FAP-α-specific tumor diagnostic imaging agent

Abstract

The present invention relates to FAP-α specific tumor diagnostic imaging agents, in particular the present invention relates to compounds of formula (I), FAP-α specific tumor imaging agents formed by coordination of compounds of formula (I) with a radionuclide, and the use of said compounds in the diagnosis of diseases characterised by overexpression of fibroblast activation protein α (FAP-α) in a subject in need thereof. [Formula 1] TIFF2025503541000052.tif43167

Description

[Technical Field]

[0001] This application claims the benefit of and priority to CN202210851703.X, filed July 20, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to diagnostic agents, and in particular to FAP-α specific tumor diagnostic imaging agents and their use in the diagnosis of diseases characterized by overexpression of fibroblast activation protein α (FAP-α) in subjects in need thereof. [Background technology]

[0003] Tumors are the second leading cause of death from disease. Early diagnosis, early treatment, and personalized comprehensive therapy are effective means to reduce tumor mortality. As the exploration of the mechanism of carcinogenesis progresses, cancer-associated fibroblasts (CAFs), one of the major stromal cells present in the tumor microenvironment (accounting for more than 90% in some tumor stroma), have been found to be involved in almost all stages of carcinogenesis and closely related to tumor growth, migration, and progression. This has made CAFs an attractive research target for tumor diagnosis and anti-tumor therapy.

[0004] A distinctive feature of CAFs is the expression of fibroblast activation protein α (FAP-α). FAP-α, a key protein in CAFs, is a type II membrane-bound glycoprotein with a molecular weight of 97 kDa that belongs to the dipeptidyl peptidase 4 (DPP4) family. FAP-α possesses both dipeptidyl peptidase and endopeptidase activities. The endopeptidase activity distinguishes FAP-α from other members of the DPP4 family. FAP-α is selectively expressed on the surface of CAFs in over 90% of epithelial cancers, including breast, ovarian, lung, colon, rectal, gastric, pancreatic, and cutaneous melanoma. FAP-α is not expressed or is expressed at low levels in fibroblasts in normal tissues, but it appears transiently in wound healing tissues and is transiently expressed during embryogenesis. Therefore, FAP-α has become an attractive target for studying tumor stromal cell biology as well as for tumor diagnosis and antitumor therapy.

[0005] In recent years, research into imaging agents targeting FAP-α in tumor diagnosis has progressed rapidly. Uwe Haberkorn et al. have developed a series of imaging agents and radiotargeted therapeutic agents for PET, MRI, and SPECT imaging based on existing FAP-α small molecule inhibitors. WO2019154886A1 discloses ligand compounds for imaging agents, such as FAPI-02, FAPI-04, FAPI-19, FAPI-34, FAPI-42, and FAPI-46, and further discloses imaging complex compounds formed from the above ligand compounds and radionuclides, such as complex compounds for PET imaging. 68 Ga-FAPI-02, 68 Ga-FAPI-04, 68 Ga-FAPI-46, Al 18 F-FAPI-42 etc. and complex compounds for SPECT imaging 99m Tc-FAPI-34, 203 Jia Bing et al. in CN111991570A disclosed the radionuclide 99m Tc-labeled complex compounds 99m Tc-HFAPi and 99m Tc-HpFAPi was disclosed.99m Tc-HFAPi and 99m Tc-HpFAPi showed the best effect in the prior art by modifying the structure of the chelating agent and linker of the complex compound. 99m It has shown superior biodistribution, higher tumor uptake and tumor / organ uptake ratio, and better tumor imaging ability than Tc-FAPI-34. 68 Ga-labeled tumor diagnostic PET imaging agents have been disclosed. 68 Ga-labeled tumor diagnostic PET imaging agents based on monomeric FAPI 68 It has shown superior tumor uptake and retention compared to Ga-FAPI-46, but because it uses lysine as the linking backbone, its molecular structure is asymmetric and its synthetic pathway is redundant. Summary of the Invention [Problem to be solved by the invention]

[0006] Although research into FAP-α-targeting imaging agents has progressed, improvements have been made in the biodistribution, tumor uptake, and imaging efficacy of this type of imaging agent, but they still fall short of meeting actual clinical needs. To date, there remains a significant and urgent clinical need for imaging agents with better biodistribution, higher tumor uptake rates, and improved imaging capabilities. To improve production efficiency and reduce production costs, it is also necessary to simplify the synthetic pathways for this type of imaging agent. [Means for solving the problem]

[0007] To achieve the above object, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] In the formula, R1, R1', R2, R2', R3, R3', and R4 are each independently H, OH, NH2, or NHC.1-6 Alkyl, Halo, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Haloalkyl, C 1-6 Alkoxyl, C 1-6 Alkylthiols and C 1-6 haloalkoxyl. R4' is H, C 2-6 Alkynyl, CN, -B(OH)2, nitro, carboxyl, -CHO, -C(O)-C 1-6 Alkyl, -C=CC(O)-C 6-10 It is selected from the group consisting of aryl, -SO3H, -SO2NH2, -PO3H2 and tetrazolyl. R5 and R5' are each independently H, halo, or C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyl, C 1-6 Alkylthiols and C 1-6 haloalkoxyl.

[0008] R6 and R7 are each independently H, OH, NH2, or NHC 1-6 Alkyl, Halo, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxyl, C 1-6 Alkylthiols and C 1-6 haloalkoxyl. A is selected from the group consisting of optionally substituted phenyl and 5- or 6-membered heteroaryl, wherein the phenyl, 5- or 6-membered heteroaryl is selected from the group consisting of OH, oxo, halo, cyano, C 1-6 Alkyl, C 1-6 Alkoxyl, C 1-6 Alkyl thiol and / or C 1-6 It may be substituted with haloalkoxyl. L1 is -X1-CO-C 1-6 Alkylene (-X2-C 1-6 alkylene) m - is a functionalized linker. L2 is -CO-C 1-6 Alkylene-(X3-C 1-6 Alkylene-) n -X4- is a functionalized linker. X1, X2, X3, and X4 are each independently selected from the group consisting of O, S, NH, and NCH3; 1-6 The alkylene may be substituted with halo, OH, NH2, oxo and / or cyano. m and n are integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

[0009] In one embodiment, A in the compounds of formula (I) of the present invention is phenyl, [ka] and these groups are selected from the group consisting of OH, oxo, halo, cyano, C 1-6 Alkyl, C 1-6 Alkoxyl, C 1-6 Alkyl thiol and / or C 1-6 It may be substituted with haloalkoxyl.

[0010] In one embodiment, the bifunctional chelating agent of the compound of formula (I) of the present invention is selected from the group consisting of 6-(2-(sulfobenzylidene)hydrazinyl)nicotinic acid (HYNIC), mercaptoacetyldiglycine (MAG2), mercaptoacetyltriglycine (MAG3), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15),11,13-triene-3, 6,9-triacetic acid (PCTA), RESCA, 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NOTA-GA), 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid (DOTA-GA), 1,4,8,11-tetraazabicyclo[6,6,2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,8- diamino-3,6,10,13,16,19-hexazabicyclo[6,6,6]eicosane (DiAmSar), 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptacosane] (DFO).

[0011] Specifically, the bifunctional chelating agent of the compound of formula (I) of the present invention is selected from the group consisting of: [ka]

[0012] In a further embodiment, the compound of the invention is a compound of formula (IA), or a pharmaceutically acceptable salt, stereoisomer thereof. [ka] wherein R3, R3', A, L1 and L2 are as defined herein.

[0013] In a further embodiment, the compound of the invention is a compound of formula (IB), or a pharmaceutically acceptable salt, stereoisomer thereof. [ka] wherein A, L1 and L2 are as defined herein.

[0014] In a further embodiment, the compound of the invention is a compound of formula (IC), or a pharmaceutically acceptable salt, stereoisomer thereof. [ka] wherein L1 and L2 are as defined herein.

[0015] In a further embodiment, the compound of the invention is a compound of formula (IC-I), (IC-II), (IC-III) or (IC-IV), or a pharmaceutically acceptable salt, stereoisomer thereof. [ka] wherein L1 and L2 are as defined herein.

[0016] In a further embodiment, the compound of the invention is a compound of formula (ID), or a pharmaceutically acceptable salt, stereoisomer thereof. [ka] wherein L1 and L2 are as defined herein.

[0017] In a further embodiment, the compound of the invention is a compound of formula (ID-I), (ID-II), (ID-III) or (ID-IV), or a pharmaceutically acceptable salt, stereoisomer thereof. [ka] wherein L1 and L2 are as defined herein.

[0018] In certain embodiments, L1 of formula (I), (IA), (IB), (IC), (IC-I), (IC-II), (IC-III), (IC-IV), (ID), (ID-I), (ID-II), (ID-III) and (ID-IV) of the present invention is -NHCO-C 1-6 Alkylene (-OC 1-6 alkylene) m -, -NHCO-C 1-6 Alkylene (-NH-C 1-6 alkylene) m -, -NHCO-C 1-6 Alkylene (-NCH3-C 1-6 alkylene) m -, -NCH3-CO-C 1-6 Alkylene (-OC 1-6 alkylene) m -, -NCH3-CO-C 1-6 Alkylene (-NH-C 1-6 alkylene) m -, -NCH3-CO-C 1-6 Alkylene (-NCH3-C 1-6 alkylene) m -, -OCO-C 1-6 Alkylene (-OC 1-6 alkylene) m -, -OCO-C 1-6 Alkylene (-NH-C 1-6 alkylene) m -, -OCO-C 1-6 Alkylene (-NCH3-C 1-6 alkylene) m -, wherein said alkylene is optionally substituted with halo, OH, NH2, oxo and / or cyano, and m is an integer of 1, 2, 3, 4, 5 or 6.

[0019] In a preferred embodiment, L1 is -NHCO-C 1-3 Alkylene (-OC 1-3 alkylene) m-, -NHCO-C 1-3 Alkylene (-NH-C 1-3 alkylene) m -, -NHCO-C 1-3 Alkylene (-NCH3-C 1-3 alkylene) m -, -NCH3-CO-C 1-3 Alkylene (-OC 1-3 alkylene) m -, -NCH3-CO-C 1-3 Alkylene (-NH-C 1-3 alkylene) m -, -NCH3-CO-C 1-3 Alkylene (-NCH3-C 1-3 alkylene) m -, -OCO-C 1-3 Alkylene (-OC 1-3 alkylene) m -, -OCO-C 1-3 Alkylene (-NH-C 1-3 alkylene) m -, -OCO-C 1-3 Alkylene (-NCH3-C 1-3 alkylene) m -, wherein said alkylene is optionally substituted with halo, OH, NH2, oxo and / or cyano, and m is an integer of 1, 2, 3, 4, 5 or 6.

