (m, 3H), 7.50 (s, 1H), 7.30–7.11 (m,5H), 5.39 (s, 1H), 5.18–4.81 (m, 2H), HRMS calcd for C 33 H 42 F2N7O8 + , 702.3057 [M+H] + ; found, 702.3055.

[0139] (3) Compound 2 (55 mg, 0.078 mmol) was dissolved in 2 mL of anhydrous DCM, and 0.4 mL of trifluoroacetic acid was added dropwise at 0 °C. The mixture was stirred at room temperature for 3 h. LC-MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 3 (37 mg, 79%) as a yellow oil. HRMS calculation for C 28 H 34 F2N7O6 + , 602.2533 [M+H] + ; found, 602.2531.

[0140] (4) Compound 3 (35 mg, 0.12 mmol) was dissolved in 3 mL of anhydrous ethanol, and TEA (0.041 mL, 0.3 mmol) was slowly added dropwise. A THF solution of 2,2-(2-(2-aminoethoxyethoxy)acetamido)-3-phenylpropionamido)-N-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide (60 mg, 0.1 mmol) was added with stirring at room temperature, and stirred at room temperature overnight. After the reaction was stopped, the mixture was purified by silica gel chromatography to obtain compound 4 (37 mg, 37%). 1 H NMR (300 MHz, CD3OD) δ 8.45 (d, J =15.1 Hz, 2H), 7.57–7.45 (m, 1H), 7.32–7.16 (m,5H), 6.85–6.21 (m, 1H), 5.58–5.28 (m, 1H), 5.12 (d, J=7.4 Hz, 1H), 5.01–4.94(m, 1H), 4.28–4.14 (m, 3H), 4.03–3.92 (m, 2H), 3.84–3.74 (m, 2H), 3.71–3.50(m, 9H), 3.35 (s, 1H), 3.27–3.19 (m, 2H), 3.13–3.02 (m, 1H), 2.98–2.71 (m,2H), 1.39 (s, 9H). HRMS calcd for C 39 H 48 F2N9O 10 + , 840.3486 [M+H] + ; found,840.3482.

[0141] (5) Compound 4 (37 mg, 0.044 mmol) was dissolved in 2 mL of anhydrous DCM, and 0.4 mL of trifluoroacetic acid was added dropwise at 0 °C. The mixture was stirred at room temperature for 4 h. LC-MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain an oily compound 5 (30 mg, 92%). HRMS calculation for C 34 H 40 F2N9O8 + , 740.2962 [M+H]+; found, 740.2960.

[0142] (6) 2,2',2''-(10-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (38 mg, 0.062 mmol) was dissolved in DMSO, TEA (0.029 mL, 0.21 mmol) was added at 0 °C, and the mixture was stirred at room temperature. Compound 5 (30 mg, 0.041 mmol) was added to the above solution, and the mixture was stirred at room temperature under nitrogen protection. The mixture was purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 75 / 25) showed a peak elution time of 18.8 min to obtain compound F15 (14 mg, 19%) as a white solid. HRMS calcd for C 53 H 70 F2N 13 O 17 + , 1198.4975 [M+H] + ; found, 1198.4974.

[0143] Example 16 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F16.

[0144] Its synthetic route is as follows:

[0145] The specific steps of synthesis are as follows: (1) Dissolve 4-(tert-Butyloxycarbonyl)amino)butyric acid (27 mg, 0.132 mmol) in 2 mL of anhydrous DMF, add HBTU (50 mg, 0.132 mmol), add DIPEA (58 μL, 0.33 mmol) dropwise under ice bath, and finally add compound 1 (30 mg, 0.066 mmol). Protect with nitrogen and stir at room temperature overnight to obtain compound 2 (30 mg, 71%). 1 H NMR (300 MHz, CDCl3) δ 9.91–9.46 (m, 1H), 8.52–8.19 (m, 2H), 8.15–8.01 (m, 1H), 7.56–7.43 (m, 1H), 7.34–7.28 (m, 1H), 7.27–7.12 (m, 5H), 5.19–5.05 (m, 1H), 5.04–4.76(m, 2H), 4.71–4.34 (m, 1H), 4.10–3.86 (m, 3H), 3.29–3.11 (m, 2H), 3.09–2.93(m, 2H), 2.86–2.64 (m, 2H), 2.28 (d, J =5.0 Hz, 2H), 1.86–1.68 (m, 2H), 1.42(s, 9H). HRMS calcd for C 31 H 38 F2N7O6 + , 642.2846 [M+H] + ; found, 642.2844.

[0146] (2) Compound 2 (30 mg, 0.047 mmol) was dissolved in 2 mL of anhydrous DCM, and 0.4 mL of trifluoroacetic acid was added dropwise at 0 °C. The mixture was stirred at room temperature for 3 h. MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain an oily compound 3 (23 mg, 90%). HRMS calculation for C 26 H 30 F2N7O4 + , 542.2321 [M+H] + ;ESI MS(m / z): 542.2318[M+H]+ .

[0147] (3) Compound 3 (40 mg, 0.065 mmol) was dissolved in DMSO, TEA (30 μL, 0.22 mmol) was added at 0 °C, and the mixture was stirred at room temperature. 2-(( S )-2-(4-aminobutyramido)-3-phenylpropionamido)-N-(2-(( S The product was stirred at room temperature overnight under nitrogen atmosphere and purified by HPLC. RP-HPLC (0.1% aqueous formic acid / acetonitrile = 82 / 18) yielded Compound F16 (9 mg, 21%) as a white solid at a retention time of 17.6 min. HRMS calculation for C 45 H 60 F2N 11 O 13 + , 1000.4334 [M+H] + ; found,1000.4330.

[0148] Example 17 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F17.

[0149] Its synthetic route is as follows:

[0150] The specific steps of synthesis are as follows: (1) The starting materials 6-fluoronicotinic acid (500 mg, 3.55 mmol) and 2,3,5,6-tetrafluorophenol (770 mg, 4.61 mmol) were dissolved in 10 mL of anhydrous DCM. A DCM solution of DCC (880 mg, 4.26 mmol) was slowly added dropwise under ice bath and stirred at room temperature for 8 h. The reaction was stopped after the starting materials were consumed as monitored by MS. The reaction solution was concentrated and redissolved in ethyl acetate. The DCU was filtered and the filtrate was purified by FC on silica gel to obtain 2,3,5,6-tetrafluorophenyl-6-fluoronicotinate (830 mg, 81%). HRMS calcd for C 12 H5F5NO2 + , 290.0235 [M+H] + ; found, 290.0232.

[0151] (2) Dissolve 2,3,5,6-tetrafluorophenyl-6-fluoronicotinate (55 mg, 0.19 mmol) in 3 mL of acetonitrile, add TEA (0.14 mL, 0.75 mmol) dropwise to the solution, and finally add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S The mixture was stirred at room temperature for 8 h under nitrogen atmosphere. LC-MS analysis confirmed the absence of residual amino groups, and the reaction was terminated. The reaction mixture was concentrated and purified by silica gel chromatography to afford compound F17 (61.5 mg, 53%). 1 H NMR (300 MHz, CDCl3) δ9.58 (s, 1H), 8.70 (s, 1H), 8.36–8.14 (m, 4H), 7.63 (d, J =7.2 Hz, 1H), 7.45(s, 1H), 7.36–7.31 (m, 1H), 7.29–7.15 (m, 5H), 6.96 (d, J =6.6 Hz, 1H), 5.14–5.03 (m, 1H), 5.02–4.91 (m, 1H), 4.50–4.36 (m, 1H), 4.20–4.04 (m, 4H), 3.74–3.51 (m, 17H), 3.39–3.24 (m, 1H), 3.18–3.05 (m, 1H), 2.94–2.71 (m, 2H). HRMScalcd for C 38 H 44 F3N8O9 + , 813.3177 [M+H] + ; found, 813.3173.

[0152] Example 18 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F18.

