Quinolinium-based FAP inhibitor, and preparation method therefor and use thereof

Abstract

Provided in the present invention is a compound targeting FAP or a pharmaceutically acceptable salt thereof, which has a structure represented by formula I. Specifically, the present invention relates to an inhibitor targeting FAP (fibroblast activating protein), and a preparation method therefor and the use thereof. The inhibitor is a quinolinium-based compound that can target the FAP protein or a pharmaceutically acceptable salt thereof. The inhibitor has the advantages of simple synthesis steps, a high yield, and simple and convenient operations. The inhibitor of the present invention can be used for preparing an FAPI probe targeting the FAP protein. The obtained FAPI probe has the advantages of low background signal, high signal-to-noise ratio, etc., in PET imaging, and provides a broad application for tumor diagnosis or treatment.

Description

A quinolinium-based FAP inhibitor and its preparation method and application Technical Field

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

[0002] Early detection and early treatment are the most effective cures for cancer. Early diagnosis and treatment of tumors are closely linked to cancer patient survival rates. Molecular imaging is currently the most effective diagnostic tool. Among various molecular imaging diagnostic techniques, positron emission tomography (PET) and single photon emission computed tomography (SPECT), a nuclear medicine molecular imaging technique, are highly effective and commonly used clinically. Using radionuclide tracer technology, PET and SPECT can clearly visualize the molecular imaging characteristics of deep tissues within a patient's body, aiding in the early diagnosis of cancer.

[0003] As the mechanisms of cancer growth continue to be explored, tumor growth, spread, and metastasis are closely linked to the tumor microenvironment (CAFs). Fibroblast activation protein (FAP), a specific marker of the tumor microenvironment, is an excellent target for cancer research. FAP itself is a 97kDa protein that is expressed at very low or near-zero levels in normal human tissues but is highly expressed in tumors.

[0004] PET probes based on FAPI have demonstrated superior performance in tumor imaging. Structural modifications of FAPI small molecules continue to deepen, including studies on linkers and attempts at dimers. However, these modification schemes are still based on the existing quinoline-coupled 2-cyanopyrrolidine core structure and no possible modifications to this core structure have been attempted.

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

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

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

[0008] in,

[0009] R1, R2 are independently H or halogen;

[0010] R3 is selected from -CN or -B(OH)2;

[0011] R4 is selected from C 1-6 Alkylamino or H;

[0012] R5 is or does not exist;

[0013] X is selected from O or methylamino;

[0014] Y is selected from C 1-6 Alkyl or -(PEG) n , n is an integer from 1 to 5;

[0015] Z is selected from the group consisting of an unsubstituted 5-10 membered N-containing heterocycloalkyl group, an unsubstituted 6-10 membered N-containing heteroaryl group;

[0016] Q is -(Ra) m -, wherein m is an integer from 0 to 40; wherein each Ra is independently selected from the following group: -CH2-, -O-, -NH-, -(CO)-, -NH(CO)-, -(CO)-NH-, 5-10 membered carbocyclic ring, 5-10 membered heterocyclic ring, and two adjacent Ra are not simultaneously non-CH2-groups;

[0017] 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 losing a hydrogen atom or a functional group of a therapeutic drug group.

[0018] In another preferred embodiment, the chelate group in L is selected from tetraazacyclododecane tetraacetic acid, triazacyclononane 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)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid (Dar), dimethyltriaminepentaacetic acid (DTPA), 32-amino-5,16,27-trihydroxy-4,12,15,23,26-pentacarbonyl-5,11,16,22,27-pentaazadotriacontanoic acid (DFO), 2-(6-hydrazinopyridin-3-yl)acetic acid (HYNIC) and mercaptoacetyl triglycine (MAG3).

[0019] In another preferred embodiment, the halogen is selected from the following group: F, Br.

[0020] In another preferred embodiment, the Q is a structure selected from the following group:

[0021] In another preferred embodiment, the Z is selected from the following structures:

[0022] In another preferred embodiment, the Z is

[0023] In another preferred embodiment, the compound or a pharmaceutically acceptable salt thereof may be an enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof.

[0024] In another preferred embodiment, 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-tetraaza-1,9(2,6)-bipyridin-5,13(1,3)-diphenylcyclohexadecane-3,7,11,15-tetrayne)tetraacetic acid)(Dar).