[0020] In a more preferred embodiment, L1 is -NHCO-CH2CH2(-O-CH2CH2) m -, -NHCO-CH2CH2(-NH-CH2CH2) m -, -NHCO-CH2CH2(-NCH3-CH2CH2) m -, -NCH3-CO-CH2CH2(-O-CH2CH2) m -, -NCH3-CO-CH2CH2(-NH-CH2CH2) m -, -NCH3-CO-CH2CH2(-NCH3-CH2CH2) m -, -OCO-CH2CH2(-O-CH2CH2) m -, -OCO-CH2CH2(-NH-CH2CH2) m-, -OCO-CH2CH2(-NCH3-CH2CH2) m wherein said CH2CH2 is optionally substituted with halo, OH, NH2, oxo and / or cyano; and m is an integer of 1, 2, 3, 4, 5, or 6. In the most preferred embodiment, L1 is a functionalized linker of -NHCO-CH2CH2-(-O-CH2CH2)4-.

[0021] In certain embodiments, L2 of formula (I), (IA), (IB), (IC), (IC-I), (IC-II), (IC-III), (IC-IV), (ID), (ID-I), (ID-II), (ID-III) or (ID-IV) of the present invention is -CO-C 1-6 Alkylene-(NH-C 1-6 Alkylene-) n -NH-, -CO-C 1-6 Alkylene-(NH-C 1-6 Alkylene-) n -NCH3-, -CO-C 1-6 Alkylene-(NH-C 1-6 Alkylene-) n -O-, -CO-C 1-6 Alkylene-(OC 1-6 Alkylene (v-) n -NH-, -CO-C 1-6 Alkylene-(OC 1-6 Alkylene-) n -NCH3-, -CO-C 1-6 Alkylene-(OC 1-6 Alkylene-) n -O-, -CO-C 1-6 Alkylene-(NCH3-C 1-6 Alkylene-) n -NH-, -CO-C 1-6 Alkylene-(NCH3-C 1-6 Alkylene-) n -NCH3-, -CO-C 1-6 Alkylene-(NCH3-C 1-6 Alkylene-) nA functionalized linker selected from the group consisting of -O-, wherein said alkylene is optionally substituted with halo, OH, NH2, oxo, and / or cyano, and n is an integer of 1, 2, 3, 4, 5, or 6.

[0022] In a preferred embodiment, L2 is -CO-C 1-3 Alkylene-(NH-C 1-3 Alkylene-) n -NH-, -CO-C 1-3 Alkylene-(NH-C 1-3 Alkylene-) n -NCH3-, -CO-C 1-3 Alkylene-(NH-C 1-3 Alkylene-) n -O-, -CO-C 1-3 Alkylene-(OC 1-3 Alkylene-) n -NH-, -CO-C 1-3 Alkylene-(OC 1-3 Alkylene-) n -NCH3-, -CO-C 1-3 Alkylene-(OC 1-3 Alkylene-) n -O-, -CO-C 1-3 Alkylene-(NCH3-C 1-3 Alkylene-) n -NH-, -CO-C 1-3 Alkylene-(NCH3-C 1-3 Alkylene-) n -NCH3-, -CO-C 1-3 Alkylene-(NCH3-C 1-3 Alkylene-) n A functionalized linker selected from the group consisting of -O-, wherein said alkylene is optionally substituted with halo, OH, NH2, oxo, and / or cyano, and n is an integer of 1, 2, 3, 4, 5, or 6.

[0023] In a more preferred embodiment, L2 is -CO-CH2CH2-(NH-CH2CH2-) n -NH-, -CO-CH2CH2-(NH-CH2CH2-) n-NCH3-, -CO-CH2CH2-(NH-CH2CH2-) n -O-, -CO-CH2CH2-(O-CH2CH2-) n -NH-, -CO-CH2CH2-(O-CH2CH2-) n -NCH3-, -CO-CH2CH2-(O-CH2CH2-) n -O-, -CO-CH2CH2-(NCH3-CH2CH2-) n -NH-, -CO-CH2CH2-(NCH3-CH2CH2-) n -NCH3-, -CO-CH2CH2-(NCH3-CH2CH2-) n A functionalized linker selected from the group consisting of -O-, wherein said CH2CH2 is optionally substituted with halo, OH, NH2, oxo and / or cyano, and n is an integer of 1, 2, 3, 4, 5 or 6. In the most preferred embodiment, L2 is a functionalized linker of -CO-CH2CH2-(O-CH2CH2-)4-NH-.

[0024] In certain embodiments, the compound of the invention is a compound selected from the following, or a pharmaceutically acceptable salt, stereoisomer thereof: [ka] TIFF0007741988000011.tif95163

[0025] In another aspect, the invention provides a kit comprising a compound described herein and a pharmaceutically acceptable carrier.

[0026] In one embodiment, the present invention provides a kit comprising a ligand compound of formula (I), (IA), (IB), (IC), (IC-I), (IC-II), (IC-III), (IC-IV), (ID), (ID-I), (ID-II), (ID-III) or (ID-IV) and a pharmaceutically acceptable carrier.

[0027] In a further embodiment, the present invention provides a kit comprising the above-mentioned H7ND, D7ND or N7ND ligand compound and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, but is not limited to, water, saline, buffered saline, etc. Preferably, the present invention provides a kit in the form of a solution or freeze-dried powder, which may contain one or more stabilizers, such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc.

[0028] In certain embodiments, the present invention provides a kit comprising the ligand compound H7ND, tris(hydroxymethyl)methylglycine (tricine), trisodium triphenylphosphine-3,3',3"-trisulfonate (TPPTS), and one or more stabilizers, and a pharmaceutically acceptable carrier. Preferably, the reagents in the kit are in the form of a freeze-dried powder, and the radionuclide (e.g., 99m Tc) is mixed with In certain embodiments, the present invention provides a kit comprising the ligand compound D7ND, optionally one or more stabilizers, and a pharmaceutically acceptable carrier. The reagents in the kit may be sensitized to a radionuclide (e.g., 68 It is mixed with Ga.

[0029] In certain embodiments, the present invention provides a kit comprising the ligand compound D7ND, optionally one or more stabilizers, and a pharmaceutically acceptable carrier. The reagents in the kit may be sensitized to a radionuclide (e.g., 177 Lu). In certain embodiments, the present invention provides a kit comprising the ligand compound D7ND, optionally one or more stabilizers, and a pharmaceutically acceptable carrier. The reagents in the kit may be sensitized to a radionuclide (e.g., 90 Y) is mixed with In certain embodiments, the present invention provides a kit comprising the ligand compound N7ND, optionally one or more stabilizers, and a pharmaceutically acceptable carrier. The reagents in the kit may be sensitized to a radionuclide (e.g., 18 F).

[0030] In another aspect, the present invention provides a method of making a kit, comprising the step of combining a ligand compound described herein with a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a method of preparing a kit comprising the step of combining a ligand compound described herein with one or more stabilizers and a pharmaceutically acceptable carrier.

[0031] In certain embodiments, the present invention provides a method for preparing a kit, comprising mixing a ligand compound H7ND described herein with tricine, TPPTS, and one or more stabilizers, and a pharmaceutically acceptable carrier to form a solution. Preferably, the method further comprises lyophilizing the solution. In certain embodiments, the present invention provides a method for preparing a kit, comprising mixing the ligand compound D7ND, optionally one or more stabilizers, and a pharmaceutically acceptable carrier to form a solution. In certain embodiments, the present invention provides a method for preparing a kit, comprising mixing the ligand compound N7ND, optionally one or more stabilizers, and a pharmaceutically acceptable carrier to form a solution.

[0032] In another aspect, the present invention provides a complex compound comprising a compound of formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to the bifunctional chelator of formula (I) by a coordinate bond. [ka] wherein R1, R1', R2, R2', R3, R3', R4, R4', R5, R5', R6, R7, A, L1 and L2 are as defined herein.

[0033] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IA), or a pharmaceutically acceptable salt, stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to a bifunctional chelator of formula (IA) by a coordinate bond. [ka] wherein R3, R3', A, L1 and L2 are as defined herein.

[0034] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IB), or a pharmaceutically acceptable salt, stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to a bifunctional chelator of formula (IB) by a coordinate bond. [ka] wherein A, L1 and L2 are as defined herein.

[0035] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IC), or a pharmaceutically acceptable salt, stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to a bifunctional chelator of formula (IC) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0036] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IC-I), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to a bifunctional chelator of formula (IC-I) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0037] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IC-II), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein the radionuclide is bound to a bifunctional chelator of formula (IC-II) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0038] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IC-III), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein the radionuclide is bound to a bifunctional chelator of formula (IC-III) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0039] In one embodiment, the present invention provides a complex compound comprising a compound of formula (IC-IV), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein the radionuclide is bound to a bifunctional chelator of formula (IC-IV) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0040] In one embodiment, the present invention provides a complex compound comprising a compound of formula (ID), or a pharmaceutically acceptable salt, stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to a bifunctional chelator of formula (ID) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0041] In one embodiment, the present invention provides a complex compound comprising a compound of formula (ID-I), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein said radionuclide is bound to a bifunctional chelator of formula (ID-I) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0042] In one embodiment, the present invention provides a complex compound comprising a compound of formula (ID-II), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein the radionuclide is bound to the bifunctional chelator of formula (ID-II) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0043] In one embodiment, the present invention provides a complex compound comprising a compound of formula (ID-III), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein the radionuclide is bound to the bifunctional chelator of formula (ID-III) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0044] In one embodiment, the present invention provides a complex compound comprising a compound of formula (ID-IV), or a pharmaceutically acceptable salt or stereoisomer thereof, and a radionuclide, wherein the radionuclide is bound to a bifunctional chelator of formula (ID-IV) by a coordinate bond. [ka] wherein L1 and L2 are as defined herein.

[0045] In one embodiment, the present invention provides a complex compound comprising a compound of H7ND or D7ND or N7ND and a radionuclide, wherein said radionuclide is bound by a coordinate bond to a bifunctional chelator of the compound H7ND or D7ND or N7ND. [ka] TIFF0007741988000026.tif95163

[0046] In certain embodiments, the complex compounds described herein contain radionuclides, including alpha-emitting isotopes, beta-emitting isotopes, gamma-emitting isotopes, Auger electron-emitting isotopes, or X-ray-emitting isotopes.

