FAP-targeting compound, and radionuclide-labeled complex based thereon, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a FAP-targeting compound, and a radionuclide-labeled complex based thereon and a preparation method therefor and the use thereof. The FAP-targeting compound has a structure as shown in formula (I), and has high affinity for a FAP target. A radiopharmaceutical obtained by means of labeling the FAP-targeting compound with a radionuclide such as 68Ga shows higher tumor uptake and longer tumor retention time, and can be used for the application and further development of FAP-targeting drugs.

Description

Compounds targeting FAP, radionuclide-labeled complexes based thereon, and preparation methods and applications thereof Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a compound targeting fibroblast activation protein (FAP) as shown in formula (I), a radionuclide-labeled complex based thereon, and methods for preparing the same, and their use in preparing drugs for diagnosing, preventing and / or treating diseases characterized by high expression of FAP. Background Art

[0002] 90% of deaths among patients with malignant tumors are caused by tumor cell metastasis, but preventive or therapeutic strategies for tumor metastasis are still scarce. Tumors are complexes composed of tumor cells, surrounding stromal cells, and non-cellular components. The occurrence and development of tumors is a dynamic process in which tumor cells and their microenvironment promote each other and evolve together. The tumor microenvironment (TME) includes cells and extracellular matrix (ECM). The formed elements are mainly cellular components, including immune cells, endothelial cells, fibroblasts, etc. Among them, cancer-associated fibroblasts (CAFs) are the most important stromal cells in the tumor microenvironment, accounting for approximately 50% of the total number of tumor tissue cells. CAFs play an important role in tumor growth, metastasis, drug resistance, and treatment resistance, and are one of the hot topics in tumor diagnosis and treatment research.

[0003] In cancer, fibroblast activation protein (FAP) has emerged as a unique marker of tumor-associated fibroblasts (CAFs), a key regulator and driver of the tumor microenvironment (TME). CAFs are one of the largest components of the TME, promoting tumor growth and cell invasion by secreting proinflammatory and growth factors and remodeling the ECM. In addition to cells within the TME, FAP expression can also be found in malignant epithelial cells. FAP promotes tumor growth through ECM remodeling, leading to the formation of an active cancer stroma, which is essential for cancer cell invasion and metastasis. Clinically, increased FAP expression has been observed in the invasive front of colorectal cancer samples, further supporting its role in invasion and metastasis. FAP also promotes the formation of an immunosuppressive TME by enabling tumor-promoting 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 melanomas. FAP expression is typically absent in benign and premalignant epithelial tumors, such as colorectal adenomas, breast phyllodes tumors, and fibroadenomas. FAP is not expressed in normal human tissues and is only found in the cervix and endometrium, where it is transiently expressed during embryonic development. Due to its important role in the TME, FAP has attracted increasing interest in using it as a target for imaging and therapy.

[0004] In recent years, many FAP inhibitors have entered clinical research, such as 68 Ga-FAPI-04, 68 Ga-FAPI-46, 68 Ga-oncoFAP-DOTAGA, 177 Lu-FAPI-04 and other therapeutic agents provide powerful tools for the precise localization and targeted destruction of various cancer lesions. However, their relatively short tumor retention time, low absolute tumor uptake, and rapid clearance from the body result in suboptimal uptake and imaging in tumors with low FAP expression. This also limits the cytotoxicity of long-half-life therapeutic radionuclides on target cells or tissues. Therefore, the development of FAP-targeted probes with enhanced tumor uptake, extended retention, low non-target tissue uptake, and rapid clearance is crucial for the advancement of integrated FAP-targeted diagnosis and treatment.

[0005] Summary of the Invention

[0006] FAP is selectively and highly expressed in over 90% of epithelial malignancies, making it an ideal target for developing tumor-targeted probes. Considering the low tumor uptake and short retention of existing FAP-targeting small molecule compounds, the present invention provides a compound represented by formula (I), which can simultaneously recognize different amino acid sites of FAP to enhance affinity and increase tumor uptake. Thus, it possesses high affinity for FAP and can be used to target FAP. Furthermore, the present invention discovered that radionuclide-labeled complexes based on this compound can specifically recognize the FAP target and increase tumor uptake, thereby improving tumor detection rate and / or therapeutic efficacy.

[0007] Based on this, an object of the present invention is to provide a compound represented by formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof.

[0008] Another object of the present invention is to provide a method for preparing the compound represented by formula (I).

[0009] Another object of the present invention is to provide a radionuclide-labeled complex, which is obtained by labeling a radionuclide with a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound as a ligand.

[0010] Another object of the present invention is to provide a method for preparing the radionuclide labeled complex.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising one or more selected from the group consisting of a compound represented by formula (I), its pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates, and isotope-labeled compounds, and the radionuclide-labeled complexes, and optionally pharmaceutically acceptable excipients.

[0012] Another object of the present invention is to provide the use of the compound shown in (I) or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound, or the above-mentioned radionuclide-labeled complex in the preparation of an agent for inhibiting FAP activity.

[0013] Another object of the present invention is to provide the use of the compound represented by formula (I) or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound, or the radionuclide-labeled complex thereof in the preparation of a drug or reagent for the diagnosis, prevention and / or treatment of diseases characterized by high expression of FAP.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] In a first aspect, a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof is provided.

[0016] wherein each R1 is independently halogen or H, preferably F;

[0017] R2 is -(W1) n1 -(W1) n2 -(W1) n3 -(W1) n4 -(W1) n5 -(W1) n6 -, n1 to n6 are each independently 0 or 1;

[0018] W1 are each independently selected from the following structures: where R a is selected from H, C1-C3 alkyl (such as methyl, ethyl, n-propyl, isopropyl), each W1 is connected to the carbonyl group and N to form an amide bond, and three or more adjacent W1 are not simultaneously

[0019] R3 is a bifunctional chelator, a fluorescent reporter group, or a therapeutic drug;

[0020] L is -(CH2) n7 -, wherein n7 is an integer from 0 to 40; wherein each CH2 is independently optionally replaced with -O-, -NH-, -(CO)-, -NH(CO)- or -(CO)-NH-, provided that no two adjacent CH2 groups are replaced simultaneously;

[0021] R4 is a cyano group (-CN) or a boronic acid group (-B(OH)2).