[0025] In another preferred embodiment, the L is selected from the following group: 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA).

[0026] In another preferred embodiment, the fluorescent group is a fluorescent group selected from the following group: a visible light band, a near-infrared band 1, or a near-infrared band 2.

[0027] In another preferred embodiment, the visible light group is selected from the following group: fluorescein, rhodamine, fluorescein isothiocyanate, cyanine fluorescent dyes (such as Cy2), green fluorescent protein, quantum dots, nanoparticles, or F16.

[0028] In another preferred embodiment, the near-infrared region 1 group is selected from the following group: cyanine dyes (such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5), BODIPYs (such as fluoroboron dipyrrole, azafluoroboron dipyrrole), rhodamines (such as rhodamine green, rhodamine 6G, tetramethylrhodamine, rhodamine B, lissamine rhodamine, X-rhodamine, Texas Red, silicon-based rhodamine), quantum dots, nanoparticles, or phthalocyanine.

[0029] In another preferred embodiment, the near-infrared zone II group is selected from the following groups: cyanine dyes (such as Cy7, Cy7.5), DADs (such as CH-1055, CH-4T, FT-TQT), BODIPYs (such as NJ960, NJ1030, NJ1060, PCP-BDP2), quantum dots, or nanoparticles.

[0030] In another preferred embodiment, the therapeutic drug is selected from the following group: small molecule inhibitors, monoclonal antibodies, biological alkylating agents, cytotoxic drugs, hormone drugs, or biological response modifiers.

[0031] In another preferred embodiment, the compound of formula (I) is selected from the following structures:

[0032] In a second aspect of the present invention, there is provided a method for preparing the compound or pharmaceutically acceptable salt as described in the first aspect, the method comprising the steps of:

[0033] ①: providing compound I to react with trans-4-methoxy-3-butene-2-one to obtain compound II;

[0034] ②: subjecting the compound II obtained in step ① to an N-arylation reaction to obtain compound III;

[0035] ③: providing compound III obtained in step ② to undergo substitution reaction to obtain compound IV;

[0036] ④: Compound IV obtained in step ③ is subjected to an amide condensation reaction with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride to obtain compound V;

[0037] ⑤: The compound V obtained in step ④ is first de-tert-butyloxycarbonylated, then condensed with DOTA-tri, and finally de-tert-butylated to obtain compound VI, which is the compound of formula (I) described in the first aspect.

[0038] In another preferred embodiment, in step ①, compound 1 and trans-4-methoxy-3-butene-2-one are reacted in a solvent, and a first solvent is added to react to produce compound II.

[0039] In another preferred embodiment, the organic solvent in step ① is selected from the group consisting of toluene, benzene, and xylene.

[0040] In another preferred embodiment, in step ①, the first solvent is selected from the group consisting of trifluoroacetic acid, trichloroacetic acid, and acetic acid.

[0041] In another preferred embodiment, in step ①, the molar ratio of the first solvent to compound I is 1.2:1.

[0042] In another preferred embodiment, the reaction temperature in step ① is 65° C.-85° C., and the reaction time is 4-6 h.

[0043] In another preferred embodiment, in step ②, compound II undergoes N-arylation reaction with 3-chloro-N-methylpropan-1-amine hydrochloride in the presence of 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl, (±)-BINAP, [1,1′-binaphthyl]-2,2′-bisdiphenylphosphine and tris(dibenzylideneinacetone)dipalladium to obtain compound III.

[0044] In another preferred embodiment, the solvent used in step ② is selected from water, toluene, and isopropanol.

[0045] In another preferred embodiment, the reaction temperature in step ② is 62° C.-82° C., and the reaction time is 3-5 h.

[0046] In another preferred embodiment, in step ③, compound III undergoes a substitution reaction with tert-butyloxycarbonylpiperazine in the presence of cesium carbonate to obtain compound IV.

[0047] In another preferred embodiment, the reaction temperature in step ③ is 52° C.-72° C., and the reaction time is 2-4 h.

[0048] In another preferred embodiment, in step ④, compound IV undergoes an amide condensation reaction with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride in the presence of an organic solvent to obtain compound V.

[0049] In another preferred embodiment, the organic solvent in step ④ is selected from the group consisting of acetonitrile, N,N-dimethylformamide, and dichloromethane.

