A compound targeting fibroblast activation protein and use thereof

Abstract

The application belongs to the technical field of biological medicine, and specifically discloses a compound targeting fibroblast activation protein and application thereof. The compound forms a new skeleton molecule targeting FAP with phenylalanine / pyridylalanine / naphthylalanine-picolinoyl-glycyl-prolyl amide as a core, and the structure is shown as formula I. The compound designed in the application has high tumor uptake, low normal tissue (such as liver and bone) uptake, and high target-to-non-target ratio, thereby greatly improving the properties of the original probe and facilitating accurate tumor contouring. Meanwhile, the compound provided in the application has strong chelating ability and can form stable chelates with most radioactive metals, thereby being conducive to the integration of tumor diagnosis and treatment.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a compound that targets fibroblast activation proteins and its applications. Background Technology

[0002] Fibroblast activating protein (FAP) is a type II transmembrane serine protease belonging to the proline-specific peptidase family, possessing dipeptidyl peptidase and endopeptidase activities. It is highly expressed in tumor-associated fibroblasts (CAFs) of the vast majority of epithelial-derived tumors, but almost non-expressed in normal tissues (appearing only in some wound healing and fibrotic lesions). CAFs, as key components of the tumor microenvironment, 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 therapy. J Biol Chem. 1999, 274, 36505-36512, Hum Pathol. 2013, 44, 2549-2557).

[0003] Current development of FAP-based radiopharmaceuticals mainly focuses on small molecule inhibitors (FAPIs), whose core structures often contain quinoline acyl-glycyl-prolyl amides (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 nuclide labeling. Among them, 68 Ga-labeled FAPIs show superior tumor / background ratios in PET / CT imaging compared to... 18 F-FDG has unique advantages in treating hypoglycemic tumors and fibrotic lesions; 64 Cu、 44 The long half-life of Sc nuclides optimized the imaging time window and pharmacokinetics studies; 177 Lu、 225 Therapeutic radionuclide-labeled FAPIs such as Ac have preliminarily confirmed the cumulative radiation dose to tumors, 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 therapies suffer from problems such as short tumor retention time and non-specific uptake (e.g., 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 treatment effectiveness, and the modification of the quinoline core of the UAMC-1110-derived probe urgently requires breakthroughs in the molecular skeleton. To address the issue of radioactivity in quinoline-ring-containing FAPs, peptide compounds such as FAP-2286 have been developed. While this compound significantly improves tumor retention, it also suffers from insufficient renal uptake. Eur J Nucl Med Mol Imaging (2023) 50:3050–3061); The compounds obtained by replacing the quinoline ring with a pyridine ring have poor affinity (in the μM range). J Med Chem. 2014, 57, 3053-3074), as a radioactive probe, has poor specificity, which has also been applied to the research of PET probes (CN202410031820.0). The compounds invented in this patent are all... 18 F-labeled compounds are only suitable for diagnosis, and their tumor uptake is low (all less than 1), with a tumor-to-cytoplasm ratio of around 2. This is significantly lower than FAPI-04 and FAPI-46, and further optimization is needed. Similarly, azacyclic compounds (Pharmaceuticals 16 (2023)449) have also been explored. 68 Ga-labeled, but with lower affinity (187 ± 52.0 and 17.1 ± 4.60 nM), compared to... J With Chem. The results from 2014, 57, 3053-3074 are consistent, leading to low tumor uptake and a low target-to-non-target ratio, which limits further clinical applications. Summary of the Invention

[0004] The purpose of this application is to address the shortcomings of existing technologies, specifically the insufficient affinity and specificity of the pyridine core in current FAPIs. It innovatively replaces quinoline with pyridine and introduces phenylalanine, pyridinealanine, and naphthylalanine next to pyridine, forming a new FAP-targeting backbone molecule with phenylalanine / pyridinealanine / naphthylalanine-pyridyl-glycyl-prolyl amide as its core. This significantly enhances the compound's affinity for FAP. Combined with the introduction of different linker groups and polymerization of the targeting molecule, novel ligands with single- and multi-target synergistic effects and their metal chelate FAP probes are innovatively designed and developed. This research addresses the dual clinical needs of solid tumor diagnosis and targeted radionuclide therapy. Through original structural optimization, a series of novel FAP-targeting ligands and their integrated diagnostic and therapeutic metal nuclide probes with high resolution and high specificity have been successfully developed. Based on this, the first aspect of this application provides a compound of Formula I or its pharmaceutically acceptable salt, diastereomer, tautomer, hydrate, solvate, or crystal.

[0005]

[0006] Among them, R1 and R2 are independently selected from H and F; R3 is selected from aromatic rings or aromatic heterocycles; R py1 R py2 R py3 R4 and R5 are independently selected from: hydrogen, deuterium, halogens, and -C. 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl group; of R6 and R7, one is H and the other is CN or B(OH)2; * indicates dextrorotatory, levorotatory, or racemic; R a and R b Independently selected from: hydrogen, a first compound, and a second compound; the first compound is a labeled precursor, a cold compound, or a radioactive compound containing a linker and a chelating agent; the second compound is a labeled precursor, a cold compound, or a radioactive compound containing a first targeting molecule, a linker, and a chelating agent; the first targeting molecule is a targeting molecule that does not target fibroblast activation proteins; R a and R b They are not both hydrogen.

[0007] In some preferred embodiments, R3 in the compound of Formula I is a first group after substitution by 1-3 first substituents, wherein the first group is a benzene ring, pyridine ring, naphthalene ring, or quinoline ring, and the first substituent is hydrogen, deuterium, halogen, or -C. 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl group, -C substituted with 1-3 second substituents 1-6 Alkyl groups, 1-3 second substituents substituted -OC 1-6 -SC with alkyl groups and 1-3 second substituents 1-6 Alkyl, alkenyl, cyclohexeneyl or alkynyl, wherein the second substituent is -OH, oxygen or halogen.

[0008] Preferably, the structure of the above-mentioned compound is as shown in Formula II or Formula III.

[0009]

[0010] Where n is 1, 2 or 3;

[0011] L is a linker, selected from substituted or unsubstituted C. 1-12 Alkylene, substituted or unsubstituted C 1-12 Cycloalkylene, substituted or unsubstituted C 1-12 heteroalkyl, substituted or unsubstituted C 1-12 heterocyclic alkyl, substituted or unsubstituted C 1-12 alkylene oxides, substituted or unsubstituted C 1-12aminoalkylene, substituted or unsubstituted C 1-12 alkenyl, substituted or unsubstituted C 1-12 heterocyclic alkenyl, substituted or unsubstituted C 1-12 alkenyl, substituted or unsubstituted C 1-12 alkyne group, substituted or unsubstituted C 1-12 aryl, substituted or unsubstituted C 1-12 One or more of the following groups: heteroaryl, polypeptide (one of dipeptides to hexapeptides), carbonyl, imine, imide, thioamide, phosphoramide, thioether, dithio, ester, thioester, carbamate, carbonate, phosphate, anhydride, hydrazone, acylhydrazone, and sugars.

[0012] G is 18 F, 18 The F-labeled leaving group or radioactive metal chelating group; the leaving group is selected from: halogen, iodonium salt, quaternary ammonium salt, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, methanesulfonyl, trifluoromethanesulfonyl, tinalkyl, silalkyl, borate, borate ester, nitrate, acetic acid, cyano, thiol; the radioactive metal chelating group is a chelating agent group suitable for radiolabeling, or a chelate containing a non-radioactive isotopic metal and a chelating agent, or a chelate containing a radioactive isotope and a chelating agent.

[0013] T is the target molecule that targets the first protein, which is at least one of PSMA, albumin, somatostatin receptor 2, integrin, MAT2A, and GPPC.

[0014] In some preferred embodiments, R1 and R2 in Equations II and III are F.

[0015] When the above compound has the structure of formula II, one end of L is connected to formula I (R). a and R b An amino derivative of formula II is attached to the first amino group (H), which can further undergo 1, 2, or 3 couplings and can be further coupled with a chelating group to obtain the structure of formula II. The structures of the above compounds are shown in any of the following:

[0016]

[0017] Where R is an amino acid residue, and I is the R in the structure of formula I. a and R b The structure is different from that of H. Q is a condensed diacid or condensed triacid of I. When G contains a chelating agent, Q can condense with the chelating agent. x is selected from integers from 0 to 10, and y is selected from 1, 2 or 3.

[0018] For example, the structures of the above compounds are shown in any of the following:

[0019]

[0020] Where * represents the dextrorotatory, levorotatory, or racemic form.

[0021] When the above compound has the structure 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 to another targeting molecule (T), and can be further coupled to a chelating agent to obtain the structure of formula III. The structures of the above compounds are shown in any of the following:

[0022]

[0023] Where R is an amino acid residue, and I is the R in the structure of formula I. a and R b The structure is different from that of H. Z is a linker that can condense with I. When G contains a chelating agent, Z can be coupled with both the chelating agent and T. x is selected from integers from 0 to 10, and y is selected from 1, 2 or 3.

[0024] For example, the structures of the above compounds are shown in any of the following:

[0025]

[0026] Where * represents the dextrorotatory, levorotatory, or racemic form.

[0027] When G has coordinating ability, the chelating agent groups suitable for radiolabeling are selected from sulfur colloids, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), and 1,4,7,10-tetraazacyclododecane-N , N ,, N ,,, N ,,,, Tetraacetic acid (TETA), iminodiacetic acid, bis(carboxymethylimidazolium)glycine, 6-cyanopyridine-3-carboxylic acid (HYNIC), HBED-CC, AAZTA, DOTA, DODAGA, NOTA, NODAGA, or DFO. Or selected from any of the following structures:

[0028] .

[0029] The radioactive isotopes mentioned above are 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 Er、 177 Lu、 47 Sc、 142 Pr、 159 Gd, 121 Bi、 123 Bi、 72 As、 72 Se、 97 Ru、 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.

[0030] Where T is selected from any of the following structures:

[0031] .

[0032] In some embodiments, the structure of the above-mentioned compound is shown in any of the following:

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049] .

[0050] A second aspect of this application provides a pharmaceutical composition comprising the above-described compounds; in some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0051] A third aspect of this application provides the use of the above-described compound or pharmaceutical composition in the preparation of a reagent for diagnosing or treating diseases characterized by overexpression of fibroblast activation protein (FAP). Diseases characterized by overexpression of fibroblast activation protein (FAP) are selected from cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and scarring. Preferably, the cancers are selected from breast cancer, pancreatic cancer, small bowel cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal carcinoma, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal cell carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (primary unknown cancer), thymic carcinoma, glioma, neuroglioma, astrocytoma, cervical cancer, and prostate cancer.

[0052] A fourth aspect of this application provides a kit comprising the above-described compound or the above-described pharmaceutical composition.

[0053] The fifth aspect of this application provides an imaging agent comprising the above-described compound or the above-described pharmaceutical composition.

