A multimeric compound targeting and binding to fibroblast activation protein and its application

Abstract

The present invention relates to the technical field of radiopharmaceuticals, and in particular to a targeted binding fibroblast activation protein polymer compound and its application. The structure of the compound represented by formula (I) is: (I); The present invention obtains a compound with a longer retention time in tumors in vivo by modifying the structure of the FAP binding compound.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceuticals, and in particular to a targeted binding fibroblast activation protein polymer compound and application thereof. Background Art

[0002] Fibroblast activation protein (FAP) is a type II transmembrane serine protease that belongs to the prolyl oligopeptidase family. The full length of FAP consists of 760 amino acids, with the intracellular domain containing only 4 amino acids, the transmembrane domain containing 21 amino acids, and the extracellular domain containing 735 amino acids. Under physiological conditions, FAP is expressed at low levels in most adult tissues and is widely expressed on fibroblasts in the tumor microenvironment, affecting tumor growth through multiple mechanisms, including promoting proliferation, invasion, angiogenesis, epithelial-mesenchymal transition, stem cell promotion, immunosuppression, and drug resistance. FAP-expressing CAFs are present in many tumors, especially epithelial tumors, as well as malignant tumors with strong connective tissue proliferative reactions, such as breast cancer, colorectal cancer, pancreatic cancer, and lung cancer. It has also been reported that FAP is expressed in tumor cells of certain tumors (Scanlan, MJ, Proc. Natl. Acad. Sci. USA1994, 91, 5657–5661; Dohi, O Histopathology 2009, 55, 432–440). In general, high FAP expression is associated with tumor aggressiveness and poor prognosis (Cohen, SJ, Pancreas 2008, 37, 154–158; Coto-Llerena, M, Front. Oncol. 2020, 10, 979), while FAP expression in normal healthy adult tissues is negligible, making it a very attractive target for tumor imaging and treatment (Lindner, T, EJNMMI Radiopharm. Chem. 2019, 4, 16). Therefore, FAP is considered to be a powerful therapeutic target for cancer treatment. The corresponding inhibitor (FAPI) molecule has been developed globally recently for this target. It is a strategy for imaging and treating cell populations in the matrix around the tumor (cancer-associated fibroblasts). This type of FAPI therapeutic drug is divided into several categories, including small molecules, cyclic peptides and targeted proteins. The fastest-progressing treatment research and development category is small molecules with a proline structure represented by FAPI-04, and the other is cyclic peptide molecules represented by FAP-2286. Both types of molecules have made certain progress in the development of the therapeutic field.

[0003] Although existing compounds are designed with high enzyme activity, they have low tumor retention and rapid metabolism, which is not conducive to radiotherapy.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] In order to solve the above problems in the prior art, the present invention provides a compound, specifically a targeted fibroblast activation protein (FAP) multimerization inhibitor or binder, a FAP-targeting nuclide probe drug based on the FAP binder, and the use of the multimerization inhibitor and probe in the preparation of tumor imaging agents and tumor therapeutic agents targeting FAP.

[0006] In the first aspect, the present invention first provides a compound represented by formula (I) or a derivative thereof, wherein the structure of the compound represented by formula (I) is:

[0007] (I);

[0008] Where x represents any integer from 2 to 4;

[0009] R represents one or more -H, -CN, -F, -B (OH) 2 , alkyl, alkyl substituted with F, and alkylene;

[0010] L each independently, identically or differently represents a substituted or unsubstituted C 1 ~C 10 Alkylene, substituted or unsubstituted C 1 ~C 10 Cycloalkylene, substituted or unsubstituted C 1 ~C 10 Heteroalkylene, substituted or unsubstituted C 1 ~C 10 Heterocycloalkylene, substituted or unsubstituted C 1 ~C 10 Oxyalkylene, substituted or unsubstituted C 1 ~C 10 aminoalkylene, substituted or unsubstituted C 1 ~C 10 Alkenylene, substituted or unsubstituted C 1 ~C 10 Cycloalkenylene, substituted or unsubstituted C 1 ~C 10 heteroalkenylene, substituted or unsubstituted C 1 ~C 10 Heterocycloalkenylene, substituted or unsubstituted C 1 ~C 10 Alkyne, substituted or unsubstituted C 1 ~C 10 heteroalkynylene, substituted or unsubstituted C 1 ~C 30 arylene, substituted or unsubstituted C 1~C 30 a heteroarylene group, a dialkylene siloxane, an imine group, an imide group, an amide group, a thioamide group, a phosphoramide group, a thioether group, a disulfide group, an ester group, a thioester group, a carbamate group, a carbonate group, a phosphate group, a diacid group, an anhydride group, a nitroimidazole group, a hydrazone group, a carbohydrate, a dipeptide, a tripeptide or a tetrapeptide, or a group consisting of one or more of the above groups; wherein, when substituted, the substituent includes one or more of an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an ester group and an amine group;

[0011] D is a multi-linking group having any of the following structural formulas or any connected groups thereof: ; m is any integer between 1 and 10;

[0012] C is selected from any one of (a) to (c): (a) a chelator group suitable for radiolabeling; (b) a radioactive group containing a radioisotope; and (c) a chelate of a radioisotope and a chelator;

[0013] The derivatives include pharmaceutically acceptable tautomers, racemates, hydrates, solvates or salts.

[0014] In the present invention, x may be 2, 3 or 4; m may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0015] In the present invention, the structural formula of the imide group is -CO-NH-, the structural formula of the thioamide group is -CS-NH-, the structural formula of the phosphoramide group is -PO-NH2-, the structural formula of the thioester group is -S-CO-, and the structural formula of the carbamate group is -NHCOO-; the phosphate group can be one or more of a monophosphate (hydrocarbon phosphate), a secondary phosphate (phosphodiester) and a tertiary phosphate (phosphotriester); the sugar can be one of the monosaccharide groups of a pentose or a hexose, the pentose is selected from ribose, and the hexose is selected from glucose, mannose or galactose.

[0016] In the present invention, the heteroatoms in the heteroalkylene group, heterocycloalkylene group, heteroalkenylene group, heterocycloalkenylene group, heteroalkynylene group and heteroarylene group include at least one of oxygen, sulfur and nitrogen.

[0017] As a preference, Selected from Any of .

[0018] More preferably, for .

[0019] In the present invention, L is an intermediate linking fragment between the active fragment and the metal complex, which comprises one or more linker groups, and each linker group is independently selected from any of the following groups.

