A PET / MR dual-modality molecular probe responsive to legumain constructed intracellularly and its application

Abstract

The present invention discloses an intracellularly constructed legumain-responsive PET / MR dual-modal molecular probe and its application, which belongs to the field of molecular imaging technology. Among them, the PET / MR dual-modal molecular probe includes a radiopharmaceutical containing 68 gallium element and / or a magnetic resonance contrast agent containing gadolinium element; the radiopharmaceutical and the magnetic resonance contrast agent are synthesized from the same precursor molecule; the structural formula of the precursor molecule is: #imgabs0# This PET / MR dual-modal molecular probe can construct 68 nanoparticle structures of gallium and gadolinium in situ in tumors to simultaneously enhance the PET signal of tumors with high legumain expression and T1-weighted magnetic resonance tumor imaging, so as to accurately diagnose legumain-related cancers.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular imaging, and in particular to a PET / MR dual-modality molecular probe responsive to legumain constructed in a cell and application thereof. Background Art

[0002] According to the German Journal of Angewandte Chemie (Angew. Chem. Int. ed. 2023, 62(22), e202302303), legumain is widely distributed in different types of cancers, such as breast cancer, colon cancer, lung cancer, gastric cancer, lymphoma, melanoma, brain cancer, etc. It is mainly involved in degrading intracellular and extracellular matrices, inducing the formation of tumor angiogenesis, promoting tumor growth, infiltration and metastasis, and plays an important role in the occurrence and development of tumors. Therefore, legumain is considered to be an attractive target for the diagnosis and treatment of tumors.

[0003] At present, there are many imaging technologies for detecting or imaging legumain. ACS Sensors (ACS Sens. 2023, 8 (12), 4473-4477) reported the use of fluorescence / photoacoustic imaging technology to detect the activity of legumain; ACS Nano (ACS Nano. 2015, 9 (5), 5117-5124) reported the use of magnetic resonance imaging technology to detect the activity of legumain; Analytical Chemistry (Anal. Chem. 2020, 92, 11627-11634) reported the use of positron emission tomography imaging technology to detect the activity of legumain. Although there are many detection methods, they all have certain limitations. For example, optical imaging technology has poor diagnostic specificity for deep tissue tumors due to tissue autofluorescence and shallow tissue penetration depth; although magnetic resonance imaging technology has high temporal and spatial resolution, it has poor sensitivity for early diagnosis of diseases; although positron emission tomography technology can dynamically detect the metabolic changes of radioactive drugs or metabolites in the human body at the molecular level, achieving earlier, more sensitive and more accurate diagnosis of diseases, it has low spatial resolution and poor diagnostic specificity for diseases.

[0004] PET-MR is an advanced device that combines positron emission tomography (PET) and magnetic resonance imaging (MRI). It has the advantages of multi-parameter, multi-sequence, and high soft tissue resolution of magnetic resonance imaging, and at the same time has the detection sensitivity and molecular targeting of PET imaging to provide information on human physiological metabolism. It can achieve time-space synchronization and obtain precise consistency in time and space, playing an important role in the early diagnosis, staging, and efficacy evaluation of tumors.

[0005] At present, most of the PET imaging agents commonly used in clinical practice are small molecules, which are easily excreted by cells and have a short retention time, thus affecting the tumor imaging effect. The click condensation reaction between 2-cyanobenzothiazole (CBT) and D-cysteine ​​(Cys) reported in Nature Chemistry (Nat.Chem.2010,2,54-60) can convert small molecules into amphiphilic oligomers after entering cells, and further self-assemble into nanostructures. Due to the hydrophobicity of the nanoparticle structure, it is not easy to be pumped out by cells. The "smart" strategy of assembling small molecule precursors into nanostructures in cells can significantly improve the molecular imaging effect and drug delivery efficiency. In addition, the self-assembly process triggered by the click condensation reaction has the advantages of mild conditions, efficient reaction, stable products and good biocompatibility, and has been widely used in the detection of intracellular biomolecules, tumor imaging and drug delivery. The journal JACS (J.Am.Chem.Soc.2022,144,7667-7675) reported that the competition between free cysteine ​​in the body and the cysteine ​​in the probe based on the Cys-CBT click reaction may affect the intracellular self-assembly process and thus affect the imaging effect of the tumor. Since the chemical amount of radioactive drugs is usually very small, the radioactive drug molecules based on Cys-CBT in the body will inevitably be affected by free cysteine ​​and reduce the click condensation reaction and self-assembly process between their own molecules, resulting in reduced uptake efficiency and retention time in the target area. Summary of the invention

[0006] The purpose of the present invention is to provide a PET / MR dual-modality molecular probe responsive to legumain constructed intracellularly to solve the problems raised in the above-mentioned background technology.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] A PET / MR dual-modality molecular probe responsive to legumain constructed in a cell, comprising 68A radiopharmaceutical containing Ga element and / or a magnetic resonance contrast agent containing Gd element; the radiopharmaceutical and the magnetic resonance contrast agent are synthesized by the same precursor molecule; the structural formula of the precursor molecule is Formula I:

[0009]

[0010] Preferably, the structural formula of the radiopharmaceutical is Formula II:

[0011]

[0012] Preferably, the method for synthesizing the radiopharmaceutical comprises the following steps:

[0013] The precursor molecule is dissolved in a vacuum bottle with sodium acetate solution, and the positron nuclide is eluted from the Ge-Ga generator with high-purity hydrochloric acid solution. 68 Ga is added into the above vacuum bottle and heated for reaction, and then purified to obtain the radioactive drug.