[0022] Bifunctional chelators refer to chelators that have the functions of simultaneously chelating radionuclides and linking targeting molecular probes, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), triethylenetetramine (TETA), 2-(4,7-biscarboxymethyl[1,4,7]triazacyclononyl-1-yl-ethyl)carbonyl-methylamino]acetic acid (NETA), diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), N,N-bis(2-hydroxyphenyl)ethylenediamine-N,N'-diacetic acid (HBED), 2,2 ′ ,2″,2″ ′ -(5 2 ,13 2 -Dihydroxy-5- 5 ,13 5 -dimethyl-3,7,11,15-tetraaza-1,9(2,6)-bipyridine-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid (DAR), etc.; preferably 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

[0023] Fluorescent reporter groups refer to groups that absorb and emit light within a certain wavelength range and release light energy, including but not limited to visible light groups, near-infrared group 1, and near-infrared group 2.

[0024] The visible light group can be selected from any one of fluorescein, rhodamine, fluorescein isothiocyanate, cyanine fluorescent dyes (such as Cy2), green fluorescent protein, quantum dots, nanoparticles, F16, etc.;

[0025] The near-infrared zone 1 group can be selected from: cyanine dyes (such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, etc.), BODIPYs (such as fluoroboron dipyrrole, azafluoroboron dipyrrole, etc.), rhodamines (such as rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lissamine rhodamine, X-rhodamine, Texas Red, silicon-based rhodamine, etc.), quantum dots, nanoparticles, and any one of phthalocyanines.

[0026] The near-infrared second zone group can be selected from: cyanine dyes (such as Cy7, Cy7.5, etc.), DADs (such as CH-1055, CH-4T, FT-TQT, etc.), BODIPYs (such as NJ960, NJ1030, NJ1060, PCP-BDP2, etc.), quantum dots, and nanoparticles.

[0027] Therapeutic drugs include small molecule inhibitors, monoclonal antibodies, biological alkylating agents, cytotoxic drugs, hormone drugs, biological response modifiers, etc.

[0028] In some embodiments, each R1 is a H or F atom.

[0029] In some embodiments, R2 is selected from Preferably:

[0030] In some embodiments, R3 is selected from the following structures:

[0031] Preferably

[0032] In some embodiments, L is -(CH2) n7 -; n7 is an integer from 0 to 30, more preferably an integer from 0 to 12, further preferably 0, 3 or 10; wherein each -CH2- is optionally replaced independently with -O-, -NH- or -(CO)-, provided that no two adjacent -CH2- groups are replaced.

[0033] In some embodiments, L is absent or selected from -NH-(CH2) n8 -(CO)-, -NH-(CH2CH2O) n9 -(CH2) n8 -(CO)-, each n8 and n9 is independently an integer of 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), preferably 1-8, 1-6, 1-4.

[0034] In some embodiments, R4 is cyano (-CN).

[0035] In some embodiments, the compound of formula (I) is selected from formula (II-1) and (II-2):

[0036] Wherein, R1 and L are defined as above.

[0037] In some embodiments, the compound of formula (I) is selected from the following structures:

[0038] The present invention further provides a method for preparing compound ZC-1, comprising the following steps:

[0039] (1) Compound 1 undergoes a demethylation reaction to obtain Compound 2; the reaction can be carried out in an aqueous solution of hydrogen bromide;

[0040] (2) Compound 2 is subjected to an esterification reaction with methanol to obtain Compound 3; the reaction can be carried out in the presence of thionyl chloride;

[0041] (3) Compound 4 undergoes bromination reaction to generate compound 5; this reaction can be carried out with carbon tetrabromide in the presence of triphenylphosphine;

[0042] (4) Compound 5 undergoes a substitution reaction with compound 3 to generate compound 6; the reaction can be carried out in the presence of potassium carbonate;

[0043] (4) Compound 6 undergoes ester hydrolysis to generate compound 7; this reaction can be carried out under LiOH conditions;

[0044] (5) Compound 7 reacts with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile to form compound 8;

[0045] (6) Compound 8 is deprotected from the Boc protecting group to form compound 9 (this reaction can be carried out under trifluoroacetic acid conditions), and then compound 9 undergoes a condensation reaction with Boc-gly-pro-OH to form compound 10;

[0046] (7) Compound 10 is deprotected from the Boc protecting group (this reaction can be carried out under trifluoroacetic acid conditions), and then reacted with DOTA-NHS to obtain compound ZC-1;

[0047] The synthetic route of the above-mentioned specific steps is as follows:

[0048] The preparation methods of other compounds in the scheme of the present invention (such as compounds ZC-2 to ZC-14) are similar to the preparation method of compound ZC-1, for example, the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile reacted with compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile, or compound 9 is reacted with a carboxylic acid compound containing an L group and an L protecting group (such as BOC) (such as BOC glycine, 6 -[(tert-Butoxycarbonyl)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-hexaoxahexadecanoic acid, etc.), after removing the L protecting group, condensing with Boc-gly-pro-OH, removing the Boc protecting group, and finally coupling with DOTA-NHS; or both can be replaced at the same time.

[0049] In a second aspect, the present invention further provides a radionuclide-labeled complex, which is obtained by labeling a radionuclide M with the compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof as a ligand. The radionuclide-labeled complex can be used as a tumor radiotherapy probe, i.e., a radionuclide diagnostic probe or a radionuclide therapeutic probe.

