Compounds targeting FAP, radioactive nucleolabeled complexes based thereon, and methods for producing and using them.

Abstract

This invention discloses compounds that target FAP, radionucleolabeled complexes based thereon, and methods for producing and using them. The FAP-targeting compounds have the structure shown in formula (I) and have high affinity for the FAP target. 68 Radiopharmaceuticals obtained by labeling with radionuclei such as Ga exhibit higher tumor uptake and longer tumor residence times, and can be used for application and further development of FAP-targeted pharmaceuticals.

Description

[Technical Field]

[0001] The present invention belongs to the biopharmaceutical field and, more specifically, relates to compounds targeting fibroblast-activating protein (FAP) represented by formula (I), radionucleolabeled complexes based thereon, methods for producing them, and their use in the manufacture of pharmaceuticals for the diagnosis, prevention and / or treatment of diseases characterized by high expression of FAP. [Background technology]

[0002] Ninety percent of deaths in patients with malignant tumors are caused by tumor cell metastasis, yet currently, preventive or therapeutic strategies against tumor metastasis remain insufficient. A tumor is a complex composed of tumor cells and their surrounding stromal cells and non-cellular components. Tumor development and progression are dynamic processes in which tumor cells and their microenvironment mutually promote and evolve together. The tumor microenvironment (TME) includes cells and extracellular matrix (ECM), with the formed components being primarily cellular components, including immune cells, endothelial cells, and fibroblasts. Among these, cancer-associated fibroblasts (CAFs) are the most prominent stromal cells in the tumor microenvironment, accounting for approximately 50% of the total number of tumor tissue cells. CAFs play a crucial role in tumor growth, metastasis, drug resistance, and treatment resistance, and are a focus of tumor diagnostic and therapeutic research.

[0003] In cancer, fibroblast-activating protein (FAP) is a unique marker for tumor-associated fibroblasts (CAFs) and a crucial regulator and driver of the tumor microenvironment (TME). CAFs are one of the largest components of the TME and promote tumor growth and cell invasion through the secretion of pro-inflammatory and growth factors, and ECM remodeling. Beyond cells within the TME, FAP expression can also be present in malignant epithelial cells. FAP promotes tumor growth through ECM remodeling, leading to the formation of activated cancer stroma necessary for cancer cell invasion and metastasis. Clinically, increased FAP expression has been observed in the invasive anterior margin of colorectal cancer tumor samples, further supporting its role in invasion and metastasis. FAP also promotes the formation of immunosuppressive TME by enabling pro-tumor inflammation. FAP is selectively expressed on the surface of stromal fibroblasts in over 90% of epithelial malignancies, including breast, ovarian, lung, colorectal, gastric, pancreatic, and cutaneous melanoma. FAP is not typically expressed in benign and precancerous epithelial tumors, such as colorectal adenomas, mammary phyllodes tumors, and fibroadenomas. On the other hand, FAP is generally not expressed in normal human tissues, being present only in the cervix and endometrium, and is expressed transiently during embryonic development. Given the important role FAP plays in TME, there is growing interest in using it as an imaging and therapeutic target.

[0004] In recent years, many FAP inhibitors have entered clinical trials. For example, 68 Ga-FAPI-04, 68 Ga-FAPI-46, 68 Ga-oncoFAP-DOTAGA, 177Lu-FAPI-04 and similar probes offer powerful tools for precise localization and targeted killing of a wide variety of cancerous lesions. However, their relatively short tumor residence time, low absolute tumor uptake, and rapid clearance from the body result in undesirable uptake and imaging effects in tumors with low FAP expression, and also limit the killing effect on target cells or tissues of therapeutic radionuclides with long half-lives. Therefore, developing FAP-targeting probes with higher tumor uptake, longer residence time, lower non-target tissue uptake, and faster clearance is of significant importance in promoting FAP-targeted integrated pharmaceutical treatments. [Overview of the project] [Problems that the invention aims to solve]

[0005] Because FAP is selectively overexpressed in over 90% of epithelial malignancies, it is an ideal target for developing tumor-targeting probes. Considering that existing FAP-targeting small molecule compounds do not show high uptake in tumors and have short residence times, the present invention provides a compound shown in formula (I). This compound can simultaneously recognize different amino acid sites of FAP to increase affinity and enhance tumor uptake, thus possessing high affinity for FAP and being usable for FAP targeting. Furthermore, the present invention has found that a radionucleolabeled complex based on this compound specifically recognizes the FAP target, increases tumor uptake rates, and thereby enhances tumor detection rates and / or therapeutic effects.

[0006] Based on this, one object of the present invention is to provide a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound.

[0007] Another object of the present invention is to provide a method for producing the compound represented by formula (I).

[0008] Another object of the present invention is to provide radionuclei-labeled complexes obtained by labeling a radionuclei with a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound thereof as a ligand.

[0009] Another object of the present invention is to provide a method for producing the radioactive nucleon-labeled complex.

[0010] Another object of the present invention is to provide a pharmaceutical composition comprising one or more compounds selected from the compound represented by formula (I), its pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropoisomers, crystalline polymorphs, solvates, isotope-labeled compounds, and the radionucleo-labeled complex, and optionally pharmaceutically acceptable excipients.

[0011] Another object of the present invention is to provide for the use of the compound represented by formula (I) or any pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound, or the radionucleon-labeled complex, in the preparation of reagents for inhibiting FAP activity.

[0012] A further object of the present invention is to provide for the use of the compound represented by formula (I) or any pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound, or the radionucleo-labeled complex, in the manufacture of pharmaceuticals or reagents for the diagnosis, prevention and / or treatment of diseases characterized by high FAP expression. [Means for solving the problem]

[0013] To achieve the above objectives, the present invention employs the following technical solutions.

[0014] As a first aspect, there is provided a compound represented by formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, crystalline polymorph, solvate, or isotope-labeled compound thereof.

Chem.

[0015] Here, each R1 is independently halogen or H, and halogen is preferably F; R2 is -(W1) n1 -(W1) n2 -(W1) n3 -(W1) n4 -(W1) n5 -(W1) n6 -, and n1 to n6 are each independently 0 or 1; each W1 is independently selected from the following structures:

Chem.

Chem.

[0016] A dual-function chelating agent refers to a chelating agent that has the function of chelating radionuclei while simultaneously linking to a target molecular probe. Examples include 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), triaethylenetetramine (TETA), 2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl)carbonylmethylaminoacetic acid (NETA), diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA), N,N-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid (HBED), and 2,2',2'',2'''-(5 2 ,13 2 -dihydroxy-5 5 ,13 5 Examples include -dimethyl-3,7,11,15-tetraaza-1,9(2,6)-dipyridine-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid (DAR). Preferably, it is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0017] A fluorescent reporter group refers to a group that absorbs and emits light in a specific wavelength range, thereby releasing light energy. This includes, but is not limited to, visible light groups, near-infrared first-region groups, and near-infrared second-region groups.

