Prostate cancer diagnostic or therapeutic radiopharmaceuticals targeting both psma and fap-alpha

Abstract

A novel compound targeting PSMA and FAP-alpha can be coupled with 99m Tc、 68 Ga、 177 Lu and other radionuclides through corresponding chelating agents to prepare radiopharmaceuticals for the diagnosis or treatment of prostate cancer. The radiopharmaceuticals show better tumor uptake and retention in tumors with different PSMA expression levels, and can be applied to the diagnosis or treatment of most prostate cancer patients, and have good therapeutic clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to a series of radionuclide-labeled drug complexes targeting PSMA and FAP-alpha, precursor compounds for preparing the drug complexes and pharmaceutical kits thereof, and a preparation method thereof, in particular to a radiopharmaceutical for diagnosing or treating different PSMA expression level prostate cancer individuals. BACKGROUND

[0002] Prostate cancer, as a high incidence of epithelial malignancies in men, its high mortality rate seriously threatens the life and health of men. Since 2008, the incidence of prostate cancer in China has ranked first and continues to rise. Currently, the diagnosis of prostate cancer relies on the comprehensive evaluation of prostate specific antigen detection, digital rectal examination and biopsy. Under this background, 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 early detection and precise treatment of prostate cancer.

[0003] With the continuous deepening of the study of prostate cancer, a specific biomarker, prostate specific membrane antigen (Prostate Specific Membrane Antigen, PSMA), expressed on the surface of prostate cancer cells was found. PSMA is a transmembrane glycoprotein composed of extracellular C-terminal, transmembrane structure and cytoplasmic N-terminal, of which the extracellular region accounts for 95% of the entire glycoprotein, which is the main site for recognition by antibodies, polypeptides and small molecule compounds, and the cytoplasmic domain contains a motif that can trigger the internalization of receptor-ligand complexes, allowing the effective deposition of drugs into cells. PSMA is selectively overexpressed on the surface of more than 90% of prostate cancer cells, which is 100-1000 times the expression amount of normal prostate epithelial tissue, and can increase with the progression of malignant tumors, making PSMA an attractive target for the study of prostate cancer diagnosis and treatment.

[0004] Currently, 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 attracted more and more attention and selection due to their excellent in vivo metabolic speed. Based on the structural characteristics of the extracellular region of PSMA protein, small molecule inhibitors mainly include three types: 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 most widely used in the diagnosis and treatment of prostate cancer. In 2014, it was first reported that 68Ga-PSMA-617 has been applied to the 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 show no uptake or very low uptake at the lesion site after injection of Ga-PSMA-617, indicating low PSMA expression. In addition, if the primary tumor lesion of a patient has low PSMA expression, it often leads to 68 Ga-PSMA-617. Moreover, for patients with PSMA-positive primary tumor lesions, although their metastatic lesions are usually positive, in some advanced metastatic castration-resistant prostate cancer (CRPC) patients who have experienced multiple chemotherapy failures, some of their metastatic lesions may be PSMA-negative. 68 Ga-PSMA-617. Moreover, for patients with PSMA-positive primary tumor lesions, although their metastatic lesions are usually positive, in some advanced metastatic castration-resistant prostate cancer (CRPC) patients who have experienced multiple chemotherapy failures, some of their metastatic lesions may be PSMA-negative.

[0005] In recent years, in order to solve the diagnostic range and detection sensitivity of PSMA-targeting imaging agents, a series of tumor diagnostic imaging agents supplemented with or based on direct modification of PSMA have been developed. Yang Xing et al. disclosed in CN114989261A a radionuclide 18 F-labeled coordination compound 18 F-AIF-ETN 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 64 Cu-labeled PET imaging diagnostic agent. Yang Zhi et al. disclosed in CN116082306A a 68 Ga-labeled dual-targeting PSMA and FAP-α tumor diagnostic PET imaging agent, which can achieve imaging of PSMA-negative tumors, but the 68 Ga-labeled tumor diagnostic PET imaging agent has an asymmetric molecular structure and a complicated synthesis process, and generally has poor tumor retention ability.

[0006] Although diverse modifications and attempts have been made by research teams worldwide in the field of targeting PSMA tumor imaging agents, these efforts are often constrained by the lagging production efficiency and suboptimal imaging results, which are manifested in complex preparation processes, high costs, and problems such as rapid metabolism in vivo, no or low uptake of tumor tissues, and short retention time. In view of this, the development of a diagnostic imaging agent that not only has a simplified production process and excellent metabolic properties, but also 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 a new imaging agent is not only expected to improve the accuracy and sensitivity of tumor diagnosis in patients, but also will provide strong technical support for early detection, precise treatment and efficacy evaluation of tumors. SUMMARY

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

[0008] To achieve the above-mentioned purpose, the present application provides a compound having the structure shown in formula (I),

[0009]

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

[0011] L1 is a functional linker -CO-C 1-6 alkylene (-X1-C 1-6 alkylene) m -;

[0012] L2 is a functional linker -CO-C 1-6 alkylene-(X2-C 1-6 alkylene-) n -NH2;

[0013] X1 and X2 are each independently selected from O, S, NH; the C 1-6 alkylene is optionally substituted with halogen, OH, NH2, oxo, cyano;

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

[0015] In an embodiment of the present application, X1 and X2 are each selected from O.

[0016] In an embodiment of the present application, the C 1-6 alkylene is selected from -C2H4-, -C3H6-.

[0017] In an embodiment of the present application, n, m are integers selected from 3-6.

[0018] In an embodiment of the present application, L1 is a functionalized linker -CO-CH2CH2- (O-CH2CH2-)4-.

