Prostate cancer diagnosis or treatment radiopharmaceutical for dual targeting of PSMA and FAP-alpha

Abstract

The invention relates to a novel double-targeting PSMA and FAP-alpha compound, which can be coupled with radionuclides such as 99mTc, 68Ga, 177Lu and the like through corresponding chelating agents to prepare radiopharmaceuticals applied to diagnosis or treatment of prostatic cancer. The radiopharmaceutical shows better tumor uptake and retention capacity in tumors with different PSMA expression levels, can be applied to diagnosis or treatment of most prostate cancer patients, and has a good clinical application prospect in treatment.

Description

Technical Field

[0001] The present invention relates to a series of radionuclide-labeled drug complexes dual-targeting PSMA and FAP-α, precursor compounds for preparing the drug complexes and drug-forming kits, and preparation methods thereof, and in particular to a radioactive drug for diagnosis or treatment of individual prostate cancers with different PSMA expression levels. Background Art

[0002] Prostate cancer, as a common epithelial malignant tumor in men, has a high mortality rate and seriously threatens men's life and health. Since 2008, the incidence of prostate cancer in my country has ranked first and continued to rise. Currently, the diagnosis of prostate cancer relies on a comprehensive evaluation of prostate-specific antigen detection, rectal examination and biopsy. In this context, molecular imaging technology based on tumor biomarkers, especially nuclear medicine molecular imaging, is increasingly becoming an important tool in the field of tumor diagnosis, providing strong support for the early detection and precise treatment of prostate cancer.

[0003] With the continuous deepening of research on prostate cancer, a biomarker specifically expressed on the surface of prostate cancer cells, Prostate Specific Membrane Antigen (PSMA), has been discovered. PSMA is a transmembrane glycoprotein, consisting of an extracellular C-terminus, a transmembrane structure, and a cytoplasmic N-terminus. The extracellular region accounts for 95% of the entire glycoprotein and is the main site for recognition by antibodies, peptides, and small molecule compounds. The cytoplasmic domain contains a motif that can trigger the internalization of the receptor-ligand complex, allowing the drug to be effectively deposited into the cell. PSMA is selectively overexpressed on the surface of more than 90% of prostate cancer cells, which is 100-1000 times the expression level of normal prostate epithelial tissue, and can increase with the progression of malignant tumors, making PSMA an attractive target for the diagnosis and treatment of prostate cancer.

[0004] At present, the ligands targeting PSMA mainly include monoclonal antibodies and small molecule inhibitors. Compared with the longer in vivo clearance time and later imaging time of antibodies, small molecule inhibitors have received more and more attention and selection due to their excellent in vivo metabolic rate. Based on the structural characteristics of the extracellular region of the PSMA protein, small molecule inhibitors mainly include three categories: sulfur-containing compounds, phosphorus-containing compounds and urea-based compounds. Among them, urea-based compounds with glutamic acid-urea-lysine as the structural basis are the most widely used in the diagnosis and treatment of prostate cancer. In 2014, the first report of PET imaging for prostate cancer patients was published. 68Ga-PSMA-617 has been used in clinical PET diagnosis of prostate cancer patients due to its excellent targeting performance and good clinical efficacy. Although PSMA is highly expressed on the surface of prostate cancer cells, less than 10% of prostate cancer patients still have 68 After Ga-PSMA-617, no uptake or very low uptake was observed in the lesion, indicating low PSMA expression. In addition, if the patient's primary tumor lesion has low PSMA expression, it often leads to 68 The sensitivity of Ga-PSMA-617 in detecting lymph node or distant metastasis in patients is reduced. Furthermore, for patients whose primary tumor lesions are PSMA-positive, although their metastatic lesions are usually also positive, in some patients with advanced metastatic castration-resistant prostate cancer (CRPC) who have failed multiple chemotherapy regimens, some of their metastatic lesions may be PSMA-negative.

[0005] In recent years, in order to solve the diagnostic range and detection sensitivity of imaging agents targeting PSMA, a series of tumor diagnostic imaging agents that supplement or are directly modified based on PSMA have been developed. Yang Xing et al. disclosed a radionuclide in CN114989261A 18 F-labeled coordination compounds 18 F-AIF-ETN is used as a diagnostic imaging agent for PSMA-negative prostate cancer patients. In general, fibroblast activation protein-α (FAP-α) ligands can be considered as ideal ligands for isomers of various tumor cell targeting ligands. Srikanth Boinapally et al. developed a method targeting PSMA and FAP-α. 64 Cu-labeled PET imaging diagnostic agent. Yang Zhi et al. disclosed in CN116082306A a method based on PSMA-617 and FAPI-04. 68 Ga-labeled dual-targeting PSMA and FAP-α tumor diagnostic PET imaging agent can achieve imaging of PSMA-negative tumors, but 68 Ga-labeled tumor diagnostic PET imaging agents have an asymmetric molecular structure and cumbersome synthesis steps, and their tumor retention ability is average.

[0006] Although scientific research teams around the world have carried out various transformations and attempts in the field of PSMA-targeted tumor imaging agents, these efforts are often constrained by lagging production efficiency and unsatisfactory imaging effects, specifically manifested in complex preparation processes, high costs, and problems such as too fast metabolism in the body, no or low uptake in tumor tissues, and short retention time. In view of this, the development of a diagnostic imaging agent that has a simplified production process, excellent metabolic characteristics, and can maximize the detection of all tumor lesions has become a key clinical problem that needs to be solved urgently, and its importance is self-evident. The development of such new imaging agents is not only expected to improve the accuracy and sensitivity of patient tumor diagnosis, but also provide strong technical support for early detection, precision treatment, and efficacy evaluation of tumors. Summary of the invention

[0007] The purpose of the present invention is to provide a PSMA and FAP dual-targeting small molecule compound with a simple labeling method, high labeling rate, strong tumor uptake and retention ability, which can be used for tumor diagnosis or treatment of nearly all prostate cancer patients, and belongs to the field of radiopharmaceuticals and nuclear medicine.

[0008] In order to achieve the above object, the present invention provides a compound having a structure shown in formula (I),

[0009]

[0010] or a pharmaceutically acceptable salt or stereoisomer thereof;

[0011] Among them, L 1 Functionalized linker -CO-C 1-6 Alkylene (-X 1 -C 1-6 Alkylene) m -;

[0012] L 2 Functionalized linker -CO-C 1-6 Alkylene-(X 2 -C 1-6 Alkylene-) n -NH 2 ;

[0013] X 1 , X 2 Each independently selected from O, S, NH; said C 1-6 The alkylene group is optionally substituted with halogen, OH, NH 2 , oxo, cyano substitution;

[0014] m and n are integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0015] In one embodiment of the present invention, X1 , X 2 Selected from O respectively.

[0016] In one embodiment of the present invention, the C 1-6 Alkylene is selected from -C 2 H 4 -、-C 3 H 6 -.

[0017] In one embodiment of the present invention, said n, m are selected from integers of 3-6.

