Cascade targeting PET molecular probe and preparation method and application thereof

Abstract

The invention is suitable for the technical field of molecular imaging, and provides a cascade targeting PET molecular probe as well as a preparation method and application thereof, the molecular formula of the molecular probe is Glu-B-Lys-Asp-Gly-Arg-Cys-Lys (DOTA-68Ga)-CBT, and the molecular formula is simply recorded as 1-68Ga. According to the present invention, the radioactive drug labeled by the cascade targeting GGT and NRP-1 positron nuclide < 68 > Ga is constructed, and the in-situ self-assembly is performed in the tumor cell to form the nanoparticle structure so as to enhance the PET signal of the GGT and NRP-1 high expression tumor, such that the GGT and NRP-1 high expression tumor can be used for preparing the instrument for accurately diagnosing the GGT and NRP-1 related cancer.

Description

Technical Field

[0001] The present invention belongs to the field of molecular imaging technology, and in particular relates to a cascade-targeted PET molecular probe and a preparation method and application thereof. Background Art

[0002] Cancer is a major global health problem. Treatments for advanced metastatic cancer are often ineffective, but early detection remains the best option for combating this disease. Early diagnosis can significantly improve patient management, enhance treatment outcomes by providing accurate prognostic information and enabling selection of the most appropriate treatment options. Molecular imaging techniques (such as magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and single-photon emission computed tomography (SPECT)) provide valuable structural and functional information in cancer diagnosis. PET, due to its high sensitivity and deep tissue penetration, is considered the most advanced non-invasive imaging technique. However, to accurately visualize target cancer cells in PET imaging, radioactive tracers are required. After injection, these radioactive tracers are selectively distributed throughout the subject's body, and their dynamic distribution within the body is recorded by the PET scanner. Therefore, highly specific tracers play a crucial role in tumor PET imaging.

[0003] The prior art discloses that neuropilin-1 (NRP-1) is widely distributed in different types of cancers, such as breast cancer, lung cancer, gastric cancer, and pancreatic cancer. It not only enhances VEGF-dependent or -independent angiogenesis, allowing tumor tissues to obtain sufficient blood supply and nutrition, but also promotes the migration, invasion, and distal metastasis of tumor cells by regulating extracellular matrix (ECM) components and extracellular signaling pathways. Therefore, NRP-1 is considered to be an attractive target for the diagnosis and treatment of tumors. The prior art also discloses that the C-terminal rule peptide (the R / KXXR / K motif must be exposed at the C-terminus of the polypeptide chain to be active) structure can specifically bind to NRP-1. Since NRP-1 is also expressed in some normal organs such as the heart, liver, and brain, the construction of a drug delivery strategy targeting NRP-1 with a C-terminal rule peptide needs further improvement and optimization. The journal Analytical Chemistry (Anal Chem. 2019, 91(21), 13639-13646) disclosed that gamma-glutamyl transpeptidase (GGT) is widely distributed in different types of cancers, including liver cancer, breast cancer, and pancreatic cancer. It is mainly involved in maintaining the homeostasis of GSH and cysteine ​​in cells and can also promote cell proliferation and metastasis. The journal Nature Nanotechnology (Nat Nanotechnol. 2019;14(8):799-809) disclosed that GGT is highly expressed in tumor vascular endothelial cells. Therefore, designing a GGT and NRP-1 cascade targeting can effectively improve the accuracy of drug delivery and reduce the uptake of normal tissues.

[0004] Currently, most of the PET imaging agents commonly used in clinical practice are small molecules, which have the problems of easy off-target effects and short retention time in the target area, thus affecting the tumor imaging effect. The prior art discloses a click condensation reaction between 2-cyanobenzothiazole (CBT) and D-cysteine ​​(Cys), which can convert small molecules into amphiphilic oligomers after entering cells, and further self-assemble into nanostructures. Due to the hydrophobicity of the formed nanostructures, they are not easily pumped out of the cell membrane, thereby increasing their concentration and retention time in the cell. Using this "smart" strategy of self-assembling small molecule precursors into nanostructures in cells can significantly improve drug delivery efficiency and molecular imaging effects. In addition, the self-assembly process triggered by this click condensation reaction has the advantages of mild conditions, high reaction efficiency, stable products and good biocompatibility. It has been widely used in the detection of intracellular biomolecules, tumor imaging and drug delivery. To date, there has been no research on the use of cascade targeting of GGT and NRP-1 to deliver positron emission tonics and the use of the click condensation reaction between 2-cyanobenzothiazole and D-cysteine ​​to form in situ based on 68 Literature reports on research work on the use of Ga nanostructures to simultaneously enhance the PET signals of tumors with high GGT and NRP-1 expression for the diagnosis of GGT and NRP-1 related cancers. Summary of the Invention

