Application of a novel 99mTc-FAPI diagnostic probe with a unique structure to the manufacture of tumor diagnostic drugs or reagents.

Abstract

This invention discloses a 99mTc-FAPI diagnostic probe having a novel skeleton and its application in the manufacture of a tumor diagnostic drug or reagent, the structural formula of the FAP-targeting dimer compound is shown in the following formula. Compared to conventional FAP inhibitor radiopharmaceuticals, the technetium-99m-labeled FAP inhibitor dimer compound having a novel skeleton exhibits a very high tumor uptake rate, high contrast between tumor and background, and good in vivo biological distribution. [C28] TIFF2026518096000031.tif51170

Description

[Technical Field]

[0001] This invention relates to a novel 99mTc-FAPI diagnostic probe and its application in the manufacture of a drug or reagent for tumor diagnosis, and belongs to the field of medicinal chemistry. [Background technology]

[0002] Fibroblast activation protein-α (FAP) is a transmembrane serine protease primarily expressed on the surface of tumor-associated fibroblasts. It is selectively overexpressed in over 90% of epithelial-derived tumors, particularly in liver cancer, colorectal cancer, pancreatic cancer, and ovarian cancer, while being absent or low in normal tissues. FAP can influence tumor growth through various mechanisms, including proliferation, invasion, angiogenesis, epithelial-mesenchymal transition, stem cell promotion, immunosuppression, and drug resistance. Given the widespread expression of FAP in tumors and its influence on tumor growth through multiple mechanisms, FAP has already become an important target for tumor imaging and therapy. Extensive studies have shown that FAPIs labeled with diagnostic radionuclides are superior to 18F-FDG in diagnostic performance as broad-spectrum tumor developers. Most conventional diagnostic FAPIs that have seen clinical advancements are based on PET probes using 18F, 68Ga, or 64Cu, while diagnostic FAP probes based on 99mTc-SPECT are less common. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] The problem that this invention aims to solve is to provide a dimer compound targeting FAP that has a high tumor uptake rate and development contrast, a method for producing the same, and its applications. [Means for solving the problem]

[0004] To solve the above problems, the present invention provides a dimer compound targeting FAP, and pharmaceutically acceptable salts, hydrates, and solvates thereof, as well as a corresponding technetium-99m radionuclide marker, wherein the compound has the structure of the following formula. [ka] (In the formula, R1 is selected from a cyano group, an aldehyde group, or a chloroacetyl group.) R2 is selected from hydrogen, deuterium, fluorine, or chlorine. R3 is selected from hydrogen, deuterium, methyl group, isopropyl group, isobutyl group, cyclopropyl group, or cyclobutyl group. R4 is selected from hydrogen, methyl group, ethyl group, propyl group, cyclopropyl group, or cyclobutyl group. R5 and R6 are independently selected from hydrogen, a methyl group, fluorine, chlorine, a hydroxyl group, or a methoxy group. R7 and R8 may be the same or different, and each may be independently selected from hydrogen, a methyl group, a halogen, or a carbonyl group. L1 and L2 may be the same or different as linking chains, and each is independently at least one selected from the group consisting of aliphatic carbon chains, polyethylene glycolic chains, and amino acid chains of different lengths, but is not limited to these, and includes side chains that are joined by different reactions, the reaction including at least one of amide condensation, ester condensation, substitution, or click chemical reaction. Y is selected from one of the following structures: [ka] Q is either a hydrogen atom or selected from any group capable of chelate-coordinating to technetium-99mTc as part of a nuclide chelate group.

[0005] In a preferred embodiment of the present invention, L1 and L2 each independently include side chains selected from the group consisting of aliphatic carbon chains, polyethylene glycol chains, or amino acid chains of different lengths, and which are joined by different reactions, the reactions comprising at least one of amide condensation, ester condensation, substitution, or click chemical reactions.

[0006] L1 and L2 are each independently selected from one of the following structures: [ka] The aforementioned Q is either a hydrogen atom or part of a nuclide chelate group, selected from one of the following structures. [ka]

[0007] Q is preferably 6-hydrazinopyridine-3-carboxylic acid (HYNIC).

[0008] In a preferred embodiment of the present invention, the compound is selected from any of the following structures. [ka] TIFF2026518096000007.tif157170

[0009] The present invention further provides dimerized compounds targeting FAP labeled with the radionuclide technetium-99m, the compounds comprising pharmaceutically acceptable salts, hydrates, solvates, and tautomers thereof.

[0010] The present invention further provides a method for synthesizing the dimer compound and a method for labeling technetium-99m, comprising the following steps (1) to (3).

[0011] Step (1) Synthesis of the targeting ligand: React piperazinylquinolone with di-tert-butyl dicarbonate to form an intermediate I protected with tert-butoxycarbonyl, React the corresponding tert-butoxycarbonyl-protected amino acid with pyrrolidine having different substituents to form a dipeptide analog, and remove the tert-butoxycarbonyl with trifluoroacetic acid to obtain intermediate II, Condense intermediate I and intermediate II. After the reaction is completed, drop the reaction solution into water to precipitate a solid, filter and dry it, and further remove the tert-butoxycarbonyl protecting group with trifluoroacetic acid to obtain the targeting ligand III.