[0153] Its synthetic route is as follows:

[0154] The specific steps of synthesis are as follows: (1) 2-(2-Fluoroethoxy)ethoxyethanol-1-ol (1 g, 5 mmol) was dissolved in 10 mL of anhydrous THF. NaH (300 mg, 7.7 mmol, 60% w / w) was slowly added under ice bath. The mixture was stirred from 0 °C to room temperature for 1 h. 2-Benzyl 2-bromoacetate (1.8 g, 7.7 mmol) was then added. The mixture was heated to 100 °C and stirred for 12 h. The reaction was stopped when the starting material no longer decreased as monitored by MS. The reaction solution was quenched with ice-saturated ammonium chloride solution and washed twice. The reaction solution was dried and concentrated, and separated by FC on silica gel. 14-Fluoro-3,6,9,12-tetrahydrotetradecanoic acid benzyl ester (640 mg, 37%) was obtained as a colorless oil. HRMS calcd for C 17 H 25 FNaO6 + ,367.1532 [M+Na] + ; found, 367.1529.

[0155] (2) Benzyl 14-fluoro-3,6,9,12-tetraoxotetradecanoate (640 mg, 1.86 mmol) was dissolved in 13 mL of ethanol, and palladium carbon (192 mg, 30% w / w) was added. The mixture was stirred at room temperature overnight under hydrogen atmosphere. MS monitoring showed no starting material, and the reaction was stopped. The palladium carbon was filtered through celite, and the filtrate was concentrated to obtain 14-fluoro-3,6,9,12-tetraoxatetradecanoic acid (340 mg, 72%) without further purification. HRMS calculation for C 10 H 18 FO6 - , 253.1093 [MH] - ; found, 253.1092.

[0156] (3) 14-Fluoro-3,6,9,12-tetraoxatetradecanoic acid (100 mg, 0.39 mmol) and 2,3,5,6-tetrafluorophenol (86 mg, 0.512 mmol) were dissolved in 5 mL of water (DCM). A DCM solution of DCC (97 mg, 0.47 mmol) was added dropwise under ice-cooling and stirred at room temperature for 10 h. The reaction was stopped after no starting material remained after LC-MS monitoring. The reaction solution was concentrated and redissolved in ethyl acetate. DCU was filtered and the filtrate was concentrated to obtain 2,3,5,6-tetrafluorophenyl 14-fluoro-3,6,9,12-tetraoxatetradecanoate without further purification. HRMS calculation for C 16 H 19 F5NaO6 + , 425.0999 [M+Na]+; found, 424.0996.

[0157] (4) 2,3,5,6-tetrafluorophenyl 14-fluoro-3,6,9,12-tetrahydrotetradecanoate (106 mg, 0.263 mmol) was dissolved in 4 mL of anhydrous THF and 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S To the above solution, 1,000 mg of (2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide (100 mg, 0.22 mmol) was added dropwise TEA (0.153 mL, 1.1 mmol). The solution was stirred at room temperature for 10 h. The reaction was terminated after LC-MS monitoring revealed no residual amino groups. The reaction solution was concentrated, re-dissolved in DCM, and purified by FC on silica gel to afford Compound F18 (26 mg, 17%) as a light yellow oil. 1 H NMR (300 MHz, CDCl3) δ 9.48 (s, 1H), 8.21 (s, 1H), 8.13–8.06 (m, 1H), 7.49 (d, J =7.1 Hz, 1H), 7.31–7.08 (m, 6H), 5.07–4.94 (m, 1H), 4.93–4.79 (m,1H), 4.56–4.50 (m, 1H), 4.44–4.27 (m, 2H), 4.15–3.83 (m, 6H), 3.76–3.43 (m,14H), 3.29–3.15 (m, 1H), 3.09–2.96 (m, 1H), 2.85–2.55 (m, 2H). HRMS calcd forC 32 H 40 F3N6O8 + , 693.2854 [M+H] + ; found, 693.2851.

[0158] Example 19 A compound targeting fibroblast activation protein, whose structure is as shown in compound F19.

[0159] Its synthetic route is as follows:

[0160] The specific steps of synthesis are as follows: (1) Dissolve tert-butyl 4-iodobenzoate (2.3 g, 8.6 mmol) and hexamethyltin (4.3 g, 13 mmol) in 35 mL of dioxane, add tetrakis(triphenylphosphine)palladium (1.99 g, 1.72 mmol), and stir at 105 °C for 5 h under nitrogen. After LC-MS monitoring, no tert-butyl 4-iodobenzoate remains, stop the reaction, concentrate the reaction solution, redissolve in dichloromethane, wash with water three times, dry and concentrate the organic phase, and purify by FC on silica gel to obtain methyl 4-(trimethyltin)benzoate (1.1 g, 43%). HRMScalcd for C 11 H 17 O2Sn + , 301.0245 [M+H] + ; found, 301.0242.

[0161] (2) Methyl 4-(trimethyltinyl)benzoate (0.5 g, 1.67 mmol) was dissolved in 10 mL of ethanol, potassium hydroxide (0.12 g, 2.17 mmol) was added at 0 °C, and the mixture was stirred at 60 °C for 10 h. The pH was adjusted to 5 with glacial acetic acid, and the mixture was diluted with ethyl acetate and extracted three times. The organic phase was dried and concentrated, and purified by FC on silica gel to obtain 4-(trimethyltinyl)benzoic acid (0.38 g, 65%). HRMS calcd for C 10 H 13 O2Sn - , 284.9943 [MH] - ; found, 284.9941.

[0162] (3) 4-(Trimethyltinyl)benzoic acid (0.38 g, 1.33 mmol) and N-hydroxysuccinimide (0.184 g, 1.6 mmol) were dissolved in anhydrous THF. A 2 mL dichloromethane solution of DCC (0.33 g, 1.6 mmol) was slowly added dropwise at 0 °C and stirred at room temperature overnight. LC-MS monitoring indicated that no starting material remained. The reaction was stopped, the reaction solution was concentrated, and FC purification was performed to obtain 2,5-dioxypyrrolidin-1-yl 4-(trimethyltinyl)benzoate (0.27 g, 54%). HRMS calculation for C 14 H 18 NO4Sn + ,284.9943 [M+H] + ;found, 284.9941.

[0163] (4) 2,5-Dioxopyrrolidin-1-yl 4-(trimethyltinyl)benzoate (0.14 g, 0.37 mmol) was dissolved in 1 mL of DMSO and 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S )-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide (0.17 g, 0.25 mmol). Finally, DIPEA (0.22 mL, 1.25 mmol) was added dropwise to the above solution and stirred at room temperature for 12 h. LC-MS monitoring of the remaining starting material was performed. DMSO was removed, and the organic phase was redissolved in dichloromethane and washed three times with water. The organic phase was dried and concentrated, and purified by FC on silica gel to obtain compound F19 (85 mg, 35%). HRMS calculation for C 42 H 54 F2N7O9Sn + , 958.2968 [M+H] + ; found, 958.2966.

[0164] Example 20 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F20.

[0165] Its synthetic route is as follows:

[0166] The specific steps of synthesis are as follows: (1) Dissolve tetraethylene glycol (15 g, 77.5 mmol) in 30 mL of anhydrous DCM, add p-toluenesulfonic acid monohydrate (59 mg, 0.311 mmol), slowly add DHP (1.4 mL, 15.5 mmol) in DCM solution dropwise under ice bath, and stir at room temperature overnight. After monitoring for no DHP residue, stop the reaction. Add water to the reaction solution, wash once with a mixed solution of saturated salt water and sodium bicarbonate, extract the aqueous phase twice with dichloromethane, concentrate and dry the organic phase, and separate by FC on silica gel to obtain compound 1 (2.2 g, 51%) as a colorless oil. HRMS calcd for C 13 H 27 O6 + , 279.1802 [M+H] + ; found, 279.1803.

[0167] (2) Compound 1 (1 g, 3.6 mmol) was dissolved in 10 mL of anhydrous THF. NaH (0.43 g, 10.8 mmol, 60% w / w) was slowly added under ice-bath. After stirring at room temperature for 1 h, 2-benzyl bromoacetate (2.45 g, 10.8 mmol) was added and stirred at room temperature overnight. After monitoring that no starting material remained, saturated ammonium chloride was added under ice-bath to quench the reaction solution to pH = 7. The reaction solution was concentrated, redissolved in ethyl acetate, washed twice with tap water, dried and concentrated, and the organic phase was separated by FC on silica gel to obtain compound 2 (1.17 g, 76%) as a colorless oil. HRMS calcd for C 22 H 34 NaO8, 449.2151 [M+Na] + ; found,449.2153.