[0005] The purpose of the embodiments of the present invention is to provide a cascade-targeted PET molecular probe, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The embodiment of the present invention is achieved as follows: a cascade-targeted PET molecular probe is formed by GGT activating an RGDK structure and combining it with NRP-1 to deliver radionuclides and self-assemble in cells, and contains a GGT-specific enzyme cleavage substrate (γ-Glu), an RGDK structure exposed by GGT cleavage, a potential cysteine ​​(Cys) motif with a disulfide bond, and a 2-cyano-benzothiazole (CBT) and a chelating agent bound to a lysine (Lys) side chain. 68 Ga 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA- 68 Ga) structure of a small molecule compound with the molecular formula of Glu-B-Lys-Asp-Gly-Arg-Cys-Lys(DOTA- 68 Ga)-CBT, abbreviated as 1- 68 Ga, the structural formula is:

[0007] .

[0008] Another object of the present invention is to provide a method for preparing a cascade targeted PET molecular probe, comprising the following steps:

[0009] Dissolve compound 1 in a vacuum bottle with sodium acetate solution and elute the positron nuclide from the Ge-Ga generator with 0.1M hydrochloric acid solution. 68 Ga was added to the above vacuum bottle and reacted at 95℃ for 10 minutes to purify the positron nuclide. 68 Ga-labeled radiopharmaceutical molecules, namely 1- 68 Ga;

[0010] The structural formula of the compound 1 is shown below:

[0011] .

[0012] Another object of an embodiment of the present invention is to provide an application of a cascade targeted PET molecular probe in the preparation of a kit for diagnosing tumors with high expression of GGT and NRP-1.

[0013] Another object of the embodiment of the present invention is to provide an application of a cascade-targeted PET molecular probe in the preparation of a kit for imaging tumors with high expression of GGT and NRP-1.

[0014] The present invention provides an intelligent PET molecular probe targeting neuropilin-1 (NRP-1) activated by glutamyl transpeptidase (GGT) to enhance the accuracy of tumor radionuclide delivery and prolong the retention time of the nuclide in the tumor. The probe contains a GGT enzyme-specific enzymatic substrate (γ-Glu), an RGDK sequence that specifically binds to NRP-1, a potential cysteine ​​motif with a disulfide bond, and a 2-cyano-benzothiazole and a chelated positron-emitting nuclide bound to a lysine side chain. 68 Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid can form a C-terminal regular peptide (RGDK) under the action of GGT, and then specifically bind to NRP-1 on the surface of tumor cells to penetrate tumor cells, and a click reaction between 2-cyano-benzothiazole and D-cysteine ​​occurs inside the cells and self-assembles in situ to form a 68 The nanoparticle structure of Ga effectively increases the positron nuclide 68 The precision of Ga delivery and the concentration in tumor cells prolong the 68 The retention time of Ga in the target area can enhance the radionuclide uptake of tumors with high expression of GGT and NRP-1, so it can be used for early and accurate diagnosis of GGT and NRP-1 related cancers. The embodiment of the present invention fills the gap between cascade targeted precise delivery of positron nuclides and in situ formation of linked nuclides by intracellular click reaction. 68 Ga nanoparticle structures can simultaneously enhance the PET signals of tumors with high expression of GGT and NRP-1, thereby diagnosing GGT and NRP-1 related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A cascade targeted PET molecular probe provided by the embodiment of the present invention is formed by in situ self-assembly in cells 68 Schematic diagram of Ga-NPs simultaneously enhancing PET signals;

[0016] Figure 2 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound A prepared in Example 1 of the present invention;

[0017] Figure 3 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound B prepared in Example 1 of the present invention;

[0018] Figure 4 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound C prepared in Example 1 of the present invention;

[0019] Figure 5 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound D prepared in Example 1 of the present invention;

[0020] Figure 6 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound E prepared in Example 1 of the present invention;

[0021] Figure 7 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound F prepared in Example 1 of the present invention;

[0022] Figure 8 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound G prepared in Example 1 of the present invention;

[0023] Figure 9 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound H prepared in Example 1 of the present invention;

[0024] Figure 10 This is a diagram showing the results of mass spectrometry (ESI-MS) analysis of pure compound I prepared in Example 1 of the present invention;

[0025] Figure 11 This is a diagram showing the mass spectrometry (ESI-MS) analysis results of pure compound J prepared in Example 1 of the present invention;

[0026] Figure 12 This is a diagram showing the results of high-resolution mass spectrometry (HR-MALDI / MS) analysis of pure compound 1 prepared in Example 1 of the present invention;

[0027] Figure 13 The hydrogen spectrum of the pure compound 1 prepared in Example 1 of the present invention ( 1 H-NMR) analysis results diagram;

