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

Abstract

The present invention relates to the field of radiopharmaceuticals, specifically, compounds that target and bind to fibroblast activation protein and their applications. The FAP-binding compounds and their metal complexes of the present invention possess novel chemical structures, enabling the use of the compounds or pharmaceutical compositions in the diagnosis or treatment of diseases characterized by overexpression of fibroblast activation protein (FAP).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410542901.7 filed on May 1, 2024, entitled “A compound targeting and binding to fibroblast activation protein (FAP) and its application”, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of radiopharmaceuticals, in particular to a compound targeting and binding to fibroblast activation protein and its application. Background Art

[0004] Fibroblast activation protein alpha (FAP-α, abbreviated as FAP) inhibitor (FAPI) for PET imaging is a strategy to target a cell population of the peritumoral stroma (cancer-associated fibroblasts). Although of different origin, cancer-associated fibroblasts can be identified by the upregulation of several surface markers, with FAP being the most specific surface marker. FAP is a membrane-bound type II serine protease belonging to the dipeptidyl peptidase 4 family. Several methods have been effectively used to target this molecule with radiolabeled ligands such as antibodies, peptides, or small molecule inhibitors. Among the FAP-based radiotracers currently under development, gallium-68[ 68 The high detection rate of Ga]-labeled FAPI in various tumors provides favorable imaging evidence, even in tumors that are considered to be resistant to conventional [ 18 F] fluorodeoxyglucose ( 18 This also applies to situations where F-FDG PET is challenging. Fibroblast activation occurs not only in peritumoral tissue but also in benign conditions such as wound healing, inflammation, or ischemia. Therefore, FAPI PET imaging may also have the potential to diagnose common benign diseases associated with a wide range of morbidity (Uwe Haberkor, Radiology, 2023, 306, 2).

[0005] FAP is a type II transmembrane glycoprotein composed of 760 amino acids. As a serine protease, unlike other members of the dipeptidyl peptidase (DPP) family, it possesses both endopeptidase and exopeptidase activities, capable of cleaving gelatin and type I collagen, and plays a crucial role in ECM remodeling. DPPIV shares approximately 50% amino acid sequence similarity with FAP, and approximately 70% homology in the catalytic domain. FAP-expressing CAFs are found in many tumors, particularly epithelial tumors and malignancies with a strong desmoplastic reaction, such as breast, colorectal, pancreatic, and lung cancers. FAP has also been reported to be expressed in tumor cells of certain tumors (Scanlan, MJ, Proc. Natl. Acad. Sci. USA 1994, 91, 5657–5661; Dohi, O Histopathology 2009, 55, 432–440). In general, high FAP expression is associated with tumor aggressiveness and poor prognosis (Cohen, SJ, Pancreas 2008, 37, 154–158; Coto-Llerena, M, Front. Oncol. 2020, 10, 979). FAP expression is negligible in normal healthy adult tissues, making it a very attractive target for tumor imaging and treatment (Lindner, T, EJNMMI Radiopharm. Chem. 2019, 4, 16). FAP overexpression makes it a target for cancer diagnosis and treatment. Currently, there are mainly strategies such as antibodies, peptides, enzyme inhibitors, vaccines, immunoconjugates and chimeric antigen receptor T cells (Xin, L, Front. Oncol. 2021, 11, 3187). In 1994, Welt et al. first reported the use of 131Clinical experience with FAP-targeted imaging in patients with metastatic colorectal cancer (CRC) using I-labeled mouse monoclonal antibody (mAb) F19 was established (Welt, S, J. Clin. Oncol. 1994, 12, 1193–1203). Subsequently, sibrotuzumab, a humanized version of mAb F-19, was developed to explore the potential of FAP-targeted therapy. However, a phase I clinical study reported that sibrotuzumab failed to produce any objective tumor responses in 26 patients with advanced FAP-expressing colorectal or lung cancer, hindering further development of this molecule (Scott, AM, Clin. Cancer Res. 2003, 9, 1639–1647). Radiolabeled antibodies suffer from the limitations of their large molecular weight, resulting in slow clearance of the tracer from the body, leading to disadvantages such as high background signal, low sensitivity for lesion detection, and increased overall radiation exposure (Garousi, J, EJNMMI Radiopharm. Chem. 2020, 5, 16). Some early work on FAPI focused on pyrrolidine-2-boronic acid derivatives, such as talabostat mesylate (PT-100), an orally available aminoboron dipeptide. However, talabostat has affinity for members of the DPP subfamily and is not very specific for FAP (Adams, S, Cancer Res. 2004, 64, 5471–5480). Finally, the researchers shifted their focus to small molecule FAP inhibitors with N-(4-quinolinyl)-gly-(2-cyanopyrrolidine) structure, which have good pharmacokinetics and higher FAP specificity. This structure was first developed by the University of Antwerp (Jansen, K, ACS Med. Chem. Lett. 2013, 4, 491–496; Jansen, K, J. Med. Chem. 2014, 57, 3053–3074). Subsequently, the Heidelberg team in Germany modified its structure and developed FAPI-01 and FAPI-02 as the first generation of FAPI based on quinoline structure, and used 125 I and 68 Ga / 177 Lu was radiolabeled. 68 Compared with Ga]Ga-FAPI-02, 125I-labeled FAPI-01 exhibits low binding and uptake into human FAPI-expressing cells in vitro and in vivo due to its time-dependent efflux and enzymatic deiodination (Loktev, A, J. Nucl. Med. 2018, 59, 1423–1429). Lindner et al. synthesized several FAPI derivatives (FAPI-03 to FAPI-15) based on a common pharmacophoric functional group (UAMC1110). Among them, FAPI-04 exhibited higher tumor uptake (3.0% vs. 1.12% ID / g, 24 hours) and longer retention than FAPI-02 (Lindner, T, J. Nucl. Med. 2018, 59, 1415–1422), making it considered the most promising tracer for therapeutic applications. Further attempts to improve tumor retention led to the discovery of FAPI-46 (Loktev, A, J. Nucl. Med. 2019, 60, 1421–1429). Most of the studies published on the clinical application of FAP targeting have used FAPI-04 and FAPI-46.

[0006] Many researchers around the world have used FAPI-04 and FAPI-46 to compare with F-FDG for diagnosis. Studies by Halil Kömek (Annals of Nuclear Medicine, 2021, 35:744–752) and Philipp Backhaus (Radiology 2022, 302:39–47) have confirmed that FAPI has better detection capabilities for in situ and metastatic breast cancer. For pancreatic ductal carcinoma, multiple studies have also shown that FAPI has a higher detection rate (Li Huo, EJNMMI, 2023 50:4036–4050; Jens T. Siveke, J. Nucl. Med, 2023, 64, 1910-1917). In addition, FAPI has also been used in colorectal cancer (Ebubekir Gündeş, EJNMMI, 2022, 49, 3898–3909), cholangiocarcinoma (Wolfgang P. Fendler, J Nucl Med, 2023, 64, 1049-1055, and other indications, all of which have demonstrated good diagnostic capabilities. The excellent diagnostic capabilities demonstrated by FAPI in various tumors make FAP targets a promising tool for future therapeutic applications. However, the rapid clearance of FAPI-04 and FAPI-46 from the body, which does not match the half-life of therapeutic nuclides, has limited their clinical application.

[0007] Moon et al. developed a new FAPI radiotracer using a bifunctional chelator (DOTA, DATA5m, DOTAGA) and an aromatic acid (SA)-based linker between the UAMC1110 structure to simplify the previously complex synthesis of FAPI (Moon, ES, EJNMMI Radiopharm. Chem. 2020, 5, 19). 68 Ga-labeled DOTA.SA.FAPI has a good imaging effect. However, using radionuclides 177 Lu-labeled drugs showed tracer shedding 48 hours after injection, limiting their clinical application. Subsequently, the same research group developed dimers such as DOTA.(SA.FAPI)2 and DOTAGA.(SA.FAPI)2 to improve tumor affinity and retention time (Moon, ES, Am. J. Nucl. Med. Mol. Imaging 2021, 11, 476–491). 177 Lu, these dimers showed good therapeutic application results in early clinical studies (Ballal, S, Thyroid. 2021, 32, 65–77). Xu et al. coupled two albumin-binding 4-(p-iodophenyl)butyric acid fragments and truncated Evans blue fragments to the parent molecule FAPI-04, respectively, to develop TEFAPI-06 and TEFAPI-07 (Xu, M, J. Nucl. Med. 2022, 63, 952–958). A research team in the United States developed a trifunctional inhibitor RPS-309, which consists of a fragment targeting FAP, an albumin binding group and DOTA for radiometal chelation. Onco-FAP is an ultra-high affinity FAP organic ligand that was successfully labeled with DOTAGA as a chelator. 68 Ga and 177 Lu. The development of these molecules has helped improve rapid blood clearance and tumor retention in vitro and in vivo (Kelly, JM, Mol. Imaging Biol. 2021, 23, 686–696; Millul, J, Proc. Natl. Acad. Sci. USA 2021, 118).