[0012] Step (2) Synthesis of the dimer: React the targeting ligand with different linking chains (L1 / L2) having protecting groups, and further obtain intermediate IV through a deprotection reaction, React intermediate IV with Y having a protecting group, and further obtain intermediate V through deprotection, React intermediate V with a ligand Q capable of chelating with technetium-99m to obtain a drug precursor targeting fibroblast activation protein.

[0013] Step (3): Label the drug precursor compound targeting fibroblast activation protein by a conventional wet method or a dry method to produce a dimer compound having a novel skeleton targeting fibroblast activation protein labeled with the radionuclide technetium-99m of the present invention.

[0014] The present invention further provides a dimer compound having a novel skeleton (piperazinylquinolone) targeting fibroblast activation protein, a dimer compound targeting fibroblast activation protein that can be labeled with the radionuclide technetium-99m, a dimer compound targeting fibroblast activation protein labeled with the radionuclide technetium-99m, or a pharmaceutical composition comprising a pharmaceutically acceptable salt, hydrate, solvate, tautomer, racemate, and any pharmaceutically acceptable carrier and / or excipient thereof.

[0015] The present invention further provides applications in the diagnosis or treatment of diseases characterized by FAP positivity for dimer compounds having a novel scaffold (piperazine quinolone) targeting fibroblast-activating proteins, dimer compounds targeting fibroblast-activating proteins labelable with the radionuclide technetium-99m, dimer compounds targeting fibroblast-activating proteins labeled with the radionuclide technetium-99m, or pharmaceutically acceptable salts, hydrates, solvates, tautomers, racemates, and any pharmaceutically acceptable carriers and / or excipients thereof.

[0016] The aforementioned diseases characterized by FAP positivity include tumors with high FAP expression.

[0017] Diseases characterized by overexpression of fibroblast-activating proteins include, but are not limited to, a variety of malignant tumors (such as breast cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, colorectal cancer, and thyroid cancer) and non-tumor diseases (such as myocardial infarction, rheumatoid arthritis, heart failure, kidney disease, pulmonary fibrosis, tissue remodeling, and scarring).

[0018] The present invention further provides a kit comprising a dimer compound targeting a fibroblast-activating protein labelable with the radionuclide technetium-99m, a ligand capable of coordinating with technetium-99m according to conventional methods, a reducing agent, an additive, a stabilizer, and instructions for diagnosing a disease. [Effects of the Invention]

[0019] Compared to the prior art, the present invention has the following remarkable advantages: 1. The present invention develops a novel skeleton based on piperazine quinolone, and the FAP-targeting probe derived based on this novel skeleton has a higher tumor uptake rate and development contrast. 2. To further improve tumor uptake and optimize metabolic distribution in vivo, the physicochemical properties of the novel molecule are balanced by screening and optimizing different linkage chains, and a dimerized probe based on this type of skeleton is designed. Compared to single probes, this dimerized probe has a significantly improved tumor uptake rate, faster tumor targeting ability, and longer retention capacity, and exhibits very low physiological uptake in non-target organs (liver, lungs, kidneys, pancreas, intestines, blood pool, thyroid, etc.), possessing good pharmacokinetic properties and future potential for clinical application. [Brief explanation of the drawing]

[0020] [Figure 1] This is the nuclear magnetic resonance hydrogen spectrum of compound a8 in Example 1 of the present invention. [Figure 2] This is the mass spectrum of compound a11 in Example 1 of the present invention. [Figure 3] This is the mass spectrum of compound a13 in Example 1 of the present invention. [Figure 4] This is the mass spectrum of compound 1 in Example 1 of the present invention. [Figure 5] This is the mass spectrum of compound c11 in Example 3 of the present invention. [Figure 6] This is the mass spectrum of compound 3 in Example 3 of the present invention. [Figure 7] This is the nuclear magnetic resonance hydrogen spectrum of compound d8 in Example 4 of the present invention. [Figure 8] This is the mass spectrum of compound f11 in Example 6 of the present invention. [Figure 9] This is the mass spectrum of compound f12 in Example 6 of the present invention. [Figure 10] This is the mass spectrum of compound 6 in Example 6 of the present invention. [Figure 11]This is the nuclear magnetic resonance hydrogen spectrum of compound i8 in Example 9 of the present invention. [Figure 12] This is the mass spectrum of compound 11 in Example 11 of the present invention. [Figure 13] This shows the development results of compound 5 labeled with 99mTc in the examples of the present invention in U87MG mice. [Figure 14] This shows the development results of compound 9 labeled with 99mTc in the examples of the present invention in U87MG mice. [Figure 15] This is an in vitro distribution statistical map of tumors and vital organs after injecting U87MG mice with compounds 5 and 9 labeled with 99mTc according to the present invention for 1 hour, with A: 99mTc-5 and B: 99mTc-9. [Figure 16] This diagram shows the tumor uptake rates in important organs after injecting U87MG mice with compounds 5 and 9 labeled with 99mTc in the present invention for 1 hour, with A: 99mTc-5 and B: 99mTc-9. [Figure 17] This is an in vitro distribution statistical chart after tumor blockade following injection of compounds 5 and 9 labeled with 99mTc in the present invention into U87MG mice for 1 hour, with A: 99mTc-5 and B: 99mTc-9. [Figure 18] This diagram shows the uptake rates of compounds 5 and 9 labeled with 99mTc in U87MG cells according to the present invention, with A: 99mTc-5 and B: 99mTc-9. [Modes for carrying out the invention]