[0028] Figure 14 The carbon spectrum of the pure compound 1 prepared in Example 1 of the present invention ( 13 C-NMR) analysis results diagram;

[0029] Figure 15 1- prepared in Example 1 of the present invention 68 Stability results of Ga incubated in physiological saline, phosphate buffer, mouse serum and 5% human serum for different time periods;

[0030] Figure 16 The experimental results of in vitro enzymatic cleavage of compound 1 prepared in Example 1 of the present invention at different time points and different enzyme concentrations are shown;

[0031] Figure 17 This is the in vitro electron microscopy result of the formation of NPs by compound 1 prepared in Example 1 of the present invention;

[0032] Figure 18 This is an intracellular electron microscopy result showing that compound 1 prepared in Example 1 of the present invention forms NPs in cells;

[0033] Figure 19 1- prepared in Example 1 of the present invention 68 Ga uptake rate results at different time points in BxPC-3 cells, BxPC-3 cells pretreated with DON inhibitor, BxPC-3 cells pretreated with NRP-1 inhibitor, and 3T3 cells;

[0034] Figure 20 1- prepared in Example 1 of the present invention 68 Results of the affinity experiment of Ga in BxPC-3 cells;

[0035] Figure 21 1- prepared in Example 1 of the present invention 68 The drug concentration-time curve results of Ga in vivo;

[0036] Figure 22 1- prepared in Example 1 of the present invention 68 The biodistribution results of Ga in normal Kunming mice;

[0037] Figure 23 A is the intravenous injection of 1- 68 Ga BxPC-3 tumor-bearing mice (top row), pretreated with GGT inhibitors and then injected with 1- 68 Ga BxPC-3 tumor-bearing mice (middle row) were pretreated with NRP-1 inhibitors and then injected with 1- 68Coronal and transverse PET / MR fusion images of Ga BxPC-3 tumor-bearing mice (bottom row) at 10, 30, 60, 90, 120, 150, 180, 210, and 240 minutes; Figure 23 B is the intravenous injection of 1- 68 Ga group, injected with GGT inhibitor DON and then injected with 1- 68 Ga group and NRP-1 inhibitor A7R were injected after 1- 68 Tumor-to-muscle ratios at different time points in the Ga group; Figure 23 C is the intravenous injection of 1- 68 Ga group, injected with GGT inhibitor DON and then injected with 1- 68 Ga group and NRP-1 inhibitor A7R were injected after 1- 68 The tumor-to-liver ratios at different times in the Ga group. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0040] Example 1: A cascade-targeted PET molecular probe, the synthesis route of which is as follows:

[0041]

[0042]

[0043]

[0044] The specific steps include:

[0045] Step 1: Dissolve 3 mmol (910 mg) of amino acid Boc-Glu-OtBu, 3 mmol (1140 mg) of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), and 6 mmol (1.04 ml) of N,N-diisopropylethylamine (DIPEA) in 20 ml of anhydrous tetrahydrofuran, add 3 mmol (369 mg) of p-aminobenzyl alcohol at 0°C and react for 10 minutes, then at room temperature for 3 hours, then remove the reaction solvent by rotary evaporation, extract with ethyl acetate three times, and finally freeze-dry to obtain the first compound, named Compound A: ;

[0046] Step 2: The peptide chain Fmoc-Cys(Trt)-Lys(Boc) was synthesized by solid phase synthesis and dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF) with 2 mmol (222 μL) of 4-methylmorpholine (MMP). The mixture was cooled to 0°C and 2 mmol (254 μL) of isobutyl chloroformate (IBCF) was added. The reaction mixture was stirred at 0°C for 40 minutes. Then, 1 mmol (17.5 mg) of 2-cyano-6-aminobenzothiazole (CBT) was dissolved in anhydrous DMF and added to the reaction. Stirring was continued at 0°C for 1 hour, and then the reaction was stirred at room temperature overnight. The second pure compound was separated and purified by high performance liquid chromatography (HPLC) to obtain compound B: ;

[0047] Step 3: Dissolve 0.5 mmol (407 mg) of compound B in 10 mL of 50% trifluoroacetic acid (TFA) solution (5 mL of trifluoroacetic acid, 4.95 mL of dichloromethane, and 50 μL of triisopropylsilane). Stir at room temperature for 3 hours to remove the protecting groups trityl (Trt) and tert-butyloxycarbonyl (Boc) on compound B. Then, remove the trifluoroacetic acid solution by rotary evaporation. Purify the product by high performance liquid chromatography to obtain a third pure compound, named compound C: ;