[0019] In an embodiment of the present application, L2 is a functionalized linker -CO-CH2CH2- (O-CH2CH2-)4-NH2.

[0020] The present application also provides a compound of the structure shown in formula (II):

[0021]

[0022] or a pharmaceutically acceptable salt, stereoisomer thereof.

[0023] wherein, L1 is a functionalized linker -CO-C 1-6 alkylene-(X1-C 1-6 alkylene) m -;

[0024] L2 is a functionalized linker -CO-C 1-6 alkylene-(X2-C 1-6 alkylene-) n -NH-;

[0025] X1, X2 are each independently selected from O, S, NH; the C 1-6 alkylene is optionally substituted with halogen, OH, NH2, oxo, cyano;

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

[0027] In an embodiment of the present application, X1, X2 are each selected from O.

[0028] In an embodiment of the present application, the C 1-6 alkylene is selected from -C2H4-, -C3H6-.

[0029] In an embodiment of the present application, n, m are integers selected from 3-6.

[0030] In an embodiment of the present application, L1 is a functionalized linker -CO-CH2CH2- (O-CH2CH2-)4-.

[0031] In an embodiment of the present application, L2 is a functionalized linker -CO-CH2CH2- (O-CH2CH2-)4-NH-.

[0032] In particular, the bifunctional chelator in the compound of formula (II) of the present application is selected from the following groups:

[0033]

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

[0035]

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

[0037] The present application also provides a coordination compound obtained by labeling the compound of formula (I) above with a radionuclide and a bifunctional chelator; wherein the bifunctional chelator is linked to the amino group at the end of L2 in the compound of formula (I) via its carboxyl group.

[0038] In the present application, the bifunctional chelator is selected from the following compounds: 6-hydrazinopyridine-3-carboxylic acid (HYNIC), mercaptoacetic acid diglycine (MAG2), mercaptoacetic acid triglycine (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]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), RESCA, 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NOTA-GA), 1,4,7,10-tetraazacyclododecane-1-glutaric 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-isothiocyanatophenyl)-3-[6,17-dihydroxy-7,10,18,21-tetraoxo-27-(N-acetyloxylamino)-6,11,17,22-tetraazahexacosyl](DFO), S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(2-carbamoylmethyl) (TCMA).

[0039] The present application also provides a coordination compound obtained by labeling the compound of formula (II) above with a radionuclide.

[0040] In one embodiment, the radionuclide in the complexing compound is selected from the group consisting of an isotope emitting alpha rays, an isotope emitting beta rays, an isotope emitting gamma rays, an isotope emitting Auger electrons, or an isotope emitting X-rays, and the like.

[0041] In a preferred embodiment, the radionuclide in the complexing compound is selected from the group consisting of: 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, and the like.

[0042] In a more preferred embodiment, the radionuclide in the complexing compound is selected from the group consisting of: 68 Ga, 99m Tc, or 177 Lu, and the like.

[0043] In one embodiment, the complexing compound of the present application is selected from the group consisting of:

[0044]

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

[0046] The present application also provides a pharmaceutical composition comprising the above-mentioned complexing compound and a pharmaceutically acceptable carrier.

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

[0048] The present application also provides a kit comprising a compound of formula (II) as described hereinbefore, optionally a 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] In particular, the kit for the labeling of the ligand compound with 99m The kit for the labeling of the ligand compound with Tc comprises the ligand compound HFaPSMA, tricine, triphenylphosphine tris-benzenesulfonic acid sodium salt (TPPTS), and one or more pharmaceutically acceptable carriers, excipients, stabilizers and / or diluents. Preferably, the kit is obtained by preparing a solution comprising the substances (i.e. the ligand compound HFaPSMA, tricine, triphenylphosphine tris-benzenesulfonic acid sodium salt (TPPTS)) and a pH buffer solution required for the radionuclide labeling. 99m The pH buffer solution is selected from the group consisting of 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 like buffer systems, which can be a buffered pair of the acid and its salt, a buffered pair of the acid and a base, or a buffered pair of two salts of the acid. The pH buffer solution is used for 99m The pH of the kit for the labeling of the ligand compound with Tc 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, the HFaPSMA, tricine, triphenylphosphine tris-benzenesulfonic acid sodium salt (TPPTS) are placed in a solution of succinic acid / sodium hydroxide, filtered and lyophilized to obtain the kit. In the kit, the HFaPSMA is present in an amount of 10 μg-1000 μg, the tricine is present in an amount of 1 mg-50 mg, and the TPPTS is present in an amount of 3 mg-30 mg. Preferably, the amounts of the three are in the range of 100-200 ug: 4-10 mg: 3-10 mg.

[0050] In particular, the kit for the labeling of the ligand compound with 68 Ga, 177 The kit for the labeling of the ligand compound with Lu comprises the ligand compound DFaPSMA and one or more stabilizers and pharmaceutically acceptable carriers. Preferably, the kit is obtained by preparing a solution.

[0051] The present application also provides the use of a compound of formula (I), a compound of formula (II), a complex, a pharmaceutical composition or a kit as described hereinbefore for the manufacture of a medicament for the diagnosis or treatment of prostate cancer lesions with different PSMA expression levels in a patient.

[0052] Beneficial effects

[0053] The compounds of this invention have a nitrogen atom as the connecting center, a symmetrical molecular skeleton, and a simple and efficient synthetic route, which greatly reduces production costs. Furthermore, this invention uses a one-step method to prepare the complexes, which is convenient to operate, has a high labeling rate, and exhibits better drug-like properties, making it suitable for industrial production and clinical application.

[0054] This invention prepares precursor compounds (I) and (II) of dual-targeted PSMA and FAP-α prostate cancer diagnostic or therapeutic radiopharmaceuticals. After structural modification, the uptake of the drug in the blood is reduced, which is beneficial for obtaining a lower background signal in imaging.