[0014] Preferably, the magnetic resonance contrast agent has the structural formula of Formula III:

[0015]

[0016] Preferably, gadolinium chloride is dissolved in water, the pH value is adjusted to 6.5-7.2 with a saturated sodium carbonate solution, and then added to the N,N-dimethylformamide solution of the precursor molecule, and the pH value of the mixed solution is adjusted to 6.5-7.2, the reaction is stirred at room temperature, and then purified to obtain the magnetic resonance contrast agent.

[0017] Preferably, the synthesis method of the precursor molecule comprises the following steps:

[0018] Put the resin into a horn tube, add anhydrous N,N-dimethylformamide to activate the resin and remove impurities;

[0019] Weigh the amino acid Fmoc-Lys(Boc)-OH, dissolve it in a centrifuge tube with anhydrous N,N-dimethylformamide, add N,N-diisopropylethylamine with a pipette, and mix evenly with ultrasound to obtain the first amino acid solution;

[0020] After the activation of the resin in the ox-horn tube is completed, N,N-dimethylformamide is removed, and then the first amino acid solution is added to react. After the reaction is completed, the unreacted amino acid is removed, and then methanol and N,N-dimethylformamide are added to react to block the unreacted chlorine active sites on the resin; then piperidine solution is added to the ox-horn tube to react, and the 9-fluorenylmethoxycarbonyl protecting group on the amino acid Lys is removed by repeating three times;

[0021] The amino acid Fmoc-Cys (StBu), anhydrous 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in a centrifuge tube with anhydrous N,N-dimethylformamide, and then N,N-diisopropylethylamine is added to obtain a second amino acid solution; the second amino acid solution is added to the above-mentioned ox horn tube for reaction, and then the amino acids in the above steps are replaced with amino acids Fmoc-Asn-OH, amino acids Fmoc-Ala-OH and amino acids Fmoc-Ala-OH in sequence to prepare an amino acid solution, and the amino acid solution is added to the above-mentioned ox horn tube in sequence for reaction; after the reaction, anhydrous N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine are added to the ox horn tube for reaction to block the amino group on the amino acid Ala at the terminal of the peptide chain;

[0022] A dichloromethane solution containing trifluoroacetic acid was added to the above-mentioned ox-horn tube in portions to cut the oligopeptide from the resin, and then ether was added, and the oligopeptide was dispersed in the ether by ultrasound, and then refrigerated centrifugation was performed, the ether was poured off, and lyophilization was performed to obtain a dry oligopeptide, which was named compound A;

[0023] Dissolve compound A and 4-methylmorpholine in anhydrous N,N-dimethylformamide, cool to 0°C, add isobutyl chloroformate, and react at 0°C; then dissolve 2-cyano-6-aminobenzothiazole in anhydrous N,N-dimethylformamide and add to the above reaction, continue stirring at 0°C, and then stir the reaction at room temperature, and then separate and purify to obtain compound B;

[0024] Dissolve compound B in trifluoroacetic acid solution, stir at room temperature, remove the amino protecting group tert-butyloxycarbonyl of the amino acid Lys side chain on compound B, then remove the trifluoroacetic acid solution by rotary evaporation, and obtain compound C after separation and purification;

[0025] Dissolve tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in anhydrous N,N-dimethylformamide, stir at room temperature for reaction, then add N,N-diisopropylethylamine to the reaction solution, and adjust the pH value of the reaction solution to 7-8; dissolve compound C in anhydrous N,N-dimethylformamide, add the compound C to the reaction solution to continue the reaction, and separate and purify to obtain compound D;

[0026] The compound D is dissolved in a trifluoroacetic acid solution and stirred at room temperature to remove the carboxyl protecting group tert-butyl on the tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid on the compound D, and then the trifluoroacetic acid solution is removed by rotary evaporation. After separation and purification, the precursor molecule is obtained.

[0027] Another object of an embodiment of the present invention is to provide a use of the above-mentioned PET / MR dual-modality molecular probe in the preparation of a kit for diagnosing legumain-related cancer.

[0028] Another object of an embodiment of the present invention is to provide a use of the above-mentioned PET / MR dual-modality molecular probe in preparing a tumor imaging kit.

[0029] The embodiment of the present invention uses radioactive drugs and magnetic resonance contrast agents with the same precursor molecules as ET / MR dual-modality molecular probes to construct a 68Ga and Gd nanoparticle structure in situ in the tumor to simultaneously enhance the PET signal of the tumor with high legumain expression and T1-weighted magnetic resonance tumor imaging, thereby accurately diagnosing legumain-related cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The molecular probe 1 of the present invention 68 In situ self-assembly of Ga / Gd 68 Schematic diagram of Ga / Gd-CBT-NPs simultaneously enhancing PET and MR signals.

[0031] Figure 2 This is a diagram showing the results of mass spectrometry (ESI-MS) analysis of the first pure compound A synthesized in Example 1.

[0032] Figure 3 This is a diagram showing the results of mass spectrometry (ESI-MS) analysis of the second pure compound B synthesized in Example 1.

[0033] Figure 4 This is a diagram showing the results of mass spectrometry (ESI-MS) analysis of the third pure compound C synthesized in Example 1.

[0034] Figure 5 This is a diagram showing the results of high-resolution mass spectrometry (HR-MALDI / MS) analysis of the fifth pure compound 1 synthesized in Example 1.

[0035] Figure 6 is the hydrogen spectrum of the fifth pure compound 1 synthesized in Example 1 ( 1 H-NMR) analysis results.

[0036] Figure 7 is the carbon spectrum of the fifth pure compound 1 synthesized in Example 1 ( 13 C-NMR) analysis results.

