Compound targeting and binding to fibroblast activation protein and use thereof

Abstract

A compound targeting and binding to a fibroblast activation protein and a use thereof. The compound comprises the structure of a compound represented by Formula (I): Formula (I). According to the compound targeting and binding to a fibroblast activation protein and the use thereof, the structural formula of the compound is optimized, so as to effectively increase the retention amount and retention time of the compound at a tumor site, slow down the clearance rate, and facilitates more effective use in tumor radiotherapy.

Description

Compound targeting fibroblast activation protein and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of compounds, in particular to a compound targeting fibroblast activation protein and application thereof. BACKGROUND

[0002] Fibroblast activation protein (FAP) is a type II transmembrane serine protease belonging to the prolyl oligopeptidase family. FAP is 760 amino acids in length, with a 4-amino acid intracellular domain, a 21-amino acid transmembrane domain, and a 735-amino acid extracellular domain. Under physiological conditions, FAP is expressed at low levels in most adult tissues, but is highly expressed on fibroblasts in the tumor microenvironment, affecting tumor growth through various mechanisms, including promoting proliferation, invasion, angiogenesis, epithelial-mesenchymal transition, stem cell promotion, immunosuppression, and drug resistance. CAFs expressing FAP are present in many tumors, especially epithelial tumors, and malignant tumors with strong connective tissue proliferation, such as breast cancer, colorectal cancer, pancreatic cancer, and lung cancer. There are also reports that FAP is expressed in some tumor cells (Scanlan, M.J, Proc. Natl. Acad. Sci. USA 1994, 91, 5657-5661; Dohi, O Histopathology 2009, 55, 432-440). Overall, high expression of FAP is associated with tumor invasiveness and poor prognosis (Cohen, S.J, Pancreas 2008, 37, 154-158; Coto-Llerena, M, Front. Oncol. 2020, 10, 979), and FAP expression in normal healthy adult tissues can be ignored, which makes it a very attractive target for tumor imaging and treatment (Lindner, T, EJNMMI Radiopharm. Chem. 2019, 4, 16). Therefore, FAP is considered a strong therapeutic target for cancer treatment, and corresponding inhibitors (FAPI) molecules have been developed globally in recent years. FAPI is a strategy for imaging and treatment of a cell population targeting the tumor stroma (cancer-associated fibroblasts), and this FAPI therapeutic drug is divided into small molecules, cyclic peptides, and targeted proteins, among others. One of the fastest developing classes of therapeutic research is represented by FAPI-04, a small molecule with a proline structure, and the other is represented by FAP-2286, a cyclic peptide molecule. Both of these molecules have made some progress in the development of the treatment field.

[0003] Although the existing compound design has high enzyme activity, the tumor retention is low and the metabolism is fast, which is not conducive to radiotherapy.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The present application provides a compound, in particular a fibroblast activation protein (FAP) binding inhibitor or binding agent, a FAP-targeted radionuclide probe drug based on the FAP binding agent, and the use of the inhibitor and the probe in the preparation of a FAP-targeted tumor imaging agent and a tumor therapeutic agent.

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

[0007] wherein x represents any integer from 1 to 4, and can be any of 1, 2, 3, or 4;

[0008] R1 represents one or more of -H, -CN, -F, -B(OH)2, alkyl, alkyl substituted with F, and alkylene;

[0009] R2, R3, R4 are independently selected from -H, -F, -Cl, -Br, -I, -C 1-6 alkyl, -O-C 1-6 alkyl, -S-C 1-6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, or -C 1-6 aralkyl, each of said -C 1-6 alkyl is optionally substituted with 1 to 3 substituents selected from -OH, oxo, halogen;

[0010] R 5 is bicyclo[1.1.1]pentane, 1-naphthyl moiety, or a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, which optionally further comprises 1, 2, or 3 heteroatoms selected from one or more of O, N, and S;

[0011] L each independently, identically or differently, represents a single bond, substituted or unsubstituted C1-C 10 alkylene, substituted or unsubstituted C1-C 10 cycloalkylene, substituted or unsubstituted C1-C 10 heteroalkylene, substituted or unsubstituted C1-C 10 heterocycloalkylene, substituted or unsubstituted C1-C 10 oxyalkylene, substituted or unsubstituted C1-C 10 aminoalkylene, substituted or unsubstituted C1-C 10 alkenylene, substituted or unsubstituted C1-C 10 cycloalkenylene, substituted or unsubstituted C1-C10 Heterene groups, substituted or unsubstituted C1-C 10 heterocyclic alkenyl, substituted or unsubstituted C1-C 10 alkyne group, substituted or unsubstituted C1-C 10 The heteroynyl group, substituted or unsubstituted C1-C 30 arylene, substituted or unsubstituted C1-C 30 The group comprises one or more of the following groups: heteroaryl, diallylsiloxane, carbonyl, imino, imide, amide, thioamide, phosphoramide, thioether, dithio, ester, thioester, urethane, carbonate, phosphate, diacid, anhydride, nitroimidazole, hydrazone, sugar, dipeptide, tripeptide, or tetrapeptide; wherein, when substituted, the substituents include one or more of alkyl, alkenyl, alkynyl, aryl, heteroaryl, ester, and amino groups.

[0012] D is a single bond or any of the following structures or any group of their connections: m is any integer from 0 to 10;

[0013] C includes any one of (a) to (c): (a) a chelating agent group suitable for radiolabeling; (b) a radioactive group containing a radioactive isotope; (c) a chelate of a radioactive isotope and a chelating agent.

[0014] The derivatives include pharmaceutically acceptable tautomers, racemates, hydrates, solvates, or salts.

[0015] According to the present invention, a compound of formula (I) or a derivative thereof has a structure of general formula (I-1):

[0016] In general formula (I-1), Selected from any one of them;

[0017] Choose from any of the following structural formulas:

[0018] Among them, R8, R9, R 10 It does not exist or independently consists of one or two heteroatoms selected from O, N, and S;

[0019] Preferably, Choose from any of the following structural formulas:

[0020] Preferably, selected from any of the following structural formulae:

[0021] According to the present application, a compound of formula (I) or a derivative thereof is provided, wherein in general formula (I-1), L is an intermediate linking moiety of the active moiety and the metal complex;

[0022] Preferably, L comprises one or more linker groups, each linker group being independently selected from (alkyl)ene, (hetero)alkyl, (hetero)cycloalkyl, heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, peptide, polyethylene glycol, amide, or ester;

[0023] Preferably, L comprises, independently or in any combination thereof, at least one linker group having the following structure:

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

[0025] More preferably, L comprises one or more linker groups selected from:

[0026] wherein R7is selected from -H, -C 1-6 alkyl, -O-C 1-6 alkyl, -S-C 1-6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, or -C 1-6 arylalkyl, said -C 1-6 alkyl, each of which is optionally substituted with 1 to 3 substituents selected from -OH, oxo, halogen; n is any integer from 1 to 10;

[0027] Preferably, C is a ligand moiety, which is a chelator forming a complex with a divalent or trivalent metal cation; preferably, the chelator 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-(carboxymethyl imidazole)glycine, or 6-hydrazinopyridine-3-carboxylic acid;

[0028] Alternatively, C is a ligand moiety, wherein the ligand moiety is a non-radioisotope, a radioisotope, a radiopharmaceutical, or a combination thereof; preferably, the radioisotope is selected from an isotope emitting alpha rays, an isotope emitting beta rays, an isotope emitting gamma rays, an isotope emitting Auger electrons, an isotope emitting X-rays, such as 18 F, 18 F-Al, 51 Cr, 67 Ga,68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 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 Ag;

[0029] Alternatively, C is a ligand moiety selected from the following classes of fluorescent dyes: xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, borondipyrromethenes and phthalocyanines and conjugates and combinations of these classes of dyes;

[0030] Alternatively, C is a contrast agent comprising or consisting of a paramagnetic agent; preferably, the paramagnetic agent comprises or consists of paramagnetic nanoparticles.

[0031] According to the present application, a compound of formula (I) or its derivative is provided, and the general formula (I-1) has any of the following structural formulae:

[0032] The present application further provides a preparation method of the compound of the general formula (I-1) as described above, and the reaction route is as follows:

[0033] (a) Substituted pyrrole derivative A and glycine derivative B undergo condensation reaction to generate amide C, which, under alkaline conditions, is condensed with another molecule of amine D through isocyanate intermediate, to prepare compound E.

[0034] (b) Terminal R5 carboxylic acid derivative F with a linker undergoes Curtius rearrangement reaction under azide (such as azide diphenyl phosphate DPPA) conditions, and is condensed with another molecule of amine C through isocyanate intermediate to generate compound G, which is condensed with carboxylic acid H to prepare compound I.

[0035] The compound of the general formula (I-1) as described in the present application can effectively increase the retention amount of the compound at the target position (for example, the tumor site) by introducing a urea group at a specific position in the structure, although the enzyme activity is slightly decreased. Specifically, the tumor uptake can be effectively improved, the retention time of the compound at the tumor site can be improved, and the compound can realize the application of diagnosis and treatment integration.

[0036] Further, the present application aims to enhance the stability of the drug in the tumor, and to replace the benzene ring with a bioisostere: the replacement of the bioisostere can improve the biological activity, physicochemical properties, PK properties and in vivo stability of the drug. In addition to the application of traditional pyridine, cyclohexane, piperidine and the like, the present application finds that certain polycyclic saturated skeletons have better effects.

[0037] The present application replaces the quinoline structure of JYT2-401 in the general formula (I-1) with bicyclo[1.1.1]pentane, optimizes the structure, carries out synthesis research and in vitro activity determination of single-chain and double-chain molecules, carries out comparison of drug metabolism, tumor uptake and the like in the body after labeling different nuclides, and protects the bicyclo[1.1.1]pentane molecules targeting FAP.

[0038] According to the present application, there is provided a compound of formula (I) or a derivative thereof, having the structure of formula (I-2):

[0039] wherein x represents any integer from 1 to 4 inclusive;

[0040] selected from any one of

[0041] and / or, each L independently, identically or differently, represents a substituted or unsubstituted C1-C5 alkylene, a substituted or unsubstituted C1-C5 heteroalkylene, a substituted or unsubstituted C1-C6 heterocycloalkylene, a substituted or unsubstituted C1-C5 heteroarylene, a substituted or unsubstituted C1-C5 arylene, a carbonyl, an alkoxy, a thioether, a disulfide, an anhydride, a carbonate, a carbamate, a sugar, a peptide, a polyethylene glycol, an amide or an ester, or a group of one or more of the foregoing; wherein when substituted, the substituents include one or more of an alkyl, an alkenyl, an alkynyl, an aryl, a heteroaryl, an ester and an amine;

[0042] C is a ligand moiety that is a chelator that forms a complex with a divalent or trivalent metal cation; the chelator 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-(carboxymethyl imidazole)glycine, or 6-hydrazinopyridine-3-carboxylic acid; preferably, the ligand moiety includes a non-radioactive isotope, a radioactive isotope, a radiopharmaceutical, or a combination thereof; the radioactive isotope is selected from an isotope that emits alpha radiation, an isotope that emits beta radiation, an isotope that emits gamma radiation, an isotope that emits Auger electrons, an isotope that emits X-rays; the isotope includes 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 Er, 177 Lu, 47 ​Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 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 Ag;

[0043] Alternatively, C is a ligand moiety selected from the following classes of fluorescent dyes: xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, borondipyrromethenes and phthalocyanines, or conjugates or combinations of the above classes of dyes.

[0044] Alternatively, C is a contrast agent comprising or consisting of a paramagnetic agent.

[0045] In the present application, x can be 2, 3 or 4; m can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In particular, m in the different structural formulae in D are each independently, the same or different.

[0046] In the present application, the structural formula of imide group is -CO-NH-, the structural formula of thioamide group is -CS-NH-, the structural formula of phosphoramide group is -PO-NH2-, the structural formula of thioester group is -S-CO-, the structural formula of carbamate group is -NHCOO-; the phosphate group can be one or more of monoester (hydrocarbyl phosphate), secondary phosphate (diester phosphate) and tertiary phosphate (triester phosphate); the sugar can be one of monosaccharide groups of five-carbon sugars selected from ribose or six-carbon sugars selected from glucose, mannose or galactose.

[0047] In the present application, the heteroatom in the heteroalkylene, heterocycloalkylene, heteroalkenylene, heterocycloalkenylene, heteroalkynylene and heteroaryl group includes at least one of oxygen, sulfur and nitrogen.

[0048] In the present application, L is an intermediate linking segment between the active fragment and the metal complex, which contains one or more linker groups, each linker group is independently selected from any of the following groups.

[0049] According to the present application, a compound represented by formula (I) or its derivative is provided, in the general formula (I-2), each L independently, identically or differently represents any of the following groups:

[0050] Wherein, n is any integer from 1 to 10; R8 is selected from -H, -C 1～6 alkyl, -O-C 1～6 alkyl, -S-C 1～6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or -C 1～6 aralkyl; each of the -C 1～6 alkyl is optionally substituted with 1 to 3 substituents selected from -OH, oxygen, halogen; n is any integer from 1 to 10.

[0051] As a preferred embodiment of the present application, L is n is any integer from 1 to 5, such as 1, 2, 3, 4 or 5.

[0052] In the present application, when there are two or more n in the group of L, different n are each independent and can be identical or different; for example The values of the two n in the above formula can be identical or different.

[0053] According to the present application, a compound represented by formula (I) or its derivative is provided, the compound represented by the general formula (I-2) has any of the following structural formulae:

[0054] The compound shown in the structure of general formula (I-2) in the application can ensure that the activity of the new FAPI molecule is not only improved to a certain extent, but also the uptake amount of the new bicyclo[1.1.1]pentane FAPI molecule in the tumor does not decrease obviously in a long time (>48h) after the bicyclo[1.1.1]pentane replaces the quinoline structure, which indicates that the bicyclo[1.1.1]pentane replacing the quinoline structure can effectively improve the stability of the FAPI molecule in the body.

[0055] According to the application, a compound or derivative thereof shown in formula (I) is provided, and the compound or derivative thereof shown in formula (I) has a structure of general formula (I-3):

[0056] wherein x represents any integer from 2 to 4;

[0057] selected from any one of ;

[0058] and / or, each L independently, identically or differently represents substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C1-C5 heteroalkylene, substituted or unsubstituted C1-C5 heterocycloalkylene, substituted or unsubstituted C1-C5 heteroarylene, substituted or unsubstituted C1-C5 arylene, alkoxy, thioether, disulfide, anhydride, carbonate, carbamate, sugar, peptide, polyethylene glycol, amide or ester, or a group of one or more of the above; when substituted, the substituents include one or more of alkyl, alkenyl, alkynyl, aryl, heteroaryl, ester and amine;

[0059] C is a ligand moiety that is a chelator that forms a complex with a divalent or trivalent metal cation; the chelator 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-(carboxymethyl imidazole)glycine, or 6-hydrazinopyridine-3-carboxylic acid;

[0060] Alternatively, C is a ligand moiety, wherein the ligand moiety is a non-radioisotope, a radioisotope, a radiopharmaceutical, or a combination thereof; the radioisotope is selected from an isotope that emits alpha radiation, an isotope that emits beta radiation, an isotope that emits gamma radiation, an isotope that emits Auger electrons, an isotope that emits X-rays; the isotope includes 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 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 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 Ag;

[0061] Alternatively, C is a ligand moiety selected from the following classes of fluorescent dyes: xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, borondipyrromethenes and phthalocyanines, or conjugates or combinations of the above classes of dyes;

[0062] Alternatively, C is a contrast agent comprising or consisting of a paramagnetic agent.

[0063] In the present application, x can be 2, 3 or 4; m can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0064] In the present application, 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-, the structural formula of the carbamate group is -NHCOO-; the phosphate group can be one or more of monoester (hydrocarbyl phosphate), secondary phosphate (diester phosphate) and tertiary phosphate (triester phosphate); the sugar can be one of monosaccharide groups of pentose or hexose, the pentose is selected from ribose, and the hexose is selected from glucose, mannose or galactose.

[0065] In the present application, the heteroatom in the heteroalkylene group, heterocycloalkylene group, heteroalkenylene group, heterocycloalkenylene group, heteroalkynylene group and heteroaryl group includes at least one of oxygen, sulfur and nitrogen.

[0066] According to the present application, a compound represented by formula (I) or a derivative thereof is provided, and in the general formula (I-2), each L independently, identically or differently represents any one of the following groups:

[0067] In the present application, n is any integer from 1 to 10.

[0068] In the present application, R1 is selected from -H, -C 1～6 alkyl, -O-C 1～6 alkyl, -S-C 1～6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or -C 1～6 aralkyl; each of the -C 1～6 alkyl is optionally substituted with 1 to 3 substituents selected from -OH, oxygen, halogen; n is any integer from 1 to 10.

[0069] As a preferred, L is connected to the 2nd, 3rd, 5th, 6th, 7th or 8th position of the quinoline group of the active fragment; preferably, L is connected to the 6th position or the 8th position of the quinoline group of the active fragment, which is specifically as follows:

[0070] According to the present application, a compound represented by formula (I) or a derivative thereof is provided, and the compound represented by general formula (I-3) has any one of the following structural formulae:

[0071] The compound of formula (I-3) in the present application is formed by introducing the intermediate linking segment L at a specific position in the structure, and combining multiple active center connecting segments with the center D to form a polymeric compound. Due to the particularity of the active center structure, the selection of the intermediate linking segment L and the combination center D is particularly important in the research process of the present application, which has different degrees of influence on the synthesis process, the stability of the product molecule, the in vivo metabolism of the compound, etc. The results show that it can effectively improve the retention amount and retention time of the compound in the tumor site, slow down the clearance rate, and be more beneficial to the application of tumor radiotherapy.

[0072] In a second aspect, the present application provides a pharmaceutical composition comprising the compound of formula (I) or a derivative thereof.

[0073] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0074] In a third aspect, the present application provides a kit comprising the compound of formula (I) or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt thereof, or the pharmaceutical composition; and instructions for diagnosing or treating a disease.

[0075] In a fourth aspect, the present application provides the use of the compound of formula (I) or a derivative thereof, or the pharmaceutical composition or the kit in the preparation of a medicament for diagnosing and / or treating a disease.

[0076] Preferably, the disease comprises 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 scarring; more preferably, the cancer is selected from one or more of small intestine cancer, head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, nasopharyngeal cancer, myeloma cells, cholangiocellular carcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, primary unknown cancer, thymus cancer, glioma, glioblastoma, astrocytoma, cervical cancer, eye cancer, rectal cancer, colon cancer, cervical cancer, prostate cancer, breast cancer, bladder cancer, oral cancer, gastric cancer, liver cancer, pancreatic cancer, lung cancer, uterine cancer, ovarian cancer, testicular cancer, kidney cancer, brain cancer, central nervous system cancer, throat cancer, skin melanoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma, Kaposi sarcoma, basal cell carcinoma, squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, nephroblastoma, neuroblastoma, esophageal cancer, laryngeal cancer, lymphoma, neurofibroma, tuberous sclerosis, hemangioma and lymphoma.

[0077] In a fifth aspect, the present application provides a fibroblast activation protein (FAP) targeting inhibitor or binding agent, which comprises the compound of formula (I) or its derivative, or the pharmaceutical composition.

[0078] In a sixth aspect, the present application provides a tumor therapeutic agent, which comprises the compound of formula (I) or its derivative, or the pharmaceutical composition.

[0079] In a seventh aspect, the present application provides a tumor imaging agent, which comprises the compound of formula (I) or its derivative, or the pharmaceutical composition.

[0080] In an eighth aspect, the present application provides a tumor diagnostic agent, which comprises the compound of formula (I) or its derivative, or the pharmaceutical composition.

[0081] The present application obtains a compound with longer retention time in tumor in vivo by modifying the structure of FAP binding multimeric compound, which is more beneficial for the application of tumor radiotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0082] In order to more clearly illustrate the technical solutions in the present application or prior art, the drawings needed to be used in the following embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0083] [According to Rule 91 Correction 11.10.2025] FIG. 1 is the HPLC result of JYT2-212-1 in Example 1A of the present application. 1 H NMR.

