Quinolinium-based FAP inhibitor as well as preparation method and application thereof

Abstract

The invention provides a compound targeting FAP (Fibroblast Activating Protein) or a pharmaceutically acceptable salt thereof, which has a structure as shown in a formula I. The invention particularly relates to an inhibitor targeting FAP (Fibroblast Activating Protein) as well as a preparation method and application thereof, the inhibitor is a compound which is based on quinolinium and can target FAP protein or a pharmaceutically acceptable salt thereof, and the compound or the pharmaceutically acceptable salt has a structure as shown in a formula I. The invention further provides a preparation method and application of the compound or the pharmaceutically acceptable salt. The inhibitor is simple in synthesis step, relatively high in yield and simple and convenient to operate. The inhibitor can be used for preparing an FAPI probe targeting FAP protein, and the obtained FAPI probe has the advantages of low background signal, high signal-to-noise ratio and the like in PET imaging, and provides wide application for tumor diagnosis or treatment. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and specifically relates to a preparation method and application of a quinolinium-based FAPI probe. Background Art

[0002] Early detection and early treatment are the most effective ways to cure cancer. The early diagnosis and treatment of tumors are closely related to the survival rate of cancer patients. Molecular imaging technology is currently the most effective diagnostic tool. Among various molecular imaging diagnostic technologies, positron emission computed tomography (PET) and single photon emission computed tomography (SPECT) in nuclear medicine molecular imaging technology are commonly used efficient diagnosis and treatment means in clinical practice. Through radionuclide tracer technology, PET and SPECT can clearly show the molecular imaging characteristics of deep tissues in the patient's body and help in the early diagnosis of cancer.

[0003] With the continuous exploration of the growth mechanism of cancer tumors, the growth, spread, and metastasis of tumors are closely related to the tumor microenvironment (CAFs). Fibroblast activation protein (FAP), as a specific marker of the tumor microenvironment, is an excellent target for cancer tumors. FAP itself is a 97kda protein, which is expressed very low or hardly expressed in normal human physiological tissues, but highly expressed in tumors.

[0004] PET probes prepared based on FAPI have shown superior performance in tumor imaging. The structural modification of FAPI small molecules is still in progress, including the research on linkers and the attempt of dimers. However, these modification schemes are still based on the existing quinoline-coupled 2-cyanopyrrolidine structural core, and no possible modification attempts have been made on this core structure.

[0005] Therefore, there is an urgent need in the art to develop a small molecule structure based on FAPI to achieve good PET imaging effects. Summary of the Invention

[0006] The object of the present invention is to develop a small molecule structure based on FAPI that can achieve good PET imaging effects, and specifically relates to a FAP inhibitor based on quinolinium, its preparation method and application.

[0007] In the first aspect of the present invention, there is provided a compound targeting FAP or a pharmaceutically acceptable salt thereof, having the structure shown in Formula I:

[0008]

[0009] Wherein,

[0010] R1 and R2 are independently H or halogen;

[0011] R3 is selected from -CN or -B(OH)2;

[0012] R4 is selected from C 1-6 alkylamino or H;

[0013] R5 is or absent;

[0014] X is selected from O or methylamino;

[0015] Y is selected from C 1-6 alkyl or -(PEG) n , where n is an integer from 1 to 5;

[0016] Z is selected from the group consisting of: unsubstituted 5- to 10-membered N-containing heterocycloalkyl, unsubstituted 6- to 10-membered N-containing heteroaryl;

[0017] Q is -(Ra) m -, where m is an integer from 0 to 40; where each Ra is independently selected from the group consisting of: -CH2-, -O-, -NH-, -(CO)-, -NH(CO)-, -(CO)-NH-, 5- to 10-membered carbocyclic ring, 5- to 10-membered heterocyclic ring, and two adjacent Ra are not simultaneously non-CH2- groups;

[0018] L is a chelating agent group capable of forming a complex with a divalent or trivalent metal cation, a fluorescent group, or a drug fragment formed by removing a hydrogen atom or a functional group from a therapeutic drug group.

[0019] In another preferred embodiment, the chelating group in L is selected from the group consisting of cyclen tetraacetic acid, trineopentylamine triacetic acid, 1,4,7-triazacyclononane-1-pentanedioic acid-4,7-diacetic acid, 1,4,7,10-tetraazacyclododecane-1-pentanedioic acid-4,7,10-triacetic acid, 2,2',2'',2''-(5 2 ,13 2 -dihydroxy-5 5 ,13 5 -dimethyl-3,7,11,15-tetraaza-1,9(2,6)-dipyridine-5,13(1,3)-dibenzocyclohexadione-3,7,1,11,15-tetrayl)tetraacetic acid (Dar), diethylenetriaminepentaacetic acid (DTPA), 32-amino-5,16,27-trihydroxy-4,12,15,23,26-pentacarbonyl-5,11,16,22,27-pentaaza-1-oic acid (DFO), 2-(6-hydrazinopyridin-3-yl)acetic acid (HYNIC), and mercaptoacetyltriglycine (MAG3).