[0018] In one embodiment of the present invention, L 1 Functionalized linker -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) 4 -.

[0019] In one embodiment of the present invention, L2 is a functionalized linker -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) 4 -NH 2 .

[0020] The present invention also provides a compound having a structure shown in formula (II):

[0021]

[0022] or a pharmaceutically acceptable salt or stereoisomer thereof;

[0023] Among them, L 1 Functionalized linker -CO-C 1-6 Alkylene-(X 1 -C 1-6 Alkylene) m -;

[0024] L 2 Functionalized linker -CO-C 1-6 Alkylene-(X 2 -C 1-6 Alkylene-) n -NH-;

[0025] X 1 , X 2 Each independently selected from O, S, NH; said C 1-6 The alkylene group is optionally substituted with halogen, OH, NH 2 , oxo, cyano substitution;

[0026] m and n are integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0027] In one embodiment of the present invention, X 1 , X 2 Selected from O respectively.

[0028] In one embodiment of the present invention, the C 1-6 Alkylene is selected from -C 2 H 4 -、-C 3 H 6 -.

[0029] In one embodiment of the present invention, said n, m are selected from integers of 3-6.

[0030] In one embodiment of the present invention, L 1 Functionalized linker -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) 4 -.

[0031] In one embodiment of the present invention, L2 is a functionalized linker -CO-CH 2 CH 2 -(O-CH 2 CH 2 -) 4 -NH-.

[0032] Specifically, the bifunctional chelating agent in the compound represented by formula (II) of the present invention is selected from the following groups:

[0033]

[0034] In one embodiment, the compound of formula (II) of the present invention is selected from:

[0035]

[0036] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0037] The present invention also provides a coordination compound obtained by labeling the compound represented by the above formula (I) with a radioactive nuclide and a bifunctional chelating agent; wherein the bifunctional chelating agent binds its carboxyl group to L in the compound represented by the formula (I) 2 The terminal amino group reacts to bond.

[0038] In the present invention, the bifunctional chelating agent is selected from the following compounds: 6-hydrazinopyridine-3-carboxylic acid (HYNIC), diglycine thioglycolate (MAG2), triglycine thioglycolate (MAG3), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), 3,6,9,15-tetraazabicyclo[9.3.1]pentadecane-1(15),11,13-triene-3,6,9-triacetic acid (PCTA) , RESCA, 1,4,7-triazacyclononane-1-pentanedioic acid-4,7-diacetic acid (NOTA-GA), 1,4,7,10-tetraazacyclododecane-1-pentanedioic acid-4,7,10-triacetic acid (DOTA-GA), 1,4,8,11-tetraazabicyclo[6,6,2]hexadecane-4,11-diacetic acid (CB-TE2A), 1-(4-isothiocyanatephenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetylhydroxyamino)-6,11,17,22-tetraazaheptacosanetane] (DFO), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraaza1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane (TCMA).

[0039] The present invention also provides a coordination compound obtained by labeling the compound having the structure shown in the above formula (II) with radioactive nuclides.

[0040] In a specific embodiment, the radioactive nuclide in the coordination compound is selected from an isotope emitting α rays, an isotope emitting β rays, an isotope emitting γ rays, an isotope emitting Auger electrons, or an isotope emitting X rays.

[0041] In a preferred embodiment, the radionuclide in the coordination compound is selected from: 18 F. 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y.90 Y. 149 Pm, 177 Lu, 47 Sc, 212 Bi, 213 Bi, 72 As, 211 At 201 Tl, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 223 Ra or 89 Sr et al.

[0042] In a more preferred embodiment, the radionuclide in the coordination compound is selected from 68 Ga, 99m Tc, or 177 Lu et al.

[0043] In one embodiment, the coordination compound of the present invention is selected from:

[0044]

[0045] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0046] The present invention also provides a pharmaceutical composition, which comprises the above coordination compound and a pharmaceutically acceptable carrier.

[0047] The pharmaceutical composition according to the present invention is an intravenous injection drug.

[0048] The present invention also provides a kit, which comprises the compound represented by formula (II) as described above, an optional synergistic ligand, and a pharmaceutically acceptable carrier, excipient, stabilizer and / or diluent. Preferably, the pharmaceutically acceptable carrier comprises a pH buffer system required for the radionuclide labeling.

[0049] Specifically, for 99m The kit of Tc-labeled ligand compounds comprises ligand compounds HFaPSMA, tris(hydroxymethyl)methylglycine (Tricine), triphenylphosphine tris(methyl)sulfonic acid sodium salt (TPPTS), and one or more pharmaceutically acceptable carriers, excipients, stabilizers and / or diluents. Preferably, the kit can be prepared by combining the substances (i.e., ligand compounds HFaPSMA, tris(hydroxymethyl)methylglycine (Tricine), triphenylphosphine tris(methyl)sulfonic acid sodium salt (TPPTS)) and the radionuclide 99mThe pH buffer solution required for Tc labeling is formed by freeze-drying. The pH buffer solution is selected from a buffer system such as succinic acid, citric acid, tartaric acid, phosphoric acid, boric acid, acetic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (i.e., HEPES), and the buffer solution can be a buffer pair of the acid and its salt, a buffer pair of the acid and a base, or a buffer pair of two salts of the acid. The buffer solution is used in 99m The pH of the Tc-labeled kit is maintained in the range of 3-6, such as 4.5-6.0, preferably 5.3-5.7, such as 5.4, 5.5, 5.6. More preferably, HFaPSMA, tris(hydroxymethyl)methylglycine (Tricine), and triphenylphosphine trissulfonic acid sodium salt (TPPTS) are placed in a succinic acid / sodium hydroxide solution, filtered and freeze-dried to obtain a kit. In the kit, the content of HFaPSMA is 10μg-1000μg, the content of Tricine is 1mg-50mg, and the content of TPPTS is 3mg-30mg. Preferably, the content range of the three is 100-200ug: 4-10mg: 3-10mg.

[0050] Used for 68 Ga, 177 The kit of Lu-labeled ligand compound comprises ligand compound DFaPSMA and one or more stabilizers and a pharmaceutically acceptable carrier. Preferably, the kit is obtained by preparing a solution.

[0051] The present invention also provides an application of the compound of formula (I), compound of formula (II), coordination compound, pharmaceutical composition or kit as described above, which is used to prepare a drug for diagnosing or treating prostate cancer lesions with different PSMA expression levels in patients.

[0052] Beneficial Effects

[0053] The compound of the present invention uses nitrogen atom as the connection center, the molecular skeleton structure is overall symmetrical, the synthesis route is simple and efficient, and the production cost is greatly reduced. At the same time, the present invention prepares the complex by a one-step method, which is easy to operate, has a high labeling rate, has better drugability, and is suitable for industrial production and clinical promotion.