[0047] In a preferred embodiment, the complex compounds described herein include, but are not limited to: 18 F, 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, 177 Lu, 47 Sc, 212 Bi, 213 Bi, 72 As, 123 I, 124 I, 131 I, 211 At, 201 Tl, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 223 Ra or 89 It contains radioactive nuclides such as Sr. In a more preferred embodiment, the complex compounds described herein include, but are not limited to: 68 Ga, 99m Tc, 177 Lu, 90 Y or 18 It contains radioactive nuclides including F.

[0048] In certain embodiments, the present invention provides a complex compound comprising the following compound, or a pharmaceutically acceptable salt or stereoisomer thereof: [ka] TIFF0007741988000028.tif242147TIFF0007741988000029.tif99167

[0049] In another aspect, the present invention provides a diagnostic kit comprising a complex compound described herein and a pharmaceutically acceptable carrier. In one embodiment, the present invention provides a diagnostic kit comprising a complex compound described herein and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes, but is not limited to, water, saline, buffered saline, etc. Preferably, the present invention provides a diagnostic kit that may also include one or more stabilizers, such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc.

[0050] In certain embodiments, the present invention provides 99m A diagnostic kit is provided which includes a complex compound of Tc-H7ND, optionally one or more stabilizers such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc., and a pharmaceutically acceptable carrier. In certain embodiments, the present invention provides 68 A diagnostic kit is provided which includes a complex compound of Ga-D7ND, optionally one or more stabilizers such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc., and a pharmaceutically acceptable carrier.

[0051] In certain embodiments, the present invention provides 177 A diagnostic kit is provided which includes a complex compound of Lu-D7ND, optionally one or more stabilizers such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc., and a pharmaceutically acceptable carrier. In certain embodiments, the present invention provides 90 A diagnostic kit is provided which includes a complex compound of Y-D7ND, optionally one or more stabilizers such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc., and a pharmaceutically acceptable carrier.

[0052] In certain embodiments, the present invention provides the Al 18 A diagnostic kit is provided which includes a complex compound of F-N7ND, optionally one or more stabilizers such as sodium chloride, silicate, phosphate buffer, acetate buffer, succinate buffer, ascorbic acid, gentisic acid, etc., and a pharmaceutically acceptable carrier. In another aspect, the present invention provides a method for diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof, comprising using a complex compound described herein or a kit comprising the same and imaging.

[0053] In a preferred embodiment, the complex compound described herein can be directly administered into the bloodstream, muscle or internal organs.Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular and subcutaneous delivery.Depending on the type of cancer described herein and / or the administration route, a wide range of acceptable dosages is contemplated herein. The acceptable dosage range includes a dosage of about 1 μg / kg to about 1 g / kg; preferably, a dosage of about 1 μg / kg to about 0.5 g / kg; more preferably, a dosage of about 1 μg / kg to about 0.1 g / kg; more preferably, a dosage of about 1 μg / kg to about 0.05 g / kg; more preferably, a dosage of about 1 μg / kg to about 0.01 g / kg; more preferably, a dosage of about 1 μg / kg to about 5 mg / kg; more preferably, a dosage of about 1 μg / kg to about 1 mg / kg; more preferably, a dosage of about 1 μg / kg to about 0.5 mg / kg; more preferably, a dosage of about 1 μg / kg to about 0.1 mg / kg; more preferably, a dosage of about 1 μg / kg to about 0.05 mg / kg; more preferably, a dosage of about 1 μg / kg to about 0.01 mg / kg.

[0054] In a preferred embodiment, imaging may be performed by any method suitable for measuring one particular radionuclide, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), etc. In a preferred embodiment, the diseases characterized by overexpression of fibroblast activation protein alpha (FAP-α) include, but are not limited to, cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders.

[0055] In a preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is cancer, including, but not limited to, breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, liver cancer, esophageal cancer, gastric cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, bladder cancer, bile duct cancer, renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, carcinoma of unknown primary, thymic carcinoma, glioma, astrocytoma, cervical cancer, prostate cancer, and testicular cancer. In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is chronic inflammation, including, but not limited to, rheumatoid arthritis, osteoarthritis, Crohn's disease, and the like.

[0056] In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is fibrosis, including, but not limited to, idiopathic pulmonary fibrosis, liver cirrhosis, and the like. In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is a keloid disorder, including but not limited to scar formation, keloid tumors, keloid scars, etc.

[0057] In another aspect, the present invention provides the use of a complex compound described herein in the manufacture of a diagnostic kit for diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof. In one embodiment, the present invention provides the use of a complex compound described herein in the manufacture of a diagnostic kit for diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof, wherein the subject is administered with reagents included in the diagnostic kit and then imaged.

[0058] In a preferred embodiment, imaging may be performed by any method suitable for measuring one particular radionuclide, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), etc. In a preferred embodiment, the diseases characterized by overexpression of fibroblast activation protein alpha (FAP-α) include, but are not limited to, cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders.

[0059] In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is cancer, including but not limited to breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, liver cancer, esophageal cancer, gastric cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, bladder cancer, bile duct cancer, renal cancer, neuroendocrine tumors, oncogenetic osteomalacia, sarcoma, cancer of unknown primary, thymic cancer, glioma, astrocytoma, cervical cancer, prostate cancer, and testicular cancer. In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is chronic inflammation, including, but not limited to, rheumatoid arthritis, osteoarthritis, Crohn's disease, and the like.

[0060] In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is fibrosis, including, but not limited to, idiopathic pulmonary fibrosis, liver cirrhosis, and the like. In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is a keloid disorder, including but not limited to scar formation, keloid tumors, keloid scars, etc. In another aspect, the present invention provides a complex compound as described herein for use in diagnosing disease.

[0061] In one embodiment, the present invention provides a complex compound as described herein for use in diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof, wherein the subject is administered with reagents included in the diagnostic kit and then imaged. In certain embodiments, the present invention provides a complex compound as described herein for use in diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof, wherein the complex compound is administered to the subject and then the subject is imaged.

[0062] In a preferred embodiment, imaging may be performed by any method suitable for measuring one particular radionuclide, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), etc. In a preferred embodiment, the diseases characterized by overexpression of fibroblast activation protein alpha (FAP-α) include, but are not limited to, cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders. In a preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is cancer, including, but not limited to, breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, liver cancer, esophageal cancer, gastric cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, bladder cancer, bile duct cancer, renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, carcinoma of unknown primary, thymic carcinoma, glioma, astrocytoma, cervical cancer, prostate cancer, and testicular cancer.

[0063] In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is chronic inflammation, including, but not limited to, rheumatoid arthritis, osteoarthritis, Crohn's disease, and the like. In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is fibrosis, including, but not limited to, idiopathic pulmonary fibrosis, liver cirrhosis, and the like. In a more preferred embodiment, the disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) is a keloid disorder, including but not limited to scar formation, keloid tumors, keloid scars, etc. [Effects of the Invention]

[0064] The FAP-α specific tumor diagnostic imaging agent of the present invention is in a dimeric form. The imaging agent of the present invention is based on the monomeric FAP-α of the prior art. 99m Tc-HFAPi or 68 Compared with Ga-FAPi-04 imaging agent, it has significantly higher FAP-α binding affinity and in vivo stability, a higher tumor / normal tissue uptake ratio, and better imaging contrast, which may be beneficial for the diagnosis of small tumor lesions, especially when SPECT imaging is sensitive to background signal noise. The FAP-α specific tumor diagnostic imaging agent of the present invention is based on monomeric FAP-α in the prior art. 99m Tc-HFAPi or 68 Compared to the Ga-FAPi-04 imaging agent, it has a significantly shorter retention time in the body, which may reduce radiation exposure to the patient and reduce the risk of side effects due to radiation.

[0065] The FAP-α-specific tumor diagnostic imaging agent of the present invention is based on the dimeric form of FAP-46 described above. 68 Compared with Ga-labeled tumor diagnostic PET imaging agents, the FAP-α-specific tumor diagnostic imaging agent of the present invention has a nitrogen atom as a linking backbone and an overall symmetrical molecular structure, which makes the synthesis simple and efficient, greatly improves the production efficiency, and significantly reduces the production cost. In addition, the FAP-α-specific tumor diagnostic imaging agent of the present invention is based on the above-mentioned dimeric FAP-46. 68 Compared with Ga-labeled tumor diagnostic PET imaging agents, significantly better technical effects are achieved in terms of improving FAP-α binding affinity and tumor uptake. [Brief explanation of the drawings]

[0066] The drawings constituting a part of this disclosure are used to provide a further understanding of the disclosure. The drawings of the present disclosure are used to explain the present invention together with the examples, and are not intended to place undue limitations on the present invention. The drawings include the following drawings: [Figure 1]The synthetic route of the complex compound H7ND is shown. [Figure 2] 1 shows the mass spectrum of the complex compound H7ND. [Figure 3] 1 shows the mass spectrum of the complex compound D7ND. [Figure 4] Figure 4 shows quality control analysis of the complex compound 68Ga-D7ND, where Figure 4A shows the measurement results of the labeling yield of 68Ga-D7ND, and Figure 4B shows the measurement results of the radiochemical purity of 68Ga-D7ND after purification. [Figure 5] 1 shows an in vitro cell binding assay of the complex compound 99mTc-H7ND. [Figure 6] Figure 6 shows the experimental results of the in vivo stability and metabolism of the complex compound 99mTc-H7ND in normal mice. Figure 6A shows the results of the in vivo metabolic stability test of the complex compounds 99mTc-HFAPi and 99mTc-H7ND in mice, and Figure 6B shows the results of the in vivo retention test of the complex compounds 99mTc-HFAPi and 99mTc-H7ND in mice. [Figure 7] 1 shows SPECT / CT imaging of the complex compound 99mTc-H7ND in a U87MG tumor-bearing mouse model. [Figure 8] Figure 8 shows a comparison of the biodistribution of the complex compounds 99mTc-H7ND and 99mTc-HFAPi in a U87MG tumor-bearing mouse model. Figure 8A shows the biodistribution of 99mTc-H7ND in a U87MG tumor-bearing mouse model, Figure 8B shows the tumor / normal organ uptake ratio of 99mTc-H7ND in a U87MG tumor-bearing mouse model, Figure 8C shows the biodistribution of 99mTc-HFAPi in a U87MG tumor-bearing mouse model, and Figure 8D shows the tumor / normal organ uptake ratio of 99mTc-HFAPi in a U87MG tumor-bearing mouse model. [Figure 9] Comparison of micro-PET imaging of complex compounds 68Ga-D7ND and 68Ga-FAPI-04 in a U87MG tumor-bearing mouse model. [Figure 10]10A and 10B show 18F-FDG-PET and 99mTc-H7ND-SPECT imaging of a lung tumor patient, of which FIG. 10A shows 18F-FDG-PET / CT imaging and FIG. 10B shows 99mTc-H7ND-SPECT imaging. [Figure 11] 99mTc-H7ND-SPECT imaging of a patient with lung tumor. DETAILED DESCRIPTION OF THE INVENTION

[0067] definition As used herein, the following words and phrases are generally intended to have the meanings set forth below, unless the context in which they are used indicates otherwise. As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0068] As used herein, the term "alkyl" refers to a straight- or branched-chain saturated hydrocarbon group having the indicated number of carbon atoms (e.g., having 1 to 6 carbon atoms, etc.). In some embodiments, examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl), 1-butyl (n-butyl), 2-methyl-1-propyl (iso-butyl), 2-butyl (sec-butyl), 2-methyl-2-propyl (tert-butyl), 1-pentyl (n-pentyl), 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, etc.

