A radionuclide drug and its preparation method and application

Abstract

The present invention provides a kind of radionuclide drug and its preparation method and application, belonging to the field of biomedical technology. The radionuclide drug provided by the present invention is based on a compound after specific structural optimization, which solves the problem that the antibody-radionuclide drug in the prior art has a long blood circulation time in the body and excessive radioactive exposure to normal tissues, and achieves the technical effects of improving the stability of radioactive labeling, enhancing tumor targeting and anti-tumor effects, and reducing non-specific radiation damage. The compound provided by the present invention maintains high stability in serum in vitro, and the labeling rate remains above 95% within 7 days, which not only reduces the possibility of in vitro labeling, but also ensures the continuous targeting and therapeutic effect of the drug in vivo, showing high uptake value and long-term retention characteristics for tumors, and low radioactive absorption to normal organs, showing better therapeutic effects and lower toxic side effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a radionuclide medicine and a preparation method and application thereof. Background Art

[0002] With the increasing incidence of cancer every year, the development of diagnostic and therapeutic methods for malignant tumors has become an important topic in the medical field. At present, radionuclide drugs play an important role in the diagnosis and treatment of cancer. Radionuclide drugs can use the energy of radioactive isotopes to release radiation at the tumor site, killing cancer cells while minimizing damage to surrounding normal tissues. The advantage of this treatment method is that it can provide more precise and effective treatment by targeting tumor cells. Radionuclide drugs have achieved good results in the diagnosis and treatment of many cancers, especially some difficult-to-treat malignant tumors, such as prostate cancer and neuroendocrine tumors.

[0003] In the development of radionuclide drugs, the stability and specificity of the compounds are two key factors. In order to effectively target tumor cells, radionuclide drugs usually need to be combined with specific targeting molecules, such as antibodies, peptide chains or small molecule compounds, which can recognize and bind to specific receptors or antigens on tumor cells. However, these conjugates usually face two major challenges: one is the problem of radioactive isotope off-labeling, that is, the radioactive isotope detaches from the targeting molecule, causing the radioactive drug to circulate in the blood and increase radiation damage to normal tissues; the second is the metabolic rate and targeting of the drug in the body, that is, how to ensure that the drug quickly reaches the tumor site after entering the body and stays in the tumor for a long time to exert its therapeutic effect.

[0004] The prior art discloses that a variety of radionuclide drugs have been developed for targeted diagnosis and treatment of tumors. For example, some researchers have developed antibody-based radionuclide drugs, which combine radioisotopes with monoclonal antibodies that can target tumor antigens to achieve the effect of precise tumor targeting; peptide-based radionuclide drugs have also been widely studied, such as radiolabeled somatostatin analogs, which are used to treat neuroendocrine tumors; however, these methods still have some problems, for example, monoclonal antibody or peptide-coupled radiopharmaceuticals usually have a large molecular weight, which may lead to insufficient drug penetration in the tumor, thereby affecting the therapeutic effect. In addition, these drugs may have a long blood circulation time in the body, increasing the radiation exposure to normal tissues. Summary of the invention

[0005] The purpose of the present invention is to provide a radionuclide drug, which improves the stability of radioactive labeling, enhances tumor targeting and anti-tumor effects, and reduces nonspecific radiation damage.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a compound having a structure shown in Formula A:

[0008]

[0009] Formula A.

[0010] The present invention also provides a method for preparing the above compound, comprising the following steps:

[0011] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((2R,3R,4R,5S,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)-L-serine and tri-tert-butyl 2,2',2''-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate were dissolved in DMF, DIEA and PyBOP were added, and the reaction was carried out at 15-25° C. for 0.5-1.5 h. After the reaction was completed, the solvent was removed and purified in sequence to obtain an intermediate product 1, as shown in the following formula 1:

[0012]

[0013] Formula 1;

[0014] The intermediate product 1 is dissolved, and lithium hydroxide monohydrate is added to react for 0.5 to 1.5 hours. After the reaction is completed, the solvent is removed, extracted with a dichloromethane-methanol solution, the organic layers are combined, concentrated, and dried under reduced pressure to obtain an intermediate product 2, as shown in Formula 2 below:

