Compound targeting FAP, radionuclide labeled complex based on compound, and preparation method and application of radionuclide labeled complex

Abstract

The invention discloses a compound for targeting FAP, a radionuclide labeled complex based on the compound, and a preparation method and application of the radionuclide labeled complex. The compound targeting FAP has a structure as shown in a formula (I) and has high affinity to an FAP target spot. A radiopharmaceutical obtained on the basis that the targeted FAP compound is labeled by radionuclide such as 68Ga shows higher tumor uptake and longer tumor residence time, and can be applied to FAP targeted drugs and further developed. # imgabs0 #

Description

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 a preparation method thereof, and their use in preparing a drug for diagnosing, preventing and / or treating a disease characterized by high expression of FAP. Background Art

[0002] 90% of deaths among patients with malignant tumors are caused by tumor cell metastasis, but there is still a lack of preventive or therapeutic strategies for tumor metastasis. Tumors are complexes composed of tumor cells, their 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, tumor-associated fibroblasts (CAFs) are the most important stromal cells in the tumor microenvironment, accounting for about 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) and a key regulator and driver of the tumor microenvironment (TME). CAFs are one of the largest components of the TME and promote tumor growth and cell invasion by secreting pro-inflammatory 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 active cancer stroma, which is required for cancer cell invasion and metastasis. It has been observed clinically that FAP expression is increased in the invasive front of colorectal cancer tumor 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 more than 90% of epithelial malignancies, including breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, skin melanoma, etc. FAP is usually not expressed in benign and precancerous epithelial tumors, such as colorectal adenomas, breast phyllodes tumors, and fibroadenomas. FAP is generally not expressed in normal human tissues, but only exists in the cervix and endometrium, where it is transiently expressed during embryonic development. Due to the important role that FAP plays in the TME, there is 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 others provide powerful tools for the precise positioning and targeted killing of various cancer lesions. However, their retention time in tumors is relatively short, the absolute value of tumor uptake is low, and they are cleared too quickly in the body, resulting in unsatisfactory uptake and imaging effects in tumors where FAP expression is not high, and also limits the killing effect of long half-life therapeutic nuclides on target cells or tissues. Therefore, the development of FAP-targeted probes with higher tumor uptake, longer retention time, low non-target tissue uptake and rapid clearance is of great significance for promoting FAP-targeted diagnosis and treatment integrated drugs. Summary of the invention

[0005] FAP is selectively highly expressed in more than 90% of epithelial malignancies, and therefore is an ideal target for developing tumor-targeted probes. Considering the low uptake and short retention time of existing FAP-targeted small molecule compounds in tumors, the present invention provides a compound represented by formula (I), which can simultaneously recognize different amino acid sites of FAP to improve affinity and increase tumor uptake, and thus has a high affinity for FAP and can be used to target FAp. Furthermore, the present invention finds that radionuclide-labeled complexes based on this compound can specifically recognize FAP targets and increase tumor uptake rates, thereby improving tumor detection rates and / or therapeutic effects.

[0006] 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.

[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 the compound represented by formula (I) or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound as a ligand.

[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 of the compounds selected from the group consisting of the compounds represented by formula (I), their pharmaceutically acceptable salts, enantiomers, diastereomers, racemates, atropisomers, polymorphs, solvates, and isotope-labeled compounds, and the radionuclide-labeled complexes, and optionally pharmaceutically acceptable excipients.

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

[0012] Another 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, or use of 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.

[0013] In order to achieve the above object, the present invention adopts the following technical solutions:

[0014] 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.

[0015]

[0016] wherein each R1 is independently halogen or H, and halogen is 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 (e.g. methyl, ethyl, n-propyl, isopropyl), each W1 is connected to N by a carbonyl group 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 cyano (-CN) or boric acid (-B(OH)2).

[0022] Bifunctional chelators refer to chelators that have the functions of simultaneously chelating radionuclides and connecting to 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 region 1 groups, and near-infrared region 2 groups.

[0024] The visible light group can be selected from any one of fluorescein, rhodamine, fluorescein isothiocyanate, cyanine fluorescent dyes (such as Cy2, etc.), 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 zone II 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 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.

[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), (II-2):

[0036]

[0037] Wherein, R1 and L are defined as 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 obtain Compound 2; the reaction can be carried out in an aqueous solution of hydrogen bromide;

[0044] (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;

[0045] (3) Compound 4 undergoes bromination reaction to generate compound 5; the 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; the reaction can be carried out in the presence of potassium carbonate;

[0047] (4) Compound 6 undergoes ester hydrolysis to generate compound 7; the 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-carbonitrile to generate compound 8;

[0049] (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;

[0050] (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;

[0051] The synthetic route of the above-mentioned specific steps is as follows:

[0052]

[0053] The preparation methods of other compounds in the scheme of the present invention (e.g., compounds ZC-2 to ZC-14) are similar to the preparation method of compound ZC-1, for example, (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile reacted with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile, or compound 9 is reacted with a carboxylic acid compound (e.g., BOC glycine, 6 -[(tert-butyloxycarbonyl)amino]hexanoic acid, N-tert-butyloxycarbonyl-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-hexaoxahexanecarboxylic 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 are replaced at the same time.

