Compounds targeting fap, radionuclide-labelled complexes based thereon, and methods of preparation and use thereof

Abstract

The application discloses a FAP-targeting compound, a radionuclide-labeled complex based on the same, and a preparation method and application thereof. The FAP-targeting compound has a structure shown in formula (I), and has high affinity for a FAP target. After being labeled with a radionuclide such as 68 Ga, the obtained radiopharmaceutical shows higher tumor uptake and longer tumor retention time, and can be applied to FAP-targeting drugs and further development.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a compound of formula (I) that targets fibroblast activation protein (FAP), a radionuclide-labeled complex based thereon, a method for preparing the same, and their use in the preparation of medicaments for the diagnosis, prevention and / or treatment of diseases characterized by high FAP expression. Background Technology

[0002] In malignant tumor patients, 90% of deaths are caused by tumor cell metastasis, but current preventive and therapeutic strategies for tumor metastasis remain scarce. A tumor is a complex composed of tumor cells, surrounding stromal cells, and non-cellular components. Tumor development and progression are a dynamic process of mutual promotion and co-evolution between tumor cells and their microenvironment. The tumor microenvironment (TME) includes cells and the extracellular matrix (ECM). Its formed elements are mainly cellular components, including immune cells, endothelial cells, and fibroblasts. Among these, cancer-associated fibroblasts (CAFs) are the most abundant 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, making them a hot topic in tumor diagnosis and treatment research.

[0003] In cancer, fibroblast activation protein (FAP) has become a unique marker of tumor-associated fibroblasts (CAF) and a key regulator and driver of the tumor microenvironment (TME). CAF is one of the largest components of the TME, promoting tumor growth and cell invasion through the secretion of pro-inflammatory and growth factors and the remodeling of the extracellular matrix (ECM). Besides 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 matrix, which is essential for cancer cell invasion and metastasis. Increased FAP expression in the pre-invasive portion of colorectal cancer tumor samples has been observed clinically, further supporting its role in invasion and metastasis. FAP also promotes the formation of an immunosuppressive TME by activating tumor-promoting inflammation. FAP is selectively expressed on the surface of stromal fibroblasts in more than 90% of epithelial malignancies, including breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, and melanoma. FAP is typically not expressed in benign and precancerous epithelial tumors, such as colorectal adenomas, phyllodes tumors of the breast, and fibroadenomas. FAP is generally not expressed in normal human tissues, but is only present in the cervix and endometrium, and is expressed transiently during embryonic development. Because FAP plays an important role in the TME (tumor endothelial encephalopathy), there is increasing interest in using it as an imaging and therapeutic target.

[0004] In recent years, many FAP inhibitors have entered clinical trials, such as 68 Ga-FAPI-04 68 Ga-FAPI-46 68 Ga-oncoFAP-DOTAGA, 177 Lu-FAPI-04 and similar probes provide powerful tools for the precise localization and targeted killing of various cancer lesions. However, their relatively short retention time in tumors, low absolute tumor uptake, and rapid clearance in vivo result in unsatisfactory uptake and imaging effects in tumors with low FAP expression. This also limits the killing effect of long-half-life therapeutic radionuclides on target cells or tissues. Therefore, developing FAP-targeting probes with higher tumor uptake, longer retention time, low uptake in non-target tissues, and rapid clearance is of great significance for promoting integrated FAP-targeted diagnosis and therapy. Summary of the Invention

[0005] FAP is selectively and highly expressed in over 90% of epithelial malignancies, making it an ideal target for developing tumor-targeting probes. Considering the low uptake and short retention time of existing FAP-targeting small molecule compounds in tumors, this invention provides a compound of formula (I) that can simultaneously recognize different amino acid sites of FAP to increase affinity and tumor uptake, thus exhibiting high affinity for FAP and can be used for FAP targeting. Furthermore, this invention discovers that radionuclide-labeled complexes based on this compound can specifically recognize FAP targets and increase tumor uptake, thereby improving tumor detection rate and / or therapeutic efficacy.

[0006] Based on this, one object of the present invention is to provide a compound of formula (I), or a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a racemic mixture, a transisomer, a polymorph, a solvate, or an isotopically labeled compound thereof.

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

[0008] Another object of the present invention is to provide a radionuclide-labeled complex, which is obtained by labeling a radionuclide with a compound of formula (I) or a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a racemic mixture, a transisomer, a polymorph, a solvate, or an isotopically labeled compound.