[0168] (3) Compound 2 (476 mg, 1.12 mmol) was dissolved in 10 mL of ethanol, and 143 mg of palladium on carbon (30% w / w) was added to the reaction mixture. The mixture was stirred at room temperature overnight under hydrogen. After MS monitoring showed that no starting material remained, the palladium on carbon was filtered through celite, and the filtrate was concentrated to obtain compound 3 (285 mg, 70%). HRMS calculation for C 15 H 27 O8 - , 335.1711 [MH] - ; found, 335.1709.

[0169] (4) Compound 3 (106 mg, 0.316 mmol) was dissolved in 3 mL of anhydrous DMF, and HBTU (150 mg, 0.395 mmol) was added. DIPEA (138 μL, 0.789 mmol) was added dropwise under an ice bath, and finally compound F1 (120 mg, 0.263 mmol) was added. The mixture was stirred at room temperature for 8 h under nitrogen. The remaining compound F1 was monitored by LC-MS, and the reaction was stopped. The reaction solution was diluted with ethyl acetate and washed three times with saturated brine. The reaction solution was dried and concentrated, and purified by FC on silica gel to obtain compound 4 (88 mg, 43%). 1 HNMR (300 MHz, CDCl3) δ 9.54 (s, 1H), 8.40–8.19 (m, 2H), 8.11 (d, J =3.8 Hz,1H), 7.54 (d, J=7.3 Hz, 1H), 7.28–7.16 (m, 5H), 5.14–4.99 (m, 1H), 4.98–4.85(m, 1H), 4.57 (s, 1H), 4.39 (dd, J =17.1, 4.8 Hz, 1H), 4.17–3.75 (m, 8H), 3.71–3.50 (m, 15H), 3.48–3.40 (m, 1H), 3.31–3.19 (m, 1H), 3.14–2.99 (m, 1H), 2.88–2.64 (m, 2H), 1.83–1.62 (m, 2H), 1.59–1.40 (m, 4H). HRMS calcd for C 37 H 49 F2N6O 10 + , 775.3472 [M+H] + ;found, 775.3475.

[0170] (5) Compound 4 (60 mg, 0.078 mmol) was dissolved in 3 mL of anhydrous methanol. A solution of p-toluenesulfonic acid monohydrate (1.5 mg, 0.008 mmol) in methanol was added under ice-cooling, and the mixture was stirred at room temperature overnight. MS monitoring indicated that no starting material remained, and the reaction was stopped. Methanolic ammonia solution was added dropwise under ice-cooling to adjust the reaction solution to pH 7. The reaction solution was concentrated and purified by FC on silica gel to obtain compound 5 (35 mg, 66%) as a colorless oil. 1 H NMR (300 MHz, CDCl3) δ 9.78–9.45 (m, 1H), 8.38–8.06 (m,3H), 7.71 (s, 1H), 7.35–7.28 (m, 1H), 7.25–7.13 (m, 4H), 5.09–4.95 (m, 1H),4.89–4.78 (m, 1H), 4.31 (d, J =14.0 Hz, 1H), 4.14–3.84 (m, 6H), 3.74–3.46 (m,16H), 3.35–3.01 (m, 3H), 2.87–2.59 (m, 2H). HRMS calcd for C 32 H 41 F2N6O9 + ,691.2897 [M+H] + ; found,691.2894.

[0171] (6) Compound 5 (35 mg, 0.051 mmol) was dissolved in 5 mL of anhydrous dichloromethane. p-Toluenesulfonyl chloride (25 mg, 0.126 mmol) was added under ice-cooling, followed by dropwise addition of TEA (35 μL, 0.255 mmol). The mixture was stirred at room temperature under nitrogen overnight. The reaction was stopped after no further reduction of the starting material was observed by LC-MS. The sample was diluted with DCM and loaded onto silica gel. Purification by FC gave Compound F20 (29.5 mg, 67%) as a yellow oil. 1 H NMR (300 MHz, CDCl3) δ 9.82–9.42 (m, 1H), 8.37–8.10 (m, 3H), 7.76 (d, J =7.6 Hz, 2H), 7.60 (d, J =5.8 Hz, 1H), 7.36–7.29 (m, 3H), 7.27–7.17 (m, 4H), 5.14–5.01 (m, 1H), 4.97–4.86 (m, 1H), 4.45–4.31 (m, 1H), 4.23–3.99 (m, HRMS calcd for C 39 H 47 F2N6O 11 S + , 845.2986 [M+H] + ; found, 845.2983.

[0172] Example 21 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F21.

[0173] Its synthetic route is as follows:

[0174] The specific steps of synthesis are as follows: (1) The starting material ( S)-4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triaza-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid (80 mg, 0.147 mmol) and 2,3,5,6-tetrafluorophenol (37 mg, 0.221 mmol) were dissolved in 10 mL of anhydrous DCM. A DCM solution of DCC (36 mg, 0.176 mmol) was slowly added dropwise under ice bath. The mixture was stirred at room temperature for 8 h. No starting material remained after LC-MS monitoring. The reaction solution was concentrated and redissolved in ethyl acetate. DCU was filtered and the filtrate was concentrated. 1.5 mL of 4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triaza-1-yl)-5-(tert-butoxy)-5-oxopentanoic acid was added dropwise under ice bath. TFA was added and stirred at room temperature overnight to remove trifluoroacetic acid to obtain 78 mg of light brown 2,2'-(7-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7-triazolin-1,4-diyl)diacetic acid. HRMS calculation for C 21 H 26 F4N3O8 + , 524.1650 [M+H] + ; found, 524.1648.

[0175] (2) 2,2'-(7-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7-triazolin-1,4-diyl)diacetic acid (78 mg, 0.15 mmol) was dissolved in 1 mL of anhydrous DMSO, and TEA (125 μL, 0.9 mmol) was added dropwise under ice bath. Finally, 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S The mixture was stirred at room temperature overnight under nitrogen atmosphere. The reaction mixture was filtered, and the filtrate was purified by HPLC. RP-HPLC (0.1% aqueous formic acid / acetonitrile = 80 / 20) yielded compound F21 (24 mg, 23%) at 26.12 min. HRMS calculation for C 47 H 65 F2N 10 O 15 + ,1047.4593 [M+H] + ; found, 1047.4592.

[0176] Example 22 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F22.

[0177] Its synthetic route is as follows:

[0178] The specific steps of synthesis are as follows: Will( E )-2-((2-(5-((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)pyridin-2-yl)hydrazone)methyl)benzenesulfonic acid (73 mg, 0.174 mmol; Synthesis reference: J. Med. Chem. 2023, 66, 7, 4952–4960 ) was dissolved in 5 mL of anhydrous acetonitrile, TEA (0.06 mL, 0.435 mmol) was slowly added dropwise, and 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S The reaction mixture was heated to 70°C and refluxed for 1 h. The reaction was terminated after the amino group was consumed, as monitored by LC-MS. The reaction mixture was concentrated and purified by silica gel chromatography (FC) to afford a yellow oil. A small amount of ethyl acetate was added and sonicated. The mother liquor was filtered to afford Compound F22 (24 mg, 14%) as a yellow solid. 1 H NMR (300 MHz, MeOD) δ 9.64(s, 1H), 8.94 (s, 1H), 8.61 (s, 1H), 8.44 (s, 1H), 8.37–8.27 (m, 2H), 8.23(d, J =7.6 Hz, 1H), 8.06–7.92 (m, 3H), 7.55–7.37 (m, 4H), 7.29–7.13 (m, 6H), 5.16–5.08 (m, 1H), 4.17 (d, J =6.9 Hz, 2H), 4.13–4.07 (m, 1H), 4.04–3.99 (m,2H), 3.71–3.53 (m, 17H), 3.27–3.10 (m, 2H), 3.08–2.99 (m, 1H), 2.98–2.85 (m,1H), 2.83–2.72 (m,1H). HRMS calcd for C 45 H 49 F2N 10 O 12 S - , 991.3225 [MH] - ; found,991.3228.