[0008] Recently, a German research group developed a novel FAP-binding peptide, FAP-2286 (Zboralski, D, Eur. J. Nucl. Med. Mol. Imaging 2022, 49, 3651–3667), which is different from FAP small molecule inhibitors. FAP-2286 has been evaluated as a radiotracer at the preclinical and clinical levels.68 The tumor uptake ID% / g of Ga-FAP-2286 in mice reached 10.58% and 10.76% at 1h and 3h, respectively, which were higher than those of FAPI-46 (10.05% and 9.23%). 177 The uptake of Lu-FAP-2286 in the tumor reached 21.1% at 3 hours and 15.8% at 24 hours, which is much higher than 177 The 24h uptake of Lu-FAPI-46 (3.8%) showed excellent specific selection for FAP, high tumor uptake, high tumor retention, potential anti-tumor activity and acceptable toxicity (Baum, RP, J. Nucl. Med. 2022, 63, 415–423). Existing clinical trial data show that 68 The uptake of Ga-FAP-2286 in various tumors was significantly higher than that in 18 F-FDG, especially low to moderate uptake 18 F-FDG expression in different cancer types, including gastric cancer (9.1 vs. 3.4), pancreatic cancer (13.0 vs. 6.5), and liver cancer (11.3 vs. 4.8) (Haojun Chen, J Nucl Med, 2023, 64, 386-394); 177 The results of the first human trial of Lu-FAP-2286 showed that patients had significant changes in tumor lesions on SPECT imaging (72h-10d after injection). 177 The uptake and retention of Lu-FAP-2286 has a systemic effective half-life of 35 hours, and 44 hours for bone metastases, showing excellent therapeutic potential (Richard P. Baum, JNucl Med, 2022, 63, 415-423).

[0009] Another alternative molecular design could be based on the boronic acid derivative N-(pyridine-4-carbonyl)-D-Ala-boroPro (3099), first discovered and characterized by Poplawski et al. 3099's nanomolar potency and high selectivity for FAP surpass those of other DPP4-like subfamily enzymes, providing a basis for its use as a FAP-targeting fragment (Poplawski SE, J MedChem. 2013, 56: 3467–3477). William W. Bachovchin et al., based on the structure of 3099, used a DOTA chelator to modify it by linking an aminobenzoic acid residue (PNT6555) or a tranexamic acid residue to D-alanine at the N-terminus (PNT6952) or a Gly-Gly-Val tripeptide (PNT6522). These three molecules showed significant inhibitory activity against human and mouse FAP. PNT6555 and PNT6952 were used to68 Ga-labeled, it showed rapid renal clearance and sustained uptake in the tumor 60 min after administration, with intratumoral uptake (ID% / g) reaching 9% and 10.5%, respectively, within 1 h; 177 Lu-PNT6555 has the highest uptake in animals within 24 hours, with ID% / g of approximately 24%, and then decreases. It has the greatest effect on delaying tumor growth, and 30MBq shows a tumor inhibitory effect, demonstrating its clinical application value (William W Bachovchin, J Nucl Med, 2024, 65, 100-108).

[0010] Although existing compounds are designed with high enzyme activity, they have low tumor retention and rapid metabolism, which is not conducive to radiotherapy.

[0011] In view of this, the present invention is proposed. Summary of the Invention

[0012] The present invention provides a compound, specifically a targeted fibroblast activation protein (FAP) inhibitor or binder, a FAP-targeting nuclide probe drug based on the FAP binder, and uses of the inhibitor and probe in the preparation of FAP-targeting tumor imaging agents and tumor therapeutic agents.

[0013] In a first aspect, the present invention first provides a compound represented by formula (I) or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt thereof, wherein the structural formula of the compound represented by formula (I) is:

[0014] (I);

[0015] in, It is a nitrogen-containing 4-10 membered cyclic compound, wherein n is any integer from 0 to 6; R2, R3, and R4 are independently selected from -H, -F, -Cl, -Br, -I, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or -C 1-6 Aralkyl, the -C 1-6 Each of the alkyl groups is optionally substituted with 1 to 3 substituents selected from -OH, oxygen, and halogen; R5 is a 1-naphthyl moiety or a 5- to 10-membered N-containing aromatic or non-aromatic monocyclic or bicyclic heterocyclic ring, which optionally further contains 1, 2 or 3 heteroatoms selected from O, N and S; L represents a bond or a linker; R6 represents any of the following functional groups: an optical dye, a photodynamic therapy agent, a radioimaging agent, a radiotherapeutic agent, a chemotherapeutic agent, an anti-fibrotic agent or an anti-cancer agent.

[0016] Preferably, n is 1, that is It is a nitrogen-containing 5-membered cyclic compound.

[0017] Preferably, the nitrogen-containing cyclic compound is a group of a fibroblast activation protein (FAP) ligand.

[0018] Preferably, R1 is selected from -H, -CN, -F, -F2, -B(OH)2, -C(O)-alkyl, -C(O)-aryl-, -C=C-C(O)-aryl, -C=C-S(O)2-aryl, -CO2H, -SO3H, -SO2NH2, -SO2F, -CH2SO2F, -PO3H2, cyclic ether or 5-tetrazolyl.

[0019] Preferably, Selected from .

[0020] Preferably, in the general formula (I), Select from any of the following structural formulas:

[0021] ;

[0022] Among them, R8, R9, R 10 There is no or independently 1 or 2 heteroatoms selected from O, N and S.

[0023] Preferably, Select from any of the following structural formulas:

[0024] .

[0025] Preferably, Select from any of the following structural formulas:

[0026] .

[0027] Preferably, L is an intermediate linking fragment between the active fragment and the metal complex.

[0028] Preferably, L comprises one or more linker groups, each linker group being independently selected from (alkylene), (heteroalkylene), (heterocycloalkylene), heteroaryl, aryl, alkoxy, thioether, disulfide, carboxylic acid, anhydride, carbonate, carbamate, thioether, sugar, peptide, polyethylene glycol (PEG), amide or ester.

[0029] Preferably, L independently or in any combination thereof comprises at least one linker group having the following structure: ; Wherein, n is any integer from 1 to 10.

[0030] More preferably, L comprises one or more of the following linker groups: ; wherein R7 is selected from -H, -C 1-6 Alkyl, -OC 1-6 Alkyl, -SC 1-6 Alkyl, alkenyl, heteroalkenyl, cycloalkenyl, cycloheteroalkenyl, alkynyl, aryl or -C 1-6 Aralkyl, the -C 1-6 Each of the alkyl groups is optionally substituted by 1 to 3 substituents selected from -OH, oxygen, and halogen; and n is any integer from 1 to 10.

[0031] Preferably, R6 is a ligand moiety, which is a chelating agent that forms a complex with a divalent or trivalent metal cation; preferably, the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), bis-(carboxymethylimidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC).

[0032] Alternatively, R6 is a ligand moiety, wherein the ligand moiety is a non-radioactive isotope, a radioactive isotope, a radiopharmaceutical, or a combination thereof; preferably, the radioactive isotope is selected from an alpha-emitting isotope, a beta-emitting isotope, a gamma-emitting isotope, an Auger electron-emitting isotope, an X-ray-emitting isotope, 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 Second, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72As、 72 Se, 97 Such as 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 et al.

[0033] Alternatively, R6 is a ligand moiety selected from the group consisting of fluorescent dyes such as xanthines, acridines, oxazines, cyanines, styryl dyes, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, boron dipyrromethenes, and phthalocyanines, as well as conjugates and combinations of these classes of dyes.

[0034] Alternatively, R6 is a contrast agent comprising or consisting of a paramagnetic agent; preferably, the paramagnetic agent comprises or consists of paramagnetic nanoparticles.

[0035] Preferably, the compound represented by formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt has any of the following structural formulas:

[0036] .