[0050] The radioactive nuclides M include radioactive diagnostic nuclides and radioactive therapeutic nuclides.

[0051] The radioactive diagnostic nuclide may be, for example, selected from: 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 I etc., preferably 86 Y、Al[ 18 F]、 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124 Any one of I, etc.;

[0052] The radioactive therapeutic nuclide may be, for example, selected from: 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 Any one of Th, etc.; preferably 67 Cu, 90 Y. 125 I. 131 I.177 Lu, 223 Ra, 225 Ac, 211 At, etc.; more preferably 68 Ga, 177 Lu or 90 Y.

[0053] In some embodiments, the structure of the radionuclide labeled complex is shown in the following formula (III):

[0054] in,

[0055] The definitions of L and R1 are as described above;

[0056] M is selected from the aforementioned radionuclides.

[0057] In some embodiments, M is selected from 68 Ga, 177 Lu or 90 Any one of Υ.

[0058] In some embodiments, the radionuclide labeled complex has the following structure:

[0059] The radionuclide-labeled complex of the present invention can be prepared by reacting a compound containing a radionuclide with the compound of formula (I) of the present invention according to various existing labeling methods. The preferred labeling method of the present invention can be the following wet method or freeze-drying method, but is not limited thereto.

[0060] The wet labeling method comprises the following steps: dissolving an appropriate amount of the compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound of the present invention in a buffer solution or deionized water to obtain a solution; adding a radioactive nuclide solution to the obtained solution, and reacting in a closed reaction for 5-40 minutes to generate a radioactive nuclide-labeled complex;

[0061] The freeze-drying labeling method comprises the following steps: dissolving an appropriate amount of the compound of formula (I) of the present invention or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound in a buffer solution or deionized water to obtain a solution; sterile filtering the obtained solution, sub-packaging it into containers, freeze-drying it, and sealing it with a stopper to obtain a freeze-dried medicine box; adding an appropriate amount of acetic acid solution or buffer solution to the freeze-dried medicine box to dissolve it, and then adding the corresponding radionuclide solution, and reacting it in a sealed manner for 5-40 minutes to generate a radionuclide labeled complex. Wherein, the sub-packaging container is preferably a cryovial or a controlled antibiotic bottle. It is also possible to add excipients such as mannitol, ascorbic acid, etc. to the medicine box according to the molding of the freeze-dried powder in the medicine box, and by adjusting the dosage of the compound of formula (I) and the excipients described in the present invention, the medicine box molding is optimized.

[0062] The products obtained from the wet labeling scheme and the freeze-drying labeling scheme can be further prepared into injection solutions through conventional treatment (such as chromatographic separation and purification, solvent removal by rotary evaporation, dissolution of the residue with PBS, water or saline, sterile filtration, etc.).

[0063] In some embodiments, the method for preparing a radioactive nuclide-labeled complex using the compound represented by formula (II) as a ligand is a wet labeling method, comprising the following steps: dissolving the compound represented by formula (II) in a buffer solution or deionized water; adding a fresh radioactive nuclide solution thereto, reacting in a sealed manner at 37-90° C. for 5-40 minutes, and cooling; diluting the reaction solution with water, separating and purifying it on a Sep-Pak Cl8 chromatographic column, rinsing the chromatographic column with a buffer solution or water to remove unreacted radioactive ions, eluting it with a hydrochloric acid ethanol solution or an ethanol solution, and then diluting it with physiological saline or PBS and sterile filtering to obtain an injection of a radioactively labeled complex having a structure as described in formula (III); wherein the radionuclide M is 68 Ga, 177 Lu or 90 Y et al.

[0064] In some embodiments, the method for preparing a radioactive nuclide-labeled complex using the compound represented by formula (II) as a ligand is a freeze-drying labeling method, comprising: dissolving the compound represented by formula (II) and other necessary reagents in a buffer solution, sterile filtering the resulting solution, and then packaging it into cryopreservation tubes, freeze-drying, and sealing to obtain a freeze-dried drug box; adding an appropriate amount of buffer solution to the freeze-dried drug box to dissolve it, then adding a freshly prepared radioactive nuclide solution, sealing and reacting at 37-120° C. for 5-40 minutes, and cooling; diluting the reaction solution with water, and then separating and purifying it on a Sep-Pak Cl8 chromatographic column, rinsing the chromatographic column with buffer or water to remove unreacted radioactive ions, eluting with a hydrochloric acid ethanol solution or an ethanol solution, and then diluting it with physiological saline or PBS and sterile filtering to obtain an injection of a radioactively labeled complex with a structure as represented by formula (III); wherein the radioactive nuclide 68 Ga, 177 Lu or 90 Y et al.

[0065] Other chemical substances used in the above synthesis steps are commercially available products.

[0066] The buffer solution is a substance that stabilizes the pH value of the reaction solution, and can be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate and phosphate, as well as mixtures thereof.

[0067] In another aspect, the present invention provides a pharmaceutical composition comprising a compound selected from the group consisting of a compound represented by formula (I), pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates, and isotope-labeled compounds thereof, one or more of the above-mentioned radionuclide-labeled complexes, and optionally pharmaceutically acceptable excipients.

[0068] In another aspect, the present invention further provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, or the above-mentioned radionuclide-labeled complex in the preparation of an agent for inhibiting FAP activity.

[0069] In another aspect, the present invention further provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, or the above-mentioned radionuclide-labeled complex in the preparation of a drug or reagent for diagnosing, preventing and / or treating a disease characterized by high expression of FAP.

[0070] In some embodiments, the present invention provides the use of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or the above-mentioned radionuclide-labeled complex in the preparation of a drug or reagent for radionuclide therapy or imaging of FAP-overexpressing tumors.

[0071] In the application of the present invention, the complex can be prepared into an injection and administered by intravenous injection, but is not limited thereto.