[0179] Example 23 A compound targeting fibroblast activation protein, whose structure is as shown in compound F23.

[0180] Its synthetic route is as follows:

[0181] The specific steps of synthesis are as follows: (1) 2,5-Dioxopyrrolidin-1-yl 6-(2-(tert-butyloxycarbonyl)hydrazinyl)nicotinate (50 mg, 0.144 mmol) was dissolved in 5 mL of anhydrous acetonitrile, and DIPEA (0.14 mL, 0.575 mmol) was added dropwise. Finally, 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S )-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isonicotinamide, heated to 40 °C and stirred for 3 h under nitrogen. The reaction solution was then concentrated, redissolved in DCM, washed twice with saturated sodium bicarbonate, extracted twice with dichloromethane, dried and concentrated, loaded onto silica gel, and purified by FC to obtain a light yellow oily product 2-(5-(( S )-2-benzyl-1-((4-((2-(( S (4,4-difluoropyrrolidin-1-yl,2-oxoethyl,4,4-difluoropyrrolidin-1-yl,2-oxoethyl,4,4-difluoropyrrolidin-1-yl,4,4-difluoropyrrolidin-2-yl ... 1 H NMR (300 MHz, CDCl3) δ 9.83(s, 1H), 8.47 (s, 1H), 8.41–8.20 (m, 2H), 8.14–8.03 (m, 1H), 7.99–7.74 (m,2H), 7.69–7.56 (m, 1H), 7.54–7.35 (m, 2H), 7.31–7.01 (m, 6H), 6.53 (d, J =7.5Hz, 1H), 5.11–4.78 (m, 2H), 4.39–4.18 (m, 1H), 4.06–3.79 (m, 5H), 3.73–3.33(m, 16H), 3.22–3.09 (m, 1H), 3.04–2.91 (m, 1H), 2.82–2.50 (m, 2H), 1.33 (s,9H). HRMS calcd for C43 H 55 F2N 10 O 11 + , 925.4014 [M+H] + ; found, 925.4017.

[0182] (2) 2-(5-(( S )-2-benzyl-1-((4-((2-(( S Tert-butyl 2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)pyridin-2-yl)amino)-1,4-dioxo-6,9,12,15-tetraoxo-3-azaheptan-17-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carboxylate (82 mg, 0.089 mmol) was dissolved in 2 mL of anhydrous DCM. 0.5 mL of trifluoroacetic acid was added dropwise under ice-cooling. The mixture was stirred at room temperature for 1 h. LC-MS analysis revealed no residual starting material. The trifluoroacetic acid was removed, and the mixture was reconstituted in 1 mL of methanol. Separation was performed by HPLC (0.1% aqueous ammonia solution / acetonitrile = 58 / 42) with a retention time of 24.3 min to give compound F23 (33.7 mg, 48%). HRMS calculation for C 40 H 50 F2N 11 O9 + , 866.3755 [M+MeCN+H] + ; found, 866.3758.

[0183] Example 24 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F24.

[0184] Its synthetic route is as follows:

[0185] The specific steps of synthesis are as follows: (1) Fmoc-L-glutamic acid-γ-benzyl ester (83 mg, 0.18 mmol) and HATU (87 g, 0.23 mmol) were dissolved in 5 mL of anhydrous DMF, and DIPEA (158 μL, 0.90 mmol) was added dropwise at 0 °C. The mixture was stirred at room temperature for 15 min, and then compound 1 (123 mg, 0.15 mmol) was added. The mixture was protected by nitrogen and stirred at room temperature overnight. The reaction solution was washed three times with saturated brine, and the mixture was extracted with ethyl acetate. The organic phases were combined and purified by FC to obtain compound 2 (120 mg, 63%). HRMS calcd for C 72 H93 N6O 14 + , 1265.6744 [M+ H] + ; found, 1265.6747.

[0186] (2) Compound 2 (110 mg, 0.087 mmol) was dissolved in 2 mL of 20% diethylamine / THF solution, stirred at room temperature for 2 h, concentrated to remove the solvent, and purified by FC. The resulting compound was dissolved in 2 mL of acetonitrile, and DIPEA (76 μL, 0.435 mmol) and tri-tert-butyl 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (64 mg, 0.096 mmol) were added. The solution was stirred at room temperature for 4 h, concentrated to remove the solvent, and purified by FC to obtain compound 3 (81 mg, 58%). HRMS calcd for C 85 H 133 N 10 O 19 + , 1597.9743 [M+H] + ; found, 1597.9747.

[0187] (3) Compound 3 (75 mg, 0.047 mmol) was dissolved in 10 mL of ethanol, and palladium carbon (22 mg, 30% w / w) was added. The mixture was stirred at room temperature overnight under hydrogen atmosphere. No starting material was detected by LC-MS. The reaction was stopped. The palladium carbon was filtered through celite and the filtrate was concentrated. The obtained compound and 2,3,5,6-tetrafluorophenol (12 mg, 0.072 mmol) were dissolved in 6 mL of anhydrous dichloromethane. A solution of DCC (15 mg, 0.073 mmol) in 2 mL of dichloromethane was slowly added dropwise under ice bath. The mixture was stirred at room temperature for 4 h. No further increase in product was detected by LC-MS. The reaction was stopped. The reaction solution was concentrated, redissolved in ethyl acetate, and DCU was filtered. The filtrate was concentrated and loaded onto silica gel. Compound 4 (59 mg, 76%) was purified by FC. HRMS calcd for C 84 H 127 F4N 10 O 19 + , 1655.9210 [M+ H] + ; found,1655.9213.

[0188] (4) 1.5 mL of TFA was added to compound 4 (55 mg, 0.033 mmol) under ice bath, stirred at room temperature overnight, trifluoroacetic acid was removed, and the mixture was redissolved in 1 mL of anhydrous DMSO. TEA (23 μL, 0.165 mmol) was added dropwise under ice bath, and finally 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S The mixture was stirred at room temperature overnight under nitrogen atmosphere. The reaction mixture was filtered, and the filtrate was purified by RP-HPLC to obtain compound F24 (14 mg, 23%). HRMS calculation for C 86 H 118 F2N 17 O 26 + , 1842.8396 [M+ H] + ; found, 1842.8395.

[0189] Example 25 A compound targeting fibroblast activation protein, whose structure is as shown in compound F25.

[0190] Compound F25 (22 mg, 19.8%) was obtained by referring to the method of Example 24. HRMS calculation for C 65 H 94 F2N 15 O 24 + , 2315.1500 [M+H] + ; found, 2315.1503.

[0191] Example 26 A compound targeting fibroblast activation protein, whose structure is as shown in compound F26.

[0192] Compound F26 (37 mg, 18.5%) was obtained by referring to the method of Example 24. HRMS calculation for C 80 H 114 F2N 21 O 25 + , 1806.8257 [M+H] + ; found, 1806.8255.

[0193] Example 27 A compound targeting fibroblast activation protein, whose structure is as shown in compound F27.

[0194] Compound F27 (22 mg, 10.4%) was obtained by referring to the method of Example 24. HRMS calculation for C 97 H 133 F2N 23 O 28 S2 + , 2169.9050 [M+H] + ; found, 2169.9052.

[0195] Example 28 A compound targeting fibroblast activation protein, whose structure is as shown in compound F28.

[0196] Compound F28 (46 mg, 20%) was obtained by referring to the method of Example 24. HRMS calculation for C 109 H 155 F2N 26 O 28 + , 2315.1500 [M+H] + ; found, 2315.1504.

[0197] Example 29 A compound targeting fibroblast activation protein, whose structure is as shown in compound F29.

[0198] Its synthetic route is as follows:

[0199] The specific steps of synthesis are as follows: (1) 4-(4-iodophenyl)butyric acid (0.5 g, 1.73 mmol) and HBTU (0.98 g, 2.6 mmol) were dissolved in 5 mL of DMF. DIPEA (1.5 mL, 8.65 mmol) was added dropwise at 0 °C, followed by (tert-butyloxycarbonyl)-L-lysine benzyl ester (0.58 g, 1.73 mmol). The mixture was stirred at room temperature for 12 h under nitrogen protection. After no raw material remained by LC-MS, the reaction was stopped. The reaction solution was diluted with ethyl acetate, washed with tap water once, and washed with saturated brine three times. The organic phase was dried over sodium sulfate, concentrated, and purified by FC on silica gel to obtain compound 1 (0.56 g, 53%). HRMS calcd for C 28 H 38 IN2O5 + , 609.1820 [M+H] +;found, 609.1822.