[0069] As used herein, the term "alkenyl" refers to a monovalent, linear or branched, unsaturated hydrocarbon group having a designated number of carbon atoms (e.g., 2 to 6 carbon atoms, etc.) and carbon-carbon double bonds (e.g., 1, 2, or 3 carbon-carbon double bonds). In some embodiments, examples of alkenyl include, but are not limited to, ethenyl (i.e., -CH=CH), propen-1-yl (i.e., -CH=CHCH), propen-3-yl (alternatively allyl, i.e., -CHCH=CH), propen-2-yl (i.e., -C(CH)=CH), butadienyl (including 1,2-butadienyl and 1,3-butadienyl), and the like.

[0070] As used herein, the term "alkynyl" refers to a monovalent linear or branched, unsaturated hydrocarbon group having a designated number of carbon atoms (e.g., 2 to 6 carbon atoms, etc.) and carbon-carbon triple bonds (e.g., 1, 2, or 3 carbon-carbon triple bonds). In some embodiments, examples of alkynyl include, but are not limited to, ethynyl (i.e., -C≡CH), propargyl (i.e., -CHC≡CH), propynyl (i.e., -C≡CCH), and the like.

[0071] As used herein, the term "heteroaryl" refers to an aromatic monocyclic ring group containing 5 to 6 ring atoms, which, in addition to carbon atoms, contain at least one heteroatom selected from oxygen, nitrogen, and / or sulfur. It should also be understood that a heteroaryl group can be bonded through any suitable ring atom of the heteroaryl, including carbon atoms and heteroatoms (e.g., nitrogen). Exemplary heteroaryls include, but are not limited to, pyridyl, pyrrolyl, pyrazinyl, pyrimidyl, pyridazinyl, pyrazolyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, furanyl, and the like.

[0072] As used herein, the term "halo" refers to fluoro or fluorine, chloro or chlorine, bromo or bromine, and iodine or iodine. As used herein, the term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms have been replaced with a halogen, where alkyl is as defined herein. As used herein, the term "alkoxyl" refers to an alkyl-O- group, where alkyl is as defined herein. As used herein, the term "alkylthiol" refers to an alkyl-S- group, where alkyl is as defined herein.

[0073] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the bioavailability and properties of a particular compound, and which is not biologically or otherwise undesirable. Pharmaceutically acceptable salts can be prepared from inorganic and organic acids. Salts derived from organic acids include hydrochloride, hydrobromide, sulfate, nitrate, phosphate, carbonate, hydrogensulfate, hydrogenphosphate, dihydrogenphosphate, bicarbonate, etc. Salts derived from organic acids include formate, acetate, propionate, glycolate, pyruvate, oxalate, malate, malonate, succinate, maleate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, mesylate, ethylsulfonate, tosylate, salicylate, etc.

[0074] As used herein, the term "amino protecting group" is well understood by those skilled in the art of synthetic organic chemistry as a moiety that can be selectively attached to and removed from an appropriate amine functional group. The field of protecting group methodology is advanced, and many amine protecting groups and methods for their use are well known in the art and are described, for example, in authoritative treatises on the subject, such as "Green's Protecting Groups in Organic Synthesis," P.G.M.Wuts, T.W.Greene, 4th Edition (Wiley, 2006).

[0075] As used herein, the terms "optional," "optionally," and "may" mean that the preceding described event or circumstance may or may not occur, and the description includes cases where said event or circumstance occurs and cases where it does not occur. As used herein, the term "stereoisomers" refers to compounds that have identical chemical composition and connectivity but differ in the orientation of their atoms in space and are not interconvertible by rotation about a single bond. "Stereoisomers" includes "diastereomers" and "enantiomers." "Diastereomers" refer to stereoisomers with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers differ in physical properties such as melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography. "Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0076] general synthesis The compounds of the present invention can be prepared using the methods disclosed herein, routine modifications thereof that are apparent from the disclosure herein, and methods well known in the art. Exemplary embodiments of the compounds of the present invention can be synthesized using the general reaction schemes set forth below. It will be apparent from the disclosure herein that this general scheme can be modified by substituting other starting materials of similar structure to yield corresponding different products. Starting materials are typically obtained from commercial sources or synthesized using published methods.

[0077] [ka] A compound of formula (Ia) is condensed with a compound of formula (Ib) under conditions suitable for the formation of an amide. For example, a condensing agent (e.g., 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), ethyl cyanoglyoxylate-2-oxime) and a base (e.g., N-methylmorpholine, 4-dimethylaminopyridine, triethylamine, diisopropylethylamine, etc.) are added to a mixture of the compound of formula (Ia) and the compound of formula (Ib) in an inert solvent (e.g., dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, methylene chloride), and the mixture is reacted to obtain the compound of formula (I). wherein the variables R1, R1', R2, R2', R3, R3', R4, R4', R5, R5', R6, R7, A, L1, and L2 are as defined herein.

[0078] [ka] The compound of formula (Ic) is converted to a compound of formula (Ia) under acidic or basic conditions or in the presence of hydrogen / a catalyst (e.g., palladium on carbon). In the formula, PG represents a common amino-protecting group such as tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), para-methoxybenzyl (PMB), triphenylmethyl (Trt), or a hydroxyl-protecting group such as methyl, ethyl, benzyl, methanesulfonyl, p-toluenesulfonyl, or a silicon-based protecting group. In the formula, the variables R, R', R, R', R, R, R', R, R, R', R, R, R, R, A, L, and L are as defined herein.

[0079] [ka] The compound of formula (Id) is condensed with the compound of formula (Ie) under appropriate conditions. For example, a mixture of the compound of formula (Id) and the compound of formula (Ie) in an inert solvent (e.g., dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, methylene chloride) is added with a condensing agent (e.g., 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (O ... and reacting with methyl methyl phosphate (HBTU), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), ethyl cyanoglyoxylate-2-oxime, and a base (e.g., N-methylmorpholine, 4-dimethylaminopyridine, triethylamine, diisopropylethylamine, etc.) to obtain a compound of formula (Ic), where the variables R1, R1', R2, R2', R3, R3', R4, R4', R5, R5', R6, R7, A, L1, and L2 are as defined herein.

[0080] [ka] The protecting group PG' is removed from the compound of formula (If) under appropriate conditions to give the compound of formula (Ie).

[0081] [ka] The compound of formula (Ig) is condensed with a compound of formula (Ih) under appropriate conditions. For example, a condensing agent (e.g., 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), ethyl cyanoglyoxylate-2-oxime) and a base (e.g., N-methylmorpholine, 4-dimethylaminopyridine, triethylamine, diisopropylethylamine, etc.) are added to a mixture of the compound of formula (Ig) and the compound of formula (Ih) in an inert solvent (e.g., dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, tetrahydrofuran, methylene chloride), and the mixture is reacted to obtain a compound of formula (If). [Example]

[0082] The following examples are offered to illustrate the preparation of compounds of the present invention and are not intended to limit the disclosure in any way.

[0083] I. Experimental Reagents and Equipment 1. Experimental Reagents Unless otherwise noted, starting materials and reagents are either commercially available or prepared by known methods.

[0084] 2. Experimental equipment and high-performance liquid chromatography (HPLC) method The HPLC equipment, Agilent 1260 HPLC system (equipped with a diode array detector) was purchased from Agilent, USA. The HPLC radioactive signal detector (Gabistar γ-ray test) was purchased from Reitest, Germany. The semi-preparative C18 reversed-phase column (ReproSil-Pur Basic C18, 5 μm, 250 × 10.0 mm) and analytical C18 reversed-phase column (ReproSil-Pur Basic C18, 5 μm, 250 × 4.6 mm) were purchased from Dr. Maisch, Germany. The freeze dryer (FD-1D-50) was purchased from Beijing Biocool Laboratory Equipment Co., Ltd. The radioactive γ-counter (Wizard-2470) was purchased from PerkinElmer, USA. The dose calibrator (CRC-25R) was purchased from Capintec, USA.

[0085] Method 1 for HPLC analysis and purification of the target product: An Agilent 1260 HPLC system was equipped with an analytical or semi-preparative C18 reverse-phase chromatography column. The flow rate was set at 1 mL / min for analytical and 4 mL / min for semi-preparative. Gradient elution lasted for 25 min. Mobile phase A was deionized water (containing 0.05% TFA) and mobile phase B was acetonitrile (containing 0.05% TFA). The gradient elution was set to 85% A and 15% B at the beginning, 85% A and 15% B at 5 min, 20% A and 80% B at 20 min, and 85% A and 15% B at 25 min.

[0086] Method 2 for analysis and purification of the target product by HPLC: The equipment and flow rate were the same as in Method 1. Gradient elution lasted for 20 min. Mobile phase A was deionized water (containing 0.05% TFA), and mobile phase B was acetonitrile (containing 0.05% TFA). The gradient elution was set to 95% A and 5% B at the start, 95% A and 5% B at 5 min, 70% A and 30% B at 10 min, 70% A and 30% B at 15 min, 95% A and 5% B at 15.1 min, and 95% A and 5% B at 20 min.

[0087] Method 3 for analysis and purification of the target product by HPLC: The equipment and flow rate were the same as in Method 1. Gradient elution lasted for 20 min, with mobile phase A being deionized water (containing 0.05% TFA) and mobile phase B being acetonitrile (containing 0.05% TFA). The gradient elution was set to 95% A and 5% B at the beginning, 55% A and 45% B at 14 min, and 95% A and 5% B at 20 min.