[0009] Another object of the present invention is to provide a method for preparing the radionuclide-labeled complex.

[0010] Another object of the present invention is to provide a pharmaceutical composition comprising one or more selected from compounds of formula (I), pharmaceutically acceptable salts thereof, enantiomers, diastereomers, racemates, transisomers, polymorphs, solvates, and isotopically labeled compounds, as well as radionuclide-labeled complexes thereof, and optionally pharmaceutically acceptable excipients.

[0011] Another object of the present invention is to provide the use of the compound shown in (I) or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, transisomer, polymorph, solvate, or isotopically labeled compound, or the radionuclide-labeled complex thereof, in the preparation of a reagent for inhibiting FAP activity.

[0012] Another object of the present invention is to provide the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, transisomer, polymorph, solvate, or isotopically labeled compound, or the radionuclide-labeled complex thereof, in the preparation of medicaments or reagents for the diagnosis, prevention and / or treatment of diseases characterized by high FAP expression.

[0013] To achieve the above objectives, the present invention adopts the following technical solution:

[0014] In a first aspect, a compound of formula (I) is provided, or a pharmaceutically acceptable salt thereof, an enantiomer, a diastereomer, a racemic mixture, a transisomer, a polymorph, a solvate, or an isotopically labeled compound thereof.

[0015]

[0016] Each R1 is independently a halogen or H, with F being the preferred halogen;

[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 is independently selected from the following structures: Where R a Selected from H, C1-C3 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl), each W1 is connected by a carbonyl group and an N phase to form an amide bond, and three or more adjacent W1 are not simultaneously...

[0019] R3 can be a bifunctional chelating agent, a fluorescent reporter group, or a therapeutic drug;

[0020] L stands for -(CH2) n7 - where n7 is an integer from 0 to 40; where each CH2 group is independently and optionally replaced by -O-, -NH-, -(CO)-, -NH(CO)- or -(CO)-NH-, provided that no two adjacent CH2 groups are replaced at the same time;

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

[0022] Bifunctional chelating agents are chelating agents that simultaneously chelate radionuclides and link targeted molecular probes. Examples include 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-di(2-hydroxyphenyl)ethylenediamine-N,N′-diacetic acid (HBED), and 2,2′,2″,2″′-(5 2 13 2 -dihydroxy-5 5 13 5 -Dimethyl-3,7,11,15-tetraaza-1,9(2,6)-dipyridin-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 are groups that absorb and emit light within a certain wavelength range, releasing light energy. These include, but are not limited to, visible light groups, near-infrared I-region groups, and near-infrared II-region groups.

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

[0025] The near-infrared I region group can be selected from any of the following: cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, etc.), BODIPY dyes (e.g., fluoroboron dipyrrole, azafluoroboron dipyrrole, etc.), rhodamine dyes (e.g., rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lissamine rhodamine, X-rhodamine, Texas red, silicon-based rhodamine, etc.), quantum dots, nanoparticles, and phthalocyanines.

[0026] The near-infrared II region group can be selected from any of the following: 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.

[0027] Treatment drugs include small molecule inhibitors, monoclonal antibodies, bioalkylating agents, cytotoxic drugs, hormonal drugs, and biological response modifiers.

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

[0029] In some implementations, R2 is selected from... Preferred options are:

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

[0031] Preferred

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

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

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

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

[0036]

[0037] R1 and L are defined as described above.

[0038] In some embodiments, the compound of formula (I) is selected from the following structures:

[0039]

[0040]

[0041]

[0042] The present invention further provides a method for preparing compound ZC-1, comprising the following steps:

[0043] (1) Compound 1 undergoes a demethylation reaction to give compound 2; this reaction can be carried out in an aqueous solution of hydrogen bromide.

[0044] (2) Compound 2 undergoes an esterification reaction with methanol to give compound 3; this reaction can be carried out in the presence of thionyl chloride.

[0045] (3) Compound 4 undergoes a bromination reaction to generate compound 5; this reaction can be carried out with carbon tetrabromide in the presence of triphenylphosphine.