[0048] Step 4: Take 0.5 g (0.55 mmol) of 2-chlorotrityl resin and put it into a glass ox horn tube, add 2 mL of anhydrous N, N-dimethylformamide (DMF) to activate the resin and remove impurities, weigh 468.5 mg (1 mmol) of amino acid Fmoc-Lys(Boc)-OH, dissolve it in 2 mL of anhydrous DMF in a 5 mL centrifuge tube, add 174 μL (1 mmol) of N, N-diisopropylethylamine (DIPEA), and mix it evenly with ultrasound to obtain the first amino acid solution. After the resin activation is completed, use an ear bulb to pressurize and remove DMF from the top of the chromatography column, then add the first amino acid solution, and react at room temperature for 8 hours. After the reaction is complete, wash the resin 3 times with 2 mL of anhydrous DMF to remove the unreacted amino acid, and then add 45 μL of methanol and 2 mL DMF was added and reacted for 40 minutes to block the unreacted chlorine active sites on the resin. Then, 20% piperidine solution was added to the ox horn tube and reacted for 10 minutes. The 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid Lys was removed three times. 345 mg (0.84 mmol) of amino acid Fmoc-Asp(OtBu)-OH, 109 mg (0.84 mmol) of anhydrous 1-hydroxybenzotriazole (HOBt) and 315 mg (0.84 mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were dissolved in 2 mL of anhydrous DMF in a 5 mL centrifuge tube, and 145 μL (0.84 mmol) of DIPEA was added to obtain a second amino acid solution. The second amino acid solution was added to the ox horn tube. The reaction was carried out for 8 hours; the amino acids in the above steps were replaced with the amino acid Fmoc-Gly-OH and the amino acid Fmoc-Arg(Pbf)-OH in sequence to prepare amino acid solutions, which were added to a ox horn tube for reaction. The amount of each solution was 0.84 mmol, corresponding to a mass of 250 mg and 545 mg, respectively. The amounts of anhydrous HOBt, HBTU, and DIPEA remained unchanged. After the reaction, a dichloromethane solution containing 1% trifluoroacetic acid (300 μL of TFA, 30 mL of DCM) was added to the ox horn tube in portions to cut the oligopeptide from the resin. About 50 ml of ether was added, and the oligopeptide was dispersed in the ether by ultrasound. The oligopeptide was refrigerated and centrifuged at 4000 r / min for 20 min. The ether in the centrifuge tube was discarded, and the oligopeptide was lyophilized in a freeze dryer to obtain a dry oligopeptide, which was named Compound D: ;

[0049] Step 5: Dissolve 0.47 mmol (514 mg) of compound D, 0.47 mmol (188 mg) of compound A, 1.41 mmol (291 mg) of dicyclohexylcarbodiimide (DCC), and 1.41 mmol (172 mg) of 4-dimethylaminopyridine (DMAP) in 10 ml of dichloromethane solution and react overnight. After the reaction, the mixture was spin-dried and purified by high performance liquid chromatography to obtain a fifth compound, named compound E: ;

[0050] Step 6: The N-fluorenylmethoxycarbonyl (Fmoc) protecting group of Compound E was reacted with a 5% piperidine-containing N,N-dimethylformamide solution at 0°C for 10 minutes with stirring, followed by the addition of 270 μL of trifluoroacetic acid to neutralize the base. Purification by HPLC afforded a sixth pure compound, designated Compound F: ;

[0051] Step 7: Chemical F was dissolved in anhydrous DMF with 1H-benzo[d][1,2,3]triazol-1-yl(2-(pyridin-2-yldisulfonyl)ethyl) carbonate and DIPEA at a ratio of 1:1:3. The mixture was reacted at room temperature for 12 hours. The mixture was separated and purified by high performance liquid chromatography to obtain the seventh pure compound, named Compound G: ;

[0052] Step 8: Dissolve chemical G, compound C, and DIPEA in anhydrous DMF at a ratio of 1:1:1, react at room temperature for 3 hours, and separate and purify by high performance liquid chromatography to obtain the eighth pure compound, named compound H: ;

[0053] Step 9: Compound H and 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid were dissolved in anhydrous DMF at a ratio of 1:1. The reaction system was then adjusted to pH 6-7 with DIPEA. The reaction was allowed to react at room temperature for 3 hours. The ninth pure compound was separated and purified by high performance liquid chromatography and named Compound I: ;

[0054] Step 10: The N-fluorenylmethoxycarbonyl (Fmoc) protecting group of Compound I was reacted with a 5% piperidine-containing N,N-dimethylformamide solution at 0°C with stirring for 10 minutes, followed by the addition of 270 μL of trifluoroacetic acid to neutralize the base. Purification by HPLC afforded a tenth pure compound, designated Compound J: ;