[0015]

[0016] Formula 2;

[0017] 4-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate (DZ-1), HOBT and EDCI were dissolved in dichloromethane and reacted for 10-50 min. Then, intermediate 2 was added and the reaction was continued for 0.5-1.5 h. After the reaction, the solvent was removed and purified in sequence to obtain intermediate 3, as shown in Formula 3 below:

[0018]

[0019] Formula 3;

[0020] The intermediate product 3 is dissolved in dichloromethane and trifluoroacetic acid and reacted for 5 to 7 hours. After the reaction is completed, the solvent is removed and purified to obtain the compound.

[0021] The present invention also provides the use of the above compound or the compound prepared by the above preparation method in the preparation of anti-tumor drugs.

[0022] The present invention also provides a radionuclide drug, comprising a compound having a structure shown in Formula B:

[0023]

[0024] Formula B;

[0025] Wherein, M is a radioactive metal.

[0026] Preferably, the radioactive metal is selected from one or more of terbium-161, lutetium-177 and copper-64.

[0027] The present invention also provides a method for preparing a radionuclide drug, comprising the following steps:

[0028] The above compound is dissolved to obtain solution 1;

[0029] Will 177 LuCl3, solution 1 and buffer solution were mixed and heated for reaction for 10-30 min to obtain a radionuclide drug;

[0030] The temperature of the heating reaction is 80-100°C.

[0031] The present invention also provides a radionuclide drug or the use of the radionuclide drug prepared by the above preparation method in the preparation of anti-tumor drugs.

[0032] Preferably, the tumor is a malignant tumor.

[0033] Preferably, the tumor is cervical cancer, brain glioma, lung cancer and / or breast cancer.

[0034] Beneficial effects of the present invention:

[0035] The compounds provided by the present invention can significantly improve the stability of radioactive labeling. Specifically, they maintain high stability in serum in vitro, and the labeling rate remains above 95% within 7 days. This high stability not only reduces the possibility of labeling off in vitro, but also ensures the continuous targeting and therapeutic effect of the drug in vivo.

[0036] The compounds of the present invention have significantly enhanced their targeting and anti-tumor effects on various tumors such as lung cancer and cervical cancer through structural optimization. They show high tumor uptake values ​​and long-term retention characteristics in SPECT / CT imaging and fluorescence imaging, and low radioactive absorption in normal organs, showing better therapeutic effects and lower toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 HPLC 177 The labeling rate of Lu-DOTA-Glucose-DZ (A) and 177 Stability of Lu-DOTA-Glucose-DZ in PBS and FBS in vitro.

[0038] Figure 2 for 177 In vitro cell killing ability test of Lu-DOTA-Glucose-DZ on Hela cervical cancer cells (A) and 177 Blood clearance rate of Lu-DOTA-Glucose-DZ (B).

[0039] Figure 3 for 177 In vivo distribution of Lu-DOTA-Glucose-DZ in a cervical cancer tumor-bearing mouse model.

[0040] Figure 4 for 177 The therapeutic effect of Lu-DOTA-Glucose-DZ on cervical cancer tumor model. A: Tumor volume change curve; B: Tumor tissue photo after treatment; C: Mouse weight change curve.

[0041] Figure 5 for 177 HE staining of important organs and tumor tissues of cervical cancer model mice after Lu-DOTA-Glucose-DZ treatment.

[0042] Figure 6 The figure is based on the research results of the first generation compound DOTA-DZ-1. A: structural diagram of DOTA-DZ-1; B: screening of labeling conditions for DOTA-DZ-1; C: HPLC detection 68 Labeling rate of Ga-DOTA-DZ-1; D: 68 Stability of Ga-DOTA-DZ-1 in FBS in vitro; E: detected by TLC 177 The labeling rate of Lu-DOTA-DZ-1; F: 177 In vitro stability of Lu-DOTA-DZ-1 in PBS and FBS.