[0054] This application addresses the technical bottlenecks in the prior art and, based on the rational drug design concept, focuses on optimizing analogs of FAPI-04 and FAPI-46, as well as the compound invented in CN202410031820.0, and has the following characteristics:

[0055] 1. Modification of the target group: By reducing the quinoline ring to a pyridine ring and introducing phenylalanine, pyridinealanine, or naphthylcycloalanine at the adjacent position of the pyridine N atom, the affinity of the target molecule for the FAP protein is increased through precise regulation.

[0056] 2. Improve pharmacokinetic properties: The aim is 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 the target molecules, the uptake and retention of tumors are improved, while the uptake by the liver and kidneys is reduced.

[0057] 3. Diversified imaging radioactivity: By introducing different chelating agents and leaving groups, it is possible to... 18 F, 68 Ga-labeled FAP probes are used in PET imaging, and 99m Tc-labeled FAP probes are used in SPECT imaging, thus providing patients with more options.

[0058] 4. Therapeutic Potential: This optimized chelation system possesses excellent coordination capabilities and can be seamlessly integrated for the treatment of therapeutic radionuclides (such as...). 177 Lu, 90 The markers (such as Y) enable precise diagnosis and efficient targeted therapy of systemic FAP-related tumors using the same molecular carrier.

[0059] This application is expected to lead to a leapfrog development of the FAP molecular imaging "integrated diagnosis and treatment platform," providing a powerful tool for precision oncology medicine. This application relates to fibroblast activation protein ligand cold compounds, radioactive compounds and labeled precursors, compositions containing such compounds, kits containing such compounds or compositions, and the use of such compounds, compositions, or kits for diagnostic imaging via positron emission tomography (PET) or single-photon emission computed tomography (SPECT), or for radionuclide therapy of tumors.

[0060] The beneficial effects of this application are as follows: the compound designed in this application has high tumor uptake, low uptake by normal tissues (such as liver and bone), and a high target-to-non-target ratio, thereby greatly improving the properties of the original probe and facilitating precise tumor delineation. At the same time, the compound provided in this application has strong chelating ability and can form stable chelates with most radioactive metals, which is beneficial for the integrated diagnosis and treatment of tumors. Attached Figure Description

[0061] Figure 1 The following is a diagram: 68 PET image of Ga] compound F1 at 1 hour, with the circled area indicating the tumor;

[0062] Figure 2 The following is a diagram: 99m SPECT image of compound F32 (Tc) at 1 hour, with the circled area indicating the tumor. Detailed Implementation

[0063] The following will provide a clear and complete description of the concept and technical effects of this application in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution and effects of this application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0064] The structures of compounds F1-F34 mentioned below are shown below:

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] .

[0082] Example 1

[0083] A compound that targets a fibroblast-activating protein, with the structure of compound F1.

[0084] The synthetic route is shown below:

[0085]

[0086] The specific steps of the synthesis are as follows:

[0087] (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-aminoisonicotinic acid (0.43 g, 2.84 mmol) under nitrogen protection and stir overnight at room temperature. Wash the reaction solution three times with saturated brine. Extract the mixture with ethyl acetate. Combine the organic phases and purify with FC to give compound 1 (0.35 g, 46% yield), a pale yellow solid. 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.

[0088] (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 at 0 °C to room temperature for 4 h. After the reaction was stopped, the reaction solution was concentrated, a small amount of water was added to dissolve the residue, and the pH was adjusted to 4 with 2 M dilute hydrochloric acid. A white solid precipitated, which was filtered to obtain compound 2 (0.18 g, 53%). HRMS calcd for C 20 H 24 N3O5 + , 386.1710 [M+H] + ; found, 386.1711.

[0089] (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. DIPEA (0.41 mL, 2.35 mmol) was added dropwise at 0 °C, and the mixture was stirred at room temperature for 15 min. Then, (…) S 1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride (0.09 g, 0.47 mmol) was stirred overnight at room temperature under nitrogen protection. The reaction mixture was washed three times with saturated brine, extracted with ethyl acetate, and the organic phases were combined and purified by FC to give 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.

[0090] (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. After stirring at 0 °C to room temperature for 6 h, LC-MS monitoring showed no remaining raw material. The reaction solution was concentrated to obtain a foamy solid compound 4 (0.14 g, 86%). HRMS calcd for C 22 H 23 F2N6O3 + , 457.1794 [M+H] + ; found, 457.1795.

[0091] (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. Then, compound 4 (0.18 g, 0.35 mmol) was added. Under nitrogen protection, the mixture was stirred overnight at 50 °C. After the reaction was stopped, 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.

[0092] (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 no residual starting material. The reaction solution was concentrated to obtain compound 6 (0.07 g, 85%) as a yellow foamy solid. HRMS calcd for C 32 H 42 F2N7O8 + , 690.3057 [M+H] + ; found, 690.3059.

[0093] (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, and TEA (16 μL, 0.12 mmol) was added at 0 °C with stirring. Then, compound 6 (16 mg, 0.02 mmol) was added. Under nitrogen protection and with stirring at room temperature, the mixture was purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 78 / 22) yielded a white solid compound F1 (6.5 mg, 28%) with a retention time of 26.3 min. HRMS calcd for C 51 H 72 F2N 11 O 17 + , 574.7571 [M+2H] 2+ ; found, 574.7569.

[0094] Example 2

[0095] A compound that targets a fibroblast-activating protein, with the structure of compound F2.

[0096] The synthetic route is shown below:

[0097]

[0098] The specific steps of the synthesis are as follows:

[0099] 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester (20 mg, 0.039 mmol) and compound 1 (17.91 mg, 0.026 mmol) were dissolved in 1.5 mL of anhydrous acetonitrile, and DIPEA (23.2 μL, 0.13 mmol) was added dropwise to the solution. The mixture was stirred overnight at room temperature under nitrogen protection. Purification was performed by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 72 / 28), retention time 21.4 min, yielded a white solid compound F2 (4.3 mg, 15%). HRMS calcd for C 48 H 68 F2N 11 O 15 + , 1076.4859 [M+H] + ; found, 1076.4856.

[0100] Example 3

[0101] A compound that targets a fibroblast-activating protein, with the structure of compound F3.

[0102] The synthetic route is shown below:

[0103]

[0104] The specific steps of the synthesis are as follows:

[0105] (1) HBTU (0.24 g, 0.62 mmol) and 5,8,11-trioxa-2-azatridecanoic acid-1-tert-butyl ester (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. Then, compound 1 (0.14 g, 0.31 mmol) was added. Under nitrogen protection, the mixture was stirred overnight at 50 °C. After the reaction was stopped, 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 a yellow oily compound 2 (0.14 g, 61%). 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.

[0106] (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, and MS monitoring showed no remaining starting material. The reaction solution was concentrated to obtain compound 3 (0.11 g, 81%). HRMScalcd for C 35 H 46 F2N7O9 + , 746.3319 [M+H] + ; found, 746.3322.

[0107] (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 then added. Under nitrogen protection, the mixture was stirred at room temperature and purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 78 / 22) yielded a white solid compound F3 (22 mg, 26%) with a retention time of 21.1 min. HRMS calcd for C 49 H 68 F2N 11 O 16 +, 1104.4808 [M+H] + ; found, 1104.4805.

[0108] Example 4

[0109] A compound that targets a fibroblast-activating protein, with the structure of compound F4.

[0110] The synthetic route is shown below:

[0111]

[0112] The specific steps of the synthesis are as follows:

[0113] (1) HBTU (0.203 g, 0.54 mmol) and 2,2-dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azadocoadecan-22-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. Then, compound 1 (0.132 g, 0.268 mmol) was added. Under nitrogen protection, the mixture was stirred overnight at 50 °C. After the reaction was stopped, 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 give compound 2 (0.15 g, 67%), which was 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.

[0114] (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 the mixture was stirred at room temperature for 3 h. MS monitoring showed no remaining starting material. The reaction solution was concentrated to obtain a yellow, foamy solid, compound 3 (58 mg, 88%). HRMS calcd for C 34 H 46 F2N7O9 + , 734.3319 [M+H] + ; found, 734.3321.

[0115] (3) Compound 3 (96 mg, 0.153 mmol) was dissolved in DMSO, and TEA (71 μL, 0.51 mmol) was added at 0 °C. The mixture was stirred at room temperature, and 2-(( S )-20-amino-2-benzyl-4-oxo-6,9,12,15,18-pentoxo-3-azaazamide)-N-(2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide (75 mg, 0.102 mmol), purified by HPLC under nitrogen protection and stirring at room temperature. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 77 / 23), retention time 21.7 min, gave white solid compound F4 (27 mg, 22%). HRMS calcd for C 53 H 76 F2N 11 O 18 + ,1192.5332 [M+H] + ; found, 1192.5335.

[0116] Example 5

[0117] A compound that targets a fibroblast-activating protein, with the structure of compound F5.

[0118] The synthetic route is shown below:

[0119]

[0120] The specific steps of the synthesis are as follows:

[0121] (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. Then, compound 4 (0.13 g, 0.28 mmol) was added. Under nitrogen protection, the mixture was stirred at 45 °C for 8 h. After the reaction was stopped, 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 a colorless oily compound 5 (0.14 g, 63%). 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.

[0122] (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, and MS monitoring showed no residual starting material. The reaction solution was concentrated to obtain a yellow, foamy solid compound 6 (0.1 g, 78%). HRMS calcd for C 31 H 41 F2N8O8 + , 691.3009 [M+H] + ; found, 691.3006.

[0123] (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 then added. Under nitrogen protection, the mixture was stirred at room temperature and purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 89 / 11) yielded a white solid compound F5 (22 mg, 26%) with a retention time of 18.7 min. HRMS calcd for C 50 H 71 F2N 12 O 17 + , 1149.5023 [M+H] + ; found, 1194.5022.

[0124] Example 6

[0125] A compound that targets a fibroblast-activating protein, with the structure of compound F6.

[0126] The synthetic route is shown below:

[0127]

[0128] The specific steps of the synthesis are as follows:

[0129] 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 1-(2,5-dioxo-1-pyrrolyl) ester (16 mg, 0.032 mmol) and compound 1 (14.7 mg, 0.021 mmol) were dissolved in 1 mL of anhydrous acetonitrile, and DIPEA (18.8 μL, 0.105 mmol) was added dropwise to the solution. The mixture was stirred overnight at room temperature under nitrogen protection. Purification was performed by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 85 / 15), retention time 19.4 min, yielded a white solid compound F6 (2.6 mg, 11.5%). HRMScalcd for C 47 H 67 F2N 12 O 15 + , 1077.4811 [M+H] + ; found, 1077.4814.

[0130] Example 7

[0131] A compound that targets a fibroblast-activating protein, with the structure of compound F7.