[0055] Step 11: Compound J was dissolved in 10 mL of 50% trifluoroacetic acid (TFA) solution (5 mL of trifluoroacetic acid, 4.95 mL of dichloromethane, and 50 μL of triisopropylsilane), and stirred at room temperature for 3 hours to remove the protecting groups tert-butyl ester (OtBu), tert-butyloxycarbonyl (Boc), and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) on compound J. The trifluoroacetic acid solution was then removed by rotary evaporation. After separation and purification by high performance liquid chromatography, the eleventh pure compound was obtained and named compound 1: ;

[0056] Step 12: Dissolve 50 μg of compound 1 in a vacuum bottle with 125 μL (1.25 M) sodium acetate solution and elute the positron nuclide from the Ge-Ga generator with 2 ml (0.1 M) high-purity hydrochloric acid solution. 68 Ga, take 1 ml and add it to the above vacuum bottle, react on a 95 degree heater for 5 minutes, take out the system with a 2 mL syringe and measure it, and then purify it with a Sep-pak column to obtain the positron nuclide 68 The radioactive molecular drug labeled with Ga is named 1- 68 Ga: .

[0057] 1- prepared in Example 1 68 Schematic diagram of the self-assembly process of Ga under the action of GGT to activate NRP-1 and its intracellular self-assembly into nanoparticles for enhanced radionuclide uptake imaging. Figure 1 As shown, from Figure 1 It can be seen that when 1- 68 After being activated by GGT highly expressed on the surface of tumor vascular endothelial cells or tumor cells, Ga binds to NRP-1 on the cell surface and enters the cell. After the self-assembly triggered by intracellular GSH reduction and CBT-Cys click condensation reaction, Ga forms in situ in cancer cells. 68 Ga nanoparticles, this process not only improves the positron nuclide 68 The precision of Ga delivery increases its intracellular concentration and prolongs 68 The retention time of Ga can be prolonged, thereby enhancing the radionuclide imaging effect of tumors in vivo.

[0058] The pure compounds AJ and 1 were qualitatively analyzed by mass spectrometry, and the structures were analyzed by nuclear magnetic resonance. The mass spectra of the pure compounds AJ were as follows: Figure 2-Figure 11 As shown, the high-resolution mass spectrum of pure compound 1 is as follows Figure 12 As shown, the mass spectrometry results are obsvd.HR-MALDI-MS[(M+H)+]: m / z 1063.5413; the nuclear magnetic resonance hydrogen spectrum is as shown Figure 13As shown, H NMR (d6-dimethyl sulfoxide, 400 MHz) δ 10.80 (s, 1H), 10.16 (s, 1H), 9.06 (s, 1H),8.75 (d, J = 3.0 Hz, 2H), 8.41 (s, 3H), 8.19 (s, 3H), 7.94 (s, 1H), 7.84 (s,2H), 7.78 (d, J = 9.0 Hz, 3H), 7.58 (d, J = 8.2 Hz, 3H), 7.46 (d, J = 8.4 Hz,3H), 7.08 (s, 1H), 5.34 – 5.29 (m, 1H), 5.03 (s, 3H), 4.62 (s, 2H), 3H), 4.52 (d, J = 6.7 Hz, 2H), 4.17 (s, 4H), 4.10 (s, 3H), 3.97 (s, 4H), 3.73 (s, 5H), 3.57(s, 5H), 3.40 (s, 5H), 3.08 (s, 9H), 2.91 (d, J = 17.9 Hz, 6H), 2.72 (s, 5H),2.23 (s, 2H), 2.07 (s, 3H), 2.00 (d, J = 8.0 Hz, 1H), 1.66 (s, 3H), 1.51–1.47 (m, 6H), 1.23 (d, J = 5.1 Hz, 8H). Figure 14 As shown, the carbon NMR spectrum (d6-dimethyl sulfoxide, 101 MHz) δ 172.60, 172.02, 171.40, 170.44, 169.14, 167.51, 162.90,159.22, 158.89, 157.43, 156.40, 148.32, 137.28, 137.15, 135.80, 129.24,121.61, 112.77, 112.05, 66.41, 62.52, 55.52 – 52.92 (m), 52.10, 51.64, 46.19, 42.72, 42.34, 40.94, 39.07, 36.35, 35.67, 32.04, 31.32, 29.59, 29.14, 26.91,26.19, 25.60, 23.43, 22.64, 20.98.

[0059] 1- 68 Ga in vitro stability analysis: Radio-HPLC was used to analyze the 1-68 Ga was used for in vitro stability analysis, and 7.4 MBq of 1- 68 Ga was incubated with normal saline, phosphate buffer, mouse serum and 5% human serum for 5 minutes, 30 minutes, 60 minutes and 120 minutes respectively, and then detected by high performance liquid chromatography to obtain 1- 68 The in vitro stability results of Ga are as follows Figure 15 As shown, it can be seen that 1- 68 Ga can maintain good stability after incubation in physiological saline, phosphate buffer, mouse serum and 5% human serum solutions for 120 minutes, and the radiochemical purity is greater than 95%.