[0020] In another preferred embodiment, the halogen is selected from the group consisting of: F, Br.

[0021] In another preferred example, Q is a structure selected from the following group:

[0022]

[0023] In another preferred example, Z is selected from the following group of structures:

[0024]

[0025] In another preferred example, Z is

[0026] In another preferred example, the compound or its pharmaceutically acceptable salt can be its enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotopically labeled compound.

[0027] In another preferred example, L is a bifunctional chelating agent group, wherein the bifunctional chelating agent is selected from the following group: 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N″-pentaacetic acid (DTPA), bis-(carboxymethylimidazole) glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC), 2,2',2″,2'″-(5 2 ,13 2 -dihydroxy-5 5 ,13 5 -dimethyl-3,7,11,15-tetraazaazacyclo-1,9(2,6)-bispyridine-5,13(1,3)-dibenzocyclohexadecane-3,7,11,15-tetrayne) tetraacetic acid) (Dar).

[0028] In another preferred example, L is selected from the following group: 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA).

[0029] In another preferred example, the fluorescent group is a fluorescent group selected from the following group: visible light band, near-infrared region I band, or near-infrared region II band.

[0030] In another preferred example, the visible light group is selected from the following group: fluorescein, rhodamine, fluorescein isothiocyanate, cyanine fluorescent dye (such as Cy2), green fluorescent protein, quantum dot, nanoparticle, or F16.

[0031] In another preferred example, the group in the first near-infrared region is selected from the following group: cyanine dyes (such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5), BODIPY dyes (such as fluoroboron dipyrrole, azaboron dipyrrole), rhodamine dyes (such as rhodamine green, rhodamine 6G, tetramethyl rhodamine, rhodamine B, Lissamine rhodamine, X-rhodamine, Texas Red, silicon-based rhodamine), quantum dots, nanoparticles, or phthalocyanine.

[0032] In another preferred example, the group in the second near-infrared region is selected from the following group: cyanine dyes (such as Cy7, Cy7.5), D-A-D dyes (such as CH-1055, CH-4T, FT-TQT), BODIPY dyes (such as NJ960, NJ1030, NJ1060, PCP-BDP2), quantum dots, or nanoparticles.

[0033] In another preferred example, the therapeutic drug is selected from the following group: small molecule inhibitors, monoclonal antibody drugs, biologic alkylating agents, cytotoxic drugs, hormone drugs, or biological response modifiers.

[0034] In another preferred example, the compound of formula (I) is selected from the following structures:

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] In the second aspect of the present invention, a method for preparing the compound or a pharmaceutically acceptable salt as described in the first aspect is provided, and the method includes the steps of:

[0041]

[0042] ①: Reacting compound I with trans-4-methoxy-3-buten-2-one to obtain compound II;

[0043] ②: Performing N-arylation on the compound II obtained in step ① to obtain compound III;

[0044] ③: Performing a substitution reaction on the compound III obtained in step ② to obtain compound IV;

[0045] ④: Provide the amide condensation reaction of the compound IV obtained in step ③ with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride to obtain compound V;

[0046] ⑤: First remove the tert-butoxycarbonyl group from the compound V obtained in step ④, then carry out a condensation reaction with DOTA-tri, and finally remove the tert-butyl ester to obtain compound VI, which is the compound of formula (I) described in the first aspect.

[0047] In another preferred example, in step ①, compound 1 and trans-4-methoxy-3-buten-2-one are reacted in a solvent in the presence of a first solvent to form compound II.

[0048] In another preferred example, the organic solvent in step ① is selected from the group consisting of toluene, benzene, and xylene.

[0049] In another preferred example, the first solvent in step ① is selected from the group consisting of trifluoroacetic acid, trichloroacetic acid, and acetic acid.

[0050] In another preferred example, the molar ratio of the first solvent to compound I in step ① is 1.2:1.

[0051] In another preferred example, the reaction temperature in step ① is 65°C - 85°C, and the reaction time is 4 - 6 h.

[0052] In another preferred example, in step ②, compound II and 3-chloro-N-methylpropan-1-amine hydrochloride undergo an N-arylation reaction in the presence of 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene, (±)-BINAP, [1,1′-binaphthalene]-2,2′-bis(diphenylphosphine) and tris(dibenzylideneacetone)dipalladium to obtain compound III.