[0018] The visible light group is any one of the following, for example: fluorescein, rhodamine, fluorescein isothiocyanate, cyanine-based fluorescent dyes (e.g., Cy2), green fluorescent protein, quantum dots, nanoparticles, F16, etc. The near-infrared first region group is any one of the following, arbitrarily selected from, for example, cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, etc.), BODIPYs (e.g., fluoroborone dipyrrometene, azafluoroborone dipyrrometene, etc.), rhodamines (e.g., rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lyssamine rhodamine, X-rhodamine, Texas Red, silylrhodamine, etc.), quantum dots, nanoparticles, phthalocyanines, etc.; The near-infrared second region group is any one of the following, arbitrarily selected from, for example, cyanine dyes (e.g., Cy7, Cy7.5, etc.), DADs (e.g., CH-1055, CH-4T, FT-TQT, etc.), BODIPYs (e.g., NJ960, NJ1030, NJ1060, PCP-BDP2, etc.), quantum dots, nanoparticles, etc.

[0019] Therapeutic agents include small molecule inhibitors, antibody drugs, bioalkylating agents, cytotoxic drugs, hormones, and biological response modifiers.

[0020] In some embodiments, each R1 is either an H or F atom.

[0021] In some embodiments, R2 is [ka] Selected from, preferably [ka] That is the case.

[0022] In some embodiments, R3 is selected from the following structures: [ka] Preferably [ka] That is the case.

[0023] In some embodiments, L is -(CH2) n7 -where n7 is an integer between 0 and 30, more preferably an integer between 0 and 12, and even more preferably 0, 3, or 10; where each -CH2- can be independently replaced with -O-, -NH-, or -(CO)-, provided that no two adjacent -CH2- groups are replaced.

[0024] In some embodiments, L is absent, or -NH-(CH2) n8 -(CO)-, -NH-(CH2CH2O) n9 -(CH2) n8 Selected from -(CO)-, each n8 and n9 is independently 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), preferably an integer between 1-8, 1-6, and 1-4.

[0025] In some embodiments, R4 is a cyano group (-CN).

[0026] In some embodiments, the compound of formula (I) is selected from formulas (II-1) and (II-2). [ka]

[0027] Here, R1 and L are defined as described above.

[0028] In some embodiments, the compound of formula (I) is selected from the following structures. [ka] [ka] [ka]

[0029] The present invention further provides a method for producing compound ZC-1, comprising the following steps: (1) Compound 1 is demethylated to obtain compound 2. This reaction can be carried out in an aqueous solution of hydrogen bromide.

[0030] (2) Compound 2 is esterified with methanol to obtain compound 3. This reaction can be carried out in the presence of thionyl chloride.

[0031] (3) Compound 4 is brominated to produce compound 5. This reaction can be carried out with carbon tetrabromide in the presence of triphenylphosphine.

[0032] (4) Compound 6 is produced by a substitution reaction between compound 5 and compound 3. This reaction can be carried out in the presence of potassium carbonate.

[0033] (4) Compound 6 is subjected to ester hydrolysis to obtain compound 7. This reaction can be carried out under LiOH conditions.

[0034] (5) Compound 7 and (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile are subjected to an amide condensation reaction to obtain compound 8.

[0035] (6) The Boc protecting group of compound 8 is removed to form compound 9 (this reaction can be carried out under trifluoroacetic acid conditions), and then compound 9 is condensed with Boc-gly-pro-OH to form compound 10.

[0036] (7) The Boc protecting group of compound 10 is removed (this reaction can be carried out under trifluoroacetic acid conditions), and then it is reacted with DOTA-NHS to obtain compound ZC-1.

[0037] The synthesis pathway for the specific steps described above is as follows: [ka]

[0038] The method for producing other compounds of the present invention (e.g., compounds ZC-2 to ZC-14) is the same as the method for producing compound ZC-1, for example, by replacing (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitriel, which is reacted with compound 7, with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitriel, or by reacting compound 9 with a carboxylic acid compound having an L group and an L protecting group (e.g., BOC) (e.g., Boc-glycine, 6-[(tert-butoxyca Coupling is performed with (e.g., [carbonyl)amino]hexanoic acid, N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid, 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester, 21-(BOC-amino)-4,7,10,13,16,19-hexaoxahenicosanoic acid), the L protecting group is removed, then condensation is performed with Boc-gly-pro-OH, the Boc protecting group is removed, and finally DOTA-NHS is coupled. Alternatively, both can be replaced simultaneously.

[0039] In a second aspect, the present invention further provides a radionucleomarker-labeled complex obtained by labeling a radionucleomarker M with a compound of formula (I) described in the present invention or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound thereof as a ligand. The radionucleomarker-labeled complex can be used as a radiopharmaceutical probe for tumors, i.e., as a radionucleomarker diagnostic probe or a radionucleomarker therapeutic probe.

[0040] The aforementioned radioactive element M includes radiodiagnostic nuclides and radiotherapeutic nuclides.

[0041] The aforementioned radioactive diagnostic radionuclides are, for example, 86 Y, 18 F, 51 Mn, 52m Mn, 52g Mn, Al[ 18 F], 64 Cu, 67 Ga, 68 Ga, 89 Zr, 99m Tc,111 In, 123 I, 124 I, 125 It is one of the following, which can be arbitrarily selected from I, and is preferably 86 Y, Al[ 18 F], 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124 It is one of any type arbitrarily selected from I, etc. The aforementioned radiotherapeutic radionuclides are, for example, 67 Cu, 90 Y, 125 I, 131 I, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 225 Ac, 227 It is any one of the following, which can be arbitrarily selected from Th, and is preferably 67 Cu, 90 Y, 125 I, 131 I, 177 Lu, 223 Ra, 225 Ac, 211 It is one of the following, which can be arbitrarily selected from At, and more preferably 68 Ga, 177 Lu or 90 It is Y.

[0042] In some embodiments, the structure of the radioactive nucleon-labeled complex is as shown in formula (III) below. [ka]

[0043] Here, L and R1 are defined as described above; M is selected from the aforementioned radioactive elements.

[0044] In some embodiments, M is 68 Ga, 177 Lu or 90 It is one of the options arbitrarily selected from Y.

[0045] In some embodiments, the radioactive nucleon-labeled complex has the following structure. [ka]

[0046] The radionuclein-labeled complex described in the present invention can be obtained by preparing a radionuclein-containing compound and a compound of formula (I) described in the present invention according to various existing labeling methods. Preferred labeling methods of the present invention include, but are not limited to, the following wet methods or freeze-drying methods.

[0047] The wet labeling method comprises the following steps: an appropriate amount of the compound of formula (I) described in the present invention or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound is dissolved in buffer or deionized water to obtain a solution. A radionucleochemical solution is added to the obtained solution, and the mixture is sealed and reacted for 5-40 minutes to produce a radionucleochemically labeled complex.