[0200] (2) Compound 1 (0.2 g, 0.33 mmol) was dissolved in 8 mL of anhydrous dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise to the above solution. The mixture was stirred at room temperature for 3 h. No starting material remained after LC-MS monitoring. The reaction was stopped, trifluoroacetic acid was removed, the reaction solution was concentrated, and 5 mL of anhydrous acetonitrile was used to redissolve it. DOTA-NHS ester (0.23 g, 0.35 mmol) was added, and DIPEA (0.29 mL, 1.65 mmol) was added dropwise to the above solution. The mixture was stirred at room temperature for 8 h, the reaction solution was concentrated, redissolved in ethyl acetate, washed once with water, washed once with saturated NaHCO3, dried and concentrated, and the organic phase was loaded on silica gel and purified by FC to obtain compound 2 (0.16 g, 46%). HRMScalcd for C 51 H 80 IN6O 10 + , 1063.4975 [M+H] + ; found, 1063.4977.

[0201] (3) Compound 2 (0.16 g, 0.15 mmol) was dissolved in 6 mL of ethanol and palladium carbon (48 mg, 30% w / w) was added. The mixture was stirred at room temperature overnight under hydrogen atmosphere. No residual starting material was detected by LC-MS. The palladium carbon was filtered through celite and the filtrate was concentrated without further purification. The filtrate was redissolved in 8 mL of anhydrous dichloromethane, 2,3,5,6-tetrafluorophenol (32.3 mg, 0.2 mmol) was added, and a 0.5 mL anhydrous dichloromethane solution of DCC (37.1 mg, 0.18 mmol) was slowly added dropwise at 0°C. The mixture was stirred at room temperature for 10 h. After the reaction was stopped, DCU was filtered, the filtrate was concentrated, and the mixture was purified by FC on silica gel to obtain compound 3 (0.95 g, 57%). HRMS calcd for C 50 H 74 F4IN6O 10 + , 1121.4442 [M+H] + ; found, 1121.4444.

[0202] (4) Compound 3 (45 mg, 0.04 mmol) was dissolved in 1 mL of trifluoroacetic acid and stirred at room temperature for 12 h. After LC-MS monitoring, the reaction was stopped after no starting material remained. The trifluoroacetic acid was removed, the reaction solution was concentrated, and re-dissolved in 1 mL of DMSO. DIPEA (35 μL, 0.2 mmol) was added, followed by the addition of 2-(( S)-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptaneamide)-N-(2-(( S The mixture was stirred at room temperature for 12 h under a nitrogen atmosphere. Compound F29 (4.7 mg, 8%) was obtained by HPLC purification. HRMS calculation for C 64 H 89 F2IN 13 O 17 + , 1476.5507 [M+H] + ; found, 1476.5503.

[0203] Example 30 A compound targeting fibroblast activation protein, whose structure is as shown in Compound F30.

[0204] Compound F30 (16 mg, 15%) was obtained by referring to the method of Example 28. HRMS calculated for, C 87 H 115 F2N 16 O 22 + , 1065.4335 [M+H] + ; found, 1065.4332.

[0205] Example 31 A compound targeting fibroblast activation protein, whose structure is as shown in compound F31.

[0206] Compound F31 (21 mg, 10%) was obtained by referring to the method of Example 25. HRMS calculation for C 97 H 127 F2N 22 O 24 + ,2022.9390 [M+H] + ; found, 2022.9393.

[0207] Example 32 A compound targeting fibroblast activation protein, whose structure is as shown in compound F33.

[0208] Its synthetic route is as follows:

[0209] The specific steps of synthesis are as follows: (1) Boc-L-phenylalanine (2.65 g, 10.00 mmol) and HATU (7.68 g, 20.20 mmol) were dissolved in 20 mL of DMF. DIPEA (3.23 g, 25.00 mmol) was added dropwise at 0 °C, followed by 2-aminoisonicotinate methyl ester (2.73 g, 18.00 mmol). Under nitrogen protection, the mixture was stirred at room temperature for 3.5 h. After no raw material remained by LC-MS, the reaction was stopped. The reaction solution was diluted with ethyl acetate and washed three times with saturated brine. The organic phases were combined, dried over sodium sulfate, concentrated, and purified by flash column chromatography to obtain compound 2 (2.53 g, 63.60%). HRMS calcd for C 21 H 24 N3O5 + , 400.1872 [M+H] + ; found, 400.1875.

[0210] (2) Compound 2 (1.35 g, 3.38 mmol) was dissolved in an appropriate amount of methanol, and lithium hydroxide aqueous solution (0.33 g, 13.52 mmol, 2 mL of water) was added. The mixture was stirred at room temperature for 4 h. The reaction was stopped after the raw material was consumed by LC-MS monitoring. After removing the methanol, an appropriate amount of water was added and the pH was adjusted to about 6 with 2M hydrochloric acid. The precipitate was filtered and dried to obtain compound 3 (1.16 g, 89.1%), which was directly subjected to the next step. HRMS calculation for C 20 H 24 N3O5 + , 386.1716 [M+H] + ; found,386.1720.

[0211] (3) Referring to the synthesis scheme of compound 2, compound 4 (730 mg, 46.0%) was obtained. HRMS calculation for C 29 H 33 N4O6 + , 533.2400 [M+H] + ; found, 533.2395.

[0212] (4) Compound 4 (730 mg, 1.36 mmol) was dissolved in 3 mL of anhydrous DCM, and 1.5 mL of trifluoroacetic acid was slowly added dropwise at 0 °C. After stirring at room temperature for 5 h, LC-MS monitoring showed that no starting material remained. The reaction solution was concentrated to obtain compound 5 (508 mg, 86.5%). HRMS calculation for C 24 H 25 N4O4 +, 433.1876 [M+H] + ; found, 433.1882.

[0213] (5) Referring to the synthesis scheme of compound 2, compound 5 (164 mg, 0.38 mmol) was condensed with BocNH-PEG5-acid (225 mg, 0.57 mmol) to obtain compound 6 (169 mg, 55.0%). HRMS calculation for C 41 H 56 N5O 12 + ,810.3925 [M+H] + ; found, 810.3933.

[0214] (6) Referring to the synthesis scheme of compound 5, trifluoroacetic acid was added to compound 6 for hydrolysis, and compound 7 was obtained after post-treatment, which was directly subjected to the next condensation step.

[0215] (7) Compound 7 (115 mg, 0.16 mmol) was condensed with DOTAGA-tetra-tert-butyl ester (168 mg, 0.24 mmol) by referring to the synthesis scheme and post-treatment method of compound 2 to obtain compound 8 (139 mg, 62.5%). HRMS calculation for C 71 H 110 N9O 19 + , 1392.7918 [M+H] + ; found, 1392.7909.

[0216] (8) Compound 8 (130 mg, 0.093 mmol) was dissolved in 10 mL of ethanol, and palladium carbon (28 mg, 20% w / w) was added. The mixture was stirred at room temperature overnight under hydrogen atmosphere. MS monitoring showed no starting material, and the reaction was stopped. The palladium carbon was filtered through celite, and the filtrate was concentrated to obtain compound 9 (105 mg, 86.5%) without further purification. HRMS calculation for C 64 H 104 N9O 19 + , 1302.7448 [M+H] + ; found, 1302.7459.

[0217] (9) Compound 9 (100 mg, 0.076 mmol) and HATU (64 mg, 0.169 mmol) were dissolved in 5 mL of DMF, and DIPEA (30 mg, 0.23 mmol) was added dropwise, followed by 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrrolidine (23 mg, 0.12 mmol). The mixture was stirred at room temperature under nitrogen protection. After LC-MS monitoring showed that no starting material remained, the reaction was stopped. The reaction solution was diluted with ethyl acetate and washed three times with saturated brine. The organic phases were combined, dried over sodium sulfate, concentrated, and purified by flash column chromatography to obtain compound 10 (66 mg, 59.1%). HRMS calcd for C 74 H 122 BN 10 O 20 + , 1481.8930 [M+H] + ;found, 1481.8922.