[0055] The dual-targeted PSMA and FAP-α radiopharmaceuticals for the diagnosis or treatment of prostate cancer of this invention are superior to those in the prior art. 99m Tc-P137, 68 Ga-PSMA-617 or 177 Lu-PSMA-617 exhibits higher tumor uptake and tumor / tissue uptake ratio, as well as better tumor retention in PSMA-positive sites, while also demonstrating excellent tumor imaging capabilities in PSMA-negative sites due to the presence of FAP-α. This is beneficial for the diagnosis of systemic lesions in prostate cancer patients, further increasing the proportion of patients who benefit, and shows promising prospects for tumor diagnosis and radiotargeted therapy.

[0056] Compared with existing technologies, the dual-targeted PSMA and FAP-α radiopharmaceuticals for the diagnosis or treatment of prostate cancer of the present invention have a lower systemic effective dose to the patient. In one embodiment of the present invention, the systemic effective dose is only 0.0016 mSv / MBq. Calculated based on a standard injection dose of 73 kg (20 mCi, 740 MBq), the systemic effective dose received by the patient is 1.2 mSv. Therefore, the radiation damage caused by imaging with the drug of the present invention is significantly less, and the safety effect is better.

[0057] Compared to existing technologies, the dual-targeted PSMA and FAP-α radiopharmaceuticals for the diagnosis or treatment of prostate cancer of this invention have better hydrophilicity and higher renal uptake. Therefore, drug metabolism can be promoted by drinking plenty of water and urinating before imaging, thereby minimizing the appearance of the drug 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. Attached image description:

[0059] Figure 1 Synthetic routes for ligand compounds HFaPSMA and DFaPSMA.

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

[0061] Figure 3 : Coordination compound 68 Quality control analysis of Ga-DFaPSMA, wherein Figure 3 A shows 68 Labeling yield determination results of Ga-DFaPSMA, Figure 3 B shows 68 Radiochemical purity determination results of Ga-DFaPSMA after purification.

[0062] Figure 4 : Determination of coordination compound 177 Radio-HPLC signal plot of Lu-DFaPSMA radiochemical purity.

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

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

[0065] Figure 7 : Coordination compound 99m SPECT / CT imaging of Tc-HFaPSMA and 99m 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 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model.

[0067] Figure 9 : Coordination compound 99m Biodistribution comparison of Tc-HFaPSMA and 99m Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse model, wherein Figure 9 A shows 99mBiodistribution results of Tc-HFaPSMA in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse models, Figure 9 Figure 6B shows the biodistribution results of Tc-HFaPSMA in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models, 99m Figure 6A shows the tumor / normal organ uptake ratios of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models.

[0068] Figure 10 : Coordination compound 99m Tc-HFaPSMA and 99m Figure 6B shows the biodistribution results of Tc-HFaPSMA in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models, Figure 10 Figure 6A shows the biodistribution results of Tc-HFaPSMA in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models, 99m Figure 6B shows the biodistribution results of Tc-HFaPSMA in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models, Figure 10 Figure 6A shows the biodistribution results of Tc-HFaPSMA in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models, 99m Figure 6A shows the tumor / normal organ uptake ratios of Tc-PSMA-617 in PC-3 / F3 (PSMA- / FAP+) tumor-bearing mouse models.

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

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

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

[0072] The compounds of the present application, their preparation methods and uses will be further described in conjunction with specific examples below. It should be understood that the following examples are illustrative and explanatory in nature only and, as such, are not to be construed as limiting the scope of the present application. Any technical within the purview of the present application as described above is encompassed within the scope intended to be protected by the present application.

[0073] I. Experimental reagents and instruments

[0074] 1. Experimental reagents

[0075] Unless otherwise indicated, the starting 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 equipped with an array diode detector) was purchased from Thermo Fisher Scientific, USA. The HPLC radioactivity signal detector (Gabi star γ-ray test) was purchased from Raytest, Germany. The semi-preparative C18 reverse phase chromatographic column (ReproSil-Pur Basic C18, 5 μm, 250 x 10.0 mm) and the analytical C18 reverse phase chromatographic column (ReproSil-Pur Basic C18, 5 μm, 250 x 4.6 mm) were purchased from Dr. Maisch, Germany. The freeze dryer (FD-1D-50) was purchased from Beijing Boyikang Experimental Instrument Co., Ltd. The radioactivity γ-counter (Wizard-2470) was purchased from PerkinElmer, USA. The radioactivity activity meter (CRC-25R) was purchased from Capintec, USA. The 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] The method 1 for analyzing and separating and purifying the target product by high performance liquid chromatography was as follows: the Thermo vanquish core HPLC system was equipped with an analytical or semi-preparative C18 reverse phase chromatographic column, the flow rate was 0.8 mL / min for analysis and 3.2 mL / min for semi-preparation. Gradient elution was performed for 25 min, in which the mobile phase A was deionized water containing 0.05% TFA and the mobile phase B was acetonitrile containing 0.05% TFA, and the elution gradient was set as 90% A and 10% B at the beginning, 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 analysis and purification of target product by HPLC: same equipment and flow rate as method 1. Gradient elution for 25 min with mobile phase A being deionized water (containing 0.05% TFA) and mobile phase B being acetonitrile (containing 0.05% TFA) with elution gradient set at 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 analysis and purification of target product by HPLC: same equipment and flow rate as method 1. Gradient elution for 25 min with mobile phase A being deionized water (containing 0.05% TFA) and mobile phase B being acetonitrile (containing 0.05% TFA) with elution gradient set at 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] II. Experimental Methods and Results

