PET (Polyethylene Terephthalate) imaging agent for covalent targeting tumor FAP (Fibroblast Amplification Protein) as well as labeled precursor, preparation method

Abstract

The invention provides a PET (Polyethylene Terephthalate) developer for covalently targeting tumor FAP (Fibroblast Amplification Protein) as well as a labeled precursor, a preparation method and application of the PET developer and belongs to the technical field of biological medicines. The structural formula of a labeled precursor of the PET developer for covalently targeting the tumor FAP is as shown in a formula I, and the structural formula of the PET developer for covalently targeting the tumor FAP is as shown in a formula II. According to the invention, DOTA-FAP-2286 is adopted as a skeleton, a SuFEx covalent targeting method is utilized, and a'linker 'is modified to synthesize a labeled precursor of a PET imaging agent of covalent targeting tumor FAP shown in a formula I. The labeled precursor is novel in compound structure, stable in physicochemical property and high in imaging efficiency. The further constructed PET imaging agent of covalent targeting tumor FAP with the structure as shown in the formula II has excellent stability, pharmacokinetic characteristics and efficient tumor retention capacity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a PET imaging agent for covalently targeting tumor FAP and a labeled precursor thereof, a preparation method and application thereof. Background Art

[0002] With the development of medicine, the diagnosis and treatment of cancer have made significant progress. However, the prognosis of many patients with advanced or refractory tumors is still poor, and there is a need to continuously develop innovative and effective diagnosis and treatment strategies. In recent years, nuclear medicine diagnosis and treatment integration (Theranostics) has played an important role in the diagnosis and treatment of cancer. Theranostics combines molecular imaging [mainly PET (positron emission tomography) and SPECT (single photon emission tomography)] with targeted radionuclide therapy. Diagnostic nuclear medicine molecular imaging can accurately display lesions, and radionuclide targeted therapy can treat discovered lesions. It combines the specificity of targeted therapy with the cell-killing ability of internal irradiation, and can target and eliminate tumor cells to achieve personalized and precise diagnosis and treatment.

[0003] Fibroblast activation protein (FAP) is a type II transmembrane serine protease that is highly expressed in cancer-associated fibroblasts in over 90% of malignant epithelial tumors. FAP plays a crucial role in tumor growth, invasion, and metastasis. Because FAP is highly expressed on the cell surface of activated cancer-associated fibroblasts and low in normal tissues, it has become a marker for active fibroblasts in tumors, granulation tissue, and fibrotic lesions. Due to its widespread and specific expression, FAP has become a novel target for the diagnosis and treatment of tumors, inflammation, and other lesions, and holds great research significance and application prospects.

[0004] Currently, dozens of radionuclide-labeled fibroblast activation protein inhibitor (FAPinhibitor, FAPI) derivatives have been used in the diagnosis and treatment of malignant tumors. The development of FAPI began with the modification of dipeptidyl peptidase IV inhibitors, and finally obtained the core structure of N-(4-quinolinyl)-GIy-(2-cyanopyrrolidine). Based on this structural motif, 68 A series of FAPI radiopharmaceuticals represented by Ga-DOTA-FAPI-04 have been developed and successfully used in PET / CT imaging of various human tumors. Compared with the most commonly used PET probe 18F-fluorodeoxyglucose ( 18 Compared with F-FDG, 68 Ga or 18F-labeled FAPI tracers usually show low background signals, enhanced tumor-specific uptake, and can be used for the diagnosis of various tumors. However, the relatively short in vivo tumor retention time limits the further application of DOTA-FAPI-04 radiopharmaceuticals in radionuclide therapy.

[0005] To enhance tumor uptake and retention, many FAPI tracers based on FAPI-04 have been developed, such as 68 Ga-DOTA-mFS-FAPI-04. In a first clinical trial involving 50 patients with medullary thyroid carcinoma, it was found that 68 Ga-DOTA-mFS-FAPI-04 was significantly superior to 18 F-FDG PET-CT in detecting primary tumors, lymph node metastases, and distant metastases of medullary thyroid carcinoma. However, in human imaging, the salivary glands, thyroid gland, and pancreas of patients all showed relatively high 68 Ga-DOTA-mFS-FAPI-04 uptake. The high uptake in the parotid gland, thyroid gland, and pancreas of patients is not conducive to the visualization of lesions in the corresponding regions on the one hand, and on the other hand, if radioligand therapy (RLT) is performed, the corresponding glands will be damaged due to excessive radiation doses. Therefore, it is necessary to further optimize the pharmacokinetic properties of this type of covalently targeted FAP radiopharmaceuticals. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a PET imaging agent for covalently targeting tumor FAP, its labeling precursor, preparation method, and application. The labeling precursor of the PET imaging agent for covalently targeting tumor FAP provided by the present invention can be synthesized with diagnostic radionuclides or / and therapeutic radionuclides for the diagnosis and treatment of tumors with high FAP expression; the obtained PET imaging agent for covalently targeting tumor FAP has good stability, pharmacokinetic properties, and high tumor retention ability.