[0072] In the application of the present invention, the FAP-high expressing tumors include but are not limited to breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer and pancreatic cancer. Beneficial effects:

[0073] The compound of formula (I) and its radionuclide complex provided by the present invention can specifically target FAP and inhibit FAP activity. Biological test results show that it has significantly prolonged blood circulation half-life and enhanced tumor uptake, enrichment and retention time effects, which are not possessed by other current FAPI imaging agents (such as FAPI-04). It is suitable for use as a radionuclide therapy and imaging of tumors with high FAP expression. Description of the drawings:

[0074] FIG1 is a HPLC chart of compound ZC-1 prepared in Example 1.

[0075] FIG2 is a diagram showing the simulated docking binding of compounds ZC-1, FAPI-04 and FAP in Test Example 1.

[0076] Figure 3 shows the results of test example 3 at different times. 68 In vitro stability of Ga-ZC-1 at 37°C.

[0077] FIG4 is a diagram of the test example 4. 68 Ga-ZC-1 and 68 PET / CT images of Ga-FAPI-04-injected U87MG tumor mice at 0.5, 1, and 2 h.

[0078] FIG5 is a diagram of the test example 4 68 Statistical diagram of the distribution of Ga-ZC-1 in U87MG tumor mice.

[0079] FIG. 6 is a diagram of the test in Example 4. 68 Ga-ZC-1 and 68 Area under the curve of time-tumor uptake of Ga-FAPI-04. DETAILED DESCRIPTION

[0080] The following will further describe in detail the compounds of the present invention, their preparation methods, and applications with reference to specific examples. It should be understood that the following examples are intended only to illustrate and explain the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above-mentioned content of the present invention are encompassed within the scope of protection intended by the present invention. Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0081] Laboratory instrument consumables

[0082] Animal Model: 5- to 7-week-old female BALB / c mice (15-20 g) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Mice were housed under specific pathogen-free conditions at 25°C, 35-45% humidity, and a 12-hour light-dark cycle. All animals had free access to water and food.

[0083] Cell model: U-87MG (human brain astroglioblastoma cells), obtained from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0084] Chemical reagents: Acetonitrile, dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine (DIPEA), dichloromethane (DCM), and N,N-dimethylformamide (DMF) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Trifluoroacetic acid (TFA) and N,N,N',N'-TETRAMETHYL-O-(7-AZABENZOTRIAZOL-1-YL)URONIUM HEXAFLUOROPHOSPATE (HATU) were purchased from San Chemical Technology (Shanghai) Co., Ltd. (S)-1-(2-Aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride was purchased from Shaoyuan Technology (Shanghai) Co., Ltd.

[0085] DOTA-NHS, Purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0086] Boc-gly-pro-OH, Purchased from Shanghai Bidex Pharmaceutical Technology Co., Ltd.

[0087] ICG-NHS, Purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0088] FAPI-04 (CAS No.: 2374782-02-0), Purchased from Wuxi Jiehua Pharmaceutical Technology Co., Ltd.

[0089] Biological reagents: DMEM medium, RPMI 1640 medium, fetal bovine serum, trypsin, phosphate buffered saline (PBS), skim milk powder, 4% paraformaldehyde, and CCK8 kit were all purchased from Dalian Meilun Biotechnology Co., Ltd.

[0090] Example 1: Preparation of compound ZC-1:

[0091] Step 1: Synthesis of compound 2

[0092] Compound 1 (2.00 g, 24.61 mmol) was dissolved in 100 mL of aqueous hydrogen bromide solution and stirred at 120° C. for 24 hours. The reaction solution was then alkalized to pH 6 with 50% aqueous sodium hydroxide solution, resulting in the precipitation of a large amount of solid, which was collected and dried to obtain compound 2. 1 H NMR (600MHz, DMSO-d6) δ13.64(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)δ168.26,157.36,147.00,144.61,133.83,131.71,126.68,122.80,122.73,106.95.LR-ESI-MS(ESI) + :m / z calcd for C 10 H8NO3 + [M+H] + :190.1,found 190.2.

[0093] Step 2: Synthesis of compound 3

[0094] Compound 2 (4.1 g, 21.67 mmol) obtained in step 1 was dissolved in 150 mL of MeOH. 5 mL of thionyl chloride was slowly added dropwise in an ice bath. After the addition was complete, the mixture was stirred at 60°C overnight, and the solvent was subsequently evaporated. The residue was precipitated with diethyl ether. The residue was stirred at 0°C for 30 min, and the solid was collected by filtration to obtain compound 3.1 H NMR(500MHz,Methanol-d4)δ8.85(d,J=5.0Hz,1H),8.18(d,J=2.7Hz,1H),8.11( d,J=4.9Hz,1H),8.05(d,J=9.2Hz,1H),7.55(dd,J=9.2,2.7Hz,1H),4.07(s,3H). 13 C NMR(126MHz,Methanol-d4)δ165.09,158.07,143.12,139.90,135.66,127.07,126.76,123.86,122.18,106.22,51.59.LR-ESI-MS(ESI) - :m / z calcd for C 11 H8NO3 - [MH] - :202.1,found.202.5

[0095] Step 3: Synthesis of compound 5

[0096] To a solution of compound 4 (5 g, 20.46 mmol) in tetrahydrofuran (120 mL) were added triphenylphosphine (5.9 g, 22.51 mmol, 1.1 eq) and carbon tetrabromide (7.46 g, 22.51 mmol, 1.1 eq), and the reaction was stirred at 25°C for 12 hours. After completion of the reaction, the reaction solution was concentrated, extracted with ethyl acetate, washed with water, and the combined organic phases were concentrated and purified on a silica gel column (PE / EA = 3:1) to obtain compound 5. 1 H NMR (400MHz, Chloroform-d) δ3.46 (dt, J=15.6, 5.8Hz, 6H), 2.57-2.31 (m, 6H), 2.10-2.00 (m, 2H), 1.47 (s, 9H).