[0082] The abbreviations used in the examples of the present application have the following respective meanings:

[0083]

[0084]

[0085] Example 1 Synthesis of compound HFaPSMA and DFaPSMA (as shown in Figure 1

[0086] Step 1: Preparation of Fmoc-NH-PEG4-CO-N-bis(PEG4-NHS) (compound 2)

[0087] ​Take 10.0 mg Fmoc-NH-PEG4-N-bis(PEG4-NHS) (compound 1) (9.3 pmol, 1.0 eq) dissolved in 200 pL DMSO. Take 4.3 mg EDC (27.9 pmol, 3.0 eq) dissolved in 86 pL DMSO. Take 3.2 mg NHS (27.9 pmol, 3.0 eq) dissolved in 64 pL DMSO. After mixing the three solutions in a 1.5 mL EP tube, oscillate at 37 °C for 4 h. Detect the reaction progress by HPLC (method 1), and after the reaction is completed, separate and purify the target product, collect the fraction corresponding to the elution peak at 14.5 min in semi-preparative HPLC (method 2), and obtain 8.3 mg (compound 2) of colorless oily liquid after freeze-drying by vacuum freeze-drying method. MALDI-TOF mass spectrometric analysis confirms that it is 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: Preparation of Fmoc-NH-PEG4-CO-N-PEG4-FaPSMA (compound 5)

[0089] Take 5.0 mg Fmoc-NH-PEG4-CO-N-bis(PEG4-NHS) (4.2 pmol, 1.0 eq) dissolved in 250 pL DMSO. Take 1.9 mg compound 3 (4.6 pmol, 1.1 eq) dissolved in 80 pL DMSO. Take 2.5 mg compound 4 (3.8 pmol, 0.9 eq) dissolved in 125 pL DMSO. After mixing the compound 2 solution and the compound 4 solution in a 1.5 mL EP tube, add 4 pL DIPEA, oscillate at 37 °C for 4 h, add the compound 3 solution and add 2 pL DIPEA, react at 37 °C for 4 h. Detect the reaction progress by HPLC (method 2), and after the reaction is completed, separate and purify the target product, collect the fraction corresponding to the elution peak at 13.6 min in semi-preparative HPLC (method 2), and obtain 1.5 mg (compound 5) of light yellow solid after freeze-drying by vacuum freeze-drying method. MALDI-TOF mass spectrometric analysis confirms that it is the expected product, 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: Preparation of NH2-PEG4-CO-N-PEG4-FaPSMA (Compound 6)

[0091] Weigh 1.5 mg Fmoc-NH-PEG4-CO-N-PEG4-FaPSMA (2.5 pmol, 1.0 eq) into 100 pL DMSO, add 25 pL piperidine, and oscillate at 37 °C for 0.5 h. Detect the reaction progress by HPLC (Method 1). After the reaction is completed, separate and purify the target product, collect the fraction corresponding to the elution peak at 14.6 min in semi-preparative HPLC (Method 3), and obtain 1.0 mg of white solid (Compound 6) after freeze-drying using the vacuum freeze-drying method. MALDI-TOF mass spectrometric analysis confirms that it is the expected product, MALDI-TOF-MS: m / z = 1790.88 [M+H] + , the theoretical molecular weight is 1789.81.

[0092] Step 4-1: Preparation of HYNIC-NH2-PEG4-CO-N-PEG4-FaPSMA (Compound 7, HFaPSMA)

[0093] Weigh 5.0 mg NH2-PEG4-CO-N-PEG4-FaPSMA (2.8 pmol, 1 eq) into 250 pL DMSO. Weigh 1.75 mg HYNIC-NHS (4.19 pmol, 1.5 eq) into 250 pL DMSO. After combining the two solutions in a 1.5 mL EP tube, add 4 pL DIPEA, and oscillate at 37 °C for 4 h. Detect the reaction progress by HPLC (Method 1). After the reaction is completed, separate and purify the target product, collect the fraction corresponding to the elution peak at 16.8 min in semi-preparative HPLC (Method 1), and obtain 0.9 mg of white solid product (Compound 7, HFaPSMA) after freeze-drying using the vacuum freeze-drying method. MALDI-TOF mass spectrometric analysis confirms that it is the expected product, MALDI-TOF-MS: m / z = 2093.77 [M+H] + , the theoretical molecular weight is 2092.85.

[0094] Step 4-2: Preparation of DOTA-NH2-PEG4-CO-N-PEG4-FaPSMA (Compound 8, DFaPSMA)

[0095] 2.7 mg of NH2-PEG4-CO-N-PEG4-FaPSMA (1.5 μmol, 1.0 eq) was weighed and dissolved in 135 μL of DMSO. 1.2 mg of DOTA-NHS (2.4 μmol, 1.5 eq) was weighed and dissolved in 60 μL of DMSO. The two solutions were combined in a 1.5 mL EP tube, and 2 μL of DIPEA was added. The mixture was shaken at 37 °C for 4 h. The reaction progress was monitored by HPLC (Method 1). After the reaction was complete, the target product was separated and purified. The fraction corresponding to the elution peak at 16.0 min in the semi-preparative HPLC was collected and combined (Method 1). The fraction was lyophilized using a vacuum freeze-drying method to obtain 1.4 mg of a white solid product (Compound 8, DFaPSMA). MALDI-TOF mass spectrometry analysis confirmed it as 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 Lyophilized Reagent Kit

[0097] Weigh 1 mg HFaPSMA and dissolve it in 1 mL of succinate buffer solution at pH 5.5, resulting in 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 the solution 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 one vial suitable for use. 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 technetium [ 99m Tc] sodium phosphate injection solution is ready for use. Take one vial of HFaPSMA lyophilized injection kit, check for damage and ensure it is within its shelf life, and use it for later use. Under aseptic conditions, use a sterile syringe to draw up the high-technetium [ 99m 20-30 mCi (740-1110 MBq) of sodium thiocyanate injection solution was added to physiological saline injection to a final volume of 1 mL. This solution was then injected into a HFaPSMA lyophilized vial, and the vial was shaken thoroughly to completely dissolve the solids until the solution was clear. The vial was then placed in a 100°C water bath for 25 minutes. After removal, the solution was cooled to room temperature. 99mTc-HFaPSMA injection.