[0043] Figure 7 Based on DZ-1-TCO 68 Results of Ga pre-targeted imaging study, including A: DZ-1-TCO and DOTA-MeTz structures; B: Comparison of traditional imaging based on DZ-1 and pre-targeted imaging based on DZ-1-TCO; C: Semi-quantitative analysis of tumor / muscle ratio of pre-targeted PET / CT imaging. D: In vivo fluorescence imaging of cervical cancer mouse model after tail vein injection of DZ-1-TCO at different time points; E: Tumor-bearing mice injected with DZ-1-TCO alone 68 PET / CT imaging after Ga-DOTA-MeTz (the arrow indicates the tumor). DETAILED DESCRIPTION

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Preparation method of DZ-Glucose-DOTA:

[0047] Synthesis of intermediate 1: tri-tert-butyl 2,2',2''-(10-((S)-5-((((2R,3R,4R,5S,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)oxy)methyl)-1-(9H-fluorenyl)-3,6,13-trioxo-2-oxa-4,7,12-triazatetradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, as shown in Formula 1 below:

[0048]

[0049] Formula 1.

[0050] N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-((2R,3R,4R,5S,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)-L-serine (200 mg, 0.30 mmol) and tri-tert-butyl 2,2',2''-(10-(2-((4-aminobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (193 mg, 0.30 mmol) were dissolved in 8 mL of DMF, DIEA (38.7 mg, 0.30 mmol) and PyBOP (312 mg, 0.6 mmol) were added, and the mixture was stirred at 20 °C for 1 hour. After the reaction, the reaction solvent was filtered and purified by preparative HPLC (column: Welch Xtimate 21.2*250 mm C18, 10 µm, mobile phase: A: water (0.2% formic acid) B: acetonitrile; B%: 30%-70% in 15.0 minutes) to obtain tri-tert-butyl 2,2',2''-(10-((S)-5-((((2R,3R,4R,5S,6R)-3-acetylamino-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)oxy)methyl)-1-(9H-fluorenyl)-3,6,13-trioxo-2-oxa-4,7,12-triazatetradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl) triacetate (200 mg, Yield 52%), white solid. LCMS: m / z 641.4 (M+H)+

[0051] Synthesis of intermediate product 2: tri-tert-butyl 2,2',2''-(10-(2-((4-((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl)oxy)-2-aminopropionylamino)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate, as shown in Formula 2 below:

[0052]

[0053] Formula 2.

[0054] To a solution of tri-tert-butyl 2,2',2''-(10-((S)-5-((((2R,3R,4R,5S,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl)oxy)methyl)-1-(9H-fluoren-9-yl)-3,6,13-trioxo-2-oxa-4,7,12-triazatetradec-14-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (150 mg, 0.117 mmol) (8 mL of methanol and 8 mL of water) was added lithium hydroxide monohydrate (39.3 mg, 0.936 mmol) and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was removed and the residue was suspended in water (20 mL). The mixture was extracted with dichloromethane / methanol (volume ratio = 20:1, 200 mL x 2) solution. The organic layers were combined and concentrated, and the residue was dried under reduced pressure to obtain tri-tert-butyl 2,2',2''-(10-(2-((4-((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl)oxy)-2-aminopropionamido)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (98 mg, 89.9% yield) as a brown solid. LCMS: m / z 467.3 [1 / 2M+H]^+.

[0055] Synthesis of intermediate 3

[0056] 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7,10-tri(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetylamino)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ylidene)ethenylidene)-2-chlorocyclohex-1-enyl)ethenyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate, as shown in the following formula 3:

[0057]

[0058] Formula 3.

[0059] 4-(2-((E)-2-((E)-3-(2-((E)-1-(5-carboxypentyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate (DZ-1) (76 mg, 0.082 mmol), HOBT (16.5 mg, 0.122 mmol) and EDCI (23.4 mg, 0.122 mmol) were dissolved in dichloromethane (DCM, 20 mL) and stirred at room temperature for 30 minutes. Then, tri-tert-butyl 2,2',2''-(10-(2-((4-((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-2-aminopropionylamino)butyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (57 mg, 0.082 mmol) was added and stirring was continued at room temperature for 1 hour. The solvent was evaporated at 30°C and the residue was purified by preparative high performance liquid chromatography (Prep-HPLC) [acetonitrile in (containing 0.1% TFA in water) in the ratio from 0% to 34%] to give 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetamido)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate (65 mg, 49.2% yield) was a green solid.