[0084] [According to Rule 91 Correction 11.10.2025] FIG. 2 is the HPLC result of JYT2-212-1 in Example 1A of the present application.

[0085] [According to Rule 91 Correction 11.10.2025] FIG. 3 is the LC-MS result of JYT2-212-1 in Example 1A of the present application.

[0086] [According to Rule 91 Correction 11.10.2025] FIG. 4 is the HPLC result of JYT2-222-1 in Example 2A of the present application. 1 H NMR result.

[0087] [According to Rule 91 Correction 11.10.2025] FIG. 5 is the HPLC result of JYT2-222-1 in Example 2A of the present application.

[0088] [Rule 91 Correction 11.10.2025] Figure 6 is the LC-MS result of JYT2-222-1 in Example 2A of the present application.

[0089] [Rule 91 Correction 11.10.2025] Figure 7 is the HPLC result of JYT2-431-1 in Example 3A of the present application. 1 H NMR result.

[0090] [Rule 91 Correction 11.10.2025] Figure 8 is the HPLC result of JYT2-431-1 in Example 3A of the present application.

[0091] [Rule 91 Correction 11.10.2025] Figure 9 is the LC-MS result of JYT2-401 in Example 11A of the present application. 1 H NMR result.

[0092] [Rule 91 Correction 11.10.2025] Figure 10 is the HPLC result of JYT2-401 in Example 11A of the present application.

[0093] [Rule 91 Correction 11.10.2025] Figure 11 is the LC-MS result of JYT2-401 in Example 11A of the present application.

[0094] Figure 12 is the result of different molecular enzyme activity tests.

[0095] Figure 13 is 68 HPLC quality control result after Ga-JYT2-401 labeling.

[0096] Figure 14 is the PET imaging result of tumor-bearing mice injected with different molecules 68 Ga-JYT2-401, 68 Ga-JYT2-411, 68 Ga-JYT2-441, 68 Ga-JY2-011( 68 Ga-PNT6555) respectively.

[0097] Figure 15 is the biodistribution result of tumor-bearing mice injected with 68 Ga-JYT2-401, 68 Ga-JYT2-411, 68 Ga-JYT2-441, 68 Ga-JY2-011( 68 Ga-PNT6555) respectively at different times.

[0098] Figure 16 is 177Quality control results of Lu-JYT2-401.

[0099] Figure 17 is an injection 177 Tissue distribution results of Lu-JYT2-401 injection in HEK293 hFAP human embryonic kidney cell Balb / c nude tumor-bearing mice.

[0100] In Figure 18, the left side is a comparison 177 SPECT imaging images of Lu-JYT2-401 at different time points, and the right side is the maximum and average tumor uptake in the SPECT images over time.

[0101] In Figure 19, the left side is a comparison 177 Lu-JYT2-401 and the control group 177 Lu-FAP2286, 177 Lu-PNT6555( 177 SPECT imaging MIP images of Lu-JY2-011 48 hours after injection, and the right side is a comparison of the maximum tumor uptake in the SPECT images.

[0102] In Figure 20, a comparison 177 Lu-JYT2-401 and the control group 177 Tumor volume inhibition of Lu-FAP2286, saline group (vehicle) injection of 15 MBq per HEK293 hFAP human embryonic kidney cell Balb / c nude tumor-bearing mice (n=5).

[0103] In Figure 21, a comparison 177 Lu-JYT2-401 and the control group 177 Survival curve of Lu-FAP2286, saline group (vehicle) injection of 15 MBq per HEK293 hFAP human embryonic kidney cell Balb / c nude tumor-bearing mice (n=5).

[0104] [According to Rule 91 Correction 11.10.2025] Figure 22 is the H NMR chart of JYQ2-452 in Example 1B of the present application. 1 H NMR chart.

[0105] [According to Rule 91 Correction 11.10.2025] Figure 23 is the HPLC result of JYQ2-452 in Example 1B of the present application.

[0106] [According to Rule 91 Correction 11.10.2025] Figure 24 is the MS result of JYQ2-452 in Example 1B of the present application.

[0107] [Rule 91 Correction 11.10.2025] Figure 25 is the HPLC results of JYT-4521 in Example 2B of the present application. 1 H NMR results.

[0108] [Rule 91 Correction 11.10.2025] Figure 26 is the HPLC results of JYT-4521 in Example 2B of the present application.

[0109] [Rule 91 Correction 11.10.2025] Figure 27 is the MS results of JYT-4521 in Example 2B of the present application.

[0110] [Rule 91 Correction 11.10.2025] Figure 28 is the results of in vitro activity testing of JYQ2-452 in Example IB and JYT-4521 in Example 2B of the present application, wherein (A) is the results of testing of JYQ2-452 and (B) is the results of testing of JYT-4521.

[0111] [Rule 91 Correction 11.10.2025] Figure 29 is the results of in vitro activity testing of JYQ2-452 in Example 2B of the present application. 68 Radioactive signal profile of HPLC detection of Ga-JYQ2-452.

[0112] [Rule 91 Correction 11.10.2025] Figure 30 is the results of in vitro activity testing of JYT-4521 in Example 2B of the present application. 68 UV signal profile of HPLC detection of Ga-JYQ2-452.

[0113] [Rule 91 Correction 11.10.2025] Figure 31 is the results of in vitro activity testing of JYT-4521 in Example 2B of the present application. 68 Radioactive signal profile of HPLC detection of Ga-JYT-4521.

[0114] [Rule 91 Correction 11.10.2025] Figure 32 is the results of in vitro activity testing of JYT-4521 in Example 2B of the present application. 68 UV signal profile of HPLC detection of Ga-JYT-4521.

[0115] [Rule 91 Correction 11.10.2025] Figure 33 is the results of in vitro activity testing of Ga-JYQ2-452 and Ga-JYT-4521 in Example 2B of the present application. 68 PET / CT imaging of Ga-JYQ2-452 and 68 PET / CT imaging of Ga-JYT-4521.

[0116] [Rule 91 Correction 11.10.2025] Figure 34 is the results of in vitro activity testing of Lu-JYQ2-452 in Example 3B of the present application. 177 Radioactive signal profile of HPLC detection of Lu-JYQ2-452.

[0117] [Rule 91 Correction 11.10.2025] Figure 35 is the UV signal profile of HPLC detection of Lu-JYQ2-452 in Experimental Example 3B of the present application. 177 HPLC detection of Lu-JYQ2-452 in Experimental Example 3B of the present application.

[0118] [Rule 91 Correction 11.10.2025] Figure 36 is the radioactivity signal profile of HPLC detection of Lu-JYT-4521 in Experimental Example 3B of the present application. 177 HPLC detection of Lu-JYT-4521 in Experimental Example 3B of the present application.

[0119] [Rule 91 Correction 11.10.2025] Figure 37 is the UV signal profile of HPLC detection of Lu-JYT-4521 in Experimental Example 3B of the present application. 177 HPLC detection of Lu-JYT-4521 in Experimental Example 3B of the present application.

[0120] [Rule 91 Correction 11.10.2025] Figure 38 is the biodistribution result of Lu-JYQ2-452 in Experimental Example 3B of the present application. 177 Biodistribution result of Lu-JYQ2-452 in Experimental Example 3B of the present application.

[0121] [Rule 91 Correction 11.10.2025] Figure 39 is the H NMR of JYT2-4013 in Example 1C of the present application. 1 H NMR of JYT2-4013 in Example 1C of the present application.

[0122] [Rule 91 Correction 11.10.2025] Figure 40 is the HPLC result of JYT2-4013 in Example 1C of the present application.

[0123] [Rule 91 Correction 11.10.2025] Figure 41 is the MS result of JYT2-4013 in Example 1C of the present application.

[0124] [Rule 91 Correction 11.10.2025] Figure 42 is the H NMR result of JYT2-4015 in Example 2C of the present application. 1 H NMR result of JYT2-4015 in Example 2C of the present application.

[0125] [Rule 91 Correction 11.10.2025] Figure 43 is the HPLC result of JYT2-4015 in Example 2C of the present application.

[0126] [Rule 91 Correction 11.10.2025] Figure 44 is the MS result of JYT2-4015 in Example 2C of the present application.

[0127] [Rule 91 Correction 11.10.2025] Figure 45 is the in vitro activity test result of JYT2-4013 in Example 1C and JYT2-4015 in Example 2C of the present application, wherein (a) is the test result of JYT2-4015, and (b) is the test result of JYT2-4013.

[0128] [Rule 91 Correction 11.10.2025] Figure 46 is a post-labeling HPLC profile of JYT2-4013 in Experimental Example 2C of the present application. 177 Lu-JYT2-4013 Radio-HPLC Quality Control Results.

[0129] [Rule 91 Correction 11.10.2025] Figure 47 is a post-labeling HPLC profile of JYT2-4013 in Experimental Example 2C of the present application. 177 Lu-JYT2-4013 UV Analysis HPLC Quality Control Results.

[0130] [Rule 91 Correction 11.10.2025] Figure 48 is a post-labeling HPLC profile of JYT2-4013 in Experimental Example 2C of the present application. 177 Lu-JYT2-4013 Biodistribution Profile.

[0131] [Rule 91 Correction 11.10.2025] Figure 49 is a post-labeling HPLC profile of JYT2-401 in Comparative Example 1C of the present application. 177 Lu-JYT2-401 SPECT Imaging Profile.

[0132] [Rule 91 Correction 11.10.2025] Figure 50 is a post-labeling HPLC profile of JYT2-4013 in Experimental Example 2C of the present application. 177 Lu-JYT2-4013 SPECT Imaging Profile. DETAILED DESCRIPTION

[0133] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be described clearly and completely below in conjunction with the drawings in the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0134] The compounds shown in relation to the structure of general formula (I-1) and their use are verified:

[0135] [Rule 91 Correction 11.10.2025] Preparation of Small Molecule Inhibitor of Example 1A

[0136] The synthesis route of JYT2-212-1 is as follows:

[0137] Compound 3: (S)-4,4-difluoro-l-glycylpyrrolidine-2-carbonitrile: To a solution of compound 1 (300 mg, 1.39 mmol) in dichloromethane (10 mL) was added HATU (1870 mg, 4.92 mmol), DIEA (2935 mg, 22.70 mmol) and compound 2 (1000 mg, 5.68 mmol) at 25 °C. The reaction was stirred at 25 °C for 1 h. The reaction was filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by column chromatography (V 石油醚 / 乙酸乙酯 = 2: 1) to give intermediate 3 (700 mg, 64%) as a white solid. LC-MS: [M+H] + = 312.7.

[0138] Compound 4: (S)-4,4-difluoro-l-glycylpyrrolidine-2-carbonitrile: To a solution of compound 3 (600 mg, 2.07 mmol) was added hydrochloric acid in ethyl acetate (10 mL) at 25 °C. The reaction was stirred at 25 °C for 0.5 h. The reaction was filtered and concentrated under reduced pressure to give intermediate 4 (350 mg, 89%) as a white solid. LC-MS: [M+H] + = 190.1.

[0139] Compound JYT2-212-1: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-l-yl)-2- oxoethyl)-3,4-dihydroquinoline-l(2H)-carboxamide: To a solution of intermediate 4 (85 mg, 0.45 mmol) and compound 5 (60 mg, 0.45 mmol) in dichloromethane (5 mL) was added BTC (93 mg, 0.31 mmol) and TEA (227 mg, 2.25 mmol) at 25 °C. The reaction was stirred at 25 °C for 1 h. The reaction was filtered and concentrated under reduced pressure to give the crude product. The crude product was purified by preparative high performance liquid chromatography (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H20 (0.1% FA); B%: 8%-15%, 10 min) to give JYT2-212-1 (10 mg, 6%) as a white solid.

[0140] JYT2-212-1 1 H NMR is shown in Figure 1, HPLC results are shown in Figure 2, and LC-MS results are shown in Figure 3.

[0141] LC-MS: [M+H] + = 349.1. 1H NMR (400 MHz, DMSO-d6) δ 7.52 (d, J = 8.0 Hz, 1H), 7.12-7.09 (m, 2H), 6.96-6.89 (m, 1H), 6.90-6.88 (m, 1H), 5.08-5.06 (m, 1H), 4.27-4.16 (m, 1H), 4.06-3.92 (m, 1H), 3.92-3.90 (m, 2H), 3.64-3.58 (m, 2H), 2.95-2.76 (m, 2H), 2.70-2.68 (m, 2H), 1.89-1.79 (m, 2H).

[0142] [According to Rule 91 correction 11.10.2025] Preparation of Example 2A small molecule inhibitor

[0143] Synthesis route of JYT2-222-1:

[0144] Preparation method of compound JYT2-222-1: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-3,4-dihydroquinoline-1(2H)-carboxamide: To a solution of intermediate 4 (50 mg, 0.26 mmol) and compound 5 (35 mg, 0.26 mmol) in dichloromethane (5 mL) was added BTC (55 mg, 0.19 mmol) and TEA (134 mg, 1.32 mmol) at 25 °C. Stirring at 25 °C for 1 h. The reaction solution was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative high performance liquid chromatography (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H2O (0.1% FA); B%: 12%-19%, 10 min) to obtain JYT2-222-1 (7 mg, 7%) as a white solid.

[0145] JYT2-222-1 1 H NMR see Figure 4, HPLC results see Figure 5, LC-MS results see Figure 6.

[0146] LC-MS: [M+H] + = 350.1. 1H NMR (400 MHz, DMSO-d6) δ 7.26 - 7.22 (m, 1H), 6.88 - 6.82 (m, 1H), 6.61-6.54 (m, 2H), 6.53 - 6.48 (m, 1H), 6.11 (s, 1H), 5.07-5.05 (m, 1H), 4.24 - 4.13 (m, 1H), 4.09 - 3.96 (m, 1H), 3.91 d, J = 5.6 Hz, 2H), 3.60 - 3.47 (m, 2H), 3.22 (d, J = 2.8 Hz, 2H), 2.82-2.76 (m, 2H).

[0147] [According to Rule 91 Correction 11.10.2025] Preparation of Example 3A small molecule inhibitor

[0148] [According to Rule 91 Correction 11.10.2025] Synthetic route of JYT2-431-1:

[0149] Step 1: 7-((tert-butyldimethylsilyl)oxy)quinoline: 7-hydroxyquinoline (5.0 g, 34.4 mmol) was dissolved in DMF (50 mL), tert-butyldimethylsilyl chloride (6.2 g, 41.3 mmol) and imidazole (3.5 g, 51.6 mmol) were added. After addition, the reaction was stirred at room temperature for 70 minutes, TLC showed that the starting material was completely reacted. The reaction solution was quenched with water (200 mL), extracted with ethyl acetate (100 mL), washed with water (50 mL), separated, dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (PE / EA = 20 / 1) to give the colorless liquid title compound 019C-069-1 (8.9 g, yield: 99.7%).

[0150] Step 2: 7-((tert-butyldimethylsilyl)oxy)-1,2-dihydroquinoline: Compound 019C-069-1 (2.0 g, 7.7 mmol) was dissolved in anhydrous tetrahydrofuran (30 mL), diisobutylaluminum hydride (15.4 mL, 15.4 mmol) was added at room temperature, after addition, the reaction temperature was increased to 25°C and stirred for 2 hours, TLC showed that most of the starting material was reacted. The reaction solution was poured into ice water to quench, extracted with ethyl acetate (100 mL), washed with water (50 mL), dried, and concentrated to give the light yellow title compound 019C-069-2 (2.1 g, crude), which was used directly in the next step. LC-MS: m / z = 262.2 [M+H]+.

[0151] Step 3: 7-((tert-butyldimethylsilyl)oxy)quinoline-1 (2H)-carboxylic acid: Compound 019C-062-2 (1.5 g, crude) was dissolved in dichloromethane (40 mL), triethylamine (567 mg, 5.6 mmol) was added, the reaction was cooled to 0 °C under nitrogen protection, then triphosgene (550 mg, 1.86 mmol) was quickly added to the reaction, the reaction was stirred at 0 °C for 10 minutes; TLC showed that the starting material was completely reacted. The reaction solution was quenched by adding dilute hydrochloric acid (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to give a red-brown solid title compound 019C-069-3 (1.4 g, crude), which was used directly in the next step.

[0152] Step 4: 7-((tert-butyldimethylsilyl)oxy)-N-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1- yl)ethyl)quinoline-1 (2H)-carboxamide: Compound 019C-062-2 (654 mg, 1.91 mmol) was dissolved in dichloromethane (20 mL), triethylamine (386 mg, 3.82 mmol) and compound 019C-069-3 (1.4 g, crude) were added, the reaction was stirred at room temperature overnight. After the reaction was completed, dilute hydrochloric acid (100 mL) was added to quench. The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude product was purified by silica gel column chromatography to give a red solid title compound 019C-069-4 (0.39 g, yield: 34%).

[0153] Step 5: 7-hydroxy-N-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro- 4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1-yl)ethyl)quinoline-1 (2H)- carboxamide: Compound 019C-069-4 (0.39 g, 0.67 mmol) was dissolved in tetrahydrofuran (10 mL), TBAF (10 mL, 10 mmol) was added, and the reaction was stirred at room temperature for 1 hour. The reaction solution was quenched by adding water (100 mL); extracted with ethyl acetate (50 mL), separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated to give a white solid title compound 019C-069-5 (205 mg, yield: 64%).

[0154] Step 6: (R)-(l-((7-hydroxy-l,2-dihydroquinoline-l-carbonyl)glycyl)pyrrolidin-2-yl)boronic acid: Compound 019C-069-5 (205 mg, 0.43 mmol) was dissolved in a mixture of methanol (3 mL) and n-hexane (3 mL), benzeneboronic acid (156 mg, 1.28 mmol) and dilute hydrochloric acid (3 N, 2 d) were added. After addition, the mixture was allowed to react at room temperature overnight, and TLC detection showed that the starting material was completely reacted. The mixture was separated, and the methanol layer was prepared by prep-HPLC to obtain 58 mg of white solid. Purification by Prep TLC (DCM / MeOH = 10 / 1) gave 25 mg of white solid of the title compound SUS019C-069 (JYT2-431-1) (yield: 17%).

[0155] The synthesis results of JYT2-431-1 are as follows, and the structure of JYT2-431-1 is shown in Figure 1. 1 The H NMR results are shown in Figure 7, and the HPLC results are shown in Figure 8.

[0156] LC-MS: m / z = 328.2 [M-H20+1] + (96.97% purity, 254 nm). 1 H NMR (400 MHz, CD3OD) δ 7.03-6.96 (m, 2H), 6.56 (dd, J = 8.0 Hz, 6.0 Hz, 1H), 6.45 (d, J = 7.1 Hz, 1H), 5.87-5.82 m, 1H), 4.32-4.22 (m, 2H), 4.07-3.96 (m, 2H), 3.63-3.46 (m, 2H), 3.13-3.00 (m, 1H), 2.21-2.12 (m, 1H), 2.07-1.92 (m, 2H), 1.71-1.58 (m, 1H).

[0157] [Corr. According to Rule 91 11.10.2025] Preparation of small molecule inhibitors of Example 4A

[0158] Synthesis results of JYT2-201-1:

[0159] Referring to the above synthesis route, the synthesis result of JYT2-201-1 is as follows, 3-{2-[(2S)-2-cyano-4,4-difluoropyrrolidin-1-yl]-2-oxoethyl}-1-(6-methoxyquinolin-4-yl)urea (900 mg, 2.3 mmol) and BBr3(1 M in DCM) (11.6 mL) were dissolved in DCM (10 mL, the reaction was stirred at 25 °C for 16 h. LCMS showed that the starting material was consumed completely, and the desired product was detected. Pre-high performance liquid chromatography (preparation conditions: column: WELCH xmax C18 21.2 x 250 mm 10 um), mobile phase: ACN-H2O (0.1% FA) gradient: 10-30%), the product (200 mg, yield 23%) was obtained as a white solid.

[0160] LC-MS: [M+H] + : 376.1. 1 H NMR (400 MHz, DMSO) δ 9.13 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 8.33 (s, 2H), 8.06 (d, J = 5.2 Hz, 1H), 7.79 (d, J = 9.0 Hz, 1H), 7.51 (s, 1H), 7.38 - 7.29 (m, 2H), 5.13 (d, J = 9.2 Hz, 1H), 4.27 - 4.03 (m, 4H), 2.87 - 2.75 (m, 2H).