[0037] Figure 8 This is a diagram showing the results of high-resolution mass spectrometry (HR-MALDI / MS) analysis of the sixth pure compound 1-Gd synthesized in Example 1.

[0038] Fig. 9 For Example 2 1- 68 Quality control result diagram of Ga; Fig. 9 a is 1- 68 Radioactivity-HPLC chromatogram of Ga, Fig. 9 b is 1- 68 The stability results of Ga after incubation in normal saline, 5% human serum and mouse serum for different time periods.

[0039] Fig.10 For Example 3 1- 68 The results of Ga uptake rate in C6 cells, C6 cells treated with legumain inhibitor and Huvec cells at different time points.

[0040] Fig.11 For Example 4 1- 68 Affinity results of Ga in C6 cells.

[0041] Fig.12 The graph shows the cell survival rate of C6 cells in Example 5 incubated in 50, 100, 200, and 400 μM 1-Gd for 4, 8, and 12 hours, respectively.

[0042] Fig.13 For Example 6 1- 68 The in vivo drug concentration-time curve of Ga.

[0043] Fig.14 For Example 7 1- 68 Figure 2 shows the in vivo biodistribution results of Ga in normal mice.

[0044] Fig.15 a is the intravenous injection of 1- 68 Ga and 1-Gd C6 tumor-bearing mice (top row), pretreated with legumain inhibitor and then co-injected with 1- 68 Ga and 1-Gd in C6 tumor-bearing mice (middle row), and 1- 68 Coronal PET / MR fusion images of C6 tumor-bearing mice (bottom row) at 10, 30, 60, 90, and 120 minutes (white circles indicate tumors); Fig.15 b is the total injection of 1- 68 Ga and 1-Gd groups, and 1- 68 The Ga and 1-Gd groups were compared with those injected with 1- 68 Tumor-to-muscle ratios at different times in the Ga group.

[0045] Fig.16 a is the intravenous injection of 1- 68 Ga and 1-Gd C6 tumor-bearing mice (top row), pretreated with legumain inhibitor and then co-injected with 1- 68 C6 tumor-bearing mice injected with Ga and 1-Gd (second row), and mice injected with 1- 68 T1-weighted coronal MR images of C6 tumor-bearing mice injected with Ga (third row) and C6 tumor-bearing mice injected with Gd-DPTA (bottom row) at 0 min (left column) and 90 min (right column) (white circles indicate tumors). Fig.16 b is the total injection of 1- 68 Ga and 1-Gd groups, injection of legumain inhibitor RR-11a and co-injection of 1- 68 Ga and 1-Gd group, simple injection of 1- 68 The tumor-to-muscle ratios of the Ga group and the Gd-DPTA injection group at different times relative to 0 minutes. DETAILED DESCRIPTION

[0046] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0047] In one embodiment of the present invention, a PET / MR dual-modality molecular probe responsive to legumain constructed in a cell is provided, which comprises 68 Ga element radiopharmaceutical and / or Gd element magnetic resonance contrast agent; the radiopharmaceutical and magnetic resonance contrast agent are synthesized by the same precursor molecule; specifically, the PET / MR dual-modality molecular probe contains legumain-specific enzyme cleavage substrate (alanine-alanine-asparagine, AAN), potential cysteine ​​(Cys) motif with disulfide bond and 2-cyano-benzothiazole (CBT) and chelating agent bound to lysine (Lys) side chain. 68 Ga or Gd 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA- 68 Ga / Gd) structure of small molecules; the general formula of radiopharmaceuticals and magnetic resonance imaging is Ala-Ala-Asn-Cys(SEt)-Lys(DOTA- 68 Ga / Gd)-CBT, the general structural formula is:

[0048]

[0049] In practical applications, the above-mentioned PET / MR dual-modality molecular probe, namely 68 The synthesis method of the radiopharmaceutical and magnetic resonance contrast agent of Ga element comprises the following steps:

[0050] S1. Put the resin into a horn tube, add anhydrous N,N-dimethylformamide to activate the resin and remove impurities;

[0051] Take the amino acid Fmoc-Lys(Boc)-OH, dissolve it in a centrifuge tube with anhydrous N,N-dimethylformamide, add N,N-diisopropylethylamine with a pipette, and mix it evenly with ultrasound to obtain the first amino acid solution;

[0052] After the activation of the resin in the ox-horn tube is completed, N,N-dimethylformamide is removed, and then the first amino acid solution is added to react. After the reaction is completed, the unreacted amino acid is removed, and then methanol and N,N-dimethylformamide are added to react to block the unreacted chlorine active sites on the resin; then piperidine solution is added to the ox-horn tube to react, and the 9-fluorenylmethoxycarbonyl protecting group on the amino acid Lys is removed by repeating three times;

[0053] The amino acid Fmoc-Cys (StBu), anhydrous 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in a centrifuge tube with anhydrous N,N-dimethylformamide, and then N,N-diisopropylethylamine is added to obtain a second amino acid solution; the second amino acid solution is added to the above-mentioned ox horn tube for reaction, and then the amino acids in the above steps are replaced with amino acids Fmoc-Asn-OH, amino acids Fmoc-Ala-OH and amino acids Fmoc-Ala-OH in sequence to prepare an amino acid solution, and the amino acid solution is added to the above-mentioned ox horn tube in sequence for reaction; after the reaction, anhydrous N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine are added to the ox horn tube for reaction to block the amino group on the amino acid Ala at the terminal of the peptide chain;