[0100] 99m After Tc-HFaPSMA labeling, one drop was applied using a sterile syringe to rapid silica gel thin-layer paper chromatography (ITLC-SG) for quality control analysis. Using rapid thin-layer chromatography paper as the support, the sample was developed using two different developing systems: acetone and a mixture of acetonitrile and physiological saline (Vacetonitrile:Vphysiological saline = 1:1). After drying the chromatography strip, data was acquired using a Bioscan radiometric thin-layer scanner. Images, percentages of radioactivity, and Rf values ​​were obtained to calculate the radiochemical purity of the drug. The main radioactive substance contained in the labeling solution was technetium-labeled material (…). 99m Tc-HFaPSMA), free technetium ( 99m TcO4 - ) and colloidal technetium ( 99m 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 the Tc-HFaPSMA labeling system f value

[0102]

[0103] 99m Quality control analysis of Tc-HFaPSMA can also be performed using Radio-HPLC. Method 1 was used to detect the components of the drug box. 99m The retention time of Tc-HFaPSMA was 17.5 min, and the radiolabeling rate was 97.34% (e.g., Figure 2 (As 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 physiological saline to prepare a 1 μg / μL solution. Wash the Sep-Pak C18 column sequentially with 5 mL of anhydrous ethanol and 10 mL of water for injection, then dry it with 10 mL of air to complete the activation process. Take 20 μL of DFaPSMA solution (containing 20 μg of DFaPSMA) into a 1.5 mL EP tube, and add 80 μL of 1M sodium acetate and 1.0 mL of... 68 GaCl3 eluent (5-15 mCi, 0.05 M hydrochloric acid medium), solution pH approximately 4.2, after mixing, incubated at 90°C for 15 min, Radio-iTLC analysis showed a labeling rate of 92.2% (e.g., ...). Figure 3The reaction mixture was incubated at 95 °C for 20 min. After the reaction was completed, the reaction solution was extracted with a 5 mL syringe which had been previously extracted with 4 mL of water for injection, and was loaded onto a C18 column which had been activated, and then the C18 column was washed with 10 mL of water for injection and the waste liquid was discarded. Finally, 0.5 mL of 80% ethanol was used to elute the C18 column to collect the label in a sterile vacuum vial, and the label was diluted with 2 mL of normal saline and filtered through a 0.22 μm microporous filter and stored in a sterile vacuum vial for use. The label was analyzed using Radio-iTLC 68 The radiochemical purity of Ga-DFaPSMA was greater than 99% (as shown in Figure 3 as shown in Figure 2B.

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

[0107] 1 mg of DFaPSMA was weighed into 1 mL of pure water to prepare a solution with a concentration of 1 μg / μL for use; a Sep-Pak C18 column was activated by sequentially washing with 5 mL of anhydrous ethanol, 10 mL of water for injection, and then dried with 10 mL of air. 20 μL of the DFaPSMA solution (containing 20 μg of DFaPSMA) was taken into a 1.5 mL EP tube, and 60 μL of 1 M sodium acetate buffer (pH 4.0), 0.1 mL of 0.1 M SnCl2solution, and 0.1 mL of 0.1 M Na2EDTA solution were sequentially added. 177 The reaction mixture was incubated at 95 °C for 20 min. After the reaction was completed, the reaction solution was extracted with a 5 mL syringe which had been previously extracted with 4 mL of water for injection, and was loaded onto a C18 column which had been activated, and then the C18 column was washed with 10 mL of water for injection and the waste liquid was discarded. Finally, 0.5 mL of 80% ethanol was used to elute the C18 column to collect the label in a sterile vacuum vial, and the label was diluted with 2 mL of normal saline and filtered through a 0.22 μm microporous filter and stored in a sterile vacuum vial for use. The label was analyzed using Radio-iTLC 177 The radiochemical purity of Lu-DFaPSMA was greater than 95% (as shown in Figure 4 as shown in Figure 2B.

[0108] Example 6 Biological experiments

[0109] Experimental cells and animals

[0110] SPF grade male BALB / c nude mice, 4-5 weeks old; SPF grade male Kunming mice, 6 weeks old; purchased from Beijing Vital River Laboratory 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), NIH-3T3 mouse embryonic fibroblasts (FAP negative) were cultured in DMEM cell culture medium containing 10% FBS. Cells were routinely subcultured in a 37°C, 5% CO2 incubator. Take 4-5 weeks old BALB / c nude mice, each was inoculated with 1×10 6 22Rv1 cells or 1×10 6 PC-3 cells and 1×10 6 F3 cell mixture, SPF conditions. When the tumor volume reached 100-300 mm 3 for imaging and biodistribution experiments. By cell mixing 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 research.

[0111] 1、 99m Tc-HFaPSMA in vitro cell binding experiment

[0112] To verify the specific binding ability of the dual-targeting small molecule 99m Tc-HFaPSMA, we prepared and used the same radioactivity of 99m Tc-HFaPSMA and 99m Tc-PSMA-617 for in vitro cell binding experiments.