[0132] The synthetic route is shown below:

[0133]

[0134] The specific steps of the synthesis are as follows:

[0135] (1) N-tert-butoxycarbonyl-L-glutamic acid (13 mg, 0.052 mmol) was dissolved in 3 mL of DMF, HBTU (68 mg, 0.18 mmol) was added, and DIPEA (46 μL, 0.26 mmol) was added dropwise under ice bath conditions. Then, compound R (95 mg, 0.13 mmol) was added, and the mixture was stirred at room temperature for 6 h under nitrogen protection. After the reaction was complete, the mixture was diluted with ethyl acetate, washed three times with saturated brine, washed once with saturated sodium bicarbonate solution, extracted twice with ethyl acetate, dried, and concentrated. After purification by FC, the orange compound 1 was obtained. HRMScalcd for C 74 H 96 F4N 15 O 20 + , 1590.6887 [M+H] + ; found, 1590.6885.

[0136] (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 conditions. The mixture was stirred at room temperature for 2 h, and MS monitoring showed no residual starting material. The reaction solution was concentrated to obtain a yellow oily compound 2 (50 mg, 92%). HRMS calcd for C 69 H 88 F4N 15 O 18 + , 1490.6362 [M+H] + ; found, 1490.6365.

[0137] (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, and TEA (24 μL, 0.17 mmol) was added at 0 °C. The mixture was stirred at room temperature, and compound 2 (50 mg, 0.034 mmol) was added. The mixture was then purified by HPLC under nitrogen protection and stirring at room temperature. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 73 / 27) yielded a white solid compound F7 (11 mg, 16%) with a retention time of 19.9 min. HRMS calcd for C 88 H 117 F4N 19 O 27 + , 1949.8409 [M+H] + ; found, 1949.8406.

[0138] Example 8

[0139] A compound that targets a fibroblast-activating protein, with the structure of compound F8.

[0140] Following the synthesis method of Example 7, compound F8 was obtained; HRMS calcd for, C 86 H 116 F4N 21 O 27 + ,1950.8280 [M+H] + ; found, 1950.8284.

[0141] Example 9

[0142] A compound that targets a fibroblast-activating protein, with the structure of compound F9.

[0143] The synthetic route is shown below:

[0144]

[0145] The specific steps of the synthesis are as follows:

[0146] (1) Aminotert-butyl ester-tris-(carbonylethoxymethyl)-methane (16 mg, 0.037 mmol) was dissolved in 2 mL DMF, HBTU (68 mg, 0.18 mmol) was added, and DIPEA (65 μL, 0.46 mmol) was added dropwise under ice bath conditions. Then, compound R (167 mg, 0.19 mmol) was added, and the mixture was stirred at room temperature for 6 h under nitrogen protection. After the reaction was completed, the mixture was purified by HPLC. RP-HPLC (water / acetonitrile = 72 / 28) yielded a colorless oily compound 1 (55 mg, 59%). HRMS calcd for C 114 H 150 F6N 22 O 32 2+ , 1227.0357 [M+2H] 2+ ; found, 1227.0355.

[0147] (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 conditions. The mixture was stirred at room temperature for 2 h, and MS monitoring showed no residual starting material. The reaction solution was concentrated to obtain a yellow oily compound 2 (48 mg, 91%). HRMS calcd for C 109 H 142 F6N 22 O 30 2+ , 1176.5078 [M+2H] 2+ ; found, 1176.5075.

[0148] (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 then added. Under nitrogen protection, the mixture was stirred overnight at room temperature. After stopping the reaction, it was purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 71 / 29) showed a peak time of 19.3 min, yielding 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.

[0149] Example 10

[0150] A compound that targets a fibroblast-activating protein, with the structure of compound F10.

[0151] The synthetic route is shown below:

[0152]

[0153] The specific steps of the synthesis are as follows:

[0154] (1) N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetyl tert-butyl ester (150 mg, 0.233 mmol) and 2,3,5,6-tetrafluorophenol (38.7 mg, 0.233 mmol) were dissolved in 10 mL of anhydrous dichloromethane. A solution of DCC (72 mg, 0.35 mmol) in dichloromethane was slowly added dropwise under ice bath conditions. The mixture was stirred at room temperature for 4 h. The reaction was stopped when no further increase in product was observed by LC-MS. The reaction solution was concentrated, reconstituted with ethyl acetate, and filtered through DCC. The filtrate was concentrated, loaded onto silica gel, and purified by FC to obtain compound 1 (113 mg, 61%). HRMS calcd for C 40 H 49 F4N2O 10 + 793.3318 [M+H] + ; found, 793.3319.

[0155] (2) Compound 1 (57 mg, 0.072 mmol) was dissolved in 1.5 mL of trifluoroacetic acid and stirred overnight at room temperature. LC-MS monitoring showed no starting material remaining, at which point the reaction was stopped, and the trifluoroacetic acid was removed to obtain an oily compound 2 (41 mg, 88%), which required no further purification. HRMS calcd for C 32 H 33 F4N2O 10 + , 681.2066 [M+H] + ; found, 681.2067.

[0156] (3) Compound 2 (43 mg, 0.063 mmol) was dissolved in 1 mL of anhydrous DMSO. TEA (29 μL, 0.21 mmol) was added dropwise under ice bath conditions, followed by compound 3 (29 mg, 0.042 mmol). The mixture was stirred overnight at room temperature 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 calcd for C 58 H 72 F2N9O 17 + , 1204.5009 [M+H] + ; found, 1204.5006.

[0157] Example 11

[0158] A compound that targets a fibroblast-activating protein, with the structure of compound F11.

[0159] The synthetic route is shown below:

[0160]

[0161] The specific steps of the synthesis are as follows:

[0162] (1) Put ( S 2-(tert-Butoxycarbonyl)amino)-3-(naphthyl-1-yl)propionic 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. Then, methyl 2-aminoisonicotinic acid (44 mg, 0.29 mmol) was added, and the mixture was stirred at room temperature for 6 h under nitrogen protection. After the reaction was stopped, the DCM was evaporated to dryness, redissolved in ethyl acetate, washed three times with saturated brine, and once with saturated sodium bicarbonate solution. The collected organic phase was dried, concentrated, and purified by FC on silica gel to give compound 1 as a white solid (60 mg, 47%). HRMS calcd for C 25 H 28 N3O5 + 450.2023 [M+H] + ; found, 451.0962.

[0163] (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 bath conditions. The mixture was stirred at room temperature for 4 h. MS monitoring showed no remaining starting material, at which point 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, resulting in the precipitation of a white solid. The solid was filtered to obtain compound 2 (20 mg, 38%) as a white solid. HRMS calcd for C 24 H 26 N3O5 + , 436.1867 [M+H] + ; found, 437.1156.

[0164] (3) Compound 2 (20 mg, 0.05 mmol) and HBTU (23 mg, 0.06 mmol) were dissolved in 0.5 mL of DMF, and DIPEA (44 μL, 0.25 mmol) was added dropwise under ice bath conditions, followed by the addition of ( S 1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride (9.5 mg, 0.05 mmol) was stirred at room temperature for 6 h under protective conditions. The reaction was stopped when no starting material remained as monitored by MS. 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 give compound 3 (13 mg, 43%) as a pale yellow solid. HRMS calcd for C 31 H 33 F2N6O5 + , 607.2475 [M+H] + ; found, 607.1223.

[0165] (4) Compound 3 (75 mg, 0.13 mmol) was dissolved in 0.5 mL of anhydrous DCM, and 1 mL of TFA was added under ice bath conditions. The mixture was stirred at room temperature for 1 h, and the reaction was stopped when no starting material remained as monitored by MS. The reaction solution was concentrated to obtain a yellow solid, which was compound 4 (58 mg, 88%). HRMS calcd for C 26 H 25 F2N6O3 + , 507.1951 [M+H] + ; found, 507.1942.

[0166] (5) 2,2-Dimethyl-4-oxo-3,8,11,14,17,20-hexaoxa-5-azadocoadecan-22-acid (60 mg, 0.15 mmol) and HBTU (57 mg, 0.15 mmol) were dissolved in 2 mL of DMF. DIPEA (0.11 mL, 0.62 mmol) was added under ice bath conditions, followed by compound 4 (63 mg, 0.13 mmol). The mixture was stirred at room temperature for 4 h under nitrogen protection. The reaction was stopped when no amino group remained as monitored by MS. The reaction solution was diluted with ethyl acetate, washed three times with saturated brine, and once with tap water. The organic phase was concentrated, dried, and purified by silica gel FC 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.

[0167] (6) Compound 5 (79 mg, 0.09 mmol) was dissolved in 0.5 mL of anhydrous DCM, and 1 mL of TFA was added under ice bath conditions. The mixture was stirred at room temperature for 1 h, and the reaction was stopped when no starting material remained as monitored by MS. The reaction solution was concentrated to obtain a yellow oily substance, which was compound 6 (61 mg, 87%). HRMS calcd for C 38 H 48 F2N7O9 + , 784.3476 [M+H] + ; found, 784.3477.

[0168] (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. Purification was performed by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 81 / 19), retention time 21.3 min, yielded a white solid compound F11 (19 mg, 17%). HRMS calcd for C 57 H 78 F2N 11 O 18 + , 1242.5489 [M+H]+ ; found, 1242.1074.

[0169] Example 12

[0170] A compound that targets a fibroblast-activating protein, with the structure of compound F12.

[0171] The synthetic route is shown below:

[0172]

[0173] The specific steps of the synthesis are as follows:

[0174] (1) N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetyl tert-butyl ester (300 mg, 0.47 mmol) and 2,3,5,6-tetrafluorophenol (154.7 mg, 0.932 mmol) were dissolved in 25 mL of anhydrous dichloromethane. A solution of DCC (144 mg, 0.7 mmol) in dichloromethane was slowly added dropwise under ice bath conditions. The mixture was stirred at room temperature for 6 h. LC-MS monitoring showed no further increase in product yield, at which point the reaction was stopped. The reaction solution was concentrated, reconstituted with ethyl acetate, and filtered through DCC. The filtrate was concentrated, loaded onto silica gel, and purified by FC to obtain compound 1 (321 mg, 73%). HRMS calcd for C 46 H 49 F8N2O 10 + , 941.3254 [M+H] + ; found, 941.3255.

[0175] (2) Compound 1 (60 mg, 0.064 mmol) was dissolved in 1.5 mL of trifluoroacetic acid and stirred overnight at room temperature. LC-MS monitoring showed no starting material remaining, at which point the reaction was stopped, and the trifluoroacetic acid was removed to obtain an oily compound 2 (44 mg, 83%), which required no further purification. HRMS calcd for C 38 H 33 F8N2O 10 + , 829.2002 [M+H] + ; found, 829.2006.

[0176] (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 conditions. Finally, 2-(( S)-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide (29 mg, 0.042 mmol) was stirred overnight at room temperature under nitrogen. The reaction solution was filtered, and the filtrate was purified by HPLC using acetonitrile / 0.1% formic acid aqueous solution, 2 mL / min (0–16 min, 10–60% acetonitrile; 16–20 min, 60% acetonitrile), retention time 17.5 min. Compound F12 (13 mg, 17%) was obtained. HRMS calcd for C 90 H 111 F4N 16 O 24 + , 1875.7888 [M+H] + ; found, 1875.7885.