[0097] Step 4: Synthesis of compound 6

[0098] In a 100 mL flask, 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 added sequentially to 50 mL of N,N-dimethylformamide. The system was heated to 60°C and stirred overnight. The solvent was then removed by distillation under reduced pressure to obtain a crude product. The product was then purified on a silica gel column (PE / EA = 1:1) to obtain compound 6. 1H NMR (600MHz, Chloroform-d) δ8.84(d,J=4.5Hz,1H),8.23(d,J=2.8Hz,1H),8.05(d,J=9.2Hz,1H),7.92(d,J=4.5Hz,1H),7.41( dd,J=9.2,2.8Hz,1H),4.20(t,J=6.2Hz,2H),4.03(s,3H),3.46(t,J=5.1Hz,4H),2.64-2.41(m,6H),2.07(m,2H),1.47(s,9H). 13 C NMR(151MHz,Chloroform-d)δ166.80,158.60,154.78,146.89,145.57,132.35,131.38,12 6.78,122.98,122.78,103.87,79.67,66.40,55.18,52.61,28.44,26.49.LR-ESI-MS(ESI) + :m / z calcd for C 23 H 32 N3O5 + [M+H] + :430.2,found.430.2.

[0099] Step 5: Synthesis of compound 7

[0100] To a solution of compound 6 (1.00 g, 2.33 mmol, 1 eq.) in 100 mL of methanol and 50 mL of tetrahydrofuran was added 100 mL of an aqueous solution of lithium hydroxide (167.26 mg, 6.98 mmol, 3 eq.). The mixture was stirred at 25°C for 1 hour and then concentrated in vacuo. The residue was diluted with water and adjusted to pH 5 with hydrochloric acid. After stirring for an additional 15 minutes, the resulting solid was isolated by filtration and dried in vacuo. Compound 7 was obtained quantitatively. 1 H NMR (500MHz, DMSO-d6) δ8.87(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). 13C NMR (126MHz, DMSO-d6) δ167.74,157.39,153.51,147.61,144.79,134.23,131.18,125. 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.

[0101] Step 6: Synthesis of compound 8

[0102] 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, followed by the addition of HATU (241 mg, 634.36 μmol, 1.2 eq.) and N,N-diisopropylethylamine (205 mg, 1.59 mmol, 3 eq.). The reaction was stirred at 25 ° C. for 12 hours. The filtrate was filtered and purified using preparative HPLC to obtain compound 8. 1 H NMR (500MHz, Methanol-d4) δ8.98(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.

[0103] Step 7: Synthesis of compound 9

[0104] Crude compound 8 (100.00 mg, 170.46 μmol) was dissolved in a solution of TFA (10 mL) in DCM (10 mL) and stirred at room temperature for 1 hour. The mixture was concentrated by evaporation and then added with diethyl ether to precipitate a solid. The solid was collected and dried under vacuum to obtain compound 9. This product was used directly in the next step without further purification. 1 H NMR(500MHz,D2O)δ8.96(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,found487.5.

[0105] Step 8: Synthesis of compound 10

[0106] 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, followed by the addition of HATU (167.57 mg, 440.69 μmol, 1 eq.) and N,N-diisopropylethylamine (237.32 mg, 1.84 mmol, 5 eq.). The reaction was stirred at 25°C for 12 hours. After the reaction, the reaction solution was concentrated and purified using preparative HPLC to obtain compound 10 as a white solid. 1H NMR(500MHz,Chloroform-d)δ8.58(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.

[0107] Step 9: Synthesis of compound 11

[0108] Crude compound 10 (103.78 mg, 161.98 μmol) was dissolved in a solution of TFA (10 mL) in DCM (10 mL) and stirred at room temperature for 1 hour. The mixture was concentrated by evaporation and then added with diethyl ether to precipitate a solid. The solid was collected and dried under vacuum to quantitatively obtain compound 11. This product was used directly in the next step without further purification.

[0109] Step 10: Synthesis of Compound ZC-1

[0110] The crude 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, concentrated by evaporation, and purified by preparative HPLC to obtain compound ZC-1 as a white solid. 1H NMR(600MHz,D2O)δ8.94(d,J=5.3Hz,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),5.10(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.

[0111] Example 2 Preparation of Compound ZC-2

[0112] The preparation method refers to Example 1, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0113] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0114] Example 3 Preparation of Compound ZC-3

[0115] The preparation method refers to Example 1, except that before performing step 8, compound 9 is reacted with BOC glycine. The specific reaction steps are as follows: compound 9 (1.0 eq) and BOC glycine (1.5 eq) are dissolved in DMF, 1.2 eq HATU and 3 eq DIPEA are added, and the mixture is stirred at room temperature for 8 hours; the obtained compound is deprotected with TFA and then reacted with Boc-gly-pro-OH, and the obtained compound is deprotected with TFA and then reacted with DOTA-NHS (the reaction conditions are the same or similar to those in Example 1) to obtain ZC-3.

[0116] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0117] Example 4 Preparation of Compound ZC-4

[0118] The preparation method refers to Example 3, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with Compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0119] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0120] Example 5 Preparation of Compound ZC-5

[0121] The preparation method is similar to that of Example 3, except that BOC glycine reacted with compound 9 is replaced by 6-[(tert-butyloxycarbonyl)amino]hexanoic acid.

[0122] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0123] Example 6 Preparation of Compound ZC-6

[0124] The preparation method refers to Example 5, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with Compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0125] Characterization data: 1 H NMR(400MHz, Methanol-d4)δ8.59(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),4.28m,1H),4.11-3.88(m,6H),3.74-2.28(m,42H),2.22-1.31(m,16H).