[0050] In another preferred embodiment, the reaction temperature in step ④ is 60°C-75°C, and the reaction time is 2-4h.

[0051] In another preferred embodiment, the compound V in step ⑤ is subjected to de-tert-butyloxycarbonylation in a first organic solution, and then undergoes a condensation reaction with DOTA-tri in a second organic solvent, and finally the tert-butyl ester is removed in a third organic solvent to obtain compound VI, which is the compound of formula (I) described in the first aspect.

[0052] In another preferred embodiment, in step ⑤, the first organic solvent is selected from the group consisting of acetonitrile / trifluoroacetic acid, dichloromethane / trifluoroacetic acid, and hydrochloric acid methanol solution.

[0053] In another preferred embodiment, in step ⑤, the second organic solvent is selected from the group consisting of acetonitrile, N,N-dimethylformamide, and dichloromethane.

[0054] In another preferred embodiment, the third organic solvent in step ⑤ is selected from the group consisting of acetonitrile / trifluoroacetic acid and dichloromethane / trifluoroacetic acid.

[0055] In another preferred embodiment, the reaction temperature in step ⑤ is 0°C-40°C, and the reaction time is 4-5h.

[0056] In a third aspect of the present invention, a FAPI probe targeting FAP is provided, wherein the probe comprises the compound or pharmaceutically acceptable salt of 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 Second, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi, 72 As、 72 Se, 97 Such as 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.

[0057] In a fourth aspect of the present invention, a method for preparing the probe according to the third aspect is provided, the method comprising the steps of:

[0058] Providing the compound or pharmaceutically acceptable salt of the first aspect, labeled with a radionuclide;

[0059] The labeling method is selected from the following group: a wet labeling method or a freeze-drying labeling method; the radionuclide is defined as described in the third aspect.

[0060] In another preferred embodiment, the compound or a pharmaceutically acceptable salt thereof may be an enantiomer, diastereomer, racemate, atropisomer, polymorph, solvate, or isotope-labeled compound thereof.

[0061] In another preferred embodiment, the wet labeling method comprises the following steps: dissolving an appropriate amount of the compound described in the first aspect in a buffer solution or deionized water to obtain a solution; adding a radioactive nuclide M solution to the obtained solution, and reacting in a closed manner for 5-40 minutes to generate a radioactive nuclide labeled complex.

[0062] In another preferred embodiment, the lyophilization labeling method comprises the following steps: dissolving an appropriate amount of the compound described in the first aspect in a buffer solution or deionized water to obtain a solution; sterile filtering the obtained solution, dividing it into containers, freeze-drying it, and sealing it with a stopper to obtain a lyophilized drug box; adding an appropriate amount of acetic acid solution or buffer solution to the lyophilized drug box to dissolve it, and then adding the corresponding radionuclide M solution, and reacting it in a sealed manner for 5-40 minutes to generate a radionuclide labeled complex.

[0063] In the fifth aspect of the present invention, a pharmaceutical composition is provided, comprising the compound or pharmaceutically acceptable salt according to the first aspect and optionally a pharmaceutically acceptable carrier or excipient.

[0064] In the sixth aspect of the present invention, there is provided a use of the compound or pharmaceutically acceptable salt as described in the first aspect, the probe as described in the third aspect, or the composition as described in the fifth aspect, for preparing a preparation for diagnosing and / or treating diseases caused by overexpression of FAP.

[0065] In another preferred embodiment, the disease in which FAP is overexpressed is selected from the group consisting of cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling and scar 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, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, glioma, glioma, astrocytoma, cervical cancer, or prostate cancer.

[0066] In a seventh aspect of the present invention, a kit is provided, comprising:

[0067] (Z1) the compound or pharmaceutically acceptable salt of the first aspect, the probe of the third aspect, or the composition of the fifth aspect, and

[0068] (Z1) Instructions.

[0069] In the eighth aspect of the present invention, there is provided a use of the compound or pharmaceutically acceptable salt as described in the first aspect, the probe as described in the third aspect, or the composition as described in the fifth aspect for preparing an agent for inhibiting FAP activity.

[0070] 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 shows the IC values ​​of FAPI-ANG for FAP, PREP, and DPPIV proteins. 50 curve chart.

[0072] FIG2 shows the HPLC purity analysis chart of FAPI-ANG.