[0037] In a second aspect, the present invention provides a method for preparing the above-mentioned compound, the reaction scheme of which is as follows:

[0038]

[0039] (a) A substituted pyrrole derivative A and a glycine derivative B undergo condensation reaction to form an amide C, which is then condensed with another amine D via an isocyanate intermediate in the presence of a carbonylating agent (such as triphosgene, BHT, etc.) under alkaline conditions to prepare compound E.

[0040] (b) The R5 formic acid derivative F with a terminal connector undergoes a Curtius rearrangement reaction in the presence of an azide (such as diphenylphosphoryl azide DPPA). Through the isocyanate intermediate, it condenses with another amine molecule C to form compound G, which then condenses with the carboxylic acid H to prepare compound I.

[0041] In a third aspect, the present invention provides a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt thereof.

[0042] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0043] In a fourth aspect, the present invention provides a targeted fibroblast activation protein (FAP) inhibitor or binder, comprising the compound represented by formula (I) or a pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt thereof, or the pharmaceutical composition.

[0044] In a fifth aspect, the present invention provides a kit comprising the compound of formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt or the pharmaceutical composition; and instructions for diagnosing or treating a disease.

[0045] In a sixth aspect, the present invention provides the use of the compound represented by formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt or the pharmaceutical composition for preparing drugs for diagnosing and / or treating diseases.

[0046] Preferably, the disease includes a disease characterized by overexpression of fibroblast activation protein (FAP) in an animal or human subject.

[0047] Further preferably, the disease is selected from cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scar disease.

[0048] 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, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, glioma, glioma, astrocytoma, cervical cancer, eye cancer, rectal cancer, colon cancer, cervical cancer, prostate cancer, breast cancer, bladder cancer, oral cancer, stomach cancer, liver cancer, pancreatic cancer, lung cancer, uterine cancer. , ovarian cancer, testicular cancer, kidney cancer, brain cancer, central nervous system cancer, pharyngeal cancer, skin melanoma, acute lymphoblastic leukemia, acute myeloid leukemia, Ewing's sarcoma, Kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms' tumor, neuroblastoma, esophageal cancer, laryngeal cancer, lymphoma, neurofibroma, tuberous sclerosis, hemangioma or lymphoma.

[0049] In a seventh aspect, the present invention provides a tumor therapeutic agent comprising the compound represented by formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt or the pharmaceutical composition.

[0050] In an eighth aspect, the present invention provides a tumor imaging agent comprising the compound represented by formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt or the pharmaceutical composition.

[0051] In a ninth aspect, the present invention provides a tumor diagnostic agent comprising the compound represented by formula (I) or its pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt or the pharmaceutical composition.

[0052] The compounds described herein, by introducing urea groups at specific locations within their structures, can effectively increase compound retention at target sites (e.g., tumors) and slow clearance, despite a slight decrease in enzymatic activity. This invention can effectively enhance tumor uptake and increase compound retention at tumor sites, enabling integrated diagnostic and therapeutic applications. In summary, by modifying the structure of FAP-binding compounds, the present invention has yielded compounds with extended tumor retention in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0054] Figure 1For JYT2-212-1 in Example 1 of this application 1 H NMR.

[0055] Figure 2 This is the HPLC result of JYT2-212-1 in Example 1 of the present application.

[0056] Figure 3 This is the LC-MS result of JYT2-212-1 in Example 1 of the present application.

[0057] Figure 4 For JYT2-222-1 in Example 2 of this application 1 H NMR results.

[0058] Figure 5 This is the HPLC result of JYT2-222-1 in Example 2 of this application.

[0059] Figure 6 This is the LC-MS result of JYT2-222-1 in Example 2 of the present application.

[0060] Figure 7 For JYT2-431-1 in Example 3 of this application 1 H NMR results.

[0061] Figure 8 This is the HPLC result of JYT2-431-1 in Example 3 of the present application.

[0062] Figure 9 For JYT2-401 in Example 4 of this application 1 H NMR results.

[0063] Figure 10 This is the HPLC result of JYT2-401 in Example 4 of the present application.

[0064] Figure 11 This is the LC-MS result of JYT2-401 in Example 4 of the present application.

[0065] Figure 12 These are the test results of enzyme activities of different molecules.

[0066] Figure 13 for 68 HPLC quality control results after Ga-JYT2-401 labeling.

[0067] Figure 14 To inject different molecules 68 Ga-JYT2-401, 68 Ga-JYT2-411, 68 Ga-JYT2-441, 68Ga-JY2-011 ( 68 PET imaging results of HEK293 hFAP-bearing mice with Ga-PNT6555 tumors 1 hour later.

[0068] Figure 15 For separate injections 68 Ga-JYT2-401, 68 Ga-JYT2-411, 68 Ga-JYT2-441, 68 Ga-JY2-011 ( 68 Biodistribution results of hFAP in HEK293 (Ga-PNT6555) tumor-bearing mice at different times.

[0069] Figure 16 for 177 Lu-JYT2-401 quality control results.

[0070] Figure 17 For injection 177 Tissue distribution results of Lu-JYT2-401 injection in HEK293 hFAP human embryonic kidney cell Balb / c nude tumor-bearing mice.

[0071] Figure 18 In the middle, on the left 177 SPECT images of Lu-JYT2-401 at different time points. The right side shows the changes in the maximum and average tumor uptake in the SPECT image over time.

[0072] Figure 19 Middle, left side is comparison 177 Lu-JYT2-401 and control group 177 Lu-FAP2286, 177 Lu-PNT6555( 177 MIP image of SPECT imaging 48 hours after Lu-JY2-011 injection. The right side shows the comparison value of the maximum tumor uptake in the SPECT image.

[0073] Figure 20 In contrast 177 Lu-JYT2-401 and control group 177 Tumor volume inhibition in HEK293 hFAP human embryonic kidney cell Balb / c nude mice (n=5) bearing tumors after injection of 15 MBq of Lu-FAP2286 and saline (vehicle) groups.

[0074] Figure 21 In contrast 177 Lu-JYT2-401 and control group 177Survival curves of Balb / c nude tumor-bearing mice (n=5) injected with 15 MBq / mouse of HEK293 hFAP human embryonic kidney cells using Lu-FAP2286 and saline groups (vehicle). DETAILED DESCRIPTION

[0075] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0076] Example 1 Preparation of small molecule inhibitors

[0077] The synthetic route of JYT2-212-1 is as follows:

[0078] .

[0079] Compound 3: ( S Preparation of 4,4-difluoro-1-glycylaminopyrrolidine-2-carbonitrile: To a solution of compound 1 (300 mg, 1.39 mmol) in dichloromethane (10 mL) at 25°C were added HATU (1870 mg, 4.92 mmol), DIEA (2935 mg, 22.70 mmol), and compound 2 (1000 mg, 5.68 mmol). The mixture was reacted at 25°C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (V 石油醚 / 乙酸乙酯 = 2:1) to obtain intermediate 3 as a white solid (700 mg, 64%). LC-MS: [M+H] + = 312.7.

[0080] Compound 4: ( S Preparation of 4,4-difluoro-1-glycylpyrrolidine-2-carbonitrile: Add hydrochloric acid in ethyl acetate (10 mL) to compound 3 (600 mg, 2.07 mmol) at 25°C. Incubate at 25°C for 0.5 hours. The reaction mixture is filtered and concentrated under reduced pressure to yield intermediate 4 (350 mg, 89%) as a white solid. LC-MS: [M+H] + = 190.1.

[0081] Compound JYT2-212-1: ( S )- NPreparation method of -(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-3,4-dihydroquinoline-1(2H)-carboxamide: o To a solution of intermediate 4 (85 mg, 0.45 mmol) and compound 5 (60 mg, 0.45 mmol) in dichloromethane (5 mL) at 4°C was added BTC (93 mg, 0.31 mmol) and TEA (227 mg, 2.25 mmol). The mixture was stirred at 25°C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H2O (0.1% FA); B%: 8%-15%, 10 min) to afford JYT2-212-1 (10 mg, 6%) as a white solid.

[0082] JYT2-212-1 1 H NMR see Figure 1 HPLC results are shown in Figure 2 , LC-MS results are shown in Figure 3 .

[0083] LC-MS: [M+H] + = 349.1. 1 H NMR (400 MHz, DMSO- d 6 ) 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).

[0084] Example 2 Preparation of small molecule inhibitors

[0085] JYT2-222-1 Synthesis Route:

[0086] .