[0007] To achieve the above object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a labeling precursor of a PET imaging agent for covalently targeting tumor FAP, having the structure shown in Formula I:

[0009]

[0010] The present invention provides a preparation method of the above labeling precursor of the PET imaging agent for covalently targeting tumor FAP, comprising the following steps:

[0011] Using the solid-phase peptide synthesis method of Fmoc, a compound 1 with the structure of Hexanoic acid-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH is prepared;

[0012] Using the Fmoc synthesis method of polypeptides, compound 2 with the structure of Mpr-Gly-Arg-Gln-Arg-Gln-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA)-NH2 was prepared;

[0013] Compound 1, compound 2 and TBMB were subjected to a substitution reaction to obtain a labeling precursor of a PET imaging agent covalently targeting tumor FAP.

[0014] The present invention provides a PET imaging agent covalently targeting tumor FAP, having the structure shown in formula II:

[0015]

[0016] The present invention provides a preparation method of the above PET imaging agent covalently targeting tumor FAP, comprising the following steps:

[0017] Mix 68 The Ga eluate, the labeling precursor of the PET imaging agent covalently targeting tumor FAP with the structure shown in formula I and a buffer solution were mixed to carry out a radiolabeling reaction to obtain a PET imaging agent covalently targeting tumor FAP with the structure shown in formula II.

[0018] Preferably, the temperature of the radiolabeling reaction is 90-100 °C and the time is 10-15 min.

[0019] Preferably, the radiolabeling reaction is carried out in an acidic environment;

[0020] The pH value of the acidic environment is 4-6.

[0021] Preferably, the buffer solution is a sodium acetate buffer solution.

[0022] Preferably, after the radiolabeling reaction, column purification is further included for the obtained radiolabeling reaction system to obtain a pure product of the PET imaging agent covalently targeting tumor FAP with the structure shown in formula II;

[0023] The elution column used for the column purification is an Oasis HLB column;

[0024] The elution phase of the column purification is ethanol with a volume concentration of 40-60%.

[0025] The present invention provides the application of the above PET imaging agent covalently targeting tumor FAP in the preparation of tumor diagnosis products or inflammation diagnosis products.

[0026] Preferably, the tumor includes one or more of lung cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, fibrosarcoma, pancreatic cancer, and glioma.

[0027] The present invention provides a labeling precursor of a covalent targeting tumor FAP PET imaging agent, having the structure shown in Formula I. The present invention uses DOTA-FAP-2286 as the molecular backbone, utilizes the SuFEx (sulfur(VI)-fluoride exchange reaction) covalent targeting method, and constructs a covalent cyclic peptide inhibitor against tumor FAP having the structure shown in Formula I, denoted as DOTA-mFS-KERERG-FAP-2286, which can be used as a labeling precursor of a covalent targeting tumor FAP PET imaging agent. The structural formula of the labeling precursor provided by the present invention is scientifically designed and ingeniously conceived. By introducing the KERERG hydrophilic polypeptide, the in vivo pharmacokinetic properties can be optimized, enabling DOTA-mFS-FAP-2286 excreted through both the liver and gallbladder and the kidneys to be excreted only through the kidneys. The labeling precursor provided by the present invention has a novel structure and stable physicochemical properties, and can be synthesized with diagnostic radionuclides or / and therapeutic radionuclides for the diagnosis and treatment of tumors with high FAP expression. The precursor compound provided by the present invention 68 After being labeled with 177 Ga, the compound of Formula II obtained has good stability, excellent pharmacokinetics, and can be used in the diagnosis of various malignant tumors (such as epithelial cell carcinoma) and inflammation; the precursor compound provided by the present invention can be combined with therapeutic radionuclides such as 225 Lu,

[0028] 68 Ac for labeling to achieve better therapeutic effects and has broad application prospects. Description of the Drawings

[0029] Figure 1 is the synthetic route diagram of DOTA-mFS-KERERG-FAP-2286 in Example 1;

[0030] Figure 2 is the mass spectrum of DOTA-mFS-KERERG-FAP-2286 in Example 1;