[0054] The present invention prepares precursor compounds of formula (I) and formula (II) for dual-targeting PSMA and FAP-α prostate cancer diagnosis or treatment radioactive drugs. After structural modification and adjustment, the drug uptake in the blood is reduced, which is conducive to imaging to obtain a lower background signal.

[0055] The dual-targeting PSMA and FAP-α prostate cancer diagnosis or treatment radiopharmaceuticals of the present invention are compared with the prior art 99m Tc-P137, 68Ga-PSMA-617 or 177 For Lu-PSMA-617, it shows higher tumor uptake and tumor / tissue uptake ratio and better tumor retention ability in PSMA-positive sites, and also shows excellent tumor imaging ability in PSMA-negative sites due to the presence of FAP-α, which is beneficial for the diagnosis of systemic lesions in prostate cancer patients, further increasing the proportion of benefited patients, and has good prospects for tumor diagnosis and radioactive targeted therapy.

[0056] The dual-targeted PSMA and FAP-α prostate cancer diagnostic or therapeutic radioactive drug of the present invention has a lower effective whole-body dose to patients compared to the prior art. In one embodiment of the present invention, the effective whole-body dose is only 0.0016 mSv / MBq. Calculated based on a standard injection dose of 73 kg (20 mCi, 740 MBq), the effective whole-body dose received by the patient is 1.2 mSv. Therefore, the radiation damage caused by the imaging of the drug of the present invention is significantly smaller, and the safety effect is better.

[0057] Compared with the prior art, the dual-targeted PSMA and FAP-α prostate cancer diagnostic or therapeutic radioactive drugs of the present invention have better hydrophilicity and higher renal uptake. Therefore, drug metabolism can be promoted by drinking a lot of water and urinating before imaging, thereby minimizing the appearance of drugs in the blood circulation of normal organs.

[0058] Therefore, the tracer according to the present invention is fully capable of diagnosing and treating the vast majority of prostate cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 :Synthesis route of ligand compounds HFaPSMA and DFaPSMA.

[0060] Figure 2 :Coordination compound 99m Radio-HPLC signal profile of Tc-HFaPSMA.

[0061] Figure 3 :Coordination compound 68 Quality control analysis of Ga-DFaPSMA, where Figure 3 A shows 68 The labeling rate of Ga-DFaPSMA was measured. Figure 3 B shows 68 Results of radiochemical purity determination of Ga-DFaPSMA after purification.

[0062] Figure 4 :Determination of coordination compounds 177 Radio-HPLC signal plot of the radio-purity of Lu-DFaPSMA.

[0063] Figure 5 :Coordination compound 99m Tc-HFaPSMA and 99m In vitro cell binding assay of Tc-PSMA-617.

[0064] Figure 6 :Coordination compound 99m Tc-HFaPSMA and 99m SPECT / CT images of Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0065] Figure 7 :Coordination compound 99m Tc-HFaPSMA and 99m SPECT / CT images of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse model.

[0066] Figure 8 :Coordination compound 99m Biodistribution of Tc-HFaPSMA at metabolic time points in the 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0067] Fig. 9 :Coordination compound 99m Tc-HFaPSMA and 99m Comparative results of biodistribution of Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model, where Fig. 9 A shows 99m The biodistribution results of Tc-HFaPSMA in the 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model. Fig. 9 B shows 99m Tumor / normal organ uptake ratio of Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0068] Fig.10 :Coordination compound 99m Tc-HFaPSMA and 99m Comparative results of biodistribution of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse model, where Fig.10 A shows 99m The biodistribution results of Tc-HFaPSMA in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse model, Fig.10 B shows 99mTumor / normal organ uptake ratio of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse model.

[0069] Fig.11 :Coordination compound 68 PET / CT imaging of Ga-DFaPSMA in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0070] Fig.12 :Coordination compound 177 Lu-DFaPSMA and 177 SPECT / CT imaging of Lu-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0071] Fig.13 :Prostate cancer patients 18 F-PSMA-1007-PET and 99m Tc-HFaPSMA-SPECT imaging. DETAILED DESCRIPTION

[0072] The following will be further described in detail with reference to specific examples of the compounds of the present invention and their preparation methods and applications. It should be understood that the following examples are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection that the present invention is intended to protect.

[0073] 1. Experimental reagents and instruments

[0074] 1. Experimental reagents

[0075] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0076] 2. Experimental instruments and high performance liquid chromatography (HPLC) method

[0077] The high performance liquid chromatograph Thermo vanquish core (with array diode detector) was purchased from Thermo Fisher Scientific, USA, and the HPLC radioactivity signal detector (Gabi starγ-raytest) was purchased from Raytest, Germany. Semi-preparative C18 reverse phase chromatographic column (ReproSil-Pur Basic C18, 5 μm, 250×10.0 mm) and analytical C18 reverse phase chromatographic column (ReproSil-Pur Basic C18, 5 μm, 250×4.6 mm) were purchased from Dr. Maisch, Germany. Freeze dryer (FD-1D-50) was purchased from Beijing Boyikang Experimental Instrument Co., Ltd. Radioactive γ-counter (Wizard-2470) was purchased from PerkinElmer, USA. Radioactivity meter (CRC-25R) was purchased from Capintec, USA. Small animal PET / SPECT / CT integrated machine (Inliview-3000B) was purchased from Beijing Yongxin Medical Equipment Co., Ltd., China. The human SPECT / CT imaging system (NM / CT 870DR) was purchased from GE, USA.

[0078] Method 1 for analyzing, separating and purifying the target product by high performance liquid chromatography: Thermo vanquishcore HPLC system is equipped with an analytical or semi-preparative C18 reverse phase column, with a flow rate of 0.8 mL / min for analysis and 3.2 mL / min for semi-preparation. Gradient elution for 25 min, wherein the mobile phase A is deionized water (containing 0.05% TFA), the mobile phase B is acetonitrile (containing 0.05% TFA), and the elution gradient is set to 90% A and 10% B at the start, 90% A and 10% B at 5 min, 20% A and 80% B at 20 min, and 90% A and 10% B at 25 min.

[0079] Method 2 for analyzing, separating and purifying the target product by high performance liquid chromatography: The equipment and flow rate are the same as those in method 1. Gradient elution was performed for 25 min, wherein the mobile phase A was deionized water (containing 0.05% TFA), the mobile phase B was acetonitrile (containing 0.05% TFA), and the elution gradient was set to 60% A and 40% B at the beginning, 60% A and 40% B at 5 min, 20% A and 80% B at 20 min, and 60% A and 40% B at 25 min.

[0080] Method 3 for analyzing, separating and purifying the target product by high performance liquid chromatography: The equipment and flow rate are the same as those in method 1. Gradient elution was performed for 25 min, wherein the mobile phase A was deionized water (containing 0.05% TFA), the mobile phase B was acetonitrile (containing 0.05% TFA), and the elution gradient was set to 80% A and 20% B at the beginning, 80% A and 20% B at 5 min, 20% A and 80% B at 20 min, and 80% A and 20% B at 25 min.