[0177] Example 13

[0178] A compound that targets a fibroblast-activating protein, with the structure of compound F13.

[0179] The synthetic route is shown below:

[0180]

[0181] Compound F13 (6.3 mg, 16%) was obtained according to the method in Example 4, and its characterization results are as follows: HRMS calcd for C 46 H 63 F2N 10 O 17 + , 1065.4335 [M+H] + ; found, 1065.4333.

[0182] Example 14

[0183] A compound that targets a fibroblast-activating protein, with the structure of compound F14.

[0184] The synthetic route is shown below:

[0185]

[0186] The specific steps of the synthesis are as follows:

[0187] Triphosgene (10 mg, 0.034 mmol) was dissolved in 3 mL of anhydrous DCM. Then, under ice bath conditions, 3 mL of anhydrous DCM solution containing deferoxamine mesylate (48 mg, 0.073 mmol) and TEA (22 μL, 0.158 mmol) was slowly added dropwise. After stirring at room temperature for 0.5 h, 2-(…)… was slowly added dropwise under ice bath conditions. S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S A solution of 2-cyano-4,4-difluoropyrrolidone-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. The solvent was removed by concentration, and the residue was redissolved in DMSO / water (V / V = 1 / 1). The residue was filtered through a membrane, and the filtrate was purified by RP-HPLC to give compound F14 (15 mg, 16.1%). HRMS calcd for C 58 H 88 F2N 13 O 17 + , 1276.6384 [M+H] + ; found, 1276.6382.

[0188] Example 15

[0189] A compound that targets a fibroblast-activating protein, with the structure of compound F15.

[0190] The synthetic route is shown below:

[0191]

[0192] The specific steps of the synthesis are as follows:

[0193] (1) 3,4-diethoxycyclobut-3-en-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 overnight at room temperature. After the reaction was stopped, the reaction solution was concentrated, redissolved in ethyl acetate, washed once with saturated ammonium chloride and once with water, dried and concentrated, and separated by silica gel using FC 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.

[0194] (2) 2,2-Dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecane-14-acid (55 mg, 0.203 mmol) was dissolved in 2 mL of DMF, and HBTU (77 mg, 0.202 mmol) was added. DIPEA (0.09 mL, 0.51 mmol) was added dropwise under ice bath, followed by compound 1 (50 mg, 0.101 mmol). 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 three times with saturated brine, washed once with saturated sodium bicarbonate solution, extracted twice with ethyl acetate, dried and concentrated, and purified by FC to obtain a colorless oily substance, compound 2 (55 mg, 77%). 1 H 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.

[0195] (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 no residual starting material. The reaction solution was concentrated to obtain a yellow oily compound 3 (37 mg, 79%). HRMS calcd for C 28 H 34 F2N7O6 + , 602.2533 [M+H] + ; found, 602.2531.

[0196] (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. While stirring at room temperature, a THF solution of 2,2-(2-(2-aminoethoxyethoxy)acetamido)-3-phenylpropamido)-N-(2-((S)-2-cyano-4,4-difluoropyrrolidone-1-yl)-2-oxoethyl)isonicotinamide (60 mg, 0.1 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was stopped, the mixture was purified by FC on silica gel 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.

[0197] (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 no residual starting material. The reaction solution was concentrated to obtain an oily compound 5 (30 mg, 92%). HRMS calcd for C 34 H 40 F2N9O8 + , 740.2962 [M+H]+; found, 740.2960.

[0198] (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 stirred at room temperature. Compound 5 (30 mg, 0.041 mmol) was added to the above solution under nitrogen protection and stirred at room temperature. The mixture was then purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 75 / 25) showed a peak time of 18.8 min, yielding a white solid compound F15 (14 mg, 19%). HRMS calcd for C 53 H 70 F2N 13 O 17 + , 1198.4975 [M+H] + ; found, 1198.4974.

[0199] Example 16

[0200] A compound that targets a fibroblast-activating protein, with the structure of compound F16.

[0201] The synthetic route is shown below:

[0202]

[0203] The specific steps of the synthesis are as follows:

[0204] (1) Dissolve 4-(tert-butoxycarbonyl)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). Under nitrogen protection, stir overnight at room temperature 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.

[0205] (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, and MS monitoring showed no residual starting material. The reaction solution was concentrated to obtain an oily compound 3 (23 mg, 90%). HRMS calcd for C 26 H 30 F2N7O4 + , 542.2321 [M+H] + ESI MS (m / z): 542.2318 [M+H] + .

[0206] (3) Compound 3 (40 mg, 0.065 mmol) was dissolved in DMSO, and TEA (30 μL, 0.22 mmol) was added at 0 °C. The mixture was stirred at room temperature, and 2-(( S )-2-(4-aminobutyramido)-3-phenylpropionamido)-N-(2-(( S2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide (23 mg, 0.043 mmol), under nitrogen protection, stirred overnight at room temperature, and purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 82 / 18), retention time 17.6 min, gave white solid compound F16 (9 mg, 21%). HRMS calcd for C 45 H 60 F2N 11 O 13 + , 1000.4334 [M+H] + ; found, 1000.4330.

[0207] Example 17

[0208] A compound that targets a fibroblast-activating protein, with the structure of compound F17.

[0209] The synthetic route is shown below:

[0210]

[0211] The specific steps of the synthesis are as follows:

[0212] (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 conditions. The mixture was stirred at room temperature for 8 h. The reaction was stopped after the starting materials were completely consumed, as monitored by MS. The reaction solution was concentrated, reconstituted with ethyl acetate, filtered through DCU, and purified by silica gel loading onto FC to obtain 2,3,5,6-tetrafluorophenyl-6-fluoronicotinic acid ester (830 mg, 81%). HRMS calcd for C 12 H5F5NO2 + , 290.0235 [M+H] + ; found, 290.0232.

[0213] (2) Dissolve 2,3,5,6-tetrafluorophenyl-6-fluoronicotinic acid ester (55 mg, 0.19 mmol) in 3 mL of acetonitrile, add TEA (0.14 mL, 0.75 mmol) dropwise to the above solution, and finally add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S(110 mg, 0.15 mmol)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide was stirred at room temperature for 8 h under nitrogen. The reaction was stopped when no amino group remained as monitored by LC-MS. The reaction solution was concentrated and purified by silica gel loading and FC to give 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.

[0214] Example 18

[0215] A compound that targets a fibroblast-activating protein, with the structure of compound F18.

[0216] The synthetic route is shown below:

[0217]

[0218] The specific steps of the synthesis are as follows:

[0219] (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 conditions. The mixture was stirred from 0 °C to room temperature for 1 h, followed by the addition of benzyl 2-bromoacetate (1.8 g, 7.7 mmol). The mixture was heated to 100 °C and stirred for 12 h. The reaction was stopped when MS monitoring showed no further decrease in the starting material. 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 chromatography on silica gel. A colorless oily substance, benzyl 14-fluoro-3,6,9,12-tetraoxotetradecanoate (640 mg, 37%), was obtained. HRMS calcd for C 17 H 25 FNaO6 + 367.1532 [M+Na] + ; found, 367.1529.

[0220] (2) Benzyl 14-fluoro-3,6,9,12-tetraoxotetradecanoate (640 mg, 1.86 mmol) was dissolved in 13 mL of ethanol, and palladium on carbon (192 mg, 30% w / w) was added. The mixture was stirred overnight at room temperature under hydrogen atmosphere. The reaction was stopped when no starting material was detected by MS. The palladium on carbon was filtered through diatomaceous earth, and the filtrate was concentrated to obtain 14-fluoro-3,6,9,12-tetraoxotetradecanoic acid (340 mg, 72%), which required no further purification. HRMS calcd for C 10 H 18 FO6 - 253.1093 [MH] - ; found, 253.1092.

[0221] (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 bath conditions, and the mixture was stirred at room temperature for 10 h. The reaction was stopped when no starting material remained as monitored by LC-MS. The reaction solution was concentrated, reconstituted with ethyl acetate, filtered through DCU, and the filtrate was concentrated to obtain 2,3,5,6-tetrafluorophenyl-14-fluoro-3,6,9,12-tetraoxatetradecanoic acid ester, which required no further purification. HRMS calcd for C 16 H 19 F5NaO6 + , 425.0999 [M+Na]+; found, 424.0996.

[0222] (4) Dissolve 2,3,5,6-tetrafluorophenyl 14-fluoro-3,6,9,12-tetraoxotetradecanoate (106 mg, 0.263 mmol) in 4 mL of anhydrous THF, and add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S 100 mg (0.22 mmol) of 2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide was added dropwise to the above solution with 0.153 mL (1.1 mmol). The mixture was stirred at room temperature for 10 h. The reaction was stopped when no amino group remained as monitored by LC-MS. The reaction solution was concentrated, redissolved in DCM, and purified by silica gel loading with FC to obtain a pale yellow oily compound F18 (26 mg, 17%). 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.

[0223] Example 19

[0224] A compound that targets a fibroblast-activating protein, with the structure of compound F19.

[0225] The synthetic route is shown below:

[0226]

[0227] The specific steps of the synthesis are as follows:

[0228] (1) 2.3 g (8.6 mmol) of 4-iodobenzoate tert-butyl ester and 4.3 g (13 mmol) of hexamethyldistin (2.3 g, 13 mmol) were dissolved in 35 mL of dioxane. Tetra(triphenylphosphine)palladium (1.99 g, 1.72 mmol) was added. The mixture was stirred at 105 °C for 5 h under nitrogen atmosphere. After LC-MS monitoring showed no 4-iodobenzoate tert-butyl ester remaining, the reaction was stopped. The reaction solution was concentrated, redissolved in dichloromethane, washed three times with water, dried and concentrated in the organic phase, and purified by silica gel FC to obtain methyl 4-(trimethylstin)benzoate (1.1 g, 43%). HRMScalcd for C 11 H 17 O2Sn + 301.0245 [M+H] + ; found, 301.0242.

[0229] (2) Methyl 4-(trimethyltinyl)benzoate (0.5 g, 1.67 mmol) was dissolved in 10 mL of ethanol, and potassium hydroxide (0.12 g, 2.17 mmol) was added at 0 °C. 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, concentrated, and purified by silica gel loading and FC to obtain 4-(trimethyltinyl)benzoic acid (0.38 g, 65%). HRMS calcd for C 10 H 13 O2Sn - 284.9943 [MH] - ; found, 284.9941.

[0230] (3) Dissolve 4-(trimethyltinyl)benzoic acid (0.38 g, 1.33 mmol) and N-hydroxysuccinimide (0.184 g, 1.6 mmol) in anhydrous THF. Slowly add 2 mL of dichloromethane solution of DCC (0.33 g, 1.6 mmol) at 0 °C and stir overnight at room temperature. LC-MS monitoring showed no remaining starter. Stop the reaction, concentrate the reaction solution, and purify by FC to obtain 2,5-dioxopyrrolidone-1-yl-4-(trimethyltinyl)benzoate (0.27 g, 54%). HRMS calcd for C 14 H 18 NO4Sn + 284.9943 [M+H] + ;found, 284.9941.