[0054] A dichloromethane solution containing trifluoroacetic acid was added to the above-mentioned ox horn tube in portions to cut the oligopeptide from the resin, and then ether was added, and the oligopeptide was dispersed in the ether by ultrasound, and then refrigerated centrifugation was performed, the ether was poured off, and lyophilization was performed to obtain a dry oligopeptide, which was named Compound A, and its structural formula is as follows:

[0055]

[0056] S2. Compound A and 4-methylmorpholine are dissolved in anhydrous N,N-dimethylformamide, cooled to 0°C, isobutyl chloroformate is added, and the reaction is carried out at 0°C; then 2-cyano-6-aminobenzothiazole is dissolved in anhydrous N,N-dimethylformamide and added to the above reaction, and stirring is continued at 0°C, and then the reaction is stirred at room temperature, and then separated and purified to obtain compound B, whose structural formula is as follows:

[0057]

[0058] S3, dissolving compound B in trifluoroacetic acid solution, stirring at room temperature, removing the amino protecting group tert-butyloxycarbonyl of the amino acid Lys side chain on compound B, and then removing the trifluoroacetic acid solution by rotary evaporation, and obtaining compound C after separation and purification, whose structural formula is as follows:

[0059]

[0060] S4. Dissolve tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in anhydrous N,N-dimethylformamide, stir at room temperature for reaction, then add N,N-diisopropylethylamine to the reaction solution, and adjust the pH value of the reaction solution to 7-8; dissolve compound C in anhydrous N,N-dimethylformamide, add the compound to the above reaction solution to continue the reaction, and separate and purify to obtain compound D, whose structural formula is as follows:

[0061]

[0062] S5. Dissolve compound D in trifluoroacetic acid solution, stir at room temperature, remove the carboxyl protecting group tert-butyl on tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid on compound D, and then remove the trifluoroacetic acid solution by rotary evaporation. After separation and purification, obtain the precursor molecule, named as compound 1, whose structural formula is as follows:

[0063]

[0064] S6. Dissolve the precursor molecule in a vacuum bottle with sodium acetate solution, and elute the positron nuclide from the Ge-Ga generator with high-purity hydrochloric acid solution. 68 Ga, added to the above vacuum bottle, and heated to react, and then purified to obtain the radioactive drug, named 1- 68 Ga, its structural formula is as follows:

[0065]

[0066] Gadolinium chloride is dissolved in water, and the pH value is adjusted to 6.5-7.2 with a saturated sodium carbonate solution, and then added to the N,N-dimethylformamide solution of the precursor molecule, and the pH value of the mixed solution is adjusted to 6.5-7.2, and the reaction is stirred at room temperature, and then purified to obtain the magnetic resonance contrast agent, named 1-Gd, whose structural formula is as follows:

[0067]

[0068] The PET / MR dual-modality molecular probe provided in the embodiment of the present invention can generate a click reaction between 2-cyano-benzothiazole and D-cysteine ​​in cancer cells with high legumain expression, thereby forming an in situ 68 The nanoparticle structure of Ga and Gd effectively improves 68 The intracellular concentrations of Ga and Gd extend 68 The retention time of Ga and Gd in the target area can enhance the radionuclide uptake of tumors with high legumain expression, and enhance the T1-weighted magnetic resonance contrast of tumors, which can be used for early and accurate diagnosis of legumain-related cancers. 68 The multifunctional nanoparticle structure of Ga and Gd can simultaneously enhance the PET signal of legumain-highly expressing tumors and T1-weighted magnetic resonance tumor imaging for the diagnosis of legumain-related cancers.

[0069] The following embodiments are some specific implementation cases of the present invention in practical applications, but are not limited thereto.

[0070] Wherein Example 1 provides a first pure compound A, a second pure compound B, a third pure compound C, a fourth pure compound D, a fifth pure compound 1, a sixth pure compound 1-Gd, a seventh pure compound 1- 68 Synthesis of Ga. Example 2 provides 1- 68 The in vitro quality control and stability test results of Ga are provided in Example 3. 68 Ga uptake experimental results of C6 cells and Huvec cells at different times. Example 4 provides 1- 68 The results of the C6 cell affinity test of Ga. Example 5 provides the results of the C6 cell toxicity test of different concentrations of 1-Gd at different times. Example 6 provides the results of the C6 cell toxicity test of 1-Gd at different times. 68 The in vivo pharmacokinetic results of Ga. Example 7 provides 1- 68 The results of the in vivo biodistribution experiment of Ga in normal mice are provided in Example 8. 68The formation of Ga / Gd-CBT-NPs enhanced the radionuclide uptake of tumors in vivo and enhanced T1 magnetic resonance contrast imaging experimental results.

[0071] Example 1: This example provides pure compounds A, B, C, D, 1, 1-Gd, 1- 68 The synthesis method of Ga is as follows:

[0072] This example specifically introduces a method for in situ formation of a 2-cyanobenzothiazole-D-cysteine ​​click reaction in cancer cells that highly express legumain. 68 The preparation method of the Ga and Gd nanostructured PET / MR dual-modality molecular probe is completed in 6 steps. The synthesis route is as follows:

[0073]