[0081] 2. Experimental Methods and Results

[0082] The abbreviations used in the embodiments of the present invention have the following respective meanings:

[0083]

[0084]

[0085] Example 1 Synthesis of compounds HFaPSMA and DFaPSMA (eg Figure 1 (shown)

[0086] Step 1: Fmoc-NH-PEG 4 -CO-N-bis(PEG 4 Preparation of -NHS) (Compound 2)

[0087] Weigh 10.0 mg Fmoc-NH-PEG 4 -N-bis(PEG 4 -NHS)(Compound 1)(9.3μmol, 1.0eq) was dissolved in 200μL DMSO. 4.3mg EDC (27.9μmol, 3.0eq) was weighed and dissolved in 86μL DMSO. 3.2mg NHS (27.9μmol, 3.0eq) was weighed and dissolved in 64μL DMSO. The three solutions were combined in a 1.5mL EP tube and shaken at 37°C for 4h. The reaction progress was detected by HPLC (method 1). After the reaction was completed, the target product was separated and purified, and the fractions corresponding to the elution peak of 14.5min in semi-preparative HPLC were collected and combined (method 2). After being freeze-dried by vacuum freeze-drying method, 8.3mg of colorless oily liquid (compound 2) was obtained. MALDI-TOF mass spectrometry confirmed that it was the expected product, MALDI-TOF-MS: m / z=1177.31[M+H] + 、1199.38[M+Na] + 、1215.34[M+K] + , the theoretical molecular weight is 1176.52.

[0088] Step 2: Fmoc-NH-PEG4 -CO-N-PEG 4 Preparation of -FaPSMA (Compound 5)

[0089] Weigh 5.0 mg Fmoc-NH-PEG 4 -CO-N-bis(PEG 4 -NHS) (4.2μmol, 1.0eq) was dissolved in 250μL DMSO. 1.9mg of compound 3 (4.6μmol, 1.1eq) was weighed and dissolved in 80μL DMSO. 2.5mg of compound 4 (3.8μmol, 0.9eq) was weighed and dissolved in 125μL DMSO. After mixing the compound 2 solution and the compound 4 solution in a 1.5mL EP tube, 4μL of DIPEA was added, and the mixture was shaken at 37°C for 4h. After the compound 3 solution was added, 2μL of DIPEA was added, and the mixture was reacted at 37°C for 4h. The reaction progress was detected by HPLC (Method 2). After the reaction was completed, the target product was separated and purified, and the fractions corresponding to the elution peak of 13.6min in the semi-preparative HPLC were collected and combined (Method 2). After being freeze-dried by vacuum freeze-drying, 1.5mg of a light yellow solid (Compound 5) was obtained. The expected product was confirmed by MALDI-TOF mass spectrometry analysis, MALDI-TOF-MS: m / z = 2012.85 [M+H] + ; 2034.87[M+Na] + ; 2050.84[M+K] + , the theoretical molecular weight is 2011.88.

[0090] Step 3: NH 2 -PEG 4 -CO-N-PEG 4 - Preparation of FaPSMA (Compound 6)

[0091] Weigh 1.5 mg Fmoc-NH-PEG 4 -CO-N-PEG 4 -FaPSMA (2.5 μmol, 1.0 eq) was dissolved in 100 μL DMSO, 25 μL piperidine was added, and the mixture was shaken at 37°C for 0.5 h. The reaction progress was detected by HPLC (method 1). After the reaction was completed, the target product was separated and purified. The fractions corresponding to the elution peak at 14.6 min in semi-preparative HPLC were collected and combined (method 3). The fractions were freeze-dried by vacuum freeze drying to obtain 1.0 mg of a white solid (compound 6). The expected product was confirmed by MALDI-TOF mass spectrometry analysis. MALDI-TOF-MS: m / z = 1790.88 [M+H] + , the theoretical molecular weight is 1789.81.

[0092] Step 4-1: HYNIC-NH 2 -PEG 4 -CO-N-PEG 4 Preparation of -FaPSMA (Compound 7, HFaPSMA)

[0093] Weigh 5.0 mg NH 2 -PEG 4 -CO-N-PEG 4 -FaPSMA (2.8 μmol, 1 eq) was dissolved in 250 μL DMSO. 1.75 mg HYNIC-NHS (4.19 μmol, 1.5 eq) was weighed and dissolved in 250 μL DMSO. The two solutions were combined in a 1.5 mL EP tube and then 4 μL DIPEA was added and shaken at 37°C for 4 h. The reaction progress was detected by HPLC (method 1). After the reaction was completed, the target product was separated and purified. The fractions corresponding to the elution peak of 16.8 min in semi-preparative HPLC were collected and combined (method 1). After being freeze-dried by vacuum freeze drying, 0.9 mg of white solid product (compound 7, HFaPSMA) was obtained. MALDI-TOF mass spectrometry confirmed that it was the expected product. MALDI-TOF-MS: m / z=2093.77[M+H] + , the theoretical molecular weight is 2092.85.

[0094] Step 4-2: DOTA-NH 2 -PEG 4 -CO-N-PEG 4 Preparation of -FaPSMA (Compound 8, DFaPSMA)

[0095] Weigh 2.7 mg NH 2 -PEG 4 -CO-N-PEG 4 -FaPSMA (1.5 μmol, 1.0 eq) was dissolved in 135 μL DMSO. 1.2 mg DOTA-NHS (2.4 μmol, 1.5 eq) was weighed and dissolved in 60 μL DMSO. The two solutions were combined in a 1.5 mL EP tube and then 2 μL DIPEA was added and shaken at 37°C for 4 h. The reaction progress was detected by HPLC (method 1). After the reaction was completed, the target product was separated and purified. The fractions corresponding to the elution peak at 16.0 min in semi-preparative HPLC were collected and combined (method 1). After being freeze-dried by vacuum freeze drying, 1.4 mg of white solid product (compound 8, DFaPSMA) was obtained. MALDI-TOF mass spectrometry confirmed that it was the expected product. MALDI-TOF-MS: m / z=2176.86[M+H] +、2198.82[M+Na] + 、2214.80[M+K] + , the theoretical molecular weight is 2175.99.

[0096] Example 2 Preparation of HFaPSMA Lyophilization Kit

[0097] Weigh 1 mg HFaPSMA and dissolve it in 1 mL pH 5.5 succinic acid buffer solution at a concentration of 1 μg / μL; then prepare 1 mL of a mixed solution containing 180 μg (180 μL) HFaPSMA, 6.5 mg Tricine, 5.0 mg TPPTS, 29.55 mg succinic acid and 17.0 mg sodium hydroxide, filter it through a 0.22 μm filter membrane, and add it to a 10 mL sterile vial. After freeze-drying the mixed solution, seal and cap it to obtain a vial that can be used for 99m Tc-labeled HFaPSMA lyophilized kit.