[0231] (4) Dissolve 0.14 g, 0.37 mmol of 2,5-dioxopyrrolidone-1-yl 4-(trimethyltinyl)benzoate in 1 mL of DMSO, and add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide (0.17 g, 0.25 mmol). Finally, DIPEA was added dropwise to the above solution (0.22 mL, 1.25 mmol), and the mixture was stirred at room temperature for 12 h. The remaining starter was monitored by LC-MS. After removing DMSO, the organic phase was redissolved in dichloromethane, washed three times with water, dried and concentrated, and purified by silica gel loading and FC to obtain compound F19 (85 mg, 35%). HRMS calcd for C 42 H 54 F2N7O9Sn + , 958.2968 [M+H] + ; found, 958.2966.

[0232] Example 20

[0233] A compound that targets a fibroblast-activating protein, with the structure of compound F20.

[0234] The synthetic route is shown below:

[0235]

[0236] The specific steps of the synthesis are as follows:

[0237] (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), and slowly add DHP (1.4 mL, 15.5 mmol) in DCM solution under ice bath conditions. Stir overnight at room temperature. Stop the reaction when no DHP remains. Add water to the reaction solution, wash once with a mixture of saturated saline and sodium bicarbonate, extract the aqueous phase twice with dichloromethane, concentrate and dry the organic phase, and separate by silica gel loading with FC to obtain a colorless oily compound 1 (2.2 g, 51%). HRMS calcd for C 13 H 27 O6 + , 279.1802 [M+H] + ; found, 279.1803.

[0238] (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 conditions. After stirring at room temperature for 1 h under ice bath conditions, benzyl 2-bromoacetate (2.45 g, 10.8 mmol) was added, and the mixture was stirred overnight at room temperature. After monitoring for any remaining starter, ice-saturated ammonium chloride was added under ice bath conditions to quench the reaction mixture, bringing the pH to 7. The reaction mixture was concentrated, reconstituted with ethyl acetate, washed twice with tap water, dried, and concentrated. The organic phase was separated by silica gel using FC chromatography to obtain a colorless oily compound 2 (1.17 g, 76%). HRMS calcd for C 22 H 34 NaO8, 449.2151 [M+Na] + ; found, 449.2153.

[0239] (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 solution. The mixture was stirred overnight at room temperature under hydrogen atmosphere. After MS monitoring showed no starting material remaining, the palladium on carbon was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound 3 (285 mg, 70%). HRMS calcd for C 15 H 27 O8 - 335.1711 [MH] - ; found, 335.1709.

[0240] (4) Compound 3 (106 mg, 0.316 mmol) was dissolved in 3 mL of anhydrous DMF, HBTU (150 mg, 0.395 mmol) was added, and DIPEA (138 μL, 0.789 mmol) was added dropwise under ice bath conditions. Finally, compound F1 (120 mg, 0.263 mmol) was added. The mixture was stirred at room temperature for 8 h under nitrogen atmosphere. The remaining compound F1 was monitored by LC-MS, and the reaction was stopped. The reaction solution was diluted with ethyl acetate, washed three times with saturated brine, dried and concentrated, and purified by silica gel FC 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.

[0241] (5) Compound 4 (60 mg, 0.078 mmol) was dissolved in 3 mL of anhydrous methanol. A methanol solution of p-toluenesulfonic acid monohydrate (1.5 mg, 0.008 mmol) was added under ice bath conditions, and the mixture was stirred overnight at room temperature. The reaction was stopped when no starting material remained as monitored by MS. Methanol-ammonia solution was added dropwise under ice bath conditions to adjust the pH of the reaction solution to 7. The reaction solution was concentrated and purified by silica gel loading and FC to obtain a colorless oily compound 5 (35 mg, 66%). 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.

[0242] (6) Compound 5 (35 mg, 0.051 mmol) was dissolved in 5 mL of anhydrous dichloromethane, and p-toluenesulfonyl chloride (25 mg, 0.126 mmol) was added under ice bath conditions, followed by the dropwise addition of TEA (35 μL, 0.255 mmol). The mixture was stirred overnight at room temperature under nitrogen. The reaction was stopped after LC-MS monitoring showed that the starting material no longer decreased further. The sample was loaded onto DCM diluted silica gel and purified by FC to obtain a yellow oily compound F20 (29.5 mg, 67%). 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.

[0243] Example 21

[0244] A compound that targets a fibroblast-activating protein, with the structure of compound F21.

[0245] The synthetic route is shown below:

[0246]

[0247] The specific steps of the synthesis are as follows:

[0248] (1) The starting material ( S4-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triaza-1-yl)-5-(tert-butoxy)-5-oxovaleric 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 solution of DCC (36 mg, 0.176 mmol) in DCM was slowly added dropwise under ice bath conditions. The mixture was stirred at room temperature for 8 h. LC-MS monitoring showed no starting material remaining. The reaction mixture was concentrated, reconstituted with ethyl acetate, filtered through DCM, and the filtrate was concentrated. 1.5 mL of the filtrate was added under ice bath conditions. TFA was stirred overnight at room temperature to remove trifluoroacetic acid, yielding 78 mg of light brownish-yellow 2,2'-(7-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7-triazolline-1,4-diyl)diacetic acid. HRMS calcd for C 21 H 26 F4N3O8 + 524.1650 [M+H] + ; found, 524.1648.

[0249] (2) Dissolve 2,2'-(7-(1-carboxy-4-oxo-4-(2,3,5,6-tetrafluorophenoxy)butyl)-1,4,7-triazolline-1,4-diyl)diacetic acid (78 mg, 0.15 mmol) in 1 mL of anhydrous DMSO, add TEA (125 μL, 0.9 mmol) dropwise under ice bath, and finally add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide (68 mg, 0.1 mmol) was stirred overnight at room temperature under nitrogen. The reaction solution was filtered, and the filtrate was purified by HPLC. RP-HPLC (0.1% formic acid aqueous solution / acetonitrile = 80 / 20) yielded compound F21 (24 mg, 23%) with a peak time of 26.12 min. HRMS calcd for C 47 H 65 F2N 10 O 15 + 1047.4593 [M+H] + ; found, 1047.4592.

[0250] Example 22

[0251] A compound that targets a fibroblast-activating protein, with the structure of compound F22.

[0252] The synthetic route is shown below:

[0253]

[0254] The specific steps of the synthesis are as follows:

[0255] Will( E )-2-((2-(5-((2,5-dioxopyrrolidone-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 Dissolve 0.06 mL of TEA in 5 mL of anhydrous acetonitrile, slowly add 0.06 mL of TEA (0.435 mmol), and add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S (60 mg, 0.087 mmol)-2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide. The mixture was then refluxed at 70 °C for 1 h. The reaction was stopped after the amino group was completely consumed, as monitored by LC-MS. The reaction solution was concentrated, purified by FC on silica gel, yielding a yellow oil. A small amount of ethyl acetate was added, and the mixture was sonicated. The mother liquor was filtered to obtain a yellow solid compound F22 (24 mg, 14%). 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.

[0256] Example 23

[0257] A compound that targets a fibroblast-activating protein, with the structure of compound F23.

[0258] The synthetic route is shown below:

[0259]

[0260] The specific steps of the synthesis are as follows:

[0261] (1) Dissolve 2,5-dioxyrrolidone-1-yl 6-(2-(tert-butyloxycarbonyl)hydrazinoyl)nicotinate (50 mg, 0.144 mmol) in 5 mL of anhydrous acetonitrile, add DIPEA (0.14 mL, 0.575 mmol) dropwise, and finally add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide was heated to 40 °C and stirred for 3 h under nitrogen atmosphere. The reaction solution was then concentrated, redissolved in DCM, washed twice with saturated sodium bicarbonate water, extracted twice with dichloromethane, dried, and concentrated. The solution was then loaded onto silica gel and purified by FC to obtain a light yellow oily substance, 2-(5-(…). S )-2-benzyl-1-((4-((2-(( S 2-Cyano-4,4-Difluoropyrrolidine-1-yl)-2-O-ethyl)carbamoyl)pyridin-2-yl)amino)-1,4-dioxo-6,9,12,15-tetraoxo-3-azaheptane-17-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carboxylic acid tert-butyl ester (82 mg, 62%). 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 C 43 H 55 F2N 10 O 11 + , 925.4014 [M+H] + ; found, 925.4017.

[0262] (2) 2-(5-( S )-2-benzyl-1-((4-((2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)carbamoyl)pyridin-2-yl)amino)-1,4-dioxo-6,9,12,15-tetraoxo-3-azaheptane-17-yl)carbamoyl)pyridin-2-yl)hydrazine-1-carboxylic acid tert-butyl ester (82 mg, 0.089 mmol) was dissolved in 2 mL of anhydrous DCM. 0.5 mL of trifluoroacetic acid was added dropwise under ice bath conditions. The mixture was stirred at room temperature for 1 h. LC-MS showed no residue. After removing the trifluoroacetic acid, the mixture was reconstituted with 1 mL of methanol. HPLC separation (0.1% ammonia / acetonitrile = 58 / 42) was performed with a retention time of 24.3 min, yielding compound F23 (33.7 mg, 48%). HRMS calcd for C 40 H 50 F2N 11 O9 + , 866.3755 [M+MeCN+H] + ; found, 866.3758.

[0263] Example 24

[0264] A compound that targets a fibroblast-activating protein, with the structure of compound F24.

[0265] The synthetic route is shown below:

[0266]

[0267] The specific steps of the synthesis are as follows:

[0268] (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. DIPEA (158 μL, 0.90 mmol) was added dropwise at 0 °C, and the mixture was stirred at room temperature for 15 min. Then, compound 1 (123 mg, 0.15 mmol) was added, and the mixture was stirred overnight at room temperature under nitrogen protection. 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 forC 72 H 93 N6O 14 + , 1265.6744 [M+ H] + ; found, 1265.6747.

[0269] (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, purified by FC, and the resulting compound was dissolved in 2 mL of acetonitrile. DIPEA (76 μL, 0.435 mmol) and 2,2',2''-(10-(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tritert-butyl ester (64 mg, 0.096 mmol) were added, stirred at room temperature for 4 h, concentrated to remove the solvent, 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.

[0270] (3) Compound 3 (75 mg, 0.047 mmol) was dissolved in 10 mL of ethanol, and palladium on carbon (22 mg, 30% w / w) was added. The mixture was stirred overnight at room temperature under hydrogen atmosphere. LC-MS showed no initial product, so the reaction was stopped. The palladium on carbon was filtered through diatomaceous earth, and the filtrate was concentrated. The resulting compound and 2,3,5,6-tetrafluorophenol (12 mg, 0.072 mmol) were dissolved in 6 mL of anhydrous dichloromethane. 2 mL of a dichloromethane solution of DCC (15 mg, 0.073 mmol) was slowly added dropwise under ice bath conditions. The mixture was stirred at room temperature for 4 h. LC-MS showed no further increase in product, so the reaction was stopped. The reaction solution was concentrated, reconstituted with ethyl acetate, and filtered through DCC. The filtrate was concentrated, loaded onto silica gel, and purified by FC to obtain compound 4 (59 mg, 76%). HRMS calcd for C 84 H 127 F4N 10 O 19 + , 1655.9210 [M+ H] + ; found, 1655.9213.