[0046] (4) Compound 5 undergoes a substitution reaction with compound 3 to generate compound 6; this reaction can be carried out in the presence of potassium carbonate;

[0047] (4) Compound 6 undergoes ester hydrolysis to generate compound 7; this reaction can be carried out under LiOH conditions;

[0048] (5) Compound 7 undergoes an amide condensation reaction with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile to generate compound 8;

[0049] (6) Compound 8 is deprotected by 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;

[0050] (7) Compound 10 was deprotected by the Boc protecting group (this reaction can be carried out under trifluoroacetic acid conditions), and then reacted with DOTA-NHS to give compound ZC-1;

[0051] The specific synthetic route for the above steps is as follows:

[0052]

[0053] The preparation methods of other compounds in this invention (e.g., compounds ZC-2 to ZC-14) are similar to those of compound ZC-1. For example, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile, or compound 9 is reacted with a carboxylic acid compound containing an L group and an L protecting group (e.g., BOC) (e.g., BOC glycine, 6...). -[(tert-butoxycarbonyl)amino]hexanoic acid, N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid, 5,8,11,14-tetraoxa-2-azaheptadecanoic acid-1-tert-butyl ester, 21-(BOC-amino)-4,7,10,13,16,19-hexaoxadocosuccinic acid, etc.) are coupled, and after removing the L protecting group, they undergo condensation with Boc-gly-pro-OH to remove the Boc protecting group, and finally couple with DOTA-NHS; or both can be replaced simultaneously.

[0054] Secondly, the present invention further provides a radionuclide-labeled complex, which is obtained by labeling a radionuclide M with a compound of formula (I) as described in the present invention or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, transisomer, polymorph, solvate, or isotopically labeled compound. The radionuclide-labeled complex can be used as a tumor radiodiagnostic probe, that is, as a radionuclide diagnostic probe or a radionuclide therapeutic probe.

[0055] The radionuclide M includes radiodiagnostic radionuclides and radiotherapy radionuclides.

[0056] The radiodiagnostic nuclide may be 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 Any one of the following is preferred: 86 Y, Al[ 18 F]、 64 Cu、 68 Ga、 89 Zr、 99m Tc, 124 Any one of the following: Grade I;

[0057] The radiotherapy nuclide may be 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.; preferred 67 Cu、 90 Y、 125 I, 131 I, 177 Lu、 223 Ra、 225 Ac、 211 Any one of At, etc.; more preferably 68 Ga、 177 Lu or 90 Y.

[0058] In some embodiments, the structure of the radionuclide-labeled complex is shown in formula (III):

[0059]

[0060] in,

[0061] The definitions of L and R1 are as described above;

[0062] M is selected from the aforementioned radionuclides.

[0063] In some implementations, M is selected from... 68 Ga、 177 Lu or 90 Any one of Y.

[0064] In some embodiments, the radionuclide-labeled complex has the following structure:

[0065]

[0066] The radionuclide-labeled complexes of the present invention can be prepared by combining a radionuclide-containing compound with the compound of formula (I) of the present invention according to various existing labeling methods. Preferred labeling methods of the present invention may be the wet method or the lyophilization method described below, but are not limited thereto.

[0067] The wet labeling method includes 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, transisomer, polymorph, solvate, or isotopically labeled compound in a buffer solution or deionized water to obtain a solution; adding a radionuclide solution to the obtained solution and reacting in a sealed manner for 5-40 minutes to generate a radionuclide-labeled complex;

[0068] The lyophilization labeling method includes the following steps: dissolving an appropriate amount of the compound of formula (I) of this invention or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemic mixture, transisomer, polymorph, solvate, or isotopically labeled compound in a buffer solution or deionized water to obtain a solution; after sterile filtration, the obtained solution is dispensed into containers, lyophilized, and then sealed to obtain a lyophilized drug box; an appropriate amount of acetic acid solution or buffer solution is added to the lyophilized drug box to dissolve it, and then the corresponding radionuclide solution is added, and the reaction is sealed for 5-40 minutes to generate a radionuclide-labeled complex. The dispensing container is preferably a cryovial or a controlled antibiotic vial. Excipients, such as mannitol or ascorbic acid, can be added to the drug box depending on the lyophilized powder forming process, and the optimal drug box forming can be achieved by adjusting the amount of the compound of formula (I) and the excipients.

[0069] The products obtained by the wet labeling scheme and the lyophilized labeling scheme can be further processed into injection solutions by conventional methods (such as chromatographic separation and purification, rotary evaporation to remove solvent, dissolving the residue with PBS, water or physiological saline, sterile filtration, etc.).