[0060] The synthesis of the final product DOTA-Glucose-DZ is shown in Formula 4 below:

[0061]

[0062] Formula 4.

[0063] 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4-(2-(4,7,10-tri(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetylamino)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate (55 mg, 0.034 mmol) was dissolved in dichloromethane (DCM, 18 mL) and trifluoroacetic acid (TFA, 6 mL) and stirred at room temperature for 6 hours. The solvent was then removed and the residue was purified by preparative high performance liquid chromatography (Prep-HPLC, TFA) to give 4-(2-((E)-2-((E)-3-(2-((E)-1-(6-(((S)-3-(((2R,3R,4R,5S,6R)-3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1-oxo-1-((4- (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl)acetamido)butyl)amino)propyl)amino)-6-oxohexyl)-3,3-dimethylindol-2-ylidene)vinyl)-2-chlorocyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-yl)butane-1-sulfonate-2,2,2-trifluoroacetic acid (1 / 1) (20.7 mg, 35.6% yield) as a green solid. LCMS: m / z 1451.7 [M+H]+ and 726.3 [1 / 2M+H]+.

[0064] Experimental example

[0065] Experimental plan for investigating the therapeutic effects of DZ-Glucose-DOTA

[0066] 1. 177 Lu-DOTA-Glucose-DZ Marking

[0067] 200ul is about 37MBq 177 LuCl3 was added with 50 μl DOTA-Glucose-DZ aqueous solution and 50 μl sodium acetate buffer (1 mmol / L). The mixture was placed in a 90°C oil bath and heated for 15-20 minutes. After natural cooling, the corresponding marker was obtained. The structural formula is shown in Formula 5 below:

[0068]

[0069] Formula 5.

[0070] Where M is 177 Lu.

[0071] Radio-HPLC was used with a Raytest Gabistar radioactivity detector, an Agilent-35900E digital-to-analog converter, and a Kinetex C18 column (4.6 × 100 mm, 3.5 μm). 177 Radiochemical Purity (RCP) of Lu-labeled drugs. The mobile phase A of HPLC was deionized water (containing 0.1% HCOOH), and the mobile phase B was acetonitrile, with a flow rate of 1 mL / min. The HPLC gradient used was: 90% A and 10% B for 0-5 minutes, 40% A and 60% B for 15 minutes, and 90% A and 10% B for 25 minutes.

[0072] 2. 177 In vitro stability of Lu-DOTA-Glucose-DZ

[0073] Take 400 μCi of the concentrated solution on a C18 column 177 Lu-DOTA-Glucose-DZ was placed in a suitable EP tube, and then 500 μL PBS and 500 μL fetal bovine serum (FBS) were added, mixed evenly, and incubated at 37 °C. Samples were taken at 0 h, 4 h, 24 h, 48 h, 120 h, and 168 h of incubation, and iTLC-SG was used as the stationary phase and 0.1 mmol / L citric acid as the mobile phase. The product was taken for radiochemical purity determination and its stability at this time point was determined.

[0074] 3. 177 Lipid-water partition coefficient of Lu-DOTA-Glucose-DZ

[0075] 177 The lipid-water partition coefficient (Log P value) of Lu-DOTA-Glucose-DZ was determined by its radioactivity distribution in n-octanol and phosphate buffer. 177Lu-DOTA-Glucose-DZ was added to a two-phase system of 0.5 mL of n-octanol and 0.5 mL of PBS buffer, vortexed for 2 min, and centrifuged at 6000 g for 20 min to ensure complete stratification of the liquid. 100 μL of the upper and lower layers were taken, and the radioactive count CPM was detected using a radioactive gamma counter Wizard. The lipid-water partition coefficient was calculated according to the formula Log P = Log (CO / CW) (CO is the radioactive count of n-octanol, CW is the radioactive count of PBS buffer).