[0098] Example 3 99m Preparation and quality control analysis of Tc-HFaPSMA

[0099] Take 20-30mCi (740-1110MBq) of high technetium [ 99m Take one HFaPSMA freeze-dried drug box for injection, check that it is not damaged and is within the shelf life. Under aseptic operation conditions, use a sterile syringe to extract the high-technetium [ 99m Tc] sodium phosphate injection 20-30mCi (740-1110MBq), add normal saline injection to 1mL; inject it into the HFaPSMA freeze-dried medicine box bottle, shake it thoroughly to make the solid in the medicine box bottle completely dissolved until clear; place the bottle in a 100℃ water bath and heat for 25min; take it out and cool it to room temperature to obtain 99m Tc-HFaPSMA injection.

[0100] 99m After Tc-HFaPSMA labeling is completed, a sterile syringe is used to take a drop of a spot on the rapid silica gel thin layer paper chromatography (ITLC-SG) for quality control analysis: the rapid thin layer chromatography paper is used as the support, and after the sample is spotted, two development systems are used for upward development, namely acetone, acetonitrile and saline mixed system (V acetonitrile: V saline = 1:1), and the chromatography paper strip is dried and then used to collect data using the Bioscan radioactive thin layer scanner. After the data collection is completed, the image, each radioactive percentage and Rf value are obtained to calculate the radiochemical purity of the drug. The main radioactive substance contained in the labeling solution is technetium marker ( 99m Tc-HFaPSMA), free technetium ( 99m T8h 4 -) and colloidal technetium ( 99m T8h 2 ). The Rf values ​​of each radioactive component obtained in the two development systems are shown in Table 1.

[0101] Table 1 99m R of each radioactive component in Tc-HFaPSMA labeling system f value

[0102]

[0103] 99m The quality control analysis of Tc-HFaPSMA can also be completed by Radio-HPLC. Method 1 is used to detect the components of the drug kit. 99m The retention time of Tc-HFaPSMA was 17.5 min, and the radiolabeling rate was 97.34% (e.g. Figure 2 shown).

[0104] Example 4 68 Preparation and quality control analysis of Ga-DFaPSMA

[0105] Weigh 1 mg of DFaPSMA and dissolve it in 1 mL of saline to prepare a solution with a concentration of 1 μg / μL for later use; rinse the Sep-PakC18 column with 5 mL of anhydrous ethanol and 10 mL of water for injection, and then blow dry with 10 mL of air to complete the activation process before use. Take 20 μL of DFaPSMA solution (containing 20 μg of DFaPSMA) in a 1.5 mL EP tube, and add 80 μL of 1 M sodium acetate, 1.0 mL 68 GaCl 3 The eluent (5-15 mCi, 0.05 M hydrochloric acid medium) was used, and the pH of the solution was about 4.2. After mixing, it was placed in an incubator at 90°C for 15 minutes. The labeling rate of Radio-iTLC analysis was 92.2% (e.g. Figure 3 A). After the reaction is completed, the reaction solution is extracted with a 5 mL syringe that has been pre-drawn with 4 mL of injection water, and the sample is loaded onto the activated C18 column. Then, the C18 column is rinsed with 10 mL of injection water and the waste liquid is discarded. Finally, 0.5 mL of 80% ethanol is used to rinse the C18 column to collect the marker in a sterile vacuum vial, and the marker is diluted with 2 mL of saline and filtered through a 0.22 μm microporous filter membrane and stored in a sterile vacuum bottle for later use. Use Radio-iTLC to analyze the marker 68 The radiochemical purity of Ga-DFaPSMA is greater than 99% (e.g. Figure 3 B).

[0106] Example 5 177 Preparation and quality control analysis of Lu-DFaPSMA

[0107] Weigh 1 mg of DFaPSMA and dissolve it in 1 mL of pure water to prepare a solution with a concentration of 1 μg / μL for later use; rinse the Sep-Pak C18 column with 5 mL of anhydrous ethanol and 10 mL of water for injection, and then blow dry with 10 mL of air to complete the activation process before use. Take 20 μL of DFaPSMA solution (containing 20 μg of DFaPSMA) in a 1.5 mL EP tube, and add 60 μL of 1 M sodium acetate buffer (pH 4.0), 0.1 mL 177 LuCl 3 Solution (10-50mCi, 0.01M hydrochloric acid medium). Place the reaction mixture in an incubator at 95°C for 20 minutes. After the reaction is completed, use a 5mL syringe that has been pre-drawn with 4mL of water for injection to extract the reaction solution, load it onto the activated C18 column, then rinse the C18 column with 10mL of water for injection and discard the waste liquid. Finally, use 0.5mL of 80% ethanol to rinse the C18 column and collect the marker in a sterile vacuum vial. Dilute the marker with 2mL of normal saline and filter it through a 0.22μm microporous filter membrane and store it in a sterile vacuum bottle for later use. Measure the radioactivity of the final product and calculate the yield to be approximately 85%. Use Radio-HPLC to analyze the marker 177 The radiochemical purity of Lu-DFaPSMA is greater than 95% (e.g. Figure 4 shown).

[0108] Example 6 Biological Experiment

[0109] Experimental cells and animals

[0110] SPF male BALB / c nude mice, 4-5 weeks old; SPF male Kunming mice, 6 weeks old; all purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 22Rv1 human prostate cancer cells (PSMA positive) were cultured in 1640 cell culture medium containing 10% FBS; PC-3 human prostate cancer cells (PSMA negative) were cultured in F12 cell culture medium containing 10% FBS; F3 mouse embryonic fibroblasts (NIH-3T3 overexpression cell line stably transfected with mFAP) and NIH-3T3 mouse embryonic fibroblasts (FAP negative) were cultured in DMEM cell culture medium containing 10% FBS. The cells were routinely subcultured in an incubator at 37°C with 5% CO2. 4-5 week old BALB / c nude mice were taken and 1×10 6 22Rv1 cells or 1×10 6 PC-3 cells and 1×10 6 A mixture of F3 cells was grown under SPF conditions. When the tumor volume reached 100-300 mm 3Through the cell hybridization method, we established PSMA-positive and FAP-positive tumor animal models (22Rv1 / F3(PSMA+ / FAP+)) and PSMA-negative and FAP-positive tumor animal models (PC-3 / F3(PSMA- / FAP+)) for later biological experimental studies.

[0111] 1. 99m Tc-HFaPSMA in vitro cell binding assay

[0112] To validate dual-targeting small molecules 99m To investigate the specific binding ability of Tc-HFaPSMA, we prepared and used 99m Tc-HFaPSMA and 99m Tc-PSMA-617 was used for in vitro cell binding assay.

[0113] In the in vitro cell binding assay, 22Rv1 human prostate cancer cells (PSMA positive) and F3 mouse embryonic fibroblasts (FAP positive) were used as positive control cells, and PC-3 human prostate cancer cells (PSMA negative) and NIH-3T3 mouse embryonic fibroblasts (FAP negative) were used as negative control cells.