[0020] Preferably, L each independently, identically or differently represents a substituted or unsubstituted C 1 ~C 5 Alkylene, substituted or unsubstituted C 1 ~C 5 Heteroalkylene, substituted or unsubstituted C 1 ~C 5 Heterocycloalkylene, substituted or unsubstituted C 1 ~C 5 Heteroarylene, substituted or unsubstituted C 1 ~C 5 The invention further comprises an arylene group, an alkoxy group, a thioether group, a disulfide group, an anhydride group, a carbonate group, a carbamate group, a sugar, a peptide, a polyethylene glycol, an amide group or an ester group, or a group of one or more of the above groups; wherein, when substituted, the substituent includes one or more of an alkyl group, an alkenyl group, an alkynyl group, an aryl group, a heteroaryl group, an ester group and an amine group.

[0021] In the present invention, when substituted, L each independently, the same or different, may be C substituted by an alkyl group. 1 ~C 30 Arylene (i.e., aralkylene), C 1 ~C 30 heteroarylene (i.e., heteroarylalkylene), C 1 ~C 30 arylene (i.e. arylene alkenyl), C substituted by alkenyl 1 ~C 30 heteroarylene (i.e., heteroarylene), thiazolidine, methylene alkoxycarbamate, etc.

[0022] More preferably, L each independently, identically or differently represents any of the following groups:

[0023] ;

[0024] Wherein, n is any integer from 1 to 10;

[0025] Among them, R 1 Selected from -H, -C 1~6 Alkyl, -OC 1~6 Alkyl, -SC 1~6 Alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or -C 1~6 Arylalkyl; the -C 1~6Each of the alkyl groups is optionally substituted by 1 to 3 substituents selected from -OH, oxygen, and halogen; n is any integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0026] Preferably, L is connected to position 2, 3, 5, 6, 7 or 8 of the quinoline group of the active fragment; preferably, L is connected to position 6 or 8 of the quinoline group of the active fragment, as shown below: .

[0027] Preferably, C represents an optical dye, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an antifibrotic agent or an anticancer agent.

[0028] Preferably, C is a ligand moiety, and C is a chelating agent that forms a complex with a divalent or trivalent metal cation; preferably, the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid, ethylenediaminetetraacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, triethylenetetramine, iminodiacetic acid, diethylenetriamine-N,N,N',N',N-pentaacetic acid, bis-(carboxymethylimidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid;

[0029] Alternatively, C is a ligand moiety, wherein the ligand moiety is a non-radioactive isotope, a radioactive isotope, a radiopharmaceutical or a combination thereof; preferably, the radioactive isotope is selected from an isotope emitting α rays, an isotope emitting β rays, an isotope emitting γ rays, an isotope emitting Auger electrons, an isotope emitting X rays; more preferably, the isotope comprises 18 F. 18 F-Al, 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y. 90 Y. 149 Pm, 165 Dy, 169 2. 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72Se, 97 Such as 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 8. 123 I. 124 I. 131 I. 149 Tb, 152 Tb, 155 Tb, 161 Tb, 197 Hg, 211 At 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au, 225 Ac, 227 Th and 199 One or more of Ag;

[0030] Alternatively, C is a ligand moiety selected from the group consisting of fluorescent dyes such as xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, boron dipyrromethenes, and phthalocyanines, or conjugates or combinations of dyes of the above types;

[0031] Alternatively, C is a contrast agent comprising or consisting of a paramagnetic agent; preferably, the paramagnetic agent comprises or consists of paramagnetic nanoparticles.

[0032] Preferably, the compound represented by formula (I) has any of the following structural formulas: .

[0033] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound represented by formula (I) or a derivative thereof.

[0034] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0035] In a third aspect, the present invention provides a kit comprising the compound represented by formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt or the pharmaceutical composition; and instructions for diagnosing or treating a disease.

[0036] In a fourth aspect, the present invention provides use of the compound represented by formula (I) or its derivatives, or the pharmaceutical composition, or the kit in the preparation of drugs for diagnosing and / or treating diseases.

[0037] Preferably, the disease includes a disease characterized by overexpression of fibroblast activation protein in an animal or human subject; further preferably, the disease is selected from one or more of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scar disease; more preferably, the cancer is selected from small intestinal cancer, head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, nasopharyngeal carcinoma, myeloma cells, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, primary unknown cancer, thymic carcinoma, glioma, glioma, astrocytoma, cervical cancer, eye cancer, rectal cancer , colon cancer, cervical cancer, prostate cancer, breast cancer, bladder cancer, oral cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, uterine cancer, ovarian cancer, testicular cancer, kidney cancer, brain cancer, central nervous system cancer, pharyngeal cancer, skin melanoma, acute lymphocytic leukemia, acute myeloid leukemia, Ewing's sarcoma, Kaposi's sarcoma, basal cell carcinoma, squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms tumor, neuroblastoma, esophageal cancer, laryngeal cancer, lymphoma, neurofibroma, tuberous sclerosis, hemangioma and lymphoma.

[0038] In a fifth aspect, the present invention provides a targeted fibroblast activation protein (FAP) inhibitor or binder, which includes the compound represented by formula (I) or its derivative, or the pharmaceutical composition.

[0039] In a sixth aspect, the present invention provides a tumor therapeutic agent, which contains the compound represented by formula (I) or its derivative or the pharmaceutical composition.

[0040] In a seventh aspect, the present invention provides a tumor imaging agent, which contains the compound represented by formula (I) or its derivative or the pharmaceutical composition.

[0041] In an eighth aspect, the present invention provides a tumor diagnostic agent, which contains the compound represented by formula (I) or its derivative or the pharmaceutical composition.

[0042] The formula (I) described in the present invention introduces an intermediate linking fragment L at a specific position in the structure, and combines multiple fragments connecting the active center with the center D to form a multimeric compound; during the research process of the present invention, due to the particularity of the active center structure, the selection of the intermediate linking fragment L and the binding center D is particularly important, which has different degrees of influence on the synthesis process, the stability of the product molecule, and the metabolism of the compound in vivo. Therefore, the present invention uses the embodiment compound molecule as a representative to demonstrate the process of this multimeric compound synthesis strategy. Compared with the active center molecule of the prior application 202410873169.1, the multimeric compound of the present invention can effectively increase the retention amount and retention time of the compound at the tumor site, slow down the clearance rate, and is more conducive to the application of tumor radiotherapy. In short, the present invention obtains a compound with a longer retention time in the tumor in vivo by modifying the structure of the FAP-bound multimeric compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 For JYT2-4013 in Example 1 of this application 1 HnmR.

[0045] Figure 2 This is the HPLC result of JYT2-4013 in Example 1 of the present application.

[0046] Figure 3 This is the MS result of JYT2-4013 in Example 1 of the present application.