[0161] [According to Rule 91 Correction 11.10.2025] Preparation of small molecule inhibitors of Example 5A

[0162] Synthesis result of JYT2-243-1:

[0163] Referring to the above synthesis route, compound JYT2-243-1: 1-(2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-acetyl)-3-(6-hydroxyquinolin-4-yl)urea was synthesized as follows: To a solution of 3-{2-[(1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl]-2-acetyl}-1-(6-methoxyquinolin-4-yl)urea (120 mg, 0.33 mmol) in dichloromethane (10 mL) was added BBr3(1 mole per liter in dichloromethane) (411.35 mg, 1.64 mmol) at 0 °C under nitrogen atmosphere. The mixture was stirred at 0 °C for 12 h. The reaction was quenched with water (1 mL) and MeOH (1 mL). The crude product was purified by preparative high performance liquid chromatography (column: Phenomenex luna C18 250 mm x 100 mm x 10 um; mobile phase: [H2O (0.1% NH4HCO3) - ACN]; B%: 15% - 35%, 9 min) to give JYT2-243-1 (2.5 mg, 2.2%) as a yellow solid.

[0164] LC-MS: [M+H] + : 352.1. 1 H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 9.10 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H), 8.08 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.31 (dd, J = 9.0, 2.5 Hz, 2H), 4.97 (dd, J = 131.4, 9.0 Hz, 1H), 4.36 (dt, J = 18.2, 9.0 Hz, 1H), 4.16 (dd, J = 17.8, 4.8 Hz, 1H), 3.73 (dd, J = 37.8, 30.0 Hz, 1H), 2.56 (d, J = 13.2 Hz, 1H), 2.28 - 2.15 (m, 1H), 1.99 - 1.82 (m, 1H), 1.08 - 0.97 (m, 1H), 0.82 - 0.71 (m, 1H).

[0165] [Corr. According to Rule 91 11.10.2025] Preparation of small molecule inhibitors of Example 6A

[0166] Synthesis results of JYT2-253-1:

[0167] Referring to the above synthesis route, compound JY2-253-1 : (S)-1-(2-(2-cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)-3-(1H-pyrrolo[2,3-b]pyridin-4-yl)urea synthesis results as follows, to the compound 3 (150 mg, 0.519 mmol) in DCM (4 mL) solution was added 2-Chloropyridine (177 mg, 1.56 mmol), Tf20 (220 mg, 0.779 mmol) at 25 °C. The reaction was stirred at 25 °C for 30 min, compound 4 (207 mg, 1.56 mmol) and TEA (315 mg, 3.11 mmol) were added to the reaction, the reaction was reacted at 25 °C for 2 h, LCMS showed that the product had been formed, vacuum filtration, reduced pressure concentration, the crude product was purified by preparative high performance liquid chromatography (column Welch 21.2 x 250 mm 10 um C18, 30 ml / min), mobile phase: ACN-H20 (0.1% NH3); B%: 30%-70%, 10 min) to give compound JYT2-253-1 (19.1 mg, 11%) as a white powder solid.

[0168] LC-MS: (ESI) m / z [M+H] + : 349.2. 1 H NMR (400 MHz, DMSO-d6) δ 10.33 (t, J = 5.4 Hz, 1H), 7.87 (d, J = 5.6 Hz, 1H), 7.58 (d, J = 3.8 Hz, 1H), 6.77 (d, J = 4.0 Hz, 1H), 6.68 (s, 2H), 6.38 (d, J = 5.8 Hz, 1H), 5.13 (dd, J = 9.2, 2.6 Hz, 1H), 4.39 - 4.18 (m, 3H), 4.18 - 4.03 (m, 1H), 2.97 - 2.75 (m, 2H).

[0169] [Corr. According to Rule 91 11.10.2025] Preparation of small molecule inhibitors of Example 7A

[0170] Synthesis results of JYT2-242-1:

[0171] Referring to the above synthetic route, compound JYT2-242-1 : (S)-1-(2-(2-cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)-3-(6-hydroxyquinolin-4-yl)-1-methylurea was synthesized as follows, to a solution of intermediate 8 (57 mg, 0.14 mmol) in dichloromethane (2 mL) was added BBr3 (177 mg, 0.71 mmol) at 0 °C. Stirring at 25 °C for 1 h. The reaction was quenched with methanol and filtered to give a crude product. The crude product was purified by preparative high performance liquid chromatography (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H2O (0.1% FA); B%: 7% - 12%, 10 min) to give JYT2-242-1 (6 mg, 9%) as a white solid.

[0172] LC-MS: [M+H] + : 390.2. 1 H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.72 (s, 1H), 8.55 (d, J = 4.2 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 4.6 Hz, 1H), 7.34 (d, J = 9.2 Hz, 2H), 5.13 (d, J = 8.4 Hz, 1H), 4.31 - 4.18 (m, 2H), 4.14 - 3.90 (m, 2H), 3.13 (s, 3H), 2.87 - 2.80 (m, 2H).

[0173] Preparation of small molecule inhibitors of Example 8A

[0174] Synthesis results of JYT2-242-1 :

[0175] Referring to the above synthetic route, compound JY2-231-1 : (S)-N-(2-(2-cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)isoindoline-2-carboxamide was synthesized as follows, to a solution of intermediate 4 (85 mg, 0.45 mmol) and compound 5 (54 mg, 0.45 mmol) in dichloromethane (5 mL) was added BTC (67 mg, 0.22 mmol) and TEA (91 mg, 0.89 mmol) at 25 °C. Stirring at 25 °C for 1 h. The reaction was filtered and concentrated under reduced pressure to give a crude product. The crude product was purified by preparative high performance liquid chromatography (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H2O (0.1% FA); B%: 10% - 15%, 10 min) to give JY2-231-1 (20 mg, 13%) as a white solid.

[0176] LC-MS: [M+H] + : 335.1. 1 H NMR (400 MHz, DMSO-d6) δ 7.37 - 7.27 (m, 4H), 6.70-6.67 (m, 1H), 5.08-5.05 (m, 1H), 4.63 (s, 4H), 4.30-4.20 (m, 1H), 4.09-3.90 (m, 1H), 3.90-3.84 (m, 2H), 2.98-2.73 (m, 2H).

[0177] [Corrected according to Rule 91 11.10.2025] Preparation of small molecule inhibitors of Example 9A

[0178] Synthesis results of JYT2-441-1:

[0179] The synthetic route is shown below:

[0180] Step 1: 7-((tert-butyldimethylsilyl)oxy)naphthalen-1-amine: 8-amino-2- naphthalenol (3.2 g, 20.0 mmol) was dissolved in DMF (20 mL), tert-butyldimethylsilyl chloride (3.6 g, 24.0 mmol) and imidazole (2.8 g, 41.0 mmol) were added. After addition, the reaction was stirred at room temperature for 30 minutes, TLC detection of the raw material reaction was complete. The reaction solution was quenched with water (100 mL), extracted with ethyl acetate (100 mL), washed with water (50 mL), separated, the organic phase was dried over anhydrous sodium sulfate, concentrated, and the crude product was purified by silica gel column chromatography (PE / EA = 20 / 1) to give the colorless liquid title compound 019C-068-1 (4.7 g, yield: 85.9%).

[0181] Step 2: tert-butyl ((8-isocyanatophenyl)oxy)dimethylsilane: Triflic anhydride (179 mg, 0.60 mmol) was dissolved in dichloromethane (10 mL) and cooled to -10 °C under nitrogen protection. Compound 019C-068-1 (0.5 g, 1.83 mmol) was dissolved in dichloromethane (15 mL), and triethylamine (407 mg, 4.03 mmol) was added. The resulting mixture was slowly added dropwise to the triflic anhydride, and the temperature was maintained at 0 °C during the addition. After the addition was completed, the reaction was stirred at room temperature for 30 minutes to give a dichloromethane solution of compound 019C-068-2, which was directly used in the next step.

[0182] Step 3: 1-(7-((tert-butyldimethylsilyl)oxy)naphthalen-1-yl)-3-(2-oxo-2-((R)-2- ((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2- yl)pyrrolidin-1-yl)ethyl)urea: Compound 019C-062-2 (400 mg, 1.16 mmol) was added to the reaction mixture above, triethylamine (281 mg, 2.78 mmol) was added and the reaction was stirred at room temperature for 50 min. The reaction was quenched by the addition of dilute hydrochloric acid (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography to give the title compound 019C-068-3 (0.65 g, yield: 92.5%) as a red solid. LC-MS: m / z = 452.2 [M-H] - .

[0183] Step 4: (R)-(1-(((7-hydroxynaphthalen-1-yl)carbamoyl)glycino)pyrrolidin-2-yl)boronic acid: Compound 019C-068-3 (300 mg, 0.50 mmol) was dissolved in a mixture of methanol (4 mL) and n-hexane (4 mL), phenylboronic acid (91 mg, 0.74 mmol) and dilute hydrochloric acid (3 N, 2 d) were added. The reaction was heated to 40 °C for 5 h. TLC showed that the starting material was completely consumed. The reaction was partitioned, and the organic phase was concentrated. The crude product was purified by Pre-TLC (DCM / MeOH = 10 / 1) and then slurried in methanol to give the title compound SUS019C-068 (JYT2-441-1) (63 mg, yield: 35.59%) as a yellow solid.

[0184] Referring to the above synthetic route, compound JYT2-441-1: (R)-(1-(((7-hydroxynaphthalen-1-yl)carbamoyl)glycino)pyrrolidin-2-yl)boronic acid; the results are as follows: LC-MS: m / z = 340.2 [M-H2O-H]-(99.92% purity, 220 nm). 1 H NMR (400 MHz, DMSO-d6 + D2O) δ 7.81 - 7.72 (m, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.25 - 7.17 (m, 1H), 7.12 (dd, J = 8.8, 2.4 Hz, 1H), 3.98 - 3.74 (m, 2H), 3.51 - 3.30 (m, 2H), 2.98 - 2.85 (m, 1H), 2.02 - 1.50 (m, 4H).

[0185] [Corr. R. 91, 11.10.2025] Preparation of small molecule inhibitors

[0186] The synthetic route of JYT2-421-1 is as follows:

[0187] Step 1: (S)-2-(R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6- methylbenzo[d][l,3,2]dioxol-2-yl)pyrrolidine-l-carbonyl)pyrrolidine-2-carboxylic acid tert-butyl ester: Compound 019C-062-2 (1.15 g, 4.65 mmol), N-Boc-L-proline (1.00 g, 4.65 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl)urea hexafluorophosphate (2.65 g, 6.97 mmol) were dissolved in tetrahydrofuran (10 mL), and N,N-diisopropylethylamine (1.80 g, 13.94 mmol) was added. After addition, the reaction was stirred at room temperature for 2 hours. TLC (ethyl acetate = 100%, KMnO4coloration) showed that a new spot was formed obviously. After the reaction was quenched with water (20 mL), the product was extracted with ethyl acetate (10 mL x 2), the combined organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give the title compound 019C-065-1 (1.07 g, yield: 52.1%) as colorless transparent oil.

[0188] Step 2: (R)-l-(L-propanoyl)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6- methylbenzo[d][l,3,2]dioxol-2-yl)pyrrolidine hydrochloride: Compound 019C-065-1 (1.07 g, 2.40 mmol) was dispersed in hydrogen chloride / 1,4-dioxane (10 mL, 4N) and stirred at 25 °C for 2 hours. TLC (ethyl acetate = 100%) showed that the starting material was consumed completely. The reaction was concentrated to give the title compound 019C-065-2 (crude product) as white solid, which was used directly in the next step.

[0189] Step 3: (S)-N-(6-hydroxyquinolin-4-yl)-2-(R)-2-(((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxol-2-yl)pyrrolidine-l- carbonyl)pyrrolidine-2-carboxamide: Compound 019C-062-5 (106 mg, 0.39 mmol), diisopropylethylamine (169 mg, 1.31 mmol) and diphenyl phosphorazide (129 mg, 0.47 mmol) were dispersed in toluene (6 mL) and warmed to 80 °C for 2 hours with stirring. TLC (dichloromethane / methanol = 9 / 1) was used to monitor the disappearance of starting material. Compound 019C-065-2 (100 mg, crude) was added. The reaction was continued to stir at 80 °C for 16 hours and TLC (dichloromethane / methanol = 9 / 1) was used to monitor the formation of new spot. The reaction was concentrated and purified by silica gel column chromatography (dichloromethane / methanol = 19 / 1) to give the title compound 019C-065-3 (95 mg, yield: 68.3%) as a light yellow oil. LC-MS: m / z = 531.2 [M-H].

[0190] Step 4: ((R)-l-(((6-hydroxyquinolin-4-yl)carbamoyl)-L-propanoyl)pyrrolidin-2- yl)boronic acid: Compound 019C-065-3 (95 mg, 0.18 mmol) and benzenboronic acid (44 mg, 0.36 mmol) were dissolved in a mixture of methanol (3 mL) and n-hexane (3 mL). Dilute hydrochloric acid (0.1 mL, 3 N) was added dropwise. The reaction was stirred at 25 °C for 3 hours and TLC (dichloromethane / methanol = 9 / 1) was used to monitor the complete reaction of starting material. The lower methanol phase was pipetted out. After concentration, the title compound SUS019C-065 (JYT2-421-1) (12 mg, yield: 16.9%) was obtained as a white solid by prep-TLC purification and pre-HPLC preparation.

[0191] Referring to the above synthetic route, compound JYT2-421-1 : ((R)-l-(((6- hydroxyquinolin-4-yl)carbamoyl)-L-propanoyl)pyrrolidin-2-yl)boronic acid; the results are as follows: LC-MS: m / z = 399.2 [M+H] + (98.98% purity, 220 nm). 1H NMR (400 MHz, D20) δ 8.54-8.47 (m, 1H), 8.02-7.95 (m, 1H), 7.93-7.86 (m, 1H), 7.61-7.54 (m, 1H), 7.51-7.41 (m, 1H), 3.86-3.65 (m, 3H), 3.49-3.33 (m, 2H), 3.01-2.88 (m, 1H), 2.42-2.23 (m, 1H), 2.15-1.80 (m, 6H), 1.66-1.52 (m, 1H).

[0192] Synthesis of JYT2-453-1: (R)-(1-(((1,7-naphthyridin-4-yl)carbamoyl)glycyl)pyrrolidin-2-yl)boronic acid; results as follows: LC-MS: m / z = 344.1 [M+H] + (98.12% purity, 220 nm). 1 H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.99 (d, J = 6.4 Hz, 1H), 8.94-8.88 (m, 2H), 8.49 (d, J = 6.0 Hz, 1H), 4.25-4.11 (m, 2H), 3.66-3.59 (m, 1H), 3.55-3.46 (m, 1H), 3.16-3.12 (m, 1H), 2.23-2.12 (m, 1H), 2.08-1.97 (m, 2H), 1.81-1.66 (m, 1H).

[0193] [Corr. R. 91, 11.10.2025] Preparation of Example 11A complete small molecule inhibitor

[0194] The synthesis route of JYT2-401 is as follows:

[0195] Step 1: Preparation method of 6-hydroxyquinoline-4-carboxylic acid methyl ester: 6-hydroxyquinoline-4-carboxylic acid hydrobromide (5.0 g, 18.51 mmol) was dispersed in methanol (50 mL), and concentrated sulfuric acid (0.5 mL) was added. The reaction was carried out at 65 °C for 15 hours. TLC detection showed that the raw material was completely reacted. The reaction solution was concentrated to remove methanol, diluted with water (20 mL), alkalized to pH = 10 with saturated sodium bicarbonate solution, and extracted with ethyl acetate (200 mL). Filtration, liquid separation. The water phase was extracted with ethyl acetate (50 mL x 3), the organic phases were combined, and concentrated to obtain a brown solid title compound 401-1 (1.5 g, yield: 39.89%).

[0196] Step 2: Preparation of methyl 6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)quinoline-4-carboxylic acid: Compound 401-1 (1.3 g, 6.40 mmol) was dissolved in DMF (15 mL), and potassium carbonate (2.65 g, 19.2 mmol) and tert-butyl 4-(3-bromopropyl)piperazin-1-carboxylic acid (1.97 g, 6.40 mmol) were added. After the addition was complete, the reaction solution was heated to 85 °C and reacted for 2 hours. TLC detection showed that the reaction of the starting material was complete. The reaction solution was quenched with water (50 mL), extracted with ethyl acetate (100 mL x 2), the organic phases were combined, concentrated, and the crude product was purified by silica gel column chromatography (PE / EA = 1 / 2) to give brown liquid title compound 401-2 (1.75 g, yield: 63.64%). LC-MS: m / z = 430.2 [M+H] + .

[0197] Step 3: Preparation of 6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)quinoline-4-carboxylic acid: Compound 401-2 (1.75 g, 4.09 mmol) was dispersed in a mixed solution of THF (20 mL) and water (10 mL), and LiOH·H2O (503 mg, 12.3 mmol) was added. After the addition was complete, the reaction solution was allowed to react at room temperature for 3 hours. TLC was used to confirm the complete reaction of the starting material. The reaction solution was acidified to pH 6 with saturated ammonium chloride, and extracted with 2-methyltetrahydrofuran (50 mL x 3). The organic phases were combined and concentrated to give a white liquid, namely, title compound 401-3 (1.64 g, yield: 97.04%). LC-MS: m / z = 416.2 [M+H] + .

[0198] Step 4: Preparation method of tert-butyl 4-(3-((4-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxol-2-yl)piperidin-l- yl)ethyl)ureido)quinolin-6-yl)oxy)propyl)piperazine-l-carboxylate: Compound 401-3 (905 mg, 2.18 mmol) and compound 066-2 (746 mg, 2.18 mmol) were dispersed in toluene (15 mL), triethylamine (661 mg, 6.54 mmol) and DPPA (660 mg, 2.4 mmol) were added. After addition, the reaction solution was heated to 110 °C for 3 hours. TLC detection showed that the reaction was complete. The reaction solution was quenched with water (50 mL), extracted with ethyl acetate (100 mL x 2), the organic phases were combined and concentrated, and the crude product was purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to give the title compound 401-4 (491 mg, yield: 31.27%) as a yellow solid. LC-MS: m / z = 719.4 [M+H] + .

[0199] Step 5: Preparation method of l-(2-oxo-2-((R)-2-(((3aS,4S,6S,7aR)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxol-2-yl)piperidin-l-yl)ethyl)-3-(6-(3- (piperazin-l-yl)propoxy)quinolin-4-yl)urea hydrochloride: Compound 401-4 (491 mg, 0.68 mmol) was dissolved in dichloromethane (2 mL), 4M hydrochloric acid-dioxane solution (2 mL) was added, and after addition, the reaction solution was reacted at room temperature for 2 hours. TLC detection showed that the reaction was complete. The reaction solution was concentrated to give the title compound 401-5 (430 mg, yield: 96.28%) as a yellow solid.

[0200] Step 6: Preparation of 2,2',2'-(10-(2-oxo-2-(4-(4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxol-2-yl)piperidin-1-yl)ethyl) ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane- 1,4,7-trisyl)triacetic acid tri-t-butyl ester: Dissolve tri-t-butyl 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetate (39 mg, 0.069 mmol) in DMF (8 mL), add PYBOP (43 mg, 0.083 mmol) and DIPEA (27 mg, 0.21 mmol). After addition, the reaction solution is stirred at room temperature for 30 minutes, and then compound 401-5 (45 mg, 0.069 mmol) is added. After addition, the reaction solution is reacted at room temperature for 3 hours. TLC detection shows that the reaction is complete. The reaction solution is quenched with water (20 mL), extracted with ethyl acetate (20 mL x 2), the organic phases are combined, concentrated, and the crude product is purified by Pre-TLC (DCM / MeOH = 10 / 1) to obtain the title compound 401-6 (21 mg, yield: 26.05%) as a yellow solid. LC-MS: m / z = 587.5 [M+2H / 2] + .