[0114] 22Rv1 and PC-3 cells were digested with 0.25% trypsin, and F3 and NIH-3T3 cells were digested with 0.05% trypsin. The cells were collected into centrifuge tubes and centrifuged at 1000 rpm for 3 min. The supernatant was discarded. 10 mL of PBS was added to resuspend and wash the cells. The cells were resuspended in PBS and counted separately using a cell counting plate and diluted with PBS to 1 × 10 cells per ml. 7 The above cell suspension was added at 1×10 6 Add 100 cells to the filter plate blocked with 5% BSA overnight. 99m Tc-HFaPSMA and 99m Tc-PSMA-617 was added to the wells containing cells at 14.8 kBq / well and incubated on ice for 30 minutes. For the study of blocking the interaction between the probe and cells, a 1000-fold molar amount of unlabeled ligand was added to the labeled radioactive probe for co-incubation. Pre-cooled ice-cold PBST (PBS containing 0.05% Tween-20) was added for 5 washes, and the radioactivity of the wells on the filter plate was collected in a γ counter. Four parallel samples were set up for all cell binding experimental groups. The binding value of cells and probes is expressed as the percentage of radioactive counts per minute to the added radioactive dose (%AD). This value has been calculated after decay correction. The experimental results are shown in Figure 5 As shown,99m The %AD value of Tc-HFaPSMA binding to 22Rv1 human prostate cancer cells was 2.94% (±0.2%); the %AD value of Tc-HFaPSMA binding to F3 mouse embryonic fibroblasts was 6.14% (±0.6%), while 99m The %AD value of Tc-PSMA-617 binding to F3 mouse embryonic fibroblasts was only 0.5% (±0.02%). 99m Tc-HFaPSMA and 99m The binding value of Tc-PSMA-617 was comparable and significantly lower than that of the positive control group. The experimental results show that 99m Tc-HFaPSMA showed 99m Tc-PSMA-617 has comparable affinity to PSMA, but 99m Tc-HFaPSMA also has the ability to specifically bind to expressed FAP-α.

[0115] 2. 99m Tc-HFaPSMA and 99m Comparison of SPECT / CT imaging of Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model

[0116] The prepared 99m Tc-HFaPSMA and 99m Tc-PSMA-617 was prepared into 37MBq / 200μL with normal saline, and 200μL (37MBq) was injected into each 22Rv1 / F3 tumor-bearing mouse via the tail vein. SPECT / CT imaging was performed 1 hour after injection. 99m The mice in the blocking group were injected with Tc-HFaPSMA. 99m 300μg FAPI or 500μg PSMA or 300μg FAPI + 500μg PSMA were injected at the same time as Tc-HFaPSMA, and imaging was performed 1 hour after administration. Mice were anesthetized with 1.5% isoflurane-oxygen during imaging. After imaging, the SPECT images were reconstructed and fused with the CT images to obtain a 3D image (posterior view). The imaging results are shown in Figure 6 As shown, the tumor location is marked with an arrow. 99m Compared with Tc-PSMA-617, 99m The tumor uptake of Tc-HFaPSMA was basically the same as that of HFaPSMA, and the two had similar imaging contrast. 99m The tumor uptake of Tc-HFaPSMA was reduced, but there was still obvious tumor imaging; after blocking both PSMA and FAP,99m The tumor uptake of Tc-HFaPSMA was significantly reduced, no tumor imaging was observed, and the kidney uptake was reduced, further demonstrating that 99m Tc-HFaPSMA has excellent dual-targeting function and tumor specificity in vivo.

[0117] 3. 99m Tc-HFaPSMA and 99m Comparison of SPECT / CT imaging of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse model

[0118] The prepared 99m Tc-HFaPSMA and 99m After Tc-PSMA-617 was prepared into 37MBq / 200μL with normal saline, 200μL (37MBq) was injected into each PC-3 / F3 tumor-bearing mouse via the tail vein, and SPECT / CT imaging was performed 1h after injection. The mice were anesthetized with 1.5% isoflurane-oxygen during the imaging process. After imaging, the SPECT image was reconstructed and fused with the CT image to obtain a 3D image (posterior view). The imaging results are shown in Figure 7 As shown, the tumor location is marked with an arrow. The experimental results show that 99m Tc-HFaPSMA can be effectively taken up by PSMA-negative and FAP-positive tumors. 99m The signal intensity of Tc-PSMA-617 in the tumor site is close to the background signal and cannot be imaged in the tumor site, which further proves that compared with 99m For Tc-PSMA-617, 99m Tc-HFaPSMA exhibits a wider detection range in the diagnosis of prostate cancer.

[0119] 4. 99m Study on the biodistribution of Tc-HFaPSMA at different time points in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model

[0120] Sixteen 22Rv1 / F3 tumor-bearing BALB / c nude mice (6 weeks old) were randomly divided into 4 groups, with 4 mice in each group. 99m After Tc-HFaPSMA was prepared into 370 kBq / 100 μL with normal saline, 100 μL (370 kBq) was injected into each mouse via the tail vein. The animals were killed at 0.5, 1, 2, and 4 hours after injection, and the blood and major organs were taken, weighed, and the radioactive cpm count was measured. The percentage injected dose per gram of tissue (%ID / g) was calculated after decay correction. The biodistribution results were expressed as mean ± standard deviation (mean ± SD, n = 4).

[0121] The experimental results are as follows Figure 8 As shown, 99m The uptake of Tc-HFaPSMA in the tumor site reached the highest value 1 hour after injection. 4 hours after injection, the tumor uptake was still higher than that of the first three groups, and there was no obvious decrease in tumor uptake, indicating that 99m Tc-HFaPSMA has excellent tumor retention ability, indicating that it has significant application potential in the field of tumor treatment. In addition, except for the kidney, a metabolic organ, there is no abnormal drug concentration imaging in organs such as the liver, gallbladder, stomach, and small intestine, suggesting that 99m Tc-HFaPSM has excellent in vivo stability and superb in vivo metabolic properties.

[0122] 5. Radiation dose experiment

[0123] The biodistribution experiments were performed using 99m The uptake values ​​of Tc-HFaPSMA in various organs of mice at four time periods (%ID / g) were estimated by combining OLINDA software with the animal weight of 25g and the adult male weight of 73kg. 99m We estimated the normal organ radiation absorbed dose of Tc-HFaPSMA. 99m The radiation damage of Tc-HFaPSMA to various organs (in mSv / MBq) is shown in Table 2. 99m The highest radiation dose of Tc-HFaPSMA in the kidney is 0.045mSv / MBq, and the radiation dose in major tissues and organs of the organism, such as the liver, spleen, stomach, and pancreas, is less than 0.0050mSv / MBq, which is much lower than its distribution in the kidney. The effective whole-body dose of the patient is 0.0016mSv / MBq. Assuming the standard injection dose of a 73kg adult in clinical practice (20mCi, 740MBq), the effective whole-body dose received by the patient is 1.2mSv, which is significantly lower than the small molecule PET imaging agent targeting PSMA reported in clinical studies. 68 Whole-body irradiation dose of Ga-PSMA-617 (0.021 mSv / MBq×203 MBq, 4.3 mSv, JNM·Vol.56·No.11·Nov.2015) and small molecule PET imaging agent targeting FAP 68 The whole-body irradiation dose of Ga-FAPI-46 (0.0078mSv / MBq×200MBq, 1.6mSv, JNM·Vol.61·No.8·Aug.2020). At the same time, it is also significantly lower than the currently commonly used PET imaging agents. 18 5.1±0.73mSv of F-FDG and SPECT imaging agent 99mThe radioactive dose of Tc-MDP is 4.7±0.97mSv, which is lower than the radioactive dose of 7.4mSv in a single abdominal and pelvic enhanced CT scan. 99m The radiation safety of Tc-HFaPSMA is guaranteed to a certain extent.