[0073] Figure 3 shows [ 68 Radioactivity purity analysis chart of [Ga]Ga-FAPI-ANG.

[0074] FIG4 shows the PET imaging results of FAPI-ANG in mice, where the white arrows indicate the tumors.

[0075] FIG5 shows a graph of a cellular uptake assay of FAPI-ANG.

[0076] FIG6 shows the ESI-HR analysis graph of FAPI-ANG-3.

[0077] FIG7 shows the ESI-HR analysis graph of Gu-2ANG-1. DETAILED DESCRIPTION

[0078] After extensive and in-depth research and numerous experimental screenings, the inventors unexpectedly developed for the first time an inhibitor targeting FAP (Fibroblast Activating Protein), its preparation method, and its application. The inhibitor is a quinolinium-based compound or a pharmaceutically acceptable salt thereof that targets the FAP protein. The inhibitor's synthesis steps are simple, with high yield and easy operation. In the present invention, the inhibitor can be used to prepare a FAP-targeting FAPI probe. The resulting FAPI probe exhibits advantages such as low background signal and high signal-to-noise ratio in PET imaging, providing broad applications in tumor diagnosis and treatment. This is the basis for the present invention.

[0079] the term

[0080] Fibroblast activation protein (FAP)

[0081] Fibroblast activation protein, also known as seprase or melanoma membrane-bound gelatinase, is a 170 kDa protein encoded by the human FAP gene (2q23). It is a homodimeric membrane gelatinase belonging to the serine protease family. It is selectively expressed in the reactive matrix of epithelial cancer fibroblasts, in the granulation tissue of wound healing, and in malignant cells of bone and soft tissue sarcomas. This protein is believed to be involved in fibroblast growth during development, the control of epithelial-mesenchymal interactions, tissue repair, and epithelial carcinogenesis.

[0082] FAP belongs to the SC protease family and the S9B proline oligopeptidase subfamily. Other members of the S9B subfamily include DPPIV, DPP8, and DPP9. FAP is highly related to DPPIV, sharing approximately 50% amino acid identity.

[0083] Compared with the prior art, the present invention has the following beneficial effects:

[0084] 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.

[0085] 2. The synthesis steps of the inhibitor of the present invention are simple, the yield is high, and the operation is easy.

[0086] 3. The inhibitor of the present invention 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.

[0087] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. Unless otherwise stated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

[0088] Laboratory instrument consumables

[0089] Animal Model: 5- to 7-week-old female BALB / c mice (15-20 g) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Mice were housed under specific pathogen-free conditions at 25°C, 35-45% humidity, and a 12-hour light-dark cycle. All animals had free access to water and food.

[0090] Cell model: U-87MG (human brain astroglioblastoma cells), from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.

[0091] Chemical reagents: All were purchased from Shanghai Bidex Pharmaceutical Technology Co., Ltd.

[0092] 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.

[0093] In all examples, the mobile phase used in C18 column purification was A: 0.1% water B: methanol, the selected C18 column was 60 g, and the flow rate was 50 ml / min; the mobile phase in HPLC purification was A: 0.1% water B: acetonitrile, and the flow rate was 3 ml / min.

[0094] Example 1: FAPI-ANG Synthesis Route

[0095] Synthesis of compound 1

[0096] 600 μL (approximately 3.65 mmol) of methyl tetrabromopyridine-2-acetate and 400 μL (3.928 mmol) of trans-4-methoxy-3-buten-2-one were dissolved in toluene in a three-necked flask. A water separator was then added, followed by the addition of 600 μL of trifluoroacetic acid. The mixture was stirred and refluxed at 75°C for 5 h. After the reaction, the toluene was removed by rotary evaporation, followed by the addition of water and dichloromethane. The aqueous layer was extracted with dichloromethane, concentrated, and purified by C18 column chromatography. 591 mg (2.11 mmol; 57.8%) of compound 1 were obtained. LC-MS (ESI+): m / z 280.23 [M] + .

[0097] 1 H NMR(600MHz,Chloroform-d)δ9.86(s,1H),9.45–9.42(m,1H),8.84(d,J=7.3Hz,1H),8.30–8.26(m,1H),8.00(d,J=7.5Hz,1H),4.07(s,3H),3.18(s,3H). 13 CNMR(126MHz,Deuterium Oxide)δ166.26,164.07,163.78,150.41,143.63,141.99,135.88,134.94,129.61,129.32,126.03,125.72,118.70,116.39,55.28,22.02.