[0201] Step 7: Preparation of 2,2',2'-(10-(2-oxo-2-(4-(4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxol-2-yl)piperidin-1-yl)ethyl) ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane- 1,4,7-trisyl)triacetic acid: Dissolve compound 401-6 (21 mg, 0.018 mmol) in dichloromethane (1 mL), and add trifluoroacetic acid (2 mL). The reaction is carried out at room temperature for 15 hours. The reaction solution is directly concentrated to obtain purple solid 401-7 (crude product), which is directly used in the next step.

[0202] Step 8: Preparation of (R)-2,2',2'-(10-(2-(4-(3-(3-(2-(2-bromopyrrolidin-1-yl)-2- oxoethyl)ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)2-oxoethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-trisyl)triacetic acid: Compound 401-1 (crude) was dissolved in a mixture of water (2 mL) and acetonitrile (1 mL), benzeneboronic acid (2 mg, 0.016 mmol), trifluoroacetic acid (2 d) and MTBE (6 mL) were added. After addition, the reaction solution was stirred at room temperature for 3 hours. The solution was separated, and the aqueous phase was washed with MTBE (10 mL). The aqueous phase was prepared into a white solid by Pre-HPLC to obtain the title compound JYT2-401 (10 mg, two-step yield: 38.77%).

[0203] JYT2-401 1 H NMR is shown in Figure 9, HPLC results are shown in Figure 10, and LC-MS results are shown in Figure 11.

[0204] LC-MS: m / z = 853.4 [M-H2O+H] + (96.74% purity, 210 nm). 1 H NMR (400 MHz, D2O) δ 8.54 (d, J = 6.8 Hz, 1H), 8.39 (d, J = 6.8 Hz, 1H), 7.94 (d, J = 9.2 Hz, 1H), 7.69 (d, J = 2.0 Hz, 1H), 7.64 (dd, J = 9.2, 2.4 Hz, 1H), 4.62 - 4.44 (m, 1H), 4.31 (t, J = 5.2 Hz, 2H), 4.20 - 4.05 (m, 2H), 4.04 - 2.97 (m, 36H), 2.42 - 2.27 (m, 2H), 2.15 - 1.88 (m, 3H), 1.79 - 1.53 (m, 1H).

[0205] [Corrected according to Rule 91 11.10.2025] Preparation of a complete small molecule inhibitor

[0206] Referring to the above synthesis route, compound JYT2-402:

[0207] 2,2',2'-(10-(4-(2-(4-(3-(2-(((R)-2-bromopyrrolidin-1-yl)-2-oxoethyl)ureido)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-trisyl)triacetic acid; the results are as follows: LC-MS: m / z = 470.7 [(M-H2O) / 2+H] +(97.43% purity, 210 nm); 1 H NMR (400 MHz, D20) δ 8.58 (d, J = 6.8 Hz, 1H), 8.39 (d, J = 6.8 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.76-7.71 (m, 1H), 4.04 (s, 2H), 3.70-3.68 (m, 5H), 3.53-3.44 (m, 4H), 3.39-3.32 (m, 2H), 3.28-3.13 (m, 10H), 2.99-2.88 (m, 6H), 2.76 (t, J = 6.4 Hz, 3H), 2.57 (t, J = 6.8 Hz, 2H), 2.23-1.54 (m, 10H).

[0208] [Rule 91 Correction 11.10.2025] Preparation of complete small molecule inhibitor of Example 13A

[0209] Referring to the above synthetic route, compound JYT2-406:

[0210] (R)-2,2',2'-(10-(2-(4-(3-(3-(2-(2-bromopyrrolidin-l-yl)-2-oxoethyl)ureido)quinolin-6-yl)(methyl)amino)propyl)piperazin-l-yl)2-oxoethyl)-l,4,7,10-tetraazacyclododecane-l,4,7-trisyl)triacetic acid; results as follows: LC-MS: m / z = 852.4 [M-H20+H]+(98.44% purity, 210 nm); 1 H NMR (400 MHz, D20) δ 8.58 (d, J = 6.8 Hz, 1H), 8.39 (d, J = 6.8 Hz, 1H), 8.28 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 7.2 Hz, 1H), 7.76-7.71 (m, 1H), 4.04 (s, 2H), 3.70-3.68 (m, 5H), 3.53-3.44 (m, 4H), 3.39-3.32 (m, 2H), 3.28-3.13 (m, 10H), 2.99-2.88 (m, 6H), 2.76 (t, J = 6.4 Hz, 3H), 2.57 (t, J = 6.8 Hz, 2H), 2.23-1.54 (m, 10H).

[0211] [Rule 91 Correction 11.10.2025] Preparation of complete small molecule inhibitor of Example 14A

[0212] Referring to the above synthetic route, compound JYT2-411:

[0213] 2,2',2'-(10-(2-(4-(3-((R)-1-(R)-2-bromopyrrolidin-1-yl)-1-oxoprop-2-yl)ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; results as follows: LC-MS: m / z = 434.3 [(M-H20) / 2+H]+ (96.81% purity, 210 nm); 1 H NMR (400 MHz, D20) δ 8.64 (d, J = 6.8 Hz, 1H), 8.46 (d, J = 6.8 Hz, 1H), 8.04 (d, J = 10.0 Hz, 1H), 7.81-7.70 (m, 2H), 4.76-4.69 (m, 1H), 4.69-4.53 (m, 1H), 4.48-4.29 (m, 2H), 4.15-3.69 (m, 13H), 3.63-2.97 (m, 23H), 2.59-2.30 (m, 2H), 2.30-1.99 (m, 3H), 1.89-1.65 (m, 1H), 1.47 (d, J = 6.8 Hz, 3H).

[0214] [Corrected according to Rule 91 11.10.2025] Preparation of complete small molecule inhibitor of Example 15A

[0215] Referring to the above synthetic route, compound JYT2-441:

[0216] 2,2',2'-(10-(2-oxo-2-(4-(3-(8-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR))-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxol-2-yl)piperidin-1-yl)ethyl)ureido)naphthalen-2- yl)oxy)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; results as follows: LC-MS: m / z = 852.4 [M-H20+H] +(98.79% purity, 220 nm);1H NMR (400 MHz, D2O) δ 7.81 (d, J = 9.2 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.43 - 7.28 (m, 2H), 7.24 (s, 1H), 7.13 (d, J = 8.8 Hz, 1H), 4.55 - 4.37 (m, 1H), 4.27 - 4.09 (m, 2H), 4.01 - 2.76 (m, 38H), 2.27 - 2.05 (m, 2H), 2.05 - 1.90 (m, 2H), 1.89 - 1.77 (m, 1H), 1.65 - 1.41 (m, 1H).

[0217] The structures of FAPI04, JY2-011 (PNT6555), FAP2286 reported in the literature are shown in Table 1.

[0218] Table 1

[0219] [According to Rule 91 correction 11.10.2025] Experimental Example 1A Inhibitor Affinity Test

[0220] 1. Refer to the literature (Bioorg. Med. Chem. Lett. 2020, 30, 127253.). The specific operation is as follows: dilute the FAP protein concentration to 0.4 μg / mL with assay buffer (25 mM Tris, 250 mM NaCl, pH 7.4); dilute the substrate GP-AMC to 40 μM with assay buffer; prepare different concentrations of inhibitor (compounds 1-20) solution with assay buffer; add 25 μL of inhibitor and 25 μL of substrate to a 96-well black plate in turn, then add 50 μL of protein, and place it in an enzyme marker for incubation at 37°C for 1 h, and measure the fluorescence intensity (Ex / Em = 380 / 460 nm); use Graphpad Prism to fit, using the sigmoidal 11 dose response model, to calculate the IC50.

[0221] 2. Determination of the inhibitory activity of the compound on hFAP:

[0222] 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 with assay buffer to a concentration gradient of 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 protected from light. 50 μL of the prepared protein solution and 25 μL of the inhibitor were added sequentially to a 96-well plate (Corning 96-well microplate, black transparent bottom, number 3904), centrifuged, and vortexed at room temperature for 30 min. Then, 25 μL of the prepared enzyme substrate was added. After incubation at room temperature for 3–5 hours, the fluorescence values ​​at excitation wavelength of 360 nm and emission wavelength of 465 nm were monitored using a multi-functional microplate reader (Agilent BioTek Synergy H1). The IC50 was calculated. 50 The value was defined as the concentration of a compound that caused a 50% decrease in enzyme activity under the assay conditions. The results are shown in Table 2 and Figure 12.

[0223] Table 2

[0224] 3. Location of other urea groups:

[0225] Molecules JYT2-311-1, JTZ2-281-1, and JYZ2-291-1 with urea groups at different positions were synthesized. The synthetic routes were basically similar to those described above, but the enzyme activity IC50 was significantly reduced, all of which were greater than 100 μM.

[0226] The structural formulas and results for molecules JYT2-311-1, JTZ2-281-1, and JYZ2-291-1 are shown below:

[0227] [Corrected to 11.10.2025 according to Detailed Rules 91] Experiment 2A: Labeling and Quality Control

[0228] 1. Preparation of test samples: Accurately measure 100 μL of 2M acetate-sodium acetate solution (pH 5.0) into a 1.5 mL centrifuge tube, and accurately add 2 μL of 20 nmol of the test sample (JYT2-401, JYT2-402, JYT2-406, JYT2-411, JYT2-441, JY2-011(PNT6555), FAPI04, FAP2286) (10 nmol / μL) precursor aqueous solution. Mix thoroughly, and add 0.3 mL of the solution. 68GaCl3 eluate (activity: ~2.28 mCi), was taken out after heating in a metal bath at 95°C for 15 min under sealed condition, and 100 μL of 0.1% DTPA (pH 5.5) was added after the reaction was completed. After cooling to room temperature, a small amount of the post-reaction solution was taken for radiochemical purity analysis.

[0229] The structure of the labeled product of JYT2-401 is:

[0230] HPLC detection: JYT2-401 was subjected to HPLC detection, and the detection conditions are shown in Table 3; and the results are shown in Figure 13.

[0231] Table 3

[0232] 2、 68 Ga-labeled product imaging and biodistribution

[0233] 2.1 PET / CT imaging

[0234] To 68 Ga-labeled 4 molecules (JYT2-401, JYT2-402, JYT2-411, JY2-011 (PNT6555), volume 350 μL) were prepared into test samples (pH ~ 6.1) by adding 500 μL of PBS buffer and 32 μL of 1 M NaOH solution (radioactivity concentration was about 2.7 μCi / μL), and 75 μL of the mixture was taken with an insulin syringe, and the activities were measured as JYT2-401: 192 μCi, JYT2-402: 193 μCi, JYT2-411: 199 μCi, and JY2-011 (PNT6555): 203 μCi, which were injected into mice through the tail vein, and the radioactivity in the mice was measured with a radioactivity meter, and each mouse was injected with only ~ 120 μCi (~ 1 nmol) of the molecule.

[0235] About 1 h after administration, 180 μL of 1% sodium pentobarbital solution was taken with a syringe and administered to the mouse through the abdominal cavity to anesthetize the mouse, and the anesthetized mouse was fixed to the PET / CT instrument bed, and after positioning by CT, PET / CT was scanned.

[0236] After the scan was completed, the data was reconstructed, and the mouse PET data was subjected to ROI circle value with software, and the radioactivity distribution %ID / g of organs and tissues such as heart, kidney, liver, muscle, bladder, and tumor was obtained.

[0237] The results are shown in Figure 14. The PET / CT 1 h imaging results were analyzed by circling the radioactivity signal (%ID / g) in the mouse organs and tumor, and the results showed that JYT2-401 was comparable to the positive control JY2-011 (PNT6555).

[0238] 2.2 Biodistribution

[0239] Injection 68 Ga-labeled different molecules FAPI04, FAP2286, JY2-011 (PNT6555), JYT2-401, JYT2-406, JYT2-411, JYT2-441 tumor-bearing mice HEK293 hFAP n = 3, after different time points, dissection of mice, blood, heart, liver, spleen, lung, kidney, stomach, pancreas, small intestine, large intestine, muscle, femur, tumor, brain, skin, the collected organs, tissues were weighed and measured by gamma counter radioactivity in organs, tissues.

[0240] The biodistribution results at different times are shown in Figure 15. Tail vein injection 68 Ga-labeled different molecules FAPI04, FAP2286, JY2-011 (PNT6555), JYT2-401, JYT2-406, JYT2-411, JYT2-441 compound injection, 2h after administration, different degrees of radioactivity enrichment in tissues and organs, 68 Ga-JYT2-401 and 68 Ga-FAPI04, 68 Ga-PNT6555, 68 Ga-FAP2286 tumor uptake is equivalent, no significant difference; enrichment in other tissues and organs is low.

[0241] 3、 177 Lu labeling and quality control

[0242] 3.1 Labeling process

[0243] Precise amount of compound (JYT2-401, FAP2286, JY2-011 (PNT6555)) solution 4 μL 20 nmol, 50 μL of 0.5M sodium acetate buffer solution pH 4.5 was added accurately, and shaken well, then ~ 2 mCi (~ 2 μL) 177 LuCl3 injection was mixed, and after heating at 95℃ for 15 min in a sealed condition, it was taken out and cooled to room temperature. 150 μL of 0.1% DTPA PBS buffer solution was added, the radioactivity concentration of the solution was detected, and a small amount was taken for radiochemical purity analysis. The radioactivity was detected before and after administration, and the test sample was stored at room temperature and in a lead tank.

[0244] The structure of the product after JYT2-401 labeling is

[0245] 3.2 Quality control analysis

[0246] Radiochemical purity: HPLC: Waters Xbridege 5 μm C-18 (4.6 x 150 mm), flow rate of 1 mL / min; column temperature of 35 °C; detection wavelength of 210 nm; injection volume of 20 μL (50-100 μCi). Gradient elution conditions are shown in Table 4.

[0247] Table 4

[0248] The results are shown in Figure 16. The radiochemical purity was calculated by area normalization method, and was greater than 95%, meeting the requirements of the test.

[0249] The radiochemical purity was calculated by area normalization method, and was greater than 95%, meeting the requirements of the test.

[0250] 3.3 177 Biodistribution of Lu-JYT2-401

[0251] Animal identification: Each animal in the cage must be hung with a cage card, indicating the animal group, animal number, etc.

[0252] Feed supply: The mouse growth maintenance feed meets GB14924.4-2001.

[0253] Feeding method: free access to food.

[0254] Drinking water: drinking water bottles supply autoclaved tap water, which is tested by the Taiyuan monitoring station of the national urban water supply quality monitoring network and meets the requirements of the "Drinking Water Health Standards" (GB5749-2006); drinking water bottles are replaced every 2 days after cleaning and autoclaving.

[0255] Tumor inoculation: The animal model was constructed by Beijing Vivotecnia Animal Technology Co., Ltd., and each experimental animal was inoculated with HEK293 tumor cells subcutaneously on the right side of the back, a total of 10 animals for later experiments.

[0256] The test was divided into 3 groups, and the mice were weighed before grouping, and were randomly grouped according to the animal weight and tumor size.

[0257] Grouping information is shown in Table 5.

[0258] Table 5

[0259] A disposable syringe was used to extract the drug solution, and the activity meter was used to detect the activity (μCi) before administration (full needle); the tail vein was injected with the drug, and the activity meter was used to detect the residual radioactivity count (μCi) after administration (empty needle). 177 The administration dose of Lu-JYT2-401 injection is shown in Table 6.

[0260] Table 6 177Dose Table of Lu-JYT2-401 Injection

[0261] Sampling time: 2h, 24h, 48h after administration.

[0262] Tissues and organs: animals were slightly anesthetized, eyeball was taken out and blood was drawn (blood sample was reserved), heart, liver, spleen, lung, kidney, stomach (contents were removed), large intestine (contents were removed), small intestine (contents were removed), femur (hindlimb), knee, muscle, pancreas, brain, bladder (no urine), tumor, skin, urine, feces (2-3 pellets in large intestine), administration site (tail) and remaining carcass were taken out.

[0263] γ counter detection: biological samples were collected at each time point, residual blood was absorbed with absorbent paper by gently squeezing, and then weighed and recorded, samples were placed in EP tubes, γ counter detection, samples were detected within 24h, (if the radioactivity count was too high, the sample could be measured after decay). The detection results were expressed as CPM (CPM: radioactivity count per minute).

[0264] Data processing and analysis: %ID / g = A tissue / [(A0-A residue )*M tissue ] x 100%.

[0265] A0: total radioactivity count of syringe before administration (CPM).

[0266] A residue : residual radioactivity count of syringe after administration (CPM).

[0267] A tissue : radioactivity count of tissues and organs (CPM).

[0268] M tissue : weight of tissue sample (g).

[0269] Physical decay correction: 177 Lu physical decay: activity dose at time T = 0.5^(T / 160.8).

[0270] The dose (A0-A residue ) multiplied by the decay coefficient of the corresponding time is the corrected CPM value, and %ID / g is calculated.

[0271] Note: within the activity range of not more than 75 μCi, the detection of activity meter (μCi) and the correction curve of γ counter (CPM) are Y = 135628X (R 2 = 0.9991).

[0272] Results are shown in Table 7 and Figure 17. HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice were injected with a single tail vein injection of 177 Lu-JYT2-401 injection, with the extension of time, the blood radioactivity uptake decreased significantly; within the range of 2h-48h after administration, there were different degrees of radioactivity enrichment in tissues and organs, the tumor, kidney and liver had the highest distribution, (the tumor %ID / g at each time point: 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, intestine, lung, bladder, spleen, etc., and the brain had the lowest distribution.

[0273] Table 7 177 Results of Lu-JYT2-401 injection in HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice

[0274] 3.4 SPECT / CT imaging

[0275] To 177 The three Lu-labeled molecules (JYT2-401, FAP2286, JY2-011 (PNT6555)) were added with an appropriate amount of PBS buffer solution to prepare the test sample (pH ~ 5.5-6), and after uniform mixing, 100 μL was drawn by an insulin syringe, and only ~ 400 μCi (1-2 nmol) of the molecule was injected into each mouse, and the activity was measured to be 177 Lu-JYT2-401: 415 μCi, 177 Lu-FAP2286: 435 μCi, JY2-011 (PNT6555): 420 μCi by tail vein injection into the mouse.

[0276] About 48h after administration, 180 μL of 1% sodium pentobarbital solution was drawn by a syringe and administered to the mouse by intraperitoneal injection to anesthetize the mouse, and the anesthetized mouse was fixed on the SPECT / CT instrument bed, and after CT positioning, SPECT / CT was scanned.

[0277] After the scan was completed, the data was reconstructed, and the mouse SPECT data was ROI circled by software to obtain the radioactivity distribution %ID / g of the tumor organs and tissues.

[0278] Results are shown in Figure 18. SPECT / CT imaging was performed at 1h, 2h, 4h, 8h, 24h, 48h, 72h, and the results showed that 177Lu-JYT2-401 remained in the tumor for a long time, with the maximum tumor uptake remaining at ~40% ID / cc. The average tumor uptake was comparable to the biodistribution data in Section 3.3. After 48 hours, it was ~15% ID / cc, and a significant decrease was observed with prolonged exposure.

[0279] The results are shown in Figure 19. (Comparison) 177 Lu-JYT2-401 and control group 177 Lu-FAP2286, 177 Comparison of MIP images from SPECT imaging 48 hours after Lu-PNT6555 injection and the maximum tumor uptake in SPECT images. 177 The maximum uptake of Lu-JYT2-401 was significantly higher than that of [previous study]. 177 Lu-FAP2286, 177 Lu-PNT6555. The molecular component is currently undergoing clinical trials for treatment. 177 Lu-FAP2286, and 177 Lu-FAP2286 tumor uptake was also higher than 177 Lu-PNT6555, therefore, a comparison will follow. 177 Lu-JYT2-401 and 177 Pharmacological study of Lu-FAP2286.

[0280] 3.5 Pharmacodynamic Study

[0281] The aim of this study was to evaluate a single injection by assessing tumor growth delay. 177 The therapeutic effects of Lu-JYT2-401.

[0282] Provide one concentration 177 Lu-JYT2-401 and 177 Lu-FAP2286 was prepared for injection (100 μL / mouse), and the injection dose was determined using a radioactivity meter (CAPINTEC calibrated).