[0076] 4. 177 In vitro cell killing ability of Lu-DOTA-Glucose-DZ

[0077] Detection by CCK-8 method 177 Lu-DOTA-Glucose-DZ cell killing ability. Hela cells were taken and 10 4 Cells / well were inoculated into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The original culture medium was discarded after the cells adhered to the wall. 177 Lu-DOTA-Glucose-DZ (0 MBq, 0.4625 MBq, 0.925 MBq, 1.85 MBq, 2.775 MBq, and 3.7 MBq) and different activities 177 LuCl3 (0 MBq, 0.4625 MBq, 0.925 MBq, 1.85 MBq, 2.775 MBq and 3.7 MBq) medium was added and incubated for 48 hours, with 5 replicate wells for each activity. Negative control group: Hela cells plus blank medium; blank control group: blank medium. After 24 hours, the 96-well plate was removed, the medium was discarded and washed 3 times with PBS, then 100 μL of fresh medium and 10 μL of CCK-8 solution were added to each well, incubated in the dark for 1-4 hours, and the absorbance (optical density, OD) of each well at a wavelength of 450 nm was measured by a microplate reader. Cell activity rate = (OD experimental group-OD blank control group) / (OD negative control group-OD blank group)×100%

[0078] 5. 177 In vivo fluorescence imaging of Lu-DOTA-Glucose-DZ

[0079] Subcutaneous model mice were constructed with Hela cell cervical cancer (n=3), and tumor-bearing mice were injected with 100uCi via the tail vein. 177Lu-DOTA-Glucose-DZ was imaged in vivo on IVIS at 4 h, 24 h, 48 h, and 96 h to detect the distribution of the probe in the mouse and its enrichment in the tumor. The fluorescence intensity change of the tumor region (ROI) and the signal-to-noise ratio of the tumor region were calculated. SBR = fluorescence intensity of the tumor region / fluorescence intensity of the tumor peritumor. The imaging instrument used was IVIS small animal in vivo imager, and the channel selected was 745-820 nm (excitation and emission wavelength channels, respectively).

[0080] 6. 177 Plasma half-life of Lu-DOTA-Glucose-DZ

[0081] Take 8 ICR mice weighing about 20 g and inject 20uCi into the tail vein of each mouse. 177 Lu-DOTA-Glucose-DZ, 10 μL of blood was collected from the tail of mice at 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h after injection using a microcapillary tube, and the wet weight of blood was recorded. The CPM value was determined using a radioactive γ counter Wizard and calculated using the pharmacokinetic software DAS 2.0 after attenuation correction. 177 Pharmacokinetic parameters of Lu-DOTA-Glucose-DZ.

[0082] 7. 177 In vivo distribution experiment of Lu-DOTA-Glucose-DZ

[0083] Twenty-four Hela tumor-bearing animal models were randomly divided into 6 groups, with 4 animals in each group. Each tumor-bearing animal model was injected with 1.11 MBq of 177 Lu-DOTA-Glucose-DZ, mice were killed at 1 h, 4 h, 24 h, 48 h, 72 h and 96 h, and blood, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, brain and tumor tissues and organs were taken and weighed. The radioactivity counts of each organ and tissue were measured with a radioactive γ counter and calculated. 177 The distribution of Lu-DOTA-Glucose-DZ in various tissues and organs of Hela tumor-bearing animal models.