[0271] (4) Add 1.5 mL of TFA to compound 4 (55 mg, 0.033 mmol) under ice bath conditions, stir overnight at room temperature to remove trifluoroacetic acid, redissolve in 1 mL of anhydrous DMSO, add TEA (23 μL, 0.165 mmol) dropwise under ice bath conditions, and finally add 2-(( S )-17-amino-2-benzyl-4-oxo-6,9,12,15-tetraoxo-3-azaheptanamide)-N-(2-(( S 18 mg (0.026 mmol) of 2-cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide was stirred overnight at room temperature under nitrogen. The reaction mixture was filtered, and the filtrate was purified by RP-HPLC to give compound F24 (14 mg, 23%). HRMS calcd for C 86 H 118 F2N 17 O 26 + , 1842.8396 [M+ H] + ; found, 1842.8395.

[0272] Example 25

[0273] A compound that targets a fibroblast-activating protein, with the structure of compound F25.

[0274] Compound F25 (22 mg, 19.8%) was obtained according to the method in Example 24. HRMS calcd for C 65 H 94 F2N 15 O 24 + , 2315.1500 [M+H] + ; found, 2315.1503.

[0275] Example 26

[0276] A compound that targets a fibroblast-activating protein, with the structure of compound F26.

[0277] Compound F26 (37 mg, 18.5%) was obtained according to the method in Example 24. HRMS calcd for C 80 H 114 F2N 21 O 25 + , 1806.8257 [M+H] + ; found, 1806.8255.

[0278] Example 27

[0279] A compound that targets a fibroblast-activating protein, with the structure of compound F27.

[0280] Compound F27 (22 mg, 10.4%) was obtained according to the method in Example 24. HRMS calcd for C 97 H 133 F2N 23 O 28 S2 + , 2169.9050 [M+H] + ; found, 2169.9052.

[0281] Example 28

[0282] A compound that targets a fibroblast-activating protein, with the structure of compound F28.

[0283] Compound F28 (46 mg, 20%) was obtained according to the method in Example 24. HRMS calcd for C 109 H 155 F2N 26 O 28 + , 2315.1500 [M+H] + ; found, 2315.1504.

[0284] Example 29

[0285] A compound that targets a fibroblast-activating protein, with the structure of compound F29.

[0286] The synthetic route is shown below:

[0287]

[0288] The specific steps of the synthesis are as follows:

[0289] (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-butoxycarbonyl)-L-lysine benzyl ester (0.58 g, 1.73 mmol). The mixture was stirred at room temperature for 12 h under nitrogen protection. The reaction was stopped after no starting material remained on LC-MS. The reaction solution was diluted with ethyl acetate, washed once with tap water, and three times with saturated brine. The organic phase was dried over sodium sulfate, concentrated, and purified by silica gel FC to obtain compound 1 (0.56 g, 53%). HRMS calcd for C 28 H 38 IN2O5 + , 609.1820 [M+H] + ;found, 609.1822.

[0290] (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. LC-MS monitoring showed no remaining starting material, so the reaction was stopped. The trifluoroacetic acid was removed, and the reaction solution was concentrated. The solution was reconstituted with 5 mL of anhydrous acetonitrile, and DOTA-NHS ester (0.23 g, 0.35 mmol) was added. DIPEA (0.29 mL, 1.65 mmol) was added dropwise to the above solution. The mixture was stirred at room temperature for 8 h, and the reaction solution was concentrated. The solution was reconstituted with ethyl acetate, washed once with water, washed once with saturated NaHCO3, dried, and concentrated. The organic phase was loaded onto 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.

[0291] (3) Compound 2 (0.16 g, 0.15 mmol) was dissolved in 6 mL of ethanol, and palladium on carbon (48 mg, 30% w / w) was added. The mixture was stirred overnight at room temperature under hydrogen atmosphere. LC-MS showed no residue of the starting material. The palladium on carbon was filtered through diatomaceous earth, and the filtrate was concentrated without further purification. The filtrate was reconstituted with 8 mL of anhydrous dichloromethane, and 2,3,5,6-tetrafluorophenol (32.3 mg, 0.2 mmol) was added. A 0.5 mL solution of DCC (37.1 mg, 0.18 mmol) in anhydrous dichloromethane was slowly added dropwise at 0 °C. The mixture was stirred at room temperature for 10 h. After the reaction was stopped, the mixture was filtered through DCC, and the filtrate was concentrated and purified by silica gel loading with FC to obtain compound 3 (0.95 g, 57%). HRMS calcd for C 50 H 74 F4IN6O 10 + , 1121.4442 [M+H] + ; found, 1121.4444.

[0292] (4) Compound 3 (45 mg, 0.04 mmol) was dissolved in 1 mL of trifluoroacetic acid and stirred at room temperature for 12 h. The reaction was stopped after LC-MS monitoring showed no starting material remaining. The trifluoroacetic acid was removed, the reaction solution was concentrated, redissolved in 1 mL of DMSO, and 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-azaheptanamide)-N-(2-(( S 2-Cyano-4,4-difluoropyrrolidine-1-yl)-2-oxoethyl)isonicotinamide (28 mg, 0.04 mmol) was stirred at room temperature under nitrogen for 12 h. Compound F29 (4.7 mg, 8%) was purified by HPLC. HRMScalcd for C 64 H 89 F2IN 13 O 17 + , 1476.5507 [M+H] + ; found, 1476.5503.

[0293] Example 30

[0294] A compound that targets a fibroblast-activating protein, with the structure of compound F30.

[0295] Compound F30 (16 mg, 15%) was obtained according to the method in Example 28. HRMS calcd for, C 87 H115 F2N 16 O 22 + , 1065.4335 [M+H] + ; found, 1065.4332.

[0296] Example 31

[0297] A compound that targets a fibroblast-activating protein, with the structure of compound F31.

[0298] Compound F31 (21 mg, 10%) was obtained according to the method in Example 25. HRMS calcd for C 97 H 127 F2N 22 O 24 + ,2022.9390 [M+H] + ; found, 2022.9393.

[0299] Example 32

[0300] A compound that targets a fibroblast-activating protein, with the structure of compound F33.

[0301] The synthetic route is shown below:

[0302]

[0303] The specific steps of the synthesis are as follows:

[0304] (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 methyl 2-aminoisonicotinic acid (2.73 g, 18.00 mmol). The mixture was stirred at room temperature for 3.5 h under nitrogen protection. The reaction was stopped when no starting material remained on LC-MS. The reaction solution was diluted with ethyl acetate, washed three times with saturated brine, and the organic phases were combined, dried over sodium sulfate, concentrated, and purified by rapid 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.

[0305] (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 water) was added. The mixture was stirred at room temperature for 4 h, and the reaction was stopped after the starting material was consumed as monitored by LC-MS. After removing methanol by rotary evaporation, an appropriate amount of water was added, and the pH was adjusted to about 6 with 2M hydrochloric acid. The precipitate was filtered, dried, and compound 3 (1.16 g, 89.1%) was obtained, which was then directly used for the next reaction. HRMS calcd for C 20 H 24 N3O5 + , 386.1716 [M+H] + ; found, 386.1720.

[0306] (3) Following the synthetic scheme of compound 2, compound 4 (730 mg, 46.0%) was obtained. HRMS calcd for C 29 H 33 N4O6 + , 533.2400 [M+H] + ; found, 533.2395.

[0307] (4) Compound 4 (730 mg, 1.36 mmol) was dissolved in 3 mL of anhydrous DCM. 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 no remaining starting material. The reaction solution was concentrated to obtain compound 5 (508 mg, 86.5%). HRMS calcd for C 24 H 25 N4O4 + , 433.1876 [M+H] + ; found, 433.1882.

[0308] (5) Referring to the synthetic 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 calcd for C 41 H 56 N5O 12 + 810.3925 [M+H] + ; found, 810.3933.

[0309] (6) Referring to the synthesis scheme of compound 5, trifluoroacetic acid was added to compound 6 for hydrolysis, and after post-treatment, compound 7 was obtained, which was then directly condensed in the next step.

[0310] (7) Compound 7 (115 mg, 0.16 mmol) was condensed with DOTAGA-tetratert-butyl ester (168 mg, 0.24 mmol) using the same synthetic scheme and post-processing method as compound 2 to obtain compound 8 (139 mg, 62.5%). HRMS calcd for C 71 H 110 N9O 19 + , 1392.7918 [M+H] + ; found, 1392.7909.

[0311] (8) Compound 8 (130 mg, 0.093 mmol) was dissolved in 10 mL of ethanol, and palladium on carbon (28 mg, 20% w / w) was added. The mixture was stirred overnight at room temperature under hydrogen atmosphere. The reaction was stopped when no starting material was detected by MS. The palladium on carbon was filtered through diatomaceous earth, and the filtrate was concentrated to obtain compound 9 (105 mg, 86.5%), which required no further purification. HRMS calcd for C 64 H 104 N9O 19 + , 1302.7448 [M+H] + ; found, 1302.7459.

[0312] (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-dioxoboron-2-yl)pyrrolidine (23 mg, 0.12 mmol). The mixture was stirred at room temperature under nitrogen protection. The reaction was stopped when no starting material remained as monitored by LC-MS. The reaction solution was diluted with ethyl acetate, washed three times with saturated brine, and the organic phases were combined, dried over sodium sulfate, concentrated, and purified by rapid 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.

[0313] (10) Following the synthetic protocol of compound 5, excess trifluoroacetic acid was added to compound 10 (60 mg, 0.04 mmol) for hydrolysis. After post-treatment, compound F33 (20 mg, 38.2%) was obtained by liquid chromatography. HRMS calcd for C 52 H 80 BN 10 O 20 + , 1175.5643 [M+H] + ; found, 1175.5633.

[0314] Example 33

[0315] A compound that targets a fibroblast-activating protein, with the structure of compound F34.

[0316] The synthetic route is shown below:

[0317]

[0318] The specific steps of the synthesis are as follows:

[0319] (1) Boc-L-glutamic acid (124 mg, 0.5 mmol) was condensed with compound 7 (780 mg, 1.1 mmol) from Example 32 and compound 2 from Example 32 using the same synthesis and post-treatment method as described in Example 32 to give compound 1 (335 mg, 41.1%). HRMS calcd for C 82 H 108 N 11 O 24 + , 1630.7569 [M+H] + ; found, 1630.7558.