[0053] In another preferred example, the solvent used in step ② is selected from water, toluene, and isopropanol.

[0054] In another preferred example, the reaction temperature in step ② is 62°C - 82°C, and the reaction time is 3 - 5 h.

[0055] In another preferred example, in step ③, compound III and tert-butoxycarbonylpiperazine undergo a substitution reaction in the presence of cesium carbonate to obtain compound IV.

[0056] In another preferred example, the reaction temperature in step ③ is 52°C - 72°C, and the reaction time is 2 - 4 h.

[0057] In another preferred example, in step ④, compound IV and (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride undergo an amide condensation reaction in the presence of an organic solvent to obtain compound V.

[0058] In another preferred example, the organic solvent described in step ④ is selected from the following group: acetonitrile, N,N-dimethylformamide, dichloromethane.

[0059] In another preferred example, the reaction temperature in step ④ is 60 °C - 75 °C, and the reaction time is 2 - 4 h.

[0060] In another preferred example, in step ⑤, the compound V removes the tert-butoxycarbonyl group in the first organic solution, then undergoes a condensation reaction with DOTA-tri in the second organic solvent, and finally removes the tert-butyl ester in the third organic solvent to obtain the compound VI, which is the compound of formula (I) described in the first aspect.

[0061] In another preferred example, the first organic solvent described in step ⑤ is selected from the following group: acetonitrile / trifluoroacetic acid, dichloromethane / trifluoroacetic acid, hydrochloric acid methanol solution.

[0062] In another preferred example, the second organic solvent described in step ⑤ is selected from the following group: acetonitrile, N,N-dimethylformamide, dichloromethane.

[0063] In another preferred example, the third organic solvent described in step ⑤ is selected from the following group: acetonitrile / trifluoroacetic acid, dichloromethane / trifluoroacetic acid.

[0064] In another preferred example, the reaction temperature in step ⑤ is 0 °C - 40 °C, and the reaction time is 4 - 5 h.

[0065] In the third aspect of the present invention, a FAPI probe targeting FAP is provided, and the probe comprises a compound or a pharmaceutically acceptable salt thereof as described in the first aspect labeled with a radionuclide; the radioactive element is selected from the following group: 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 161 Tb, 153 Sm, 166 Ho, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101 mRh, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au, 225 Ac, 227 Th.

[0066] In a fourth aspect of the present invention, there is provided a method for preparing the probe as described in the third aspect, the method comprising the steps of:

[0067] Providing the compound or a pharmaceutically acceptable salt thereof as described in the first aspect, and labeling with a radionuclide;

[0068] The labeling method is selected from the group consisting of: a wet labeling method or a freeze-drying labeling method; the radionuclide is defined as described in the third aspect.

[0069] In another preferred embodiment, the compound or its pharmaceutically acceptable salt may be an enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotopically labeled compound thereof.

[0070] In another preferred embodiment, the wet labeling method comprises the following steps: dissolving an appropriate amount of the compound as described in the first aspect in a buffer solution or deionized water to obtain a solution; adding a radionuclide M solution to the obtained solution, and reacting in a sealed manner for 5 - 40 min to form a radionuclide-labeled complex.

[0071] In another preferred embodiment, the freeze-drying labeling method comprises the following steps: dissolving an appropriate amount of the compound according to the first aspect in a buffer solution or deionized water to obtain a solution; after sterile filtration of the obtained solution, aliquoting it into a container, freeze-drying it and then plugging and sealing it to obtain a freeze-dried medicine kit; adding an appropriate amount of acetic acid solution or buffer to dissolve the freeze-dried medicine kit, and then adding a corresponding solution of radionuclide M, and reacting it in a sealed manner for 5 - 40 min to generate a radionuclide-labeled complex.

[0072] In the fifth aspect of the present invention, there is provided a pharmaceutical composition, which comprises the compound according to the first aspect or a pharmaceutically acceptable salt and optionally a pharmaceutically acceptable carrier or excipient.

[0073] In the sixth aspect of the present invention, there is provided the use of a compound according to the first aspect or a pharmaceutically acceptable salt or a probe according to the third aspect or a composition according to the fifth aspect for preparing a preparation for diagnosing and / or treating diseases with overexpression of FAP.

[0074] In another preferred embodiment, the diseases with overexpression of FAP are selected from the following group: cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and keloid disease. Preferably, the cancer is selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic carcinoma, glioma, neuroglioma, astrocytoma, cervical cancer, or prostate cancer.

[0075] In the seventh aspect of the present invention, there is provided a kit, which comprises:

[0076] (Z1) a compound according to the first aspect or a pharmaceutically acceptable salt or a probe according to the third aspect or a composition according to the fifth aspect, and

[0077] (Z1) instructions.