[0070] In some embodiments, the method for preparing radiolabeled complexes using the compound shown in formula (II) as a ligand is a wet labeling method, comprising the following steps: dissolving the compound shown in formula (II) in a buffer solution or deionized water; adding a fresh radionuclide solution, reacting at 37-90℃ for 5-40 min in a sealed environment, and cooling; diluting the reaction solution with water and then separating and purifying it using a Sep-Pak Cl8 chromatographic column; rinsing the chromatographic column with buffer or water to remove unreacted radioactive ions; rinsing with hydrochloric acid ethanol solution or ethanol solution; and then diluting with physiological saline or PBS and sterilely filtering to obtain an injection solution of the radiolabeled complex with the structure described in formula (III); wherein the radionuclide M is... 68 Ga、 177 Lu or 90 Y et al.

[0071] In some embodiments, the method for preparing radiolabeled complexes using the compound shown in formula (II) as a ligand is the lyophilization labeling method, which includes: dissolving the compound shown in formula (II) and other necessary reagents in a buffer solution; sterile filtering the resulting solution and dispensing it into cryovials; lyophilizing and sealing the cryovials to obtain a lyophilized kit; adding an appropriate amount of buffer solution to the lyophilized kit to dissolve the radiolabeled complex, then adding a freshly prepared radiolabeled solution; reacting at 37-120℃ for 5-40 min under sealed conditions; cooling; diluting the reaction solution with water and purifying it using a Sep-Pak Cl8 chromatographic column; rinsing the column with buffer or water to remove unreacted radioactive ions; washing with hydrochloric acid-ethanol solution or ethanol solution; and then diluting with physiological saline or PBS and sterilely filtering to obtain an injection solution of the radiolabeled complex with the structure shown in formula (III); wherein the radiolabeled complex is... 68 Ga、 177 Lu or 90 Y et al.

[0072] The other chemical substances used in the above synthesis steps are commercially available products.

[0073] The buffer solution is a substance that stabilizes the pH of the reaction solution, and may be acetate, lactate, tartrate, malate, maleate, succinate, ascorbate, carbonate, phosphate, or mixtures thereof.

[0074] In another aspect, the present invention provides a pharmaceutical composition comprising one or more of the following: compounds represented by formula (I), pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, transisomers, polymorphs, solvates, and isotopically labeled compounds, radiolabeled complexes thereof, and optionally pharmaceutically acceptable excipients.

[0075] In another aspect, the present invention also provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, transisomer, polymorph, solvate, or isotopically labeled compound, or the above-mentioned radionuclide-labeled complex, in the preparation of a reagent for inhibiting FAP activity.

[0076] In another aspect, the present invention also provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer, racemate, transisomer, polymorph, solvate, or isotopically labeled compound, or the above-mentioned radionuclide-labeled complex, in the preparation of medicaments or reagents for the diagnosis, prevention and / or treatment of diseases characterized by high FAP expression.

[0077] In some embodiments, the present invention provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof, or the radionuclide-labeled complex thereof, in the preparation of a medicament or reagent for radionuclide therapy or imaging of tumors with high FAP expression.

[0078] In the applications described in this invention, the complex can be prepared as an injection and administered via intravenous injection, but is not limited thereto.

[0079] In the applications described in this invention, the tumors with high FAP expression include, but are not limited to, breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer.

[0080] Beneficial effects:

[0081] The compound of formula (I) and its radionuclide complex provided by this invention can specifically target FAP and inhibit FAP activity. Biological test results show that it has a significantly prolonged blood circulation half-life, enhanced tumor uptake and enrichment, and retention time effect, which are not present in other FAPI imaging agents (such as FAPI-04). It is suitable for use as a radionuclide therapy and imaging agent for tumors with high FAP expression. Attached image description:

[0082] Figure 1 The image shows the HPLC chromatogram of compound ZC-1 prepared in Example 1.

[0083] Figure 2 The simulated docking diagram of compounds ZC-1, FAPI-04 and FAP in Example 1 is shown.

[0084] Figure 3 To test different times in Example 3 68 In vitro stability diagram of Ga-ZC-1 at 37℃.

[0085] Figure 4 For testing Example 4 68 Ga-ZC-1 and 68 PET / CT images of Ga-FAPI-04 in U87MG tumor mice at 0.5, 1, and 2 hours.

[0086] Figure 5 For testing Example 4 68 Statistical distribution of Ga-ZC-1 in U87MG tumor mice.

[0087] Figure 6 For testing Example 4 68 Ga-ZC-1 and 68 Area under the curve of time-tumor uptake of Ga-FAPI-04. Detailed Implementation

[0088] The compounds of the present invention, their preparation methods, and applications will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of 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 content of the present invention are covered within the scope of protection intended by the present invention. Unless otherwise stated, the raw materials and reagents used in the following embodiments are commercially available products or can be prepared by known methods.