[0021] The technical means of the present invention will be further described below with reference to the drawings.

[0022] The synthesis method in the examples of the present invention comprises two parts: the synthesis of a targeted ligand and the synthesis of a dimer. Compounds a1, a2, a5, a9, Boc-Glu, HYNIC-NHS, b9, c9, d5, i5, 4-pentic acid, k8, k9, and o5 are purchased from Shanghai BiDe Technology Co., Ltd. Compound f9 is purchased from Jiangsu Aikang Biotechnology Co., Ltd., U87MG cells are purchased from Nanjing Kebai Biotechnology Co., Ltd., nude mice (balb / c-nu) are purchased from Nanjing Annuokang Biotechnology Co., Ltd., and sodium pertechnetate solution is purchased from Nanjing Atomic High-Tech Pharmaceutical Co., Ltd.

[0023] Example 1 Synthesis of Compound 1 1. Production of targeted ligand compound a8 Synthesis pathway: [ka] (1) Synthesis of compound a3: N-Boc glycine a1 (175 mg, 1 mmol) and (S)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride a2 (132 mg, 1 mmol) were dissolved in 5 mL of N,N dimethylformamide (DMF). 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 418 mg, 1.1 mmol) and N-ethyldiisopropylamine (DIPEA, 521 μL, 3 mmol) were added, and the mixture was reacted at room temperature for 2 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and it was subsequently washed with water and saturated saline solution. The organic layer was dried over anhydrous sodium sulfate and concentrated.

[0024] (2) Synthesis of compound a4: Compound a3, obtained by concentration, was added to a mixed solution (DCM:TFA = 10:1, v / v) and reacted at room temperature for 1 hour. The reaction mixture was then concentrated and separated and purified by C18 reversed-phase preparative chromatography (by increasing the acetonitrile content by 1% per minute at a rate of 2 mL / min, with a 10% aqueous acetonitrile solution increasing proportionally to 50%). The mixture was then freeze-dried to obtain compound a4 (171 mg, 60% yield). MS(ESI): 190.20[M+H] + .

[0025] (3) Synthesis of compound a6: 1-Ethyl-6-fluoro-1,4-dihydro-4-oxo-7-piperazine-3-quinolinecarboxylic acid (a5, 1 g, 3.13 mmol) was dissolved in 100 mL of a mixed solution (tetrahydrofuran:water = 1:1, v / v), 1.75 mL of 2 M NaOH was added, and the mixture was stirred at room temperature until clarified. (Boc)2O (0.75 g, 3.44 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was concentrated under vacuum to remove THF, the pH was adjusted to 7 with citric acid, a white solid was precipitated, filtered by suction, washed three times with 30 mL of water, and dried under vacuum to obtain 1.3 g of a white solid. The yield was 98%.

[0026] (4) Synthesis of compound a7: Compound a6 (419 mg, 1 mmol) and compound a4 (287 mg, 1 mmol) were dissolved in 5 mL of DMF, and HATU (418 mg, 1.1 mmol) and DIPEA (521 μL, 3 mmol) were added. The mixture was reacted at room temperature for 2 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and the mixture was sequentially washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The sample was loaded by wet method, and the compound was separated and purified by column chromatography to obtain compound a7 (319 mg, yield 54%).

[0027] (5) Synthesis of compound a8: Compound a7 (590 mg, 1 mmol) was added to a mixed solution (dichloromethane:trifluoroacetic acid = 10:1, v / v), reacted at room temperature for 1 hour, the reaction mixture was concentrated, and separated and purified by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min). The mixture was then freeze-dried to obtain compound a8 (490 mg, 100% yield). The hydrogen spectrum of compound a8 is shown in Figure 1. MS(ESI): 491.35[M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 10.30 (t, J = 5.2 Hz, 1H), 8.94 (s, 2H), 8.81 (s, 1H), 7.95 (d, J = 13.2 Hz, 1H), 7.19 (d, J = 7.2 Hz, 1H), 5.11 (dd, J = 9.0, 2.9 Hz, 1H), 4.54 (q, J = 7.0 Hz, 2H), 4.24 (qd, J = 17.8, 5.2 Hz, 3H), 4.09 (dt, J = 21.2, 10.3 Hz, 1H), 3.49 (dd, J = 6.7, 3.6 Hz, 4H), 3.33 (s, 4H), 2.99 - 2.73 (m, 2H), 1.40 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 174.50, 168.61, 164.71, 158.30, 154.10, 151.64, 148.07, 143.84, 136.92, 122.69, 118.24, 112.33, 112.11, 110.71, 106.78, 52.05, 48.90, 47.19, 44.60, 43.22, 41.86, 36.92, 14.94.