[0283] Provided as described in Section 3.1 above 177 Lu-JYT2-401 and 177 Lu-FAP2286. Prepare the following therapeutic compositions:

[0284] 1. Medium (physiological saline, 100 μL) per animal

[0285] 2. 177 Lu-JYT2-40115MBq (100μL) per animal

[0286] 3. 177 Lu-FAP228615MBq (100μL) per animal

[0287] A total of 15 HEK293 human embryonic kidney cells Balb / c nude tumor-bearing mice were used for single tail vein injection 177 Lu-JYT2-401 and 177 Lu-FAP2286 injection. Throughout the study, mice were examined for health, including body weight measurements, weekly. Tumor growth was monitored weekly with caliper measurements (tumor volume = length x width2x 0.5). Study endpoints included any size tumor >2 cm, tumor ulceration formation, mouse moribundity, and >15% body weight loss from last measurement. Mice were housed 5 / cage with ad libitum access to food and water in an environment temperature of 20 °C, 40-50% humidity, and 12 hours light / 12 hours dark cycle. Injected doses were determined using a radioactivity meter (CAPINTEC calibrated). Tumor growth was monitored weekly with caliper measurements, and mice survival was tracked.

[0288] Results: Data were collected as tumor volume graph 20 and survival analysis graph 21.

[0289] By comparing tumor volume, survival rate, and TGI analysis, 177 Lu-JYT2-401 tumor inhibition is superior to 177 Lu-FAP2286, and survival is also longer than 177 Lu-FAP2286.

[0290] Table 8

[0291] The compounds shown in relation to the structure of general formula (I-2) and their application verification:

[0292] [According to Rule 91 correction 11.10.2025] Preparation of small molecule inhibitors of Example IB

[0293] The synthesis route of JYQ2-452 is as follows:

[0294] Step one: tert-butyl (3-(methoxy(methyl)carbamoyl)bicyclo[1.1.1]pentan-1- yl)carbamate

[0295] Dissolve 3-((tert-butoxycarbonyl)amino)bicyclo[l. l. l]pentane-l -carboxylic acid (300 mg, 1.32 mmol) in dichloromethane (10 mL), add dimethylhydroxylamine hydrochloride (154 mg, 1.58 mmol, 1.2 eq), HATU (602 mg, 1.58 mmol, 1.2 eq) and DIPEA (512 mg, 3.96 mmol, 3.0 eq). React at room temperature for 2 hours. TLC detection shows that the reaction is complete. Add water (50 mL), extract with dichloromethane (100 mL x 2), concentrate the organic phase, and purify the crude product by silica gel column chromatography (PE / EA = 1 / 1) to obtain white solid JYQ2-452-1 (354 mg, 1.31 mmol, 99.2% yield). MS (ES+) m / z 271.2 [M+H] + .

[0296] Step two: tert-butyl (3-formylbicyclo[l. l. l]pentan-l -yl)carbamate

[0297] Dissolve JYQ2-452-1 (441 mg, 1.63 mmol) in anhydrous tetrahydrofuran (8 mL), slowly add DIBAL-H (3.26 mL, 1M, 2.0 eq) dropwise at -70°C under nitrogen protection. React at -70°C for 1 hour. TLC detection shows that the reaction is complete. Add methanol (10 mL) to the reaction solution and stir for 10 minutes. Add dilute hydrochloric acid to adjust the pH to 3, add water (10 mL), extract with ethyl acetate (50 mL x 2), combine the organic layers and wash with saturated sodium chloride solution (20 mL), dry the organic layer with anhydrous sodium sulfate, filter and concentrate to obtain white solid JYQ2-452-2 (279 mg, 1.32 mmol, 81.0% yield). MS (ES+) m / z 212.2 [M+H] + .

[0298] Step three: (E)-ethyl 3-(3-((tert-butoxycarbonyl)amino)bicyclo[l. l. l]pentan-l - yl)acrylate

[0299] Dissolve JYQ2-452-2 (279 mg, 1.32 mmol) in tetrahydrofuran (8 mL), add ethoxycarbonylmethylidene triphenylphosphonium (460 mg, 1.32 mmol, 1.0 eq). React at room temperature for 15 hours. TLC detection shows that the reaction is complete. Concentrate the reaction solution, and purify the crude product by silica gel column chromatography (PE / EA = 2 / 1) to obtain white solid JYQ2-452-3 (317 mg, 1.13 mmol, 85.3% yield). MS (ES+) m / z 282.3 [M+H] + .

[0300] Step four: methyl 3-(3-((tert-butoxycarbonyl)amino)bicyclo[l. l. l]pentan- 1 - yl)propanoate

[0301] JYQ2-452-3 (317 mg, 1.13 mmol) was dissolved in methanol (8 mL), Pd / C (100 mg, 10%) was added, and the reaction was allowed to proceed at room temperature for 3 hours. TLC detection showed that the reaction was complete. Filtration and concentration of the mother liquor yielded white solid JYQ2-452-4 (283 mg, 1.05 mmol, 93.0% yield).

[0302] Step five: tert-butyl (3-(3-hydroxypropyl)bicyclo[l. l. l]pentan-l -yl)carbamate

[0303] JYQ2-452-4 (280 mg, 1.04 mmol) was dissolved in tetrahydrofuran (10 mL), and LAH (79 mg, 2.08 mmol, 2.0 equivalents) was added portionwise at 0 °C. After addition, the reaction was allowed to proceed at 0 °C for 1 hour. TLC detection showed that the reaction was complete. The reaction solution was stirred for 10 minutes after the addition of sodium sulfate decahydrate, and then filtered. Concentration of the mother liquor yielded colorless solid JYQ2-452-5 (246 mg, 1.02 mmol, 98.0% yield). MS (ES+) m / z 186.1 [M-56+H] + .

[0304] Step six: 3-(3-((tert-butoxycarbonyl)amino)bicyclo[l. l. l]pentan-l -yl)propyl methanesulfonate

[0305] JYQ2-452-5 (246 mg, 1.02 mmol) was dissolved in dichloromethane (10 mL), and triethylamine (309 mg, 3.06 mmol, 3.0 equivalents) was added. Methylsulfonyl chloride (152 mg, 1.33 mmol, 1.3 equivalents) was added at 0 °C. After addition, the reaction solution was allowed to warm to room temperature and react for 1 hour. TLC detection showed that the reaction was complete. The reaction solution was added to water (50 mL), and dichloromethane (50 mL x 2) was extracted. Concentration of the organic phase yielded white solid JYQ2-452-6 (305 mg, 0.95 mmol, 93.6% yield).

[0306] Step seven: benzyl 4-(3-((tert-butoxycarbonyl)amino)bicyclo[l. l. l]pentan-l -yl)propyl)piperazine- 1 -carboxylate

[0307] JYQ2-452-6 (305 mg, 0.95 mmol) was dissolved in DMF (10 mL), benzyl-1-piperazine carbonate (210 mg, 0.95 mmol, 1.0 eq), potassium carbonate (262 mg, 1.9 mmol, 2.0 eq) and sodium iodide (142 mg, 0.95 mmol, 1.0 eq) were added. After addition, the reaction was heated to 80 °C for 3 h. TLC detection showed that the starting material was completely reacted. The reaction was added with water (50 mL), extracted with ethyl acetate (50 mL x 2), the organic phase was concentrated, and the crude product was purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to give yellow liquid JYQ2-452-7 (419 mg, 0.94 mmol, yield 99.4%). MS (ES+) m / z 444.3 [M+H] + .

[0308] Step eight: 4-(3-(3-aminobicyclo[l. l. l]pentan- l-yl)propyl)piperazine- l-carboxylic acid benzyl ester

[0309] JYQ2-452-7 (1.05 g, 2.37 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added, and the reaction was stirred at room temperature for 1 h. TLC detection showed that the starting material was completely reacted. The crude product was concentrated, added with water (20 mL), and the pH was alkalized to 9 with saturated sodium bicarbonate solution. Dichloromethane (50 mL) was added for extraction, the organic phase was dried and concentrated to give yellow liquid JYQ2-452-8 (755 mg, 2.20 mmol, yield 92.7%).

[0310] Step nine: 4-(3-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6- methylbenzo[d][l,3,2]dioxol-2-yl)piperidin-l-yl)ethyl)ureido)bicyclo[l. l. l]pentan- l-yl)propyl)piperazine- l-carboxylic acid benzyl ester

[0311] To a solution of JYQ2-452-8 (378 mg, 1.10 mmol) and triethylamine (245 mg, 2.42 mmol, 2.2 eq) in dichloromethane (20 mL) was added a solution of triphosgene (108 mg, 0.36 mmol, 0.33 eq) in dichloromethane (20 mL) dropwise at 0 °C. After the addition, the reaction was continued at 0 °C for 30 min. TLC detection showed that the starting material was consumed completely. To the reaction mixture was added JY2-111-1-5 (303 mg, 0.99 mmol, 0.9 eq) and the mixture was stirred at room temperature for 1 h. TLC detection showed that the starting material was consumed completely. The reaction mixture was diluted with water (30 mL) and extracted with dichloromethane (50 mL x 2). The combined organic layers were washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography on silica gel (DCM / MeOH = 20 / 1) to give JYQ2-452-9 (320 mg, 0.47 mmol, 43.1% yield) as a colorless solid. MS (ES+) m / z 676.4 [M+H] + .

[0312] Step Ten: 1-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6- methanobenzo [1,3,2]dioxol-2-yl)pyrrolidin-1 -yl)ethyl)-3-(3-(3-(piperazin-1 -yl)propyl) bicyclo[1.1.1]pentan-1-yl)urea

[0313] JYQ2-452-9 (320 mg, 0.47 mmol) was dissolved in methanol (10 mL) and Pd / C (100 mg, 10%) was added. The mixture was stirred at room temperature for 1 h. TLC detection showed that the starting material was consumed completely. The mixture was filtered and the filtrate was concentrated to give JYQ2-452-10 (251 mg, 0.46 mmol, 98.6% yield) as a white solid.

[0314] Step Eleven: 2,2',2'-(10-(2-oxo-2-(4-(3-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5- trimethylhexahydro-4,6-methanobenzo [1,3,2]dioxol-2-yl)pyrrolidin-1 -yl)ethyl)ureido) bicyclo[1.1.1]pentan-1-yl)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane- 1,4,7-triyl)tris (tert-butyl) acetate

[0315] Tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (191 mg, 0.33 mmol, 1.0 eq) was dissolved in DMF (10 mL), PYBOP (206 mg, 0.40 mmol, 1.2 eq) and DIPEA (128 mg, 0.99 mmol, 3.0 eq) were added. The reaction was continued at room temperature for 30 min. JYQ2-452-10 (180 mg, 0.33 mmol) was added and the reaction was continued at room temperature for 3 h. TLC detection showed that the starting material was completely reacted. The reaction solution was added with water (50 mL) and extracted with ethyl acetate (50 mL x 2). The organic phase was concentrated and the crude product was purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to obtain yellow viscous liquid JYQ2-452-11 (203 mg, 0.19 mmol, yield 56.1%).

[0316] Step twelve: 2,2',2'-(10-(2-oxo-2-(4-(3-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxol-2-yl)pyrrolidin-1-yl)ethyl)ureido) bicyclo[1.1.1]pentan-1-yl)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid

[0317] JYQ2-452-11 (203 mg, 0.19 mmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (2 mL) was added. The reaction was continued at room temperature for 15 h. Directly concentrated to obtain purple solid JYQ2-452-12 (crude product), which was directly used in the next step.

[0318] Step thirteen: (R)-2,2',2'-(10-(2-(4-(3-(3-(2-(2-bromopyrrolidin-1-yl)-2-oxoethyl)ureido) bicyclo[1.1.1]pentan-1-yl)propyl)piperazin-1-yl)2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7- triyl)triacetic acid

[0319] The above crude JYQ2-452-12 was dissolved in water (6 mL) and acetonitrile (3 mL), benzeneboronic acid (29 mg, 0.24 mmol, 1.3 eq), trifluoroacetic acid (2d) and MTBE (18 mL) were added. The reaction was stirred at room temperature for 3 hours. The mixture was partitioned and the aqueous phase was washed with MTBE (10 mL). The aqueous phase was purified by RP-HPLC (Waters preparative chromatograph equipped with a SunFire® Prep C18 OBD 5um 30x250mm column using a gradient: 95:5 to 20:80 water / acetonitrile + 0.1% TFA, 20 minutes) and the fractions were collected and lyophilized to give JYQ2-452 as a white solid (5 mg, 0.0037 mmol, 1.9% yield over two steps). MS (ES+) m / z 776.4 [M - 5 x CF3COOH (molecular weight 114.02) - H2O (molecular weight 18) + H] + (90.91% purity, 210 nm).

[0320] 1 H NMR (400 MHz, D2O) δ 4.54 - 4.42 (m, 1H), 3.99 - 3.64 (m, 8H), 3.60 (s, 2H), 3.55 - 3.25 (m, 13H), 3.21 - 2.85 (m, 14H), 2.09 - 1.94 (m, 3H), 1.92 - 1.73 (m, 7H), 1.71 - 1.43 (m, 5H).

[0321] HPLC results for JYQ2-452 are shown in Table 9 and Figure 23. 1 H NMR see Figure 22.

[0322] HPLC results for JYQ2-452 are shown in Table 9 and Figure 23.

[0323] Wherein, the liquid phase detection conditions include: chromatographic column: YMC-Triart C18, 3um 4.6x150mm; flow rate 1ml / min; column temperature: 30°C; mobile phase: D: 0.1% HCIO4 solution of H2O pH=1.5, C: ACN.

[0324] Table 9

[0325] MS results for JYQ2-452 are shown in Figure 24.

[0326] [Corr. According to Rule 91 11.10.2025] Preparation of small molecule inhibitors of Example 2B

[0327] The synthesis route for JYT-4521 is shown below:

[0328] Step one: N-[2-(4-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-7,10-bis{2-[(2- methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododec-1-yl)acetyl]-L- glutamic acid dibenzyl ester

[0329] Dissolve L-glutamic acid dibenzyl ester hydrochloride (0.83 g, 2.27 mmol, 1.3 eq) in DCM (20 mL), add TEA (0.53 g, 5.24 mmol, 3.0 eq), DOTA-(OtBu)3(1.00 g, 1.75 mmol) successively, stir for 5 min at room temperature, add BOPCl (0.67 g, 2.62 mmol, 1.5 eq) at 0-5 °C, after addition, raise to room temperature and react for 16 h. TLC shows that the reaction is completed. Add saturated ammonium chloride (10 mL) to the reaction solution, wash the aqueous phase with DCM (10 mL x 2), combine the organic phases, wash with saturated sodium chloride (20 mL), dry over anhydrous sodium sulfate, filter, concentrate, and purify the crude product by silica gel column chromatography to obtain JYT-4521-1 (1.04 g, 1.18 mmol, yield 67.4%) as colorless oil. MS (ES+) m / z 441.8 [M / 2+H] + .

[0330] Step two: N-[2-(4-{1-[(2-methylpropan-2-yl)oxy]-1-oxoethyl-2-yl}-7,10-bis{2-[(2- methylpropan-2-yl)oxy]-2-oxoethyl}-1,4,7,10-tetraazacyclododec-1-yl)acetyl]-L- glutamic acid

[0331] Dissolve JYT-4521-1 (0.41 g, 0.46 mmol) in methanol (5 mL), add 10% Pd / C (40 mg), replace with a hydrogen balloon for 3 times, stir for 16 h at room temperature, filter the reaction solution, wash the filter cake with methanol (3 mL), and concentrate the filtrate to obtain JYT-4521-2 (0.32 g, 0.46 mmol, yield 99.1%) as white foamy solid. MS (ES+) m / z 702.4 [M+H] + .

[0332] Step three: [10-(2-{[(2S)-5-{4-[3-(1-{[({2-[(2R)-2-[(1S,2S,6R,8S)-2,9,9- trimethyl-4-bora-3,5-dioxatricyclo[6.1.1.02,6]dec-4-yl]tetrahydro-1H-pyrrol-1-yl]-2- oxovinyl}amino)carbonyl]amino}bicyclo[1.1.1]pent-3-yl)propyl]piperazin-1-yl}-1-{4-[3-(3-{ [({2-[(2R)-2-[(1S,2S,6R,8S)-2,10,10-trimethyl-4-bora-3,5-dioxatricyclo[6.1.1.02,6]dec-4- yl]tetrahydro-1H-pyrrol-1-yl]-2-oxovinyl}amino)carbonyl]amino}bicyclo[1.1.1]pent-1- yl)propyl]piperazin-1-yl}-1,5-dioxovinyl]amino}-2-oxovinyl)ethyl)-4,7-bis{2-[(2- methylprop-2-yl)oxy]-2-oxovinyl}-1,4,7,10-tetraazacyclododecan-1-yl]acetate-2- methylprop-2-yl ester

[0333] JYT-4521-2 (95 mg, 0.14 mmol) was dissolved in DMF (8 mL), DMTMM (97 mg, 0.35 mmol, 2.5 eq) and DIPEA (72 mg, 0.56 mmol, 4.0 eq) were added. Stirring at room temperature for 30 min, JYQ2-452-10 (150 mg, 0.28 mmol, 2.0 eq) was added. Reaction at room temperature for 5 h. TLC detection of the raw material reaction was complete. Add water (10 mL), precipitate solid, filter and dry to get yellow solid JYT-4521-3 (crude), directly into the next step.

[0334] Step four: [4,7-bis(carboxymethyl)-10-(2-{[(2S)-5-{4-[3-(1-{[({2-[(2R)-2-[(1S,2S,6R,8S)-2,9,9- trimethyl-4-bora-3,5-dioxabicyclo[6.1.1.02'6]dec-4-yl]tetrahydro-1H-pyrrol-1-yl]-2- oxovinyl}amino)carbonyl]amino}bicyclo[1.1.1]pent-3-yl)propyl]piperazin-1-yl}-1-{4-[3-(3-{[({2- [(2R)-2-[(1S,2S,6R,8S)-2,10,10-trimethyl-4-bora-3,5-dioxabicyclo[6.1.1.02'6]dec-4-yl]tetrahydro- 1H-pyrrol-1-yl]-2-oxovinyl}amino)carbonyl]amino}bicyclo[1.1.1]pent-1-yl)propyl]piperazin-1- yl}-1,5-dioxovinyl]amino}-2-oxovinyl)ethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid

[0335] Dissolve the above crude JYT-4521-3 in dichloromethane (2 mL) and add trifluoroacetic acid (1 mL). Stir at room temperature for 15 hours. TLC shows the reaction is complete. Concentrate the reaction mixture directly to give brown liquid JYT-4521-4 (crude) which is used directly in the next step.

[0336] Step four: [4,7-bis(carboxymethyl)-10-(2-{[(2S)-5-{4-[3-(1-{[({2-[(2R)-2-[(1S,2S,6R,8S)-2,9,9- trimethyl-4-bora-3,5-dioxabicyclo[6.1.1.02'6]dec-4-yl]tetrahydro-1H-pyrrol-1-yl]-2- oxovinyl}amino)carbonyl]amino}bicyclo[1.1.1]pent-3-yl)propyl]piperazin-1-yl}-1-{4-[3-(3-{[({2- [(2R)-2-[(1S,2S,6R,8S)-2,10,10-trimethyl-4-bora-3,5-dioxabicyclo[6.1.1.02'6]dec-4-yl]tetrahydro- 1H-pyrrol-1-yl]-2-oxovinyl}amino)carbonyl]amino}bicyclo[1.1.1]pent-1-yl)propyl]piperazin-1- yl}-1,5-dioxovinyl]amino}-2-oxovinyl)ethyl)-1,4,7,10-tetraazacyclododecan-1-yl]acetic acid

[0337] Dissolve the above crude JYT-4521-4 in a mixture of water (4 mL), acetonitrile (2 mL) and MTBE (12 mL). Add phenylboronic acid (34 mg, 0.28 mmol, 2.0 eq) and trifluoroacetic acid (1 drop). Stir at room temperature for 3 hours. Add water (8 mL) to the reaction mixture and separate the layers. Wash the aqueous layer with MTBE (6 mL). Lyophilize the aqueous layer and purify by HPLC to give white solid JYT-4521 (6 mg, 0.003 mmol, 2.1% over three steps). MS (ES+) m / z 629.9 [(M-3xH2O (molecular weight 18)-6xCF3COOH (molecular weight 114.02)) / 2+H] + (99.82% purity, 210 nm).