[0124] Table 2. 99m Absorbed dose of Tc-HFaPSMA in various tissues (mSv / MBq)

[0125]

[0126] 6. 99m Tc-HFaPSMA and 99m Comparative study of biodistribution of Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model

[0127] Eight 22Rv1 / F3 tumor-bearing BALB / c nude mice (6 weeks old) were randomly divided into two groups, with 4 mice in each group. 99m Tc-HFaPSMA and 99m Biodistribution experiment of Tc-PSMA-617. 99m Tc-HFaPSMA or 99m After Tc-PSMA-617 was prepared into 370 kBq / 100 μL with normal saline, 100 μL (370 kBq) was injected into each mouse via the tail vein. The animals were killed 1 hour after injection, and blood and major organs were collected, weighed, and radioactive cpm counts were measured. The percentage injected dose per gram of tissue (%ID / g) was calculated after decay correction. The biodistribution results were expressed as mean ± standard deviation (mean ± SD, n = 4).

[0128] The experimental results are as follows Fig. 9 As shown, compared to 99m For Tc-PSMA-617, 99m The tumor uptake of Tc-HFaPSMA was improved. 99m Tc-HFaPSMA uptake was 99m Tc-PSMA-617 increased the spleen's 99m Tc-HFaPSMA uptake was 99m Tc-PSMA-617 was reduced by 75%, and the gallbladder and submandibular gland were both reduced. Moreover, for SPECT imaging, the uptake of close normal tissues and organs increased the tumor / normal organ uptake ratio, which is conducive to the diagnosis of tiny tumor lesions.

[0129] 7. 99m Tc-HFaPSMA and 99mComparative study of biodistribution of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse model

[0130] Eight PC-3 / F3 tumor-bearing BALB / c nude mice (6 weeks old) were randomly divided into two groups, with 4 mice in each group. 99m Tc-HFaPSMA and 99m Biodistribution experiment of Tc-PSMA-617. 99m Tc-HFaPSMA or 99m After Tc-PSMA-617 was prepared into 370 kBq / 100 μL with normal saline, 100 μL (370 kBq) was injected into each mouse via the tail vein. The animals were killed 1 hour after injection, and blood and major organs were collected, weighed, and radioactive cpm counts were measured. The percentage injected dose per gram of tissue (%ID / g) was calculated after decay correction. The biodistribution results were expressed as mean ± standard deviation (mean ± SD, n = 4).

[0131] The experimental results are as follows Fig.10 As shown, 99m Tc-HFaPSMA uptake in tumors 99m Tc-PSMA-617 significantly increased by 834.22% (17.47% ID / g vs 1.87% ID / g) and showed a higher tumor / normal organ uptake ratio, suggesting 99m Tc-HFaPSMA may play a better role in the diagnosis of PSMA-negative tumors, helping patients to discover as many tumor lesions as possible.

[0132] 8. 68 Ga-DFaPSMA and 68 PET / CT imaging of Ga-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0133] The prepared 68 Ga-DFaPSMA and 68 After Ga-PSMA-617 was prepared into 18.5MBq / 100μL with normal saline, each 22Rv1 / F3 tumor-bearing mouse was injected with 100μL (18.5MBq) via the tail vein. Micro-PET / CT imaging was performed 0.5h after injection. The mice were anesthetized with 1.5% isoflurane-oxygen during the imaging process. After imaging, the PET image was reconstructed and fused with the CT image to obtain a 3D image (whole body MIP image). The imaging results are shown in Fig.11 As shown, the tumor location is marked with an arrow. The experimental results show that in the PET imaging of tumor-bearing mouse models, the present invention obtains 68Ga-DFaPSMA is based on PSMA monomer form. 68 Compared with Ga-PSMA-617, tumor uptake was improved, and both had good imaging contrast.

[0134] 9. 177 Lu-DFaPSMA and 177 SPECT / CT imaging of Lu-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0135] The prepared 177 Lu-DFaPSMA and 177 Lu-PSMA-617 was prepared into 37MBq / 200μL with saline, and each 22Rv1 / F3 tumor-bearing mouse was injected with 200μL (37MBq) via the tail vein. SPECT / CT imaging was performed at 4, 24, 48, 72, 96, 120, and 144h after injection. The mice were anesthetized with 1.5% isoflurane-oxygen during the imaging process. After imaging, the SPECT image was reconstructed and fused with the CT image to obtain a 3D image (posterior view). The imaging results are shown in Figure 2. Fig.12 As shown, the tumor location is marked with an arrow. The experimental results show that the two sets of images at the same time point are different from 177 Lu-PSMA-617, 177 Lu-DFaPSMA has higher tumor uptake. In addition, 177 Lu-DFaPSMA still had clear tumor images 144 hours after injection. 177 Lu-PSMA-617 could not obtain clear tumor imaging 96 hours after injection. Higher tumor uptake and longer tumor retention time can be effectively used in radio-targeted therapy of prostate cancer.

[0136] Example 7 Prostate cancer patients 18 F-PSMA-1007-PET and 99m Tc-HFaPSMA-SPECT imaging