[0098] Synthesis of compound 2

[0099] 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'-binaphthyl-2,2'-bisdiphenylphosphine, 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. 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 was obtained. LC-MS (ESI+): m / z 293.25 [M] + . 1H NMR(600MHz,Deuterium Oxide)δ8.54(d,J=8.0Hz,1H),8.09(d,J=7.2Hz,1H),7.89(s,1H),7.31(s,1H),7.08(d,J=7.4Hz,1H),3.75 (s,1H),3.66(t,J=5.9Hz,3H),3.61(s,1H),3.21(s,2H),3.13(s,2H),2.71(s,3H),2.12(d,J=17.4Hz,2H). 13 C NMR (126MHz,Deuterium Oxide)δ166.51,163.14–155.97(m),151.56,145.25,141.19,132.61,121.51,117 .07–112.95(m),109.89,100.37,48.60(d,J=55.6Hz),41.75,37.35,27.70,19.37.

[0100] Synthesis of compound 3

[0101] 300 mg (1.02 mmol) of compound 2 was dissolved in N,N-dimethylformamide, and 180 mg (1.06 mmol) of potassium iodide, 317 mg (1.7 mmol) of tert-butyl piperazine-1-carboxylate, and 700 mg (2.15 mmol) of cesium carbonate were added. The mixture was refluxed at 62°C for 3 h. Purification by C18 column chromatography afforded 312 mg (0.7 mmol; 69.0%) of compound 3. LC-MS (ESI+): m / z 443.57 [M] + . 1 H NMR(600MHz,Deuterium Oxide)δ8.69(d,J=7.9Hz,1H),8.33(dd,J=7.6,2.3Hz,1H),8.16(s,1H),7.44(s,1H),7.22(t,J=6.9Hz,1H),4.24(d,J=14.6Hz,2 H),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=2.5Hz,3H),2.26–2.04(m,2H),1.45(s,7H). 13C NMR (126MHz,Deuterium Oxide)δ167.35,162.34(d,J=35.6Hz),155.01,151.72,145.42,141.79,137.77,132.90,121.98,1 16.18(d,J=8.9Hz),82.42,53.84,53.49,51.02,48.28,48.05,40.22,37.42,29.11,26.95,19.38.

[0102] Synthesis of precursor 1

[0103] 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 Bidex Pharmaceuticals Co., Ltd.) were dissolved in acetonitrile, 80 mg (0.21 mmol) of HATU was added, and an appropriate amount of N,N-diisopropylethylamine was added to adjust the pH of the system to 8-9. The mixture was stirred for 3 h. HPLC purification yielded 31 mg (0.05 mmol; 56.1%) of precursor 1. LC-MS (ESI+): m / z 614.60 [M] + . 1 H NMR(600MHz,Deuterium Oxide)δ8.74(dd,J=8.1,4.1Hz,1H),7.86(dd,J=7.4,1.6Hz,1H),7.48(s,2H),7.28–7.20(m,1H),5.18(dt,J=8.8,4.3Hz,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 C NMR (126MHz,Deuterium Oxide)δ169.07,168.87,155.45,152.15,143.93,141.69,133.32,133.01,127.76,116.63,115. 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.

[0104] Synthesis of FAPI-ANG

[0105] 12 mg (0.02 mmol) of precursor 1 was dissolved in a 1:1 acetonitrile:trifluoroacetic acid solution and stirred for 1 hour. The product was then dissolved in acetonitrile with 20 mg (0.034 mmol) of DOTA-tri and 32 mg (0.084 mmol) of HATU. N,N-diisopropylethylamine was added to adjust the pH to 8-9 and stirred for 3-4 hours. The product was then dissolved in a 1:2 acetonitrile:trifluoroacetic acid solution and stirred overnight. Purification by HPLC yielded 3.4 mg (0.0038 mmol; 18.9%) of FAPI-ANG. ESI-HR: m / z 900.4540 [M]. + . 1 H NMR(600MHz,Deuterium Oxide)δ8.64(d,J=8.2Hz,1H),7.76(d,J=7.3Hz,1H),7.42–7.36(m,2H),7.17–7. 12(m,1H),5.09(dd,J=9.1,3.8Hz,1H),4.47(s,1H),4.28(d,J=17.1Hz,1H),4.25 –4.16(m,2H),4.07(dt,J=20.1,10.4Hz,1H),3.92(s,8H),3.87–3.37(m,6H),3.3 8–2.82(m,22H),2.73(d,J=10.1Hz,3H),2.14(q,J=8.1Hz,3H),1.28–1.23(m,2H). 13 C NMR (126MHz,Deuterium Oxide)δ168.45,168.22,164.39,162.48,162.20,161.91,161.63,151.61,143.35,141.16,132.78,132.51,127.35,125.35,119.16,116.85 ,116.19,114.53,112.21,53.55,51.16,50.64,48.03,44.22,44.18,4 1.69,40.78,38.41,37.49,37.44,36.42,36.04,35.84,30.90,18.93.