[0028] 2. Synthesis of dimer compound 1: Synthesis pathway: [ka] (1) Synthesis of compound a10: Compound a8 (100 mg, 0.2 mmol) and compound a9 (47 mg, 0.2 mmol) were dissolved in 2 mL of DMF, and HATU (83 mg, 0.24 mmol) and DIPEA (104 μL, 0.6 mmol) were added. The mixture was reacted at room temperature for 2 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and the mixture was sequentially washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated.

[0029] (2) Synthesis of compound a11: Compound a10, obtained by concentration, was added to a mixed solution (DCM:TFA = 10:1, v / v) and reacted at room temperature for 1 hour. The reaction mixture was concentrated and separated and purified by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute at a flow rate of 2 mL / min). It was then freeze-dried to obtain compound a11 (96.68 mg, yield 80%). The mass spectrum of compound a11 is shown in Figure 2. MS: Ms = 604.37.

[0030] (3) Synthesis of compound a12: Compound a11 (60.4 mg, 0.1 mmol) and Boc-Glu (12.35 mg, 0.05 mmol) were dissolved in 1 mL of DMF, and HATU (47.5 mg, 0.125 mmol) and DIPEA (52.16 μL, 0.3 mmol) were added. The mixture was reacted at room temperature for 14 hours. The sample was loaded using a wet method, and separated and purified by column chromatography (DCM:MeOH = 100:2) to obtain compound a12 (86.48 mg, yield 61%).

[0031] (4) Synthesis of compound a13: Compound a12, obtained by concentration, was added to a mixed solution (DCM:TFA = 10:1, v / v) and reacted at room temperature for 1 hour. The reaction mixture was concentrated and separated and purified by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute at a flow rate of 2 mL / min). It was then freeze-dried to obtain compound a13 (120.53 mg, yield 85%). The mass spectrum of compound a13 is shown in Figure 3. MS:Ms / 2 = 660.3.

[0032] (5) Synthesis of compound 1: Compound a13 (131.7 mg, 0.1 mmol) obtained by lyophilization and 6-hydrazinyl nicotinate succinimimidyl hydrochloride (HYNIC-NHS, 7 mg, 0.2 mmol) were dissolved in 2 mL of dimethyl sulfoxide (DMSO). DIPEA (69.54 μL, 0.4 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. Separation and purification were performed by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute at a flow rate of 2 mL / min). The mixture was then lyophilized to obtain compound 1 (110.9 mg, yield 70%). The mass spectrum of compound 1 is shown in Figure 4. MS: Ms / 2 = 793.36.

[0033] Example 2 Synthesis of Compound 2 Synthesis pathway: [ka] 1. Synthesis of compound b10: Compound a8 (49 mg, 0.1 mmol) and 5,8,11,14-tetraoxa-2-azaheptadecadicarboxylic acid 1-tert-butyl b9 (36.8 mg, 0.1 mmol) were dissolved in 1 mL of DMF, and HATU (45.60 mg, 0.12 mmol) and DIPEA (52.16 μL, 0.3 mmol) were added. The mixture was reacted at room temperature for 2 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and the mixture was sequentially washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrated solid was added to a mixed solution (DCM:TFA = 10:1, v / v), reacted at room temperature for 2 hours, concentrated the reaction mixture, separated and purified by C18 reversed-phase preparative chromatography (increasing the acetonitrile content by 1% per minute at a rate of 2 mL / min, while increasing the acetonitrile content proportionally to 50%), and freeze-dried to obtain compound b10 (44.4 mg, yield 60%).

[0034] 2. Synthesis of compound b11: Compound b10 (159.1 mg, 0.1 mmol) and Boc-Glu (12.4 mg, 0.05 mmol) were dissolved in 1 mL of DMF, and HATU (47.5 mg, 0.125 mmol) and DIPEA (52.2 μL, 0.3 mmol) were added. The mixture was reacted overnight at room temperature. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and the mixture was sequentially washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The sample was loaded by dry method, separated and purified by column chromatography (DCM:MeOH = 100:2), concentrated to obtain a white solid, and the white solid was added to a mixed solution (DCM:TFA = 10:1, v / v). The mixture was reacted at room temperature for 1 hour, and the reaction mixture was concentrated. Separation and purification were performed by C18 reversed-phase preparative chromatography, and the mixture was freeze-dried to obtain compound b11 (95.5 mg, yield 61%). MS:Ms / 2 = 794.33.