[0031] Figure 3 For Example 2 68 Radio-HPLC detection result graph of Ga-DOTA-mFS-KERERG-FAP-2286;

[0032] Figure 4 Cell time gradient binding experiment results for Example 4;

[0033] Figure 5 For 68 Graph of the in vivo distribution of Ga-DOTA-mFS-KERERG-FAP-2286 in the U87MG nude mouse model;

[0034] Figure 6 For nude mice bearing the U87MG tumor model injected with 100 μCi 68 Whole-body Micro-PET / CT MIP imaging graphs at 30 min, 1 h, 2 h, and 4 h after injection of Ga-DOTA-mFS-KERERG-FAP-2286;

[0035] Figure 7 For transfected HEK-293T tumor model mice injected with 100 μCi 68 Whole-body Micro-PET / CT MIP imaging graphs at 30 min, 1 h, 2 h, and 4 h after injection of Ga-DOTA-mFS-KERERG-FAP-2286. Detailed implementation manners

[0036] The present invention provides a labeling precursor of a PET imaging agent covalently targeting tumor FAP, having the structure shown in Formula I:

[0037]

[0038] In the present invention, the structural formula of the structure shown in Formula I is DOTA-mFS-Lys-Glu-Arg-Glu-Arg-Gly-FAP-2286; the chemical name is 2,2',2''-(10-((10S,13S,16S,19S,22S,39S)-1-((12S,32S,5R,13R,16S,19S,22S)-19-(3-amino-3-oxopropyl)-16-benzyl-13-carboxy-5-hexanamido-22-((R)-1-hydroxyethyl)-2,4,15,18,21,24-hexaoxo-7,11-dithia-14,17,20,23-tetraaza-1(1,2),3(2,1)-dipyrrolidino-9(1,3)-benzonacyclotetracosane-95-yl)-22-carbamoyl-13,19-bis(2-carboxyethyl)-39-(4-(3-((fluorosulfonyl)oxy)benzamido)butyl)-10,16-bis(3-aminopropyl)-5,8,11,14,17,20,28,38,41-nonaoxo-31,34-dioxa-2-thia-6,9,12,15,18,21,27,37,40-nonaazatetratetracontane-42-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid.

[0039] The present invention provides a preparation method of a labeling precursor of the above-mentioned covalently targeted tumor FAP PET imaging agent, comprising the following steps:

[0040] Using the polypeptide Fmoc synthesis method, prepare Compound 1 with the structure of Hexanoic acid-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH;

[0041] Using the polypeptide Fmoc synthesis method, prepare Compound 2 with the structure of Mpr-Gly-Arg-Gln-Arg-Gln-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA)-NH2;

[0042] Perform a substitution reaction on Compound 1, Compound 2, and TBMB to obtain a labeling precursor of the covalently targeted tumor FAP PET imaging agent.

[0043] Using the polypeptide Fmoc synthesis method, prepare Compound 1 with the structure of Hexanoic acid-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH. In the present invention, the structural formula of Compound 1 is as shown in Formula 1;

[0044]

[0045] The present invention has no special requirements for the Fmoc synthesis method of the polypeptide. The compound 1 can be synthesized using the Fmoc synthesis method of the polypeptide well-known to those skilled in the art. As a specific embodiment of the present invention, the method for preparing compound 1 by the Fmoc synthesis method of the polypeptide preferably includes the following steps:

[0046] (1) Start the synthesis using Fmoc-Cys(Trt)-CTC resin;

[0047] (2) By the SPPS (solid-phase peptide synthesis) method, add Hexanoic acid, Cys(Trt), Pro, Pro, Thr(tBu), Gln(Trt) and Phe to form a peptide chain on the resin;

[0048] (3) Release compound 1 through a cleavage reaction: Hexanoic acid-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH.

[0049] In the present invention, Hexanoic acid (HeX) represents hexanoic acid.

[0050] The present invention adopts the Fmoc synthesis method of the polypeptide to prepare compound 2 with the structure of Mpr-Gly-Arg-Gln-Arg-Gln-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA)-NH2. In the present invention, the structural formula of the compound 2 is shown in Formula 2:

[0051]

[0052] The present invention has no special requirements for the Fmoc synthesis method of the polypeptide. The compound 2 can be synthesized using the Fmoc synthesis method of the polypeptide well-known to those skilled in the art. As a specific embodiment of the present invention, the method for preparing compound 2 by the Fmoc synthesis method of the polypeptide preferably includes the following steps:

[0053] (1) Start the synthesis using Fmoc-Linker-AM resin;