[0113] In the in vitro cell binding experiment, 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, while F3 and NIH-3T3 cells were digested with 0.05% trypsin. Cells were collected into centrifuge tubes and centrifuged at 1000 rpm for 3 min, discarding the supernatant. Cells were resuspended in 10 mL of PBS to wash the cells. Cells were then counted separately using a cell counting chamber and diluted with PBS to a concentration of 1 × 10⁻⁶ cells / mL. 7 Cells. The above cell suspension was prepared at a ratio of 1 × 10⁶ cells per well. 6 Add cells to a filter plate sealed with 5% BSA overnight. Remove the labeled cells. 99m Tc-HFaPSMA and 99m Tc-PSMA-617 was added at a rate of 14.8 kBq / well to wells containing cells and incubated on ice for 30 min. For studies on blocking the interaction between the probe and cells, 1000 molar amounts of unlabeled ligand were added to the labeled radioactive probe for co-incubation. Five washes were performed with pre-chilled ice-cold PBST (PBS containing 0.05% Tween-20), and the radioactivity was measured in the wells of the filter plate using a gamma counter. All cell binding assays were performed in quadruplicate. Cell-probe binding values ​​are expressed as the percentage of radioactive counts per minute relative to the added radioactive dose (%AD). This value has been decay-corrected. Experimental results are as follows: 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 binding to F3 mouse embryonic fibroblasts was 6.14% (±0.6%). 99m The %AD value of Tc-PSMA-617 binding to F3 mouse embryonic fibroblasts was only 0.5% (±0.02%). For the negative control group and the blocked group, 99m Tc-HFaPSMA and 99m The binding values ​​measured for Tc-PSMA-617 were comparable to and significantly lower than those in the positive control group. These experimental results indicate that... 99m Tc-HFaPSMA exhibited the same performance as 99m Tc-PSMA-617 has a similar affinity to PSMA, but 99m Tc-HFaPSMA also has the additional ability to specifically bind to FAP-α expression.

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

[0116] The prepared99m Tc-HFaPSMA and 99m Tc-PSMA-617 was prepared in physiological saline at a concentration of 37 MBq / 200 μL. Each 22Rv1 / F3 tumor-bearing mouse was injected with 200 μL (37 MBq) via the tail vein. SPECT / CT imaging was performed 1 hour after injection. 99m Tc-HFaPSMA-blocked mice were injected with 99m Simultaneous injection of 300 μg FAPI, 500 μg PSMA, or 300 μg FAPI + 500 μg PSMA with Tc-HFaPSMA followed by imaging 1 hour later. Mice were anesthetized with 1.5% isoflurane-oxygen during imaging. After imaging, SPECT images were reconstructed and fused with CT images to obtain 3D images (posterior view). Imaging results are shown below. Figure 6 As shown, the tumor location is marked with an arrow. Experimental results indicate that, compared to... 99m Compared to Tc-PSMA-617, 99m The tumor uptake of Tc-HFaPSMA was essentially the same as that of its counterpart, and both exhibited similar imaging contrast. In the closed group experiment, after single-target blockade... 99m The tumor uptake of Tc-HFaPSMA was reduced, but significant tumor contrast was still observed; while blocking both PSMA and FAP, 99m Tc-HFaPSMA showed significantly reduced tumor uptake, with no tumor contrast observed; renal uptake was also reduced, further demonstrating... 99m Tc-HFaPSMA exhibits 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 models

[0118] The prepared 99m Tc-HFaPSMA and 99m Tc-PSMA-617 was prepared with physiological saline to a concentration of 37 MBq / 200 μL. Each PC-3 / F3 tumor-bearing mouse received 200 μL (37 MBq) via the tail vein. SPECT / CT imaging was performed 1 hour post-injection. Mice were anesthetized with 1.5% isoflurane-oxygen during the imaging process. After imaging, the SPECT images were reconstructed and fused with the CT images to obtain a 3D imaging image (posterior view). The imaging results are shown below. Figure 7 As shown, the tumor location is marked with an arrow. Experimental results indicate that... 99mTc-HFaPSMA can be effectively taken up by tumors that are negative for PSMA but positive for FAP, while 99m The signal intensity of Tc-PSMA-617 at the tumor site is close to the background signal, making it impossible to image the tumor site. This further proves that, compared to 99m Regarding Tc-PSMA-617, 99m Tc-HFaPSMA demonstrates a broader detection range in the diagnosis of prostate cancer.

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

[0120] Sixteen 6-week-old BALB / c nude mice bearing tumors (22Rv1 / F3) were randomly divided into four groups of four. The prepared... 99m Tc-HFaPSMA was prepared with physiological saline to a concentration of 370 kBq / 100 μL. Each mouse was injected with 100 μL (370 kBq) via the tail vein. Animals were sacrificed at 0.5, 1, 2, and 4 hours post-injection. Blood and major organs were collected, weighed, and radioactive cpm counts were measured. After decay correction, the percentage dose per gram of tissue (%ID / g) was calculated. Biodistribution results are expressed as mean ± standard deviation (mean ± SD, n = 4).

[0121] Experimental results are as follows Figure 8 As shown, 99m Tc-HFaPSMA uptake at the tumor site reached its peak 1 hour after injection, and remained at a higher level 4 hours after injection compared to the previous three groups, without a significant decrease in tumor uptake, indicating... 99m Tc-HFaPSMA exhibits excellent tumor retention capabilities, indicating significant application potential in the field of cancer treatment. Furthermore, apart from the kidneys (a metabolic organ), no abnormal drug concentration was observed in organs such as the liver, gallbladder, stomach, and small intestine, suggesting... 99m Tc-HFaPSM exhibits excellent in vivo stability and superior in vivo metabolic properties.