[0078] In the eighth aspect of the present invention, there is provided the use of a compound according to the first aspect or a pharmaceutically acceptable salt or a probe according to the third aspect or a composition according to the fifth aspect for preparing a reagent for inhibiting FAP activity.

[0079] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0080] Figure 1 shows the IC of FAPI-ANG against FAP, PREP, and DPPIV proteins 50 curves.

[0081] Figure 2 shows the HPLC purity analysis chart of FAPI-ANG.

[0082] Figure 3 shows 68 the radioactivity purity analysis chart of [Ga]Ga-FAPI-ANG.

[0083] Figure 4 shows the PET imaging results of FAPI-ANG in mice, where the tumors are indicated by the white arrows.

[0084] Figure 5 shows the cell uptake assay curves of FAPI-ANG.

[0085] Figure 6 shows the ESI-HR analysis chart of FAPI-ANG-3.

[0086] Figure 7 shows the ESI-HR analysis chart of Gu-2ANG-1. Detailed implementation mode

[0087] Through extensive and in-depth research and a large number of experimental screenings, the present inventors unexpectedly developed for the first time an inhibitor targeting FAP (Fibroblast activating protein), its preparation method and application. The inhibitor is a quinolinium-based compound capable of targeting FAP protein or a pharmaceutically acceptable salt thereof. The synthesis steps of this inhibitor are simple, with a high yield and convenient operation. In the present invention, the inhibitor can be used to prepare FAPI probes targeting FAP protein. The obtained FAPI probes have advantages such as low background signal and high signal-to-noise ratio in PET imaging, providing broad applications for tumor diagnosis or treatment. Based on this, the present invention was completed.

[0088] Term

[0089] Fibroblast activating protein (FAP)

[0090] Fibroblast activation protein, also known as seprase protein or melanoma membrane-bound gelatinase, is a 170 kDa protein encoded by the FAP gene (2q23) in the human body. It is a homodimer and belongs to the membrane gelatinase of the serine protease family. It is selectively expressed in the reaction matrix of epithelial cancer fibroblasts, the granulation tissue of healing wounds, and the malignant cells of bone and soft tissue sarcomas. This protein is considered to be related to fibroblast growth during development, the control of epithelial-mesenchymal interactions, tissue repair, and epithelial carcinogenesis.

[0091] FAP belongs to the SC protease family and is a member of the S9B proline oligopeptidase subfamily. Other members of the S9B subfamily include DPPIV, DPP8, and DPP9. FAP has a high correlation with DPPIV, and they have approximately 50% identical amino acids.

[0092] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0093] 1. The FAP-targeting inhibitor of the present invention is a quinolinium-based compound capable of targeting the FAP protein or a pharmaceutically acceptable salt thereof, and the inhibitor has specific affinity for the FAP protein.

[0094] 2. The synthesis steps of the inhibitor of the present invention are simple, the yield is high, and the operation is convenient.

[0095] 3. The inhibitor of the present invention can be used to prepare FAPI probes targeting the FAP protein. The obtained FAPI probes have advantages such as low background signal and high signal-to-noise ratio in PET imaging, providing broad applications for tumor diagnosis or treatment.

[0096] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the raw materials and reagents used in the following embodiments are commercially available products or can be prepared by known methods.

[0097] Experimental instruments and consumables

[0098] Animal model: 5- to 7-week-old female BALB / c mice (15-20 g) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The mice were raised under specific pathogen-free conditions. The breeding environment was 25°C, the humidity was 35-45%, and there was a 12-hour light-dark cycle. All animals had free access to water and food.

[0099] Cell model: U-87MG (human astrocytoma cells), obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0100] Chemical reagents: All were purchased from Shanghai Bide Pharmatech Co., Ltd.

[0101] Biological reagents: DMEM medium, RPMI 1640 medium, fetal bovine serum, trypsin, phosphate buffered saline (PBS), skim milk powder, 4% paraformaldehyde, and CCK8 kit were all purchased from Dalian Meilun Biotechnology Co., Ltd.

[0102] In all examples, for C18 column purification, the mobile phase was A: 0.1% water, B: methanol, the selected C18 column was 60 g, and the flow rate was 50 ml / min; for HPLC purification, the mobile phase was A: 0.1% water, B: acetonitrile, and the flow rate was 3 ml / min.