[0054] (2) By the SPPS (solid-phase peptide synthesis) method, add Trt-Mpr, Gly, Arg(Pbf), Glu(OtBu), Arg(Pbf)-Glu(OtBu), Lys(Dde);

[0055] (3) Deprotection was carried out by adding 3% N2H4·H2O / DMF to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys-AM Resin;

[0056] (4) Again by the SPPS method, Fmoc-NH-PEG2-CH2CH2COOH, Fmoc-Lys(Dde)-OH, and DOTA(OtBu)3 were coupled to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys(PEG2CH2CH2CO-Lys-DOTA(OtBu)3)-AM Resin;

[0057] (5) Again by the SPPS method, 3-((fluorosulfonyl)oxy)benzoic acid was coupled to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA(OtBu)3)-AM;

[0058] (6) Finally, compound 2 was released by a cleavage reaction: Mpr-Gly-Arg-Glu-Arg-Glu-Lys(PEG2CH2CH2CO-Lys-(fluorosulfonyl)oxy)benzoic acid-DOTA-NH2.

[0059] In the present invention, 3-((fluorosulfonyl)oxy)benzoic acid refers to 3-((fluorosulfonyl)oxy)benzoic acid.

[0060] After obtaining compound 1 and compound 2, the present invention carried out a substitution reaction on compound 1, compound 2, and TBMB to obtain a labeling precursor of a covalent targeting tumor FAP PET imaging agent. In the present invention, the TBMB is 1,3,5-tribromomethylbenzene.

[0061] In the present invention, compound 1 is preferably provided in the form of a solution. The solution for dissolving compound 1 is preferably a mixed solution of ammonium bicarbonate aqueous solution and acetonitrile. The concentration of the ammonium bicarbonate aqueous solution is preferably 50 mM, and the volume ratio of the ammonium bicarbonate aqueous solution to acetonitrile is preferably 1:1.

[0062] In the present invention, compound 2 is preferably provided in the form of a solution. The solvent for dissolving compound 2 is preferably acetonitrile.

[0063] In the present invention, the temperature of the substitution reaction is preferably room temperature, and the time is preferably 2 h.

[0064] After the substitution reaction, the present invention preferably adds acetic acid to the obtained substitution reaction system, followed by freeze-drying and HPLC purification in sequence. In the present invention, the chromatographic column for the HPLC purification is preferably Huapu C18, the particle size of the packing material is preferably 10 μm, the pore diameter is preferably 100 Å, the column size is preferably 20 * 250 mm, and the mobile phase for the HPLC purification is preferably: A aqueous solution of 0.1% TFA; B ACN solution of 0.1% TFA, linear gradient: 25-55% for 60 minutes, and the wavelength is preferably 220 nm.

[0065] The present invention provides a PET imaging agent for covalently targeting tumor FAP, having the structure shown in Formula II:

[0066]

[0067] The present invention provides a preparation method of the above PET imaging agent for covalently targeting tumor FAP, comprising the following steps:

[0068] Mix the 68 Ga eluate, the labeling precursor of the PET imaging agent for covalently targeting tumor FAP having the structure shown in Formula I, and a buffer solution, and carry out a radiolabeling reaction to obtain the PET imaging agent for covalently targeting tumor FAP having the structure shown in Formula II.

[0069] In the present invention, the method for preparing the 68 Ga eluate preferably comprises the following steps:

[0070] Use a hydrochloric acid solution to 68 Ge- 68 Ga generator for elution to obtain the 68 Ga eluate.

[0071] In the present invention, the molar concentration of the hydrochloric acid solution is preferably 0.05 M ～ 0.5 M, more preferably 0.1-0.4 M, and even more preferably 0.25 M.

[0072] In the present invention, the labeling precursor of the PET imaging agent for covalently targeting tumor FAP having the structure shown in Formula I is preferably provided in the form of an aqueous solution; in the aqueous solution, the mass ratio of the labeling precursor of the PET imaging agent for covalently targeting tumor FAP having the structure shown in Formula I to water is preferably 1:1-4, and more preferably 1:2-3.

[0073] In the present invention, the mass of the labeling precursor of the covalent targeting tumor FAP PET imaging agent having the structure shown in Formula I is 68 The volume ratio with the

[0074] Ga eluate is preferably 5-25 μg: 3 mL. In the present invention, the buffer is preferably sodium acetate buffer, and the concentration of the sodium acetate buffer is preferably 0.25 M. In the present invention, the 68 Volume ratio of the Ga eluate to the buffer is preferably 1 ～ 4:1 ， More preferably 2-3:1.