[0047] Figure 4 For JYT2-4015 in Example 2 of this application 1 HnmR results.

[0048] Figure 5 This is the HPLC result of JYT2-4015 in Example 2 of the present application.

[0049] Figure 6 This is the MS result of JYT2-4015 in Example 2 of this application.

[0050] Figure 7These are the in vitro activity test results of JYT2-4013 in Example 1 and JYT2-4015 in Example 2 of the present application, wherein (a) is the test result of JYT2-4015, and (b) is the test result of JYT2-4013.

[0051] Figure 8 The JYT2-4013 labeled in Example 1 of the present application 177 Lu-JYT2-4013 radio-HPLC quality control results.

[0052] Fig. 9 The JYT2-4013 labeled in Example 1 of the present application 177 Lu-JYT2-4013 UV analysis HPLC quality control results.

[0053] Fig.10 This is the biodistribution diagram of JYT2-4013 in Example 1 of this application.

[0054] Fig.11 The JYT2-401 labeled in Comparative Example 1 of this application 177 Lu-JYT2-401 SPECT imaging.

[0055] Fig.12 The JYT2-4013 labeled in Example 1 of the present application 177 Lu-JYT2-4013 SPECT imaging. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] Unless otherwise specified, the various raw materials used in the examples and comparative examples are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0058] Example 1 Preparation of small molecule inhibitors

[0059] The synthetic route of JYT2-4013 is as follows:

[0060]

[0061] Step 1: Synthesis of 4-methyl-8-nitroquinoline

[0062]

[0063] At 0℃ and nitrogen, 4-methylquinoline (12.5g, 87.3mmol) was slowly added to sulfuric acid (38mL) in batches. After the addition, a mixture of sulfuric acid (3.3mL) and nitric acid (9.3mL) was slowly added dropwise. The mixture was kept warm for 2 hours and then heated to room temperature for 16 hours. The reaction solution was poured into ice water to quench, and ammonia water was slowly added at 0℃ until solids precipitated. The solids were filtered, washed with water, and the filter cake was dried. The crude product was dispersed in boiling ethanol and stirred for 30 minutes. It was filtered while hot and the filter cake was washed with ethanol. Drying gave a yellow solid 019C-084-1 (6.51g, 34.5mmol, 40% yield).

[0064] Step 2: Synthesis of 8-nitroquinoline-4-carboxylic acid

[0065]

[0066] 019C-084-1 (6.00 g, 27.5 mmol) and selenium dioxide (6.10 g, 55.0 mmol, 2.0 equivalents) were dispersed in pyridine (49.01 g, 619.5 mmol, 22.5 equivalents) and reacted at 80 °C for 16 hours. The reaction solution was concentrated, ethanol was added and refluxed for 30 minutes, and filtered while hot. The pH of the crude product was adjusted by KHSO 4 / H 2 SO 4 The buffer solution was adjusted to 5-6, filtered, the filter cake was washed with ethanol, and dried to obtain a yellow solid 019C-084-2 (5.51 g, 25.2 mmol, 92% yield). MS (ES+) m / z 219.1 [M+H] + .

[0067] Step 3: Synthesis of 1-(8-nitroquinolin-4-yl)-3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanebenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)urea

[0068]

[0069] 019C-084-2 (0.81g, 3.7mmol), 019C-062-5 (1.14g, 3.7mmol, 1.0 equivalent), triethylamine (1.13g, 11.1mmol, 3.0 equivalent) and diphenylphosphoryl azide (1.53g, 5.6mmol, 1.5 equivalent) were dissolved in toluene (15mL) and reacted at 110°C for 2 hours under nitrogen protection. The reaction solution was poured into ice water for quenching, extracted with ethyl acetate (100mL), washed with brine (100mL), dried, filtered and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to obtain an orange solid 019C-084-3 (1.32g, 2.5mmol, 67% yield). MS (ES+) m / z 522.2 [M+H] + .

[0070] Step 4: Synthesis of 1-(8-aminoquinolin-4-yl)-3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanebenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)urea

[0071]

[0072] Disperse 019C-084-3 (1.30 g, 2.5 mmol), iron powder (2.78 g, 49.8 mmol, 20 equivalents), ammonium chloride (2.66 g, 49.8 mmol, 20 equivalents) and water (18 mL) in ethanol (35 mL) and react for 1 hour at 40 ° C under nitrogen protection. The reaction solution was cooled to room temperature, extracted with ethyl acetate (100 mL), washed with brine (100 mL), dried, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 25 / 1) to obtain an orange solid 019C-084-4 (613 mg, 1.3 mmol, 50% yield).

[0073] Step 5: Synthesis of 4-oxo-4-((4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanebenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-8-yl)amino)butyric acid

[0074]

[0075] 019C-084-4 (613 mg, 1.3 mmol), succinic anhydride (1.25 g, 12.5 mmol, 10 equivalents) and 4-dimethylaminopyridine (76 mg, 0.63 mmol, 0.5 equivalents) were dissolved in tetrahydrofuran (15 mL) and reacted at 50°C for 3 hours under nitrogen protection. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate and dried to obtain a white solid 019C-084-5 (530 mg, 0.89 mmol, 72% yield).

[0076] Step 6: Synthesis of 2-oxo-2-(3,3,7,7-tetrafluoro-4-hydroxy-1-azaspiro[4.4]nonan-1-yl)acetic acid

[0077]

[0078] 019C-084-5 (130 mg, 0.22 mmol, 2.2 equivalents), 019C-084-10 (86 mg, 0.099 mmol), 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (69 mg, 0.25 mmol, 2.5 equivalents) and N,N-diisopropylethylamine (65 mg, 0.50 mmol, 5.0 equivalents) were dispersed in N,N-dimethylformamide (6 mL) and reacted at room temperature under nitrogen protection for 2 hours. The reaction solution was added with water (20 mL) to precipitate solids, which were filtered, and the filter cake was washed with water and dried to obtain a light brown solid 019C-084-6 (80 mg, 0.04 mmol, 40% yield). MS (ES+) m / z 1009.6 [M / 2+H] + .

[0079] Step 7: Synthesis of tri-tert-butyl 2,2',2"-(10-(14,14-dimethyl-3,8,12-trioxo-1-phenyl-2,13-dioxa-4,7-diazapentadecan-11-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate

[0080]

[0081] 5-(tert-Butoxy)-5-oxo-4-(4,7,10-tri(2-(tert-Butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanoic acid (400 mg, 0.57 mmol) and benzyl N-(2-aminoethyl)carbamate (130 mg, 0.68 mmol, 1.2 equivalents) were dissolved in DMF (6 mL), and HATU (650 mg, 1.71 mmol, 3.0 equivalents) and DIPEA (220 mg, 1.71 mmol, 3.0 equivalents) were added. After the addition, the reaction solution was stirred at room temperature for 2 hours. The reaction solution was quenched with water (50 mL), extracted with ethyl acetate (50 mL x 2), and the organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 15 / 1) to give a light yellow solid 019C-084-7 (480 mg, 0.55 mmol, 96% yield).