[0089] Experimental instruments and consumables

[0090] Animal model: Female BALB / c mice (15-20g) aged 5-7 weeks were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The mice were housed under specific pathogen-free conditions. The housing environment was 25℃, humidity 35-45%, with 12-hour light-dark cycles. All animals had free access to water and food.

[0091] Cell model: U-87MG (human brain astroblastoma cells), from the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0092] 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 O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethylurea hexafluorophosphate (N,N,N′,N′-TETRAMETHYL-O-(7-AZABENZOTRIAZOL-1-YL)URONIUMHEXAFLUOROPHOSPATE (HATU) were purchased from Saen Chemical Technology (Shanghai) Co., Ltd. (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride were purchased from Shaoyuan Technology (Shanghai) Co., Ltd.

[0093] DOTA-NHS, Purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0094] Boc-gly-pro-OH, Purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.

[0095] ICG-NHS, Purchased from Shanghai Bailingwei Chemical Technology Co., Ltd.

[0096] FAPI-04 (CAS No.: 2374782-02-0), Purchased from Wuxi Jiehua Pharmaceutical Technology Co., Ltd.

[0097] Biological reagents: DMEM medium, RPMI 1640 medium, fetal bovine serum, trypsin, phosphate-buffered saline (PBS), skim milk powder, 4% paraformaldehyde, and CCK8 reagent kit were all purchased from Dalian Meilun Biotechnology Co., Ltd.

[0098] Example 1: Preparation of compound ZC-1:

[0099]

[0100] Step 1: Synthesis of Compound 2

[0101] 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% sodium hydroxide aqueous 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) 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 forC 10 H8NO3 + [M+H] + : 190.1, found 190.2.

[0102] Step 2: Synthesis of Compound 3

[0103] 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 under ice bath conditions. After the addition was complete, the mixture was stirred overnight at 60 °C, and then the solvent was 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 give 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

[0104] Step 3: Synthesis of Compound 5

[0105] 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 tetrahydrofuran (120 mL) solution of compound 4 (5 g, 20.46 mmol), and the reaction 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 the combined organic phases were concentrated and purified by silica gel column chromatography (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).

[0106] Step 4: Synthesis of Compound 6

[0107] 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 50 mL of N,N-dimethylformamide in a 100 mL flask. The system was heated to 60 °C and stirred overnight at 60 °C. The solvent was removed by vacuum distillation to obtain the crude product. Compound 6 was purified by silica gel column chromatography (PE / EA = 1:1) to obtain compound 6. 1 H 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.

[0108] Step 5: Synthesis of Compound 7

[0109] To a solution of compound 6 (1.00 g, 2.33 mmol, 1 eq.) in 100 mL of methanol and 50 mL of tetrahydrofuran, 100 mL of an aqueous solution of lithium hydroxide (167.26 mg, 6.98 mmol, 3 eq.) was added. 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 with hydrochloric acid, stirred for another 15 minutes, and the resulting solid was separated by filtration and dried under vacuum. Compound 7 was finally quantitatively obtained. 1H 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). 13 C 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.

[0110] Step 6: Synthesis of Compound 8

[0111] Compound 7 (100 mg, 528.64 μmol, 1.00 eq) and (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile 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 h. The filtrate was filtered and purified by preparative HPLC to give compound 8. 1H 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.

[0112] Step 7: Synthesis of Compound 9

[0113] The crude compound 8 (100.00 mg, 170.46 μmol) was dissolved in a solution of TFA (10 mL) and DCM (10 mL). The solution was stirred at room temperature for 1 hour, concentrated by evaporation, and then ether was added. The solid precipitated was collected and dried under vacuum to obtain compound 9. This product can be 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 forC 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.

[0114] Step 8: Synthesis of Compound 10

[0115] 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 mixture was stirred at 25 °C for 12 hours. After the reaction was completed, the reaction solution was concentrated and purified by preparative HPLC. Compound 10 was obtained as a white solid. 1 H 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.

[0116] Step 9: Synthesis of Compound 11

[0117] The crude compound 10 (103.78 mg, 161.98 μmol) was dissolved in a solution of TFA (10 mL) and DCM (10 mL). The solution was stirred at room temperature for 1 hour, concentrated by evaporation, and then ether was added. The solid precipitated was collected and dried under vacuum to obtain compound 11. This product can be used directly in the next step without further purification.