[0338] 1 H NMR (400 MHz, D20) δ 4.58 - 4.22 (m, 3H), 4.19 - 4.02 (m, 1H), 3.96 - 2.80 (m, 52H), 2.61 - 2.44 (m, 2H), 2.08 - 1.92 (m, 5H), 1.88 - 1.73 (m, 15H), 1.73 - 1.41 (m, 9H).

[0339] Figure 25 is the HPLC result of JYT-4521. 1 H NMR result.

[0340] The HPLC result of JYT-4521 is shown in Figure 26 and Table 10.

[0341] Table 10

[0342] Figure 27 is the MS result of JYT-4521.

[0343] [Rule 91 Correction 11.10.2025] Comparative Example 1B

[0344] [Rule 91 Correction 11.10.2025] Comparative Example 1B provides the structural formula of JYT2-401, which is shown in Table 11.

[0345] Table 11

[0346] [Rule 91 Correction 11.10.2025] Inhibitor affinity test of the examples of Experimental Example 1B

[0347] 1.1 Refer to the literature (Bioorg. Med. Chem. Lett. 2020, 30, 127253.) for details. The specific operation is as follows: dilute the FAP protein to a concentration of 0.4 μg / mL with assay buffer (25 mM Tris, 250 mM NaCl, pH 7.4); dilute the substrate GP-AMC to 40 μM with assay buffer; prepare a solution of the inhibitor (compounds 1-20) at different concentrations with assay buffer; sequentially add 25 μL of the inhibitor and 25 μL of the substrate to a 96-well black plate, then add 50 μL of the protein, and place it in an enzyme marker for incubation at 37°C for 1 h, and measure the fluorescence intensity (Ex / Em = 380 / 460 nm); use Graphpad Prism to fit, using the sigmoidal 11 dose response model, to calculate the IC50.

[0348] 1.2 Determination of the hFAP inhibitory activity of the compounds:

[0349] hFAP protein was diluted to 0.3 pg / mL using assay buffer (25 mM Tris, 250 mM NaCl, pH = 7.4) and stored on ice. Compounds were diluted to a certain concentration gradient using assay buffer, 40 mM, 4 mM, 400 nM, 40 nM, 4 nM, 400 pM, 40 pM and 4 pM, respectively. The enzyme substrate Z-Gly-Pro-AMC was diluted to 40 mM using assay buffer and stored in the dark. The above prepared 50 pL protein solution and 25 pL inhibitor were added into 96-well plates (Corning 96-well enzyme plate black transparent bottom, No. 3904) in turn, and after centrifugation, they were shaken at room temperature for 30 min. Then, 25 pL of the above prepared enzyme substrate was added. After incubation at room temperature for 3-5 hours, the fluorescence value at an excitation wavelength of 360 nm and an emission wavelength of 465 nm was monitored using a multifunctional enzyme marker (Agilent BioTek Synergy H1). The IC 50 value, defined as the concentration of the compound that causes a 50% reduction in enzyme activity under the assay conditions, is shown in Table 12 and Figure 28.

[0350] Table 12

[0351] [Corrected according to Rule 91 11.10.2025] Experimental Example 2B 68 Ga labeling example for PET imaging experiment

[0352] 2. 68 Ga labeling and quality control

[0353] 2.1 Preparation process

[0354] The labeling process was carried out in duplicate: 1-4 pL of 20 nmol of compound (JYQ2-452, JYT-4521) solution was accurately measured, 500 pL of 1 M acetic acid-sodium acetate solution (pH 5.0) and 200 pL of 100 mg / mL N-acetyl-L-tyrosine solution (1 M acetic acid-sodium acetate, pH 5.0) were taken into a 2 mL centrifuge tube, mixed thoroughly, and 0.75 ml 68 GaCl3 eluent was heated at 95°C in a metal bath for 15 min under sealed conditions. After cooling to room temperature, the two reaction solutions were passed through a C18 column, and 1 mL of 50% ethanol-saline solution was used for elution 68 Ga labeled product 68 Ga-JYQ2-452, 68 Ga-JYT-4521), the eluate was collected, diluted with 4 mL of saline after adding to the eluate, and used for animal administration. The radioactivity was detected before and after administration, and the test sample was stored at room temperature and in a lead tank, and was prepared and used immediately.

[0355] The labeled products of JYQ2-452 and JYT-4521 are, respectively:

[0356] 2.2 Quality control analysis

[0357] Radiochemical purity: HPLC: YMC Triart C18, 3 μm, 4.6 mm x 150 mm Column, flow rate of 1 mL / min; column temperature of 35 °C; detection wavelength of 210 nm; injection volume of 20 μL (50-100 μCi). The gradient elution conditions are shown in Table 13.

[0358] Table 13

[0359] The results are shown in Figures 29-32 and Tables 14-17. The radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 90%, which met the requirements of the test. The ultraviolet absorption peaks of the precursor and related impurities, and the ultraviolet absorption peaks of the excipients and related impurities were not counted before data statistics.

[0360] Table 14 68 Results of Ga-JYQ2-452 HPLC radioactivity signals

[0361] Note: The radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 90%, which met the requirements of the test.

[0362] Table 15 68 Results of Ga-JYQ2-452 HPLC ultraviolet signals (absorption wavelength 210 nm)

[0363] Note: The ultraviolet absorption peaks of JYQ2-452 and related impurities were counted.

[0364] Table 16 68 Results of Ga-JYT-4521 HPLC radioactivity signals

[0365] Note: The radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 90%, which met the requirements of the test.

[0366] Table 17 68 Results of Ga-JYT-4521 HPLC ultraviolet signals (absorption wavelength 210 nm)

[0367] Note: The ultraviolet absorption peaks of JYT-4521 and related impurities were counted.

[0368] 2.3 Animal model and PET imaging process

[0369] Animal identification: Each cage of animals must be hung with cage cards, indicating animal group, animal number, etc.

[0370] Provide feed: The mouse growth maintenance feed meets GB14924.4-2001.

[0371] Feeding method: free feeding.

[0372] Drinking water: drinking water bottles supply high-pressure sterilized tap water, which is tested by Taiyuan monitoring station of national urban water supply water quality monitoring network and meets the requirements of “Drinking Water Health Standards” (GB5749-2006); drinking water bottles are replaced every 2 days and used after cleaning and high-pressure sterilization.

[0373] Tumor inoculation: The animal model construction was completed by Beijing Weitong Lihua Animal Technology Co., Ltd. Each experimental animal was inoculated with HEK293T hFAP human embryonic kidney cell transfected human FAP high expression cell line tumor cells subcutaneously on the right side of the back, a total of 3 animals, for PET / CT imaging experiment.

[0374] Experimental design: The drug solution was drawn by a disposable syringe, and the activity (μCi) before administration (full needle) was detected by an activity meter; the drug was injected through the tail vein, and the residual radioactivity count (μCi) after administration (empty needle) was detected by an activity meter. The tumor-bearing mice were anesthetized at 0.5h, 2h, 4h after administration, and PET / CT imaging was performed, and the mouse PET imaging data were circled by software to obtain 68 Ga-JYQ2-452, 68 Ga-JYT-4521 tissue distribution and metabolism data of mice at different time points %ID / cc. Figure 33 is 68 PET / CT imaging of Ga-JYQ2-452 and 68 Ga-JYT-4521.

[0375] Table 18 68 PET image values of Ga-JYQ2-452 in the region of interest at different time points

[0376] Table 19 68 PET image values of Ga-JYT-4521 in the region of interest at different time points

[0377] 2.4 Results and discussion

[0378] Single tail vein injection of HEK293T hFAP human embryonic kidney cell transfected human FAP high expression cell line Balb / c nude inoculated tumor-bearing mice68 Ga-JYQ2-452 and 68 Ga-JYT-4521 compound injection, PET / CT imaging was performed at 0.5h, 2h, 4h after administration, and different degrees of radioactive enrichment were present in tissue organs. 68 Ga-JYQ2-452 and 68 Ga-JYT-4521 has low uptake value in normal organ tissues (<2% ID / cc), 68 Ga-JYQ2-452 is mainly enriched in tumors, and at 4h after administration, the average uptake value in tumors is 11.59±1.48% ID / cc, and the maximum value is 20.73±2.24% ID / cc, which is slightly higher than 68 Ga-JYT-4521 in tumors.

[0379] [According to Rule 91 correction 11.10.2025] Experimental Example 3B 177 Lu labeled examples for biodistribution experiments

[0380] 3.1 177 Process of Lu labeled examples

[0381] Precise amount of compound (JYQ2-452, JYT-4521) solution 1-4 μL 20 nmol, add 10 μL 100 mg / mL N-acetyl-L-methionine, 40 μl 100 mg / mL N-acetyl-L-tyrosine and 5 μL 100 mg / mL Vc to 1.5 mL centrifuge tube, mix well, then add 1-2 μL 177 LuCl3 (activity: 1-2 mCi, 0.1M HCl solution), heat at 90℃ in a sealed condition in a metal bath for 15 min, then take out, add 5 μL DTPA (50 mg / mL) after the reaction is completed. After cooling to room temperature, detect HPLC. Add 200 μL 0.5M HAc-NaAc (pH 5.5) and 2 mL deionized water, adjust the pH of the solution to 5.0, complete the preparation of the product 177 Lu-JYQ2-452, 177 Lu-JYT-4521) for animal administration.

[0382] The labeled products of JYQ2-452 and JYT-4521 are respectively:

[0383] 3.2 Quality control analysis

[0384] Radiochemical purity: HPLC: YMC Triart C18, 3 μm, 4.6 mm x 150 mm Column, flow rate of 1 mL / min; column temperature of 35 °C; detection wavelength of 210 nm; injection volume of 20 μL (50-100 μCi). Gradient elution conditions are shown in Table 20.

[0385] Table 20

[0386] Results are shown in Figures 34-37 and Tables 21-24. Radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 90%, which met the requirements of the test. The UV absorption peaks of the precursor and related impurities, and the UV absorption peaks of the excipients and related impurities were not counted before data statistics.

[0387] Table 21 177 Results of Lu-JYQ2-452 HPLC radioactivity signals

[0388] Note: Radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 90%, which met the requirements of the test.

[0389] Table 22 177 Results of Lu-JYQ2-452 HPLC UV signals (absorption wavelength 210 nm)

[0390] Note: The UV absorption peaks of JYQ2-452 and related impurities were counted.

[0391] Table 23 177 Results of Lu-JYT-4521 HPLC radioactivity signals

[0392] Note: Radiochemical purity was calculated by area normalization method, and the radiochemical purity was greater than 90%, which met the requirements of the test.

[0393] Table 24 177 Results of Lu-JYT-4521 HPLC UV signals (absorption wavelength 210 nm)

[0394] Note: The UV absorption peaks of JYT-4521 and related impurities were counted.

[0395] 3.3 Animal model and biodistribution results

[0396] Animal identification: Each animal in the cage must be hung with a cage card, indicating the animal group, animal number, etc.

[0397] Provide feed: The mouse growth maintenance feed meets GB14924.4-2001.

[0398] Dosing method: free access.

[0399] Drinking water: drinking bottle supply autoclaved tap water, tap water inspection by the national urban water supply water quality monitoring network Taiyuan monitoring station, in line with the requirements of "Drinking Water Health Standards" (GB5749-2006); every 2 days to replace the drinking water bottle, drinking water bottle after cleaning, autoclaving use.

[0400] Tumor inoculation: animal model construction was completed by Beijing VitoLiva Animal Technology Co., Ltd., each experimental animal was inoculated with HEK293 tumor cells subcutaneously on the right side of the back, a total of 10 animals for later experiments.

[0401] Disposable syringe to extract liquid medicine, activity meter to detect activity (μCi) before dosing (full needle); tail vein injection for dosing, activity meter to detect residual radioactivity count (μCi) after dosing (empty needle).

[0402] Sampling time: 2h, 24h, 48h, 144h after dosing;

[0403] Tissue organs: animals were slightly anesthetized, eyeball was removed and blood was drawn (blood sample was retained), heart, liver, spleen, lung, kidney, stomach (contents removed), large intestine (contents removed), small intestine (contents removed), femur (hindlimb), knee, muscle, pancreas, brain, bladder (no urine), tumor, skin, urine, feces (2-3 grains in large intestine), dosing site (tail) and remaining carcass were removed.

[0404] γ counter detection: biological samples were collected at each time point, residual blood was absorbed with absorbent paper by gently squeezing, weighed and recorded, samples were placed in EP tubes, γ counter detection, sample detection within 24h, (if radioactivity count is too high, it can be measured after decay). The detection results are expressed in CPM (CPM: radioactivity count per minute).

[0405] Data processing and analysis: %ID / g = A tissue / [(A0-A residue )×M tissue ]×100%.

[0406] A0: total radioactivity count (CPM) of the syringe before dosing.

[0407] A residue : residual radioactivity count (CPM) of the syringe after dosing.

[0408] A tissue : radioactivity count (CPM) of tissue organs.

[0409] M tissue : weight (g) of the detected tissue sample.

[0410] Physical attenuation correction: 177 Lu physical decay: Activity dose at time T = 0.5^(T / 160.8).

[0411] Dosage (A0-A) residue Multiply by the decay coefficient for the corresponding time to get the corrected CPM value, and calculate %ID / g.

[0412] Note: Within an activity range not exceeding 75 μCi, the calibration curve between the activity meter detection (μCi) and the γ counter (CPM) is Y = 135628X(R). 2 =0.9991).

[0413] Table 25 177 Detailed data on the biological distribution of Lu-JYQ2-452 at different time points

[0414] Table 26 177 Lu-JYQ2-452 and 177 Comparison of Lu-JYT-4521 injection 177 Lu-JYT2-401

[0415] Tissue distribution of HEK293 human embryonic kidney cells in Balb / c nude tumor-bearing mice after 48 hours.

[0416] 2.4 Results and Discussion

[0417] The results are shown in Tables 25-26 and Figure 38. HEK293T hFAP human embryonic kidney cells transfected with human FAP-highly-expressing cell lines were seeded into Balb / c nude tumor-bearing mice via a single tail vein injection. 177 The biodistribution experiment of Lu-JYQ2-452 compound injection was conducted at 2h, 24h, 48h and 144h after administration. Different degrees of radioactive enrichment were observed in tissues and organs. After 144h of administration, normal organs such as liver, spleen and skin showed a small amount of uptake (1-4% ID / g). 177 Lu-JYQ2-452 was mainly enriched within tumors. Two hours after administration, intratumoral uptake was 20.48 ± 1.06% ID / g, reaching a peak of 26.64 ± 1.81% ID / g at 48 hours, and remaining at 22.55 ± 5.05% ID / g after 144 hours. (Compared to the reference compound.) 177 Lu-JYT2-401 and 177 Biodistribution data of Lu-JYT-4521 48 hours after injection showed that the uptake values ​​of the three compounds in normal tissues and organs were basically the same 48 hours after injection. 177The tumor uptake of Lu-JYQ2-452 was 26.64±1.81%ID / g, which was higher than 177 Lu-JYT2-401 (13.64±1.85%ID / g) and 177 Lu-JYT-4521 (12.05±0.58%ID / g), so the series of molecules of the bicyclo[1.1.1]pentane substituted quinoline structure FPAI compounds designed by the present application have certain advantages for treatment.

[0418] It should be noted that in the present application, JYT-4521 is a dimeric compound of JYQ2-452, but does not improve the tumor uptake and retention time, which is a characteristic of the bicyclo[1.1.1]pentane substituted quinoline structure FPAI molecule.

[0419] The compounds represented by the structure of general formula (I-3) and their application verification:

[0420] [Corrected according to Rule 91 11.10.2025] Preparation of small molecule inhibitors of Example 1C

[0421] The synthesis route of JYT2-4013 is as follows:

[0422] Step one: synthesis of 4-methyl-8-nitroquinoline

[0423] At 0°C and under nitrogen, 4-methylquinoline (12.5 g, 87.3 mmol) was slowly added in batches to sulfuric acid (38 mL), after addition, a mixture of sulfuric acid (3.3 mL) and nitric acid (9.3 mL) was slowly added dropwise, and the reaction was incubated for 2 hours, then the temperature was raised to room temperature for 16 hours. The reaction liquid was poured into ice water for quenching, ammonia was slowly added at 0°C until solid precipitated, filtered, washed with water, and the filter cake was dried. The crude product was dispersed in boiling ethanol and stirred for 30 minutes, then filtered hot, and the filter cake was washed with ethanol. After drying, yellow solid 019C-084-1 (6.51 g, 34.5 mmol, 40% yield) was obtained.

[0424] Step two: synthesis of 8-nitroquinoline-4-carboxylic acid

[0425] Dissolve 019C-084-1 (6.00 g, 27.5 mmol) and selenium dioxide (6.10 g, 55.0 mmol, 2.0 eq) in pyridine (49.01 g, 619.5 mmol, 22.5 eq) and react at 80 °C for 16 h. Concentrate the reaction, add ethanol and reflux for 30 min, filter hot, adjust the pH of the crude to 5-6 with KHSO4 / H2SO4 buffer, filter, wash the filter cake with ethanol, and dry to give 019C-084-2 as a yellow solid (5.51 g, 25.2 mmol, 92% yield). MS (ES+) m / z 219.1 [M+H] +

[0426] Step Three: Synthesis of 1-(8-nitroquinolin-4-yl)-3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxepin-2-yl)pyrrolidin-1-yl)ethyl)urea

[0427] Dissolve 019C-084-2 (0.81 g, 3.7 mmol), 019C-062-5 (1.14 g, 3.7 mmol, 1.0 eq), triethylamine (1.13 g, 11.1 mmol, 3.0 eq), and diphenyl phosphorazide (1.53 g, 5.6 mmol, 1.5 eq) in toluene (15 mL) and react at 110 °C for 2 h under nitrogen. Pour the reaction into ice water, extract with ethyl acetate (100 mL), wash with brine (100 mL), dry, filter, and concentrate. Purify the crude product by silica gel column chromatography (dichloromethane / methanol = 50 / 1) to give 019C-084-3 as an orange solid (1.32 g, 2.5 mmol, 67% yield). MS (ES+) m / z 522.2 [M+H] +

[0428] Step Four: Synthesis of 1-(8-aminoquinolin-4-yl)-3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxepin-2-yl)pyrrolidin-1-yl)ethyl)urea

[0429] A mixture of 019C-084-3 (1.30 g, 2.5 mmol), iron powder (2.78 g, 49.8 mmol, 20 eq), ammonium chloride (2.66 g, 49.8 mmol, 20 eq) and water (18 mL) was dispersed in ethanol (35 mL) and stirred at 40 °C for 1 h under nitrogen atmosphere. The reaction mixture 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 column chromatography on silica gel (dichloromethane / methanol = 25 / 1) to give 019C-084-4 (613 mg, 1.3 mmol, 50% yield) as an orange solid.

[0430] Step five: synthesis of 4-oxo-4-((4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pyrrolidin-1- yl)ethyl)ureido)quinolin-8-yl)amino)butanoic acid

[0431] A mixture of 019C-084-4 (613 mg, 1.3 mmol), succinic anhydride (1.25 g, 12.5 mmol, 10 eq) and 4-dimethylaminopyridine (76 mg, 0.63 mmol, 0.5 eq) was dissolved in tetrahydrofuran (15 mL) and stirred at 50 °C for 3 h under nitrogen atmosphere. The reaction mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate and dried to give 019C-084-5 (530 mg, 0.89 mmol, 72% yield) as a white solid.