[0122] 5. Radiation Dosage Experiment

[0123] The above-mentioned biodistribution experiments were used to obtain 99m The uptake values ​​(%ID / g) of Tc-HFaPSMA in various organs of mice at four time points were estimated using OLIDA software, based on an animal body weight of 25g and an adult male body weight of 73kg. 99m We obtained the normal organ radiation absorbed dose of Tc-HFaPSMA through estimation. 99mThe radiation damage of Tc-HFaPSMA to each organ (in mSv / MBq) is shown in Table 2. 99m The highest radiation dose of Tc-HFaPSMA in the kidney is 0.045 mSv / MBq, and the radiation dose in the main organs of the body such as liver, spleen, stomach, pancreas, etc. is less than 0.0050 mSv / MBq, which is much lower than its distribution in the kidney. The effective dose of the patient is 0.0016 mSv / MBq, assuming a standard injection dose of 20 mCi (740 MBq) for a 73 kg adult in the clinic, the effective dose of the patient is 1.2 mSv, which is significantly lower than the reported small molecule PET imaging agent targeting PSMA 68 The whole body radiation dose of Ga-PSMA-617 (0.021 mSv / MBq x 203 MBq, 4.3 mSv, JNM·Vol. 56·No. 11·Nov. 2015) and small molecule PET imaging agent targeting FAP 68 The whole body radiation dose of Ga-FAPI-46 (0.0078 mSv / MBq x 200 MBq, 1.6 mSv, JNM·Vol. 61·No. 8·Aug. 2020). At the same time, it is also significantly lower than the commonly used PET imaging agent 18 5.1 ± 0.73 mSv of F-FDG and SPECT imaging agent 99m 4.7 ± 0.97 mSv of Tc-MDP. Lower than the radiation dose of 7.4 mSv of one abdominal and pelvic enhanced CT. Therefore, 99m Tc-HFaPSMA has a certain guarantee of radiation safety.

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

[0125]

[0126] 6、 99m Tc-HFaPSMA and 99m Comparison of biodistribution of Tc-HFaPSMA and Tc-PSMA-617 in 22Rv1 / F3 (PSMA+ / FAP+) tumor-bearing mouse models

[0127] Take 8 22Rv1 / F3 tumor-bearing BALB / c nude mice (6 weeks old), randomly divided into 2 groups, 4 in each group, respectively for 99m Tc-HFaPSMA and 99m Biodistribution experiment of Tc-PSMA-617. The prepared 99m Tc-HFaPSMA or 99mTc-PSMA-617 was prepared with physiological saline to a concentration of 370 kBq / 100 μL. Each mouse was injected with 100 μL (370 kBq) via the tail vein. One hour after injection, the animals were sacrificed, and blood and major organs were collected, weighed, and radioactive cpm counts were measured. After decay correction, the percentage dose per gram of tissue (%ID / g) was calculated. Biodistribution results are expressed as mean ± standard deviation (mean ± SD, n = 4).

[0128] Experimental results are as follows Figure 9 As shown, compared to 99m Regarding Tc-PSMA-617, 99m Tc-HFaPSMA uptake by tumors was increased, and tumors exhibited increased uptake of Tc-HFaPSMA. 99m Tc-HFaPSMA intake is relatively 99m Tc-PSMA-617 increased by 40%, while the spleen's effect on 99m Tc-HFaPSMA intake is relatively 99m Tc-PSMA-617 levels decreased by 75%, with reductions observed in the gallbladder and submandibular glands. Furthermore, for SPECT imaging, the near-normal tissue uptake resulted in an increased tumor / normal organ uptake ratio, which is beneficial for the diagnosis of small tumor lesions.

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

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

[0131] Experimental results are as follows Figure 10 As shown, 99m Tc-HFaPSMA uptake at tumor sites is relatively high. 99mTc-PSMA-617 significantly increased 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 have better performance in the diagnosis of PSMA-negative tumors, which is beneficial to help patients find as many tumor lesions as possible.

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

[0133] The prepared 68 Ga-DFaPSMA and 68 Ga-PSMA-617 were respectively prepared into 18.5 MBq / 100 μL with normal saline, and each 22Rv1 / F3 tumor-bearing mouse was injected with 100 μL (18.5 MBq) through the tail vein. Micro-PET / CT imaging was performed at 0.5 h 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 imaging graph (whole body MIP graph), and the imaging results are shown in Figure 11 The tumor location is marked with an arrow. The experimental results show that, in the PET imaging of tumor-bearing mouse models, the 68 Ga-DFaPSMA obtained by the application has improved tumor uptake compared with 68 Ga-PSMA-617 based on the monomer form of PSMA, and both have good imaging contrast.

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

[0135] The prepared 177 Lu-DFaPSMA and 177 Lu-PSMA-617 were prepared into 37 MBq / 200 μL with normal saline, and each 22Rv1 / F3 tumor-bearing mouse was injected with 200 μL (37 MBq) through the tail vein. SPECT / CT imaging was performed at 4, 24, 48, 72, 96, 120, and 144 h 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 imaging graph (posterior view). The imaging results are shown inFigure 12 The tumor locations are marked with arrows. The experimental results show that, for the same time point, the two groups of images, compared with 177 Lu-PSMA-617, 177 Lu-DFaPSMA has higher tumor uptake. In addition, 177 Lu-DFaPSMA still has a clear tumor image at 144h after injection, while 177 Lu-PSMA-617 cannot obtain a clear tumor image at 96h after injection. Higher tumor uptake and longer tumor retention time can be effectively used in the radiotherapy of prostate cancer.