[0118] Step 10: Synthesis of compound ZC-1

[0119] The crude compound 11 (100.00 mg, 156.08 μmol, 1 eq) was dissolved in 10 mL of LDMF, 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. The resulting compound ZC-1 was a white solid. 1 H 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.

[0120] Example 2 Preparation of compound ZC-2

[0121]

[0122] The preparation method is the same as in Example 1, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0123] Characterization 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).

[0124] Example 3: Preparation of compound ZC-3

[0125]

[0126] The preparation method is the same as in Example 1, except that before 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 resulting compound is deprotected by TFA and then reacted with Boc-gly-pro-OH. The resulting compound is deprotected by TFA and then reacted with DOTA-NHS (the reaction conditions are the same or similar to those in Example 1) to obtain ZC-3.

[0127] 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).

[0128] Example 4: Preparation of compound ZC-4

[0129]

[0130] The preparation method is the same as in Example 3, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0131] 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).

[0132] Example 5: Preparation of compound ZC-5

[0133]

[0134] The preparation method is the same as in Example 3, except that the BOC glycine that reacts with compound 9 is replaced with 6-[(tert-butoxycarbonyl)amino]hexanoic acid.

[0135] Characterization data: 1H 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).

[0136] Example 6 Preparation of compound ZC-6

[0137]

[0138] The preparation method is the same as in Example 5, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0139] 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).

[0140] Example 7 Preparation of compound ZC-7

[0141]

[0142] The preparation method is the same as in Example 3, except that before step 8, compound 9 is reacted with N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid. The specific reaction steps are as follows: compound 9 (1 eq) and N-tert-butoxycarbonyl-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 resulting compound is deprotected by TFA and then reacted with Boc-gly-pro-OH; the resulting compound is deprotected by TFA and then reacted with DOTA-NHS (the reaction conditions are the same or similar to those in Example 1) to obtain ZC-7.

[0143] Characterization data: 1H 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).

[0144] Example 8 Preparation of compound ZC-8

[0145]

[0146] The preparation method is the same as in Example 7, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0147] 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).

[0148] Example 9 Preparation of compound ZC-9

[0149]

[0150] The preparation method is the same as in Example 7, except that the N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid that reacts with compound 9 is replaced with 5,8,11,14-tetraoxa-2-azaheptadecanoic acid-1-tert-butyl ester.

[0151] 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).

[0152] Example 10 Preparation of compound ZC-10

[0153]

[0154] The preparation method is the same as in Example 9, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0155] 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).

[0156] Example 11 Preparation of compound ZC-11

[0157]

[0158] The preparation method is the same as in Example 7, except that the N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid that reacts with compound 9 is replaced with 21-(BOC-amino)-4,7,10,13,16,19-hexaoxadocosuccinic acid.

[0159] 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).

[0160] Example 12 Preparation of compound ZC-12

[0161]

[0162] The preparation method is the same as in Example 11, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0163] Characterization data: 1H 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).

[0164] Example 13 Preparation of compound ZC-13

[0165]

[0166] The preparation method is the same as in Example 7, except that the N-tert-butoxycarbonyl-diethylene glycol-carboxylic acid that reacts with compound 9 is replaced with N-tert-butoxycarbonyl-heptapolyethylene glycol-carboxylic acid.

[0167] 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).

[0168] Example 14 Preparation of compound ZC-14

[0169]

[0170] The preparation method is the same as in Example 13, except that the (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carboxynitrile hydrochloride that reacts with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carboxynitrile hydrochloride.

[0171] 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).

[0172] Example 15: Preparation of compound ZC-ICG-1:

[0173]

[0174] Step 1: Synthesis of compound ZC-ICG-1

[0175] 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. The obtained compound ZC-ICG-1 was 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).

[0176] Example 16: 68 Preparation of Ga-ZC-1

[0177]

[0178] Take 0.1M hydrochloric acid from 68 Ge- 68 Elution in Ga generator 68 344 μL of GaCl was mixed with 0.5 μL of an aqueous solution of compound ZC-1 at a concentration of 10 μg / μL. Then, 400 μL of sodium acetate solution was added to adjust the pH to 4-5, and the mixture was reacted in a metal bath at 95°C for 10 minutes. After the reaction, the reactant was diluted with 1 mL of physiological saline and injected into a C18 desalting column using a syringe. The C18 desalting column was then washed multiple times with 10 mL of physiological saline to remove unbound compounds of compound ZC-1. 68 Ga 3+ Continue this process until the activity difference between two consecutive washes is less than 10 μCi. Finally, elute the C18 desalting column with 100–200 μL of ethanol to obtain... 68 Ga-ZC-1.