[0432] Step six: synthesis of 2-oxo-2-(3,3,7,7-tetrafluoro-4-hydroxy-1- azaspiro[4.4]non-1-yl)acetic acid

[0433] A mixture of 019C-084-5 (130 mg, 0.22 mmol, 2.2 eq), 019C-084-10 (86 mg, 0.099 mmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (69 mg, 0.25 mmol, 2.5 eq) and N,N-diisopropylethylamine (65 mg, 0.50 mmol, 5.0 eq) was dispersed in N,N-dimethylformamide (6 mL) and stirred at room temperature for 2 h under nitrogen atmosphere. The reaction mixture was poured into water (20 mL) to precipitate the solid, which was filtered, washed with water and dried to give 019C-084-6 (80 mg, 0.04 mmol, 40% yield) as a light brown solid. MS (ES+) m / z 1009.6 [M / 2+H] +

[0434] Step 7: Synthesis of tri-tert-butyl triacetate of 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)

[0435] 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecano-1-yl)valeric acid (400 mg, 0.57 mmol) and N-(2-aminoethyl)carbamate benzyl ester (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 was complete, the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with water (50 mL), extracted with ethyl acetate (50 mL x 2), 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 pale yellow solid 019C-084-7 (480 mg, 0.55 mmol, 96% yield).

[0436] Step 8: Synthesis of tri-tert-butyl triacetate (2,2',2"-(10-(5-((2-aminoethyl)amino)-1-(tert-butoxy)-1,5-dioxolane-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate):

[0437] Compound 019C-084-7 (773 mg, 0.88 mmol) and palladium on 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 give a white solid 019C-084-8 (579 mg, 0.78 mmol, 89% yield).

[0438] Step Nine: Synthesis of tri-tert-butyl triacetate of 2,2',2"-(10-(9-(((benzyloxy)carbonyl)amino)-21,21-dimethyl-3,10,15,19-tetraoxo-1-phenyl-2,20-dioxa-4,11,14-triazadodecane-18-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate:

[0439] Compound 019C-084-6 (30 mg, 0.015 mmol) was dissolved in formic acid (6 mL) and reacted at 50 °C for 16 h. The reaction was directly concentrated to give colorless oil 019C-084-11 (80 mg, crude) which was directly used for the next step.

[0440] Step Ten: Synthesis of 2,2',2"-(10-(1-(tert-butoxy)-5-((2-(2,6-diaminohexanoylamino)ethyl)amino)-1,5-dioxan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-trisyl)triacetic acid tri-t-butyl ester

[0441] Compound 019C-084-6 (30 mg, 0.015 mmol) was dissolved in formic acid (6 mL) and reacted at 50 °C for 16 h. The reaction was directly concentrated to give colorless oil 019C-084-11 (80 mg, crude) which was directly used for the next step.

[0442] Step Eleven: 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-methanobenzo[d][1,3,2]dioxaboran-2-yl)pyrrolidin-1- yl)ethyl)ureido)quinolin-8-yl)amino)butanamido)hexanamido)ethyl)amino)-1- carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-trisyl)triacetic acid

[0443] Compound 019C-084-6 (30 mg, 0.015 mmol) was dissolved in formic acid (6 mL) and reacted at 50 °C for 16 h. The reaction was directly concentrated to give colorless oil 019C-084-11 (80 mg, crude) which was directly used for the next step.

[0444] Step twelve: synthesis of 2,2',2"-(10-(4-((2-(2,6-di(4-((4-(3-(2-((R)-2-boropyrrolidin-1-yl)-2-oxoethyl)ureido)quinolin-8-yl)amino)-4-oxobutyl)hexan-1-aminyl)ethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid

[0445] Dissolve 019C-084-11 (80 mg, crude), benzeneboronic acid (14 mg, 0.11 mmol, 7.3 eq), methanol (3 mL), n-hexane (3 mL), and 1 drop of dilute hydrochloric acid (1 N) in dichloromethane (1 mL) and react for 5 hours at room temperature under nitrogen protection. Concentrate the reaction, wash with n-hexane (5 mL x 2), filter, and dry the filter cake. Purify the crude product by RP-HPLC (HANBON NP7000 series system with YMC-Actus Triart 5 pm Polar-RP 20 x 250 mm C18 column using a gradient: 100:0 to 75:25 water / acetonitrile + 0.1% TFA over 30 minutes), collect fractions, and lyophilize to obtain JYT2-4013 (16 mg, 0.01 mmol, 70% yield over two steps) as a white solid.

[0446] MS (ES+) m / z 509.3 [M / 3+H] + ; MS (ES-) m / z 743.4 [(M-2H2O) / 2-H] -

[0447] 1 H NMR (400 MHz, DMSO-d6 + D2O) δ 8.69 (d, J = 5.2 Hz, 2H), 8.51 (d, J = 6.8 Hz, 2H), 8.37 (d, J = 5.2 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.58 (t, J = 8.0 Hz, 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).

[0448] HPLC results for JYT2-4013 are shown in Table 27 and Figure 40. 1 H NMR is shown in Figure 39.

[0449] HPLC results for JYT2-4013 are shown in Table 27 and Figure 40.

[0450] The liquid phase detection conditions include: a chromatographic column: YMC-Triart C18, 3um 4.6*150mm; flow rate 1ml / min; column temperature: 30 DEG C; mobile phase: D: 0.1% HCIO4 solution of H2O pH = 1.5, C: ACN.

[0451] Table 27

[0452] The MS result of JYT2-4013 is shown in FIG. 41.

[0453] [According to Rule 91 Correction 11.10.2025] Preparation of Example 2C Small Molecule Inhibitor

[0454] The synthesis route of JYT2-4015 is as shown below:

[0455] Step one: synthesis of dimethyl 5-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)- 1,4,7,10-tetraazacyclododecan-1-yl)acetamido)isophthalate

[0456] Tris-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (2.00 g, 3.49 mmol) and dimethyl 5-aminoisophthalate (0.80 g, 3.84 mmol, 1.1 eq) were dissolved in DMF (20 mL), and HATU (1.59 g, 4.19 mmol, 1.2 eq) and DIPEA (1.35 g, 10.45 mmol, 3.0 eq) were added. The reaction was carried out at room temperature for 4 hours. TLC detection showed that the raw material was completely reacted. The reaction solution was added with water (100 mL), and extracted with ethyl acetate (100 mL x 2). The combined organic layer was washed with saturated sodium chloride solution (50 mL), and 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 yellow solid JYT2-4015-1 (1.48 g, 1.94 mmol, yield 55.5%).

[0457] Step two: synthesis of 5-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10- tetraazacyclododecan-1-yl)acetamido)isophthalic acid

[0458] 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 eq) was added. The reaction was stirred at room temperature for 1 h. TLC detection showed that the reaction was complete. The tetrahydrofuran was removed by concentration, and the residue was diluted with water (50 mL). The solution was acidified to pH = 5 with potassium bisulfate solution, and extracted with ethyl acetate (100 mL x 2). The organic phase was dried and concentrated to give a yellow foaming solid, JYT2-4015-2 (900 mg, 1.22 mmol, 63.0% yield). MS (ES+) m / z 734.3 [M-H] – .

[0459] Step three: Synthesis of methyl 6-(2-((tert-butoxycarbonyl)amino)ethoxy)quinoline-4- carboxylate

[0460] Methyl 6-hydroxyquinoline-4-carboxylate (3.99 g, 19.64 mmol) was dissolved in N,N-dimethylformamide (40 mL), and N-Boc-2-bromoethylamine (6.22 g, 27.76 mmol, 1.4 eq) and potassium carbonate (8.15 g, 58.97 mmol, 3.0 eq) were added sequentially at room temperature. After the addition was complete, the reaction solution was warmed to 80 °C and stirred for 16 h. TLC detection showed that the reaction was complete. The reaction solution was diluted with water (320 mL), and extracted with ethyl acetate (80 mL x 2). The combined organic phase was washed with saturated sodium chloride solution (160 mL), 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 give white solid JYT2-4015-3 (6.46 g, 18.65 mmol, 94.9% yield). MS (ES+) m / z 347.1 [M+H]+.

[0461] Step four: Synthesis of 6-(2-((tert-butoxycarbonyl)amino)ethoxy)quinoline-4-carboxylic acid

[0462] JYT2-4015-3 (1.00 g, 2.89 mmol) was dissolved in methanol (3 mL), and water (3 mL) and sodium hydroxide solid (0.46 g, 11.56 mmol, 4.0 eq) were added sequentially at room temperature. After the addition was complete, the reaction solution was warmed to 50 °C and stirred for 2 h. TLC detection showed that the reaction was complete. The methanol was removed by distillation under reduced pressure, and hydrochloric acid (1 M) was added dropwise to the solution until the pH was 3. After stirring for 10 min, the solution was filtered, and the filter cake was washed with hydrochloric acid (1 M, 1 mL) and dried to give white solid JYT2-4015-4 (708 mg, 2.13 mmol, 73.7% yield). MS (ES+) m / z 333.1 [M+H] +

[0463] Step five: Synthesis of tert-butyl (2-((4-(3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aS)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxaborol-2-yl)piperidin-l- yl)ethyl)ureido)quinolin-6-yl)oxy)ethyl)carbamate

[0464] JYT2-4015-4 (508 mg, 1.53 mmol) and 2-amino-l-((R)-2-((3aS,4S,6S,7aS)-3a,5,5- trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxaborol-2-yl)piperidin-l-yl)ethan-l- one (468 mg, 1.53 mmol, 1.0 eq) were dissolved in toluene (10 mL), and triethylamine (386 mg, 3.82 mmol, 2.5 eq) and diphenyl phosphorazide (463 mg, 1.68 mmol, 1.1 eq) were added successively at room temperature. The reaction solution was heated to 110 °C under nitrogen protection for 3 h. TLC detection showed that the reaction was complete. The reaction solution was cooled to room temperature, quenched with water (50 mL), extracted with ethyl acetate (50 mL x 2), the combined organic phase was 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 give JYT2-4015-5 (545 mg, 0.86 mmol, 56.0% yield) as a light brown solid.

[0465] Step six: Synthesis of l-(6-(2-aminoethoxy)quinolin-4-yl)-3-(2-oxo-2-((R)-2-((3aS,4S,6S,7aS)- 3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][l,3,2]dioxaborol-2-yl)piperidin-l- yl)ethyl)urea

[0466] 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 h. TLC detection showed that the reaction was complete. The reaction solution was adjusted to pH 9 with saturated sodium bicarbonate solution, extracted with dichloromethane (30 mL x 3), the combined organic phase was washed with saturated sodium chloride solution (90 mL), dried over anhydrous sodium sulfate, and concentrated to give JYT2-4015-6 (597 mg, 1.11 mmol, 93.7% yield) as a white solid. MS (ES+) m / z 536.3 [M+H] + .

[0467] Step Seven: Synthesis of 2-(tert-butoxycarbonyl)-l,2,3,4-tetrahydroisoquinoline-6- carboxylic acid

[0468] Dissolve 2-tert-butyl-6-methyl-dihydroisoquinoline-2,6(lH)-dicarboxylate (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 eq). Warm the reaction to 50 °C for 2 hours. TLC indicates the reaction is complete. Cool the reaction to 0-5 °C, adjust the pH to 3 with 1 N dilute hydrochloric acid, and extract with ethyl acetate (100 mL x 2). Dry the combined organic layers over anhydrous sodium sulfate, filter, and concentrate the filtrate to give brown oil JYT2-4015-7 (3.50 g, 12.6 mmol, 97.2% yield).

[0469] Step Eight: Synthesis of 6-benzyl-2-(tert-butyl) 3,4-dihydroisoquinoline-2,6(lH)-dicarboxylate

[0470] Dissolve JYT2-4015-7 (4.50 g, 16.23 mmol) in DMF (50 mL), and add cesium carbonate (7.91 g, 24.35 mmol, 1.5 eq) and benzyl bromide (4.16 g, 24.35 mmol, 1.5 eq). Stir at room temperature for 1 hour. TLC indicates the reaction is complete. Quench the reaction with water (300 mL), and extract with ethyl acetate (200 mL x 2). Concentrate the organic phase, and purify the crude product by silica gel column chromatography (PE / EA = 2 / 1) to give white solid JYT2-4015-8 (4.45 g, 12.11 mmol, 74.6% yield).

[0471] Step Nine: Synthesis of benzyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate hydrochloride

[0472] Dissolve JYT2-4015-8 (4.45 g, 12.11 mmol) in dichloromethane (50 mL), and add hydrochloric acid-dioxane (10 mL). Stir at room temperature for 2 hours. TLC indicates the reaction is complete. Concentrate the reaction directly to give white solid JYT2-4015-9 (3.51 g, 11.55 mmol, 95.4% yield).

[0473] Step Ten: Synthesis of benzyl 2-((tert-butoxycarbonyl)glycyl)-l,2,3,4-tetrahydroisoquinoline- 6-carboxylate

[0474] Boc-glycine (1.15 g, 6.58 mmol, 1.0 eq) was dissolved in dichloromethane (30 mL), HATU (3.00 g, 7.90 mmol, 1.2 eq) and DIPEA (2.55 g, 19.74 mmol, 3.0 eq) were added. After 30 min of reaction at room temperature, JYT2-4015-9 (2.00 g, 6.58 mmol) was added, and the reaction was continued at room temperature for 2 h. TLC detection showed that the reaction was complete. The reaction solution was quenched with water (100 mL), extracted with dichloromethane (100 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 white solid JYT2-4015-10 (2.61 g, 6.13 mmol, yield: 93.2%). MS (ES+) m / z 369.1 [M-56+H] + .

[0475] Step eleven: Synthesis of 2-glycyl-1,2,4-tetrahydroisoquinoline-6-carboxylic acid benzyl ester

[0476] JYT2-4015-10 (1.00 g, 2.36 mmol) was dissolved in dichloromethane (10 mL), and hydrochloric acid-dioxane (2 mL) was added. The reaction was continued at room temperature for 2 h, and TLC detection showed that the reaction was complete. The reaction solution was concentrated, diluted with water (20 mL), and the pH was adjusted to 9 with saturated sodium bicarbonate solution. The reaction solution was extracted with dichloromethane (100 mL x 2), and the organic phase was concentrated to obtain white solid JYT2-4015-11 (762 mg, 2.35 mmol, yield: 99.5%).

[0477] Step twelve: Synthesis of 2,2',2'-(10-(2-(3,5-bis((2-(6-(benzyloxy)carbonyl)-3,4- dihydroisoquinolin-2(1H)-yl)-2-oxoethyl)aminocarbonyl)phenyl)amino)-2-oxoethyl)-1,4,7,10- tetraazacyclododecane-1,4,7-triyl)triacetate

[0478] JYT2-4015-2 (786 mg, 1.06 mmol) was dissolved in dichloromethane (10 mL), Pyclop (983 mg, 2.33 mmol, 2.2 eq) and DIPEA (411 mg, 3.18 mmol, 3.0 eq) were added, after stirring at room temperature for 30 minutes, compound JYT2-4015-11 (756 mg, 2.33 mmol, 2.2 eq) was added, the reaction continued at room temperature for 2 hours. TLC detection of raw materials completely reacted. The reaction was quenched with water (50 mL), extracted with dichloromethane (50 mL x 2), the combined organic layers were washed with saturated sodium chloride solution (20 mL), the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, the crude product was purified by Pre-TLC (DCM / MeOH = 20 / 1) to give white solid JYT2-4015-12 (243 mg, 0.18 mmol, yield 17.0%); MS (ES+) m / z 675.0 [M / 2+H] + .

[0479] Step Thirteen: Synthesis of 2,2'-(2,2'-((5-(2-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetamido)isophthaloyl)bis(nitrilo)bis(acetyl))bis(1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid)

[0480] JYT2-4015-12 (165 mg, 0.12 mmol) was dissolved in methanol (10 mL), Pd / C (50 mg, 10%) was added, and hydrogenation reaction was carried out at room temperature for 2 hours. TLC detection of raw materials completely reacted. Filtration, the mother liquor was concentrated to give colorless solid JYT2-4015-13 (120 mg, 0.10 mmol, yield 85.6%).

[0481] Step Fourteen: Synthesis of 2,2',2'-(10-(2-(3,5-bis(2-oxo-2-(6-(2-(4-(3-(2-oxo-2-(R)-2-((3aS,4S,6S,7aS)-3a,5,5-trimethylhexahydro-4,6-methanoben[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

[0482] JYT2-4015-13 (105 mg, 0.09 mmol) was dissolved in DMF (8 mL), DMTMM (62 mg, 0.23 mmol, 2.5 eq), DIPEA (46.53 mg, 0.36 mmol, 4.0 eq) and JYT2-4015-6 (96 mg, 0.18 mmol, 2.0 eq) were added successively. The reaction was stirred at room temperature for 5 hours. TLC detection showed that the starting material was completely reacted. The reaction solution was quenched with water (10 mL), and a solid was precipitated, which was filtered. The filter cake was slurried with ethyl acetate (5 mL), filtered, and the filter cake was dried to obtain white solid JYT2-4015-14 (63 mg, 0.03 mmol, yield 31.8%).

[0483] Step fifteen: 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-methanobenzo[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)triacetate

[0484] JYT2-4015-14 (63 mg, 0.03 mmol) was dissolved in dichloromethane (5 mL), and TFA (1 mL) was added. The reaction was stirred at room temperature for 15 hours. TLC detection showed that the starting material was completely reacted. The reaction solution was concentrated to remove TFA, water was added, and a solid was precipitated. The solid was filtered, and the filter cake was slurried with ethyl acetate (5 mL), filtered, and dried to obtain white solid JYT2-4015-15 (60 mg, crude), which was directly used in the next step.

[0485] Step sixteen: 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)triacetate

[0486] JYT2-4015-15 (60 mg, crude) was dissolved in methanol (3 mL) and dichloromethane (3 mL), benzeneboronic acid (9 mg, 0.073 mmol, 2.5 equiv) was added, 3N dilute hydrochloric acid (1 d). The reaction was stirred at room temperature for 3 hours. LC-MS indicated the reaction was complete. The crude was concentrated, methanol (0.2 mL) was added, and the mixture was slurried with n-hexane (5 mL). The mixture was filtered, the filter cake was dried, and the crude was purified by RP-HPLC (HANBON NP7000 series system with YMC-Actus Triart 5 pm Polar-RP 20 x 250 mm C18 column using a gradient: 95:5 to 20:80 water / acetonitrile + 0.35% TFA in 40 minutes), fractions were collected and lyophilized to give JYT2-4015 (12 mg, 0.007 mmol, 22.7% yield over two steps) as a white solid.

[0487] MS (ES+) m / z 577.6 [(M-2H20) / 3+H] + .

[0488] 1 H NMR (400 MHz, CD3OD) 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).

[0489] Figure 42 is the HPLC result of JYT2-4015. 1 H NMR result.

[0490] The HPLC result of JYT2-4015 is shown in Figure 43 and Table 28.

[0491] Table 28

[0492] Figure 44 is the MS result of JYT2-4015.

[0493] [Corr. According to Rule 91 11.10.2025] Comparative Example 1C

[0494] [Corr. According to Rule 91 11.10.2025] Comparative Example 1C provides the structural formula of JYT2-401, which is shown in Table 29.

[0495] Table 29

[0496] [Corrected according to Rule 91 11.10.2025] Experimental Example 1C Inhibitor Affinity Test

[0497] 1. Refer to literature (Bioorg. Med. Chem. Lett. 2020, 30, 127253.). The specific operation is as follows: dilute the FAP protein to a concentration of 0.4 μg / mL with assay buffer (25 mM Tris, 250 mM NaCl, pH 7.4); dilute the substrate GP-AMC to 40 μM with assay buffer; prepare inhibitor (compound 1-20) solutions of different concentrations with assay buffer; add 25 μL of inhibitor and 25 μL of substrate to a 96-well black plate in turn, then add 50 μL of protein, and place it in an enzyme marker at 37°C for 1 h, and measure the fluorescence intensity (Ex / Em = 380 / 460 nm); use Graphpad Prism to fit, using the sigmoidal 11 dose response model, to calculate the IC50.