[0126] Example 7 Preparation of Compound ZC-7

[0127] The preparation method refers to Example 3, except that, before performing step 8, compound 9 is reacted with N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid. The specific reaction steps are: compound 9 (1 eq) and N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid (1.1 eq) are dissolved in DMF, 1.2 eq HATU and 3 eq DIPEA are added, and the mixture is stirred at room temperature for 8 hours; the obtained compound is deprotected with TFA and then reacted with Boc-gly-pro-OH; the obtained compound is deprotected with TFA and then reacted with DOTA-NHS (the reaction conditions are the same or similar to those in Example 1) to obtain ZC-7.

[0128] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0129] Example 8 Preparation of Compound ZC-8

[0130] The preparation method refers to Example 7, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with Compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0131] Characterization data:1 H NMR(400MHz,Methanol-d4)δ8.59(d,1H),8.19(d,Hz,1H),7.75-7.67(m,1H),7.61(d,1H) ,7.28(dd,1.5Hz,1H),4.65(m,1H),4.34(m,1H),4.12-2.40(m,56H),2.23-1.71(m,10H).

[0132] Example 9 Preparation of Compound ZC-9

[0133] The preparation method is similar to that of Example 7, except that the N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid reacted with Compound 9 is replaced with 1-tert-butyl 5,8,11,14-tetraoxa-2-azaheptadecanedioate.

[0134] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0135] Example 10 Preparation of Compound ZC-10

[0136] The preparation method refers to Example 9, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with Compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0137] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(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).

[0138] Example 11 Preparation of Compound ZC-11

[0139] The preparation method is similar to that of Example 7, except that the N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid reacted with compound 9 is replaced with 21-(BOC-amino)-4,7,10,13,16,19-hexaoxaheneicosanoic acid.

[0140] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(d,1H),8.19(d,1H),7.80-7.68(m,1H),7.61(d,1H), 7.28(dd,1.5Hz,1H),4.75(m,1H),4.34(m,1H),4.11-2.36(m,74H),2.14-1.72(m,6H).

[0141] Example 12 Preparation of Compound ZC-12

[0142] The preparation method is as shown in Example 11, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0143] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(d,1H),8.19(d,1H),7.81-7.66(m,1H),7.61(d,1 H),7.28(dd,1H),4.65(m,1H),4.34(m,1H),4.14-2.40(m,72H),2.23-1.70(m,10H).

[0144] Example 13 Preparation of Compound ZC-13

[0145] The preparation method is similar to that of Example 7, except that the N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid reacted with Compound 9 is replaced by N-tert-butyloxycarbonyl-heptapeglycol-carboxylic acid.

[0146] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(d,1H),8.19(d,1H),7.78-7.69(m,1H),7.61(d,1 H),7.28(dd,1H),4.75(m,1H),4.34(m,1H),4.16-2.41(m,82H),2.08-1.68(m,6H).

[0147] Example 14 Preparation of Compound ZC-14

[0148] The preparation method refers to Example 13, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with Compound 7 is replaced by (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile hydrochloride.

[0149] Characterization data: 1 H NMR(400MHz,Methanol-d4)δ8.59(d,1H),8.19(d,1H),7.78-7.67(m,1H),7.61(d,1 H),7.28(dd,1H),4.65(m,1H),4.34(m,1H),4.17-2.38(m,80H),2.23-1.68(m,10H).

[0150] Example 15: Preparation of compound ZC-ICG-1:

[0151] Step 1: Synthesis of compound ZC-ICG-1

[0152] The crude 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, concentrated by evaporation, and purified by preparative HPLC to obtain compound ZC-ICG-1 as a green solid. 1 H NMR(400MHz, Methanol-d4)δ8.72-8.65(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),4.75m,1H),4.52(dt,3H),4.18-1.26(m,48H).

[0153] Example 16: 68 Preparation of Ga-ZC-1

[0154] Take 0.1M hydrochloric acid from 68 Ge- 68 Elution in Ga generator68 44 μL of GaCl3 was mixed with 0.5 μL of a 10 μg / μL aqueous solution of compound ZC-1. 400 μL of sodium acetate solution was then added to adjust the pH to 4-5. The mixture was then reacted in a 95°C metal bath for 10 minutes. After the reaction, the reactant was diluted with 1 mL of saline and injected into a C18 desalting column using a syringe. The C18 desalting column was then washed several times with 10 mL of saline to remove the unbound compounds ZC-1. 68 Ga 3+ , until the activity difference between two adjacent washes is less than 10μCi. Finally, 100-200μL ethanol was used to elute the C18 desalting column to obtain 68 Ga-ZC-1.

[0155] Test Example 1: Docking Simulation Analysis of ZC-1, FAPI-04 and FAP

[0156] The three-dimensional structure of FAP protein was downloaded from the RCSB protein database with PDB ID 1Z68. Compounds ZC-1 and FAPI-04 were modeled using the homology modeling method. The docking simulation analysis results are shown in Figure 2, where the red part represents the interaction between the FAP protein residues and the small molecule, and the potential hydrogen bonds are represented by yellow dotted lines. As shown in the docking analysis in Figure 2, the reference docking score of ZC-1 and FAP is 7.6177, while that of FAPI-04 is 6.3464. Compared with FAPI-04, ZC-1 can bind to Tyr 745 Trp 623 、Arg 123 、Ser 548 、Arg 550 、Gln 547 and Asn 399 interact with Trp 623 and Ser 624 Compared to FAPI-04, the Gly-Pro sequence of ZC-1 enhances the interaction between the compound and FAP, enabling it to bind to more amino acid residues in the FAP protein, thereby strengthening the interaction between ZC-1 and the FAP protein.