[0087] Compound JYT2-222-1: ( S ) -N Preparation method of -(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-3,4-dihydroquinoline-1(2H)-carboxamide: o To a solution of intermediate 4 (50 mg, 0.26 mmol) and compound 5 (35 mg, 0.26 mmol) in dichloromethane (5 mL) at 4°C was added BTC (55 mg, 0.19 mmol) and TEA (134 mg, 1.32 mmol). The mixture was stirred at 25°C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H2O (0.1% FA); B%: 12%-19%, 10 min) to afford JYT2-222-1 (7 mg, 7%) as a white solid.

[0088] JYT2-222-1 1 H NMR see Figure 4 HPLC results are shown in Figure 5 , LC-MS results are shown in Figure 6 .

[0089] LC-MS: [M+H] + = 350.1. 1 H NMR (400 MHz, DMSO- d 6 ) 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).

[0090] Example 3 Preparation of small molecule inhibitors

[0091] JYT2-431-1 synthetic route:

[0092] .

[0093] Step 1: 7-((tert-Butyldimethylsilyl)oxy)quinoline: Dissolve 7-hydroxyquinoline (5.0 g, 34.4 mmol) in DMF (50 mL). Add tert-Butyldimethylsilyl chloride (6.2 g, 41.3 mmol) and imidazole (3.5 g, 51.6 mmol). After addition, react at room temperature for 70 minutes. TLC confirms complete reaction. The reaction mixture is quenched with water (200 mL), extracted with ethyl acetate (100 mL), washed with water (50 mL), separated, dried over anhydrous sodium sulfate, and concentrated. The crude product is purified by silica gel column chromatography (PE / EA = 20 / 1) to obtain the title compound 019C-069-1 as a colorless liquid (8.9 g, yield: 99.7%).

[0094] 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 raised to 25°C and stirred for 2 hours. TLC indicated that most of the starting material had reacted completely. The reaction mixture was poured into ice water to quench the reaction, extracted with ethyl acetate (100 mL), washed with water (50 mL), dried, and concentrated to afford the pale yellow title compound 019C-069-2 (2.1 g, crude product), which was used directly in the next step. LC-MS: m / z = 262.2 [M+H]+.

[0095] Step 3: 7-((tert-Butyldimethylsilyl)oxy)quinoline-1(2H)-carbonyl chloride: Dissolve compound 019C-062-2 (1.5 g, crude) in dichloromethane (40 mL) and add triethylamine (567 mg, 5.6 mmol). After cooling the reaction mixture to 0°C under nitrogen, triphosgene (550 mg, 1.86 mmol) was quickly added to the reaction mixture. The reaction mixture was stirred at 0°C for 10 minutes. TLC indicated complete reaction. The reaction mixture was quenched by adding dilute hydrochloric acid (100 mL). The organic phase was separated, dried over anhydrous sodium sulfate, and concentrated to afford the title compound 019C-069-3 (1.4 g, crude) as a reddish-brown solid, which was used directly in the next step.

[0096] Step 4: 7-((tert-Butyldimethylsilyl)oxy)-N-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylenebenzo[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 completion, the reaction was quenched by adding dilute hydrochloric acid (100 mL). The organic phase was separated and dried over anhydrous sodium sulfate and concentrated. The crude product was purified by silica gel column chromatography to obtain the title compound 019C-069-4 as a red solid (0.39 g, yield: 34%).

[0097] 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: Dissolve compound 019C-069-4 (0.39 g, 0.67 mmol) in tetrahydrofuran (10 mL). Add TBAF (10 mL, 10 mmol). After addition, stir the reaction at room temperature for 1 hour. Quench the reaction mixture with water (100 mL) and extract with ethyl acetate (50 mL). The layers are separated, and the organic phase is dried over anhydrous sodium sulfate and concentrated to afford the title compound 019C-069-5 as a white solid (205 mg, yield: 64%).

[0098] Step 6: (R)-(1-((7-Hydroxy-1,2-dihydroquinoline-1-carbonyl)glycylamino)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). Phenylboronic acid (156 mg, 1.28 mmol) and dilute hydrochloric acid (3N, 2 d) were added. After addition, the mixture was allowed to react at room temperature overnight. TLC confirmed complete reaction. The layers were separated, and the methanol layer was purified by preparative liquid chromatography (prep-HPLC) to afford 58 mg of a white solid. This was then purified by preparative thin-layer chromatography (prep TLC) (DCM / MeOH = 10 / 1) to afford the title compound SUS019C-069 (JYT2-431-1) as a white solid (25 mg, yield: 17%).

[0099] The synthesis results of JYT2-431-1 are as follows: 1 H NMR results are shown in Figure 7 HPLC results are shown in Figure 8 .

[0100] LC-MS: m / z = 328.2 [M-H2O+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, 6.0Hz, 1H), 6.45 (d, J = 7.1Hz, 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).

[0101] Example 4 Preparation of small molecule inhibitors

[0102] JYT2-201-1 synthesis results:

[0103] Referring to the above synthetic route, the synthesis results of JYT2-201-1 are 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 min DCM) (11.6 mL) were dissolved in DCM (10 mL), and the reaction was stirred at 25 °C for 16 h. LCMS showed that the starting material was consumed and the desired product was detected. Pre-HPLC (preparative conditions: column: WELCH xmax C18 21.2 x250mm 10um, mobile phase: ACN-H2O (0.1% FA), gradient: 10-30%) was used to obtain the product (200 mg, 23% yield) as a white solid.

[0104] LC-MS: [M+H] + : 376.1. 1H 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).

[0105] Example 5 Preparation of small molecule inhibitors

[0106] JYT2-243-1 synthesis results:

[0107] Compound JYT2-243-1 was synthesized using the synthetic route above: 1-(2-((1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hexan-2-yl)-2-acetyl)-3-(6-hydroxyquinolin-4-yl)urea. 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 BBr (1 mol / L in dichloromethane) (411.35 mg, 1.64 mmol) at 0°C under a nitrogen atmosphere. The mixture was stirred at 0°C for 12 hours. The reaction was quenched with water (1 mL) and MeOH (1 mL). The crude product was purified by preparative HPLC (column: Phenomenex luna C18 250 mm x100 mm x 10 um; mobile phase: [H2O (0.1% NH4HCO3)-ACN]; B%: 15%-35%, 9 minutes) to give a yellow solid JYT2-243-1 (2.5 mg, 2.2%).

[0108] LC-MS: [M+H] + : 352.1. 1H 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 = 9.0Hz, 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).

[0109] Example 6 Preparation of small molecule inhibitors

[0110] JYT2-253-1 synthesis results:

[0111] Referring to the above synthetic 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 was synthesized as follows: 2-Chloropyridine (177 mg, 1.56 mmol) and Tf2O (220 mg, 0.779 mmol) were added to a solution of compound 3 (150 mg, 0.519 mmol) in DCM (4 mL) at 25°C. The reaction solution was stirred at 25°C for 30 minutes. Compound 4 (207 mg, 1.56 mmol) and TEA (315 mg, 3.11 mmol) were added to the reaction solution, and the reaction was continued at 25°C for 2 hours. LCMS showed that the product had formed. The mixture was vacuum filtered and concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography (column Welch 21.2 x 250mm10um C18, 30ml / min), mobile phase: ACN-H2O (0.1% NH3); B%: 30%-70%, 10 min) to obtain white powdery solid compound JYT2-253-1 (19.1 mg, 11%).

[0112] 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).

[0113] Example 7 Preparation of small molecule inhibitors

[0114] JYT2-242-1 synthesis results:

[0115] 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 using the following synthesis route: 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. The mixture was stirred at 25°C for 1 hour. The reaction mixture was quenched with methanol, filtered, and concentrated under reduced pressure to yield the crude product. The crude product was purified by preparative HPLC (column: Gemini 5u C18 100 x 21.2 mm; mobile phase: ACN-H2O (0.1% FA); B%: 7%-12%, 10 min) to obtain JYT2-242-1 (6 mg, 9%) as a white solid.

[0116] LC-MS: [M+H] + : 390.2. 1H 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 = 4.6Hz, 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).

[0117] Example 8 Preparation of small molecule inhibitors

[0118] JYT2-231-1 synthesis results:

[0119] Compound JY2-231-1: (S)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)isoindoline-2-carboxamide was synthesized using the following synthesis route: BTC (67 mg, 0.22 mmol) and TEA (91 mg, 0.89 mmol) were added to a dichloromethane solution (5 mL) of intermediate 4 (85 mg, 0.45 mmol) and compound 5 (54 mg, 0.45 mmol) at 25°C. The mixture was stirred at 25°C for 1 hour. The reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative HPLC (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.

[0120] 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).