[0075] In the present invention, the 68 After mixing the Ga eluate, the labeling precursor of the covalent targeting tumor FAP PET imaging agent having the structure shown in Formula I and the buffer, the pH value of the resulting mixture is preferably 4-6, that is, the radiolabeling reaction is carried out in an acidic environment, and the pH value of the acidic environment is preferably 4-6.

[0076] In the present invention, the temperature of the radiolabeling reaction is preferably 90-100 °C, specifically it can be 90 °C, 95 °C or 100 °C; the time is preferably 10-15 min, specifically it can be 10 min, 12 min or 15 min.

[0077] In the present invention, the process of the radiolabeling reaction is as shown in Formula A:

[0078]

[0079] In the present invention, after the radiolabeling reaction, it further includes column purification of the resulting radiolabeling reaction system to obtain a pure product of the covalent targeting tumor FAP PET imaging agent having the structure shown in Formula II.

[0080] In the present invention, the chromatographic column used for column purification is Oasis HLB column; the elution phase for column purification is ethanol with a volume concentration of 40-60%.

[0081] The present invention provides the application of the above-mentioned covalent targeting tumor FAP PET imaging agent in the preparation of tumor diagnosis products or inflammation diagnosis products.

[0082] In the present invention, the tumor is preferably a malignant tumor, specifically preferably including one or more of lung cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, fibrosarcoma, pancreatic cancer and glioma.

[0083] In the present invention, the inflammation preferably includes one or more of gastritis, sinusitis, gingivitis, enteritis, pharyngolaryngitis, prostatitis, vaginitis, cervicitis, scapulohumeral periarthritis and vertebrocostal arthritis.

[0084] The following is a detailed description of the covalent targeting tumor FAP PET imaging agent provided by the present invention, its labeling precursor, preparation method and application in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0085] Example 1

[0086] Synthesis of DOTA-mFS-KERERG-FAP-2286 with the structure shown in Formula I:

[0087] Preparation of Compound 1:

[0088] (1) Weigh Fmoc-Cys(Trt)-CTC Resin with a substitution degree of 0.712 mol / g, place it in a reactor, and soak it with DMF for 2 h;

[0089] (2) Drain DMF, add 20% Pip / DMF, purge with nitrogen for deprotection for 0.5 h, wash with DMF 5 times, and detect with ninhydrin to show dark blue;

[0090] (3) Input raw materials according to the equivalent ratio (resin obtained in step (2): Fmoc-AA-OH: DIC: HOBt = 1:3:3:3), add an appropriate amount of DMF, purge with nitrogen and react until the detection with ninhydrin is transparent, drain and wash with DMF 3 times;

[0091] (4) Repeat steps (2) and (3) using the solid-phase peptide synthesis method to connect Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, and n-hexanoic acid to obtain Hexanoic acid-Cys(Trt)-Pro-Pro-Thr(tBu)-Gln(Trt)-Phe-Cys(Trt)-CTC Resin.

[0092] (5) After draining, wash with DMF, DCM, and MeOH 2 times in sequence, drain the resin in the reactor, transfer it to a cutting tube, add Solution E [TFA: TIS: EDT: water = 92.5:2.5:2.5:2.5 (volume ratio)], control the temperature on a shaker for 2 hours, add the obtained cutting solution to 6 times the volume of methyl tert-butyl ether, precipitate with a centrifuge to collect the solid, wash the precipitate crude product with methyl tert-butyl ether 3 times, and place the crude product in a vacuum dryer and dry it overnight under vacuum to obtain the crude product.

[0093] (6) The crude product was purified by HPLC preparation (separated by preparative chromatography, column: Huapu C18, 10μm, 100A, 20*250mm, mobile phase: A aqueous solution of 0.1% TFA; B ACN solution of 0.1% TFA, linear gradient: 28 - 68% for 60 minutes, wavelength: 220nm). After lyophilization, compound 1, Hexanoic acid-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH was obtained.

[0094] Preparation of compound 2:

[0095] (1) Weigh Fmoc-LinkerAM Resin with a substitution degree of 0.39 mol / g, place it in a reactor, and add DMF to soak for 2 h.

[0096] (2) Drain the DMF, add 20% Pip / DMF, purge with nitrogen for deprotection for 0.5 h, wash with DMF 5 times, and detect with ninhydrin to show dark blue.

[0097] (3) Charge the raw materials according to the equivalent ratio (resin: Fmoc-AA-OH: DIC: HOBt = 1:3:3:3), add an appropriate amount of DMF, purge with nitrogen for reaction until the ninhydrin detection is transparent, drain and wash with DMF 3 times.