[0106] Example 2: FAPI-ANG-3 Synthesis Route

[0107] 10 mg (0.016 mmol) of precursor 1 was dissolved in a 1:1 solution of trifluoroacetic acid and acetonitrile and stirred for 1 h. The solvent was then removed and the product was dissolved in acetonitrile with 7 mg (0.032 mmol) of Boc-5-aminopentanoic acid and 20 mg (0.052 mmol) of HATU. The product was stirred for 3 h. HPLC purification yielded 4.4 mg (0.006 mmol; 38.6%) of product precursor 2. LC-MS (ESI+): m / z 627.62 [M-BOC] +

[0108] 4.4 mg (0.006 mmol) of precursor 2 was dissolved in a 1:1 acetonitrile:trifluoroacetic acid solution and stirred for 1 h. After the reaction, the trifluoroacetic acid was rotary evaporated and 10 mg (0.017 mmol) of DOTA-tri and 15 mg (0.039 mmol) of HATU were added. N,N-diisopropylethylamine was then added dropwise and the pH was adjusted to 7-8. The product was then dissolved in a 1:2 acetonitrile:trifluoroacetic acid solution and stirred for 8 h. The trifluoroacetic acid was removed and the product was dissolved in a small amount of water. Purification by HPLC yielded 0.41 mg (0.0005 mmol; 8.3%) of the product FAPI-ANG-3. ESI-HR: m / z 507.2725 [(M+H)] 2+ , the ESI-HR analysis graph of FAPI-ANG-3 is shown in Figure 6 .

[0109] Example 3: Synthesis of Gu-2ANG-1

[0110] 40 mg (0.212 mmol) of intermediate 1 and 25 mg (0.101 mmol) of BOC-L-glutamic acid were dissolved in acetonitrile. 130 mg (0.404 mmol) of TBTU was added dropwise, followed by 500 μL of N,N-diisopropylethylamine. The mixture was stirred for 3 h. HPLC purification afforded 76 mg (1.288 mmol; 60.76%) of compound 4. LC-MS (ESI+): m / z 612.28 [M+Na]+

[0111] 40 mg (0.068 mmol) of compound 4 was dissolved in a 1:1 solution of trifluoroacetic acid and acetonitrile and stirred for 1 hour. After removing the solvent, the mixture was dissolved in acetonitrile with 44 mg (0.098 mmol) of compound 3. 60 mg (0.187 mmol) of TBTU was added, followed by the dropwise addition of 500 μL of triethylamine, and the mixture was stirred for 3 hours. HPLC purification afforded 31 mg (0.034 mmol; 49.8%) of compound 5. LC-MS (ESI+): m / z 915.03 [M]+

[0112] 20 mg (0.021 mmol) of compound 5 was dissolved in a 1:1 trifluoroacetic acid:acetonitrile solution and stirred for 1 hour. After removing the solvent, the compound was dissolved in acetonitrile with 24 mg (0.040 mmol) of DOTA-tri, 30 mg (0.094 mmol) of TBTU, and 500 μL of N,N-diisopropylethylamine were added dropwise. The mixture was stirred for 3 hours. After HPLC purification, the compound was dissolved in a 2:1 trifluoroacetic acid:acetonitrile solution to remove the tert-butyl ester protecting group. Finally, 3.2 mg (0.034 mmol; 49.8%) of Gu-2ANG-1 was obtained. ESI-HR analysis: m / z 1200.5575 [M]+. The ESI-HR analysis of Gu-2ANG-1 is shown in Figure 7.