[0103] Example 1: Synthesis route of FAPI-ANG

[0104]

[0105] Synthesis of Compound 1

[0106] Dissolve 600 μL (about 3.65 mmol) of methyl tetrabromopyridine-2-acetate and 400 μL (3.928 mmol) of trans-4-methoxy-3-buten-2-one in toluene in a three-necked flask. Connect a water separator above, then add 600 μL of trifluoroacetic acid, and stir and reflux at 75 °C for 5 h. After the reaction, evaporate toluene by rotary evaporation, then add water and dichloromethane solution, take the aqueous layer, extract the aqueous layer with dichloromethane, and purify by C18 column chromatography after concentration. 591 mg (2.11 mmol; 57.8%) of Compound 1 was obtained. LC-MS (ESI+): m / z 280.23 [M] + 。

[0107] 1 1H NMR (600 MHz, Chloroform-d) δ 9.86 (s, 1H), 9.45–9.42 (m, 1H), 8.84 (d, J = 7.3 Hz, 1H), 8.30–8.26 (m, 1H), 8.00 (d, J = 7.5 Hz, 1H), 4.07 (s, 3H), 3.18 (s, 3H). 13CNMR (126 MHz, Deuterium Oxide) δ 166.26, 164.07, 163.78, 150.41, 143.63, 141.99, 135.88, 134.94, 129.61, 129.32, 126.03, 125.72, 118.70, 116.39, 55.28, 22.02.

[0108] Synthesis of Compound 2

[0109] Dissolve 400 mg (1.43 mmol) of Compound 1 in a small amount of water. Then add 94 mg (0.151 mmol) of 1,1'-binaphthalene-2,2'-bis(diphenylphosphine), 70 mg (0.07 mmol) of tris(dibenzylideneacetone)dipalladium, 1.1 g (3.38 mmol) of cesium carbonate, and 230 mg (1.5 mmol) of 3-chloropropylamine hydrochloride in sequence. Reflux at 72 °C for 4 h. Purify by C18 column chromatography to obtain a mixture of carboxylic acid and methyl carboxylate. Adjust the pH to 13 and stir for 1 h. A total of 326 mg (1.11 mmol; 77.8%) of Compound 2 is obtained. LC-MS (ESI+): m / z 293.25 [M] + 。 1 H NMR (600 MHz, Deuterium Oxide) δ 8.54 (d, J = 8.0 Hz, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.89 (s, 1H), 7.31 (s, 1H), 7.08 (d, J = 7.4 Hz, 1H), 3.75 (s, 1H), 3.66 (t, J = 5.9 Hz, 3H), 3.61 (s, 1H), 3.21 (s, 2H), 3.13 (s, 2H), 2.71 (s, 3H), 2.12 (d, J = 17.4 Hz, 2H). 13 C NMR (126 MHz, Deuterium Oxide) δ 166.51, 163.14–155.97 (m), 151.56, 145.25, 141.19, 132.61, 121.51, 117.07–112.95 (m), 109.89, 100.37, 48.60 (d, J = 55.6 Hz), 41.75, 37.35, 27.70, 19.37.

[0110] Synthesis of Compound 3

[0111] Dissolve 300 mg (1.02 mmol) of Compound 2 in N,N-dimethylformamide, add 180 mg (1.06 mmol) of potassium iodide, 317 mg (1.7 mmol) of tert-butyl piperazine-1-carboxylate, 700 mg (2.15 mmol) of cesium carbonate, and reflux at 62 °C for 3 h. Purify by C18 column chromatography to obtain 312 mg (0.7 mmol; 69.0%) of Compound 3. LC-MS (ESI+): m / z 443.57 [M] + 。 1 H NMR (600 MHz, Deuterium Oxide) δ 8.69 (d, J = 7.9 Hz, 1H), 8.33 (dd, J = 7.6, 2.3 Hz, 1H), 8.16 (s, 1H), 7.44 (s, 1H), 7.22 (t, J = 6.9 Hz, 1H), 4.24 (d, J = 14.6 Hz, 2H), 3.75 (s, 2H), 3.62–3.54 (m, 2H), 3.34–3.17 (m, 7H), 3.12–2.94 (m, 2H), 2.80 (d, J = 2.5 Hz, 3H), 2.26–2.04 (m, 2H), 1.45 (s, 7H). 13 C NMR (126 MHz, Deuterium Oxide) δ 167.35, 162.34 (d, J = 35.6 Hz), 155.01, 151.72, 145.42, 141.79, 137.77, 132.90, 121.98, 116.18 (d, J = 8.9 Hz), 82.42, 53.84, 53.49, 51.02, 48.28, 48.05, 40.22, 37.42, 29.11, 26.95, 19.38.