[0082] Step 8: Synthesis of tri-tert-butyl 2,2',2"-(10-(5-((2-aminoethyl)amino)-1-(tert-butoxy)-1,5-dioxolan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate:

[0083]

[0084] Compound 019C-084-7 (773 mg, 0.88 mmol) and palladium carbon (94 mg, 0.88 mmol, 1.0 equivalent) were dispersed in methanol (15 mL) and reacted at room temperature under hydrogen protection for 16 hours. The reaction solution was filtered through diatomaceous earth, washed with methanol, and concentrated to obtain a white solid 019C-084-8 (579 mg, 0.78 mmol, 89% yield).

[0085] Step 9: Synthesis of tri-tert-butyl 2,2',2"-(10-(9-(((benzyloxy)carbonyl)amino)-21,21-dimethyl-3,10,15,19-tetrahydro-1-phenyl-2,20-dioxa-4,11,14-triazadodecane-18-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate:

[0086]

[0087] 2,6-di{[(benzyloxy)carbonyl]amino}hexanoic acid (139 mg, 0.34 mmol), compound 019C-084-8 (250 mg, 0.34 mmol, 1.0 equivalent), triethylamine (130 mg, 1.02 mmol, 3.0 equivalent), EDCI (130 mg, 0.68 mmol, 2.0 equivalent) and HOBt (46 mg, 0.34 mmol, 1.0 equivalent) were dispersed in DMF (6 mL) and reacted for 16 hours at room temperature under nitrogen protection. The reaction solution was quenched with water (20 mL), extracted with ethyl acetate (50 mL x2), washed with saturated brine (30 mL x2), dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography to obtain a light yellow solid 019C-084-9 (380 mg, 0.33 mmol, 97% yield).

[0088] Step 10: Synthesis of tri-tert-butyl 2,2',2"-(10-(1-(tert-butoxy)-5-((2-(2,6-diaminohexaneamido)ethyl)amino)-1,5-dioxolane-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate

[0089]

[0090] 019C-084-9 (380 mg, 0.33 mmol) and palladium carbon (74 mg, 0.70 mmol, 2.1 equivalents) were dispersed in methanol (12 mL) and reacted at room temperature under hydrogen protection for 16 hours. The reaction solution was filtered through diatomaceous earth, washed with methanol, and the filtrate was concentrated to obtain a white solid 019C-084-10 (227 mg, 0.26 mmol, 79% yield).

[0091] Step 11: Synthesis of 2,2',2"-(10-(4-((2-(2,6-bis(4-oxo-4-((4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-8-yl)amino)butyramido)hexamethyleneamido)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0092]

[0093] Compound 019C-084-6 (30 mg, 0.015 mmol) was dissolved in formic acid (6 mL) and reacted at 50° C. for 16 hours. The reaction solution was directly concentrated to obtain colorless oil 019C-084-11 (80 mg, crude product), which was directly used for the next step.

[0094] Step 12: Synthesis of 2,2',2"-(10-(4-((2-(2,6-bis(4-((4-(3-(2-((R)-2-boropyrrolidin-1-yl)-2-oxoethyl)ureido)quinolin-8-yl)amino)-4-oxobutylamine)hexaneaminepoly)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0095]

[0096] 019C-084-11 (80 mg, crude), phenylboronic acid (14 mg, 0.11 mmol, 7.3 equivalents), methanol (3 mL), n-hexane (3 mL) and 1 drop of dilute hydrochloric acid (1 N) were dissolved in dichloromethane (1 mL) and reacted for 5 hours at room temperature under nitrogen protection. The reaction solution was concentrated, washed with n-hexane (5 mL x2), filtered, and the filter cake was dried. The crude product was purified by RP-HPLC (Hanbang Technology NP7000 series system equipped with YMC-ActusTriart 5μm Polar-RP 20 x 250mm C18 column, using a gradient of 100:0 to 75:25 water / acetonitrile + 0.1% TFA, within 30 minutes), and the fractions were collected and lyophilized to obtain a white solid JYT2-4013 (16 mg, 0.01 mmol, 70% two-step yield).

[0097] MS (ES+) m / z 509.3 [M / 3+H] + ; MS (ES-) m / z 743.4 [(M-2H 2 O) / 2-H] -

[0098] 1 HnmR (400mHz, DMSO-d6 + D 2O) δ 8.69 (d, J = 5.2hz, 2H), 8.51 (d, J =6.8hz, 2H), 8.37 (d, J = 5.2hz, 2H), 7.99 (d, J = 8.4hz, 2H), 7.58 (t, J =8.0hz, 2H), 4.12-3.78 (m, 12H), 3.28-2.87 (m, 22H), 2.85-2.62 (m, 6H), 2.41-2.30 (m, 4H), 2.19-1.71 (m, 12H), 1.70-1.26 (m, 10H).

[0099] JYT2-4013 1 HnmR Figure 1 .

[0100] The HPLC results of JYT2-4013 are shown in Tables 1 and Figure 2 .

[0101] The liquid phase detection conditions include: chromatographic column: YMC-Triart C18, 3um 4.6×150mm; flow rate 1ml / min; column temperature: 30℃; mobile phase: D: 0.1%hClO 4 H 2 O solution pH = 1.5, C: ACN.

[0102] Table 1

[0103]

[0104] The MS results of JYT2-4013 are shown in Figure 3 .

[0105] Example 2 Preparation of small molecule inhibitors

[0106] The synthetic route of JYT2-4015 is as follows:

[0107]

[0108] Step 1: Synthesis of dimethyl 5-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)isophthalate

[0109]

[0110] Dissolve tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (2.00 g, 3.49 mmol) and dimethyl 5-aminoisophthalate (0.80 g, 3.84 mmol, 1.1 equivalent) in DMF (20 mL), add HATU (1.59 g, 4.19 mmol, 1.2 equivalent) and DIPEA (1.35 g, 10.45 mmol, 3.0 equivalent). React at room temperature for 4 hours. TLC detection shows that the reaction of the raw material is complete. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL x 2). The organic layers were combined and washed with saturated sodium chloride solution (50 mL). The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography (DCM / EA = 20 / 1) to obtain a yellow solid JYT2-4015-1 (1.48 g, 1.94 mmol, yield 55.5%).