[0088] II. Experimental Methods and Results The abbreviations used in the examples below have the following meanings:

[0089] [Table 1]

[0090] Example 1 Synthesis of intermediate compound Fmoc-NH-PEG4-CO-N-bis(PEG4-carboxylic acid) (compound 4) [ka]

[0091] 1. Synthesis of Fmoc-NH-PEG4-CO-N-bis(PEG4-COOtBu) A 50 mL reaction flask was charged with 10.00 g of NH-bis(PEG4-COOtBu), 7.79 g of Fmoc-NH-PEG4-carboxylic acid, 6.08 g of HATU, and 40 mL of DMF, and the mixture was stirred at 20–30°C until dissolved. DIPEA was then added dropwise, resulting in a significant exotherm and a temperature rise of approximately 50°C. After the addition was complete, the reaction mixture was stirred at 20–30°C for 2.0 h. TLC (methylene chloride:methanol = 10:1, with one drop of ammonium hydroxide added) indicated the reaction was complete. 200 mL of water was added to the reaction mixture, which was then extracted with methylene chloride (30 mL x 4). The combined organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by silica column chromatography (eluent: methylene chloride to methylene chloride / methanol (10:1)) to obtain 17.5 g of product in 100% yield.

[0092] 2. Synthesis of Fmoc-NH-PEG4-CO-N-bis(PEG4-carboxylic acid) (Compound 4) A 50 ml flask was charged with 17.50 g of Fmoc-NH-PEG4-CO-N-bis(PEG4-COOtBu), 14 ml of trifluoroacetic acid, and 7.0 ml of methylene chloride, and the mixture was stirred at 20-30°C for 20-22 hours. TLC (methylene chloride:methanol = 10:1, with one drop of acetic acid added) indicated the reaction was complete. After adding 200 ml of water, the reaction mixture was extracted with methylene chloride (30 ml x 4). The combined organic phase was concentrated to dryness under reduced pressure and then purified by preparative HPLC (eluent: water-acetonitrile). The fractions were concentrated under reduced pressure to give 14.14 g of product in 90% yield.

[0093] Example 2 Synthesis of Compound HYNIC-PEG4-CO-N-bis(PEG4-7N) (hereinafter referred to as H7ND) Step 1: Synthesis of (S)-7-amino-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide (compound 3, hereinafter referred to as 7N) 84 mg (444 μmol, 1.1 equivalents) of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (compound 1) was dissolved in 1000 μL of ultra-dry DMF, and 76 mg (404 μmol, 1.0 equivalents) of 7-aminoquinoline-4-carboxylic acid (compound 2) was dissolved in 2000 μL of ultra-dry DMF. The DMF solutions of compound 1 and compound 2 were combined in a 10 mL reaction tube, and then 1315 μL of HATU (dissolved in ultra-dry DMF, 0.3 μmol / μL) (404 μmol, 1.0 equivalents) was added. 320 μL (1616 μmol, 4.0 equivalents) of DIEA was added to the mixture at room temperature in two portions with an interval of 0.5 hours. The mixture was allowed to react for 1 hour with gentle shaking. The reaction was monitored by HPLC (Method 1). After the reaction was completed, the target product was isolated and purified (Method 2). Fractions corresponding to the elution peak at 14.8 minutes in semi-preparative HPLC were collected, combined, and lyophilized using a vacuum freeze-drying method to obtain 131 mg of compound 7N (compound 3). A small amount of the product was dissolved and confirmed by HPLC, revealing a purity of 99.2%. ESI-MS mass spectrometry confirmed the identity of the desired product. ESI-MS: m / z = 360.13 [M+H] + , theoretical molecular weight: 359.12.

[0094] Step 2: Synthesis of Fmoc-NH-PEG-CO-N-bis(PEG-7N) (Compound 5) 22.5 mg (62.66 μmol, 3.0 equiv.) of 7N (compound 3) was dissolved in 500 μL of ultra-dry DMF. 26.8 mg (27.2 μmol, 1.3 equiv.) of Fmoc-NH-PEG4-CO-N-bis(PEG4-carboxylic acid) (compound 4) was dissolved in 200 μL of ultra-dry DMF. The DMF solutions of compound 3 and compound 4 were combined in a 1.5 mL EP tube, and 142 μL of HATU (0.59 μmol / μL, dissolved in ultra-dry DMF) (83.8 μmol, 4.0 equiv.) was added. 27.8 μL (166.8 μmol, 8.0 equiv.) of DIPEA was added in one portion to the mixed solution. The mixture was shaken at room temperature and reacted for 48 hours. The reaction was monitored by HPLC (Method 1). After completion of the reaction, the target product was isolated and purified (Method 3). Fractions corresponding to the peak eluting at 11.8 minutes in semi-preparative HPLC were collected, combined, and lyophilized using a vacuum freeze-drying method to obtain 14.8 mg of a saffron-colored solid (compound 5). A small amount of the product was dissolved and confirmed by HPLC, revealing a purity of 98.1%. MALDI-TOF mass spectrometry confirmed the identity of the desired product. MALDI-TOF-MS: m / z = 1665.77 [M+H] + , 1687.75[M+Na] + , 1703.73[M+K] + , theoretical molecular weight: 1664.71.

[0095] Step 3: Synthesis of NH2-PEG4-CO-N-bis(PEG4-7N) (Compound 6) 12.8 mg of Fmoc-NH-PEG4-CO-N-bis(PEG4-7N) (Compound 5) was dissolved in 200 μL of acetonitrile. 50 μL of piperidine was added and the mixture was allowed to react at room temperature for 20 minutes. The reaction process was monitored by HPLC (Method 3). After the reaction was completed, the target product was isolated and purified (Method 3). The fractions corresponding to the elution peak at 8.4 minutes in semi-preparative HPLC were collected, combined, and lyophilized by vacuum freeze-drying to obtain 9.2 mg of a yellow solid (Compound 6). A small amount of the product was dissolved and confirmed by HPLC, revealing a purity of 99.5%.

[0096] Step 4: Synthesis of HYNIC-PEG4-CO-N-bis(PEG4-7N) (Compound 8, H7ND) 8.0 mg of NH2-PEG4-CO-N-bis(PEG4-7N) (compound 6) was dissolved in 200 μL of ultra-dry DMF. 6.0 mg of NHS-HYNIC (compound 7) was dissolved in 200 μL of ultra-dry DMF. The DMF solutions of compound 6 and compound 7 were combined in a 1.5 mL EP tube, and then 10 μL of DIPEA was added. The reaction was allowed to proceed with shaking at 30 °C for 2 hours. The reaction process was monitored by HPLC (Method 3). After completion of the reaction, the target product was isolated and purified (Method 3). Fractions corresponding to the elution peak at 9.5 minutes in semi-preparative HPLC were collected, combined, and lyophilized by vacuum freeze-drying to obtain 6.5 mg of a pale yellow solid (compound 8, H7ND). A small amount of the product was dissolved and confirmed by HPLC, revealing a purity of 99.2%. MALDI-TOF mass spectrometry confirmed the identity of the desired product. MALDI-TOF-MS: m / z=1746.48 [M+H] + , 1768.46 [M+Na] + Theoretical molecular weight: 1746.81 (the results of mass spectrometry are shown in Figure 2).

[0097] Example 3 Synthesis of Compound DOTA-PEG4-CO-N-bis(PEG4-7N) (hereinafter referred to as D7ND) NH2-PEG4-CO-N-bis(PEG4-7N) (Compound 6) was obtained by referring to Steps 1 to 3 of Example 2. 6.0 mg of NH2-PEG4-CO-N-bis(PEG4-7N) (compound 6) was dissolved in 200 μL of ultra-dry DMF. 6.0 mg of NHS-DOTA was dissolved in 200 μL of ultra-dry DMF. The solutions of the two compounds were combined in a 1.5 mL EP tube, and then 10 μL of DIPEA was added. The reaction was allowed to proceed for 2 hours with shaking at 30°C. The reaction process was monitored by HPLC (Method 3). After completion of the reaction, the target product was isolated and purified (Method 3). Fractions corresponding to the elution peak at 8.4 minutes in semi-preparative HPLC were collected, combined, and lyophilized by vacuum freeze-drying to obtain 8.0 mg of a pale yellow solid (D7ND). A small amount of the product was dissolved and confirmed by HPLC, revealing a purity of 99.4%. TOF MS (ES+) mass spectrometry confirmed the identity of the desired product. m / z = 1830.77 [M+H] + , 915.41[M+2H] 2+ , 610.94[M+3H] 3+ Theoretical molecular weight: 1829.92 (the results of mass spectrometry are shown in Figure 3).

[0098] Example 4: Preparation of H7ND freeze-dried kit 1 mg of H7ND was dissolved in 1 mL of 35% ethanol / water to a concentration of 1 μg / μL. Next, 1 mL of a mixed solution containing 40 μg (40 μL) of H7ND, 2.0 mg of tricine, 3.0 mg of TPPTS, 29.55 mg of succinic acid, and 17.0 mg of sodium hydroxide was prepared, filtered through a 0.22 μm filter membrane, and placed in a 10 mL sterile vial. The mixed solution was freeze-dried and then capped. 99m The H7ND lyophilized kit for Tc labeling was obtained.

[0099] Example 5 99m Manufacturing and quality control analysis of Tc-H7ND 1 mg of H7ND was dissolved in 1 mL of 35% ethanol / water to a concentration of 1 μg / μL. Next, 1 mL of a mixed solution containing 40 μg (40 μL) of H7ND, 2.0 mg of tricine, 3.0 mg of TPPTS, 29.55 mg of succinic acid, and 17.0 mg of sodium hydroxide was prepared, filtered through a 0.22 μm filter membrane, and placed in a 10 mL sterile vial. The mixed solution was freeze-dried and then capped. 99m A freeze-dried kit for Tc labeling was obtained.

[0100] 20-35 mCi (740-1300 MBq) of sodium pertechnetate [ 99m [Tc] injection solution was prepared. The H7ND lyophilized kit for injection, which had been inspected for integrity and stored within the expiration date, was prepared under aseptic conditions. 20-35 mCi (740-1300 MBq) of sodium pertechnetate [ 99m The [Tc] injection solution was drawn into a sterile syringe, and saline injection was added to make the total volume 1 mL. This was then poured into the H7ND freeze-dried kit and shaken thoroughly until the solids in the kit were completely dissolved. The kit bottle was heated in a 100°C water bath for 20 minutes, then removed from the water bath and cooled to room temperature. 99m Tc-H7ND injection was obtained.

[0101] 99m After Tc-H7ND labeling was completed, a drop was collected with a sterile syringe and spotted onto a flash silica gel thin-layer paper chromatography (ITLC-SG) column for quality control analysis. Fast thin-layer chromatography paper was used as the support, and the column was chromatographed in acetone and acetonitrile / saline mixtures (V). アセトニトリル / V 生理食塩水 The samples were developed using two different development systems: 1 / 1 (=1 / 1). After drying the chromatography paper in air, data was collected using a Bioscan radioactivity thin-layer scanner. After data collection, images, radioactivity percentages, and Rf values ​​were acquired to calculate the radiochemical purity of the drug. The main radioactive material contained in the labeling solution was a technetium-labeled compound ( 99m Tc-H7ND), free technetium ( 99m TcO4 - ) and colloidal technetium (99m The Rf values ​​obtained for each radioactive component in the two development systems are shown in Table 1.