[0035] 3. Synthesis of Compound 2: Compound b11 (159.1 mg, 0.1 mmol) and HYNIC-NHS (76 mg, 0.2 mmol) obtained by lyophilization were dissolved in 2 mL of DMSO, DIPEA (69.54 μL, 0.4 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. Separation and purification were performed by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min), and lyophilization was performed to obtain compound 2 (157.8 mg, yield 75%). MS: Ms / 2 = 927.91.

[0036] Example 3 Synthesis of Compound 3 Synthesis pathway: [ka] 1. Synthesis of compound c10: Compound a8 (49 mg, 0.1 mmol) and compound c9 (45.8 mg, 0.1 mmol) were dissolved in 1 mL of DMF, and HATU (45.60 mg, 0.12 mmol) and DIPEA (52.16 μL, 0.3 mmol) were added. The mixture was reacted at room temperature for 3 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and the mixture was sequentially washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The concentrated solid was added to the mixed solution and reacted at room temperature for 2 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated and separated and purified by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute at a flow rate of 2 mL / min). The mixture was then freeze-dried to obtain compound c10 (48.97 mg, yield 59%).

[0037] 2. Synthesis of compound c11: Compound c10 (187.1 mg, 0.1 mmol) and Boc-Glu (12.4 mg, 0.05 mmol) were dissolved in 1 mL of DMF, and HATU (47.5 mg, 0.125 mmol) and DIPEA (52.2 μL, 0.3 mmol) were added. The mixture was allowed to react overnight at room temperature. After detecting completion of the reaction by TLC, the reaction mixture was concentrated. Ethyl acetate was added to dissolve the mixture, and the mixture was sequentially washed with water and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The sample was loaded by dry method, separated and purified by column chromatography (DCM:MeOH = 100:2, v / v), concentrated to obtain a white solid, and the white solid was added to a mixed solution (DCM:TFA = 10:1, v / v). The mixture was reacted at room temperature for 1.5 hours, and after detecting completion of the reaction by TLC, the reaction mixture was concentrated. Compound c11 (112.3 mg, 60% yield) was obtained by separation and purification using C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute at a flow rate of 2 mL / min), and then lyophilized. The mass spectrum of compound c11 is shown in Figure 5. MS:Ms / 2 = 882.61.

[0038] 3. Synthesis of Compound 3: Compound c11 (187.1 mg, 0.1 mmol) and HYNIC-NHS (76 mg, 0.2 mmol), obtained by lyophilization, were dissolved in 2 mL of DMSO. DIPEA (69.54 μL, 0.4 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. Separation and purification were performed by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min). The mixture was then lyophilized to obtain compound 3 (158.4 mg, yield 78%). The mass spectrum of compound 3 is shown in Figure 6. MS: Ms / 2 = 10¹⁵.50.

[0039] Example 4 Synthesis of Compound 4 Synthesis pathway: [ka] 1. The synthesis of compound d8 follows the synthesis route of compound a8 in Example 1. Compound a5 was substituted with compound d5 in equimolar proportions, and the other raw materials and proportions remained the same to obtain compound d8. The hydrogen spectrum of compound d8 is shown in Figure 7. 1 H NMR (400 MHz, DMSO-d6) δ 10.13 (t, J = 5.1 Hz, 1H), 9.27 (d, J = 8.0 Hz, 1H), 8.97 - 8.81 (m, 1H), 8.77 (s, 1H), 7.89 (d, J = 11.0 Hz, 1H), 5.12 (dd, J = 9.1, 2.8 Hz, 1H), 4.62 - 4.45 (m, 2H), 4.25 (qd, J = 17.8, 5.2 Hz, 3H), 4.09 (dt, J = 21.1, 10.2 Hz, 1H), 3.60 - 3.51 (m, 2H), 3.50 - 3.36 (m, 3H), 3.32 - 3.08 (m, 2H), 2.99 - 2.72 (m, 2H), 1.43 (t, J = 6.8 Hz, 3H), 1.27 (d, J = 6.5 Hz, 3H). 2. Synthesis of Compound 4: The synthesis of Compound 4 followed the synthesis route of Example 2, with compound a8 being replaced by compound d8 in equimolar proportions, and the other raw materials and proportions being the same. (Compound 4, MS: Ms / 2 = 959.42)

[0040] Example 5 Synthesis of Compound 5 Compound 5 was synthesized by referring to the synthesis route of Example 3, substituting compound a8 with compound d8 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound 5. (Compound 5, MS:Ms / 2 = 1047.47) [ka]

[0041] Example 6 Synthesis of Compound 6 Synthesis pathway: [ka] 1. Synthesis of compound f10: Compound a8 (49 mg, 0.1 mmol) and compound f9 (51.48 mg, 0.11 mmol) were taken and dissolved in 1 mL of DMF. HATU (49.4 mg, 0.13 mmol) and DIPEA (52.16 μL, 0.3 mmol) were added, and the mixture was reacted at room temperature for 6 hours. After detecting completion of the reaction by TLC, the reaction mixture was added dropwise to 20 mL of ice water to precipitate the solid, which was then filtered by suction and dried under reduced pressure to obtain compound f10 (65.8 mg, yield 70%).