[0048] The freeze-drying labeling method includes the following steps: an appropriate amount of the compound of formula (I) described in the present invention or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound is dissolved in buffer or deionized water to obtain a solution. The obtained solution is aseptically filtered, dispensed into a container, freeze-dried, and then stoppered and sealed to obtain a freeze-dried drug kit. An appropriate amount of acetic acid solution or buffer is added to the freeze-dried drug kit to dissolve it, and then the corresponding radionucleoin solution is added, and the mixture is sealed and reacted for 5-40 minutes to produce a radionucleoin-labeled complex. Here, the dispensing container is preferably a freeze-storage tube or vial. Depending on the molding state of the freeze-dried drug kit powder, excipients such as mannitol and ascorbic acid may be added to the drug kit, and the molding of the drug kit can be optimized by adjusting the dosage of the compound of formula (I) described in the present invention and the excipients.

[0049] The products obtained by the wet labeling method and the freeze-drying labeling method can both be processed by conventional methods (e.g., separation and purification by chromatography, removal of the solvent by reduced pressure distillation, dissolution of the residue in PBS, water, or physiological saline, sterile filtration, etc.) to be prepared as injectable preparations.

[0050] In some embodiments, the method for producing a radionucleolabeled complex using the compound shown in formula (II) as a ligand is a wet labeling method, comprising the following steps: Dissolve the compound shown in formula (II) in buffer or deionized water. Add fresh radionucleosolution thereto, seal the mixture, and react at 37-90°C for 5-40 minutes, then cool. Dilute the reaction mixture with water, separate and purify it using a Sep-Pak C18 chromatography column, wash the column with buffer or water to remove unreacted radioactive ions, elute with hydrochloric acid ethanol solution or ethanol solution, and further dilute with physiological saline or PBS and filtration to obtain an injectable solution of the radionucleolabeled complex having the structure described in formula (III). Here, the radionucleostate M is 68 Ga, 177 Lu or 90 Examples include Y.

[0051] In some embodiments, the method for producing a radionucleolabeled complex using the compound shown in formula (II) as a ligand is a lyophilization labeling method, which includes the following: Dissolve the compound shown in formula (II) and other necessary reagents in a buffer solution, filter the resulting solution sterilely, dispense it into a cryopreservation tube, lyophilize it, and seal it to obtain a lyophilized drug kit. Add an appropriate amount of buffer solution to the lyophilized drug kit to dissolve it, then add a freshly prepared radionucleosolution, seal the tube, and react it at 37-120°C for 5-40 minutes, followed by cooling. Dilute the reaction solution with water, separate and purify it using a Sep-Pak C18 chromatography column, wash the column with buffer solution or water to remove unreacted radioactive ions, elute with hydrochloric acid ethanol solution or ethanol solution, and then dilute with physiological saline or PBS and filter sterilely to obtain an injectable solution of a radiolabeled complex having the structure shown in formula (III). Here, the radionucleostate is 68 Ga, 177 Lu or 90 Examples include Y.

[0052] Other chemical substances used in the above synthesis process are commercially available.

[0053] The buffer solution is a substance that stabilizes the pH of the reaction solution and may be an acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, or mixture thereof.

[0054] In yet another embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds selected from the compound represented by formula (I), pharmaceutically acceptable salts thereof, enantiomers, diastereomers, racemates, atropoisomers, crystalline polymorphs, solvates, isotope-labeled compounds, and the radionucleo-labeled complex, and optionally pharmaceutically acceptable excipients.

[0055] In yet another aspect, the present invention further provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound, or the radionucleo-labeled complex, in the preparation of a reagent for inhibiting FAP activity.

[0056] In yet another aspect, the present invention further provides the use of a compound represented by formula (I) or any pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound, or the radionucleo-labeled complex, in the manufacture of a pharmaceutical or reagent for the diagnosis, prevention and / or treatment of a disease characterized by high FAP expression.

[0057] In some embodiments, the present invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or the radionucleolabeled complex, in the manufacture of a pharmaceutical or reagent for radioisotope therapy or imaging of FAP-high-expression tumors.

[0058] In the use of the present invention as described above, the complex may be prepared as an injectable preparation and administered by intravenous injection, but is not limited thereto.

[0059] In the use of the present invention as described above, the FAP-highly expressing tumors include, but are not limited to, breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer. [Effects of the Invention]

[0060] The compounds described in formula (I) and their radionucleoenometric complexes provided by the present invention can specifically target FAP and inhibit FAP activity. Biological test results have shown that they have significantly extended circulating half-lives, enhanced tumor uptake enrichment, and residence time effects, features not seen in other FAPI imaging agents (e.g., FAPI-04). These are suitable for radionuclide therapy and imaging of FAP-highly expressing tumors. [Brief explanation of the drawing]

[0061] [Figure 1] Figure 1 is the HPLC chart of compound ZC-1 produced in Example 1. [Figure 2] Figure 2 shows a simulated docking diagram of compounds ZC-1, FAPI-04, and FAP in Test Example 1. [Figure 3] Figure 3 shows the in vitro stability of 68Ga-ZC-1 at different time intervals under 37°C in Test Example 3. [Figure 4] Figure 4 shows PET / CT images of 68Ga-ZC-1 and 68Ga-FAPI-04 in U87MG tumor mice at 0.5, 1, and 2 hours in Test Example 4. [Figure 5] Figure 5 shows the statistical distribution of U87MG tumors in mice in Test Example 4. [Figure 6] Figure 6 shows the area under the time-tumor uptake curve for 68Ga-ZC-1 and 68Ga-FAPI-04 in Test Example 4. [Modes for carrying out the invention]

[0062] The compounds of the present invention, their production methods, and uses will be described in more detail below with reference to specific examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of protection of the present invention. Any techniques realized based on the above-mentioned aspects of the present invention are included within the scope that the present invention seeks to protect. Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0063] Laboratory equipment and consumables Animal model: 5-7 week old BALB / c female mice (15-20g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. The mice were raised under conditions free from specific pathogens. The rearing environment was 25°C, 35-45% humidity, and a 12-hour light-dark cycle. All animals had free access to water and food.

[0064] The cell model, U-87MG (human astrocytoma cells), was obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0065] Chemical reagents: Acetonitrile, dimethyl sulfoxide, N,N-diisopropylethylamine, dichloromethane, and N,N-dimethylformamide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Trifluoroacetic acid and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate were purchased from Saen Chemical Technology (Shanghai) Co., Ltd. (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride was purchased from Shaoyuan Technology (Shanghai) Co., Ltd.

[0066] DOTA-NHS, [ka] It was purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0067] Boc-gly-pro-OH, [ka] It was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.

[0068] ICG-NHS, [ka] It was purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0069] FAPI-04 (CAS number: 2374782-02-0), [ka] It was purchased from Wuxi Jiehua Pharmaceutical Technology Co., Ltd.

[0070] The biological reagents—DMEM medium, RPMI 1640 medium, fetal bovine serum, trypsin, phosphate-buffered saline, skim milk powder, 4% paraformaldehyde, and the CCK8 reagent kit—were all purchased from Dalian Meilun Biotechnology Co., Ltd.