[0074] Step 1: Take 0.5g (0.55mmol) of 2-chlorotrityl resin and put it into a glass ox-horn tube, add 2mL of anhydrous N,N-dimethylformamide (DMF) to activate the resin and remove impurities. Weigh 468.5mg (1mmol) of amino acid Fmoc-Lys(Boc)-OH, dissolve it in 2mL of anhydrous DMF in a 5mL centrifuge tube, add 174μL (1mmol) of N,N-diisopropylethylamine (DIEPA) with a pipette, and mix evenly by ultrasonication to obtain the first amino acid solution. After the resin activation is completed, use an ear bulb to 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 2mL of anhydrous DMF to remove the unreacted amino acid. After washing, add 45μL of methanol and 2mL DMF, and react for 40 minutes to block the unreacted chlorine active sites on the resin. Then add 20% piperidine solution to the ox horn tube to react for 10 minutes, repeat three times to remove the 9-fluorenylmethoxycarbonyl protecting group (Fmoc) on the amino acid Lys. 360mg (0.84mmol) of amino acid Fmoc-Cys (StBu), 109mg (0.84mmol) of anhydrous 1-hydroxybenzotriazole (HOBt) and 315mg (0.84mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) were dissolved in 2mL of anhydrous DMF in a 5mL centrifuge tube, and 145μL (0.84mmol) of DIEPA was added to obtain a second amino acid solution. The second amino acid solution was added to the ox horn tube to react for 8 hours. The amino acids in the above steps were replaced with amino acid Fmoc-Asn-OH (N), amino acid Fmoc-Ala-OH (A2) and amino acid Fmoc-Ala-OH (A1) in order to prepare amino acid solutions, which were added to the ox horn tube for reaction in sequence; the dosage was 0.84 mmol, and the corresponding masses were m (N) = 295 mg, m (A2) = m (A1) = 260 mg, and the amounts of anhydrous HOBt, HBTU and DIPEA remained unchanged. After the reaction, 2 mL of anhydrous DMF and 335 μL of acetic anhydride and 620 μL of DIEPA (1:1; volume ratio 100:185) were added to the ox horn tube, wherein the acetic anhydride was greatly excessive, and the amino group on the amino acid Ala at the end of the peptide chain was capped for 30 minutes. A dichloromethane solution containing 1% trifluoroacetic acid (300 μL of TFA, 30 mL of DCM) was added to a horn tube in portions to cut the oligopeptide from the resin, and about 50 ml of ether was added. The oligopeptide was dispersed in the ether by ultrasound, and refrigerated centrifuged at 4000 r / min for 20 min. The ether in the centrifuge tube was poured out, and the oligopeptide was freeze-dried in a freeze dryer to obtain a dry oligopeptide, which was named Compound A.

[0075] Step 2: Dissolve 1mmol (735.32mg) of compound A and 2mmol (222μL) of 4-methylmorpholine (MMP) in 3mL of anhydrous N,N-dimethylformamide (DMF), cool to 0°C, add 2mmol (254μL) of isobutyl chloroformate (IBCF), and stir the reaction mixture at 0°C for 40 minutes. Then, dissolve 1mmol (17.5mg) of 2-cyano-6-aminobenzothiazole (CBT) in anhydrous DMF and add it to the reaction, continue stirring at 0°C for 2 hours, and then stir the reaction at room temperature overnight. The second pure compound was obtained by separation and purification by high performance liquid chromatography (HPLC), named compound B.

[0076] Step 3: Dissolve 1 mmol (892 mg) of compound B in 10 ml of 95% trifluoroacetic acid (TFA) solution (9.5 mL of trifluoroacetic acid, 0.5 mL of dichloromethane and 50 μL of triisopropylsilane), stir at room temperature for 3 hours to remove the amino protecting group tert-butyloxycarbonyl (Boc) of the amino acid Lys side chain on compound B, then remove the trifluoroacetic acid solution by rotary evaporation, and separate and purify by high performance liquid chromatography to obtain a third pure compound, named compound C.

[0077] Step 4: 0.4 mmol (229 mg) of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA(OtBu)3), 0.48 mmol (56 mg) of N-hydroxysuccinimide and 0.48 mmol (92 mg) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) were dissolved in 4 mL of anhydrous N,N-dimethylformamide (DMF), stirred at room temperature for 12 hours, then 0.8 mmol (139 μL) of N,N-diisopropylethylamine (DIEPA) was added to the reaction solution, the pH of the reaction solution was adjusted to 7-8, 0.4 mmol (317 mg) of compound C was dissolved in 3 mL of anhydrous N,N-dimethylformamide (DMF), added to the above reaction solution and continued to react at room temperature for 3 hours. After separation and purification by high performance liquid chromatography, the fourth pure compound was obtained, named compound D.

[0078] Step 5: Dissolve 0.2 mmol (269 mg) of compound D in 10 mL 95% trifluoroacetic acid (TFA) solution (9.5 mL trifluoroacetic acid, 0.5 mL dichloromethane and 50 μL triisopropylsilane), stir at room temperature for 3 hours to remove the carboxyl protecting group tert-butyl on tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA(OtBu)3) on compound D, then remove the trifluoroacetic acid solution by rotary evaporation, and separate and purify by high performance liquid chromatography to obtain the fifth pure compound, named compound 1.

[0079] Step 6: Dissolve 0.01mmol (8.9mg) of gadolinium chloride (GdCl3·6H2O) in water, adjust the pH value to 6.5-7.2 with saturated sodium carbonate solution, then add it to 0.01mmol (11.8mg) of N,N-dimethylformamide solution of compound 1, and adjust the pH value of the mixed solution to 6.5-7.2, and stir the reaction at room temperature for 3 hours. After separation and purification by high performance liquid chromatography, a small molecule T1 contrast agent based on Gd can be obtained, named 1-Gd.

[0080] Dissolve 50 μg of the above 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 3.5 mL (0.1 M) high-purity hydrochloric acid solution. 68 Ga, take 1mL and add it to the above vacuum bottle, react on a heater at 95℃ for 10 minutes, take out the system with a 2mL syringe and measure it, then add it to the activated Sep-pak column, rinse the impurities with 3.0ml pure water and discard them. Add a 0.2μm microporous filter membrane, collect the product with 1.0mL 80% ethanol solution into a sterile vacuum bottle to obtain positron-based nuclides. 68 Ga radioactive molecular drug, named 1- 68 Ga.