[0102] [Table 2]

[0103] 99 m Quality control analysis of Tc-H7ND can also be performed by radio-HPLC. An HPLC system equipped with a Latest Gavista radioactivity detector and an Agilent 35900E digital-to-analog converter was used. A C18 reversed-phase column (ReproSil-Pur Basic C18, 5 μm, 250 × 4.6 mm) was used. The gradient elution lasted for 25 min at a flow rate of 1 mL / min. Mobile phase A was 0.01 M PBS buffer, pH 7.4, and mobile phase B was acetonitrile. The elution gradient was set to 90% A and 10% B at the beginning, 60% A and 40% B at 20 min, and 90% A and 10% B at 25 min. 99m The retention time of Tc-H7ND was 16.1 min. 99m TcO4 - The retention time was 3.5 minutes.

[0104] Example 6 68 Production and purification of Ga-D7ND 1 mg of D7ND was dissolved in 1 mL of pure water to prepare a solution with a concentration of 1 μg / μL. The Sep-Pak C18 cartridge was washed sequentially with 5 mL of absolute ethanol and 10 mL of water for injection, and then dried with 10 mL of air to complete the activation. 20 μL of D7ND solution (containing 20 μg of D7ND) in a 1.5 mL EP tube was added with 97 μL of 1 M sodium acetate. 681.0 mL of GaCl3 eluent (5-15 mCi, 0.05 M hydrochloric acid medium) was added sequentially. The mixture was reacted in an incubator at 90°C for 15 minutes. Radio-iTLC analysis indicated a labeling rate of 77% (shown in Figure 4A). After the reaction was completed, the reaction solution was aspirated into a 5 mL syringe pre-filled with 2 mL of water for injection and loaded onto an activated C18 cartridge. The C18 cartridge was washed with 10 mL of water for injection, and the waste liquid was discarded. Finally, the C18 cartridge was eluted with 0.5 mL of 80% ethanol, and the labeled compound was collected in a sterile vial. The labeled compound was diluted with 2 mL of saline, filtered through a 0.22 μm microfiltration membrane, and stored in a sterile vacuum flask. Radio-iTLC analysis revealed that 68 The radiochemical purity of Ga-D7ND was greater than 98% (shown in Figure 4B).

[0105] Example 7 177 Production and purification of Lu-D7ND 1 mg of D7ND was dissolved in 1 mL of pure water to prepare a solution with a concentration of 1 μg / μL. The Sep-Pak C18 cartridge was washed sequentially with 5 mL of absolute ethanol and 10 mL of water for injection, and then dried with 10 mL of air to complete activation. 40 μL of D7ND solution (containing 40 μg of D7ND) in a 1.5 mL EP tube was added to 0.2 mL of 4 M sodium acetate buffer (pH 4.5). 177 0.1 mL of LuCl3 solution (10-50 mCi, 0.01 M hydrochloric acid medium) was added sequentially. The mixture was reacted in an incubator at 95°C for 20 minutes. After the reaction was completed, the reaction solution was aspirated into a 5 mL syringe pre-filled with 2 mL of water for injection and loaded onto an activated C18 cartridge. The C18 cartridge was washed with 10 mL of water for injection, and the waste liquid was discarded. Finally, the C18 cartridge was eluted with 0.5 mL of 80% ethanol, and the labeled compound was collected in a sterile vial. The labeled compound was diluted with 2 mL of saline, filtered through a 0.22 μm microfiltration membrane, and then stored in a sterile vacuum flask. Measurement by radio-HPLC analysis revealed that 177 The radiochemical purity of Lu-D7ND was greater than 95%.

[0106] Example 890 Production and purification of Y-D7ND 1 mg of D7ND was dissolved in 1 mL of pure water to prepare a solution with a concentration of 1 μg / μL. The Sep-Pak C18 cartridge was washed sequentially with 5 mL of absolute ethanol and 10 mL of water for injection, and then dried with 10 mL of air to complete activation. 40 μL of D7ND solution (containing 40 μg of D7ND) in a 1.5 mL EP tube was added to 0.2 mL of 4 M sodium acetate buffer (pH 4.5). 90 0.1 mL of YCl3 solution (10-50 mCi, 0.01 M hydrochloric acid medium) was added sequentially. The mixture was reacted in an incubator at 95°C for 20 minutes. After the reaction was completed, the reaction solution was aspirated into a 5 mL syringe pre-filled with 2 mL of water for injection and loaded onto an activated C18 cartridge. The C18 cartridge was washed with 10 mL of water for injection, and the waste liquid was discarded. Finally, the C18 cartridge was eluted with 0.5 mL of 80% ethanol, and the labeled compound was collected in a sterile vial. The labeled compound was diluted with 2 mL of saline, filtered through a 0.22 μm microfiltration membrane, and stored in a sterile vacuum flask. The radioactivity of the final product was measured, and the yield was calculated to be approximately 75%. Radio-HPLC analysis revealed that 90 The radiochemical purity of Y-D7ND was greater than 99%.

[0107] Example 9 Al 18 Production and purification of F-N7ND A solution of 1 mg of D7ND and 1 mg of AlCl3 was prepared at a concentration of 1 μg / μL by dissolving each in 1 mL of a solution containing 50 mM potassium hydrogen phthalate (pH 4.0) and 100 mg / mL trehalose. The Sep-Pak C18 cartridge was washed sequentially with 5 mL of absolute ethanol and 10 mL of water for injection, and then dried with 10 mL of air to complete activation. The cartridge was placed in a 1.5 mL EP tube. 18 F -To 100 μL of the solution (approximately 3.2 GBq), 3 μL of AlCl3 solution (containing 3 μg of AlCl3) was added, and the reaction was continued at room temperature for 5 minutes. Next, 40 μL of N7ND solution (containing 4 μg of N7ND) was added, and the reaction was continued at 90°C for 15 minutes. After the reaction was completed, the reaction solution was aspirated into a 5 mL syringe prefilled with 2 mL of water for injection and loaded onto an activated C18 cartridge. The C18 cartridge was washed with 10 mL of water for injection, and the waste liquid was discarded. Finally, the C18 cartridge was eluted with 0.5 mL of 80% ethanol, and the labeled compound was collected in a sterile vial. The labeled compound was diluted with 2 mL of saline, filtered through a 0.22 μm microfiltration membrane, and stored in a sterile vacuum flask. The radioactivity of the final product was measured, and the yield was calculated to be approximately 27%. Radio-HPLC analysis revealed that Al 18 The radiochemical purity of F-N7ND was greater than 95%.

[0108] Example 10 Biological Experiments Experimental cells and animals Four- to five-week-old SPF-grade female BALB / c nude mice and six-week-old SPF-grade female Kunming mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. HT-1080 human fibrosarcoma cells (FAP-negative), HT-1080-hFAP human fibrosarcoma cells (a cell line stably transfected with hFAP overexpression), and U87MG human glioma cells (FAP is highly expressed in tumor tissue after carcinogenesis) were cultured in DMEM medium containing 10% fetal bovine serum (FBS). Cells were routinely subcultured at 37°C in a 5% CO2-containing incubator. 5 × 10 6 U87MG cells were subcutaneously inoculated into the right frontal region of 4-5 week-old BALB / c nude mice, and the mice were reared under SPF conditions. 3 When the mice reached 100 mg / kg, they were used for imaging and biodistribution experiments.

[0109] 1. 99m In vitro cell binding assay of Tc-H7ND Based on monomeric FAP-α 99mIt has a dimeric structure compared to Tc-HFAPi (compound of patent CN111991570B). 99m To verify that Tc-H7ND has a high affinity for FAP-α, we performed a 100-well platelet count assay using 100-well platelets containing 1000 ribonucleotides (1000 ribonucleotides) ... 99m Tc-H7ND and 99m Tc-HFAPi was prepared separately and subjected to in vitro cell binding assay. In vitro cell-binding assays were performed using HT-1080-hFAP human fibrosarcoma cells stably transfected with hFAP as a positive control, and HT-1080 human fibrosarcoma cells (FAP-negative) as a negative control. The in vitro cell-binding assay was performed as follows: The culture medium of HT-1080-hFAP human fibrosarcoma cells and the culture medium of HT-1080 human fibrosarcoma cells were transferred to 15 mL centrifuge tubes, centrifuged at 150 × g for 5 minutes, and the supernatant was discarded. 10 mL of PBS was added to resuspend and wash the cells. After centrifugation and discarding the supernatant, the cells were resuspended in PBS and counted using a cell counting board. Then, 2 × 10 cells were added to each tube. 6 The cells / tube were collected in 1.5 mL EP tubes and blocked with 2% BSA solution at room temperature for 0.5 hours. 99m Tc-H7ND and 99m Tc-HFAPi was added to the EP tubes containing cells at 11 kBq per tube, mixed gently, and incubated in an ice bath for 20 minutes. To study blocking of probe-cell interactions, 1,000-fold molar excess of HFAPi was added to the corresponding EP tubes for co-incubation. After incubation, the cells were resuspended in ice-cold PBS and centrifuged at 150 × g for 5 minutes, and the supernatant was discarded. This washing process was repeated five times. Finally, the washed cells were lysed with 2 mol / L NaOH solution. The cell lysates from each tube were collected and their radioactivity was measured using a gamma counter. Four samples were used for each cell binding assay. The probe binding values ​​were expressed as a percentage of the radioactive counts per minute relative to the applied radiation dose (%AD, decay-corrected value). The experimental results are shown in Figure 5. 99m The %AD value for the binding of Tc-H7ND to HT-1080-hFAP human fibrosarcoma cells was 5.8% (±0.2%).99m The %AD value of binding of Tc-HFAPi to HT-1080-hFAP human fibrosarcoma cells was 0.3% (±0.12%). 99m Tc-H7ND binding value and 99m The binding values ​​of Tc-HFAPi were comparable and significantly lower than those of the positive control group. The experimental results showed that both drugs can specifically bind to the FAP-α protein at the same binding site. 99m Tc-H7ND 99m It showed a significantly higher binding affinity, approximately 18 times that of Tc-HFAPi.