[0071] Example 1: Preparation of compound ZC-1: [ka]

[0072] Step 1: Synthesis of Compound 2 Compound 1 (2.00 g, 24.61 mmol) was dissolved in 100 mL of aqueous hydrogen bromide solution and stirred for 24 hours under conditions of 120°C. Subsequently, the pH of the reaction solution was alkalized to 6 using a 50% aqueous sodium hydroxide solution, resulting in the precipitation of a large amount of solid. The solid was collected and dried to obtain Compound 2. 1 H-NMR(600MHz,DMSO-d6) δ 13.64(s,1H),10.31(s,1H),8.81(d,J=4.4Hz,1H),8.11(d,J=2.8Hz,1H), 7.99(d,J=9.1Hz,1H),7.88(d,J=4.4Hz,1H),7.41(dd,J=9.1,2.8Hz,1H). 13 C-NMR(126MHz,DMSO-d6) δ 168.26,157.36,147.00,144.61,133.83,131.71,126.68,122.80,122.73,106.95. LR-ESI-MS(ESI) + : m / z calcd for C 10 H8NO3 + [M+H] + : 190.1, found 190.2.

[0073] Step 2: Synthesis of Compound 3 The compound 2 (4.1 g, 21.67 mmol) obtained in Step 1 was dissolved in 150 mL of methanol, and 5 mL of thionyl chloride was slowly added dropwise under ice bath conditions. After the addition was complete, the mixture was stirred overnight at 60 °C, and then the solvent was evaporated. Diethyl ether was added to the residue to precipitate. The residue was stirred at 0 °C for 30 minutes, filtered to recover the solid, and Compound 3 was obtained. 1 1H-NMR (500 MHz, Methanol-d4) δ 8.85 (d, J = 5.0 Hz, 1H), 8.18 (d, J = 2.7 Hz, 1H), 8.11 (d, J = 4.9 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 7.55 (dd, J = 9.2, 2.7 Hz, 1H), 4.07 (s, 3H). 13 13C-NMR (126 MHz, Methanol-d4) δ 165.09, 158.07, 143.12, 139.90, 135.66, 127.07, 126.76, 123.86, 122.18, 106.22, 51.59. LR-ESI-MS (ESI) - : m / z calcd for C 11 9H8NO3 - [M-H] - : 202.1, found. 202.5

[0074] Step 3: Synthesis of Compound 5 Triphenylphosphine (5.9 g, 22.51 mmol, 1.1 eq) and carbon tetrabromide (7.46 g, 22.51 mmol, 1.1 eq) were added to a solution of Compound 4 (5 g, 20.46 mmol) in tetrahydrofuran (120 mL), and then the mixture was stirred at 25 °C for 12 hours. After the reaction was completed, the reaction solution was concentrated, extracted with ethyl acetate, washed with water, and then the organic phases were combined and concentrated, and purified by silica gel column (PE / EA = 3:1) to obtain Compound 5. 1 1H-NMR (400 MHz, Chloroform-d) δ 3.46 (dt, J = 15.6, 5.8 Hz, 6H), 2.57 - 2.31 (m, 6H), 2.10 - 2.00 (m, 2H), 1.47 (s, 9H).

[0075] Step 4: Synthesis of Compound 6 Compound 3 (4 g, 19.69 mmol), compound 5 (6.05 g, 19.69 mmol), and potassium carbonate (5.44 g, 39.37 mmol) were sequentially added to a 100 mL flask containing 50 mL of N,N-dimethylformamide. The system was heated to 60 °C and stirred at 60 °C overnight. The solvent was then removed by distillation under reduced pressure to obtain a crude product. The crude product was purified by silica gel column (PE / EA = 1:1) to obtain compound 6. 1 1H-NMR (600 MHz, Chloroform-d) δ 8.84 (d, J = 4.5 Hz, 1H), 8.23 (d, J = 2.8 Hz, 1H), 8.05 (d, J = 9.2 Hz, 1H), 7.92 (d, J = 4.5 Hz, 1H), 7.41 (dd, J = 9.2, 2.8 Hz, 1H), 4.20 (t, J = 6.2 Hz, 2H), 4.03 (s, 3H), 3.46 (t, J = 5.1 Hz, 4H), 2.64 - 2.41 (m, 6H), 2.07 (m, 2H), 1.47 (s, 9H). 13 13C-NMR (151 MHz, Chloroform-d) δ 166.80, 158.60, 154.78, 146.89, 145.57, 132.35, 131.38, 126.78, 122.98, 122.78, 103.87, 79.67, 66.40, 55.18, 52.61, 28.44, 26.49. LR-ESI-MS (ESI) + : m / z calcd for C 23 H 32 N3O5 + [M + H] + : 430.2, found. 430.2.

[0076] Step 5: Synthesis of compound 7 An aqueous solution of lithium hydroxide (167.26 mg, 6.98 mmol, 3 eq.) in 100 mL of water was added to a solution of compound 6 (1.00 g, 2.33 mmol, 1 eq.) in 100 mL of methanol and 50 mL of tetrahydrofuran. The mixture was stirred at 25 °C for 1 hour and then concentrated under vacuum. The residue was diluted with water, adjusted to pH 5 by adding hydrochloric acid, and stirred for an additional 15 minutes. The precipitated solid was separated by filtration and dried in vacuo. Finally, compound 7 was obtained quantitatively. 1 H-NMR(500MHz,DMSO-d6) δ 8.87(d,J=4.5Hz,1H),8.19(d,J=2.8Hz,1H),8.04(d,J=9.2Hz,1H),7.93(d,J=4.4Hz,1H),7.50(dd,J=9. 2,2.8Hz,1H),4.20(t,J=6.1Hz,2H),3.46(t,J=5.1Hz,4H),2.98-2.76(m,6H),2.11(m,2H),1.42(s,9H). 13 C-NMR(126 MHz,DMSO-d6) δ 167.74,157.39,153.51,147.61,144.79,134.23,131.18,125.90,122.60,122.06,104.56,79.30,65.60,53.74,51.64,27.98,24.59. LR-ESI-MS(ESI) - : m / z calcd for C 22 H 28 N3O5 - [MH] - : 414.2, found. 414.4.

[0077] Step 6: Synthesis of Compound 8 Compound 7 (100 mg, 528.64 μmol, 1.00 eq.) and (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (220 mg, 528.64 μmol, 1.0 eq.) were dissolved in DMF, and then HATU (241 mg, 634.36 μmol, 1.2 eq.) and N,N-diisopropylethylamine (205 mg, 1.59 mmol, 3 eq.) were added, and the mixture was stirred at 25°C for 12 hours. The mixture was filtered, and the filtrate was purified using a preparative HPLC to obtain compound 8. 1H-NMR(500MHz,Methanol-d4) δ 8.98(d,J=5.0Hz,1H),8.24(d,J=2.7Hz,1H),8.14(d,J=9.3Hz,1H),7.87(d,J=5.0Hz,1H),7.71(dd,J=9.3,2.7Hz,1H),5.16(dd,J =9.4,3.4Hz,1H),4.46(t,J=6.0Hz,2H),4.44-4.08(m,6H),3.74-3.43(m,4H),3.31-2.73(m,6H),2.48-2.34(m,2H),1.50(s,9H). LR-ESI-MS(ESI) + : m / z calcd for C 24 H 29 F2N6O3 + [M+H] + : 487.3, found 487.5.