[0081] Figure 1 For the present invention 1- 68 Schematic diagram of the self-assembly process of Ga / Gd under the action of legumain and its intracellular self-assembly into nanoparticles for simultaneous enhanced radionuclide uptake and T1 magnetic resonance contrast imaging. Figure 1 It can be seen that when 1- 68 After Ga / Gd enters cancer cells that overexpress legumain, they form in situ in cancer cells through self-assembly guided by intracellular legumain and triggered by CBT-Cys click condensation reaction. 68 Ga / Gd nanoparticles. This process not only improves 68 The intracellular concentrations of Ga and Gd and the 68 The retention time of Ga and Gd is increased, thereby enhancing the radionuclide uptake of tumors in vivo and enhancing T1 magnetic resonance contrast imaging.

[0082] The pure compounds A, B, C, D, 1, and 1-Gd were qualitatively analyzed by mass spectrometry, and the structure of compound 1 was analyzed by nuclear magnetic resonance. The results are as follows: Figure 2-Figure 8 shown.

[0083] Figure 2 is the mass spectrum of the first pure compound A synthesized in Example 1; Figure 3is the mass spectrum of the second pure compound B synthesized in Example 1; Figure 4 is the mass spectrum of the third pure compound C synthesized in Example 1; Figure 5 is the mass spectrum of the fifth pure compound 1 synthesized in Example 1; Figure 6 is the hydrogen nuclear magnetic resonance spectrum of the fifth pure compound 1 synthesized in Example 1; Figure 7 is the carbon NMR spectrum of the fifth pure compound 1 synthesized in Example 1; Figure 8 is the mass spectrum of the sixth pure compound 1-Gd synthesized in Example 1. Figure 2 It can be seen that the mass spectrum result of the first pure compound A is obsvd.ESI-MS[(M+H) + ]:m / z 736.08; Figure 3 It can be seen that the mass spectrum result of the second pure compound B is obsvd.ESI-MS[(M+H) + ]:m / z 892.96; Figure 4 It can be seen that the mass spectrum result of the third pure compound C is obsvd.ESI-MS[(M+H) + ]:m / z 793.20; Figure 5 It can be seen that the mass spectrum result of the fifth pure compound 1 is obsvd.HR-MALDI-MS[(M+H) + ]:m / z 1179.4749; Figure 6 It can be seen that the fifth pure compound 1 has a hydrogen nuclear magnetic resonance spectrum (d6-dimethyl sulfoxide, 400MHz) δ (ppm) 10.20 (s, 1H), 8.51 (s, 1H), 8.24–8.18 (m, 3H), 8.12 (d, J = 6.1 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 7.80 (d, J = 6.5 Hz, 1H), 7.52 (d, J = 18.7 Hz, 2H), 7.02 (s, 2H), 4.24 (s, 2H), 4.12 (s, 2H), 3.92 (s ,1H),3.61(s,3H),3.41(s,9H),3.28(s,2H),3.10(s,14H),2.88(s,0H),2.72(s,0H),2.69–2.54(m,1H),2.52(d,J=3 .7Hz,1H),1.83(s,5H),1.44(s,2H),1.28(s,3H),1.25(s,6H),1.22(s,2H),1.21(s,2H),1.19(s,2H),1.18(s,2H); by Figure 7It can be seen that the fifth pure compound 1 has a carbon NMR spectrum (d6-dimethyl sulfoxide, 101MHz) δ (ppm): 172.65, 171.93, 169.47, 159.00, 158.65, 158.30, 147.81, 139.37, 136.76, 124.91, 113.68, 111.61, 54.75, 54.05, 52.76, 52.51, 50.66, 49.80, 48.39, 47.87, 47.79, 41.81, 36.75, 29.61, 23.13, 22.56, 18.04, 18.02, 17.97; Figure 8 It can be seen that the mass spectrum result of the sixth pure compound 1-Gd is obsvd.HR-MALDI-MS[(M+H) + ]:m / z 1333.4946.

[0084] Example 2: 1- 68 The in vitro quality control and stability analysis experiments of Ga are as follows:

[0085] In this example, Radio-HPLC was used to analyze 1- 68 Ga performed in vitro quality control. Fig. 9 a is 1- 68 The HPLC chromatogram of Ga shows that its retention time is 4.48 minutes. 68 Ga was incubated with normal saline, 5% human serum and mouse serum for 15 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, such as Fig. 9 As shown in b. Fig. 9 b see 1- 68 Ga can maintain stability after incubation for 120 minutes in physiological saline, 5% human serum and mouse serum solutions, and the radiochemical purity is greater than 89%, which are 92.5%, 93% and 89.25% respectively.

[0086] Example 3: 1- 68 The Ga uptake experiment in C6 cells and Huvec cells is as follows:

[0087] C6 cells were divided into two groups: experimental group: C6 cells with 37 kBq 1- 68 Ga were co-incubated for 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively; control group: 100 μM RR-11a (a legumain inhibitor) was pretreated for 30 minutes, and then incubated with 37 kBq 1- 68Ga were co-incubated for 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively; Huvec cells were incubated with 37 kBq of 1- 68 Ga were co-incubated for 5 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes and 120 minutes respectively, and then the radioactivity of the above three groups of cells was counted using a gamma counter to obtain the radioactivity of C6 cells and Huvec cells at different times. 68 The uptake rate of Ga is shown in Fig.10 As shown. Fig.10 It can be seen that the radioactivity uptake in C6 cells was the highest at 60 minutes and was higher than that in C6 cells and Huvec cells pretreated with the inhibitor.