[0084] 8. 177 Therapeutic Study of Lu-DOTA-Glucose-DZ

[0085] 4-6 week old BALB / c nude mice were randomly divided into 4 groups (5 mice in each group) and anesthetized with 2.5% isoflurane. 5 × 10 5Hela cells. After 10 days, when the tumor volume was about 50 ~ 70mm3, each group of tumor-bearing animal models was injected with the following radioactive labeled DZ-1 solutions with different specific activities through the tail vein: 1) 177 Lu-DOTA-Glucose-DZ high-activity treatment group: 37 MBq (two doses, 5 days apart, 37 MBq each time); (2) 177 Lu-DOTA-Glucose-DZ low-activity treatment group: 11.1 MBq (two administrations, 5 days apart, 11.1 MBq each time); (3) precursor group; (4) PBS group. After drug injection, the mental state and diet of mice in each group were observed every day. The body weight and tumor volume of H1975 tumor-bearing animal models were measured every 2 days, and the tumor size was measured with a vernier caliper. The tumor volume was calculated using the formula: volume = (length × width2) × 1 / 2. The tumor-bearing animal models were killed on the 12th day after administration. Hematoxylin-eosin (HE) staining was performed on the main organs (heart, liver, spleen, lung and kidney) of each group of tumor-bearing models to evaluate whether radionuclide therapy has toxic effects on normal organs. Finally, the tumor tissues of each group of tumor-bearing animal models were obtained and HE and Tunel staining were performed to evaluate the necrosis and tumor of tumor tissues.

[0086] Experimental results:

[0087] 1. 177 Labeling and stability of Lu-DOTA-Glucose-DZ

[0088] HPLC detection, such as Figure 1 As shown, 177 The labeling rate of Lu-DOTA-Glucose-DZ was 99%; the labeling rates after 7 days in FBS and PBS were 97.83 ± 0.21% and 98.90 ± 0.63%, respectively.

[0089] 2. 177 Lipid-water partition coefficient of Lu-DOTA-Glucose-DZ

[0090] 177 The LogD of Lu-DOTA-Glucose-DZ was -1.07 ± 0.08, indicating that the probe was hydrophilic.

[0091] 3. 177 In vitro cell killing ability of Lu-DOTA-Glucose-DZ

[0092] like Figure 2 As shown in A, the results show 177After 48h of Lu-DOTA-Glucose-DZ treatment, the survival rate of Hela cells was 10.98 ± 2.78%, while that of the control group was 177 The cell viability of Lu was 68.5%, indicating that the probe had a strong killing ability against cervical cancer cells in vitro. Figure 2 B 177 Lu-DOTA-Glucose-DZ is cleared slowly from the blood in a two-phase pattern, with a fast half-life in the blood of 0.6 minutes and a short half-life in the blood of 387.9 minutes.

[0093] 4. 177 In vivo distribution experiment of Lu-DOTA-Glucose-DZ

[0094] like Figure 3 As shown, the results of the in vivo distribution experiment showed 177 Lu-DOTA-Glucose-DZ has excellent targeting performance for cervical cancer. The drug is mainly metabolized by the kidneys. After injection, the drug gradually accumulates in the tumor site, reaching a peak at 72 hours, with the highest uptake value of 20.49±4.89%ID / g. The drug stays in the tumor site for a long time, and the tumor uptake value at 96 hours is 9.69±3.42%ID / g.

[0095] 5. 177 Therapeutic Experiments of Lu-DOTA-Glucose-DZ

[0096] Figure 4 The treatment experiment showed that both the high-dose group and the low-dose group could effectively inhibit tumor growth. The relative tumor inhibition rate (TGI) of the high-dose group was 91.4%, and the relative tumor inhibition rate of the low-dose group was 85.4%. The weight of the treatment group decreased to a certain extent, indicating the existence of damage related to high-dose radiation. Figure 5 HE staining showed no obvious abnormalities in the heart, liver, spleen, lung, and kidney of the treatment group and the control group.

[0097] Comparative Example 1

[0098] Direct coupling of DZ-1 68 Ga / 177 Lu, designed and synthesized 68 Ga / 177 Lu-DOTA-DZ-1. By screening the labeling conditions, a preliminary 68 Ga / 177 Lu-DOTA-DZ-1 labeling process. However, the research results ( Figure 6 )show 68 Ga / 177 Lu-DOTA-DZ-1 is unstable in vitro. 68The labeling rate of Ga-DOTA-lys-DZ-1 compound in FBS decreased from 94.9% to 90.04% after 4 h. 177 The Lu-DOTA-lys-DZ-1 compound is extremely unstable, and the 24h labeling rate in FBS dropped from 88.85% to 22.83%. The results showed that direct coupling with DZ-1 as the core 177 The radionuclide drugs constructed by Lu have serious labeling problems.