[0078] Step 7: Synthesis of Compound 9 Crude compound 8 (100.00 mg, 170.46 μmol) was dissolved in a solution of TFA (10 mL) in DCM (10 mL), stirred at room temperature for 1 hour, and concentrated by evaporation. Diethyl ether was then added, causing a solid precipitate. The solid was collected and vacuum-dried to quantitatively obtain compound 9. This product does not require further purification and can be used directly in the next step. 1 H-NMR(500 MHz,D2O) δ 8.96(d,J=5.5Hz,1H),8.16(d,J=9.3Hz,1H),8.03(d,J=5.5Hz,1H),7.81(d,J=2.6Hz,1H),7.77(d,J=9.3Hz,1H),5.10( dd,J=8.9,4.1Hz,1H),4.40-4.29(m,4H),4.29-4.02(m,2H),3.82-3.47(m,10H),2.99-2.82(m,2H),2.38-2.28(m,2H). LR-ESI-MS(ESI) + : m / z calcd for C 29 H 37 F2N6O5 + [M+H] + : 587.3,found 587.5.LR-ESI-MS(ESI) +: m / z calcd for C 24 H 29 F2N6O3 + [M+H] + : 487.3, found 487.5.

[0079] Step 8: Synthesis of compound 10 Boc-gly-pro-OH (100 mg, 367.24 μmol, 1 eq) and compound 9 (178.67 mg, 367.24 μmol, 1 eq) were dissolved in DMF, and then HATU (167.57 mg, 440.69 μmol, 1 eq) and N,N-diisopropylethylamine (237.32 mg, 1.84 mmol, 5 eq) were added, and the mixture was stirred at 25°C for 12 hours. After the reaction was complete, the reaction mixture was concentrated and purified by preparative HPLC to obtain compound 10 as a white solid. 1 1H-NMR (500MHz, Chloroform-d) δ 8.58(d,J=7.5Hz,1H),8.17(t,J=8.7Hz,1H),8.12(d,J=7.6Hz,1H),7.72(d ,J=7.4Hz,1H),7.66-7.65(m,1H),7.24(dd,J=7.5,1.5Hz,1H),6.50(t,J=8. 4Hz,1H),4.75(t,J=7.0Hz,1H),4.55(t,J=7.0Hz,1H),4.09-3.82(m,8H),3 .53(m,6H),2.73-2.48(m,8H),2.03(m,2H),1.94-1.80(m,4H),1.40(s,9H). LR-ESI-MS(ESI) + : m / z calcd for C 36 H 47 F2N8O7 + [M+H] + : 741.35, found 741.5.

[0080] Step 9: Synthesis of Compound 11 Crude compound 10 (103.78 mg, 161.98 μmol) was dissolved in a solution of TFA (10 mL) in DCM (10 mL), stirred at room temperature for 1 hour, and concentrated by evaporation. Diethyl ether was then added, causing a solid precipitate. The solid was collected and vacuum-dried to quantitatively obtain compound 11. This product does not require further purification and can be used directly in the next step.

[0081] Step 10: Synthesis of compound ZC-1 Crude product compound 11 (100.00 mg, 156.08 μmol, 1 eq) was dissolved in 10 mL of DMF, and DOTA-NHS (391.37 mg, 780.41 μmol, 5 eq) and DIPEA (201.73 mg, 1.56 mmol, 10 eq) were added. The mixture was stirred at room temperature for 12 hours. After evaporation and concentration, the mixture was purified by preparative HPLC to obtain compound ZC-1 as a white solid. 1 H-NMR(600 MHz,D2O) δ 8.94(d,J=5.3 Hz,1H),8.15(d,J=3.2Hz,1H),8.00(d,J=5.0Hz,1H),7.79(d,J=9.3,Hz,1H),7.75(d,J=9.3Hz,1H),5.1 0(dd,J=8.9,4.1Hz,1H),4.34-3.94(m,9H),3.85-2.79(m,38H),2.32(s,2H),1.98(s,2H),1.81(s,2H). HRMS calcd. for C 47 H 65 F2N 12 O 12 + [M+H] + : 1027.4807, found 1027.4805.

[0082] Example 2: Preparation of compound ZC-2 [ka]

[0083] The manufacturing method was as described in Example 1. However, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.75-7.70(m,1H),7.61(d,1H),7.28(dd,1H),4.65( m,1H),4.55(m,1H),4.11-3.88(m,6H),3.76-3.18(m,16H),2.82-1.68(m,32H).

[0084] Example 3: Preparation of compound ZC-3 [ka]

[0085] The manufacturing method was as described in Example 1. As a distinction, compound 9 was reacted with BOC-glycine before step 8. The specific reaction steps were as follows: Compound 9 (1.0 eq) and BOC-glycine (1.5 eq) were dissolved in DMF, 1.2 eq HATU and 3 eq DIPEA were added, and the mixture was stirred at room temperature for 8 hours. The resulting compound was deprotected with TFA and reacted with Boc-gly-pro-OH, and the resulting compound was deprotected with TFA and reacted with DOTA-NHS (the reaction conditions were the same or similar as in Example 1) to obtain ZC-3.

[0086] Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.75-7.69(m,1H),7.61(d,1H),7.28(dd,1H),4.75( m,1H),4.35(m,1H),4.11-3.79(m,11H),3.60-2.43(m,38H),2.08-1.71(m,6H).

[0087] Example 4: Preparation of compound ZC-4 [ka]

[0088] The manufacturing method was as described in Example 3. For distinction, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.73-7.70(m,1H),7.61(d,1H),7.28(dd,1H),4.65( m,1H),4.35(m,1H),4.10-3.89(m,8H),3.74-2.43(m,38H),2.23-1.72(m,10H).

[0089] Example 5: Preparation of Compound ZC-5 [ka]

[0090] The manufacturing method was as described in Example 3. For distinction, BOC-glycine reacted with compound 9 was replaced with 6-[(tert-butoxycarbonyl)amino]hexanoic acid. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.74-7.69(m,1H),7.61(d,1H),7.28(dd,1H),4.75( m,1H),4.28(m,1H),4.10-3.79(m,8H),3.61-2.27(m,42H),2.08-1.32(m,12H).

[0091] Example 6: Preparation of compound ZC-6 [ka]

[0092] The manufacturing method was as described in Example 5. For distinction, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,J=7.5Hz,1H),8.19(d,1H),7.75-7.70(m,1H),7.61(d,1H),7.28(dd,1H),4.65(m,1H),4.28 m,1H),4.11-3.88(m,6H),3.74-2.28(m,42H),2.22-1.31(m,16H).