[0042] 2. Synthesis of compound f11: Compound f10, obtained by vacuum drying, was added to 2 mL of a mixed solution (piperidine:DCM = 1:5, v / v) and reacted at room temperature for 3 hours. After detecting completion of the reaction by TLC, the reaction mixture was concentrated to remove piperidine. Sample loading was performed by dry method, and separation and purification were carried out by column chromatography (DCM:MeOH = 100:2) to obtain compound f11 (42.69 mg, yield 85%). The mass spectrum of compound f11 is shown in Figure 8. MS:Ms = 719.34.

[0043] 3. Synthesis of compound f12: Compound f11 (71.7 mg, 0.1 mmol) and Boc-Glu (12.4 mg, 0.05 mmol) were dissolved in 1 mL of DMF, and HATU (47.5 mg, 0.125 mmol) and DIPEA (52.2 μL, 0.3 mmol) were added. The mixture was reacted at room temperature for 14 hours. The sample was loaded by wet method, separated and purified by column chromatography, and concentrated to obtain a white solid. The white solid was added to a mixed solution (DCM:TFA = 10:1, v / v) and reacted at room temperature for 1 hour, after which the reaction mixture was concentrated. Separation and purification were performed by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min), and lyophilized to obtain compound f12 (94.25 mg, yield 61%). The mass spectrum of compound f12 is shown in Figure 9. MS:Ms / 2 = 775.26

[0044] 4. Synthesis of compound 6: Compound f12 (154.5 mg, 0.1 mmol) and HYNIC-NHS (76 mg, 0.2 mmol) obtained by lyophilization were dissolved in 2 mL of DMSO, DIPEA (69.5 μL, 0.4 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. Separation and purification were performed by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min), and lyophilization was performed to obtain compound 6 (108.2 mg, yield 70%). The mass spectrum of compound 6 is shown in Figure 10. MS: Ms / 2 = 908.33.

[0045] Example 7 Synthesis of Compound 7 Compound 7 was synthesized by referring to the synthesis route of Example 6, substituting compound a8 with compound d8 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound 7. (Compound 7, MS:Ms / 2 = 940.84) [ka]

[0046] Example 8 Synthesis of Compound 8 Compound 8 was synthesized by referring to the synthesis route of Example 3, substituting compound a8 with compound d8 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound 8. (Compound 8, MS:Ms / 2 = 1054.48) [ka]

[0047] Example 9 Synthesis of Compound 9 Synthesis pathway: [ka] Compound i8 was synthesized by referring to the synthesis route of Example a8, substituting compound a5 with an equimolar amount of compound i5, while keeping the other raw materials and proportions the same, to obtain compound i8. The hydrogen spectrum of compound i8 is shown in Figure 11. 1H NMR (400 MHz, DMSO-d6) δ 10.22 (t, J = 5.2 Hz, 1H), 9.02 (s, 2H), 8.65 (s, 1H), 7.92 (d, J = 13.2 Hz, 1H), 7.54 (d, J = 7.4 Hz, 1H), 5.11 (dd, J = 9.1, 2.8 Hz, 1H), 4.24 (qd, J = 17.8, 5.2 Hz, 3H), 4.08 (dt, J = 21.2, 10.2 Hz, 1H), 3.77 (tt, J = 7.2, 4.1 Hz, 1H), 3.49 (d, J = 6.4 Hz, 4H), 3.35 (s, 4H), 2.98 - 2.74 (m, 2H), 1.31 (d, J = 6.5 Hz, 2H), 1.12 (d, J = 3.6 Hz, 2H). The synthesis of compound 9 follows the synthesis route of Example 6. Compound a8 is substituted with compound i8 in equimolar proportions, and the other starting materials and proportions remain the same to obtain compound 9. (Compound 9, MS:Ms / 2 = 920.83)

[0048] Example 10 Synthesis of Compound 10 The synthesis of compound 10 follows the synthesis route of Example 3. Compound a8 was substituted with compound i8 in equimolar proportions, and the other starting materials and proportions remained the same to obtain compound 10. (Compound 10, MS:Ms / 2 = 1052.47) [ka]

[0049] Example 11 Synthesis of Compound 11 Synthesis pathway: [ka] 1. Synthesis of compound k10: Compound k8 (24.7 mg, 0.1 mmol) and compound k9 (52.4 mg, 0.2 mmol) were dissolved in 1 mL of DMF, and HATU (45.60 mg, 0.12 mmol) and DIPEA (52.16 μL, 0.3 mmol) were added. The mixture was reacted at room temperature for 3 hours. The compounds were separated and purified by C18 reversed-phase preparative chromatography, and lyophilized to obtain solid compounds. The lyophilized solid compounds were added to a mixed solution (DCM:TFA = 10:1, v / v) and reacted at room temperature for 1 hour. The reaction mixture was then concentrated, separated and purified by C18 reversed-phase preparative chromatography (by increasing the 10% acetonitrile aqueous solution proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min), and lyophilized to obtain compound k10 (40.6 mg, yield 64%).