[0060] The experimental results of in vitro enzymatic cleavage of compound 1: 200 μL of compound 1 (200 μM) prepared in Example 1 was incubated with 0.05U, 0.1U, 0.2U, 0.3U and 0.4U of GGT enzyme, respectively; 200 μL of compound 1 (200 μM) prepared in Example 1 was incubated with 0.4UGGT enzyme for 0.5, 1, 1.5 and 2 hours, respectively; and the results of in vitro enzymatic cleavage of compound 1 prepared in Example 1 using 0.4U of GGT were obtained. The enzymatic cleavage process was detected by high performance liquid chromatography, and the results were as follows. Figure 16 shown.

[0061] The experimental results of compound 1 forming NPs in vitro: Compound 1 (200 μM) prepared in Example 1 was incubated with 0.4 U of GGT enzyme at 37°C for 2 hours, and then 8 μL of tris(2-carboxyethyl)phosphine (100 mM) was added and incubated at 37°C for 30 minutes. After that, the solution was dripped onto a copper grid to prepare an electron microscopy sample. The results are shown in FIG. Figure 17 shown.

[0062] Intracellular electron microscopy experimental results of compound 1 forming NPs in cells: Complete culture medium containing 200 μM compound 1 was incubated with BxPC-3 cells at 37°C and 5% CO2 for 4 hours. The BxPC-3 cells collected after centrifugation were resuspended in electron microscopy fixative, and cell electron microscopy sections were prepared and photographed using a 120 kV transmission electron microscope (Talo L120C G2). The results are as follows: Figure 18 shown.

[0063] 1- 68 Ga uptake in BxPC-3 cells and 3T3 cells:

[0064] BxPC-3 cells were divided into two groups:

[0065] Experimental group: 74kBq of 1- 68 Ga was co-incubated with BxPC-3 cells for 5 min, 15 min, 30 min, 60 min, 90 min, and 120 min, respectively;

[0066] Control group: 2mM DON (a GGT inhibitor) or 100μM A7R (an NRP-1 inhibitor) were pretreated for 30 minutes, and then 74kBq 1- 68 Ga was co-incubated for 5 min, 15 min, 30 min, 60 min, 90 min, and 120 min;

[0067] 74kBq of 1- 68 Ga was co-incubated with 3T3 cells for 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively, and then the radioactivity of the above four groups of cells was counted using a gamma counter to obtain the radioactivity of BxPC-3 cells and 3T3 cells at different times. 68 The Ga uptake rate is as follows: Figure 19 As shown, it can be seen that BxPC-3 cells are sensitive to radiopharmaceuticals 1- 68 The uptake of Ga increased gradually from 5 min to reach a peak at 60 min, and was higher than that of BxPC-3 cells and 3T3 cells pretreated with inhibitors at the same time point.

[0068] 1- 68 The affinity of Ga in BxPC-3 cells: 0.1nM, 1nM, 5nM, 10nM, 50nM, 100nM of 1- 68 Ga was co-incubated with BxPC-3 cells for 1 hour, and then radioactivity was counted using a gamma counter. The results were as follows: Figure 20 As mentioned above, it can be seen that 1- 68 The affinity curve of Ga for BxPC-3 cells was calculated, and the affinity constant Kd value was 7.05, indicating that 1- 68 Ga has a high affinity for BxPC-3 cells.

[0069] 1- 68 Pharmacokinetic analysis of Ga in normal mice: 1- 68 Ga (200 μL, 1.85 MBq) was injected into Kunming mice (n=5) via the tail vein. Blood was collected from the orbital venous plexus at 2, 5, 10, 15, 30, 45, 60, 90, and 120 minutes, weighed, and radioactivity was measured using a γ counter. %ID / g was calculated. The results are shown in Figure 2. Figure 21 As shown, it can be seen that the distribution half-life of the curve is 4.75 minutes and the elimination half-life is 19.63 minutes.

[0070] 1- 68 Analysis of the biodistribution of Ga in normal mice: 12 Kunming mice were randomly divided into 4 groups, 3 mice in each group, and 1- 68Ga (200 μL, 1.85 MBq) was injected into Kunming mice via the tail vein. Mice were killed 5, 30, 60, and 120 minutes after injection. The heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, bone, muscle, brain, and blood were collected after dissection. The weights were weighed and the radioactivity was measured using a γ counter. The %ID / g was calculated and the results were obtained as shown below. Figure 22 As shown, it can be seen that at 5, 30, 60, and 120 minutes, 1- 68 The distribution of Ga in normal mice showed that 1- 68 Ga in mice is mainly metabolized through the urinary system, and a very small amount is metabolized through the liver.