[0218] (10) Referring to the synthesis scheme of compound 5, excess trifluoroacetic acid was added to compound 10 (60 mg, 0.04 mmol) for hydrolysis, and compound F33 (20 mg, 38.2%) was obtained by liquid phase separation after post-treatment. HRMS calculation for C 52 H 80 BN 10 O 20 + , 1175.5643 [M+H] + ; found, 1175.5633.

[0219] Example 33 A compound targeting fibroblast activation protein, whose structure is as shown in compound F34.

[0220] Its synthetic route is as follows:

[0221] The specific steps of synthesis are as follows: (1) Boc-L-glutamic acid (124 mg, 0.5 mmol) and compound 7 (780 mg, 1.1 mmol) from Example 32 were condensed with the synthesis and post-treatment method of compound 2 from Reference Example 32 to obtain compound 1 (335 mg, 41.1%). HRMS calcd for C 82 H 108 N 11 O 24 + , 1630.7569 [M+H]+ ; found, 1630.7558.

[0222] (2) Compound 1 (320 mg, 0.2 mmol) was hydrolyzed by adding excess trifluoroacetic acid, and the synthesis scheme of compound 5 in Example 32 was referred to. After post-treatment, compound 2 (245 mg, 80.2%) was obtained. HRMS calculation for C 77 H 100 N 11 O 22 + ,1530.7044 [M+H] + ; found, 1530.7038. Proceed directly to the next step of condensation. (3) Compound 2 (150 mg, 0.098 mmol) was weighed and separated by acid-amine condensation according to the synthesis scheme of compound 8 in Example 32 to obtain compound 3 (100 mg, 46.3%). HRMS calculation for C 112 H 162 N 15 O 31 + , 2213.1561 [M+H] + ; found, 2213.1553.

[0223] (4) Referring to the synthesis scheme of compound 9 in Example 32, compound 3 (100 mg, 0.045 mmol) was weighed and reduced to obtain compound 4 (82 mg, 90.0%). HRMS calculation for C 98 H 150 N 15 O 31 + , 2033.0622 [M+H] + ; found,2033.0610.

[0224] (5) Referring to the synthesis scheme of compound 10 in Example 32, compound 4 (65 mg, 0.032 mmol) was weighed and reduced to obtain compound 5 (50 mg, 66.3%). HRMS calculation for C 118 H 186 B2N 17 O 33 + , 2391.3585 [M+H] + ;found, 2391.3596.

[0225] (5) Referring to the synthesis scheme of compound F33, excess trifluoroacetic acid was added to compound 5 (40 mg, 0.016 mmol) for hydrolysis, and compound F34 (15 mg, 44.9%) was obtained by liquid phase separation after post-treatment. HRMS calculation for C 90 H 134 B2N 17 O 33 + , 2002.9516 [M+H] + ; found, 2002.9523.

[0226] Example 34 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F1 with gallium 68, and the radiolabeling route is as follows:

[0227] The specific steps are as follows: Use 4 mL of 0.05 M dilute hydrochloric acid to 68 Ge / 68 Ga generator elution [ 68 A Ga]GaCl3 solution was added to a reaction tube, and 200 μL of 1.25 M sodium acetate solution was added to adjust the pH to 4. Finally, compound F1 (25 μg, 0.022 μmol) was added and mixed thoroughly. The mixture was heated at 80°C for 5 min. After cooling, 4 mL of water was added, mixed thoroughly, and the mixture was rinsed into a SEP-PAK C18 cartridge. The cartridge was rinsed with 5 mL of water, and air was injected to remove excess liquid. Finally, the product was rinsed with 1 mL of ethanol, filtered through a 0.22 μm sterile filter, collected in a reaction flask, and concentrated. Physiological saline was added to dilute the ethanol content to 5% to obtain a standard Ga-68 labeled injection solution. The total radiosynthesis time was 30 min (other embodiments may require 30-40 min).

[0228] Compounds F2-F11, F15-F16, F21, F24-F31, and F33-F34 labeled with gallium-68 radioactivity can be prepared by referring to the above method.

[0229] Example 35 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F1 with copper 64, and the radiolabeling route is as follows:

[0230] The specific steps are as follows: 64 Cu]CuCl2 was dissolved in 0.05 M HCl to prepare a solution, and 17.5 μL (37 MBq) [64 A Cu]CuCl2 solution was placed in a reaction flask and the pH adjusted to 4 by adding ammonium acetate. 50 μg of compound F1 was dissolved in 1 mL of sodium ascorbate and added to the solution. The reaction was heated at 80°C for 5 minutes. After stopping the reaction, the solution was cooled, diluted with 4 mL of water, mixed thoroughly, and then rinsed onto a SEP-PAK C18 cartridge. The cartridge was rinsed with 5 mL of water, and air was injected to remove excess liquid. Finally, the product was rinsed with 1 mL of ethanol, filtered through a 0.22 μm sterile filter, collected in a reaction flask, and concentrated. Physiological saline was added to dilute the ethanol content to 5% to obtain a standard copper-64-labeled injection solution. The total radiosynthesis time was 30 minutes (other embodiments may require 30-40 minutes).

[0231] Compounds F2-F11, F15-F16, F21, and F24-F31 labeled with copper-64 radioactivity can be prepared by referring to the above method.

[0232] Example 36 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F1 with lutetium-177, and the radiolabeling route is as follows:

[0233] The specific steps are as follows: Take 0.25 mL of 0.05 M HCl [ 177 A Lu]LuCl3 solution (37 MBq) was added to a reaction vial, and 15 μL of 2 M NaOAc was added to adjust the pH to 4. 25 μg of compound F1 was dissolved in 25 μL of DMSO and added to the reaction vial. The reaction was heated at 80°C for 10 min. After stopping the reaction, the solution was cooled, diluted with 4 mL of water, mixed, and then eluted into a SEP-PAK C18 cartridge. The cartridge was rinsed with 5 mL of water, and air was injected to remove excess liquid. Finally, the product was eluted with 1 mL of ethanol, filtered through a 0.22 μm sterile filter, collected in a reaction vial, and concentrated. Physiological saline was added to dilute the ethanol content to 5% to obtain a standard Lu-177-labeled injection solution. The total radiosynthesis time was 30 min (other embodiments may require 30-40 min).

[0234] Compounds F2-F11, F15-16, F21, and F24-F31 labeled with lutetium-177 radioactivity can be prepared by referring to the above method.

[0235] Example 37 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F1 with yttrium-86, and the radiolabeling route is as follows:

[0236] The specific steps are as follows: take 37 MBq 86 A 0.1 M hydrochloric acid solution of YCl₃ was prepared in a reaction flask. 3 M sodium acetate was added to adjust the pH to 4.5. 25 μg of Compound F1 was added to the reaction solution and heated at 80°C for 10 minutes. After stopping the reaction, the solution was cooled and diluted with 4 mL of water. Mix thoroughly and rinse the solution onto a SEP-PAK C₁ₒ cartridge. The cartridge was rinsed with 5 mL of water, and air was injected to remove excess liquid. Finally, the product was rinsed with 1 mL of ethanol, filtered through a 0.22 μm sterile filter, collected in a reaction flask, and concentrated. Physiological saline was added to dilute the ethanol content to 5% to obtain a standard Y₁₆-labeled injection solution. The total radiosynthesis time was 30 minutes (other embodiments may require 30-40 minutes).

[0237] Compounds F2-F11, F15-F16, F21, and F24-F31 labeled with yttrium-86 radioactivity can be prepared by referring to the above method.

[0238] Example 38 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F21 with fluoroaluminum, and the radiolabeling route is as follows:

[0239] The specific steps are as follows: 10 μL of AlCl₃ solution (2 mM, 0.02 μmol) is added to a reaction vial, 300 μL of 0.5 M sodium acetate is added, the pH is adjusted to 4, and finally, compound F21 (25 μg, 0.024 mmol) is added and mixed. The mixture is reacted at 90°C for 15 min. After cooling, 5 mL of water is added, and the mixture is washed into a SEP-PAK C₁ₒ column. The column is rinsed with 5 mL of deionized water, air is injected, and excess liquid is removed. Finally, the product is washed with 1 mL of ethanol, filtered through a 0.22 μm sterile filter, collected in a reaction vial, and concentrated. Physiological saline is added to dilute the ethanol content to 5% to obtain a fluorinated aluminum-labeled injection solution that meets the standards. The total radiosynthesis time is 30 min (other embodiments may be 30-40 min).