[0112] Synthesis of Precursor 1

[0113] Dissolve 40 mg (0.09 mmol) of Compound 3 and 22 mg (0.11 mmol) of (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride (purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.) in acetonitrile, add 80 mg (0.21 mmol) of HATU, add an appropriate amount of N,N-diisopropylethylamine to make the system pH 8 - 9, and stir for 3 h. Purify by HPLC to obtain 31 mg (0.05 mmol; 56.1%) of (Precursor 1). LC-MS (ESI+): m / z 614.60 [M] + 。 11H NMR (600 MHz, Deuterium Oxide) δ 8.74 (dd, J = 8.1, 4.1 Hz, 1H), 7.86 (dd, J = 7.4, 1.6 Hz, 1H), 7.48 (s, 2H), 7.28–7.20 (m, 1H), 5.18 (dt, J = 8.8, 4.3 Hz, 1H), 4.53–3.99 (m, 4H), 3.92–3.50 (m, 7H), 3.46–3.15 (m, 6H), 3.15–2.85 (m, 3H), 2.82 (s, 3H), 2.39–1.97 (m, 1H), 1.66–1.09 (m, 9H). 13 13C NMR (126 MHz, Deuterium Oxide) δ 169.07, 168.87, 155.45, 152.15, 143.93, 141.69, 133.32, 133.01, 127.76, 116.63, 115.15, 82.86, 53.99, 51.68, 51.46, 48.57, 44.71, 42.21, 37.95, 36.51, 36.32, 36.12, 27.41, 19.44.

[0114] Synthesis of FAPI-ANG

[0115] Dissolve 12 mg (0.02 mmol) of precursor 1 in a solution of acetonitrile:trifluoroacetic acid = 1:1 and stir for 1 h; then dissolve 20 mg (0.034 mmol) of DOTA-tri in acetonitrile, add 32 mg (0.084 mmol) of HATU, and then add N,N-diisopropylethylamine to adjust the pH of the system to 8 - 9, and stir for 3 - 4 h; subsequently dissolve it in a solution of acetonitrile:trifluoroacetic acid = 1:2 and stir overnight, and purify by HPLC to obtain 3.4 mg (0.0038 mmol; 18.9%) of FAPI-ANG. ESI-HR: m / z 900.4540 [M] + 。 11H NMR (600 MHz, Deuterium Oxide) δ 8.64 (d, J = 8.2 Hz, 1H), 7.76 (d, J = 7.3 Hz, 1H), 7.42–7.36 (m, 2H), 7.17–7.12 (m, 1H), 5.09 (dd, J = 9.1, 3.8 Hz, 1H), 4.47 (s, 1H), 4.28 (d, J = 17.1 Hz, 1H), 4.25–4.16 (m, 2H), 4.07 (dt, J = 20.1, 10.4 Hz, 1H), 3.92 (s, 8H), 3.87–3.37 (m, 6H), 3.38–2.82 (m, 22H), 2.73 (d, J = 10.1 Hz, 3H), 2.14 (q, J = 8.1 Hz, 3H), 1.28–1.23 (m, 2H). 13 13C NMR (126 MHz, Deuterium Oxide) δ 168.45, 168.22, 164.39, 162.48, 162.20, 161.91, 161.63, 151.61, 143.35, 141.16, 132.78, 132.51, 127.35, 125.35, 119.16, 116.85, 116.19, 114.53, 112.21, 53.55, 51.16, 50.64, 48.03, 44.22, 44.18, 41.69, 40.78, 38.41, 37.49, 37.44, 36.42, 36.04, 35.84, 30.90, 18.93.

[0116] Example 2: Synthesis Route of FAPI-ANG-3

[0117]

[0118] Dissolve 10 mg (0.016 mmol) of precursor 1 in a solution of trifluoroacetic acid:acetonitrile = 1:1, and stir for 1 h; then remove the solvent, dissolve it with 7 mg (0.032 mmol) of Boc-5-aminovaleric acid in acetonitrile, add 20 mg (0.052 mmol) of HATU, and stir for 3 h. Purify by HPLC to obtain 4.4 mg (0.006 mmol; 38.6%) of product precursor 2. LC-MS (ESI+): m / z 627.62 [M-BOC] +

[0119] Dissolve 4.4 mg (0.006 mmol) of precursor 2 in an acetonitrile:trifluoroacetic acid 1:1 solution and stir for 1 h; after the reaction is completed, rotary evaporate the trifluoroacetic acid and add 10 mg (0.017 mmol) of DOTA-tri and 15 mg (0.039 mmol) of HATU, then dropwise add N,N-diisopropylethylamine to adjust the pH to 7 - 8; then dissolve it in an acetonitrile:trifluoroacetic acid 1:2 solution, stir for 8 h, remove the trifluoroacetic acid and dissolve it in a small amount of water, and purify by HPLC to obtain 0.41 mg (0.0005 mmol; 8.3%) of the product FAPI-ANG-3. ESI-HR: m / z 507.2725 [(M + H)] 2+ , and the ESI-HR analysis chart of FAPI-ANG-3 is as Figure 6 shown.