[0050] 2. Synthesis of compound k11: Compound k10 (63.5 mg, 0.1 mmol) obtained by lyophilization and HYNIC-NHS (7 mg, 0.2 mmol) were dissolved in 2 mL of DMSO, DIPEA (69.54 μL, 0.4 mmol) was added dropwise, and the mixture was stirred overnight at room temperature. Separation and purification were performed by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min), and lyophilization was performed to obtain compound k11 (63.1 mg, yield 70%).

[0051] 3. Synthesis of compound k12: Compound a8 (49.0 mg, 0.1 mmol) and 4-pentic acid (9.8 mg, 0.1 mmol) were dissolved in 1 mL of DMF, and HATU (41.8 mg, 0.11 mmol) and DIPEA (52.1 μL, 0.3 mmol) were added. The mixture was reacted at room temperature for 2 hours. After detecting completion of the reaction by TLC, the mixture was separated and purified by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute, at a flow rate of 2 mL / min).

[0052] 4. Synthesis of compound 11: Using CuSO4·5H2O, Cu + Aqueous solutions were prepared. Compound k11 (90.2 mg, 0.1 mmol) and compound k12 (114 mg, 0.2 mmol) were dissolved in 1.5 mL of a mixed solution (DMSO:H2O = 1:1, v / v), and a catalytic amount of Cu was added. + An aqueous solution was added, and the mixture was reacted at room temperature for 4 hours. Completion of the reaction was detected by TLC, and the compound was separated and purified by C18 reversed-phase preparative chromatography (10% acetonitrile aqueous solution was increased proportionally to 50%, increasing the acetonitrile content by 1% per minute at a flow rate of 2 mL / min), and then freeze-dried to obtain compound 11. The mass spectrum of compound 11 is shown in Figure 12. MS:Ms / 2 = 1022.88.

[0053] Example 12 Synthesis of Compound 12 Compound 12 was synthesized by referring to the synthesis route of Example 11, substituting compound a8 with compound d8 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound 12. (Compound 12, MS:Ms / 2 = 1054.39) Structural formula of compound 12: [ka]

[0054] Example 13 Synthesis of Compound 13 Compound 13 was synthesized by referring to the synthesis route of Example 11, substituting compound a8 with compound i8 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound 13. (Compound 13, MS:Ms / 2 = 1034.88) Structural formula of compound 13: [ka]

[0055] Example 14 Synthesis of Compound 14 1. Compound o8 was synthesized by referring to the synthesis route of Example a8, substituting compound a5 with compound o5 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound o8. 2. Compound 14 was synthesized by referring to the synthesis route of Example 11, substituting compound a8 with compound o8 in equimolar proportions, with the same other raw materials and proportions, to obtain compound 14. (Compound 14, MS:Ms / 2 = 1052.33) Structural formula of compound 14: [ka]

[0056] Example 15 Synthesis of Compound 15 Synthesis pathway: [ka] 1. Compound o8 was synthesized by referring to the synthesis route of Example a8, substituting compound a5 with compound o5 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound o8. 2. Compound 15 was synthesized by referring to the synthesis route of Example 2, substituting compound a8 with compound o8 in equimolar proportions, with the same other raw materials and proportions, to obtain compound 15. (Compound 15, MS:Ms / 2 = 957.37)

[0057] Example 16 Synthesis of Compound 16 Synthesis pathway: [ka] Compound 16 was synthesized by referring to the synthesis route of Example 3, substituting compound a8 with compound o8 in equimolar proportions, while keeping the other raw materials and proportions the same, to obtain compound 16. (Compound 16, MS:Ms / 2 = 1045.42)

[0058] Example 17 Preparation of radioactive 99mTc-labeled complex 10 μL of an aqueous solution of trisodium triphenylphosphine-3,3’,3’’-trisulfonate (150 mg / mL), 10 μL of an aqueous solution of tris(hydroxymethyl)methylglycine (200 mg / mL), and 1 μL of a DMSO solution of labeled precursor compound 5 or compound 9 (1 mg / mL) were added to a vial. Then, 2 mCi of sodium pertechnetate washing solution was added. The vial was placed in a metal bath and heated at 100 °C for 20 min for a reaction, and then cooled to room temperature. An 18-column was taken and activated by washing with 10 mL of absolute ethanol and 10 mL of physiological saline. The reaction solution was diluted with 2 mL of physiological saline, filtered through the C18 column, further washed with 5 mL of physiological saline, washed with 75% ethanol, diluted with physiological saline, and filtered through a sterile filter membrane to obtain injection solutions 99mTc-5 or 99mTc-9 composed of 99mTc-labeled complexes, respectively.