[0240] Example 39 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F15 with zirconium-89, and the radiolabeling route is as follows:

[0241] The specific steps are as follows: take 37 MBq 89 Zr 4＋The solution was diluted to 300 μL with 1.0 M oxalic acid solution, and the pH was adjusted to 6.8-7.5 by adding 1.0 M Na₂CO₃. 1 mg of compound F15 was dissolved in 200 μL of 0.5 M HEPES buffer (pH 7.5), and the above solution was added. The mixture was reacted at 37°C for 30 min (in other embodiments, this may be 30-60 min). After stopping the reaction, the mixture was cooled and diluted with 4 mL of water. The mixture was mixed and then rinsed into a SEP-PAK C18 cartridge. The cartridge was rinsed with 5 mL of water, and air was injected to remove excess liquid. Finally, the product was rinsed with 1 mL of ethanol, filtered through a 0.22 μm sterile filter, collected in a reaction flask, concentrated, and diluted to 5% ethanol with physiological saline to obtain a standard Zr-89 labeled injection solution. The total radiosynthesis time was 80 min (in other embodiments, this may be 80-120 min).

[0242] Example 40 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F19 with iodine-131, and the radiolabeling route is as follows:

[0243] The specific steps are as follows: 131 I-NaI solution (37 MBq), 5 μL of acetic acid, and 2.5 μL of NIS methanol solution (4 mg / mL) were added to a reaction vial. 30 μL of compound F19 methanol solution (5 mg / mL) was added to the reaction mixture and allowed to react at room temperature for 15 min (in other embodiments, this may be 15-20 min). After stopping the reaction, the reaction solution was diluted with 5 mL of deionized water, mixed, and rinsed into a SEP-PAK C18 cartridge. Air was injected, and excess liquid was removed. The cartridge was eluted with 0.5 mL of ethanol, filtered through a 0.22 μm sterile filter membrane, and then collected in a reaction vial, concentrated. Physiological saline was added to dilute the ethanol content to 5% to obtain an iodine-131 injection solution that met the standards. The total radiosynthesis time was 30 min (in other embodiments, this may be 30-60 min).

[0244] Example 41 A compound targeting fibroblast activation protein is obtained by radiolabeling compound F19 with astatine 211, and the radiolabeling route is as follows:

[0245] The specific steps are as follows: take 200 μL of NCS 211A methanol solution of At (37 MBq, 0.2 mg / mL NCS) was added to a reaction vial. 25 μg of compound F19 and 5 μL of acetic acid were then added to the reaction mixture and allowed to react at room temperature for 30 minutes. After stopping the reaction, excess methanol was removed with nitrogen gas and the product was reconstituted in 20% acetonitrile. After HPLC purification, the reaction mixture was diluted with 5 mL of deionized water, mixed thoroughly, and then rinsed onto a SEP-PAK C18 cartridge. Air was injected, and excess liquid was removed. The cartridge was then eluted with 0.5 mL of ethanol, filtered through a 0.22 μm sterile filter, and collected in a reaction vial. The product was concentrated and diluted to a 5% ethanol content with physiological saline to obtain a standard astatine 211 injection solution. The total radiosynthesis time was 70 minutes.

[0246] Example 42 A compound targeting fibroblast activation protein, whose structure is similar to compound F32, is obtained by radiolabeling compound F22 with technetium-99m. The radiolabeling route is as follows:

[0247] The specific steps are as follows: Compound F22 was dissolved in DMSO to prepare a 1 mg / mL solution, 10 μL of the ligand solution was dissolved in 0.5 mL of saline, succinate buffer (pH = 5.0) was added to adjust the pH value to 5, 2 mg of TPPTS and 1 mg of tris(hydroxymethyl)methylglycine were added to the above solution, and finally [ 99m After adding 2% Tc] NaTcO4 solution (37 MBq), the mixture was shaken and reacted at 100 °C for 30 min to obtain the technetium-99m labeled FAPI radioligand.

[0248] Compound F23 was radiolabeled with technetium-99m according to the above method.

[0249] Effect test (1) Affinity test Preparation buffer (25 mM Tris, 250 mM NaCl, pH = 7.4) was used for dilution and reaction. 0.2 mg / mL recombinant human FAP protein was diluted to 0.4 μg / mL on ice. The substrate Gly-Pro-AMC was diluted to 40 μM. The concentration of the candidate compound probe was diluted in eight steps from 40 μM to 4 pM. 25 μL of probe and 25 μL of substrate were evenly added to a 96-well plate, followed by 50 μL of FAP protein and incubation at 37°C for 1 hour. Each concentration was repeated three times. Fluorescence intensity (E) was measured. x / E m =380 / 460 nm). Data were processed by GraphPad Prism, and IC was calculated.50 The results are shown in Table 1. From the results, it can be seen that the compound of the present application and the reported FAP inhibitors UAMC-1110 and FAP-04 all have high affinity for FAP.

[0250] Table 1 IC values ​​of compounds 50

[0251] (2) Biodistribution Eight-week-old BALB / c nude mice were selected as experimental subjects and injected into their right axilla with a dose of 5×10 6 A549-FAP cells. When the tumor grows to about 0.5 cm 3 Biodistribution experiments can be performed by injecting [ 68 Ga]、[ 18 F] or [ 99m The radioactive compound (10 MBq) was labeled with Tc. Animals were sacrificed at 0.5 h, 1 h, and 4 h (n = 4 per time point). Radioactivity in all dissected organs and blood was measured using a gamma counter (Cobra Autogamma, Packard). Values ​​were expressed as the percentage of injected dose per gram of tissue (%ID / g), and the tumor / normal tissue ratio was calculated. The results are shown in Table 2. As shown in Table 2, the tumor / normal tissue ratio was similar to that reported in the classical [ 68 Compared with [Ga]FAP04, the compound of the present application has a greater target to non-target ratio advantage in tumor flesh, tumor liver, tumor kidney and tumor blood.

[0252] Table 2. Tumor / normal tissue ratio in A549-FAP xenografted mice at 1 hour

[0253] (3) PET imaging Eight-week-old BALB / c nude mice were selected as experimental subjects and injected into their right axilla with a dose of 5×10 6 A549-FAP cells. When the tumor grows to about 0.5 cm 3 PET imaging can be performed when the volume is larger than that of the tumor. Mice are anesthetized with isoflurane and radiolabeled compounds (10 MBq) are injected through the tail vein. PET imaging is performed within 240 minutes after injection. Images are iteratively reconstructed using the 3D-OSEM+MAP method, and the average radioactivity concentration in the tumor or organ of interest is semi-quantitatively calculated as the mean standard uptake value (SUV). Attenuation-corrected whole-body coronal images are obtained by processing with Inevon Research Workplace 4.1 software (Siemens, Erlan). Regions of interest (ROIs) of tumors and major organs are then manually drawn. See Table 3. Representative [68 Ga] PET image of compound F1 at 1 h, see Figure 1 . Compared with the reported classic [ 68 Compared with Ga]FAP-04, the compound of the present application has the advantage of a larger target to non-target ratio. At 4 h, the probe of the compound of the present application retains longer in the tumor.

[0254] Table 3. Tumor / normal tissue ratio of A549-FAP xenograft model mice at 4 h

[0255] (4) SPECT imaging Eight-week-old BALB / c nude mice were selected as experimental subjects and injected into their right axilla with a dose of 5×10 6 A549-FAP cells. When the tumor grows to about 0.5 cm 3 SPECT imaging experiments can be performed when the volume is large. Before imaging, mice were anesthetized with isoflurane and then radiolabeled compounds (10 MBq) were injected through the tail vein. Images were collected between 0.5 h and 4 h after injection. SPECT images were reconstructed with the help of 3D iterative algorithm software (Tera-Tomo), and the radioactivity counts in the tumor, kidney, muscle, heart, and liver were further analyzed in detail by drawing the volume of interest. One hour after injection, the tumor-to-meat ratio, tumor-to-liver ratio, and tumor-to-kidney ratio were [ 99m Tc] compound F32 (19.4, 11.3, 8.4), which is higher than the reported [ 99m Tc]FAPI-34 (6.2, 5.2, 3.1) has high tumor-to-skin ratio, tumor-to-liver ratio, and tumor-to-kidney ratio. 99m Tc] SPECT pattern of compound F32 at 1 h, see Figure 2 .