[0121] Example 9 Preparation of small molecule inhibitors

[0122] JYT2-441-1 synthesis results:

[0123] The synthetic route is as follows:

[0124] .

[0125] Step 1: 7-((tert-Butyldimethylsilyl)oxy)naphthalene-1-amine: Dissolve 8-amino-2-naphthol (3.2 g, 20.0 mmol) in DMF (20 mL), add tert-butyldimethylsilyl chloride (3.6 g, 24.0 mmol) and imidazole (2.8 g, 41.0 mmol). After addition, react at room temperature for 30 minutes. TLC confirms complete reaction. The reaction mixture is quenched with water (100 mL), extracted with ethyl acetate (100 mL), and washed with water (50 mL). The organic phase is separated, dried over anhydrous sodium sulfate, and concentrated. The crude product is purified by silica gel column chromatography (PE / EA = 20 / 1) to obtain the title compound 019C-068-1 as a colorless liquid (4.7 g, yield: 85.9%).

[0126] Step 2: tert-Butyl((8-isocyanatonaphthalen-2-yl)oxy)dimethylsilane: Dissolve triphosgene (179 mg, 0.60 mmol) in dichloromethane (10 mL) and cool to -10°C under nitrogen. Dissolve compound 019C-068-1 (0.5 g, 1.83 mmol) in dichloromethane (15 mL) and add triethylamine (407 mg, 4.03 mmol). Slowly add the resulting mixture dropwise to the triphosgene, maintaining the temperature below 0°C. After the addition is complete, stir the reaction at room temperature for 30 minutes to yield a dichloromethane solution of compound 019C-068-2, which is used directly in the next step.

[0127] 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-methylenebenzo[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 above reaction mixture, followed by triethylamine (281 mg, 2.78 mmol). The reaction was stirred at room temperature for 50 minutes. The mixture was quenched by the addition of dilute hydrochloric acid (100 mL). The layers were separated, the organic phase was dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography to afford the title compound 019C-068-3 as a red solid (0.65 g, yield: 92.5%). LC-MS: m / z = 452.2 [MH] - .

[0128] Step 4: (R)-(1-(((7-Hydroxynaphthalen-1-yl)carbamoyl)glyamino)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 (3N, 2d) were added. The reaction mixture was heated to 40°C for 5 hours. TLC confirmed the complete reaction. The organic phase was separated, concentrated, and the crude product was purified by pre-TLC (DCM / MeOH = 10 / 1). The product was then slurried in methanol to obtain the title compound SUS019C-068 (JYT2-441-1) as a yellow solid (63 mg, yield: 35.59%).

[0129] Referring to the above synthetic route, compound JYT2-441-1: (R)-(1-(((7-hydroxynaphthalen-1-yl)carbamoyl)glycylamino)pyrrolidin-2-yl)boronic acid; the results are as follows: LC-MS: m / z = 340.2 [M-H2O-H] - (99.92% purity, 220 nm). 1H 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).

[0130] Example 10 Preparation of small molecule inhibitors

[0131] The synthetic route of JYT2-421-1 is as follows:

[0132] .

[0133] Step 1: Tert-butyl (S)-2-(R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxolan-2-yl)pyrrolidine-1-carbonyl)pyrrolidine-2-carboxylate: 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-1-yl)uronium hexafluorophosphate (2.65 g, 6.97 mmol) were dissolved in tetrahydrofuran (10 mL). N,N-diisopropylethylamine (1.80 g, 13.94 mmol) was added. After the addition, the reaction was stirred at room temperature for 2 hours. TLC (ethyl acetate = 100%, potassium permanganate colorimetry) revealed the formation of distinct new spots. The reaction was quenched by the addition of water (20 mL), followed by extraction with ethyl acetate (10 mL x 2). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to afford the title compound 019C-065-1 as a colorless, transparent oil (1.07 g, yield: 52.1%).

[0134] Step 2: (R)-1-(L-propionyl)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]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 h. The reaction was complete as monitored by TLC (ethyl acetate = 100%). The reaction solution was concentrated to give the title compound 019C-065-2 as a white solid (crude product), which was used directly in the next reaction.

[0135] Step 3: (S)-N-(6-Hydroxyquinolin-4-yl)-2-(R)-2-(((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxolan-2-yl)pyrrolidine-1-carbonyl)pyrrolidine-2-carboxamide: Compound 019C-062-5 (106 mg, 0.39 mmol), diisopropylethylamine (169 mg, 1.31 mmol), and diphenylphosphoryl azide (129 mg, 0.47 mmol) were dispersed in toluene (6 mL), heated to 80°C, and stirred for 2 hours. The disappearance of the starting material was monitored by TLC (dichloromethane / methanol = 9 / 1). Compound 019C-065-2 (100 mg, crude) was added. The reaction was stirred at 80°C for 16 hours. TLC (dichloromethane / methanol = 9 / 1) revealed the formation of a new spot. The reaction solution was concentrated and purified by silica gel column chromatography (dichloromethane / methanol = 19 / 1) to afford the title compound 019C-065-3 as a pale yellow oil (95 mg, yield: 68.3%). LC-MS: m / z = 531.2 [MH]-.

[0136] Step 4: ((R)-1-(((6-Hydroxyquinolin-4-yl)carbamoyl)-L-propionyl)pyrrolidin-2-yl)boronic acid: Compound 019C-065-3 (95 mg, 0.18 mmol) and phenylboronic 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, 3N) was added dropwise. The reaction was stirred at 25°C for 3 hours. TLC (dichloromethane / methanol = 9 / 1) confirmed complete reaction. The lower methanol phase was removed with a pipette. After concentration, the product was purified by prep-TLC and pre-HPLC to yield the title compound SUS019C-065 (JYT2-421-1) (12 mg, yield: 16.9%) as an off-white solid.

[0137] Compound JYT2-421-1 ((R)-1-(((6-hydroxyquinolin-4-yl)carbamoyl)-L-propionyl)pyrrolidin-2-yl)boronic acid was synthesized using the above synthetic route. The results are as follows: LC-MS: m / z = 399.2 [M+H] + (98.98% purity, 220 nm). 1 H NMR (400 MHz, D2O) 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).

[0138] JYT2-453-1 synthesis results: Referring to the above synthesis route, compound JYT2-453-1: (R)-(1-(((1,7-naphthyridin-4-yl)carbamoyl)glycol)pyrrolidin-2-yl)boronic acid; the results are 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).

[0139] Example 11 Preparation of complete small molecule inhibitors

[0140] The synthetic route of JYT2-401 is as follows:

[0141] .

[0142] Step 1: Preparation of methyl 6-hydroxyquinoline-4-carboxylate: Disperse 6-hydroxyquinoline-4-carboxylic acid hydrobromide (5.0 g, 18.51 mmol) in methanol (50 mL) and add concentrated sulfuric acid (0.5 mL). React at 65°C for 15 hours. TLC confirms complete reaction. Concentrate the reaction mixture to remove methanol, dilute with water (20 mL), alkalize to pH 10 with saturated sodium bicarbonate solution, and extract with ethyl acetate (200 mL). Filter and separate the layers. Extract the aqueous phase with ethyl acetate (50 mL x 3). Combine the organic phases and concentrate to obtain the title compound 401-1 as a brown solid (1.5 g, yield: 39.89%).

[0143] Step 2: Preparation of methyl 6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)quinoline-4-carboxylate: Compound 401-1 (1.3 g, 6.40 mmol) was dissolved in DMF (15 mL). Potassium carbonate (2.65 g, 19.2 mmol) and tert-butyl 4-(3-bromopropyl)piperazine-1-carboxylate (1.97 g, 6.40 mmol) were added. After the addition, the reaction mixture was heated to 85°C for 2 hours. TLC confirmed the complete reaction. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (100 mL x 2). The organic phases were combined and concentrated. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 2) to afford the title compound 401-2 (1.75 g, yield: 63.64%) as a brown liquid. LC-MS: m / z = 430.2 [M+H] + .

[0144] Step 3: Preparation of 6-(3-(4-(tert-Butoxycarbonyl)piperazin-1-yl)propoxy)quinoline-4-carboxylic acid: Disperse compound 401-2 (1.75 g, 4.09 mmol) in a mixture of THF (20 mL) and water (10 mL). Add LiOH.H2O (503 mg, 12.3 mmol). After addition, the reaction mixture was allowed to react at room temperature for 3 hours. TLC confirmed complete reaction. The reaction mixture was acidified to pH 6 by adding saturated ammonium chloride and extracted with 2-methyltetrahydrofuran (50 mL x 3). The organic phases were combined and concentrated to afford the title compound 401-3 (1.64 g, yield: 97.04%) as a white liquid. LC-MS: m / z = 416.2 [M+H] + .