[0179] Test Example 1: Dating Simulation Analysis of ZC-1, FAPI-04 and FAP

[0180] The three-dimensional structure of the FAP protein was downloaded from the RCSB protein database, PDBID 1Z68. Compounds ZC-1 and FAPI-04 were modeled using the homology model method. The docking simulation results are shown below. Figure 2 The red portion represents the interaction between FAP protein residues and small molecules, while potential hydrogen bonds are indicated by yellow dashed lines. Figure 2 Docking analysis shows that the reference docking score for ZC-1 with FAP is 7.6177, while that for FAPI-04 is 6.3464. Compared to FAPI-04, ZC-1 is more capable of docking with Tyr. 745 Trp 623 Arg 123 Ser 548 Arg 550 、Gln 547 and Asn 399 They interact, while FAPI-04 can only interact with Trp. 623 and Ser 624 Interaction. 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, thus making the interaction between ZC-1 and the FAP protein stronger.

[0181] Test Example 2: 68 Ga-ZC-1 in vitro FAP enzyme saturation binding assay

[0182] 1×10 5 One U87MG cell was seeded in each well of a 96-well cell plate and cultured for 24 hours. The cells prepared in Example 16 were then... 68 Ga-ZC-1 and 68 Ga-labeled FAPI-04 solutions were diluted with culture medium to different concentrations of 1000 nM, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, and 0.78 nM. Simultaneously, FAPI-04 was diluted with complete culture medium to 100 μM to prepare inhibitor solutions. The upper layer of culture medium in the wells was aspirated from the plate and washed twice with 0.2 mL of PBS. The experiment was divided into several parts. 68 Ga-ZC-1 or 68 The Ga-FAPI-04 group included both a control group and an experimental group. The experimental group received 0.1 mL of blank culture medium, while the control group received 0.1 mL of FAPI-04 solution (100 μM). After incubation at 37°C for 30 minutes, 0.1 mL of different concentrations of FAPI-04 solution were added to both groups. 68 Ga-ZC-1 or 68Incubate in Ga-FAPI-04 solution at 37°C for 2 hours. Aspirate the solution and wash twice with PBS. Lyse cells with 0.2 mL NaOH solution (1M) and count cells using a gamma counter. All measurements were performed in duplicate. Results are shown in Table 1.

[0183] Table 1. 68 Ga-ZC-1 and 68 Ga-FAPI-04 binds to FAP protein (K) d value

[0184]

[0185] As shown in Table 1, the compounds 68 Ga-ZC-1 has good FAP protein binding ability.

[0186] Test Example 3: 68 In vitro stability of Ga-ZC-1 at 37℃

[0187] 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 mixture was incubated at 37°C for 0 min, 30 min, 60 min, and 120 min, and samples were taken for thin-layer chromatography. Stability was calculated after detection with a gamma detector. The results are shown in the figure. Figure 3 .

[0188] Figure 3 show 68 Ga-ZC-1 showed no significant demetallization within two hours in PBS and BSA buffer, indicating high stability, and can be used for further experiments.

[0189] Test Example 4: 68 Ga-ZC-1 and 68 PET / CT imaging of Ga-FAPI-04 in rats bearing U87MG human brain gliomas.

[0190] Experimental animals and administration method: U87MG human glioma-bearing mice, administered via tail vein injection.

[0191] Dosage grouping and dosage:

[0192] 68 Ga-ZC-1 group: 150 μCi / 200 μL injected via tail vein. 68 Ga-ZC-1.

[0193] Blocking group: Injected via tail vein 68Thirty minutes before the onset of Ga-ZC-1, compound FAPI-04 (500 times excess of probe substance) was injected via the tail vein.

[0194] Control group: 150 μCi / 200 μL injected via tail vein 68 Ga-FAPI-04.

[0195] For injection 68 PET / CT imaging was performed at 0.5, 1, and 2 hours after Ga-ZC-1 administration to observe the probe distribution in mice and its enrichment in tumor regions. Results are shown in [Figure number missing]. Figure 4-6 .