[0088] Example 4: 1- 68 The affinity detection experiment of Ga in C6 cells is as follows:

[0089] C6 cells were treated with 1.06nM, 5.3nM, 10.44nM, 52.75nM, 103.89nM, 527.47nM 1- 68 Ga were co-incubated for 1.5 hours, and then the radioactivity was counted using a gamma counter. The results are shown in Fig.11 As shown. Fig.11 As you can see, 1- 68 The affinity curve of Ga for C6 cells shows that the Kd value is 13.63, indicating that 1- 68 Ga has a strong affinity for C6 cells.

[0090] Example 5: Cytotoxicity detection experiment of 1-Gd in C6 cells, as follows:

[0091] C6 cells were plated in 96-well plates at 5 × 10 4 The cells were divided into 4 groups, each with 5 replicate wells, and 3 96-well plates were plated and incubated overnight at 37°C. The old culture medium was removed the next day, and 100 μL of fresh culture medium containing 1-Gd at concentrations of 50 μM, 100 μM, 200 μM, and 400 μM was added to each group. After incubation for 4, 8, and 12 hours, 10 μL of CCK-8 was added and incubated for 1.5 hours. The absorbance of each well was analyzed at a wavelength of 450 nm using an ELISA reader. The results are shown in the figure. Fig.12 As shown. Fig.12 It can be seen that under the action of 400 μM 1-Gd, 84.13% of the cells are still alive after 12 hours, indicating that 1-Gd has little toxicity to cells.

[0092] Example 6: 1- 68 The pharmacokinetic experiments of Ga in normal mice are as follows:

[0093] Take 200 μL 1- 68 Ga was injected into Kunming mice 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. The experiment was repeated 4 times at each time point, and %ID / g was calculated. Fig.13 As shown. Fig.13 It can be seen that after analysis of the curve, the distribution half-life is 6.082 minutes and the elimination half-life is 23.60 minutes.

[0094] Example 7: 1- 68 The biodistribution analysis experiment of Ga in normal mice is as follows:

[0095] Twenty Kunming mice were randomly divided into five groups, with four mice in each group. 200 μL of 1- 68 Ga was injected into Kunming mice via the tail vein. Mice were killed at 5, 30, 60, 120, and 240 minutes after injection. Blood, heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, bone, muscle, and brain were collected and weighed. The radioactivity was measured using a γ counter and %ID / g was calculated. Fig.14 As shown. Fig.14 As you can see, 5, 30, 60, 120, 240 minutes 1- 68 Distribution of Ga in normal mice, 1- 68 Ga in mice is mainly metabolized through the urinary system, and a very small amount is metabolized through the liver.

[0096] Example 8: legumain-guided 68 The formation of Ga / Gd nanoparticles simultaneously enhances in vivo tumor radionuclide uptake and T1 MR contrast as follows:

[0097] Two million C6 cells overexpressing legumain were transplanted subcutaneously in the right axilla of each Balb / c nude mouse. When the tumor diameter reached 5-8 mm, the nude mice were randomly divided into 4 groups: experimental group 1- 68 Mice in the Ga+1-Gd group were intravenously injected with 2.96-3.7 MBq (200 μL) of 1- 68 Ga and 0.06mmol / kg 1-Gd; control group 1- 68 Mice in Ga+1-Gd+block were first intravenously injected with 0.004mmol / kg of RR-11a (a legumain inhibitor), and 1.5 hours later, a total of 2.96-3.7MBq (200μL) of 1- 68 Ga and 0.06mmol / kg 1-Gd; control group 1- 68Mice in Ga were intravenously injected with 2.96-3.7 MBq (200 μL) of 1- 68 Ga; mice in the control group Gd-DPTA were intravenously injected with 0.06mmol / kg of Gd-DPTA. Dynamic PET-MR coronal imaging was performed on the four groups of mice on a PET-MR scanner (among which mice in the control group Gd-DPTA were only subjected to MR imaging). Fig.15 a is the intravenous injection of 1- 68 Ga and 1-Gd C6 tumor-bearing mice (top row), pretreated with legumain inhibitor and then injected with 1- 68 Ga and 1-Gd in C6 tumor-bearing mice (middle row), injected with 1- 68 Coronal PET / MR fusion images of Ga C6 tumor-bearing mice (bottom row) at 10 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes (white circles indicate tumors). Fig.15 B can be seen injection 1- 68 The maximum radioactivity uptake in the tumors of mice injected with Ga and 1-Gd occurred at 60 minutes, and the retention time of radioactivity uptake was significantly longer than that injected with legumain inhibitor RR-11a and 1- 68 Ga and 1-Gd in mice tumors and injected with 1- 68 Mouse tumors of Ga. Fig.16 a is the intravenous injection of 1- 68 Ga and 1-Gd C6 tumor-bearing mice (top row), pretreated with legumain inhibitor and then injected with 1- 68 Ga and 1-Gd C6 tumor-bearing mice (second row), injected with 1- 68 T1-weighted coronal MR images of C6 tumor-bearing mice injected with Ga (third row) and C6 tumor-bearing mice injected with Gd-DPTA (bottom row) at 0 minutes (left column) and 90 minutes (right column). The white circles indicate the tumors, and the Fig.16 B can be seen injection 1- 68 The T1-weighted MRI contrast changes in the tumors of mice injected with Ga and 1-Gd were significantly greater than those injected with the legumain inhibitor RR-11a and 1- 68 Ga and 1-Gd in mice tumors, injected with 1- 68 Ga mice tumors and Gd-DPTA injected mice tumors. 68 Ga / Gd formed under the guidance of legumain 68 Ga / Gd-CBT-NPs can stay in legumain-overexpressing tumors for a longer time, enhancing the PET signal while improving its T1 MRI contrast.