[0098] (4) Repeat steps (2) and (3) using the solid-phase peptide synthesis method to connect Fmoc-Lys(Dde)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Trt-Mpr to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys(Dde)-AMResin.

[0099] (5) Add 3% hydrazine hydrate / DMF solution to the above resin, purge with nitrogen for 10 min, then filter out the solution. After repeating the operation 2 times, wash with 5 mL DMF 5 times to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys-AM Resin.

[0100] (6) Repeat steps (2) and (3) using solid-phase peptide synthesis to link Fmoc-NH-PEG2-CH2CH2COOH, Fmoc-Lys(Dde)-OH, and DOTA(OtBu)3 to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys(PEG2CH2CH2CO-Lys(Dde)-DOTA(OtBu)3)-AM Resin.

[0101] (7) Add a 3% hydrazine hydrate / DMF solution to the resin, purge with nitrogen for 10 min, filter out the solution, repeat the operation twice, and wash with 5 mL of DMF five times to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys(PEG2CH2CH2CO-Lys-DOTA(OtBu)3)-AM Resin.

[0102] (8) Repeat steps (2) and (3) using solid-phase peptide synthesis to link 3-((fluorosulfonyl)oxy)benzoic acid to obtain Trt-Mpr-Gly-Arg(Pbf)-Glu(OtBu)-Arg(Pbf)-Glu(OtBu)-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA(OtBu)3)-AM Resin.

[0103] (9) After drying by suction, wash twice with DMF, DCM, and MeOH successively, dry the resin in the reactor by suction, transfer it to a cleavage tube, add Solution E (TFA:TIS:EDT:water = 92.5:2.5:2.5:2.5, by volume), control the temperature on a shaker for 3 hours, add the cleavage solution to 6 volumes of methyl tert-butyl ether, collect the solid by centrifugation, wash the crude product with methyl tert-butyl ether three times, place the crude product in a vacuum desiccator, and dry it under vacuum overnight to obtain the crude product.

[0104] (10) The crude product was purified by HPLC preparation (separated by preparative chromatography, column: Huapu C18, 10μm, 100A, 20*250mm, mobile phase: A aqueous solution of 0.1% TFA; B ACN solution of 0.1% TFA, linear gradient: 15 - 45% for 60 minutes, wavelength: 220nm). After lyophilization, compound 2, Mpr-Gly-Arg-Gln-Arg-Gln-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA)-NH2, was obtained.

[0105] Preparation of DOTA-mFS-KERERG-FAP-2286:

[0106] Compound 1 was dissolved in 50mM aqueous ammonium bicarbonate solution and acetonitrile (volume ratio 1:1). An acetonitrile solution of TBMB was added, and the reaction was carried out at room temperature for 1h. Then an acetonitrile solution of compound 2 was added, and stirring was continued for 2h. 1mL of acetic acid was added. After concentration and lyophilization, it was purified by HPLC (separated by preparative chromatography, column: Huapu C18, 10μm, 100A, 20*250mm, mobile phase: A aqueous solution of 0.1% TFA; B ACN solution of 0.1% TFA, linear gradient: 25 - 55% for 60 minutes, wavelength: 220nm). After lyophilization, the final product of DOTA-mFS-KERERG-FAP-2286 was obtained.

[0107] The synthetic route of the obtained DOTA-mFS-KERERG-FAP-2286 is as Figure 1 shown.

[0108] The mass spectrometry detection chart of the obtained DOTA-mFS-KERERG-FAP-2286 is as Figure 2 shown.

[0109] Example 2

[0110] Of the structure shown in Formula II 68 Synthesis of Ga-DOTA-mFS-KERERG-FAP-2286:

[0111] 25μg of the compound DOTA-mFS-KERERG-FAP-2286 of Formula I dissolved in 100μL of distilled water was placed in a reaction flask. 750μL of 0.25M sodium acetate and 3mL of freshly rinsed 68 Ga eluent ( 68 Ga elution was carried out in 0.05M hydrochloric acid solution) were added to the reaction flask. The pH value of the reaction solution was 4.0. After mixing, the radiolabeling reaction was carried out at 90°C for 10min. After the reaction was completed, purification was carried out using an Oasis HLB column.