[0157] Test Example 2: 68 In vitro FAP enzyme saturation binding assay with Ga-ZC-1

[0158] 1×10 5 U87MG cells were plated in each well of a 96-well cell plate and cultured for 24 hours. 68 Ga-ZC-1 and 68Ga-labeled FAPI-04 solution was diluted with culture medium to different concentrations of 1000nM, 500nM, 250nM, 100nM, 50nM, 25nM, and 0.78nM. Simultaneously, FAPI-04 was diluted with complete culture medium to 100μM to prepare the inhibitor solution. The upper layer of culture medium in the wells was aspirated from the well plate and washed twice with 0.2mL PBS. The experiment was divided into 68 Ga-ZC-1 or 68 Ga-FAPI-04 group, each group was set up with blank group and experimental group, the experimental group was added with 0.1mL blank culture medium, the blank group was added with 0.1mL FAPI-04 solution (100μM), and after incubation at 37℃ for 30 minutes, the experimental group and blank group were added with 0.1mL of different concentrations of 68 Ga-ZC-1 or 68 The Ga-FAPI-04 solution was incubated at 37°C for 2 hours. The solution was aspirated and the cells were washed twice with PBS. The cells were lysed with 0.2 mL of 1 M NaOH solution and counted using a gamma counter. All measurements were performed in duplicate. The results are shown in Table 1.

[0159] Table 1. 68 Ga-ZC-1 and 68 K of Ga-FAPI-04 binding to FAP protein d value

[0160] As shown in Table 1, the compound 68 Ga-ZC-1 has good FAP protein binding ability.

[0161] Test Example 3: 68 In vitro stability of Ga-ZC-1 at 37°C

[0162] Will 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 thoroughly. After labeling, the sample was placed at 37°C for 0 min, 30 min, 60 min, and 120 min. Samples were then taken for thin-layer chromatography (TLC) and analyzed using a gamma detector. Stability was calculated, as shown in Figure 3.

[0163] Figure 3 shows 68 Ga-ZC-1 showed no obvious demetallation phenomenon in PBS and BSA buffer within two hours, and had high stability and could be used for the next experiment.

[0164] Test Example 4: 68 Ga-ZC-1 and 68In vivo PET / CT imaging of Ga-FAPI-04 in mice bearing U87MG human brain glioma

[0165] Experimental animals and administration method: U87MG human brain glioma-bearing mice were injected via tail vein.

[0166] Dosage group and dosage:

[0167] 68 Ga-ZC-1 group: 150 μCi / 200 μL injected into the tail vein 68 Ga-ZC-1.

[0168] Blockage group: injection into tail vein 68 30 minutes before Ga-ZC-1, compound FAPI-04 (500-fold excess of the probe substance) was injected via the tail vein.

[0169] Control group: 150 μCi / 200 μL injected into the tail vein 68 Ga-FAPI-04.

[0170] Injection 68 PET / CT imaging was performed 0.5, 1, and 2 hours after Ga-ZC-1 to observe the distribution of the probe in the mouse body and its enrichment in the tumor area. The results are shown in Figures 4-6.

[0171] The results are shown in Figure 4. 68 Ga-ZC-1 PET imaging showed high tumor uptake and strong contrast between tumor and background. Even at 0.5 hours after injection, the accumulation in the tumor quickly reached 4.467±0.379% ID / g, and the signal gradually decreased after 1 hour after injection. 68 Ga-FAPI-04, a comparative and clinically used agent, showed much lower signal in tumors. 68 After 10.5 hours of Ga-ZC-1, the U87MG tumor in the left anterior armpit of the mouse was clearly visible. Blockade experiments showed that the uptake of the tumor was significantly reduced after the pre-injection of non-radioactive FAPI-04. This result shows that 68 The specific accumulation of Ga-ZC-1 in the body. 68 Ga-FAPI-04 was visible in the tumor 0.5 h after injection, but the uptake was almost the same as that in the liver. After that, the uptake in the tumor area decreased significantly, and there was no signal at 2 h. 68 Compared with Ga-FAPI-04, 68 The uptake of Ga-ZC-1 in the tumor area was significantly improved, and the tumor retention time was longer. 68 Ga-ZC-1 has the effect of in vivo tumor imaging.