[0120] Example 3: Synthesis route of Gu-2ANG-1

[0121]

[0122] Dissolve 40 mg (0.212 mmol) of intermediate raw material 1 and 25 mg (0.101 mmol) of BOC-L-glutamic acid in acetonitrile, add 130 mg (0.404 mmol) of TBTU, dropwise add 500 μL of N,N-diisopropylethylamine, and stir for 3 h. Purify by HPLC to obtain 76 mg (1.288 mmol; 60.76%) of compound 4. LC-MS (ESI+): m / z 612.28 [M + Na]+

[0123] Dissolve 40 mg (0.068 mmol) of compound 4 in a trifluoroacetic acid:acetonitrile = 1:1 solution and stir for 1 h; after removing the solvent, dissolve it with 44 mg (0.098 mmol) of compound 3 in acetonitrile, add 60 mg (0.187 mmol) of TBTU, dropwise add 500 μL of triethylamine, and stir for 3 h. Purify by HPLC to obtain 31 mg (0.034 mmol; 49.8%) of compound 5. LC-MS (ESI+): m / z 915.03 [M]+

[0124] Dissolve 20 mg (0.021 mmol) of Compound 5 in a solution of trifluoroacetic acid:acetonitrile = 1:1, and stir for 1 h; after removing the solvent, dissolve it in 24 mg (0.040 mmol) of DOTA-tri in acetonitrile, add 30 mg (0.094 mmol) of TBTU, dropwise add 500 μL of N,N-diisopropylethylamine, and stir for 3 h. After purification by HPLC, dissolve it in a solution of trifluoroacetic acid:acetonitrile = 2:1 to remove the tert-butyl ester protecting group. Finally, 3.2 mg (0.034 mmol; 49.8%) of Gu-2ANG-1 was obtained after purification. ESI-HR: m / z 1200.5575 [M]+, and the ESI-HR analysis chart of Gu-2ANG-1 is as Figure 7 shown.

[0125] Example 4: Protein Affinity Determination of FAPI-ANG

[0126] First, prepare the buffer (25 mM Tris (tris(hydroxymethyl)aminomethane), 250 mM NaCl, pH 7.4). Dilute FAP, DPPIV, and PREP proteins to 0.4 μg / ml with the buffer. Dilute the chromogenic substrate GP-AMC (H-Gly-Pro-7-amino-4-methylcoumarin) to 40 mM with the buffer. Dilute FAPI-ANG in gradients. Add equal volumes of FAPI-ANG and the substrate to a multi-well plate, then add twice the volume of the protein, incubate at 37 °C for 1 h, measure the absorbance at 380 nm, and fit with GraphPad to obtain the IC 50 curve, as Figure 1 shown. Calculate the IC 50 value for FAP to be 8 ± 0.97 nM, and the IC 50 values for DPPIV and PREP are both greater than 1000 nM, demonstrating that FAPI-ANG has specific affinity for FAP protein. In addition, the purity of FAPI-ANG was analyzed by HPLC, and the results are shown in Table 1 and Figure 2 shown, with a purity of over 95%.

[0127] Table 1: HPLC Purity Analysis of FAPI-ANG

[0128]

[0129] Example 5: 68 Preparation of

[0130]

[0131] Dissolve 50 μg of FAPI-ANG in 200 μL of sodium acetate (4 M), and then add 400 μL of the eluent ( 68GaCl3 hydrochloric acid solution (about 5 mCi), heated at 90 °C for 10 minutes. Subsequently, it was purified using a C18 reverse-phase column to obtain approximately 2 mCi 68 Ga]Ga-FAPI-ANG. The purity was determined by radio-HPLC as Figure 3 shown, with a radiochemical purity > 95%.

[0132] Example 6: 68 Cell uptake experiment of Ga]Ga-FAPI-ANG

[0133] U87 cells (human glioma cells) were seeded in 6-well plates and incubated for 24 h (n = 3). The next day, the 6-well plates were taken out of the incubator, and different concentrations of unlabeled FAPI-ANG were added to each well and incubated for half an hour. Subsequently, 20 μCi 68 Ga]Ga-FAPI-ANG was added to each well and incubated for 2 h. After washing the medium with PBS, the cells were lysed with NaOH (1 M), and the cells were collected for counting. The uptake curve was fitted with GraphPad as Figure 5 shown. The calculated IC 50 was 23 nM.