[0498] 2. Determination of the inhibitory activity of the compound on hFAP:

[0499] Dilute the hFAP protein to 0.3 μg / mL with assay buffer (25 mM Tris, 250 mM NaCl, pH = 7.4) and keep it on ice. Dilute the compound with assay buffer to a certain concentration gradient, which is 40 μM, 4 μM, 400 nM, 40 nM, 4 nM, 400 pM, 40 pM and 4 pM, respectively. Dilute the enzyme substrate Z-Gly-Pro-AMC with assay buffer to 40 μM, and store it in the dark. Add the above prepared 50 μL protein solution and 25 μL inhibitor to a 96-well plate (Corning 96-well enzyme plate black transparent bottom, No. 3904) in turn, centrifuge and shake at room temperature for 30 min. Then, add 25 μL of the above prepared enzyme substrate. After incubation at room temperature for 3-5 hours, use a multifunctional enzyme marker (Agilent BioTek Synergy H1) to monitor the fluorescence value at an excitation wavelength of 360 nm and an emission wavelength of 465 nm. Calculate the IC 50 value, which is defined as the concentration of the compound that reduces the enzyme activity by 50% under the assay conditions. The results are shown in Table 30 and Figure 45.

[0500] Table 30

[0501] [Corrected according to Rule 91 11.10.2025] Experimental Example 2C Labeling and Quality Control

[0502] 1、 177 Lu Labeling and Quality Control

[0503] 1.1 Labeling process

[0504] Precise pipetting compound (JYT2-401, JYT2-4013) solution 1-4 μL 20 nmol, precise addition of 50 μl 0.5M acetic acid-sodium acetate solution (pH 5.0, 4 mg N-acetyl-L-tyrosine, 1 mg N-acetyl-L-methionine) to 1.5 ml centrifuge tube, thoroughly mixed, 2 μl 177 LuCl3 solution (activity: ~2 mCi), under sealed conditions, upright in a metal bath at 95℃ (JYT2-401) or 70℃ (JYT2-4013) heated for 15 min, then removed, cooled to room temperature, added 150 μL 0.1% DTPA in PBS buffer solution, detected the radioactivity concentration of the solution, a small amount was taken for radiochemical purity analysis. The radioactivity was detected before and after administration, and the test sample was stored at room temperature and stored in a lead tank, and was prepared and used immediately.

[0505] wherein the labeled product of JYT2-4013 is:

[0506] 3.2 Quality control analysis

[0507] Radiochemical purity: HPLC: Waters Xbridege 5 μm C-18 (4.6 x 150 mm), flow rate of 1 mL / min; column temperature of 35℃; detection wavelength of 210 nm; injection volume of 20 μL (50-100 μCi). The gradient elution conditions are shown in Table 31.

[0508] Table 31

[0509] The results are shown in Figures 46-47 and Table 32. The area normalization method was used to calculate the radiochemical purity, and the radiochemical purity was greater than 95%, meeting the test requirements.

[0510] Table 32

[0511] The area normalization method was used to calculate the radiochemical purity, and the radiochemical purity was greater than 95%, meeting the test requirements.

[0512] 3.3 177 Lu-JYT2-401 biodistribution

[0513] Animal identification: Each cage of animals must be hung with a cage card indicating the animal group, animal number, etc.

[0514] Provide feed: The mouse growth maintenance feed meets GB14924.4-2001.

[0515] Feeding method: free access to food.

[0516] Drinking water: Drinking water bottles supply high-pressure sterilized tap water, which is tested by the Taiyuan Monitoring Station of the National Urban Water Supply Quality Monitoring Network and meets the requirements of the "Drinking Water Health Standards" (GB5749-2006); drinking water bottles are replaced every 2 days and used after being cleaned and high-pressure sterilized.

[0517] Tumor inoculation: The animal model construction was completed by Beijing Vantoll Life Animal Technology Co., Ltd. Each experimental animal was inoculated with HEK293 tumor cells subcutaneously on the right side of the back, and a total of 10 animals were used for later experiments.

[0518] The drug solution was extracted with a disposable syringe, the activity meter was used to detect the activity (μCi) before administration (full needle), and the tail vein was injected with the drug, and the activity meter was used to detect the residual radioactivity count (μCi) after administration (empty needle). 177 The administration dose of Lu-JYT2-401 injection is shown in Table 33.

[0519] Table 33 177 Administration dose table of Lu-JYT2-401 injection

[0520] 177 The administration dose of Lu-JYT2-4013 injection is shown in Table 34.

[0521] Table 34 177 Administration dose table of Lu-JYT2-4013 injection

[0522] Sampling time: 2h, 24h, 48h after administration.

[0523] Tissue organs: The animals were lightly anesthetized, the eyeballs were removed and blood was drawn (blood samples were retained), the heart, liver, spleen, lung, kidney, stomach (contents removed), large intestine (contents removed), small intestine (contents removed), femur (hind limb), knee, muscle, pancreas, brain, bladder (no urine), tumor, skin, urine, feces (2-3 grains in the large intestine), administration site (tail), and remaining carcass were removed.

[0524] Gamma counter detection: Biological samples were collected at each time point, gently squeezed and the residual blood was absorbed with absorbent paper, weighed and recorded, the samples were placed in EP tubes, and the gamma counter was used for detection, which was detected within 24h (if the radioactivity count is too high, it can be measured after decay). The detection results are expressed in CPM (CPM: radioactivity count per minute).

[0525] Data processing and analysis: %ID / g = A tissue / [(A0-A residue ) × M tissue ] × 100%.

[0526] A0: Total radioactivity count (CPM) of the syringe before drug administration.

[0527] A residue Post-administration residual radioactivity count (CPM) in syringe.

[0528] A tissue Radioactivity count of tissues and organs (CPM).

[0529] M tissue : Detect the weight (g) of the tissue sample.

[0530] Physical attenuation correction: 177 Lu physical decay: Activity dose at time T = 0.5^(T / 160.8).

[0531] Dosage (A0-A) residue Multiply by the decay coefficient for the corresponding time to get the corrected CPM value, and calculate %ID / g.

[0532] Note: Within an activity range not exceeding 75 μCi, the calibration curve between the activity meter detection (μCi) and the γ counter (CPM) is Y = 135628X(R). 2 =0.9991).

[0533] The results are shown in Table 35 and Figure 48. HEK293 human embryonic kidney cells were administered via a single tail vein injection to Balb / c nude tumor-bearing mice. 177 Lu-JYT2-401 injection showed a significant decrease in blood radioactive uptake over time. Within 2-48 hours after administration, varying degrees of radioactive enrichment were observed in tissues and organs, with the highest concentrations in tumors, kidneys, and liver (%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). Subsequent areas included skin, intestines, lungs, bladder, and spleen, with the lowest concentration in the brain. A single tail vein injection of HEK293T hFAP-transfected human embryonic kidney cells into Balb / c nude tumor-bearing mice was administered. 177The biodistribution experiment was performed at 2h, 24h, 48h after administration of Lu-JYT2-4013 compound injection, and different degrees of radioactivity enrichment existed in the tissues and organs. The normal organs, such as liver, knee bone, tibia, skin, thyroid, and salivary gland, had a small amount of uptake (1-3% ID / g). The uptake amount of the mouse tumor reached the maximum of 35.89±4.23 ID% / g after 2h of injection, and the retention time was long, and the radioactivity signal in the tumor remained at 33.15±5.43% ID / g after 48h of injection. The tumor uptake amount and retention time were obviously better than 177 Lu-JYT2-401, so the series of molecules designed by the present application have better advantages for treatment.

[0534] Table 35 177 Lu-JYT2-401 and 177 Tissue distribution results of Lu-JYT2-4013 injection in HEK293 human embryonic kidney cells Balb / c nude tumor-bearing mice

[0535] 3.4 SPECT / CT imaging

[0536] To 177 After Lu labeling, appropriate amount of PBS buffer solution was added to two molecules (JYT2-401, JYT2-4013) to prepare the test sample (pH ~ 5.5-6), and 100 μL of the mixture was uniformly absorbed by an insulin syringe. Each mouse was injected with only ~ 400 μCi (1-2 nmol) of molecules through the tail vein.

[0537] After administration, 180 μL of 1% sodium pentobarbital solution was injected into the mouse body through the abdominal cavity to anesthetize the mouse. The anesthetized mouse was fixed on the SPECT / CT instrument bed, and after CT positioning, SPECT / CT was scanned. After the scan was completed, the data was reconstructed, and the ROI value of the mouse SPECT data was obtained by software to obtain the radioactivity distribution %ID / g of the tumor organs and tissues.

[0538] The results are shown in Tables 36-37 and Figure 49. SPECT / CT imaging was performed at 2h, 8h, 24h, 48h, and the radioactivity signal in the mouse tumor was obtained by circle value (%ID / cc), and the results showed that 177 Lu-JYT2-401 remained in the tumor for a long time, and the maximum average value of tumor uptake was 30.51±3.83% ID / cc at 2h, which was comparable to the biodistribution data in section 3.3, and gradually decreased to 16.72±1.79% ID / cc after 48h. Compared with 177The average tumor uptake of Lu-JYT2-4013 at 2h, 8h, 24h, 48h, 72h, 144h was compared, and the results are shown in Figure 50, 177 The maximum average tumor uptake of Lu-JYT2-4013 was 33.02±1.67%ID / cc at 8h after injection, which was slightly higher than 177 The tumor uptake value of Lu-JYT2-401 at 2h remained at 27.50±3.2%ID / cc at 48h, which was significantly higher than 177 The tumor uptake value of Lu-JYT2-401 at 48h, and 177 The average tumor uptake of Lu-JYT2-4013 at 144h was still greater than ~20%ID / cc. This result shows that 177 Lu-JYT2-4013, such FAP multimeric compounds have the advantages of higher tumor uptake and longer retention time for tumor treatment.

[0539] Table 36

[0540] Table 37

[0541] It should be finally pointed out that the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial applicability

[0542] The present application provides a kind of targeted binding fibroblast activation protein compound and application, by optimizing the structure formula of compound, can effectively improve the retention amount and retention time of compound in tumor site, slow down the rate of removal, more conducive to the application of tumor radiotherapy.

Claims

1. A compound of formula (I) or a derivative thereof, characterized in that, The structure of the compound shown in formula (I) is: wherein x represents any integer from 1 to 4 inclusive; R1represents one or more of -H, -CN, -F, -B(OH)2, alkyl, alkyl substituted with F, and alkylene; R2, R3, R4are independently selected from -H, -F, -CI, -Br, -I, -C 1-6 alkyl, -O-C 1-6 alkyl, -S-C 1-6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, or -C 1-6 aralkyl, said -C 1-6 each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from -OH, oxo, halogen; R 5 is bicyclo[1.1.1]pentane, 1-naphtyl moiety or a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocycle, said heterocycle optionally further comprising 1, 2 or 3 heteroatoms selected from one or several of O, N and S; L, individually or differently, represents single bonds, substituted or unsubstituted C1 to C2 bonds. 10 Alkylene, substituted or unsubstituted C1-C 10 Cycloalkylene, substituted or unsubstituted C1-C 10 heteroalkyl, substituted or unsubstituted C1-C 10 heterocyclic alkyl, substituted or unsubstituted C1-C 10 alkylene oxides, substituted or unsubstituted C1-C1 alkylene oxides 10 aminoalkylene, substituted or unsubstituted C1-C 10 alkenyl, substituted or unsubstituted C1-C 10 Cycloalkenyl, substituted or unsubstituted C1-C 10 Heterene groups, substituted or unsubstituted C1-C 10 heterocyclic alkenyl, substituted or unsubstituted C1-C 10 alkyne group, substituted or unsubstituted C1-C 10 The heteroynyl group, substituted or unsubstituted C1-C 30 arylene, substituted or unsubstituted C1-C 30 The group comprises one or more of the following groups: heteroaryl, diallylsiloxane, carbonyl, imino, imide, amide, thioamide, phosphoramide, thioether, dithio, ester, thioester, urethane, carbonate, phosphate, diacid, anhydride, nitroimidazole, hydrazone, sugar, dipeptide, tripeptide, or tetrapeptide; wherein, when substituted, the substituents include one or more of alkyl, alkenyl, alkynyl, aryl, heteroaryl, ester, and amino groups. D is a single bond or any one of the following structural formulae or any connected group thereof: m is any integer from 0 to 10 inclusive; C comprises any one of (a) to (c): (a) a chelator group suitable for radiolabelling; (b) a radioactive group comprising a radioisotope; (c) a chelate of a radioisotope with a chelator; The derivative includes a pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt.

2. The compound of formula (I) according to claim 1, characterized in that, The compound of formula (I) or its derivative has a structure of general formula (I-1): wherein, in the general formula (I-1), selected from the group consisting of any one of the foregoing; selected from any of the following structural formulae: wherein R8, R9, R 10 one or two heteroatoms selected from O, N and S; Preferably, selected from any of the following structural formulae: Preferably, selected from any of the following structural formulae:

3. The compound of formula (I) according to claim 2, characterized in that, L is an intermediate linking moiety between the active moiety and the metal complex; Preferably, L comprises one or more linker groups, each linker group being independently selected from (hetero)alkylene, (hetero)heteroalkylene, heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, peptide, polyethylene glycol, amide or ester; Preferably, L independently or any combination thereof comprises at least one linker group having the structure: wherein n is any integer from 1 to 10 inclusive; More preferably, L comprises one or more linker groups selected from: wherein R7is selected from -H, -C 1-6 alkyl, -O-C 1-6 alkyl, -S-C 1-6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, or -C 1-6 aralkyl, said -C 1-6 each of which is optionally substituted with 1 to 3 substituents selected from -OH, oxo, halogen; n is any integer from 1-10; Preferably, C is a ligand moiety, being a chelator that forms a complex with a divalent or trivalent metal cation; preferably the chelator 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-(carboxymethyl imidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid; Alternatively, C is a ligand moiety, wherein the ligand moiety is a non-radioisotope, a radioisotope, a radiopharmaceutical, or a combination thereof; preferably, the radioisotope is selected from the group consisting of an isotope emitting alpha rays, an isotope emitting beta rays, an isotope emitting gamma rays, an isotope emitting Auger electrons, an isotope emitting X-rays, for example 18 F, 18 F-Al, 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 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 Ag; Alternatively, C is a ligand moiety selected from the following classes of fluorescent dyes: xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal-ligand complexes, fluorescent proteins, nanocrystals, perylenes, borondipyrromethenes and phthalocyanines, and conjugates and combinations of these classes of dyes; Alternatively, C is a contrast agent comprising or consisting of a paramagnetic agent; preferably the paramagnetic agent comprises or consists of paramagnetic nanoparticles.

4. The compound of formula (I) or its derivative according to claim 2, characterized in that, General formula (I-1) has any one of the following structural formulae:

5. The compound of formula (I) according to claim 2, characterized in that, The compound of formula (I) or derivative thereof has the structure of general formula (I-2): wherein x represents any integer from 1 to 4 inclusive; selected from the group consisting of any one of the foregoing; and / or, each L independently, identically or differently represents substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C1-C5 heteroalkylene, substituted or unsubstituted C1-C6 heterocycloalkylene, substituted or unsubstituted C1-C5 heteroarylene, substituted or unsubstituted C1-C5 arylene, carbonyl, alkoxy, thioether, disulfide, anhydride, carbonate, carbamate, sugar, peptide, polyethylene glycol, amide or ester, or a group of one or more of the foregoing; wherein when substituted, the substituents include one or more of alkyl, alkenyl, alkynyl, aryl, heteroaryl, ester and amine groups; C is a ligand moiety that is a chelator that forms a complex with a divalent or trivalent metal cation; the chelator 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-(carboxymethyl imidazole)glycine, or 6-hydrazinopyridine-3-carboxylic acid; preferably, the ligand moiety comprises a non-radioactive isotope, a radioactive isotope, a radiopharmaceutical, or a combination thereof; the radioactive isotope is selected from an isotope that emits alpha radiation, an isotope that emits beta radiation, an isotope that emits gamma radiation, an isotope that emits Auger electrons, an isotope that emits X-rays; 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 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 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 Ag. or C is a ligand moiety selected from the following classes of fluorescent dyes: xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, borondipyrromethenes and phthalocyanines, or conjugates or combinations of the above classes of dyes; or C is a contrast agent comprising or consisting of a paramagnetic agent.

6. The compound of formula (I) according to claim 5, characterized in that, In the general formula (I-2), L each independently, identically or differently, represents any one of the following groups: wherein n is any integer from 1 to 10; R8is selected from -H, -C 1～6 alkyl, -O-C 1～6 alkyl, -S-C 1～6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, or -C 1～6 aralkyl; each of said -C 1～6 alkyl is optionally substituted with 1 to 3 substituents selected from -OH, oxo, halogen; n is any integer from 1 to 10.

7. The compound of formula (I) according to claim 5, characterized in that, The compound of the general formula (I-2) has any one of the following structural formulae:

8. The compound of formula (I) according to claim 2, characterized in that, said compound of formula (I) or derivative thereof has the structure of general formula (I-3): wherein x represents any integer from 2 to 4 inclusive; selected from the group consisting of any one of the above; and / or each L independently, identically or differently represents a substituted or unsubstituted C1-C5 alkylene, substituted or unsubstituted C1-C5 heteroalkylene, substituted or unsubstituted C1-C5 heterocycloalkylene, substituted or unsubstituted C1-C5 heteroarylene, substituted or unsubstituted C1-C5 arylene, alkoxy, thioether, disulfide, anhydride, carbonate, carbamate, sugar, peptide, polyethylene glycol, amide or ester group, or a group of one or more of the above; wherein when substituted, the substituents include one or more of alkyl, alkenyl, alkynyl, aryl, heteroaryl, ester and amine groups; C is a ligand moiety which is a chelator forming a complex with a divalent or trivalent metal cation; said chelator 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-(carboxymethyl imidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid; Alternatively, C is a ligand moiety, wherein the ligand moiety is a non-radioisotope, a radioisotope, a radiopharmaceutical, or a combination thereof; the radioisotope is selected from the group consisting of an isotope that emits alpha radiation, an isotope that emits beta radiation, an isotope that emits gamma radiation, an isotope that emits Auger electrons, an isotope that emits X-rays; the isotope includes 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 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 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 Ag. or C is a ligand moiety selected from the following classes of fluorescent dyes: xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, borondipyrromethenes and phthalocyanines, or conjugates or combinations of the above classes of dyes; or C is a contrast agent comprising or consisting of a paramagnetic agent.

9. The compound of formula (I) according to claim 8, characterized in that, In the general formula (I-3), L each independently, identically or differently, represents any one of the following groups: wherein n is any integer from 1 to 10; wherein R1is selected from -H, -C 1～6 alkyl, -O-C 1～6 alkyl, -S-C 1～6 alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl, or -C 1～6 aralkyl; each of said -C 1～6 alkyl is optionally substituted with 1 to 3 substituents selected from -OH, oxo, halogen; n is any integer from 1 to 10; and / or L is attached to the 2, 3, 5, 6, 7 or 8 position of the quinoline group of the active fragment.

10. The compound of formula (I) according to claim 8, characterized in that, The compound of the general formula (I-3) has any one of the following structural formulae:

11. A pharmaceutical composition, characterized by, A compound of formula (I) or derivative thereof according to any one of claims 1 to 10.

12. A kit comprising a compound of formula (I) or derivative thereof according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 11; preferably, further comprising instructions for the diagnosis or treatment of a disease.

13. Use of a compound of formula (I) or derivative thereof according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 12 or a kit according to claim 13 for the manufacture of a medicament for the diagnosis and / or treatment of a disease; said disease comprising a disease characterized by overexpression of activated fibroblast protein in an animal or human subject.

14. Use according to claim 13, characterized in that, The disease is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scarring; more preferably, the cancer is selected from the group consisting of small intestine cancer, head and neck cancer, hepatocellular carcinoma, hypopharyngeal cancer, nasopharyngeal cancer, myeloma cells, cholangiocellular carcinoma, clear cell kidney cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, primary unknown cancer, thymus cancer, glioma, glioblastoma, 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, throat cancer, skin melanoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing sarcoma, Kaposi sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, nephroblastoma, neuroblastoma, esophageal cancer, laryngeal cancer, lymphoma, neurofibroma, tuberous sclerosis, hemangioma or lymphatic cancer.

15. A targeted binding fibroblast activation protein inhibitor or binding agent, characterized in that, The compound of formula (I) or its derivative according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11.

16. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. A tumor therapeutic or diagnostic agent comprising the compound of formula (I) or its derivative according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11.

17. A tumor imaging agent, characterized by, The compound of formula (I) or its derivative according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 11.

Images