[0111] Step 2: Synthesis of 5-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetamido)isophthalic acid

[0112]

[0113] JYT2-4015-1 (1.48 g, 1.94 mmol) was dissolved in tetrahydrofuran (20 mL) and water (10 mL), and lithium hydroxide monohydrate (0.32 g, 7.76 mmol, 4.0 equivalents) was added. The reaction was allowed to proceed at room temperature for 1 hour. TLC indicated that the reaction of the raw material was complete. The tetrahydrofuran was removed by concentration, and the residue was diluted with water (50 mL), and potassium hydrogen sulfate solution was added to acidify to pH = 5. Ethyl acetate (100 mL x2) was added for extraction, and the organic phase was dried and concentrated to obtain a yellow foaming solid JYT2-4015-2 (900 mg, 1.22 mmol, yield 63.0%). MS (ES+) m / z 734.3 [MH] – .

[0114] Step 3: Synthesis of methyl 6-(2-((tert-butyloxycarbonyl)amino)ethoxy)quinoline-4-carboxylate

[0115]

[0116] 6-Hydroxyquinoline-4-carboxylic acid methyl ester (3.99g, 19.64mmol) was dissolved in N,N-dimethylformamide (40mL), and N-Boc-2-bromoethylamine (6.22g, 27.76mmol, 1.4 equivalents) and potassium carbonate (8.15g, 58.97mmol, 3.0 equivalents) were added in sequence at room temperature. After the addition, the reaction solution was heated to 80°C for 16 hours. TLC detected that the reaction was complete, and the reaction solution was added with water (320mL), extracted with ethyl acetate (80mL x2), and the organic phases were combined, washed with saturated sodium chloride solution (160mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10 - 1 / 4) to obtain a white solid JYT2-4015-3 (6.46g, 18.65mmol, yield 94.9%). MS(ES+)m / z 347.1 [M+H] + .

[0117] Step 4: Synthesis of 6-(2-((tert-butyloxycarbonyl)amino)ethoxy)quinoline-4-carboxylic acid

[0118]

[0119] JYT2-4015-3 (1.00 g, 2.89 mmol) was dissolved in methanol (3 mL), and water (3 mL) and solid sodium hydroxide (0.46 g, 11.56 mmol, 4.0 equivalents) were added at room temperature. After the addition, the reaction solution was heated to 50 ° C for 2 hours. TLC showed that the reaction was complete. The reaction solution was distilled under reduced pressure to remove methanol, and hydrochloric acid (1 M) was added dropwise to pH = 3. After stirring for 10 minutes, it was filtered, and the filter cake was rinsed with hydrochloric acid (1 M, 1 mL) and dried to obtain a white solid JYT2-4015-4 (708 mg, 2.13 mmol, yield 73.7%). MS (ES+) m / z 333.1 [M+H] + .

[0120] Step 5: Synthesis of tert-butyl (2-((4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aS)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxaborolan-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-6-yl)oxy)ethyl)carbamate

[0121]

[0122] JYT2-4015-4 (508 mg, 1.53 mmol) and 2-amino-1-((R)-2-((3aS, 4S, 6S,7aS)-3a,5,5-trimethylhexahydro-4,6-methanebenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethan-1-one (468 mg, 1.53 mmol, 1.0 equivalent) were dissolved in toluene (10 mL). Triethylamine (386 mg, 3.82 mmol, 2.5 equivalent) and diphenylphosphoryl azide (463 mg, 1.68 mmol, 1.1 equivalent) were added successively at room temperature. Under nitrogen protection, the reaction solution was heated to 110°C for 3 hours. The reaction was complete by TLC detection. The reaction solution was cooled to room temperature, quenched by adding water (50 mL), extracted with ethyl acetate (50 mL x 2), the organic phases were combined, washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (methanol / dichloromethane = 1 / 40 - 1 / 20) to obtain a light brown solid JYT2-4015-5 (545 mg, 0.86 mmol, yield 56.0%).

[0123] Step 6: Synthesis of 1-(6-(2-aminoethoxy)quinolin-4-yl)-3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aS)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxaborolan-2-yl)pyrrolidin-1-yl)ethyl)urea

[0124]

[0125] JYT2-4015-5 (759 mg, 1.19 mmol) was dissolved in hydrochloric acid-dioxane solution (7.5 mL) and reacted at room temperature for 1 hour. TLC detected that the reaction was complete, and the reaction solution was added with saturated sodium bicarbonate solution to adjust the pH to 9, extracted with dichloromethane (30 mL x 3), combined organic phases, washed with saturated sodium chloride solution (90 mL), dried over anhydrous sodium sulfate, and concentrated to obtain white solid JYT2-4015-6 (597 mg, 1.11 mmol, yield 93.7%). MS (ES+) m / z 536.3 [M+H] + .

[0126] Step 7: Synthesis of 2-(tert-butoxycarbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid

[0127]

[0128] Dissolve 2-tert-butyl-6-methyl-dihydroisoquinoline-2,6(1H)-dimethyl ester (3.79 g, 13.00 mmol) in methanol (10 mL) and water (10 mL), and add sodium hydroxide (2.6 g, 65.0 mmol, 5.0 equivalents). The reaction solution was heated to 50°C for 2 hours. TLC detected that the raw material reacted completely. The resulting mixture was cooled to 0-5°C, adjusted to pH 3 with 1N dilute hydrochloric acid, and extracted with ethyl acetate (100 mL x2). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a brown oily liquid JYT2-4015-7 (3.50 g, 12.6 mmol, yield 97.2%).

[0129] Step 8: Synthesis of 6-benzyl-2-(tert-butyl) 3,4-dihydroisoquinoline-2,6(1H)-dicarboxylate

[0130]

[0131] JYT2-4015-7 (4.50 g, 16.23 mmol) was dissolved in DMF (50 mL), and cesium carbonate (7.91 g, 24.35 mmol, 1.5 equivalents) and benzyl bromide (4.16 g, 24.35 mmol, 1.5 equivalents) were added. The reaction was allowed to react at room temperature for 1 hour. TLC showed that the reaction of the raw material was complete. The reaction solution was quenched with water (300 mL), extracted with ethyl acetate (200 mL x 2), and the organic phase was concentrated. The crude product was purified by silica gel column chromatography (PE / EA = 2 / 1) to obtain a white solid JYT2-4015-8 (4.45 g, 12.11 mmol, yield 74.6%).