[0071] Analysis of GGT-induced NRP-1 activation 68 The formation of Ga nanoparticles improves the accuracy and efficiency of radionuclide delivery, enhances the uptake of radionuclide in tumors in vivo and prolongs the retention time: 2 million BxPC-3 cells overexpressing GGT and NRP-1 were implanted subcutaneously in the right armpit of each Balb / c nude mouse. When the tumor diameter reached 5-8 mm, the nude mice were randomly divided into 3 groups: experimental group 1- 68 Mice in Ga were intravenously injected with 7.4 MBq (200 μL) of 1- 68 Ga; control group 1- 68 Mice in the Ga+DON group were first intravenously injected with 0.25 mmol / kg DON (a GGT inhibitor), and half an hour later, 7.4 MBq (200 μL) of 1- 68 Ga; control group 1- 68 Mice in Ga+A7R were first intravenously injected with 10 mg / kg of A7R (an NRP-1 inhibitor), and half an hour later, 7.4 MBq (200 μL) of 1- 68 Ga; Dynamic PET-MR coronal and cross-sectional imaging of mice was performed on a PET-MR scanner, and the results were as follows Figure 23 As shown;

[0072] Figure 23 A is intravenous injection of 1- 68 Ga BxPC-3 tumor-bearing mice (top row), pretreated with GGT inhibitors and then intravenously injected with 1- 68 Ga BxPC-3 tumor-bearing mice (middle row) were pretreated with NRP-1 inhibitors and then intravenously injected with 1- 68Coronal and transverse PET / MR fusion images of Ga-treated BxPC-3 tumor-bearing mice (bottom row) at 10, 30, 60, 90, 120, 150, 180, 210, and 240 minutes. The radioactive drug was significantly taken up by the tumors of the experimental group of mice and retained in the tumors for a long time. The radioactive drug was less taken up by the tumors of the mice in the GGT blockade and NRP-1 blockade groups, and was cleared more quickly.

[0073] Depend on Figure 23 B shows that injection 1- 68 The ratio of tumor to muscle radioactivity uptake in mice injected with Ga gradually increased from 10 minutes to the highest at 120 minutes, reaching 5.21 ± 0.14, and then slowly decreased to 3.92 ± 0.05 at 240 minutes. The ratio of tumor to muscle in mice injected with GGT blockade and NRP-1 blockade groups gradually decreased from 10 minutes to 1.53 ± 0.05 and 2.53 ± 0.10 at 120 minutes, respectively. 68 The ratio of radioactive uptake in tumor and muscle of Ga mice at 120 minutes was 3.41 and 2.06 times that of GGT-blocked and NRP-1-blocked groups, respectively. Figure 23 C can be seen that injection 1- 68 The ratio of radioactive uptake of tumor to liver in mice injected with Ga was the highest at 120 minutes, which was 2.05 ± 0.19. The ratio of radioactive uptake of tumor to liver in mice injected with GGT blockade and NRP-1 blockade groups was 0.76 ± 0.06 and 1.15 ± 0.16 at 120 minutes, respectively. 68 The ratio of radioactive uptake in tumor and liver of Ga mice at 120 minutes was 2.70 and 1.78 times that of GGT blockade and NRP-1 blockade groups, respectively. These results indicate that 1- 68 Ga is formed under the guidance of GGT activation of NRP-1 68 Ga-NPs can be precisely delivered to tumors overexpressing GGT and NRP-1, significantly improving tumor uptake, prolonging the retention time of radionuclides, and enhancing PET signals.