[0113] Example 4: Protein affinity determination of FAPI-ANG

[0114] First, prepare a buffer solution (25mM Tris (tris(hydroxymethyl)aminomethane), 250mM NaCl, pH 7.4). Dilute FAP, DPPIV, and PREP proteins to 0.4ug / ml in the buffer. Dilute the chromogenic substrate GP-AMC (H-Gly-Pro-7-amino-4-methylcoumarin) to 40mM in the buffer. Dilute FAPI-ANG in a serial dilution series. Add equal volumes of FAPI-ANG and substrate to a multiwell plate, then add two volumes of protein. Incubate at 37°C for 1 hour. Measure absorbance at 380nm and use GraphPad to calculate the IC. 50 The curve is shown in Figure 1. The IC of FAP was calculated 50 The value is 8±0.97nM, and the IC value for DPPIV and PREP is 50 The values ​​were all greater than 1000 nM, demonstrating that FAPI-ANG has a 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, showing a purity of over 95%.

[0115] Table 1: HPLC purity analysis of FAPI-ANG

[0116] Example 5: 68 Preparation of Ga]Ga-FAPI-ANG

[0117] 50 μg of FAPI-ANG was dissolved in 200 μl of sodium acetate (4 M), and then 400 μl of eluent ( 68 GaCl3 hydrochloric acid solution) (about 5mCi), heated at 90 degrees Celsius for 10 minutes. Then purified with C18 reverse phase column to obtain about 2mCi [ 68The purity of the prepared product was determined by radio-HPLC, as shown in FIG3 , and the radio-purity was >95%.

[0118] Example 6: 68 Cellular uptake experiment of Ga]Ga-FAPI-ANG

[0119] U87 cells (human glioma cells) were seeded into 6-well plates and incubated for 24 h (n=3). The next day, the 6-well plates were removed from 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 incubated for 2 h. After washing the culture medium with PBS, the cells were lysed with NaOH (1 M), and the cells were collected and counted. The uptake curve was fitted using GraphPad and is shown in Figure 5. The IC 50 is 23nM.

[0120] Example 7: 68 In vivo PET / CT imaging of Ga]Ga-FAPI-ANG in mice bearing U87MG brain glioma

[0121] Animal model establishment: BALB / c female nude mice (6-8 weeks, ~20 g) were purchased from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences. Mice were placed in a sterile environment at 25°C, 35-45% humidity, with alternating light and dark conditions for 12 hours. Food and water were available ad libitum. In the tumor transplantation model, 6-8 week-old nude mice were injected subcutaneously in the left shoulder with 200 μL of 2 × 10 6 U87MG cells. Tumors were grown for one month. Before imaging, mice were anesthetized using a rodent ventilator with 3% isoflurane in air. The injection volume was 200 μL of the indicated concentration of saline solution. During imaging, mice were anesthetized using a nose cone that delivered 2% isoflurane in air.

[0122] PET / CT scan: About 200uCi of [ 68 Ga]Ga-FAPI-ANG, PET / CT imaging was performed 1 hour later, and the results are shown in Figure 4, where the white arrow indicates the tumor. The experiment showed [ 68 Ga]Ga-FAPI-ANG has strong imaging capability and a high tumor-to-background ratio, which can clearly image the tumor site.

[0123] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound targeting FAP or a pharmaceutically acceptable salt thereof, characterized in that, 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 is absent; 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 group consisting of: 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 removing a hydrogen atom or a functional group from a therapeutic drug group.

2. The compound or its pharmaceutically acceptable salt according to claim 1, wherein, The Z described is selected from the following group of 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 includes the steps of: ①: React compound I with trans-4-methoxy-3-buten-2-one to obtain compound II; ②: Subject compound II obtained in step ① to N-arylation reaction to obtain compound III; ③: Subject compound III obtained in step ② to a substitution reaction to obtain compound IV; ④: React compound IV obtained in step ③ with (S)-1-(2-aminoacetyl)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride to undergo an amide condensation reaction to obtain compound V; ⑤: First deprotect the tert-butoxycarbonyl group of 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) 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 of: 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 group consisting of: a wet labeling method or a 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 the preparation of a preparation for the diagnosis and / or treatment of 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 the preparation of a reagent for inhibiting FAP activity.

Images