[0134] Example 7: 68 PET / CT imaging of Ga]Ga-FAPI-ANG in nude mice bearing U87MG brain gliomas

[0135] Establishment of the animal model: BALB / c female nude mice (6 - 8 weeks, ~20 g) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences. The mice were placed in a sterile environment at 25 °C, 35 - 45% humidity, with a 12-h light / dark cycle, and food and water were available ad libitum. In the tumor transplantation model, 200 μL of 2 × 10 6 U87MG cells were subcutaneously injected into the left shoulder of 6 - 8-week-old nude mice. The tumor growth cycle was 1 month. Before imaging, the mice were anesthetized using a rodent ventilator with 3% isoflurane mixed in the air. The injection dose was 200 μL, which was the indicated concentration in the saline solution. During imaging, the mice were anesthetized with a nose cone, and the nose cone delivered air containing 2% isoflurane.

[0136] PET / CT scan: Approximately 200 uCi of 68 Ga]Ga-FAPI-ANG was injected into the tail vein of the tumor-bearing mice, and PET / CT imaging was performed 1 h later. The results are shown as Figure 4 shown, where the tumor is indicated by the white arrow. The experiment shows that 68 Ga]Ga-FAPI-ANG has strong imaging ability, a high tumor-to-background ratio, and can clearly visualize the tumor site.

[0137] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A compound targeting FAP or a pharmaceutically acceptable salt thereof, characterized in that, It has the structure shown in Formula I: Wherein, R1 and R2 are independently H or halogen; R3 is selected from -CN or -B(OH)2; R4 is selected from C 1-6 alkylamino or H; R5 is or does not exist; X is selected from O or methylamino; Y is selected from C 1-6 alkyl or -(PEG) n , where n is an integer from 1 to 5; Z is selected from the following group: unsubstituted 5- to 10-membered N-containing heterocycloalkyl, unsubstituted 6- to 10-membered N-containing heteroaryl; Q is -(Ra) m -, where m is an integer from 0 to 40; where each Ra is independently selected from the group consisting of: -CH2-, -O-, -NH-, -(CO)-, -NH(CO)-, -(CO)-NH-, 5- to 10-membered carbocyclic rings, 5- to 10-membered heterocyclic rings, and two adjacent Ra's are not simultaneously non-CH2- groups; L is a chelating agent group capable of forming a complex with a divalent or trivalent metal cation, a fluorescent group, or a drug fragment formed by a therapeutic drug group losing a hydrogen atom or a functional group.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The Z is selected from the following structures:

3. The compound according to claims 1-2, characterized in that, The compound of Formula (I) is selected from the following structures:

4. A method for preparing the compound or a pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that, The method comprises the steps: ①: Reacting compound I with trans-4-methoxy-3-buten-2-one to obtain compound II; ②: Subjecting compound II obtained in step ① to N-arylation reaction to obtain compound III; ③: Subjecting compound III obtained in step ② to a substitution reaction to obtain compound IV; ④: Reacting compound IV obtained in step ③ with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride to carry out an amide condensation reaction to obtain compound V; ⑤: First removing the tert-butoxycarbonyl group from compound V obtained in step ④, then carrying out a condensation reaction with DOTA-tri, and finally removing the tert-butyl ester to obtain compound VI, which is the compound of Formula (I) as claimed in claim 1.

5. A FAPI probe targeting FAP, characterized in that, The probe comprises the compound or pharmaceutically acceptable salt as described in claim 1 labeled with a radionuclide; the radioactive element is selected from the following group: 18 F, 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 161 Tb, 153 Sm, 166 Ho, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101 mRh, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 188 Re, 186 Re, 198 Au, 225 Ac, 227 Th.

6. A method for preparing the probe according to claim 5, characterized in that, The method comprises the steps: Providing the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof, and labeling with a radionuclide; The labeling method is selected from the following group: wet labeling method or lyophilization labeling method; the radionuclide is defined as claimed in claim 5.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and optionally a pharmaceutically acceptable carrier or excipient.

8. Use of a compound or a pharmaceutically acceptable salt as claimed in any one of claims 1 to 3, or a probe as claimed in claim 5, or a composition as claimed in claim 7, characterized in that For preparing a preparation for diagnosing and / or treating diseases with overexpression of FAP.

9. A kit, characterized in that, The kit comprises: (Z1) The compound as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the probe as claimed in claim 5, or the composition as claimed in claim 7, and (Z1) Instructions.

10. Use of a compound or a pharmaceutically acceptable salt as claimed in any one of claims 1 to 3, or a probe as claimed in claim 5, or a composition as claimed in claim 7, characterized in that, For preparing a reagent for inhibiting FAP activity.

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