[0093] Example 7: Preparation of compound ZC-7 [ka]

[0094] The manufacturing method was as described in Example 3. As a distinction, compound 9 was reacted with N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid before step 8. The specific reaction steps were as follows: Compound 9 (1 eq) and N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid (1.1 eq) were dissolved in DMF, 1.2 eq HATU and 3 eq DIPEA were added, and the mixture was stirred at room temperature for 8 hours. The resulting compound was deprotected with TFA and then reacted with Boc-gly-pro-OH. The resulting compound was deprotected with TFA and then reacted with DOTA-NHS (the reaction conditions were the same or similar as in Example 1) to obtain ZC-7. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.74-7.69(m,1H),7.61(d,1H),7.28(dd,1.5Hz,1H),4. 75(m,1H),4.34(m,1H),4.12-3.77(m,10H),3.73-2.39(m,48H),2.10-1.65(m,6H).

[0095] Example 8: Preparation of compound ZC-8 [ka]

[0096] The manufacturing method was as described in Example 7. For distinction, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400 MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,Hz,1H),7.75-7.67(m,1H),7.61(d,1H),7.28(dd,1.5 Hz,1H),4.65(m,1H),4.34(m,1H),4.12-2.40(m,56H),2.23-1.71(m,10H).

[0097] Example 9: Preparation of compound ZC-9 [ka]

[0098] The manufacturing method was as described in Example 7. For distinction, N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid reacted with compound 9 was replaced with 5,8,11,14-tetraoxa-2-azaheptadecanedioic acid-1-tert-butyl ester. Identification data: 1 H-NMR(400 MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,Hz,1H),7.79-7.68(m,1H),7.61(d,1H),7.28(dd,1. 5Hz,1H),4.75(m,1H),4.34(m,1H),4.12-2.36(m,66H),2.08-1.70(m,6H).

[0099] Example 10: Preparation of Compound ZC-10 [ka]

[0100] The manufacturing method was as described in Example 9. For distinction, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,Hz,1H),7.78-7.66(m,1H),7.61(d,7.28(dd,1H),4.65(m,1H),4.34(m,1H),4.14-2.40(m,64H),2.25-1.69(m,10H).

[0101] Example 11: Preparation of Compound ZC-11 [ka]

[0102] The manufacturing method was as described in Example 7. For distinction, N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid reacted with compound 9 was replaced with 21-(BOC-amino)-4,7,10,13,16,19-hexaoxahenicosanoic acid. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.80-7.68(m,1H),7.61(d,1H),7.28(dd,1.5H z,1H),4.75(m,1H),4.34(m,1H),4.11-2.36(m,74H),2.14-1.72(m,6H).

[0103] Example 12: Preparation of Compound ZC-12 [ka]

[0104] The manufacturing method was as described in Example 11. For distinction, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.81-7.66(m,1H),7.61(d,1H),7.28(dd,1H),4.65(m,1H),4.34(m,1H),4.14-2.40(m,72H),2.23-1.70(m,10H).

[0105] Example 13: Preparation of Compound ZC-13 [ka]

[0106] The manufacturing method was as described in Example 7. For distinction, N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid reacted with compound 9 was replaced with N-tert-butoxycarbonyl-heptaethylene glycol-carboxylic acid. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.78-7.69(m,1H),7.61(d,1H),7.28(dd,1H),4.75(m,1H),4.34(m,1H),4.16-2.41(m,82H),2.08-1.68(m,6H).

[0107] Example 14: Preparation of Compound ZC-14 [ka]

[0108] The manufacturing method was as described in Example 13. For distinction, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, which reacts with compound 7, was replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride. Identification data: 1 H-NMR(400MHz,Methanol-d4) δ 8.59(d,1H),8.19(d,1H),7.78-7.67(m,1H),7.61(d,1H),7.28(dd,1H),4.65(m,1H),4.34(m,1H),4.17-2.38(m,80H),2.23-1.68(m,10H).

[0109] Example 15: Preparation of compound ZC-ICG-1: [ka]

[0110] Step 1: Synthesis of compound ZC-ICG-1 Crude product compound 11 (100.00 mg, 156.08 μmol, 1 eq) was dissolved in 10 mL of DMF, and ICG-NHS (387 mg, 468.2 μmol, 3 eq) and DIPEA (201.73 mg, 1.56 mmol, 10 eq) were added. The mixture was stirred at room temperature for 12 hours. After evaporation and concentration, the mixture was purified by preparative HPLC to obtain compound ZC-ICG-1 as a green solid. 1 H-NMR(400MHz,Methanol-d4) δ 8.72-8.65(m,1H),8.59(d,1H),8.27-8.16(m,2H),8.06(d,1H),7.99-7.92(m,1H),7.82-7.71(m,3H),7.61(d,1H),7 .57-7.18(m,8H),6.71-6.47(m,4H),6.42-6.33(m,1H),6.01(d,1H),4.75(m,1H),4.52(dt,3H),4.18-1.26(m,48H).

[0111] Example 16: 68 Manufacturing of Ga-ZC-1 [ka]

[0112] 68 Ge- 68 Elution was performed using 0.1M hydrochloric acid from a Ga generator. 68 44 μL of GaCl3 was mixed with 0.5 μL of a 10 μg / μL aqueous solution of compound ZC-1, and then 400 μL of sodium acetate solution was added to adjust the pH to 4-5. The mixture was then reacted in a metal bath at 95°C for 10 minutes. After the reaction was complete, the reaction mixture was diluted with 1 mL of physiological saline and injected into a C18 desalination column using a syringe. The C18 desalination column was then washed multiple times with 10 mL of physiological saline to remove any residue that was not bound to compound ZC-1. 68 Ga 3+ Removed (until the difference in radioactivity between the two connected washes was less than 10 μCi). Finally, eluted the C18 desalting column with 100-200 μL ethanol. 68 I obtained Ga-ZC-1.

[0113] Test Example 1: Docking Simulation Analysis of ZC-1, FAPI-04 and FAP The three-dimensional structure of the FAP protein was downloaded from the RCSB Protein Databank, with PDB ID 1Z68. Homology modeling was used for the modeling of compounds ZC-1 and FAPI-04. The results of the docking simulation analysis are shown in Figure 2. The red areas in the figure indicate interactions between FAP protein residues and small molecules, and potential hydrogen bonds are indicated by yellow dotted lines. From the docking analysis in Figure 2, it can be seen that the reference docking score between ZC-1 and FAP is 7.6177, and for FAPI-04 it is 6.3464. Compared to FAPI-04, ZC-1 is Tyr 745 , Trp 623 Arg 123 , Ser 548 Arg 550 , Gln 547 Asn 399 It can interact with Trp 623 and Ser 624It can only interact with FAPI-04. Compared to FAPI-04, the Gly-Pro sequence of ZC-1 enhances the interaction between the compound and FAP, allowing it to bind to more amino acid residues in the FAP protein, thereby strengthening the interaction between ZC-1 and the FAP protein.