[0136] Example 7 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 blood PSA level after radical prostatectomy. First, the 18 F-PSMA-1007-PET nuclear medicine imaging, 18 F-PSMA-1007 is a PSMA-targeted positron emission tomography (PET) tracer, which has a higher labeling yield, a longer half-life, and better energy characteristics than 68 Ga-labeled PSMA-targeted tracers, 18 F-PSMA-1007 makes it a widely used PSMA PET imaging agent in the clinic (Giesel FL, et al. F-18 labelled 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.). After the first chemotherapy, the 99m Tc-HFaPSMA-SPECT imaging was performed again, and the imaging results are shown in Figure 13 18 ​PET results of F-PSMA-1007 showed that there was obvious accumulation of radioactivity in liver, kidney and gastrointestinal tract; there was slight uptake in salivary gland. There was a little radioactivity accumulation in bladder. The background was low in heart, lung, brain and abdomen. There was no obvious increase in nuclide uptake in the prostate surgery area. There were increased nuclide uptake in skull, right clavicle, bilateral scapula, left humerus, cervical spine, thoracic spine, lumbar spine, sacral spine, multiple vertebral bodies and accessories, multiple ribs, sternum, bilateral ischium, pubis, right ilium and left femoral shaft, with SUVmax of 5.1-43.3. Part of the CT showed bone destruction. The F-PSMA-1007 of the application 99m Tc-HFaPSMA SPECT results showed that there was obvious accumulation of radioactivity in kidney and bladder. There was a little radioactivity distribution in liver and gastrointestinal tract. There was slight uptake in salivary gland. The background was low in heart, lung, brain and abdomen. There was abnormal nuclide concentration in skull, right clavicle, bilateral scapula, left humerus, cervical spine, thoracic spine, lumbar spine, sacral spine, bilateral multiple ribs, sternum, bilateral ischium, pubis, right ilium and left femoral shaft, with SUVmax of 4.11-45.77. There was obvious nuclide uptake in right supraclavicular lymph node, with SUVmax of 16.64. 99m Tc-HFaPSMA-SPECT in this case has the same detection ability as F-PSMA-1007-PET in detecting the primary lesion and metastatic lesion of prostate cancer. 18 F-PSMA-1007-PET is equivalent, and shows lower liver and gastrointestinal uptake.

[0138] The above describes the embodiments of the application. However, the application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A compound having the structure shown in Formula (I), or a pharmaceutically acceptable salt, stereoisomer thereof; wherein L1 is -CO-CH2CH2- (O-CH2CH2-)4-. L2 is -CO-CH2CH2- (O-CH2CH2-)4-NH2.

2. A compound having the structure of Formula (II), or a pharmaceutically acceptable salt, stereoisomer thereof; wherein L1 is -CO-CH2CH2- (O-CH2CH2-)4-. L2 is -CO-CH2CH2- (O-CH2CH2-)4-NH-.

3. The compound of formula (II) according to claim 2, wherein the bifunctional chelator is selected from the following groups:

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

5. A complexing compound, which is obtained by labeling the compound of formula (I) according to claim 1 with a radionuclide and a bifunctional chelator, wherein the bifunctional chelator is linked via its carboxyl group to the amino group at the end of L2 in the compound of formula (I); or, which is obtained by labeling the compound of formula (II) according to claim 2 with a radionuclide.

6. The complexing compound according to claim 5, The radionuclide is selected from the group consisting of 68 Ga, 99m Tc, or 177 Lu.

7. The complexing compound according to claim 5, which is selected from: 99m Tc-HFaPSMA),​ 68 Ga-DFaPSMA), or​ 177 Lu-DFaPSMA),​ or a pharmaceutically acceptable salt, stereoisomer thereof.

8. A pharmaceutical composition comprising the complexing compound according to any one of claims 5 to 7 and a pharmaceutically acceptable carrier.

9. A kit comprising the compound of formula (II) according to claim 2, optionally a synergistic ligand, and a pharmaceutically acceptable carrier, excipient, stabilizer and / or diluent.

10. The kit according to claim 9, for use in 99m The kit of the Tc-labelled ligand compound comprises the ligand compound HF aPSMA according to claim 4 in an amount of 10 pg to 1000 pg, tricine in an amount of 1 mg to 50 mg, triphenylphosphine tris-mesylate sodium salt (TPPTS) in an amount of 3 mg to 30 mg, and optionally one or more pharmaceutically acceptable carriers, excipients, stabilizers and / or diluents.

11. The kit according to claim 10, which is formed by lyophilizing a solution containing HFaPSMA, tricine, triphenylphosphine tris-benzenesulfonic acid sodium salt (TPPTS) and a pH buffer solution selected from the group consisting of succinic acid, citric acid, tartaric acid, phosphoric acid, boric acid, acetic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (i.e. HEPES) solution required for labeling with the radionuclide.

12. The kit according to claim 11, wherein HFaPSMA, tricine, triphenylphosphine tris-benzenesulfonic acid sodium salt (TPPTS) are dissolved in a solution of succinic acid / sodium hydroxide, filtered and lyophilized to obtain the kit.

13. The kit according to claim 9, for use in 68 Ga, 177 The kit of Ga- and / or Lu-labeled ligand compounds comprises the ligand compound DFaPSMA according to claim 4, and one or more stabilizers and a pharmaceutically acceptable carrier.

14. The kit according to claim 13, which is obtained by preparing a solution.

15. Use of the compound of formula (I) according to claim 1, the compound of formula (II) according to any one of claims 2 to 4, the complexing compound according to any one of claims 5 to 7, the pharmaceutical composition according to claim 8, or the kit according to any one of claims 9 to 14 for the manufacture of a medicament for the diagnosis or treatment of prostate cancer lesions with different levels of PSMA expression in a patient.

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