[0256] (5) Treatment Eight-week-old BALB / c nude mice were selected as experimental subjects and injected into their right axilla with a dose of 5×10 6 A549-FAP cells were grown. Treatment was given when the average tumor diameter reached 3-4 mm. The mice were divided into 4 different groups (n=4). Group 1 was treated with normal saline (negative control group), Group 2 was treated with 18.5 MBq of lutetium-177-labeled compound F3 (low-dose group), Group 3 was treated with 37 MBq of lutetium-177-labeled compound F1 (high-dose group), and Group 4 was treated with [ 177Lu]Lu-FAPI-46 (positive control), 37 MBq. Treatment was administered via tail vein injection on day 0. Tumor volume and mouse body weight were measured on day 0 (the day of the first administration of radiotracer) and then repeated three times weekly until the completion of the study. Once the mouse body weight had decreased by 40% or the tumor diameter exceeded 1500 mm, the tumor volume and weight of the mouse were measured. 3 , the mice were euthanized. It was verified that after three cycles of treatment with lutetium-177 labeled compound F1, the tumor volume was reduced to 30%, while no significant changes were found in normal organs after dissection and HE staining. 177 The volume of the control tumor was reduced to 70% by Lu]Lu-FAPI-46, indicating that the lutetium-177 labeled compound F1 had a better therapeutic effect.

[0257] Other metal-labeled compounds were also subjected to therapeutic experiments referring to the above experimental procedures. The results showed that these compounds all had inhibitory effects on tumors.

[0258] The above is only a preferred embodiment of the present application. The present application is not limited to the above-mentioned embodiments. As long as the technical effects of the present application are achieved by the same means, they shall fall within the scope of protection of the present application. Within the scope of protection of the present application, the technical solutions and / or implementation methods may be modified and varied in various ways.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, diastereomer, tautomer, hydrate, solvate, or crystal thereof, in, R1 and R2 are independently selected from H, F; R3 is selected from an aromatic ring or an aromatic heterocyclic ring; R py1 、R py2 、R py3 , R4 and R5 are independently selected from: hydrogen, deuterium, halogen, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 alkyl; *It is the dextrorotatory, levorotatory or racemic isomer; Of R6 and R7, one is H and the other is CN or B(OH)2; R a and R b Independently selected from: hydrogen, a first compound, a second compound; the first compound is a labeling precursor, a cold compound or a radioactive compound containing a linker and a chelator; the second compound is a labeling precursor, a cold compound or a radioactive compound containing a first targeting molecule, a linker and a chelator, and the first targeting molecule is a targeting molecule of non-targeted fibroblast activation protein; R a and R b Not hydrogen at the same time.

2. The compound according to claim 1, wherein R3 is a first group substituted by 1-3 first substituents, wherein the first group is a benzene ring, a pyridine ring, a naphthalene ring or a quinoline ring, and the first substituent is hydrogen, deuterium, halogen, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, 1-3 second substituents substituted -C 1-6 Alkyl, -OC substituted with 1-3 second substituents 1-6 Alkyl, 1-3 second substituents substituted -SC 1-6 Alkyl, alkenyl, cycloheteroalkenyl or alkynyl, the second substituent is -OH, oxygen or halogen.

3. The compound according to claim 1, wherein The structure of the compound is shown in Formula II or Formula III, Wherein, n is 1, 2 or 3; L is a linking group selected from substituted or unsubstituted C 1-12 Alkylene, substituted or unsubstituted C 1-12 Cycloalkylene, substituted or unsubstituted C 1-12 Heteroalkylene, substituted or unsubstituted C 1-12 Heterocycloalkylene, substituted or unsubstituted C 1-12 Oxyalkylene, substituted or unsubstituted C 1-12 aminoalkylene, substituted or unsubstituted C 1-12 Alkenylene, substituted or unsubstituted C 1-12 Heterocycloalkenylene, substituted or unsubstituted C 1-12 Alkenylene, substituted or unsubstituted C 1-12 Alkynylidene, substituted or unsubstituted C 1-12 Arylene, substituted or unsubstituted C 1-12 one or a combination of heteroarylene groups, polypeptide groups, carbonyl groups, imine groups, imide groups, thioamide groups, phosphoramide groups, thioether groups, disulfide groups, ester groups, thioester groups, carbamate groups, carbonate groups, phosphate groups, anhydride groups, hydrazone groups, acylhydrazone groups and sugars; G is 18 F. 18 a leaving group or a radioactive metal chelating group labeled with F; the leaving group is selected from the group consisting of: halogen, iodonium salt, quaternary ammonium salt, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, methanesulfonyl, trifluoromethanesulfonyl, stannyl, silyl, boronic acid, borate ester, nitrate, acetate, cyano, and thiol; the radioactive metal chelating group is a chelating agent group suitable for radiolabeling, or a chelate comprising a non-radioactive isotope metal and a chelating agent, or a chelate comprising a radioactive isotope and a chelating agent; T is a targeting molecule targeting a first protein, wherein the first protein is at least one of PSMA, albumin, somatostatin receptor 2, integrin, MAT2A and GPPC.

4. The compound according to claim 3, wherein R1 and R2 are F.

5. The compound according to claim 3, wherein When the compound is of formula II, the structure of the compound is as shown below: Wherein, R is an amino acid residue, I is R in the structure of formula I a and R b The structure when they are not H at the same time, Q is a condensed diacid or condensed triacid of I, and when G contains a chelating agent, Q can be condensed with the chelating agent, x is selected from an integer of 0-10, and y is selected from 1, 2 or 3.

6. The compound according to claim 5, wherein The structure of the compound is shown as any one of the following: Wherein, * represents the dextrorotatory isomer, levorotatory isomer or racemic isomer.

7. The compound according to claim 3, wherein When the compound is of formula III, the structure of the compound is as shown below: Wherein, R is an amino acid residue, I is R in the structure of formula I a and R b In the structure when they are not H at the same time, Z is a linker that can condense with I, and when G contains a chelating agent, Z can be coupled with the chelating agent and T at the same time, x is selected from an integer of 0-10, and y is selected from 1, 2 or 3.

8. The compound according to claim 7, wherein The structure of the compound is shown as any one of the following: Wherein, * represents the dextrorotatory isomer, levorotatory isomer or racemic isomer.

9. The compound according to claim 3, wherein When G has coordination ability, the chelating agent group suitable for radiolabeling is selected from sulfur colloid, diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid, 1,4,7,10-tetraazacyclododecane-N , N ,, N ,,, N ,,,, -tetraacetic acid, iminodiacetic acid, bis(carboxymethylimidazole)glycine, 6-cyanopyridine-3-carboxylic acid, HBED-CC, AAZTA, DOTA, DODAGA, NOTA, NODAGA, or DFO.

10. The compound according to claim 3, wherein The radioisotope is selected from 18 F. 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re、 188 Re、 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y. 88 Y. 90 Y. 149 Pm, 165 Dy, 169 Second, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 121 Bi, 123 Bi, 72 As、 72 Se, 97 Such as 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I. 124 I. 131 I. 197 Hg, 211 At 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 227 Th, 199 Ag.

11. The compound according to claim 3, wherein T is selected from any of the following structures: 。 12. The compound according to any one of claims 1 to 11, characterized in that The structure of the compound is shown as any one of the following: 。 13. A pharmaceutical composition, characterized in that The invention comprises the compound according to any one of claims 1 to 12.

14. Use of the compound according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13 in the preparation of an agent for diagnosing or treating a disease characterized by overexpression of fibroblast activation protein (FAP).

15. A kit, characterized in that The invention comprises the compound according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13.

16. An imaging agent, characterized in that The invention comprises the compound according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13.

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