[0114] Test Example 2: 68 In vitro FAP enzyme saturation binding assay for Ga-ZC-1 U87MG cells 1 × 10⁶ 5 Cells were seeded individually into each well of a 96-well cell plate and cultured for 24 hours. 68 Ga-ZC-1 and 68 Ga-labeled FAPI-04 solution was diluted in culture medium to different concentrations of 1000 nM, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, and 0.78 nM. Simultaneously, inhibitor solutions were prepared by diluting FAPI-04 to 100 μM in complete medium. The supernatant medium was removed from the plate wells and washed twice with 0.2 mL PBS. The experiment was conducted as follows: 68 Ga-ZC-1 group or 68 The samples were divided into Ga-FAPI-04 groups, and each group was divided into a blank group and a test group. 0.1 mL of blank medium was added to the test group, and 0.1 mL of FAPI-04 solution (100 μM) was added to the blank group. After incubation at 37°C for 30 minutes, 0.1 mL of different concentrations were added to both the test group and the blank group. 68 Ga-ZC-1 or 68 Ga-FAPI-04 solution was added and incubated at 37°C for 2 hours. The solution was removed and the cells were washed twice with PBS. Cells were lysed with 0.2 mL of NaOH solution (1 M) and counted using a gamma counter. All measurements were performed in duplicate. The results are shown in Table 1.

[0115] Table 1. 68 Ga-ZC-1 and 68 Binding K between Ga-FAPI-04 and FAP protein d value [Table 1]

[0116] From Table 1, compounds 68 Ga-ZC-1 is found to have good FAP protein binding ability.

[0117] Test Example 3: 68 In vitro stability of Ga-ZC-1 at 37°C 68 Ga-ZC-1 was dissolved in FBS or BSA buffer at a concentration of 10 μg / μL. Then, 45 μL of 1.5 M sodium acetate solution and 430 μL of gallium chloride eluent were added and mixed uniformly. After labeling was complete, the samples were left at 37°C for 0, 30, 60, and 120 minutes. Samples were taken and thin-layer chromatography was performed, and the stability was calculated after detection with a gamma detector. The results are shown in Figure 3.

[0118] From Figure 3, 68 This study demonstrates that Ga-ZC-1 exhibits high stability in PBS and BSA buffers, showing no apparent demetallation within 2 hours, and can be used in subsequent experiments.

[0119] Test Example 4: In U87MG human glioma-bearing mice 68 Ga-ZC-1 and 68 PET / CT imaging of Ga-FAPI-04 Test animals and administration method: U87MG human glioma-bearing mice, administered via tail vein injection; Treatment groups and dosages: 68 Ga-ZC-1 group: 150 μCi / 200 μL via tail vein injection 68 Ga-ZC-1; Blocking group: 68 Administer compound FAPI-04 via tail vein injection 30 minutes before administering Ga-ZC-1 (500 times the amount of probe substance); Control group: 150 μCi / 200 μL via tail vein injection 68 Ga-FAPI-04; 68PET / CT imaging was performed 0.5, 1, and 2 hours after Ga-ZC-1 injection to observe the in-mice distribution of the probe and its accumulation in tumor areas. The results are shown in Figure 4-6.

[0120] The results are shown in Figure 4 for the U87MG heterologous transplant model. 68 Ga-ZC-1 PET imaging showed high tumor uptake and strong contrast between tumor and background. Even 0.5 hours after injection, tumor accumulation rapidly reached 4.467 ± 0.379% ID / g, and after 1 hour, the signal gradually decreased. Furthermore, it was compared to control and clinically used drugs. 68 Ga-FAPI-04 showed a much lower signal in tumors. Furthermore, 68 0.5 hours after Ga-ZC-1 injection, the U87MG tumor in the left anterior axilla of the mice was clearly visualized, and in the blockade experiment, pre-administration of non-radioactive FAPI-04 significantly reduced uptake into the tumor. This result indicates that 68 This shows specific accumulation of Ga-ZC-1 in the body. In the control group, 68 With Ga-FAPI-04, tumors were observed 0.5 hours after injection, but there was little difference compared to hepatic uptake. Subsequently, uptake in the tumor area was clearly reduced, and the signal disappeared after 2 hours. This is widely accepted. 68 Compared to Ga-FAPI-04, 68 Ga-ZC-1 showed significantly improved uptake in the tumor region and longer tumor residence time. The experimental results indicated that the probe 68 This study demonstrates that Ga-ZC-1 has efficacy in in vivo tumor imaging.

[0121] Figure 5 is a biodistribution statistics diagram. From the data in the figure, it can be seen that after pre-injection of FAPI-04 as an inhibitor, tail vein injection... 68 When Ga-ZC-1 is administered, 68 This shows a clear decrease in Ga-ZC-1 uptake. 68 This shows that Ga-ZC-1 specifically targets tumors with high FAP expression, achieving a tumor / brain distribution ratio of 13. Figure 6 shows that 68 Ga-ZC-1 and68 This shows the tumor uptake of Ga-FAPI-04 and the area under the curve (AUC) over time. 68 Ga-ZC-1 and 68 Used to compare tumor uptake levels of Ga-FAPI-04, they showed 422.5 and 98.14, respectively. 68 This indicates that Ga-ZC-1 has a longer tumor retention time.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound thereof, 【Chemistry 1】 Here, each R １ is independently a halogen or H, and the halogen is preferably F; R ２ is -(W １ ) ｎ１ -(W １ ) ｎ２ -(W １ ) ｎ３ -(W １ ) ｎ４ -(W １ ) ｎ５ -(W １ ) ｎ６ - and n1 to n6 are each independently 0 or 1; W １ Each is independently selected from the following structures: 【Chemistry 2】 Here, R ａ H is selected from C1-C3 alkyl groups, and each W １ Between them, the carbonyl group and N bond to form an amide bond, and three or more adjacent W １ at the same time 【Transformation 3】 Rather; R ３ is a bifunctional chelating agent, a fluorescent reporter group, or a therapeutic agent; L is - (CH ２ ) ｎ７ - where n7 is an integer from 0 to 40; where each CH ２ These can be independently replaced by -O-, -NH-, -(CO)-, -NH(CO)- or -(CO)-NH-, provided that two adjacent CH ２ The fact that the bases cannot be replaced simultaneously; R ４ -CN or -B(OH) ２ That is Compounds, or pharmaceutically acceptable salts thereof, enantiomers, diastereomers, racemates, atropoiomers, crystalline polymorphs, solvates, or isotope-labeled compounds.