[0054] 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 of the present invention as a ligand. The radionuclide labeled complex can be used as a tumor radiotherapy probe, that is, a radionuclide diagnostic probe or a radionuclide therapeutic probe.

[0055] The radioactive nuclides M include radioactive diagnostic nuclides and radioactive therapeutic nuclides.

[0056] The radioactive diagnostic 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 I, etc., preferably 86 Y、Al[ 18 F], 64 Cu, 68 Ga, 89 Zr, 99m Tc, 124 Any one of I, etc.;

[0057] The radioactive therapeutic 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.; 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.

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

[0059]

[0060] in,

[0061] L and R1 are defined as above;

[0062] M is selected from the aforementioned radionuclides.

[0063] In some embodiments, 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 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.

[0067] 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 manner for 5-40 minutes to generate a radioactive nuclide labeled complex;

[0068] The freeze-drying labeling method comprises the following steps: dissolving an appropriate amount of the compound of formula (I) or its pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound described in the present invention in a buffer solution or deionized water to obtain a solution; the obtained solution is sterile filtered, divided into containers, and sealed with a stopper after freeze drying 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, and then adding the corresponding radionuclide solution, and reacting in a closed manner for 5-40 minutes to generate a radionuclide labeled complex. Among them, the container for subpackaging is preferably a cryopreservation tube or a controlled antibiotic bottle. It is also possible to choose to add excipients to the medicine box according to the molding of the freeze-dried powder in the medicine box, such as mannitol, ascorbic acid, etc., and by adjusting the dosage of the compound of formula (I) and the excipient described in the present invention, the molding of the medicine box is optimized.

[0069] The products obtained by the wet labeling scheme and the freeze-drying labeling scheme can be further prepared into injection solutions through conventional treatments (such as purification by chromatography, removal of solvent by rotary evaporation, dissolution of the residue with PBS or water or saline, sterile filtration, etc.).

[0070] 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 at 37-90° C. in a sealed manner for 5-40 minutes, and cooling; diluting the reaction solution with water, separating and purifying it through a Sep-Pak Cl8 chromatographic column, washing 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 filtering it aseptically to obtain an injection of a radioactive labeled complex with a structure as described in formula (III); wherein the radioactive nuclide M is 68 Ga, 177 Lu or 90 Y et al.

[0071] 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, filtering the resulting solution aseptically and then dispensing it into cryopreservation tubes, freeze-drying and sealing to obtain a freeze-dried medicine box; adding an appropriate amount of buffer solution to the freeze-dried medicine 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 separating and purifying it through a Sep-Pak Cl8 chromatographic column, washing 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 filtering it aseptically to obtain an injection of a radioactive labeled complex with a structure as represented by formula (III); wherein the radioactive nuclide 68 Ga, 177 Lu or 90 Y et al.

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

[0073] 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.

[0074] 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), a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, and isotope-labeled compound thereof, one or more of the above-mentioned radionuclide-labeled complexes, and optionally a pharmaceutically acceptable excipient.

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

[0076] In another aspect, the present invention also provides a compound represented by formula (I) or a pharmaceutically acceptable salt, enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof, or the use of 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.

[0077] 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 tumors with high FAP expression.

[0078] 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.

[0079] 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.

[0080] Beneficial effects:

[0081] The compound of formula (I) and its radionuclide complex provided by the present invention can specifically target FAP and inhibit FAP activity. The biological test results show that it has a significantly prolonged blood circulation half-life, enhanced tumor uptake enrichment and retention time effect, which is not possessed by other current FAPI imaging agents (such as FAPI-04). It is suitable for use as a radionuclide therapy and imaging of FAP-highly expressed tumors. Description of the drawings:

[0082] Figure 1 This is the HPLC chart of compound ZC-1 prepared in Example 1.

[0083] Figure 2 This is a simulated docking binding diagram of compounds ZC-1, FAPI-04 and FAP in Test Example 1.

[0084] Figure 3 To test the different time of Example 3 68 In vitro stability of Ga-ZC-1 at 37°C.

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

[0086] Figure 5 For testing example 4 68 Statistical diagram of the 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 DESCRIPTION

[0088] The following will further describe in detail the compounds of the present invention and their preparation methods and applications in conjunction with specific examples. It should be understood that the following examples are only exemplary illustrations and explanations 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 included in the scope of protection of the present invention. Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products, or can be prepared by known methods.