[0145] Step 4: Preparation of tert-butyl 4-(3-((4-(2-oxo-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxolan-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-6-yl)oxy)propyl)piperazine-1-carboxylate: Disperse compound 401-3 (905 mg, 2.18 mmol) and compound 066-2 (746 mg, 2.18 mmol) in toluene (15 mL), add triethylamine (661 mg, 6.54 mmol) and DPPA (660 mg, 2.4 mmol). After the addition, heat the reaction mixture to 110°C for 3 hours. TLC confirms the complete reaction of the starting material. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (100 mL x 2). The organic phases were combined and concentrated. The crude product was purified by silica gel column chromatography (DCM / MeOH = 15 / 1) to obtain the title compound 401-4 as a yellow solid (491 mg, yield: 31.27%). LC-MS: m / z = 719.4 [M+H] + .

[0146] Step 5: Preparation of 1-(2-oxo-2-((R)-2-(((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxol-2-yl)pyrrolidin-1-yl)ethyl)-3-(6-(3-(piperazin-1-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. After the addition was complete, the reaction mixture was reacted at room temperature for 2 hours. TLC confirmed the complete reaction of the starting material. The reaction mixture was concentrated to afford the title compound 401-5 as a yellow solid (430 mg, yield: 96.28%).

[0147] Step 6: Preparation of tri-tert-butyl 2,2',2'-(10-(2-oxo-2-(4-(4-(3-(2-oxy-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxolan-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate: Dissolve tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (39 mg, 0.069 mmol) in DMF (8 mL) and add PYBOP (43 mg, The reaction mixture was stirred at room temperature for 30 minutes, followed by the addition of compound 401-5 (45 mg, 0.069 mmol). The reaction mixture was allowed to react at room temperature for 3 hours. TLC confirmed the complete reaction. The reaction mixture was quenched with water (20 mL) and extracted with ethyl acetate (20 mL x 2). The organic phases were combined and concentrated. The crude product was purified by Pre-TLC (DCM / MeOH = 10 / 1) to afford the title compound 401-6 as a yellow solid (21 mg, yield: 26.05%). LC-MS: m / z = 587.5 [M+2H / 2] + .

[0148] Step 7: Preparation of 2,2',2'-(10-(2-oxo-2-(4-(4-(3-(2-oxy-2-((R)-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxolan-2-yl)pyrrolidin-1-yl)ethyl)ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)ethyl ester)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid: Compound 401-6 (21 mg, 0.018 mmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (2 mL) was added. The mixture was reacted at room temperature for 15 hours. The reaction solution was concentrated to obtain a purple solid 401-7 (crude product), which was used directly in the next reaction.

[0149] 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-triyl)triacetic acid: Compound 401-1 (crude product) was dissolved in a mixture of water (2 mL) and acetonitrile (1 mL). Phenylboronic acid (2 mg, 0.016 mmol), trifluoroacetic acid (2d), and MTBE (6 mL) were added. After addition, the reaction mixture was reacted at room temperature for 3 hours. The layers were separated, and the aqueous phase was washed with MTBE (10 mL). The aqueous phase was purified by Pre-HPLC to yield the title compound JYT2-401 as a white solid (10 mg, two-step yield: 38.77%).

[0150] JYT2-401 1 H NMR see Figure 9 HPLC results are shown in Figure 10 , LC-MS results are shown in Figure 11 .

[0151] 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).

[0152] Example 12 Preparation of complete small molecule inhibitors

[0153] Refer to the above synthetic route compound JYT2-402:

[0154] 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-triyl)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, D2O) 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).

[0155] Example 13 Preparation of complete small molecule inhibitors

[0156] Refer to the above synthetic route compound JYT2-406:

[0157] (R)-2,2',2'-(10-(2-(4-(3-(3-(2-(2-bromopyrrolidin-1-yl)-2-oxoethyl)ureido)quinolin-6-yl)(methyl)amino)propyl)piperazin-1-yl)2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; the results are as follows: LC-MS: m / z = 852.4 [M-H2O+H] + (98.44% purity, 210 nm); 1H NMR (400 MHz, D2O) δ 8.31 (dd, J = 18.4, 6.8 Hz, 2H), 7.80 (d, J = 9.2 Hz, 1H), 7.61 (dd, J = 9.6, 2.0 Hz, 1H), 6.96 (s, 1H), 4.65-4.44 (m, 1H), 4.40-4.08 (m, 2H), 4.12-2.99 (m, 41H), 2.35-1.90 (m, 5H), 1.91-1.53 ​​(m, 1H).

[0158] Example 14 Preparation of complete small molecule inhibitors

[0159] Refer to the above synthetic route compound JYT2-411:

[0160] 2,2',2'-(10-(2-(4-(3-((R)-1-(R)-2-bromopyrrolidin-1-yl)-1-oxopropyl-2-yl)ureido)quinolin-6-yl)oxy)propyl)piperazin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; the results are as follows: LC-MS: m / z = 434.3 [(M-H2O) / 2+H] + (96.81% purity, 210 nm); 1 H NMR (400 MHz, D2O) 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=6.8Hz, 3H).

[0161] Example 15 Preparation of complete small molecule inhibitors

[0162] Refer to the above synthetic route compound JYT2-441:

[0163] 2,2',2'-(10-(2-oxyl-2-(4-(3-(8-(3-(2-oxyl-2-((R)-2-((3aS,4S,6S,7aR))-3a,5,5-trimethylhexahydro-4,6-methylbenzo[d][1,3,2]dioxol-2-yl)pyrrolidin-1-yl)ethyl)ureido)naphthalen-2-yl)oxy)propyl)piperazin-1-yl)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid; the result is as follows: LC-MS: m / z = 852.4 [M-H2O+H] + (98.79% purity, 220 nm); 1H NMR (400MHz, 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).

[0164] The structural formulas of FAPI04, JY2-011 (PNT6555), and FAP2286 reported in the literature are shown in Table 1.

[0165] Table 1

[0166]

[0167] Experimental Example 1 Inhibitor affinity test

[0168] 1. Refer to the literature (Bioorg. Med. Chem. Lett. 2020, 30, 127253). Specific procedures are as follows: dilute FAP protein to 0.4 μg / mL in assay buffer (25 mM Tris, 250 mM NaCl, pH 7.4); dilute the substrate GP-AMC to 40 μM in assay buffer; prepare inhibitor solutions (compounds 1-20) at varying concentrations in assay buffer; sequentially add 25 μL of inhibitor and 25 μL of substrate to a 96-well black plate, followed by 50 μL of protein. Place the plate in a microplate reader and incubate at 37°C for 1 hour. Fluorescence intensity (Ex / Em = 380 / 460 nm) is measured; and IC50 values ​​are calculated using Graphpad Prism fitting using the sigmoidal 11-dose response model.

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

[0170] hFAP protein was diluted to 0.3 μg / mL in assay buffer (25 mM Tris, 250 mM NaCl, pH 7.4) and kept on ice. Compounds were diluted in 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 in assay buffer and stored in the dark. 50 μL of the protein solution and 25 μL of the inhibitor were sequentially added to a 96-well plate (Corning 96-well microtiter plate, black, clear bottom, No. 3904). After centrifugation, the plate was shaken at room temperature for 30 minutes. Then, 25 μL of the prepared enzyme substrate was added. After incubation at room temperature for 3–5 hours, fluorescence was monitored at an excitation wavelength of 360 nm and an emission wavelength of 465 nm using an Agilent BioTek Synergy H1 microplate reader. IC was calculated. 50 The value is defined as the concentration of compound that causes 50% reduction in enzyme activity under the assay conditions. The results are shown in Tables 2 and Figure 12 .

[0171] Table 2

[0172]

[0173] 3. Location of other urea groups:

[0174] The 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 the above, but the enzyme activity IC50 was much lower, all greater than 100uM.

[0175] The structural formulas and results of the molecules JYT2-311-1, JTZ2-281-1 and JYZ2-291-1 are shown below:

[0176] .