[0320] (2) Compound 1 (320 mg, 0.2 mmol) was hydrolyzed with excess trifluoroacetic acid, following the synthetic protocol for compound 5 in Example 32. After post-treatment, compound 2 (245 mg, 80.2%) was obtained. HRMS calcd for C 77 H 100 N 11 O 22 + 1530.7044 [M+H] + ; found, 1530.7038. Proceed directly to the next step of condensation.

[0321] (3) Compound 2 (150 mg, 0.098 mmol) was weighed and, following the synthetic protocol for compound 8 in Example 32, was isolated by acid-amine condensation to obtain compound 3 (100 mg, 46.3%). HRMS calcd for C 112 H 162 N 15 O 31 + , 2213.1561 [M+H] + ; found, 2213.1553.

[0322] (4) Referring to the synthesis protocol 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 calcd for C 98 H 150 N 15 O 31 + , 2033.0622 [M+H] + ; found, 2033.0610.

[0323] (5) Following the synthetic protocol for 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 calcd for C 118 H 186 B2N 17 O 33 + , 2391.3585 [M+H] + ;found, 2391.3596.

[0324] (5) Following the synthetic protocol for compound F33, excess trifluoroacetic acid was added to compound 5 (40 mg, 0.016 mmol) for hydrolysis. After post-treatment, compound F34 (15 mg, 44.9%) was obtained by liquid chromatography. HRMS calcd forC 90 H 134 B2N 17 O 33 + , 2002.9516 [M+H] + ; found, 2002.9523.

[0325] Example 34

[0326] A compound targeting a fibroblast-activating protein was obtained by radiolabeling compound F1 with gallium-68, and the radiolabeling route is as follows:

[0327]

[0328] The specific steps are as follows: Use 4 mL of 0.05 M dilute hydrochloric acid from... 68 Ge / 68 Elution in Ga generator [ 68 Add GaCl3 solution to the reaction tube, and then add 200 μL of 1.25 M sodium acetate solution to adjust the pH to 4. Finally, add compound F1 (25 μg, 0.022 μmol) and mix well. Heat the mixture at 80 °C for 5 min. After cooling, add 4 mL of water, mix well, and rinse into a SEP-PAK C18 column. Rinse the column with 5 mL of water, inject air to remove excess liquid, and finally rinse the product with 1 mL of ethanol. Filter the product through a 0.22 μm sterile filter and collect it in a reaction flask for concentration. Dilute the ethanol content to 5% with physiological saline to obtain a standard gallium-68 labeled injection solution. The total radioactive synthesis time is 30 min (in other embodiments, it may be 30-40 min).

[0329] The radioactivity of gallium-68 labeled compounds F2-F11, F15-F16, F21, F24-F31, and F33-F34 can be prepared using the methods described above.

[0330] Example 35

[0331] A compound targeting a fibroblast activation protein was obtained by radiolabeling compound F1 with copper-64, and the radiolabeling route is as follows:

[0332]

[0333] 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) was taken. 64A CuCl2 solution was placed in a reaction flask, and ammonium acetate was added to adjust the pH to 4. 50 μg of compound F1 was dissolved in 1 mL of sodium ascorbate and added to the above solution. The reaction was heated at 80 °C for 5 min. After stopping the reaction and cooling, 4 mL of water was added for dilution. The mixture was then rinsed into a SEP-PAK C18 column, rinsed with 5 mL of water, air was injected to remove excess liquid, and finally the product was rinsed with 1 mL of ethanol. After filtration through a 0.22 μm sterile filter membrane, the product was 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 radioactive synthesis time was 30 min (in other embodiments, it may be 30-40 min).

[0334] The radioactivity of copper-64 labeled compounds F2-F11, F15-F16, F21, and F24-F31 can be prepared using the methods described above.

[0335] Example 36

[0336] A compound targeting a fibroblast activation protein was obtained by radiolabeling compound F1 with lutetium-177, and the radiolabeling route is as follows:

[0337]

[0338] The specific steps are as follows: Take 0.25 mL of 0.05 M HCl... 177 Add LuCl3 solution (37 MBq) to the reaction flask, and adjust the pH to 4 by adding 15 μL of 2 M NaOAc. Dissolve 25 μg of compound F1 in 25 μL of DMSO and add it to the reaction flask. Heat at 80 °C for 10 min. After stopping the reaction, cool, dilute with 4 mL of water, mix well, and rinse into a SEP-PAK C18 column. Rinse the column with 5 mL of water, inject air to remove excess liquid, and finally rinse the product with 1 mL of ethanol. Filter through a 0.22 μm sterile filter membrane and collect in a reaction flask for concentration. Dilute the ethanol content to 5% with physiological saline to obtain a standard lutetium-177 labeled injection solution. The total radiosynthesis time is 30 min (in other embodiments, it may be 30-40 min).

[0339] The radioactivity of compounds F2-F11, F15-16, F21, and F24-F31 labeled with lutetium-177 can be prepared using the methods described above.

[0340] Example 37

[0341] A compound targeting a fibroblast activation protein is obtained by radiolabeling compound F1 with yttrium-86, and the radiolabeling route is as follows:

[0342]

[0343] The specific steps are as follows: Obtain 37 MBq 86 Add 0.1 M hydrochloric acid solution of YCl3 to the reaction flask, and adjust the pH to 4.5 by adding 3 M sodium acetate. Add 25 μg of compound F1 to the above reaction solution and react at 80 °C for 10 min. After stopping the reaction, cool, dilute with 4 mL of water, mix well, and rinse into a SEP-PAK C18 column. Rinse the column with 5 mL of water, inject air to remove excess liquid, and finally rinse the product with 1 mL of ethanol. Filter through a 0.22 μm sterile filter membrane and collect in a reaction flask for concentration. Dilute the ethanol content to 5% with physiological saline to obtain a standard yttrium-86 labeled injection solution. The total radioactive synthesis time is 30 min (in other embodiments, it may be 30-40 min).

[0344] The radioactivity of yttrium-86 labeled compounds F2-F11, F15-F16, F21, and F24-F31 can be prepared using the methods described above.

[0345] Example 38

[0346] A compound targeting a fibroblast activation protein was obtained by radiolabeling compound F21 with aluminum fluoride, and the radiolabeling route is as follows:

[0347]

[0348] The specific steps are as follows: Take 10 μL of AlCl3 solution (2 mM, 0.02 μmol) into a reaction flask, add 300 μL of 0.5 M sodium acetate, adjust the pH to 4, and finally add compound F21 (25 μg, 0.024 mmol) and mix well. React at 90 °C for 15 min. After cooling, add 5 mL of water, mix well, and rinse into a SEP-PAK C18 column. Rinse the column with 5 mL of deionized water, inject air to remove excess liquid, and finally rinse the product with 1 mL of ethanol. Filter through a 0.22 μm sterile filter membrane and collect in a reaction flask for concentration. Dilute the ethanol content to 5% with physiological saline to obtain a standard fluorine-aluminum labeled injection solution. The total radioactive synthesis time is 30 min (in other embodiments, it may be 30-40 min).

[0349] Example 39

[0350] A compound targeting a fibroblast activation protein was obtained by radiolabeling compound F15 with zirconium-89, and the radiolabeling route is as follows:

[0351]

[0352] The specific steps are as follows: Obtain 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 (30-60 min in other embodiments). After stopping the reaction, the mixture was cooled, diluted with 4 mL of water, mixed, and rinsed into a SEP-PAKC18 column. The column was rinsed with 5 mL of water, air was injected to remove excess liquid, and finally the product was rinsed with 1 mL of ethanol. After filtration through a 0.22 μm sterile filter, the product was collected in a reaction flask and concentrated. Physiological saline was added to dilute the ethanol content to 5% to obtain a standard zirconium-89 labeled injection solution. The total radiosynthesis time was 80 min (80-120 min in other embodiments).

[0353] Example 40

[0354] A compound targeting a fibroblast-activating protein was obtained by radiolabeling compound F19 with iodine-131, and the radiolabeling route is as follows:

[0355]

[0356] The specific steps are as follows: 131 A solution of 1-NaI (37 MBq), 5 μL of acetic acid, and 2.5 μL of NIS in methanol (4 mg / mL) were placed in a reaction flask. 30 μL of compound F19 in methanol (5 mg / mL) was added to the above reaction solution. The reaction was carried out at room temperature for 15 min (15-20 min in other embodiments). After stopping the reaction, the reaction solution was diluted with 5 mL of deionized water, mixed, and rinsed into a SEP-PAK C18 column. Air was injected to remove excess liquid. 0.5 mL of ethanol was used to elute the column, and the solution was collected in a reaction flask after filtration through a 0.22 μm sterile filter. The solution was concentrated, and physiological saline was added to dilute the ethanol content to 5% to obtain a standard iodine-131 injection solution. The total radioactive synthesis time was 30 min (30-60 min in other embodiments).

[0357] Example 41

[0358] A compound targeting a fibroblast activation protein was obtained by radiolabeling compound F19 with astatine-211, and the radiolabeling route is as follows:

[0359]

[0360] The specific steps are as follows: Take 200 μL of NCS-containing... 211 A methanol solution of At (37 MBq, 0.2 mg / mL NCS) was added to the reaction flask. 25 μg of compound F19 and 5 μL of acetic acid were added to the reaction solution, and the reaction was carried out at room temperature for 30 min. After the reaction was stopped, excess methanol was removed with nitrogen, and the solution was reconstituted with 20% acetonitrile aqueous solution. After HPLC purification, the reaction solution was diluted with 5 mL of deionized water, mixed, and rinsed into a SEP-PAK C18 column. Air was injected to remove excess liquid, and 0.5 mL of ethanol was used to elute the column. The solution was then filtered through a 0.22 μm sterile filter and collected in a reaction flask for concentration. Physiological saline was added to dilute the ethanol content to 5% to obtain an injection solution of Astatine-211 that meets the standards. The total radiosynthesis time was 70 min.

[0361] Example 42

[0362] A compound targeting a fibroblast activation protein, with the structure of compound F32, was obtained by radiolabeling compound F22 with technetium-99m, and the radiolabeling route is as follows:

[0363]

[0364] The specific steps are as follows: Dissolve compound F22 in DMSO to prepare a 1 mg / mL solution. Dissolve 10 μL of the ligand solution in 0.5 mL of physiological saline. Add succinate buffer (pH=5.0) to adjust the pH to 5. Add 2 mg TPPTS and 1 mg tris(hydroxymethyl)glycine to the above solution. Finally, add [ 99m After mixing and shaking the Tc]NaTcO4 solution (37 MBq), the reaction was carried out at 100 °C for 30 min to obtain technetium-99m-labeled FAPI radioligands.

[0365] The technetium-99m radiolabeling of compound F23 was performed using the method described above.