[0172] Figure 5 is a statistical diagram of the distribution in vivo. The data in the figure show that after the injection of FAPI-04 as a blocking agent in advance, the 68 Ga-ZC-1, tumor part 68 The uptake of Ga-ZC-1 was significantly reduced, indicating that 68 Ga-ZC-1 can specifically target tumors with high FAP expression, and the tumor / brain distribution ratio is as high as 13. 68 Ga-ZC-1 and 68 The area under the curve (AUC) of Ga-FAPI-04 based on tumor uptake versus time was used for comparison 68 Ga-ZC-1 and 68 The size of the tumor uptake of Ga-FAPI-04 over time was 422.5 and 98.14, respectively, indicating that 68 Ga-ZC-1 retained tumors for a longer time.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, in, Each R1 is independently halogen or H, halogen is preferably F; R2 is -(W1) n1 -(W1) n2 -(W1) n3 -(W1) n4 -(W1) n5 -(W1) n6 -, n1 to n6 are each independently 0 or 1; W1 are each independently selected from the following structures: Where R a selected from H, C1-C3 alkyl, each W1 is connected to N by a carbonyl group to form an amide bond, and three or more adjacent W1 are not simultaneously R3 is a bifunctional chelator, a fluorescent reporter group or a therapeutic drug; L is -(CH2) n7 -, wherein n7 is an integer from 0 to 40; wherein each CH2 is independently optionally replaced with -O-, -NH-, -(CO)-, -NH(CO)- or -(CO)-NH-, provided that no two adjacent CH2 groups are replaced simultaneously; R4 is -CN or -B(OH)2.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, characterized in that: The bifunctional chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, triethylenetetramine, 2-(4,7-biscarboxymethyl[1,4,7]triazacyclononyl-1-yl-ethyl)carbonyl-methylamino]acetic 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 -dimethyl-3,7,11,15-tetraaza-1,9(2,6)-bipyridine-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 includes a visible light group, a near infrared zone 1 group, and a near infrared zone 2 group; The visible light group can be selected from any one of fluorescein, rhodamine, fluorescein isothiocyanate, cyanine fluorescent dye (such as Cy2), green fluorescent protein, quantum dots, nanoparticles, F16, etc.; The near-infrared region group can be selected from: cyanine dyes (e.g. Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5), Any one of BODIPY (e.g., fluoroboron dipyrrole, azafluoroboron dipyrrole), rhodamine (e.g., rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lissamine rhodamine, X-rhodamine, Texas Red, silicon-based rhodamine), quantum dots, nanoparticles, and phthalocyanine; The near-infrared zone II group can be selected from any one of: cyanine dyes (e.g., Cy7, Cy7.5), DADs (e.g., CH-1055, CH-4T, FT-TQT), BODIPYs (e.g., NJ960, NJ1030, NJ1060, PCP-BDP2), quantum dots, and nanoparticles; The therapeutic drugs include small molecule inhibitors, monoclonal antibody drugs, biological alkylating agents, cytotoxic drugs, hormone drugs, and biological response modifiers; or In formula (I), Each R1 is a H or F atom; and / or R2 is selected from Preferably: and / or R3 is selected from the following structures: Preferably and / or L is -(CH2) n7 -; n7 is an integer of 0-30, more preferably an integer of 0-12, further preferably 0, 3 or 10; wherein each -CH2- is independently optionally replaced with -O-, -NH- or -(CO)-, provided that no two adjacent -CH2- groups are replaced; or L is absent or selected from -NH-(CH2) n8 -(CO)-, -NH-(CH2CH2O) n9 -(CH2) n8 -(CO)-, each n8 and n9 is independently an integer of 1-10, preferably 1-8, 1-6, 1-4; and / or R4 is -CN.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, characterized in that: The compound of formula (I) is selected from formula (II-1) and (II-2): Wherein, R1 and L are defined as in the corresponding claims.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, characterized in that: The compound of formula (I) is selected from the following structures:

5. A method for preparing compound ZC-1, comprising the following steps: (1) Compound 1 undergoes a demethylation reaction to obtain compound 2; (2) Compound 2 is subjected to an esterification reaction with methanol to obtain compound 3; (3) Compound 4 undergoes bromination reaction to generate compound 5; (4) Compound 5 undergoes substitution reaction with compound 3 to generate compound 6; (4) Compound 6 undergoes ester hydrolysis to generate compound 7; (5) Compound 7 undergoes an amide condensation reaction with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile to generate compound 8; (6) Compound 8 is freed from the Boc protecting group to form compound 9; compound 9 then undergoes a condensation reaction with Boc-gly-pro-OH to form compound 10; (7) Compound 10 is deprotected from the Boc protecting group and then reacted with DOTA-NHS to obtain compound ZC-1.

6. A radionuclide labeled complex, which is obtained by labeling a radionuclide M with the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof as a ligand; In particular, the radionuclides M include radiodiagnostic nuclides and radiotherapeutic nuclides; In particular, the radioactive diagnostic nuclide is selected from: 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 I. 44 Sc, 47 Any one of Sc, preferably 86 Y、Al[ 18 F], 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124 Any one of I; In particular, the radioactive therapeutic nuclide is selected from: 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 of Th; preferably 67 Cu, 90 Y. 125 I. 131 I. 177 Lu, 223 Ra, 225 Ac, 211 Any one of At; more preferably 68 Ga, 177 Lu or 90 Y; Preferably, the structure of the radionuclide labeled complex is shown in the following formula (III): in, L, R1 as defined in any one of claims 1 to 4; M is as defined above; preferably, M is selected from 68 Ga, 177 Lu and 90 Any one of Y; More preferably, the radionuclide labeled complex has the following structure:

7. A method for preparing a radionuclide labeled complex according to claim 6, wherein the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof is used as a ligand to label the radionuclide M; In particular, the labeling method is a wet labeling method or a freeze-drying labeling method; In particular, the wet labeling method comprises the following steps: dissolving an appropriate amount of the compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound according to any one of claims 1 to 4 in a buffer solution or deionized water to obtain a solution; adding a radioactive nuclide M solution to the obtained solution, and reacting in a closed manner for 5 to 40 minutes to generate a radioactive nuclide labeled complex; In particular, the freeze-drying labeling method comprises the following steps: dissolving an appropriate amount of the compound of formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound according to any one of claims 1 to 4 in a buffer solution or deionized water to obtain a solution; aseptically filtering the obtained solution, dispensing it into containers, freeze-drying it, and sealing it with a stopper to obtain a freeze-dried medicine box; adding an appropriate amount of acetic acid solution or buffer solution to the freeze-dried medicine box to dissolve it, and then adding the corresponding radionuclide M solution, and reacting it in a closed manner for 5-40 minutes to generate a radionuclide labeled complex; The radionuclide M is defined as in claim 6.

8. A pharmaceutical composition comprising one or more of the compound selected from any one of claims 1 to 4, its pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates, isotope-labeled compounds, and the above-mentioned radionuclide-labeled complexes, and optionally pharmaceutically acceptable excipients.

9. Use of the compound according to any one of claims 1 to 4 or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound, or the above-mentioned radionuclide-labeled complex in the preparation of an agent for inhibiting FAP activity.

10. Use of the compound according to any one of claims 1 to 4 or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound, or the radionuclide-labeled complex thereof in the preparation of a drug or agent for diagnosing, preventing and / or treating a disease characterized by high expression of FAP; Preferably, the invention is used in the preparation of drugs or reagents for radionuclide therapy or imaging of tumors with high FAP expression; Preferably, the FAP-highly expressing tumors include breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer and pancreatic cancer.

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