[0132] Step 9: Synthesis of 1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid benzyl ester hydrochloride

[0133]

[0134] Dissolve JYT2-4015-8 (4.45 g, 12.11 mmol) in dichloromethane (50 mL), add hydrochloric acid-dioxane (10 mL), and react at room temperature for 2 hours. TLC detection shows that the raw material reacts completely. The reaction solution is directly concentrated to obtain a white solid JYT2-4015-9 (3.51 g, 11.55 mmol, yield 95.4%).

[0135] Step 10: Synthesis of benzyl 2-((tert-butyloxycarbonyl)glycylamino)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid

[0136]

[0137] Boc-glycine (1.15 g, 6.58 mmol, 1.0 equivalent) was dissolved in dichloromethane (30 mL), and HATU (3.00 g, 7.90 mmol, 1.2 equivalent) and DIPEA (2.55 g, 19.74 mmol, 3.0 equivalent) were added. After reacting at room temperature for 30 minutes, JYT2-4015-9 (2.00 g, 6.58 mmol) was added, and the reaction was continued at room temperature for 2 hours. TLC detected that the raw material reaction was complete. The reaction solution was quenched with water (100 mL), extracted with dichloromethane (100 mL x2), and the organic phase was concentrated. The crude product was purified by silica gel column chromatography (PE / EA = 2 / 1) to obtain a white solid JYT2-4015-10 (2.61 g, 6.13 mmol, yield: 93.2%). MS(ES+)m / z 369.1 [M-56+H] + .

[0138] Step 11: Synthesis of 2-glycyl-1,2,4-tetrahydroisoquinoline-6-carboxylic acid benzyl ester

[0139]

[0140] Dissolve JYT2-4015-10 (1.00 g, 2.36 mmol) in dichloromethane (10 mL), add hydrochloric acid-dioxane (2 mL), react at room temperature for 2 hours, and TLC shows that the reaction of the raw material is complete. Concentrate, dilute the residue with water (20 mL), add saturated sodium bicarbonate solution to adjust the pH to 9, add dichloromethane (100 mL x 2) for extraction, and concentrate the organic phase to obtain a white solid JYT2-4015-11 (762 mg, 2.35 mmol, yield: 99.5%).

[0141] Step 12: Synthesis of 2,2',2'-(10-(2-(3,5-bis((2-(6-(benzyloxy)carbonyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)carbamoyl)phenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate

[0142]

[0143] Dissolve JYT2-4015-2 (786 mg, 1.06 mmol) in dichloromethane (10 mL), add chlorotripyrrolidino hexafluorophosphate (Pyclop) (983 mg, 2.33 mmol, 2.2 equivalents) and N,N-diisopropylethylamine (DIPEA) (411 mg, 3.18 mmol, 3.0 equivalents), stir at room temperature for 30 minutes, then add compound JYT2-4015-11 (756 mg, 2.33 mmol, 2.2 equivalents), and continue to react at room temperature for 2 hours. TLC detection shows that the raw material reaction is complete. The reaction solution was quenched with water (50 mL), extracted with dichloromethane (50 mL x2), the organic layers were combined and washed with saturated sodium chloride solution (20 mL), the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by Pre-TLC (DCM / MeOH = 20 / 1) to obtain a white solid JYT2-4015-12 (243 mg, 0.18 mmol, yield 17.0%); MS (ES+) m / z 675.0 [M / 2+H] + .

[0144] Step 13: Synthesis of 2,2'-(2,2'-)((5-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1-yl)acetylamino)isophthaloyl)bis(nitrogendiyl)bis(acetyl))bis(1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid)

[0145]

[0146] Dissolve JYT2-4015-12 (165 mg, 0.12 mmol) in methanol (10 mL), add Pd / C (50 mg, 10%), and hydrogenate at room temperature for 2 hours. TLC shows that the reaction of the raw material is complete. Filter and concentrate the mother liquor to obtain a colorless solid JYT2-4015-13 (120 mg, 0.10 mmol, yield 85.6%).

[0147] Step 14: Synthesis of 2,2',2'-(10-(2-(3,5-bis(2-oxo-2-(6-(2-(4-(3-(2-oxy-2-(R)-2-((3aS,4S,6S,7aS)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxol-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-6-yl)oxy)ethylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)ethyl-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate

[0148]

[0149] JYT2-4015-13 (105 mg, 0.09 mmol) was dissolved in DMF (8 mL), and DMTMM (62 mg, 0.23 mmol, 2.5 equivalents), DIPEA (46.53 mg, 0.36 mmol, 4.0 equivalents) and JYT2-4015-6 (96 mg, 0.18 mmol, 2.0 equivalents) were added in sequence. The reaction was carried out at room temperature for 5 hours. TLC detected that the raw material reaction was complete. The reaction solution was quenched by adding water (10 mL), and solid precipitated and filtered. The filter cake was slurried by adding ethyl acetate (5 mL), filtered, and the filter cake was dried to obtain a white solid JYT2-4015-14 (63 mg, 0.03 mmol, yield 31.8%).

[0150] Step 15: Synthesis of 2,2',2'-(10-(2-((3,5-di(2-oxo-2-((R)-2-((3aS,4S,6S,7aS)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxol-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-6-yl)oxy)ethylcarbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)ethyl 7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0151]

[0152] Dissolve JYT2-4015-14 (63 mg, 0.03 mmol) in dichloromethane (5 mL) and add TFA (1 mL). React at room temperature for 15 hours. TLC detects that the raw material reacts completely. The reaction solution is concentrated to remove TFA, and water is added to precipitate solids. Filter, add ethyl acetate (5 mL) to the filter cake, beat, and filter. Dry the filter cake to obtain white solid JYT2-4015-15 (60 mg, crude product), which is directly carried out to the next step.

[0153] Step 16: Synthesis of 2,2',2'-(10-(2-(3,5-bis((2-(6-(2-(4-(3-(2-(((R)-2-boropyrrolidin-1-yl)-2-oxoethyl)ureido)quinolin-6-yl)oxy)ethyl)carbamoyl)-3,4-dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)aminoacetyl)phenyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0154]

[0155] JYT2-4015-15 (60 mg, crude product) was dissolved in methanol (3 mL) and dichloromethane (3 mL), and phenylboronic acid (9 mg, 0.073 mmol, 2.5 equivalents) and 3N dilute hydrochloric acid (1 d) were added. The reaction was allowed to react at room temperature for 3 hours. The reaction was complete when detected by LC-MS. After concentration, methanol (0.2 mL) and n-hexane (5 mL) were added to the crude product for slurrying. The crude product was filtered and dried. The crude product was purified by RP-HPLC (Hanbang Technology NP7000 series system equipped with a YMC-Actus Triart 5μm Polar-RP 20 x 250mm C18 column, using a gradient of 95:5 to 20:80 water / acetonitrile + 0.35% TFA, within 40 minutes), and the fractions were collected and lyophilized to obtain a white solid JYT2-4015 (12 mg, 0.007 mmol, 22.7% two-step yield).