[0089] Experimental instrument consumables

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

[0091] Cell model: U-87MG (human brain glioblastoma 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′-tetramethyluronium hexafluorophosphate (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.

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

[0094] Boc-gly-pro-OH, Purchased from Shanghai BiDe 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 culture medium, RPMI 1640 culture 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.

[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% aqueous sodium hydroxide solution, and a large amount of solid precipitated. 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 calculation 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 MeOH, and 5 mL 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 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

[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 solution of compound 4 (5 g, 20.46 mmol) in tetrahydrofuran (120 mL), and the mixture was stirred at 25° C. for 12 hours. After the reaction, the reaction solution was concentrated, extracted with ethyl acetate, washed with water, combined with the organic phases, concentrated, and purified by 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).

[0106] Step 4: Synthesis of compound 6

[0107] 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 to 50 mL of N, N-dimethylformamide. The system was heated to 60°C, and the system was stirred at 60°C overnight. The solvent was removed by distillation under reduced pressure to obtain a crude product. Compound 6 was obtained by purification on a silica gel column (PE / EA=1:1). 1 H NMR (600MHz, Chloroform-d) δ 8.84 (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 was added 100 mL of lithium hydroxide (167.26 mg, 6.98 mmol, 3 eq.) aqueous solution. The mixture was stirred at 25 ° C for 1 hour and then concentrated in vacuo. The residue was diluted with water, hydrochloric acid was added to adjust the pH to 5, and after stirring for another 15 minutes, the solid formed was separated by filtration and dried in vacuo. Finally, compound 7 was obtained quantitatively. 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-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.), N,N-diisopropylethylamine (205 mg, 1.59 mmol, 3 eq.), and the reaction was stirred at 25 ° C for 12 hours. The filtrate was filtered and purified using 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) in DCM (10 mL), stirred at room temperature for 1 hour, evaporated and concentrated, and ether was added to precipitate a solid, which was collected and vacuum dried to quantitatively obtain compound 9. The 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 calculation 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.), N, N-diisopropylethylamine (237.32 mg, 1.84 mmol, 5 eq.), and the reaction was stirred at 25°C for 12 hours. After the reaction was completed, the reaction solution was concentrated and purified using preparative HPLC to obtain compound 10 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,found741.5.

[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) in DCM (10 mL), stirred at room temperature for 1 hour, evaporated and concentrated, and ether was added to precipitate a solid, which was collected and vacuum dried to quantitatively obtain compound 11. The product was directly used 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 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, evaporated and concentrated, and purified using preparative HPLC to obtain compound ZC-1 as 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 refers to Example 1, 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.

[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 refers to Example 1, except that before performing step 8, compound 9 is reacted with BOC glycine. The specific reaction steps are: 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 stirred at room temperature for 8 hours; the obtained compound is deprotected by TFA and then reacted with Boc-gly-pro-OH, and the obtained 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 refers to Example 3, 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, 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 similar to that of Example 3, except that BOC glycine reacted with compound 9 is replaced by 6-[(tert-butyloxycarbonyl)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 refers to Example 5, 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.

[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 refers to Example 3, except that before performing step 8, compound 9 is reacted with N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid, and 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 stirred at room temperature for 8 hours; the obtained compound is deprotected by TFA and then reacted with Boc-gly-pro-OH; the obtained 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 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.

[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 refers to Example 7, except that the N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid reacted with compound 9 is replaced by 5,8,11,14-tetraoxa-2-azaheptadecanedioic 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 refers to Example 9, 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.

[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 refers to Example 7, except that the N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid reacted with compound 9 is replaced by 21-(BOC-amino)-4,7,10,13,16,19-hexaoxaheneicosanoic 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 as in Example 11, except that (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride reacted with compound 7 is replaced with (S)-1-(2-aminoacetyl)pyrrolidine-2-carbonitrile 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 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.

[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 refers to Example 13, 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.

[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, and stirred at room temperature for 12 hours. After evaporation and concentration, the mixture was purified by preparative HPLC to obtain compound ZC-ICG-1 as a green solid. 1 H NMR (400MHz, Methanol-d4) 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] Embodiment 16: 68 Preparation of Ga-ZC-1

[0177]

[0178] Take 0.1M hydrochloric acid from 68 Ge- 68 Elution from Ga generator 68 44 μL of GaCl3 was mixed with 0.5 μL of 10 μg / μL aqueous solution of compound ZC-1, and then 400 μL of sodium acetate solution was added to adjust the pH value to 4-5, and then reacted in a 95°C metal bath for 10 minutes. After the reaction was completed, the reactant was diluted with 1 mL of saline and injected into the C18 desalting column with 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 consecutive washes is less than 10μCi. Finally, the C18 desalting column was eluted with 100-200μL ethanol to obtain 68 Ga-ZC-1.