2. The aforementioned bifunctional chelating agent is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, triaethylenetetramine, 2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl)carbonylmethylaminoacetic acid, diethylenetriamine-N,N,N',N',N''-pentaacetic acid, N,N-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid, 2,2',2'',2'''-(5 ２ ,13 ２ -dihydroxy-5 ５ ,13 ５ Selected from -dimethyl-3,7,11,15-tetraaza-1,9(2,6)-dipyridine-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid, preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, or The fluorescent reporter group comprises a visible light group, a near-infrared first region group, and a near-infrared second region group; The visible light group is any one of the following, arbitrarily selected from fluorescein, rhodamine, fluorescein isothiocyanate, cyanine-based fluorescent dyes (e.g., Cy2), green fluorescent protein, quantum dots, nanoparticles, F16, etc. The near-infrared first region group is any one of the following: cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5), BODIPYs (e.g., fluoroborone dipyrrometene, azafluoroborone dipyrrometene), rhodamines (e.g., rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lyssamine rhodamine, X-rhodamine, Texas Red, silylrhodamine), quantum dots, nanoparticles, and phthalocyanines; The near-infrared second region group is any one of the following, arbitrarily selected from cyanine dyes (e.g., Cy7, Cy7.5), D-A-D dyes (e.g., CH-1055, CH-4T, FT-TQT), BODIPY dyes (e.g., NJ960, NJ1030, NJ1060, PCP-BDP2), quantum dots, and nanoparticles; The therapeutic agents include small molecule inhibitors, antibody drugs, bioalkylating agents, cytotoxic agents, hormones, and biological response modifiers; or In equation (I), Each R １ All are H or F atoms; and / or R ２ teeth 【Chemistry 4】 Selected from, preferably 【Transformation 5】 and / or R ３ The following structures are selected: 【Transformation 6】 Preferably 【Transformation 7】 and / or L is - (CH ２ ) ｎ７ - where n7 is an integer between 0 and 30, more preferably an integer between 0 and 12, and even more preferably 0, 3, or 10; where each -CH ２ The - can be independently replaced by -O-, -NH-, or -(CO)-, provided that two adjacent -CH ２ - The fact that the base cannot be replaced, or, L does not exist, or -NH-(CH ２ ) ｎ８ -(CO)-, -NH-(CH ２ CH ２ O) ｎ９ - (CH ２ ) ｎ８ Selected from -(CO)-, where each n8 and n9 is independently 1-10, preferably an integer between 1-8, 1-6, 1-4, and / or R ４ is -CN, The compound described in claim 1, or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound.

3. The compound of formula (I) is selected from formulas (II-1) and (II-2), 【Transformation 8】 Here, R １ The definition of L is as defined in the corresponding claim. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound.

4. Compound (I) is selected from the following structures: 【Chemistry 9-1】 【Chemistry 9-2】 【Chemistry 9-3】 A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound.

5. A method for producing compound ZC-1, 【Chemistry 10】 (1) A step of demethylating compound 1 to obtain compound 2, (2) A step of esterifying compound 2 with methanol to obtain compound 3, (3) A step of obtaining compound 5 by brominating compound 4, (4) A step of obtaining compound 6 by a substitution reaction between compound 5 and compound 3, (4) A step of obtaining compound 7 by ester hydrolysis of compound 6, (5) A step of obtaining compound 8 by amide condensation reaction of compound 7 and (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile, (6) The step of removing the Boc protecting group from compound 8 to form compound 9, and then condensing compound 9 with Boc-gly-pro-OH to form compound 10, (7) The step of removing the Boc protecting group from compound 10, then reacting it with DOTA-NHS to obtain compound ZC-1, Methods that include...

6. A radionucleoid-labeled complex obtained by labeling a radionucleoid M with a compound described in any one of claims 1 to 4 or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound as a ligand; In particular, the radioactive element M includes radiodiagnostic nuclides and radiotherapeutic nuclides; In particular, the radioactive diagnostic nuclide is ８６ Y, １８ F, ５１ Mn, ５２ｍ Mn, ５２ｇ Mn, Al[ １８ F], ６４ Cd, ６７ Ga, ６８ Ga, ８９ Zr, ９９ｍ Tc, １１１ In, １２３ I, １２４ I, １２５ I, ４４ Sc, ４７ It is any one of the following that can be arbitrarily selected from Sc, preferably, ８６ Y, Al[ １８ F], ６４ Cd, ６８ Ga, ８９ Zr, ９９ｍ Tc, １２４ It is one of any two types arbitrarily selected from I; In particular, the radioactive therapeutic radionuclide is ６７ Cu, ９０ Y, １２５ I, １３１ I, １５３ Sm, １６６ Ho, １７７ Lu, １８６ Re, １８８ Re, ２１１ At, ２１２ Pb, ２０３ Pb, ２１２ Bi, ２１３ Bi, ２２３ Ra, ２２５ Ac, ２２７ any one arbitrarily selected from Th, and preferably ６７ Cu, ９０ Y, １２５ I, １３１ I, １７７ Lu, ２２３ Ra, ２２５ Ac, ２１１ any one arbitrarily selected from At, and more preferably ６８ Ga, １７７ Lu or ９０ Y; Preferably, the structure of the radioactive nucleon-labeled complex is as shown in formula (III) below, 【Chemistry 11】 Here, L, R １ is defined as described in any one of claims 1 to 4; M is defined as described above, and preferably M is ６８ Ga, １７７ Lu and ９０ It is one of the following, which can be arbitrarily selected from Y. More preferably, the radioactive nucleon-labeled complex has the following structure: 【Chemistry 12】 Radionuclear labeled complex.

7. A method for producing a radionucleomarker-labeled complex according to claim 6, comprising labeling a radionucleomarker M using a compound or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound according to any one of claims 1 to 4 or thereof as a ligand, In particular, the labeling method is either a wet labeling method or a freeze-drying labeling method; In particular, the wet labeling method comprises the steps of: dissolving an appropriate amount of the compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound described in any one of claims 1 to 4 in buffer or deionized water to obtain a solution; and adding a radioactive nucleogen M solution to the obtained solution, sealing it, and reacting it for 5-40 minutes to produce a radioactive nucleogen-labeled complex. In particular, the freeze-drying labeling method comprises the steps of: dissolving an appropriate amount of the formula (I) compound or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound described in any one of claims 1 to 4 or therein in buffer or deionized water to obtain a solution; sterile filtering of the obtained solution, dispensing it into a container, freeze-drying it, and then sealing it to obtain a freeze-dried drug kit; and adding an appropriate amount of acetic acid solution or buffer to the freeze-dried drug kit to dissolve it, further adding the corresponding radionucleotype M solution, sealing it, and allowing it to react for 5-40 minutes to produce a radionucleotype-labeled complex. The radioactive element M is defined as described in claim 6. Manufacturing method.

8. A pharmaceutical composition comprising one or more compounds selected from the compounds described in any one of claims 1 to 4, pharmaceutically acceptable salts thereof, enantiomers, diastereomers, racemates, atropoiomers, crystalline polymorphs, solvates, isotope-labeled compounds, and the radionucleo-labeled complexes, and optionally pharmaceutically acceptable excipients.

9. Use of a compound, a pharmaceutically acceptable salt, enantiomer, diastereomer, racemic mixture, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound, or the radionucleon-labeled complex described in any one of claims 1 to 4, in the preparation of a reagent for inhibiting FAP activity.

10. Use of a compound, enantiomer, diastereomer, racemic mixture, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound or radionucleo-labeled complex described in any one of claims 1 to 4, or any pharmaceutically acceptable salt, enantiomer, diastereomer, racemic mixture, atropoisomer, crystalline polymorph, solvate, or isotope-labeled compound, or the radionucleo-labeled complex, in the manufacture of a pharmaceutical or reagent for the diagnosis, prevention and / or treatment of a disease characterized by high FAP expression; Preferably, its use in the manufacture of pharmaceuticals or reagents for radionuclide therapy or imaging of FAP-high-expression tumors; Preferably, the tumors that express high levels of FAP include breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer. use.

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