[0099] The preparation method of DOTA-DZ-1 is as follows:

[0100] Step 1:

[0101] DZ-1 (200 mg, 1.0 eq, 0.28 mmol) was dissolved in 1 mL of dichloromethane (DCM), and ethyl chloroformate (54 μL, 2.0 eq, 0.56 mmol, molecular weight: 108.52, density: 1.14 g / mL) and triethylamine (78 μL, 2.0 eq, 0.56 mmol, molecular weight: 101.19, density: 0.728 g / mL) were added, and stirred at room temperature for 1.5 hours. Subsequently, N-α-tert-butyloxycarbonyl-L-lysine (104 mg, 1.5 eq, 0.42 mmol, CAS number: 13734-28-6, molecular weight: 246.3) was added, and stirring was continued at the same temperature for 3 hours. The reaction process was monitored by thin layer chromatography (TLC, developing solvent: DCM / methanol = 7:1 + 10 μL formic acid). After the reaction was completed, 5 mL of water and 200 μL of formic acid were added to wash the organic phase once. The obtained organic layer was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (silica gel column: 25 g, flow rate: 20 mL / min), using 5% methanol (1% acetic acid solution) to elute the residual raw material, and 8% methanol (1% acetic acid solution) to elute the target product. Liquid chromatography-mass spectrometry (LC-MS) showed that the retention time of the product was 3.9 minutes (m / z = 933). After the crude product was obtained, the removal reaction of the Boc protecting group was continued. The obtained green solid was dissolved in dichloromethane (DCM), and an appropriate amount of trifluoroacetic acid (TFA) was added to achieve the removal of the Boc group. The reaction was stirred at room temperature for 1 hour. After the reaction was completed, the solvent and excess TFA were removed by rotary evaporation. The obtained product was dissolved in DCM, 5 mL of water was added for washing, and then the organic phase was dried over anhydrous sodium sulfate, filtered and then the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography, and the developing solvent was DCM / methanol = 8:1. Finally, the target compound after Boc removal - Compound 1 was obtained. Mass spectrometry analysis (LC-MS) showed that the molecular weight after Boc removal was 833.53, and the product was a green solid with the structural formula shown in Formula 6 below:

[0102]

[0103] Formula 6.

[0104] Step 2:

[0105] DOTA was activated by EDC at pH 5.5 for 30 minutes at 4 °C. The molar ratio of DOTA, EDC and Sulfo-NHS was 10:5:4. DOTA (24.2 mg, 48 μmol) was dissolved in 500 μL of water, and EDC (4.6 mg, 24 μmol) was dissolved in 130 μL of water. After the two were mixed, 0.1 N NaOH solution was added to adjust the pH to 5. Subsequently, Sulfo-NHS (4.2 mg, 19.2 μmol) was added under ice bath conditions and stirred continuously, and 0.1 N NaOH solution was used to further adjust the pH to 5.5. The reaction continued at 4 °C for 40 minutes to obtain the target product-compound 2, the structural formula of which is shown in Formula 7 below:

[0106]

[0107] Formula 7.

[0108] LC-MS analysis showed that the molecular weight of the target product was 603.53 [M+H]+

[0109] Step 3:

[0110] Compound 2 (10 mg, 12 μmol) was dissolved in 200 μL of 50% acetonitrile aqueous solution and added to the reaction mixture of compound 2. The pH was adjusted to 8.5 with 0.1 N NaOH. The reaction was incubated at 4 °C overnight. After the reaction was completed, the DZ-1-DOTA conjugate was purified by HPLC using an Apollo C18 semi-preparative column (5 μm, 250×10 mm). Two mobile phases were used for HPLC purification: mobile phase A was 0.1% trifluoroacetic acid (TFA) in water, and mobile phase B was 0.1% TFA in 80% acetonitrile in water. The mobile phase gradient changed from 40% B to 100% B in 30 minutes, the flow rate was 3 mL / min, and the monitoring wavelengths were 254 nm and 780 nm. The product (DOTA-DZ-1) was collected and freeze-dried to obtain 4 mg of the product with a yield of 28%. Mass spectrometry (ESI) analysis showed that the molecular ion peak was m / z = 1219.585 [M+H]+.