[0196] The results are as follows Figure 4 As shown, the U87MG xenograft model 68 Ga-ZC-1 PET imaging revealed high tumor uptake and strong contrast between tumor and background. Even 0.5 hours post-injection, tumor accumulation rapidly reached 4.467 ± 0.379% ID / g, with signal gradually decreasing after 1 hour. Furthermore, 68 Ga-FAPI-04, as a comparative and clinically used agent, showed a much lower signal intensity in tumors. Furthermore, during injection... 68 0.5 hours after Ga-ZC-1 administration, the U87MG tumor in the left anterior axilla of mice was clearly visible. The blockade experiment showed that pre-injection of non-radioactive FAPI-04 significantly reduced tumor uptake. This result indicates... 68 Specific accumulation of Ga-ZC-1 in vivo. In the control group, 68 Ga-FAPI-04 was visible in tumors 0.5 hours after injection, but its uptake was similar to that of the liver. Subsequently, uptake in the tumor area decreased significantly, and no signal was detected after 2 hours. This demonstrates that, compared to widely accepted... 68 Compared with Ga-FAPI-04, 68 Ga-ZC-1 uptake in tumor regions was significantly enhanced, resulting in longer tumor retention time. Experimental results demonstrate that the probe... 68 Ga-ZC-1 has the effect of in vivo tumor imaging.

[0197] Figure 5 This is a distribution chart in the body. The data in the chart show that after prior injection of FAPI-04 as a blocking agent, the drug was administered via tail vein injection. 68 Ga-ZC-1, tumor component against 68 The significantly reduced uptake of Ga-ZC-1 indicates 68 Ga-ZC-1 can specifically target tumors with high FAP expression, and the tumor / brain distribution ratio is as high as 13. Figure 6 for 68 Ga-ZC-1 and 68Ga-FAPI-04 is used for comparison based on the area under the curve (AUC) of tumor uptake versus time. 68 Ga-ZC-1 and 68 The tumor uptake of Ga-FAPI-04 over time is shown to be 422.5 and 98.14, respectively, indicating that... 68 Ga-ZC-1 has a longer tumor retention time.

Claims

1. Compounds selected from the following structures, or pharmaceutically acceptable salts, solvates, or isotopically labeled compounds thereof: 。 2. A radionuclide-labeled complex, comprising labeling radionuclide M with the compound of claim 1 or its pharmaceutically acceptable salt, solvate, or isotopically labeled compound as a ligand, wherein, The radionuclide M is selected from... 68 Ga、 64 Cu、 67 Cu、 177 Lu、 225 Ac、 212 Pb, 90 Y.

3. The radionuclide-labeled complex according to claim 2, wherein, The structure of the radionuclide-labeled complex is shown in formula (III) below: in, L and R1 are as defined in claim 1; M is defined as in claim 2.

4. The radionuclide-labeled complex according to claim 3, having the following structure: 。 5. A method for preparing a radionuclide-labeled complex according to any one of claims 2-4, wherein the radionuclide M is labeled using the compound of claim 1 or its pharmaceutically acceptable salt, solvate, or isotopically labeled compound as a ligand.

6. The preparation method according to claim 5, wherein, The labeling method is either a wet labeling method or a freeze-drying labeling method.

7. The preparation method according to claim 6, wherein, The wet labeling method includes the following steps: dissolving an appropriate amount of the compound of claim 1 or its pharmaceutically acceptable salt, solvate, or isotopically labeled compound in a buffer solution or deionized water to obtain a solution; adding a solution of radioactive nuclide M to the obtained solution, and reacting in a sealed environment for 5-40 min to generate a radioactive nuclide-labeled complex; The lyophilization labeling method includes the following steps: dissolving an appropriate amount of the compound of claim 1 or its pharmaceutically acceptable salt, solvate, or isotopically labeled compound in a buffer solution or deionized water to obtain a solution; after sterile filtration, dispensing the obtained solution into containers, lyophilizing it, and then sealing it to obtain a lyophilized drug box; adding an appropriate amount of acetic acid solution or buffer solution to the lyophilized drug box to dissolve it, then adding the corresponding radioactive nuclide M solution, and reacting in a sealed environment for 5-40 minutes to generate a radioactive nuclide-labeled complex.

8. A pharmaceutical composition comprising one or more of the radiolabeled complexes according to any one of claims 2-4, and optionally pharmaceutically acceptable excipients.

9. The use of the compound of claim 1 or a pharmaceutically acceptable salt, solvate, or isotopically labeled compound thereof, or the radionuclide-labeled complex of any one of claims 2-4, in the preparation of a medicament or reagent for radionuclide therapy or imaging of tumors expressing FAP high levels, wherein, The tumors with high FAP expression were selected from breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer.

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