[0074] In summary, GGT activation of NRP-1 targeting can greatly improve the accuracy and efficiency of positron emission tomography delivery, and the intracellular in situ self-assembly formed 68 Ga-CBT-NPs increased 68 The local concentration and retention time of Ga in the tumor site significantly enhance the PET signal of tumors overexpressing GGT and NRP-1 in vivo. Therefore, the cascade response and intracellular click reaction-based smart PET molecular probe 1- 68 The Ga imaging method is expected to be used to prepare clinical equipment for diagnosing malignant tumors with overexpression of GGT and NRP-1.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cascade targeted PET molecular probe, characterized in that: The molecular formula of the molecular probe is Glu-B-Lys-Asp-Gly-Arg-Cys-Lys (DOTA- 68 Ga)-CBT, abbreviated as 1- 68 Ga, the structural formula is: 。 2. A method for preparing a cascade-targeted PET molecular probe according to claim 1, characterized in that: The following steps are involved: Dissolve compound 1 in a vacuum bottle with sodium acetate solution and elute the positron nuclide from the Ge-Ga generator with hydrochloric acid solution. 68 Ga was added to the above vacuum bottle, heated to react, and purified to obtain the positron nuclide 68 Ga-labeled radiopharmaceutical molecules, namely 1- 68 Ga; The structural formula of the compound 1 is shown below: 。 3. The method for preparing the cascade targeted PET molecular probe according to claim 2, characterized in that: The preparation method of compound 1 comprises the following steps: Amino acid Boc-Glu-OtBu, O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were dissolved in anhydrous tetrahydrofuran, p-aminobenzyl alcohol was added and reacted at low temperature, then at room temperature, rotary evaporated, extracted and freeze-dried to obtain compound A; The peptide chain Fmoc-Cys(Trt)-Lys(Boc) was synthesized by a solid-phase synthesis method and dissolved with 4-methylmorpholine in anhydrous N,N-dimethylformamide. The mixture was cooled, and isobutyl chloroformate was added and stirred. Then, 2-cyano-6-aminobenzothiazole was dissolved in anhydrous DMF and added to the reaction mixture. The mixture was stirred and reacted at room temperature overnight. Compound B was isolated and purified. Compound B was dissolved in 50% trifluoroacetic acid solution, stirred at room temperature, and subjected to rotary evaporation for separation and purification to obtain compound C; 2-Chlorotrityl resin was placed in a glass ox horn tube, and anhydrous N,N-dimethylformamide was added for activation. The amino acid Fmoc-Lys(Boc)-OH was weighed and dissolved in a centrifuge tube with anhydrous DMF. N,N-diisopropylethylamine was added and ultrasonically mixed to obtain the first amino acid solution. After the resin activation was completed, DMF was removed and the first amino acid solution was added to react at room temperature. After the reaction was completed, methanol and DMF were added to cap the unreacted resin. The resin was washed with anhydrous DMF. After washing, 20% piperidine solution was added to the ox horn tube for reaction. The reaction was repeated several times to remove the Fmoc protecting group on the amino acid. The amino acid Fmoc-Asp(OtBu)-OH, anhydrous 1-hydroxy Benzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in anhydrous DMF in a centrifuge tube, and then DIPEA is added to obtain a second amino acid solution, which is added to a cow horn tube for reaction; the amino acids in the above steps are replaced with amino acids Fmoc-Gly-OH and Fmoc-Arg(Pbf)-OH in sequence to prepare amino acid solutions, which are sequentially added to cow horn tubes for reaction; after the reaction, a dichloromethane solution containing 1% trifluoroacetic acid is added to the cow horn tubes in portions, and the oligopeptide is cut from the resin, dried by spin drying, added with ether, and the oligopeptide is dispersed in the ether by ultrasonication, refrigerated centrifuged, and freeze-dried to obtain a dry oligopeptide, which is compound D; Compound D, compound A, dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in dichloromethane solution and reacted overnight. After the reaction, the mixture was dried by rotary evaporation and separated and purified to obtain compound E. Compound E was reacted with N,N-dimethylformamide solution containing 5% piperidine under stirring at low temperature, and then trifluoroacetic acid was added for neutralization, and compound F was obtained after separation and purification; Compound F was dissolved in anhydrous DMF with 1H-benzo[d][1,2,3]triazol-1-yl(2-(pyridin-2-yldisulfonyl)ethyl) carbonate and DIPEA, and the mixture was reacted at room temperature. Compound G was then isolated and purified. Compound G, compound C and DIPEA were dissolved in anhydrous DMF, reacted at room temperature, and separated and purified to obtain compound H; Compound H and 2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid were dissolved in anhydrous DMF, the pH was adjusted to neutral, the reaction was carried out at room temperature, and compound I was obtained after separation and purification; The N-fluorenylmethoxycarbonyl protecting group of compound I was reacted with N,N-dimethylformamide solution containing 5% piperidine under low temperature stirring, and then trifluoroacetic acid was added for neutralization, and compound J was obtained after separation and purification; Compound J was dissolved in 50% trifluoroacetic acid solution, stirred at room temperature, and subjected to rotary evaporation to obtain compound 1 after separation and purification.

4. The method for preparing the cascade targeted PET molecular probe according to claim 4, characterized in that: The structural formula of the compound A is: ; The structural formula of compound B is: ; The structural formula of compound C is: ; The structural formula of compound D is: ; The structural formula of compound E is: ; The structural formula of compound F is: ; The structural formula of compound G is: ; The structural formula of compound H is: ; The structural formula of compound I is: ; The structural formula of compound J is: .

5. Use of the cascade-targeted PET molecular probe according to claim 1 in the preparation of a kit for diagnosing tumors with high expression of GGT and NRP-1.

6. Use of the cascade-targeted PET molecular probe according to claim 1 in preparing a kit for imaging tumors with high expression of GGT and NRP-1.

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