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

[0180] The three-dimensional structure of FAP protein was downloaded from the RCSB protein database with PDBID 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 indicates the interaction between FAP protein residues and small molecules, and the potential hydrogen bonds are represented by yellow dotted lines. Figure 2 The docking analysis showed that the reference docking score of ZC-1 and FAP was 7.6177, while that of FAPI-04 was 6.3464. Compared with FAPI-04, ZC-1 was able to dock with Tyr 745 Trp 623 , Arg 123 , Ser 548 , Arg 550 , Gln 547 and Asn 399 interact with Trp 623 and Ser 624 Interaction. Compared with 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 making the interaction between ZC-1 and the FAP protein stronger.

[0181] Test Example 2: 68 In vitro FAP enzyme saturation binding experiment with Ga-ZC-1

[0182] 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 68 The Ga-labeled FAPI-04 solution was diluted with culture medium to different concentrations of 1000nM, 500nM, 250nM, 100nM, 50nM, 25nM, and 0.78nM. At the same time, 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 a blank group and an experimental group, the experimental group was added with 0.1 mL of blank culture medium, the blank group was added with 0.1 mL of FAPI-04 solution (100 μM), and after culturing at 37°C for 30 minutes, the experimental group and the blank group were added with 0.1 mL of different concentrations of 68 Ga-ZC-1 or 68Ga-FAPI-04 solution was incubated at 37°C for 2 hours. The solution was aspirated and washed twice with PBS. The cells were lysed with 0.2 mL of NaOH solution (1 M) and counted by a gamma counter. All measurements were performed in duplicate. The results are shown in Table 1.

[0183] Table 1. 68 Ga-ZC-1 and 68 The K of Ga-FAPI-04 binding to FAP protein d value

[0184]

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

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

[0187] Will 68 Ga-ZC-1 was dissolved in FBS or BSA buffer at a concentration of 10 μg / μL. Then 45 μL of 1.5M sodium acetate solution and 430 μL of gallium chloride eluent were added and mixed evenly. After labeling, the mixture was placed at 37°C for 0 min, 30 min, 60 min, and 120 min. Samples were taken for thin layer chromatography and the stability was calculated after detection by a γ detector. The results are shown in the table below. Figure 3 .

[0188] Figure 3 show 68 Ga-ZC-1 showed no obvious demetallation phenomenon in PBS and BSA buffer within two hours, and had high stability, so it could be used for the next experiment.

[0189] Test Example 4: 68 Ga-ZC-1 and 68 In vivo PET / CT imaging of Ga-FAPI-04 in mice bearing U87MG human brain glioma

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

[0191] Dosage group and dosage:

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

[0193] Blockage group: injection into tail vein 6830 minutes before Ga-ZC-1, compound FAPI-04 (500-fold excess of the probe substance) was injected via the tail vein.

[0194] Control group: Tail vein injection of 150μCi / 200μL 68 Ga-FAPI-04.

[0195] For injection 68 PET / CT imaging was performed at 0.5, 1, and 2 h 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 Figure 4-6 .

[0196] The results are as follows Figure 4 As shown, the U87MG xenograft model 68 Ga-ZC-1 PET imaging showed high tumor uptake and strong contrast between tumor and background. Even at 0.5 h after injection, the accumulation in the tumor quickly reached 4.467 ± 0.379% ID / g, and the signal gradually decreased after 1 h after injection. In addition, 68 Ga-FAPI-04, a comparative and clinically used agent, showed much lower signal in tumors. 68 After 0.5 hours of Ga-ZC-1, the U87MG tumor in the left anterior armpit of the mouse was clearly visible. The blocking experiment showed that the tumor uptake 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 vivo. 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. The experimental results show that the probe 68 Ga-ZC-1 has the effect of in vivo tumor imaging.

[0197] Figure 5 The data in the figure show that after the FAPI-04 blocker was injected in advance, the 68 Ga-ZC-1, tumor part 68 The uptake of Ga-ZC-1 was significantly reduced, indicating 68 Ga-ZC-1 can specifically target tumors with high FAP expression, with a tumor / brain distribution ratio as high as 13. Figure 6 for 68 Ga-ZC-1 and 68The area under the curve (AUC) of Ga-FAPI-04 based on tumor uptake versus time was used to compare 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 had a longer tumor retention 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 1 group can be selected from: cyanine dyes (such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5), BODIPYs (such as fluoroboron dipyrrole, azafluoroboron dipyrrole), rhodamines (such as rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lissamine rhodamine, X-rhodamine, Texas Red, silicon-based rhodamine), quantum dots, nanoparticles, any one of phthalocyanines; 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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