[0112] The radioactive labeling reaction equation is shown as Equation B:

[0113]

[0114] The obtained product was detected by radio-HPLC, and the results are as Figure 3 shown. Figure 3 Among them, the upper figure is the result of the radioactive detector. The radioactive peak of Ga-DOTA-mFS-KERERG-FAP-2286 is at the elution time Rt = 8.3 min. The lower figure is the result of the ultraviolet detector. The ultraviolet peak of the excessive unlabeled precursor DOTA-mFS-KERERG-FAP-2286 is at the elution time Rt = 7.6 min, and the detection wavelength is 254 nm. 68 68

[0115] Example 3

[0116] The in vitro stability experiment of Ga-DOTA-mFS-KERERG-FAP-2286 having the structure shown in Formula II was carried out as follows: 68 68

[0117] About 100 μCi of Ga-DOTA-mFS-KERERG-FAP-2286 obtained in Example 2 was respectively placed in 100 μL of 0.9% normal saline and 0.1% BSA, and after thorough mixing, it was stored at 37 °C. Samples were taken at 30 min, 1 h, and 2 h respectively, and the change in radiochemical purity was examined using analytical Radio-HPLC. 68 68

[0118] The results showed that under the above conditions, 68 the radiochemical purity of Ga-DOTA-mFS-KERERG-FAP-2286 exceeded 98% after 2 h, indicating good stability and almost no decomposition.

[0119] Example 4

[0120] The cell time-gradient binding experiment of Ga-DOTA-mFS-KERERG-FAP-2286 having the structure shown in Formula II was carried out as follows: 68 68

[0121] Take 68Ga-DOTA-mFS-KERERG-FAP-2286 was added into a 24-well plate paved with FAP-transfected HEK-293T cell line. At different time points from 30 min to 4 h, the culture medium and cell lysate were collected respectively, and the corresponding radioactivity counts were measured with a gamma counter. The cell binding rate (%) and cell binding (mol / cell) were calculated according to the following formulas: Cell binding rate (%) = CPM value of cell binding / [CPM value of cell binding + CPM value of unbound cells] × 100%; Cell binding (mol / cell) = [concentration (mol / L) × volume (L) × cell binding rate] / cell number. Compound of Formula II 68 Ga-DOTA-mFS-KERERG-FAP-2286 was subjected to a cell saturation binding experiment. 68 Ga-DOTA-mFS-KERERG-FAP-2286 was added into a 24-well plate paved with FAP-transfected HEK-293T cell line. The cells were divided into total binding and non-specific binding groups for experiments. The culture medium and cell lysate were collected respectively, and the corresponding radioactivity counts were measured with a gamma counter. The specific binding curve, its equilibrium dissociation constant Kd and maximum binding value Bmax were obtained by processing with GraphPad Prism software, and 68 the cell time-gradient binding results of Ga-DOTA-mFS-KERERG-FAP-2286 were compared with the 68 previously reported Ga-DOTA-mFS-FAPI-04 and 68 Ga-DOTA-FAP-2286 ( 68 the structure of Ga-DOTA-mFS-FAPI-04 is shown in Formula E, 68 the structure of Ga-DOTA-FAP-2286 is shown in Formula F). The results obtained are as Figure 4 shown. According to the results, with the passage of time, 68 the cell binding rate of Ga-DOTA-FAP-2286 gradually decreased, but 68 the cell binding rates of Ga-DOTA-mFS-KERERG-FAP-2286 and 68 Ga-DOTA-mFS-FAPI-04 showed an increasing trend around 60 min and began to slowly decrease around 120 min. Among them, 68 the cell binding rate of Ga-DOTA-mFS-KERERG-FAP-2286 decreased more 68 slowly than that of Ga-DOTA-mFS-FAPI-04, indicating that 68 Ga-DOTA-mFS-KERERG-FAP-2286 of the present invention is beneficial to enhancing the uptake and retention of tumors.

[0122]

[0123] Example 5

[0124] having the structure shown in Formula II 68 The biodistribution experiment of Ga-DOTA-mFS-KERERG-FAP-2286 is as follows:

[0125] Nine nude mice with U87MG tumor models were randomly divided into 3 groups, with 3 mice in each group. The compound of Formula II obtained in Example 2 68 Ga-DOTA-mFS-KERERG-FAP-2286 was intravenously injected via the tail vein at 100 μCi. The mice were decapitated and sacrificed at 30 min, 60 min, and 120 min respectively according to the groups. Organs such as the heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, salivary gland, blood, tumor, and brain were taken to measure their radioactivity, and then immediately weighed. Finally, the percentage of injected dose per gram of tissue (%ID / g) was calculated. 68 The in vivo distribution of Ga-DOTA-mFS-KERERG-FAP-2286 in the U87MG nude mouse model is as Figure 5 shown, indicating that the compound of Formula II is highly distributed in tumors and can optimize the in vivo pharmacokinetic properties.