[0177] Experimental Example 2: Labeling and Quality Control

[0178] 1. Preparation of test samples: Accurately measure 100 μL of 2M acetic acid-sodium acetate solution (pH 5.0) and place it in a 1.5ml centrifuge tube. Accurately add 2 μL of 20 nmol of the test sample (JTY2-401, JTY2-402, JTY2-406, JTY2-411, JTY2-441, JY2-011 (PNT6555), FAPI04, FAP2286) (10 nmol / μL) precursor aqueous solution, mix thoroughly, and add 0.3mL 68 Heat the GaCl3 eluent (activity: ~2.28 mCi) upright in a sealed metal bath at 95°C for 15 minutes. After the reaction is complete, add 100 μL of 0.1% DTPA (pH 5.5). After cooling to room temperature, remove a small amount of the reaction solution for radiochemical purity analysis.

[0179] HPLC detection: JYT2-401 was tested by HPLC. The test conditions are shown in Table 3. The results are shown in Figure 13 .

[0180] Table 3

[0181]

[0182] 2. 68 Imaging and biodistribution of Ga-labeled products

[0183] 2.1 PET / CT imaging

[0184] Towards 68The four Ga-labeled molecules (JYT2-401, JYT2-402, JYT2-411, and JY2-011 (PNT6555), volume 350 μL) were added with 500 μL PBS buffer and 32 μL 1M NaOH solution to prepare the test sample (pH ~6.1) (radioactivity concentration was approximately 2.7 μCi / μL). After mixing evenly, 75 μL was aspirated with an insulin syringe and the activity was measured: JYT2-401: 192 μCi, JYT2-402: 193 μCi, JYT2-411: 199 μCi, and JY2-011 (PNT6555): 203 μCi. The samples were injected into mice through the tail vein and the radioactivity in the mice was measured using an activity meter. Only ~120 μCi (~1 nmol) of molecules were injected into each mouse.

[0185] About 1 hour after administration, 180 μL of 1% sodium pentobarbital solution was drawn up by a syringe and administered intraperitoneally into the mouse to anesthetize it. The anesthetized mouse was fixed on the PET / CT instrument bed, and the PET / CT was scanned after CT positioning.

[0186] After the scan is completed, the data is reconstructed and the ROI value of the mouse PET data is circled using software to obtain the radioactivity distribution %ID / g of organs and tissues such as the heart, kidney, liver, muscle, bladder, and tumor.

[0187] See the results Figure 14 Analysis of PET / CT 1-hour imaging results, by circumscribing the radioactive signals (%ID / g) within mouse organs and tumors, showed that JYT2-401 was comparable to the positive control JY2-011 (PNT6555).

[0188] 2.2 Biodistribution

[0189] injection 68 HEK293 hFAP tumor-bearing mice (n=3) were labeled with different molecules, including FAPI04, FAP2286, JY2-011 (PNT6555), JTY2-401, JTY2-406, JTY2-411, and JTY2-441. The mice were then dissected at different time points, and blood, heart, liver, spleen, lung, kidney, stomach, pancreas, small intestine, large intestine, muscle, femur, tumor, brain, and skin were collected. The collected organs and tissues were weighed, and the radioactivity in the organs and tissues was measured using a gamma counter.

[0190] The biodistribution results at different times are shown in Figure 15 Tail vein injection 68Ga-labeled injections of different molecules, including FAPI04, FAP2286, JY2-011 (PNT6555), JTY2-401, JTY2-406, JTY2-411, and JTY2-441, showed varying degrees of radioactivity enrichment in tissues and organs 2 hours after administration. 68 Ga- JTY2-401 and 68 Ga-FAPI04, 68 Ga-PNT6555, 68 The uptake of Ga-FAP2286 in tumors was comparable, with no significant difference; its enrichment in other tissues and organs was low.

[0191] 3. 177 Lu labeling and quality control

[0192] 3.1 Marking process

[0193] Accurately measure 4 μL (20 nmol) of compound (JYT2-401, FAP2286, JY2-011 (PNT6555)) solution, accurately add 50 μL of 0.5 M sodium acetate buffer solution at pH 4.5, shake thoroughly, and then add ~2 mCi (~2 μL) 177 Mix the LuCl3 injection solution thoroughly and heat it upright in a sealed metal bath at 95°C for 15 minutes. Remove the solution, cool it to room temperature, and add 150 μL of 0.1% DTPA in PBS buffer. Measure the radioactivity concentration of the solution and remove a small amount for radiochemical purity analysis. Measure the radioactivity before and after administration. Store the sample at room temperature in a lead container and use immediately after preparation.

[0194] 3.2 Quality Control Analysis

[0195] Radiochemical purity: HPLC: Waters Xbridege 5 μm C-18 column (4.6 × 150 mm), flow rate: 1 mL / min; column temperature: 35°C; detection wavelength: 210 nm; injection volume: 20 μL (50–100 μCi). Gradient elution conditions are shown in Table 4.

[0196] Table 4

[0197]

[0198] See the results Figure 16 The radiochemical purity was calculated by area normalization method and was greater than 95%, meeting the test requirements.

[0199] The radiochemical purity was calculated by area normalization method and was greater than 95%, meeting the test requirements.

[0200] 3.3177 Lu-JYT2-401 biodistribution

[0201] Animal identification: Each cage of animals must be hung with a cage tag indicating the animal group, animal number, etc.

[0202] Feed provided: Mouse growth maintenance feed complies with GB14924.4-2001.

[0203] Feeding method: free feeding.

[0204] Drinking water: The drinking water bottles are supplied with high-pressure sterilized tap water. The tap water is inspected by the Taiyuan Monitoring Station of the National Urban Water Supply Quality Monitoring Network and meets the requirements of the "National Drinking Water Quality Standard" (GB5749-2006). The drinking water bottles are replaced every two days and are cleaned and high-pressure sterilized before use.

[0205] Tumor inoculation: The animal model was constructed by Beijing Weitonglihua Animal Technology Co., Ltd. HEK293 tumor cells were subcutaneously inoculated on the right side of the back of each experimental animal. A total of 10 animals were used for subsequent experiments.

[0206] The experiment was divided into 3 groups. The mice were weighed before grouping and randomly divided into groups according to their body weight and tumor size.

[0207] See Table 5 for grouping information.

[0208] Table 5

[0209]

[0210] The drug solution was drawn up with a disposable syringe, and the activity (μCi) before administration (full needle) was detected by an activity meter. The drug was injected into the tail vein, and the residual radioactivity count (μCi) after administration (empty needle) was detected by an activity meter. 177 The dosage of Lu-JYT2-401 injection is shown in Table 6.

[0211] Table 6 177 Lu-JYT2-401 Injection Dosage List

[0212]

[0213] Sampling time: 2 h, 24 h, and 48 h after administration.

[0214] Tissues and organs: The animals were lightly anesthetized, their eyeballs were removed and blood was drawn (blood samples were retained), and the heart, liver, spleen, lungs, kidneys, stomach (contents removed), large intestine (contents removed), small intestine (contents removed), femur (hind limbs), kneecap, muscle, pancreas, brain, bladder (no urine), tumor, skin, urine, feces (2-3 grains in the large intestine), administration site (tail) and the rest of the carcass were removed.

[0215] Gamma counter testing: Collect biological samples at various time points, gently squeeze, and remove any residual blood with absorbent paper. Weigh and record the weight. Place the sample in an EP tube and test with a gamma counter. Load the sample within 24 hours. (If the radioactivity count is too high, allow it to decay and then measure it.) Test results are expressed in CPM (counts per minute).

[0216] Data processing and analysis:

[0217] %ID / g=A tissue / [(A0-A residue )*M tissue ]×100%.

[0218] A0: Total radioactivity count in the syringe before administration (CPM).

[0219] A residue : Residual radioactivity count in the syringe after administration (CPM).

[0220] A tissue : Radioactivity counts in tissues and organs (CPM).

[0221] M tissue : Weight of the tested tissue sample (g).

[0222] Physical attenuation correction: 177 Lu physical decay: activity dose at time T = 0.5^(T / 160.8).

[0223] Dosage (A0-A residue ) multiplied by the decay coefficient at the corresponding time is the corrected CPM value, and %ID / g is calculated.

[0224] Note: Within the activity range of no more than 75 μCi, the calibration curve of the activity meter (μCi) and the gamma counter (CPM) is Y=135628X(R 2 =0.9991).

[0225] The results are shown in Table 7 and Figure 17 HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice were injected into the tail vein once 177Lu-JYT2-401 injection showed a significant decrease in radioactive uptake in the blood over time. Within 2 to 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). This was followed by skin, intestines, lungs, bladder, spleen, and the lowest concentration in the brain.