[0156] MS(ES+)m / z 577.6 [(M-2H 2 O) / 3+H] + .

[0157] 1 HnmR (400mHz, CD 3 OD) δ 8.61-8.26 (m, 4H), 8.16-7.95 (m, 2H), 7.93-7.81 (m, 1H), 7.77-7.44 (m, 9H), 7.41-6.99 (m, 3H), 4.56-3.99 (m, 17H), 3.97-3.39 (m, 25H), 3.20-2.62 (m, 14H), 2.27-1.54 (m, 10H).

[0158] Figure 4 For JYT2-4015 1 HnmR results.

[0159] The hPLC results of JYT2-4015 are shown in Figure 5 and Table 2.

[0160] Table 2

[0161]

[0162] Figure 6 These are the MS results of JYT2-4015.

[0163] Comparative Example 1

[0164] Comparative Example 1 provides the structural formula of JYT2-401, see Table 3.

[0165] Table 3

[0166]

[0167] Experimental Example 1 Inhibitor affinity test

[0168] 1. Refer to the literature (Bioorg. Med. Chem. Lett. 2020, 30, 127253.). The specific operation is as follows: dilute the FAP protein concentration to 0.4μg / mL with assay buffer (25mM Tris, 250mM NaCl, pH 7.4); dilute the substrate GP-AMC to 40μM with assay buffer; prepare different concentrations of inhibitor (compound 1-20) solutions with assay buffer; add 25μL inhibitor and 25μL substrate to a 96-well black board in sequence, then add 50μL protein, place it in an ELISA reader, incubate at 37℃ for 1h, and measure its fluorescence intensity (Ex / Em=380 / 460nm); use Graphpad Prism fitting and sigmoidal 11dose response model to calculate IC50.

[0169] 2. Determination of the inhibitory activity of compounds on hFAP:

[0170] The hFAP protein was diluted to 0.3 μg / mL using assay buffer (25 mM Tris, 250 mM NaCl, pH = 7.4) and kept on ice. The compound was diluted to a certain concentration gradient with assay buffer, namely 40 μM, 4 μM, 400 nm, 40 nm, 4 nm, 400 pM, 40 pM and 4 pM. The enzyme substrate Z-Gly-Pro-AMC was diluted to 40 μM with assay buffer and stored in the dark. The above-prepared 50 μL protein solution and 25 μL inhibitor were added to a 96-well plate (Corning 96-well ELISA plate with black transparent bottom, No. 3904) in sequence, centrifuged and shaken at room temperature for 30 minutes. Then, 25 μL of the above-prepared enzyme substrate was added. After incubation at room temperature for 3 to 5 hours, the fluorescence values ​​with an excitation wavelength of 360 nm and an emission wavelength of 465 nm were monitored using a multi-function microplate reader (Agilent BioTek Synergyh1). IC was calculated. 50 The value is defined as the concentration of the compound that causes a 50% decrease in enzyme activity under the assay conditions. The results are shown in Tables 4 and Figure 7 .

[0171] Table 4

[0172]

[0173] Experimental Example 2 Labeling and Quality Control

[0174] 1. 177 Lu labeling and quality control

[0175] 1.1 Marking process

[0176] Accurately measure 1-4 μL 20 nmol of compound (JYT2-401, JYT2-4013) solution, accurately add 50 μL 0.5M acetic acid-sodium acetate solution (pH 5.0, 4 mg N-acetyl-L-tyrosine, 1 mg N-acetyl-L-methionine) into a 1.5 ml centrifuge tube, mix thoroughly, and add 2 μL 177 LuCl 3 The solution (activity: ~2mCi) was heated upright in a metal bath at 95℃ (JYT2-401) or 70℃ (JYT2-4013) under sealed conditions for 15 minutes, then taken out, cooled to room temperature, and 150μL of 0.1% DTPA PBS buffer solution was added to detect the radioactivity concentration of the solution, and a small amount was taken for radiochemical purity analysis. The radioactivity was tested before and after administration, and the test sample was stored at room temperature and in a lead can, and was prepared and used immediately.

[0177] 3.2 Quality Control Analysis

[0178] Radiochemical purity: HPLC: Waters Xbridege 5μm C-18 (4.6 × 150mm), flow rate 1mL / min; column temperature 35℃; detection wavelength 210nm; injection volume 20μL (50~100μCi). Gradient elution conditions are shown in Table 5.

[0179] Table 5

[0180]

[0181] Results Figure 8~Figure 9 and Table 6. The radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 95%, meeting the test requirements.

[0182] Table 6

[0183]

[0184] The radiochemical purity was calculated by area normalization method and the radiochemical purity was greater than 95%, meeting the test requirements.

[0185] 3.3 177 Lu-JYT2-401 biodistribution

[0186] Animal identification: Each cage of animals must be hung with a cage card indicating the animal group, animal number, etc.

[0187] Feed provided: Mouse growth maintenance feed complies with GB14924.4-2001.

[0188] Feeding method: free feeding.

[0189] Drinking water: The drinking water bottles are supplied with high-pressure sterilized tap water. The tap water is inspected by the Taiyuan Monitoring Station of the National Urban Water Supply Quality Monitoring Network and meets the requirements of the "Sanitary Standards for Drinking Water" (GB5749-2006). The drinking water bottles are replaced every 2 days and are cleaned and sterilized under high pressure before use.

[0190] Tumor inoculation: The animal model was constructed by Beijing Weitonglihua Animal Technology Co., Ltd. HEK293 tumor cells were subcutaneously inoculated on the right side of the back of each experimental animal. A total of 10 animals were used for later experiments.

[0191] The drug solution was drawn with a disposable syringe, and the activity (μCi) was detected by an activity meter before administration (full needle). The drug was injected into the tail vein, and the residual radioactivity count (μCi) was detected by an activity meter after administration (empty needle). 177 The dosage of Lu-JYT2-401 injection is shown in Table 7.

[0192] Table 7 177 Lu-JYT2-401 Injection Dosage Table

[0193]

[0194] 177 The dosage of Lu-JYT2-4013 injection is shown in Table 8.

[0195] Table 8 177 Lu-JYT2-4013 Injection Dosage Table

[0196]

[0197] Sampling time: 2h, 24h, 48h after administration.