[0366] Effect test

[0367] (1) Affinity test

[0368] Prepare a buffer (25 mM Tris, 250 mM NaCl, pH 7.4) for dilution and reaction. Dilute 0.2 mg / mL of recombinant human FAP protein to 0.4 μg / mL on ice. Dilute the substrate Gly-Pro-AMC to 40 μM. Dilute the candidate compound probe concentrations from 40 μM to 4 pM in eight gradients. Add 25 μL of probe and 25 μL of substrate evenly to a 96-well plate, then add 50 μL of FAP protein and incubate at 37 °C for 1 h. Repeat each concentration three times. Measure fluorescence intensity (E). x / E m =380 / 460 nm). Data was processed by GraphPad Prism to calculate IC. 50 The results are shown in Table 1. The results show that the compound in this application has a high affinity for FAP compared to the reported FAPI inhibitors UAMC-1110 and FAP-04.

[0369] Table 1 IC50 of the compounds 50

[0370]

[0371] (2) Biological distribution

[0372] Eight-week-old BALB / c nude mice were selected as experimental subjects, and a dose of 5 × 10⁻⁶ was injected into their right axilla. 6 A549-FAP cells. The tumor was allowed to grow to approximately 0.5 cm. 3 Biodistribution experiments can be conducted at a volume of [amount], via tail vein injection. 68 Ga]、[ 18 F] or [ 99m Animals were sacrificed at 0.5 h, 1 h, and 4 h (n=4 at each time point) using a radiolabeled compound (10 MBq) of Tc. Radioactivity in all dissected organs and blood was measured using a gamma counter (Cobra Autogamma, Packard). Values ​​were expressed as a percentage of the injected dose per gram of tissue (%ID / g), and the tumor / normal tissue ratio was obtained. The results are shown in Table 2. Table 2 shows that, compared with the previously reported classic […] 68 Compared to Ga]FAP04, the compounds in this application have a greater target-to-non-target ratio advantage in tumor granulation, tumor liver, tumor kidney, and tumor blood.

[0373] Table 2. Tumor / normal tissue ratio in A549-FAP xenograft model mice at 1 h

[0374]

[0375] (3) PET imaging

[0376] Eight-week-old BALB / c nude mice were selected as experimental subjects, and a dose of 5 × 10⁻⁶ was injected into their right axilla. 6 A549-FAP cells. The tumor was allowed to grow to approximately 0.5 cm. 3 When the volume is small enough, PET imaging can be performed. Mice are anesthetized with isoflurane, and a radiolabeled compound (10 MBq) is injected via the tail vein. PET imaging is performed within 240 min after injection. Images are iteratively reconstructed using the 3D-OSEM+MAP method, and the mean radioactivity concentration within the tumor or organ of interest is semi-quantitatively calculated as the mean standard uptake value (SUV). After processing with Inevon Research Workplace 4.1 software (Siemens, Erlan), attenuation-corrected whole-body coronal images are obtained. Subsequently, the regions of interest (ROIs) of the tumor and major organs are manually drawn, as shown in Table 3. Representative [ 68 PET image of Ga] compound F1 at 1 h, see Figure 1 Compared with the classics already reported [ 68 Compared to Ga]FAP-04, the compound of this application has the advantage of a larger target-to-non-target ratio. At 4 h, the probe of the compound of this application has a longer retention time in the tumor.

[0377] Table 3. Tumor / normal tissue ratio in A549-FAP xenograft model mice at 4 h

[0378]

[0379] (4) SPECT imaging

[0380] Eight-week-old BALB / c nude mice were selected as experimental subjects, and a dose of 5 × 10⁻⁶ was injected into their right axilla. 6 A549-FAP cells. The tumor was allowed to grow to approximately 0.5 cm. 3 When the volume is [value missing], SPECT imaging can be performed. Before imaging, mice are anesthetized with isoflurane, and then injected with a radiolabeled compound (10 MBq) via the tail vein. Images are acquired between 0.5 h and 4 h after injection. The SPECT images are reconstructed using 3D iterative algorithm software (Tera-Tomo), and further detailed analysis of radioactivity counts in the tumor, kidney, muscle, heart, and liver is performed by plotting the volume of interest. One h after injection, the tumor-to-muscle ratio, tumor-to-liver ratio, and tumor-to-kidney ratio were [value missing]. 99m [Tc] compound F32 (19.4, 11.3, 8.4), which is better than previously reported [ 99m Tc]FAPI-34 (6.2, 5.2, 3.1) showed high tumor-to-liver ratio, tumor-to-nephropathy ratio, and tumor-to-kidney ratio. Representative [ 99mSPECT image of compound F32 at 1 h, see [Tc] Figure 2 .

[0381] (5) Treatment

[0382] Eight-week-old BALB / c nude mice were selected as experimental subjects, and a dose of 5 × 10⁻⁶ was injected into their right axilla. 6 A549-FAP cells. Treatment was administered when the average tumor diameter reached 3-4 mm. Mice were divided into four groups (n=4): Group 1 received saline (negative control); Group 2 received lutetium-177-labeled compound F3 at 18.5 MBq (low-dose group); Group 3 received lutetium-177-labeled compound F1 at 37 MBq (high-dose group); and Group 4 received […]. 177 Lu-FAPI-46 (positive control), 37 MBq. Treatment was administered via tail vein injection on day 0. Tumor volume and mouse weight were measured on day 0 (the day of the first administration of the radiotracer), and repeated three times weekly thereafter until the study was completed. Once mouse weight decreased to 40% or tumor diameter exceeded 1500 mm, the tumor was considered positive. 3 The mice were then euthanized. After three cycles of treatment, the lutetium-177-labeled compound F1 reduced tumor volume by 30%, while normal organs showed no significant changes upon anatomical HE staining. [Positive drug...] 177 The volume of the control tumor was reduced by 70% with Lu]Lu-FAPI-46, therefore, the lutetium-177 labeled compound F1 has a good therapeutic effect.

[0383] Other metal-labeled compounds were also subjected to therapeutic experiments following the same experimental procedures. The results showed that these compounds all had inhibitory effects on tumors.

[0384] The above description is merely a preferred embodiment of this application. This application is not limited to the above-described embodiments. Any embodiment that achieves the technical effect of this application using the same means should fall within the protection scope of this application. Within the protection scope of this application, the technical solutions and / or implementation methods can have various modifications and variations.

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 and F; R3 is selected from aromatic rings or heterocyclic aromatic rings; R py1 R py2 R py3 R4 and R5 are independently selected from: hydrogen, deuterium, halogens, and -C. 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 alkyl; * indicates a 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, and a second compound; the first compound is a labeled precursor, a cold compound, or a radioactive compound containing a linker and a chelating agent; the second compound is a labeled precursor, a cold compound, or a radioactive compound containing a first targeting molecule, a linker, and a chelating agent, wherein the first targeting molecule is a targeting molecule that does not target fibroblast activation proteins; R a and R b They are not both hydrogen.

2. The compound according to claim 1, characterized in that, The structure of the compound is shown in Formula II or Formula III. Where n is 1, 2 or 3; L is the linker, selected from substituted or unsubstituted C. 1-12 Alkylene, substituted or unsubstituted C 1-12 Cycloalkylene, substituted or unsubstituted C 1-12 heteroalkyl, substituted or unsubstituted C 1-12 heterocyclic alkyl, substituted or unsubstituted C 1-12 alkylene oxides, substituted or unsubstituted C 1-12 aminoalkylene, substituted or unsubstituted C 1-12 alkenyl, substituted or unsubstituted C 1-12 heterocyclic alkenyl, substituted or unsubstituted C 1-12 alkenyl, substituted or unsubstituted C 1-12 alkyne group, substituted or unsubstituted C 1-12 aryl, substituted or unsubstituted C 1-12 One or more of the following groups: heteroaryl, polypeptide, carbonyl, imino, imide, thioamide, phosphoramide, thioether, dithio, ester, thioester, carbamate, carbonate, phosphate, anhydride, hydrazone, acylhydrazone, and sugars. G is 18 F, 18 The F-labeled leaving group or radioactive metal chelating group; the leaving group is selected from: halogen, iodonium salt, quaternary ammonium salt, benzenesulfonyl, p-toluenesulfonyl, p-nitrobenzenesulfonyl, methanesulfonyl, trifluoromethanesulfonyl, tinalkyl, silalkyl, borate, borate ester, nitrate, acetate, cyano, thiol; the radioactive metal chelating group is a chelating agent group suitable for radiolabeling, or a chelate containing a non-radioactive isotopic metal and a chelating agent, or a chelate containing a radioactive isotope and a chelating agent; T is the target molecule that targets the first protein, which is at least one of PSMA, albumin, somatostatin receptor 2, integrin, MAT2A, and GPPC.

3. The compound according to claim 2, characterized in that, R1 and R2 are F.

4. The compound according to claim 2, characterized in that, When the compound has the structure of Formula II, the structure of the compound is as shown in any of the following: Where R is an amino acid residue, and I is the R in the structure of formula I. a and R b The structure is different from that of H. Q is a condensed diacid or condensed triacid of I. When G contains a chelating agent, Q can condense with the chelating agent. x is selected from integers from 0 to 10, and y is selected from 1, 2 or 3.

5. The compound according to claim 4, characterized in that, The structure of the compound is shown in any of the following: Where * represents the dextrorotatory, levorotatory, or racemic form.

6. The compound according to claim 2, characterized in that, When the compound has a structure of Formula III, the structure of the compound is as shown in any of the following: Where R is an amino acid residue, and I is the R in the structure of formula I. a and R b The structure is different from that of H. Z is a linker that can condense with I. When G contains a chelating agent, Z can be coupled with both the chelating agent and T. x is selected from integers from 0 to 10, and y is selected from 1, 2 or 3.

7. The compound according to claim 6, characterized in that, The structure of the compound is shown in any of the following: Where * represents the dextrorotatory, levorotatory, or racemic form.

8. The compound according to claim 2, characterized in that, When G has coordinating ability, the chelating agent group suitable for radiolabeling is selected from sulfur colloids, diethylenetriaminepentaacetic acid, ethylenediaminetetraacetic acid, 1,4,7,10-tetraazacyclododecane-N , N ,, N ,,, N ,,,, -Tetraacetic acid, iminodiacetic acid, bis(carboxymethylimidazolium)glycine, 6-cyanopyridine-3-carboxylic acid, HBED-CC, AAZTA, DOTA, DODAGA, NOTA, NODAGA, or DFO.

9. The compound according to claim 2, characterized in that, The 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 Er、 177 Lu、 47 Sc、 142 Pr、 159 Gd, 121 Bi、 123 Bi、 72 As、 72 Se、 97 Ru、 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.

10. The compound according to claim 2, characterized in that, T is selected from any of the following structures: 。 11. The compound according to any one of claims 1 to 10, characterized in that, The structure of the compound is shown in any of the following: 。 12. A pharmaceutical composition, characterized in that, Includes the compounds according to any one of claims 1 to 11.

13. The use of the compound of any one of claims 1 to 11 or the pharmaceutical composition of claim 12 in the preparation of a reagent for diagnosing or treating a disease characterized by overexpression of fibroblast activating protein (FAP); wherein the disease characterized by overexpression of fibroblast activating protein (FAP) is a tumor.

14. A reagent kit, characterized in that, Includes the compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12.

15. A developer, characterized in that, Includes the compound according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12.

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