[0226] Table 7 177 Tissue distribution results of Lu-JYT2-401 injection in HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice

[0227]

[0228] 3.4 SPECT / CT imaging

[0229] Towards 177 The test samples (pH ~5.5-6) ​​were prepared by adding appropriate amount of PBS buffer solution to the three Lu-labeled molecules (JYT2-401, FAP2286, and JY2-011 (PNT6555)). After mixing evenly, 100 μL was drawn with an insulin syringe. Each mouse was injected with only ~400 μCi (1-2 nmol) of the molecules. The measured activities were 177 Lu-JYT2-401: 415 μCi, 177 Lu-FAP2286: 435 μCi, JY2-011 (PNT6555): 420 μCi were injected into mice via the tail vein.

[0230] About 48 hours after administration, 180 μL of 1% sodium pentobarbital solution was injected into the mouse via the intraperitoneal route to anesthetize the mouse. The anesthetized mouse was fixed on the bed of the SPECT / CT instrument, and the SPECT / CT was scanned after CT positioning.

[0231] After the scan is completed, the data is reconstructed and the ROI value of the mouse SPECT data is circled using software to obtain the radioactivity distribution %ID / g of tumor organs and tissues.

[0232] See the results Figure 18 SPECT / CT imaging was performed at 1h, 2h, 4h, 8h, 24h, 48h, and 72h, and the radioactive signal (%ID / cc) in the mouse tumor was circled. 177Lu-JYT2-401 was retained in the tumor for a long time, with the maximum tumor uptake maintained at ~40% ID / cc. The mean value of tumor uptake was comparable to the biodistribution data in Section 3.3. After 48 h, it was ~15% ID / cc and a significant decrease was observed with prolonged time.

[0233] See the results Figure 19 .contrast 177 Lu-JYT2-401 and control group 177 Lu-FAP2286, 177 Comparison of the MIP image of 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 177 Lu-FAP2286, 177 Lu-PNT6555. Since the molecule undergoing clinical trials for the treatment of 177 Lu-FAP2286, and 177 Lu-FAP2286 tumor uptake was also higher than 177 Lu-PNT6555, so the subsequent comparison 177 Lu-JYT2-401 and 177 Efficacy study of Lu-FAP2286.

[0234] 3.5 Pharmacological Efficacy Studies

[0235] The purpose of this study was to evaluate the effect of a single injection of 177 The therapeutic efficacy of Lu-JTY2-401.

[0236] Available in 1 concentration 177 Lu-JTY2-401 and 177 Lu-FAP2286 was prepared for injection (100 μL / mouse), and the injected dose was determined using a radioactivity meter (calibrated by CAPINTEC).

[0237] Provided as described in Section 3.1 above 177 Lu-JTY2-401 and 177 Lu-FAP2286. The following therapeutic compositions were prepared:

[0238] 1. Vehicle (physiological saline, 100 μL) per

[0239] 2. 177 Lu-JTY2-401 15 MBq (100 μL) each

[0240] 3. 177Lu-FAP2286 15MBq (100 μL) each

[0241] A total of 15 HEK293 human embryonic kidney cell Balb / c nude tumor-bearing mice were injected with a single tail vein injection. 177 Lu-JYT2-401 and 177 Lu-FAP2286 injection. Throughout the study, mice underwent weekly health checks, including body weight measurements. Tumor growth was monitored weekly by caliper measurements (tumor volume = length × width × 0.5). Study endpoints included tumors >2 cm in any dimension, tumor ulceration, mouse moribundity, and body weight loss >15% from the last measurement. Mice were housed in cages of five with ad libitum access to food and water at an ambient temperature of 20°C, 40%-50% humidity, and a 12-hour light / 12-hour dark cycle. Injection doses were determined using a radioactivity meter (calibrated by CAPINTEC). Tumor growth was monitored weekly by caliper measurements, and mouse survival was followed.

[0242] Results: Data were collected as tumor volume Figure 20 and survival analysis Figure 21 .

[0243] By comparing tumor volume, survival rate and TGI, 177 Lu-JTY2-401 tumor suppression is superior to 177 Lu-FAP2286, the survival period is also longer than 177 Lu-FAP2286 lasts longer.

[0244] Table 8

[0245]

[0246] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A compound represented by formula (I) or a pharmaceutically acceptable tautomer, hydrate, solvate or salt thereof, characterized in that: The structural formula of the compound represented by formula (I) is: 。 2. The compound of formula (I) according to claim 1 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt 68 Ga labeled products.

3. The compound of formula (I) according to claim 1 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt 177 Lu-labeled product.

4. A pharmaceutical composition comprising the compound of formula (I) according to claim 1 or a pharmaceutically acceptable tautomer, hydrate, solvate or salt thereof.

5. A pharmaceutical composition comprising the compound of formula (I) according to claim 2 or a pharmaceutically acceptable tautomer, hydrate, solvate or salt thereof 68 Ga labeled products.

6. A pharmaceutical composition comprising the compound of formula (I) according to claim 3 or a pharmaceutically acceptable tautomer, hydrate, solvate or salt thereof 177 Lu-labeled product.

7. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

8. A targeted binding fibroblast activation protein inhibitor or binder, characterized in that: The compound of formula (I) according to claim 1 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt, or the compound of formula (I) according to claim 2 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt 68 Ga-labeled products, or The compound represented by formula (I) according to claim 3 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt 177 A Lu-labeled product, or the pharmaceutical composition according to any one of claims 4 to 7.

9. A kit comprising the compound of formula (I) according to claim 1 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt, or the compound of formula (I) according to claim 2 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt. 68 Ga labeled product, or, the compound represented by formula (I) according to claim 3 or its pharmaceutically acceptable tautomer, hydrate, solvate or salt 177 A Lu-labeled product, or a pharmaceutical composition according to any one of claims 4 to 7; and instructions for use in diagnosing or treating a disease.

10. The compound according to claim 2 68 The use of a Ga-labeled product or the pharmaceutical composition according to claim 5 for preparing a drug for diagnosing a disease; the disease is selected from any one or more of head and neck cancer, eye cancer, esophageal cancer, liver cancer, kidney cancer, bile duct cell carcinoma, neuroendocrine tumors, sarcoma, glioma, uterine cancer, rectal cancer, colon cancer, prostate cancer, breast cancer, bladder cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, testicular cancer, skin melanoma, basal cell carcinoma, and squamous cell carcinoma.

11. The use according to claim 10, characterized in that The head and neck cancer is any one or more of oral cancer and pharyngeal cancer; the liver cancer is hepatocellular carcinoma; the kidney cancer is clear cell renal carcinoma; the glioma is astrocytoma; the uterine cancer is cervical cancer; the sarcoma is any one or more of Ewing's sarcoma, Kaposi's sarcoma, rhabdomyosarcoma, and angiosarcoma.

12. The use according to claim 11, characterized in that The pharyngeal cancer is any one or more of hypopharyngeal cancer, nasopharyngeal cancer, and laryngeal cancer.

13. The compound according to claim 3 177 The use of the Lu labeled product or the pharmaceutical composition according to claim 6 for preparing a drug for treating a disease; the disease is selected from any one or more of head and neck cancer, eye cancer, esophageal cancer, liver cancer, kidney cancer, bile duct cell carcinoma, neuroendocrine tumors, sarcoma, glioma, uterine cancer, rectal cancer, colon cancer, prostate cancer, breast cancer, bladder cancer, gastric cancer, pancreatic cancer, lung cancer, ovarian cancer, testicular cancer, skin melanoma, basal cell carcinoma, and squamous cell carcinoma.

14. The use according to claim 13, characterized in that The head and neck cancer is any one or more of oral cancer and pharyngeal cancer; the liver cancer is hepatocellular carcinoma; the kidney cancer is clear cell renal carcinoma; the glioma is astrocytoma; the uterine cancer is cervical cancer; the sarcoma is any one or more of Ewing's sarcoma, Kaposi's sarcoma, rhabdomyosarcoma, and angiosarcoma.

15. The use according to claim 14, characterized in that The pharyngeal cancer is any one or more of hypopharyngeal cancer, nasopharyngeal cancer, and laryngeal cancer.

16. A tumor therapeutic agent, characterized in that: A pharmaceutical composition comprising the compound of formula (I) according to claim 3 or a pharmaceutically acceptable tautomer, hydrate, solvate or salt thereof, or the pharmaceutical composition according to claim 6.

17. A tumor imaging agent, characterized in that: A pharmaceutical composition comprising the compound represented by formula (I) according to claim 2 or a pharmaceutically acceptable tautomer, hydrate, solvate or salt thereof, or the pharmaceutical composition according to claim 5.

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