[0198] Tissues and organs: The animals were lightly anesthetized, their eyeballs were removed and blood was drawn (blood samples were retained), and the heart, liver, spleen, lungs, kidneys, stomach (contents removed), large intestine (contents removed), small intestine (contents removed), femur (hind limbs), kneecap, muscle, pancreas, brain, bladder (no urine), tumor, skin, urine, feces (2-3 grains in the large intestine), administration site (tail) and the rest of the carcass were removed.

[0199] γ counter detection: biological samples are collected at various time points, gently squeezed and residual blood is absorbed with absorbent paper, weighed and recorded, and the samples are placed in EP tubes and tested with a γ counter. The samples are tested within 24 hours (if the radioactive count is too high, it can be measured after decay). The test results are expressed in CPM (CPM: radioactive counts per minute).

[0200] Data processing and analysis:

[0201] %ID / g=A tissue / [(A 0 -A residue )×M tissue ]×100%.

[0202] A 0 : Total radioactivity count in the syringe before administration (CPM).

[0203] A residue : Residual radioactivity count in the syringe after administration (CPM).

[0204] A tissue : Radioactivity counts in tissues and organs (CPM).

[0205] M tissue : Weight of tested tissue sample (g).

[0206] Physical attenuation correction: 177 Lu physical decay: activity dose at time T = 0.5^(T / 160.8).

[0207] Dosage (A 0 -A residue ) multiplied by the decay coefficient of the corresponding time is the corrected CPM value and %ID / g is calculated.

[0208] Note: Within the activity range of no more than 75μCi, the calibration curve of the activity meter detection (μCi) and the γ counter (CPM) is Y=135628X(R 2 =0.9991).

[0209] The results are shown in Table 9 and Fig.10 HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice were injected with a single tail vein injection 177Lu-JYT2-401 injection, with the extension of time, the radioactive uptake in the blood decreased significantly; within 2h to 48h after administration, there were different degrees of radioactive enrichment in tissues and organs, with the highest distribution in tumors, kidneys, and livers (%ID / g at each time point: tumor 34.70 ± 10.56, 18.37 ± 7.97 and 13.64 ± 1.85; kidney 4.16 ± 1.49, 2.26 ± 0.24 and 1.16 ± 0.36, liver 2.03 ± 0.76, 1.57 ± 0.10 and 1.02 ± 0.19); followed by skin, intestines, lungs, bladder, spleen, etc., with the lowest distribution in the brain. Single tail vein injection of Balb / c nude tumor-bearing mice inoculated with HEK293ThFAP human embryonic kidney cells transfected with human FAP high-expressing cell line 177 Lu-JTY2-4013 compound injection, biodistribution experiments were conducted at 2h, 24h, and 48h after administration. There was different degrees of radioactive enrichment in tissues and organs. Normal organs such as liver, knee bone, tibia, skin, thyroid gland, and salivary gland had a small amount of uptake (1-3%ID / g). The mouse tumor uptake reached the highest level of 35.89±4.23 ID% / g 2h after injection, and the retention time was long. The radioactive signal in the tumor remained at 33.15±5.43%ID / g 48h after injection. The tumor uptake and retention time were significantly better than 177 Lu-JYT2-401, therefore a series of molecules designed by the present invention have better advantages for treatment.

[0210] Table 9 177 Lu-JYT2-401 and 177 Results of tissue distribution of Lu-JYT2-4013 injection in HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice

[0211]

[0212] 3.4 SPECT / CT imaging

[0213] Towards 177 An appropriate amount of PBS buffer solution was added to the two Lu-labeled molecules (JYT2-401, JYT2-4013) to prepare the test sample (pH ~5.5-6). After mixing evenly, 100 μL was drawn up with an insulin syringe. Only ~400 μCi (1-2 nmol) of the molecule was injected into each mouse through the tail vein.

[0214] After the administration, 180 μL of 1% sodium pentobarbital solution was drawn from the syringe and intraperitoneally administered into the mouse to anesthetize the mouse. The anesthetized mouse was fixed to the SPECT / CT instrument bed and scanned by CT positioning. After the scan was completed, the data was reconstructed, and the ROI value of the mouse SPECT data was circled using software to obtain the radioactivity distribution %ID / g of the tumor organs and tissues.

[0215] The results are shown in Tables 10 to 11 and Fig.11 SPECT / CT imaging was performed at 2h, 8h, 24h, and 48h, and the radioactive signal (%ID / cc) in the mouse tumor was circled. The results showed 177 Lu-JYT2-401 remained in the tumor for a long time, and the maximum value of tumor uptake was 30.51±3.83%ID / cc at 2h, which was comparable to the biodistribution data in Section 3.3. After 48h, it gradually decreased to 16.72±1.79%ID / cc. 177 The average tumor uptake values ​​of Lu-JYT2-4013 in SPECT / CT imaging at 2h, 8h, 24h, 48h, 72h, and 144h were compared. The results are shown in Fig.12 , 177 The maximum value of the mean tumor uptake of Lu-JYT2-4013 was 33.02±1.67 %ID / cc at 8 h after injection, slightly higher than 177 The tumor uptake value of Lu-JYT2-401 at 2 h was maintained at 27.50±3.2%ID / cc as time extended to 48 h, which was significantly higher than 177 The 48-hour tumor uptake value of Lu-JYT2-401, and 177 The average tumor uptake of Lu-JYT2-4013 was still greater than ~20% ID / cc at 144h. This result indicates that 177 FAP polymers such as Lu-JYT2-4013 have the advantages of higher tumor uptake and longer retention time and are used for tumor treatment.

[0216] Table 10

[0217]

[0218] Table 11

[0219]

[0220] It should be noted that in the present invention, the effects of JYT2-4015 and JYT2-4013 are equivalent, which will not be further described due to space limitations.

[0221] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound, characterized in that The structure of the compound is: 。 2. A pharmaceutical composition, characterized in that Comprising the compound described in claim 1.

3. A kit comprising the compound according to claim 1 or the pharmaceutical composition according to claim 2; and instructions for diagnosing or treating a disease.

4. Use of the compound according to claim 1, the pharmaceutical composition according to claim 2, or the kit according to claim 3 in the preparation of a drug for diagnosing and / or treating a disease characterized by overexpression of fibroblast activation protein in an animal or human subject.

5. A targeted binding fibroblast activation protein inhibitor or binding agent, characterized in that: Comprising the compound according to claim 1, or the pharmaceutical composition according to claim 2.

6. An anti-tumor drug, characterized in that: It comprises a tumor therapeutic agent or a tumor diagnostic agent, wherein the tumor therapeutic agent or the tumor diagnostic agent contains the compound according to claim 1 or the pharmaceutical composition according to claim 2.

7. A tumor imaging agent, characterized in that: Containing the compound according to claim 1 or the pharmaceutical composition according to claim 2.

Images