[0126] Example 6

[0127] having the structure shown in Formula II 68 The dynamic in vivo imaging experiment of Ga-DOTA-mFS-KERERG-FAP-2286:

[0128] 1. Micro-PET / CT imaging of nude mice with U87MG model

[0129] The U87MG model mice were placed on the Micro PET bedplate, anesthetized by inhaling isoflurane, and fixed with tape. 100 μCi and 0.1 mL of physiological saline solution of 68 Ga-DOTA-mFS-KERERG-FAP-2286 were intravenously injected via the tail vein in front of the bed. Static small animal PET / CT acquisitions were performed at 30 min, 1 h, 2 h, and 4 h respectively. The imaging results Figure 6 are shown. After injecting 100 μCi 68 Ga-DOTA-mFS-KERERG-FAP-2286, whole-body micro-PET / CT MIP imaging was performed at 30 min, 1 h, 2 h, and 4 h respectively, and the tumor was clearly visualized.

[0130] 2. Micro-PET / CT imaging of nude mice with transfected HEK-293T model

[0131] Place the transfected HEK-293T model mice on the Micro PET bedplate, anesthetize them by inhaling isoflurane, and fix them with tape. Inject 100 μCi, 0.1 mL of 68 physiological saline solution of Ga-DOTA-mFS-KERERG-FAP-2286, and then perform static small animal PET / CT acquisitions at 30 min, 1 h, 2 h, and 4 h respectively. The imaging results are as Figure 7 shown. After injecting 100 μCi 68 Ga-DOTA-mFS-KERERG-FAP-2286, perform whole body micro-PET / CT MIP imaging at 30 min, 1 h, 2 h, and 4 h respectively, and the tumor is clearly visualized.

[0132] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A labeling precursor of a covalent targeting tumor FAP PET imaging agent, characterized in that, It has the structure shown in Formula I:

2. The preparation method of the labeling precursor of the covalent targeting tumor FAP PET imaging agent according to claim 1, characterized in that, It includes the following steps: Using the polypeptide Fmoc synthesis method, prepare Compound 1 with the structure of Hexanoic acid-Cys-Pro-Pro-Thr-Gln-Phe-Cys-OH; Using the polypeptide Fmoc synthesis method, prepare Compound 2 with the structure of Mpr-Gly-Arg-Gln-Arg-Gln-Lys(PEG2CH2CH2CO-Lys(3-((fluorosulfonyl)oxy)benzoic acid)-DOTA)-NH2; Perform a substitution reaction on Compound 1, Compound 2, and TBMB to obtain a precursor for labeling a covalent targeting tumor FAP PET imaging agent.

3. A PET imaging agent for covalently targeting tumor FAP, characterized in that, It has the structure shown in Formula II:

4. The preparation method of the covalent targeting tumor FAP PET imaging agent according to claim 3, characterized in that, It includes the following steps: Mix 68 a Ga eluent, a labeling precursor of a PET imaging agent that covalently targets tumor FAP having the structure shown in Formula I, and a buffer solution, and conduct a radiolabeling reaction to obtain a PET imaging agent that covalently targets tumor FAP having the structure shown in Formula II.

5. The preparation method according to claim 4, characterized in that, The temperature of the radioactive labeling reaction is 90 - 100 °C, and the time is 10 - 15 min.

6. The preparation method according to claim 4 or 5, characterized in that The radioactive labeling reaction is carried out in an acidic environment; The pH value of the acidic environment is 4 - 6.

7. The preparation method according to claim 4, wherein, The buffer solution is sodium acetate buffer solution.

8. The preparation method according to claim 4, characterized in that, After the radioactive labeling reaction, it also includes column purification of the obtained radioactive labeling reaction system to obtain a pure product of a covalent targeting tumor FAP PET imaging agent with the structure shown in Formula II; The elution column used for the column purification is Oasis HLB column; The elution phase of the column purification is ethanol with a volume concentration of 40 - 60%.

9. Use of the covalent targeting tumor FAP PET imaging agent described in claim 3 or the covalent targeting tumor FAP PET imaging agent prepared by the preparation method described in any one of claims 4 - 8 in the preparation of tumor diagnostic products or inflammatory diagnostic products.

10. The application according to claim 9, wherein, The tumors include one or more of lung cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, fibrosarcoma, pancreatic cancer, and glioma; The inflammations include one or more of gastritis, sinusitis, gingivitis, enteritis, pharyngolaryngitis, prostatitis, vaginitis, cervicitis, scapulohumeral periarthritis, and vertebrocostal arthritis.

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