[0059] Example 18 Cell Uptake Experiment U87MG cells (1×10 5 cells) were inoculated into a 6-well plate. 17.5 kBq of 99mTc-5 or 99mTc-9 was added to each well. After incubation at 4 °C for 1 h, the cells were washed three times with cold PBS and collected. The cell-associated radiation dose was measured using a γ-counter and the results were expressed as a percentage of the total added dose per 10 5 cells. In the blocking experiment, U87MG cells were incubated with a 100-fold excess amount of FAP inhibitor (UAMC1110) for 1 h, and then the probe 99mTc-5 or 99mTc-9 was added and incubated, respectively. As shown in Figure 18, the 99mTc-5 or 99mTc-9 probe showed a high tumor cell uptake rate (~15% ID).

[0060] Example 19 Animal Imaging Experiment Probes 99mTc-5 or 99mTc-9 were injected into U87MG tumor-bearing mice via the tail vein, with each mouse receiving 300 μCi of technetium-labeled probe. Micro-SPECT / CT was used to scan the tumor-bearing mice after injection. Anesthesia was maintained by continuous inhalation of 2% isoflurane, and images were acquired at 1, 2, 6, and 12 hours post-injection. As shown in Figures 13 and 14, the 99mTc-5 / 99mTc-9 probes exhibited excellent tumor targeting and long retention capabilities. Furthermore, to evaluate the biodistribution of 99mTc-5 or 99mTc-9 in the major organs and tumor tissues of tumor-bearing mice, 1.85 MBq of probe 99mTc-5 or 99mTc-9 was injected into U87MG tumor-bearing mice. After 1 hour, the mice were separated, weighed, and the radiation dose in each organ was measured using a gamma counter. As shown in Figure 15, the tumor uptake rate after 1 hour for probe 99mTc-5 reached 38.1% ID / g, and the tumor uptake rate after 1 hour for 99mTc-9 reached 35.7% ID / g. As shown in Figure 16, this type of probe has excellent development contrast. In blockade experiments, probe 99mTc-5 or 99mTc-9 was injected into U87MG tumor-bearing mice with a 100-fold excess of the FAP inhibitor (UAMC1110), and in vitro distribution was performed to statistically analyze the changes in tumor uptake rate. As shown in Figure 17, the 99mTc-5 / 99mTc-9 probes exhibit specificity in targeting fibroblast-activating proteins.

Claims

1. A dimer compound targeting FAP or a pharmaceutically acceptable salt thereof, characterized in that the dimer compound targeting FAP has the structure of the following formula. 【Chemistry 23】 (In the formula, R １ It is selected from the cyano group, R ２ It is selected from hydrogen, fluorine, or chlorine. R ３ It is selected from hydrogen or a methyl group, R ４ This is selected from hydrogen, methyl group, ethyl group, propyl group, cyclopropyl group, or cyclobutyl group. R ５ and R ６ Each of these is independently selected from hydrogen, methyl group, fluorine, or chlorine. R ７ and R ８ Each is independently selected from either a hydrogen or a methyl group. L １ and L ２ are each independently selected from any of the following structures: 【Chemistry 24】 Y is selected from one of the following structures: 【Chemistry 25】 Q is either a hydrogen atom or part of a nuclide chelate group, selected from one of the following structures: 【Chemistry 26】

2. A dimer compound targeting FAP or a pharmaceutically acceptable salt thereof, characterized in that the dimer compound is selected from any of the following compounds. 【Chemistry 27】 【change】

3. The pharmaceutically acceptable salt is selected from hydrochloride, sulfate, trifluoroacetate, fumarate, succinate, sulfonate, maleate, acetate, phosphate, or citrate, characterized in that the FAP-targeting dimer compound or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

4. A compound that targets a fibroblast-activating protein labeled with the radionuclide technetium-99m, characterized by reacting a dimer compound that targets FAP as described in claim 1 or 2 or a pharmaceutically acceptable salt thereof with a compound containing the radionuclide technetium-99m, according to a conventional wet labeling method or freeze-drying labeling method.

5. A 99m-Tc labeling kit characterized by comprising a dimer compound targeting FAP according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or a compound targeting a fibroblast-activating protein labeled with the radionuclide technetium-99m according to claim 4, a ligand that forms coordination with technetium-99m, a reducing agent, an additive, and a stabilizer.

6. Application of a dimer compound targeting FAP according to any one of claims 1 to 3, or a compound targeting a fibroblast-activating protein labeled with the radionuclide technetium-99m according to claim 4, or a kit according to claim 5, in the manufacture of a drug or reagent for treating and / or diagnosing a disease characterized by FAP positivity.

7. The application according to claim 6, characterized in that the disease characterized by FAP positivity includes tumors with high FAP expression.

Images