[0137] A 63-year-old male patient was found to have a significantly elevated PSA level in his blood after radical prostatectomy. 18 F-PSMA-1007-PET Nuclear Medicine Imaging, 18 F-PSMA-1007 is a PSMA-targeted positron emission tomography (PET) tracer that 68 Compared with Ga-labeled PSMA-targeted tracers, 18F-PSMA-1007 has a higher labeling yield, longer half-life and better energy characteristics, making it a widely used PSMA PET imaging agent in clinical practice (Giesel FL, et al. F-18 labeled PSMA-1007: biodistribution, radiation dosimetry and histopathological validation of tumor lesions in prostate cancer patients. Eur J Nucl Med Mol Imaging. 2017 Apr; 44 (4): 678-688.). 99m Tc-HFaPSMA-SPECT imaging, the imaging results are as follows Fig.13 shown. 18 The PET results of F-PSMA-1007 showed obvious accumulation of radioactivity in the liver, kidneys and gastrointestinal tract; mild uptake in the salivary glands. A small amount of radioactivity accumulated in the bladder. The background in the heart, lungs, brain and abdomen was low. No obvious increased radionuclide uptake was found in the prostate surgery area. Increased radionuclide uptake was seen in the skull, right clavicle, bilateral scapulae, left humerus, multiple vertebrae and accessories of the cervical, thoracic, lumbar and sacral spine, multiple ribs, sternum, bilateral ischia, pubis, right ilium, left femoral shaft, and right femoral head. The SUVmax was 5.1-43.3, and bone destruction was seen in some CT scans of the same machine. The present invention 99m Tc-HFaPSMA SPECT results showed that radioactivity was significantly accumulated in the kidneys and bladder. A small amount of radioactivity was distributed in the liver and gastrointestinal tract. Mild uptake was observed in the salivary glands. The background in the heart, lungs, brain, and abdomen was low. Abnormal radionuclide concentration was observed in the skull, right clavicle, bilateral scapulae, left humerus, multiple vertebrae and accessories of the cervical, thoracic, lumbar, and sacral spine, multiple ribs on both sides, sternum, bilateral ischia, pubis, right ilium, left femoral shaft, and right femoral head, with SUVmax of 4.11-45.77. Obvious radionuclide uptake was observed in the enlarged lymph nodes in the right supraclavicular fossa, with SUVmax = 16.64. 99m The ability of Tc-HFaPSMA-SPECT to detect primary and metastatic prostate cancer in this case is comparable to that of 18 F-PSMA-1007-PET was comparable to F-PSMA-1007-PET, while showing lower liver and gastrointestinal uptake.

[0138] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound having a structure represented by formula (I), or a pharmaceutically acceptable salt or stereoisomer thereof; in, L1 is a functionalized linker -CO-C 1-6 Alkylene (-X1-C 1-6 Alkylene) m -; L2 is a functionalized linker -CO-C 1-6 Alkylene-(X2-C 1-6 Alkylene-) n -NH2; X1 and X2 are each independently selected from O, S, and NH; 1-6 The alkylene group is optionally substituted with halogen, OH, NH2, oxo, cyano; m and n are integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment of the present invention, X1 and X2 are independently selected from O. In one embodiment of the present invention, the C 1-6 The alkylene group is selected from -C2H4-, -C3H6-. In one embodiment of the present invention, said n, m are selected from integers of 3-6. In one embodiment of the present invention, L1 is a functionalized linker -CO-CH2CH2-(O-CH2CH2-)4-. In one embodiment of the present invention, L2 is a functionalized linker -CO-CH2CH2-(O-CH2CH2-)4-NH2.

2. A compound having a structure represented by formula (II), or a pharmaceutically acceptable salt or stereoisomer thereof; in, L1 is a functionalized linker -CO-C 1-6 Alkylene-(X1-C 1-6 Alkylene) m -; L2 is a functionalized linker -CO-C 1-6 Alkylene-(X2-C 1-6 Alkylene-) n -NH-; X1 and X2 are each independently selected from O, S, and NH; 1-6 The alkylene group is optionally substituted with halogen, OH, NH2, oxo, cyano; m and n are integers selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In one embodiment of the present invention, X1 and X2 are independently selected from O. In one embodiment of the present invention, the C 1-6 The alkylene group is selected from -C2H4-, -C3H6-. In one embodiment of the present invention, said n, m are selected from integers of 3-6. In one embodiment of the present invention, L1 is a functionalized linker -CO-CH2CH2-(O-CH2CH2-)4-. In one embodiment of the present invention, L2 is a functionalized linker -CO-CH2CH2-(O-CH2CH2-)4-NH-.

3. According to the compound of formula (II) according to claim 2, the bifunctional chelating agent is selected from the following groups:

4. The compound of formula (II) according to claim 2, which is selected from: or a pharmaceutically acceptable salt or stereoisomer thereof.

5. A coordination compound obtained by labeling the compound of formula (I) according to claim 1 with a radionuclide and a bifunctional chelating agent, wherein the bifunctional chelating agent reacts and bonds with the amino group at the end of L2 in the compound of formula (I) through its carboxyl group; or, obtained by labeling the compound of formula (II) according to claim 2 with a radionuclide.

6. The coordination compound according to claim 5, wherein the radioactive nuclide is selected from an isotope emitting α rays, an isotope emitting β rays, an isotope emitting γ rays, an isotope emitting Auger electrons, or an isotope emitting X rays. Preferably, the radionuclide is selected from: 18 F. 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 88 Y. 90 Y. 149 Pm, 177 Lu, 47 Sc, 212 Bi, 213 Bi, 72 As, 211 At 201 Tl, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 223 Ra or 89 Sr. More preferably, the radionuclide is selected from 68 Ga, 99m Tc, or 177 Lu.

7. The coordination compound according to claim 5, which is selected from: or or a pharmaceutically acceptable salt or stereoisomer thereof.

8. A pharmaceutical composition comprising the coordination compound according to any one of claims 5 to 7 and a pharmaceutically acceptable carrier; preferably, the pharmaceutical composition is an intravenous injection drug.

9. A kit comprising the compound of formula (II) according to claim 2, an optional synergistic ligand, and a pharmaceutically acceptable carrier, excipient, stabilizer and / or diluent. Preferably, for 99m The kit of Tc-labeled ligand compound comprises the ligand compound HFaPSMA as claimed in claim 4, as well as tris(hydroxymethyl)methylglycine (Tricine), triphenylphosphine tris(sulfonic acid) sodium salt (TPPTS), and optionally one or more pharmaceutically acceptable carriers, excipients, stabilizers and / or diluents. Preferably, the content of HFaPSMA is 10 μg-1000 μg, the content of Tricine is 1 mg-50 mg, and the content of TPPTS is 3 mg-30 mg. Preferably, the kit can be formed by freeze-drying HFaPSMA, tris(hydroxymethyl)methylglycine (Tricine), triphenylphosphine tris(sulfonic acid) sodium salt (TPPTS) and a pH buffer solution required for radionuclide labeling. The pH buffer solution is selected from a buffer system such as succinic acid, citric acid, tartaric acid, phosphoric acid, boric acid, acetic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (i.e., HEPES). Preferably, HFaPSMA, tris(hydroxymethyl)methylglycine (Tricine), and triphenylphosphine tris(sulfonic acid) sodium salt (TPPTS) are placed in a succinic acid / sodium hydroxide solution, filtered, and then freeze-dried to obtain a kit. Preferably, for 68 Ga, 177 The Lu-labeled ligand compound kit comprises the ligand compound DFaPSMA according to claim 4, and one or more stabilizers and pharmaceutically acceptable carriers. Preferably, the kit is obtained by preparing a solution.

10. Use of the compound of formula (I) according to claim 1, the compound of formula (II) according to any one of claims 2-4, the coordination compound according to any one of claims 5-7, the pharmaceutical composition according to claim 8, or the kit according to claim 9 for preparing a drug for diagnosing or treating prostate cancer lesions with different PSMA expression levels in patients.

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