[0110] 2. 99m In vivo stability and metabolism of Tc-H7ND in normal BALB / c mice Based on monomeric FAP-α 99m It has a novel structure compared to Tc-HFAPi. 99m To investigate the excellent pharmacokinetic properties of Tc-H7ND in vivo, we divided normal BALB / c mice into two groups, with three mice per group. The two groups of mice were administered 100 μL (approximately 37 MBq) of Tc-H7ND. 99m Tc-H7ND and 99m Tc-HFAPi was injected into the tail vein, and the radiochemical purity of the drug in mouse urine 0.5 and 1 hour after injection was analyzed by radioactive high-performance liquid chromatography. While the mice were allowed to exercise, eat, drink, and excrete normally, whole-body radioactivity was measured and recorded at 0, 0.5, 1, 2, and 4 hours after injection. The experimental results of in vivo stability and metabolism are shown in Figure 6A. After metabolism, the drug in mice 99m Tc-HFAPi 99m It showed more significant degradation than Tc-H7ND, and only about 50% of it was excreted from the body in the form of the prototype drug one hour after injection, but the novel dimeric form 99m Tc-H7ND maintained good in vivo stability with no observed decomposition during the observation period. Figure 6B shows the difference in the in vivo retention of the two drugs, with the remaining amount in the body 4 hours after injection. 99m Less than 10% of Tc-H7ND remained in the body 4 hours after injection. 99mThe Tc-HFAPi was approximately 35%, and in the case of radiopharmaceuticals, this undoubtedly increased the unnecessary radiation dose absorbed by the patient. 99m Tc-H7ND 99m It has significantly higher metabolic stability in the body than Tc-HFAPi, and showed almost no degradation during the observation period. 99m Approximately 50% of Tc-HFAPi was converted to metabolites. 99m Tc-H7ND is 99m It has a significantly shorter retention time in the body than Tc-HFAPi, which reduces patient radiation exposure and the risk of side effects from radiation.

[0111] 3. U87MG tumor-bearing mouse model 99m SPECT / CT imaging of Tc-H7ND 99m Tc-H7ND was dissolved in saline at a concentration of 37 MBq / 100 μL. 100 μL (37 MBq) of the above solution was injected into the tail vein of each U87MG tumor-bearing mouse, and SPECT / CT imaging was performed 0.5, 1, 2, 4, 8, 12, and 24 hours after injection. 99m Simultaneously with the injection of Tc-H7ND, mice in the blocking group were injected with 100 μL (500 μg) of HFAPi and imaged 0.5 hours after injection. During imaging, mice were anesthetized by inhalation of 1.5% isoflurane-oxygen. After imaging, the SPECT images were reconstructed and fused with the CT images to obtain 3D images (posterior view). The imaging results are shown in Figure 7, with the location of the tumor marked with an arrow. The experimental results were as follows: 99m This indicates that Tc-H7ND was effectively taken up into the tumor with high imaging contrast. 99m Tc-H7ND demonstrated excellent tumor-specific targeting. In the blocking group experiment, tumor-specific 99m The uptake of Tc-H7ND was significantly reduced, and no tumor imaging was observed. 99m The superior tumor-specific targeting of Tc-H7ND was further demonstrated.

[0112] 4. U87MG tumor-bearing mouse model 99m Tc-H7ND and 99m Comparison of biodistribution of Tc-HFAPi Sixteen U87UM tumor-bearing BALB / c nude mice (6 weeks old) were randomly divided into four groups, with four mice per group. 99m Tc-H7ND and 99m Two groups were used for the biodistribution experiment of Tc-HFAPi. 99m Tc-H7ND and 99m Tc-HFAPi was dissolved in saline at a concentration of 370 kBq / 100 μL and injected via the tail vein of each mouse at 100 μL (370 kBq). At 1 and 4 hours post-injection, the mice were sacrificed, and blood and major organs were collected, weighed, and radioactivity counts in cpm were determined. The percentage of injected dose per gram of tissue (%ID / g) was calculated after decay correction. Biodistribution results were expressed as mean ± standard deviation (mean ± SD, n = 4).

[0113] The experimental results are shown in Figure 8. 99m Tc-H7ND is 99m Compared with Tc-HFAPi, the absolute value of tumor uptake was significantly higher, and 99m Tc-H7ND uptake 99m 300% higher than Tc-HFAPi, and 99m Tc-H7ND uptake 99m 60% lower than Tc-HFAPi. 99m The metabolism and clearance of Tc-H7ND 99m It is faster than Tc-HFAPi, so it depends on the tumor / normal organs. 99m The uptake ratio of Tc-H7ND is 99m This is significantly higher than that of Tc-HFAPi, which is beneficial for nuclear medicine imaging of tumors. In particular, because SPECT imaging is very sensitive to background signal noise, a higher tumor / normal organ uptake ratio is beneficial for diagnosing minute tumor foci.

[0114] 5. U87MG tumor-bearing mouse model 68 Ga-D7ND and 68 PET imaging comparison of Ga-FAPi-04 68 Ga-D7ND and 68 Ga-FAPi-04 was dissolved in saline at a concentration of 18.5 MBq / 100 μL. 100 μL (18.5 MBq) of the above solution was injected into each U87MG tumor-bearing mouse via the tail vein, and micro-PET / CT imaging was performed 0.5, 1, and 2 hours after injection. During imaging, the mice were anesthetized by inhalation of 1.5% isoflurane-oxygen. After imaging, the PET images were reconstructed and fused with the CT images to obtain 3D images (whole-body MIP images). The imaging results are shown in Figure 9, with the tumor location marked by an arrow. The experimental results are indicative of the present invention. 68 Ga-D7ND is based on the monomeric FAPI-α 68 It has a tumor SUVmax value that is approximately 220% higher than Ga-FAPi-04, which indicates that 68 Ga-D7ND is based on the monomeric FAPI-α 68 It was shown to have significantly better tumor uptake, significantly higher imaging contrast, and higher tumor specificity than Ga-FAPi-04, which is beneficial for imaging resolution and diagnostic accuracy. 68 The dose of Ga-D7ND is 68 This is significantly lower than Ga-FAPi-04, which reduces radiation exposure to patients and mitigates the risk of side effects from radiation.

[0115] 6. Lung tumor patients 18 F-FDG-PET and 99m Tc-H7ND-SPECT imaging A 71-year-old male patient presented with a 2-month persistent cough and a 2-week history of a pulmonary nodule. Chest CT scan revealed an irregular nodule at the posterior apex of the right upper lobe. This suggested a high possibility of peripheral lung cancer, and a needle biopsy was recommended. 18 F-FDG-PET / CT imaging and 99mTc-H7ND-SPECT / CT imaging was performed, respectively. 18 The results of F-FDG-PET / CT imaging are shown in Figure 10A. A nodule measuring approximately 18.5 mm × 15.9 mm × 18.6 mm was observed at the apical end of the right upper lobe of the lung, with lobulation, spinous processes, and spicules. The adjacent bronchus was severed, and the uptake of radionuclides by the nodule was significantly increased, with an SUVmax of 7.0. 99m Tc-H7ND-SPECT / CT imaging is shown in Figure 10B. The nodular shadow in the right upper lobe of the lung showed increased radionuclide distribution, with a maximum T / N of 4.13, and was considered to be a malignant lung lesion. Finally, intraoperative pathological examination confirmed invasive poorly differentiated adenocarcinoma in the right upper lobe of the lung. The results showed that for lung cancer detection in this case, 99m The effectiveness of Tc-H7ND-SPECT 18 It was shown that the F-FDG-PET was not lower than that of F-FDG-PET.

[0116] A 65-year-old female patient with lung adenocarcinoma underwent targeted therapy and immunotherapy after surgery, and tumor markers gradually increased. Chest CT revealed chest asymmetry, right thoracic collapse, diffuse thickening of the right pleura, and multiple subpleural nodules. Mediastinal lymph node enlargement with calcification was observed, and there was evidence of a small amount of pleural effusion on the right side. 99m Nuclear medicine imaging was performed using Tc-H7ND-SPECT / CT, and the imaging results are shown in Figure 11. The right pleura showed diffuse thickening and multiple locally scattered nodular shadows with abnormally dense radionuclide distribution, with a maximum T / N of 4.58. The right hilum showed nodular shadows with abnormally dense radionuclide distribution, with a maximum T / N of 3.45. The right lower lobe and left lobe of the liver showed irregular, slightly hypodense foci with abnormally dense radionuclide distribution, with a maximum T / N of 2.83-3.51. Possible pleural metastasis, hilar lymph node metastasis, and liver metastasis were considered. A final needle biopsy of the pleural nodule confirmed infiltration of poorly differentiated adenocarcinoma with fibrous tissue. The results in this case were as follows: 99m It was shown that Tc-H7ND-SPECT / CT has excellent detection efficacy for pleural metastasis, lymph node metastasis, and liver metastasis of lung cancer.

[0117] All references mentioned herein are incorporated herein by reference. It will be understood that many variations and modifications can be made to the embodiments of the present invention without departing from the spirit and scope of the disclosure.

Claims

1. A ligand compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer thereof. 【Chemical 1】 In the formula, R １ , R １ ', R ２ , R ２ ', R ３ , R ３ ', R ４ are each independently H, OH, or NH ２ , N.H.C. １－６ Alkyl, halo, C １－６ Alkyl, C ２－６ Alkenyl, C ２－６ Alkynyl, C １－６ Haloalkyl, C １－６ Alkoxyl, C １－６ Alkylthiol and C １－６ haloalkoxyl; R ４ ' is H, C ２－６ Alkynyl, CN, -B(OH) ２ , nitro, carboxyl, —CHO, —C(O)—C １－６ Alkyl, —C═C—C(O)—C ６－１０ Aryl, —SO ３ H, -SO ２ NH ２ , -PO ３ H ２ and tetrazolyl; R ５ , R ５ ' are each independently H, halo, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Alkoxyl, C １－６ Alkylthiol and C １－６ haloalkoxyl; R ６ , R ７ are each independently H, OH, or NH ２ , N.H.C. １－６ Alkyl, halo, C １－６ Alkyl, C １－６ Haloalkyl, C １－６ Alkoxyl, C １－６ Alkylthiol and C １－６ haloalkoxyl; A is phenyl, said phenyl being selected from the group consisting of OH, halo, cyano, C １－６ Alkyl, C １－６ Alkoxyl, C １－６ Alkylthiol and / or C １－６ optionally substituted with haloalkoxyl; L １ is -X １ -CO-C １－６ Alkylene (-X ２ -C １－６ alkylene) ｍ - a functionalized linker of the formula: L ２ is -CO-C １－６ Alkylene-(X ３ -C １－６ alkylene-) ｎ -X ４ - a functionalized linker of the formula: X １ , X ２ , X ３ , X ４ are each independently O, S, NH, and NCH ３ C is selected from the group consisting of １－６ Alkylene is halo, OH, NH ２ , optionally substituted with oxo and / or cyano; m and n are integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively.