[0111] The structural formula of DOTA-DZ-1 is shown in Formula 8 below:

[0112]

[0113] Formula 8.

[0114] Comparative Example 2

[0115] The preparation method of DZ-TCO is as follows:

[0116] Step 1

[0117] The raw material DZ-1 (50 mg, 1.0 eq) was dissolved in 3 mL of dichloromethane (DCM), and ethyl chloroformate (13 μL, 2.0 eq) and triethylamine (19 μL, 2.0 eq) were added. After stirring at room temperature for 1.5 hours, N-α-tert-butyloxycarbonyl-L-lysine (26 mg, 1.5 eq) was added. The reaction was monitored by thin layer chromatography (TLC, developing solvent: DCM / methanol = 6:1). The reaction solution was washed twice with water and once with a saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum drying. The crude product was purified by silica gel column chromatography (developing solvent: DCM / methanol = 6:1) to finally obtain a green solid compound-compound 1 (the structural formula is shown in Formula 9 below). Mass spectrometry electrospray ionization (ESI) showed a molecular ion peak of m / z = 468 [2+].

[0118]

[0119] Formula 9.

[0120] Step 2:

[0121] Compound 1 (31 mg, 1.1 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 9 mg, 1.5 eq) and 1-hydroxybenzotriazole (HoBt, 6 mg, 1.2 eq) were dissolved in dichloromethane (DCM). After the mixture was stirred at room temperature for 30 minutes, the reaction substrate trans-cyclooctene (TCO, 7 mg, 1.0 eq) was added. Stirring was continued at room temperature for 1 hour, and the reaction progress was monitored by thin layer chromatography (TLC, developing solvent: DCM / methanol = 7:1). The reaction was complete after 3 hours. After the reaction was completed, the reaction solution was transferred to a separatory funnel, 2 mL of water was added, and DCM was used for extraction twice. The organic phases were combined and dried over anhydrous sodium sulfate. Subsequently, the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (developing solvent: DCM / methanol = 8:1), and the target compound was scraped and rotary evaporated to obtain pure compound 2 (structural formula shown in Formula 10 below). The molecular ion peak m / z = 538.9 [2+] was measured by mass spectrometry electrospray ionization (ESI).

[0122]

[0123] Formula 10.

[0124] Step 3:

[0125] Compound 2 (13 mg) was dissolved in 800 μL dichloromethane (DCM), trifluoroacetic acid (TFA, 75 μL) was added dropwise, and then diluted with 300 μL DCM. The reaction mixture was stirred at room temperature for 3 hours. Thin layer chromatography (TLC, developing solvent: DCM / methanol = 7:1) showed that the reactants were almost completely converted. After the reaction was completed, the pH of the reaction solution was adjusted to weak alkalinity using N,N-diisopropylethylamine (DIPEA, 75 μL) diluted in 300 μL DCM. The aqueous phase was extracted twice with DCM, the organic layers were combined, dried and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography using a developing solvent DCM / methanol = 6.5:1 to obtain 8 mg of a green solid (DZ-TCO, the structural formula is shown in Formula 11 below).

[0126]

[0127] Formula 11.

[0128] Based on DZ-1 68 Ga pre-targeted PET / CT imaging, preparation of DZ-1-TCO targeting molecules, and design and synthesis of 68 Ga-DOTA-MeTz was used as a radionuclide effector molecule to study the effects of DZ-1-TCO / 68 Ga-DOTA-MeTz pre-targeted PET / CT imaging study, the results are as follows Figure 7 As shown in the results, 68 The stability, bioorthogonal reaction rate, membrane permeability and other properties of Ga-DOTA-MeTz need to be improved.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A radionuclide drug, characterized in that: Contains a compound having a structure shown in formula B: Formula B; Wherein, M is the radioactive metal; The radioactive metal is lutetium-177.

2. Use of the radionuclide drug according to claim 1 in the preparation of anti-cervical cancer drugs.

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