2. The bifunctional chelating agent is 6-(2-(sulfobenzylidene)hydrazinyl)nicotinic acid (HYNIC), mercaptoacetyldiglycine (MAG ２ ), mercaptoacetyltriglycine (MAG ３ ), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N',N''-pentaacetic acid (DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), RESCA, 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NOTA-GA) , 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid (DOTA-GA), 1,4,8,11-tetraazabicyclo[6,6,2]hexadecane-4,11-diacetic acid (CB-TE2A), 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6,6,6]eicosane (DiAmSar), and 1-(4-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxylamino)-6,11,17,22-tetraazaheptacosane] (DFO).

3. 3. The ligand compound of claim 2, wherein the bifunctional chelating agent is selected from the group consisting of: 【Chemistry 2】

4. 2. The ligand compound of claim 1, which is a compound of formula (IA), or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 3】 In the formula, R 2 , R 2 ', L １ , L ２ is as defined in claim 1.

5. 2. The ligand compound of claim 1, which is a compound of formula (IC), or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 4】 In the ceremony, L １ , L ２ is as defined in claim 1.

6. The ligand compound according to claim 5, which is a compound of formula (IC-I), (IC-II), (IC-III) or (IC-IV), or a pharmaceutically acceptable salt or stereoisomer thereof. 【Chemistry 5】 In the ceremony, L １ , L ２ is as defined in claim 1.

7. 2. The ligand compound of claim 1, which is a compound of formula (ID): or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 6】 In the ceremony, L １ , L ２ is as defined in claim 1.

8. The ligand compound according to claim 7, which is a compound of formula (ID-I), (ID-II), (ID-III) or (ID-IV), or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 7】 In the ceremony, L １ , L ２ is as defined in claim 1.

9. L １ is -NHCO-C １－６ Alkylene (—O—C １－６ alkylene) ｍ -, -NHCO-C １－６ Alkylene (-NH-C １－６ alkylene) ｍ -, -NHCO-C １－６ Alkylene (-NCH ３ -C １－６ alkylene) ｍ -, -NCH ３ -CO-C １－６ Alkylene (—O—C １－６ alkylene) ｍ -, -NCH ３ -CO-C １－６ Alkylene (-NH-C １－６ alkylene) ｍ -, -NCH ３ -CO-C １－６ Alkylene (-NCH ３ -C １－６ alkylene) ｍ -, -OCO-C １－６ Alkylene (—O—C １－６ alkylene) ｍ -, -OCO-C １－６ Alkylene (-NH-C １－６ alkylene) ｍ - and / or -OCO-C １－６ Alkylene (-NCH ３ -C １－６ alkylene) ｍ -, wherein the alkylene is selected from halo, OH, NH ２ , oxo and / or cyano, and m is an integer selected from 1, 2, 3, 4, 5 or 6; L ２ is -CO-C １－６ Alkylene-(NH-C １－６ alkylene-) ｎ -NH-, -CO-C １－６ Alkylene-(NH-C １－６ alkylene-) ｎ -NCH ３ -, -CO-C １－６ Alkylene-(NH-C １－６ alkylene-) ｎ —O—, —CO—C １－６ Alkylene-(O-C １－６ alkylene-) ｎ -NH-, -CO-C １－６ Alkylene-(O-C １－６ alkylene-) ｎ -NCH ３ -, -CO-C １－６ Alkylene-(O-C １－６ alkylene-) ｎ —O—, —CO—C １－６ Alkylene-(NCH ３ -C １－６ alkylene-) ｎ -NH-, -CO-C １－６ Alkylene-(NCH ３ -C １－６ alkylene-) ｎ -NCH ３ - and / or -CO-C １－６ Alkylene-(NCH ３ -C １－６ alkylene-) ｎ -O- functionalized linker, wherein the alkylene is selected from halogen, OH, NH ２ 2. The ligand compound of claim 1, wherein n is an integer of 1, 2, 3, 4, 5, or 6, and n is optionally substituted with oxo and / or cyano.

10. L 1 is —NHCO—C 1-3 alkylene(—O—C 1-3 alkylene) m —, —NHCO—C 1-3 alkylene(—NH—C 1-3 alkylene) m —, —NHCO—C 1-3 alkylene(—NCH 3 —C 1-3 alkylene) m —, —NCH 3 —CO—C 1-3 alkylene(—O—C 1-3 alkylene) m —, —NCH 3 —CO—C 1-3 alkylene(—NH—C 1-3 alkylene) m —, —NCH 3 —CO—C 1-3 alkylene(—NCH 3 —C 1-3 alkylene) m —, —OCO—C 1-3 alkylene(—O—C 1-3 alkylene) functionalized linkers of m -, -OCO-C 1-3 alkylene(-NH-C 1-3 alkylene) m - and / or -OCO-C 1-3 alkylene(-NCH 3 -C 1-3 alkylene) m -, said alkylene optionally substituted with halo, OH, NH 2 , oxo and / or cyano, and m is an integer of 1, 2, 3, 4, 5 or 6; L 2 is —CO—C 1-3 alkylene-(NH—C 1-3 alkylene-) n -NH—, —CO—C 1-3 alkylene-(NH—C 1-3 alkylene-) n -NCH 3 —, —CO—C 1-3 alkylene-(NH—C 1-3 alkylene-) n -O—, —CO—C 1-3 alkylene-(O—C 1-3 alkylene-) n -NH—, —CO—C 1-3 alkylene-(O—C 1-3 alkylene-) n -NCH 3 —, —CO—C 1-3 alkylene-(O—C 1-3 alkylene-) n -O—, —CO—C 1-3 alkylene-(NCH 3 —C 1-3 alkylene-) n 10. The ligand compound according to claim 9, which is a functionalized linker of -NH-, -CO-C 1-3 alkylene-(NCH 3 -C 1-3 alkylene-) n -NCH 3 - and / or -CO-C 1-3 alkylene-(NCH 3 -C 1-3 alkylene-) n -O-, wherein said alkylene is optionally substituted with halo, OH, NH 2 , oxo and / or cyano, and n is an integer of 1, 2, 3, 4, 5 or 6.

11. L 1 is -NHCO-CH 2 CH 2 (-O-CH 2 CH 2 ) m -, -NHCO-CH 2 CH 2 (-NH-CH 2 CH 2 ) m -, -NHCO-CH 2 CH 2 (-NCH 3 -CH 2 CH 2 ) m -, -NCH 3 -CO-CH 2 CH 2 (-O-CH 2 CH 2 ) m -, -NCH 3 -CO-CH 2 CH 2 (-NH-CH 2 CH 2 ) m -, -NCH 3 -CO-CH 2 CH 2 (-NCH 3 -CH 2 CH 2 ) m -, -OCO-CH 2 CH 2 (-O-CH 2 CH 2 ) functionalized linkers of m -, -OCO-CH 2 CH 2 (-NH-CH 2 CH 2 ) m - and / or -OCO-CH 2 CH 2 (-NCH 3 -CH 2 CH 2 ) m -, wherein said CH 2 CH 2 may be substituted with halo, OH, NH 2 , oxo and / or cyano, and m is an integer of 1, 2, 3, 4, 5 or 6; L 2 is -CO-CH 2 CH 2 -(NH-CH 2 CH 2 -) n -NH-, -CO-CH 2 CH 2 -(NH-CH 2 CH 2 -) n -NCH 3 -, -CO-CH 2 CH 2 -(NH-CH 2 CH 2 -) n -O-, -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) n -NH-, -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) n -NCH 3 -, -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) n -O-, -CO-CH 2 CH 2 -(NCH 3 -CH 2 CH 2 -) n 11. The ligand compound according to claim 10, which is a functionalized linker of -NH-, -CO-CH 2 CH 2 -(NCH 3 -CH 2 CH 2 -) n -NCH 3 -, -CO-CH 2 CH 2 -(NCH 3 -CH 2 CH 2 -) n -O-, wherein said CH 2 CH 2 may be substituted with halo, OH, NH 2 , oxo and / or cyano, and n is an integer of 1, 2, 3, 4, 5 or 6.

12. L １ is -NHCO-CH ２ CH ２ -(-O-CH ２ CH ２ ) ４ - is a functionalized linker of the formula: ２ is -CO-CH ２ CH ２ -(O-CH ２ CH ２ -) ４ The ligand compound according to claim 11, which is a functionalized linker of -NH-.

13. 2. The ligand compound of claim 1, which is a compound selected from the following, or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 8】

14. 10. A complex compound comprising the compound of claim 1 and a radionuclide, said radionuclide being bound to the bifunctional chelator of the compound of claim 1 by a coordinate bond.

15. 15. The complex compound of claim 14, wherein the radionuclide is selected from the group consisting of an alpha-emitting isotope, a beta-emitting isotope, a gamma-emitting isotope, an Auger electron-emitting isotope, and an X-ray-emitting isotope.

16. The radioactive nuclide is 18F, 51Cr, 67Ga, 68Ga, 111In, 99mTc, 186Re, 188Re, 139La, 140La, 175Yb, 153Sm, 166Ho, 88Y, 90Y, 149Pm, 177Lu, 47Sc, 212Bi, 213Bi, 72As, 123I, 124I, 131I, 211At, 201Tl, 212Pb, 64Cu, 67Cu, 198Au, 225Ac, 15. The complex compound of claim 14, selected from the group consisting of 223Ra or 89Sr.

17. 17. The complex compound of claim 16, which is a compound selected from the following, or a pharmaceutically acceptable salt or stereoisomer thereof: 【Chemistry 9】 【change】 【change】

18. A kit comprising the complex compound according to any one of claims 14 to 17 and a pharmaceutically acceptable carrier.

19. The complex compound of any one of claims 14 to 17 for use in a method for diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof.

20. 20. The complex compound of claim 19, wherein the disease is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disorders.

21. The complex compound described in claim 19, wherein the disease is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, liver cancer, esophageal cancer, gastric cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, bladder cancer, bile duct cancer, renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, cancer of unknown primary, thymic cancer, glioma, astrocytoma, cervical cancer, prostate cancer and testicular cancer.

22. 20. Use of a compound according to any one of claims 1 to 17 in the manufacture of a medicament for diagnosing a disease characterized by overexpression of fibroblast activation protein alpha (FAP-α) in a subject in need thereof.

23. 23. The use according to claim 22, wherein the disease is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorders.

24. The use according to claim 22, wherein the disease is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, liver cancer, esophageal cancer, gastric cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, bladder cancer, bile duct cancer, renal cancer, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, cancer of unknown primary, thymic cancer, glioma, astrocytoma, cervical cancer, prostate cancer and testicular cancer.

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