[0098] The above experimental data show that legumain-induced intracellular 68 The formation of Ga / Gd-CBT-NPs increased 68 The local concentration and retention time of Ga and Gd at the tumor site significantly enhance the PET and MRI signals of the tumor overexpressing legumain in vivo. 68 Ga / Gd PET / MR dual-modality molecular probe 1- 68 Ga / 1-Gd and imaging methods have the potential to be used as a new type of imaging agent in future clinical applications for PET-MR imaging diagnosis of malignant tumors with overexpression of legumain.

[0099] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A PET / MR dual-modality molecular probe constructed intracellularly and responsive to legumain, characterized in that: Including 68 A radiopharmaceutical containing Ga element and a magnetic resonance contrast agent containing Gd element; the radiopharmaceutical and the magnetic resonance contrast agent are synthesized by the same precursor molecule; the structural formula of the precursor molecule is Formula I: ; The structural formula of the radiopharmaceutical is Formula II: The synthesis method of the radiopharmaceutical comprises the following steps: The precursor molecule is dissolved in a vacuum bottle with sodium acetate solution, and the positron nuclide is eluted from the Ge-Ga generator with high-purity hydrochloric acid solution. 68 Ga is added into the vacuum bottle, and heated to react, and then purified to obtain the radioactive drug; The structural formula of the magnetic resonance contrast agent is Formula III: ; Gadolinium chloride is dissolved in water, and the pH value is adjusted to 6.5-7.2 with a saturated sodium carbonate solution, and then added to the N,N-dimethylformamide solution of the precursor molecule, and the pH value of the mixed solution is adjusted to 6.5-7.2, and the reaction is stirred at room temperature, and then purified to obtain the magnetic resonance contrast agent.

2. The intracellular legumain-responsive PET / MR dual-modality molecular probe according to claim 1, characterized in that: The synthesis method of the precursor molecule comprises the following steps: Put the resin into a horn tube, add anhydrous N,N-dimethylformamide to activate the resin and remove impurities; Weigh the amino acid Fmoc-Lys(Boc)-OH, dissolve it in a centrifuge tube with anhydrous N,N-dimethylformamide, add N,N-diisopropylethylamine with a pipette, and mix evenly with ultrasound to obtain the first amino acid solution; After the activation of the resin in the ox-horn tube is completed, N,N-dimethylformamide is removed, and then the first amino acid solution is added to react. After the reaction is completed, the unreacted amino acid is removed, and then methanol and N,N-dimethylformamide are added to react to block the unreacted chlorine active sites on the resin; then piperidine solution is added to the ox-horn tube to react, and the 9-fluorenylmethoxycarbonyl protecting group on the amino acid Lys is removed by repeating three times; The amino acid Fmoc-Cys (StBu), anhydrous 1-hydroxybenzotriazole and benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate are dissolved in a centrifuge tube with anhydrous N,N-dimethylformamide, and then N,N-diisopropylethylamine is added to obtain a second amino acid solution; the second amino acid solution is added to the above-mentioned ox horn tube for reaction, and then the amino acids in the above steps are replaced with amino acids Fmoc-Asn-OH, amino acids Fmoc-Ala-OH and amino acids Fmoc-Ala-OH in sequence to prepare an amino acid solution, and the amino acid solution is added to the above-mentioned ox horn tube in sequence for reaction; after the reaction, anhydrous N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine are added to the ox horn tube for reaction to block the amino group on the amino acid Ala at the terminal of the peptide chain; A dichloromethane solution containing trifluoroacetic acid was added to the above-mentioned ox-horn tube in portions to cut the oligopeptide from the resin, and then ether was added, and the oligopeptide was dispersed in the ether by ultrasound, and then refrigerated centrifugation was performed, the ether was poured off, and lyophilization was performed to obtain a dry oligopeptide, which was named compound A; Dissolve compound A and 4-methylmorpholine in anhydrous N,N-dimethylformamide, cool to 0°C, add isobutyl chloroformate, and react at 0°C; then dissolve 2-cyano-6-aminobenzothiazole in anhydrous N,N-dimethylformamide and add to the above reaction, continue stirring at 0°C, and then stir the reaction at room temperature, and then separate and purify to obtain compound B; Dissolve compound B in trifluoroacetic acid solution, stir at room temperature, remove the amino protecting group tert-butyloxycarbonyl of the amino acid Lys side chain on compound B, then remove the trifluoroacetic acid solution by rotary evaporation, and obtain compound C after separation and purification; Dissolve tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in anhydrous N,N-dimethylformamide, stir at room temperature for reaction, then add N,N-diisopropylethylamine to the reaction solution, and adjust the pH value of the reaction solution to 7-8; dissolve compound C in anhydrous N,N-dimethylformamide, add the compound C to the reaction solution to continue the reaction, and separate and purify to obtain compound D; The compound D is dissolved in a trifluoroacetic acid solution and stirred at room temperature to remove the carboxyl protecting group tert-butyl on the tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid on the compound D, and then the trifluoroacetic acid solution is removed by rotary evaporation. After separation and purification, the precursor molecule is obtained.

3. Use of the PET / MR dual-modality molecular probe according to any one of claims 1 to 2 in the preparation of a kit for diagnosing legumain-related cancer.

4. Use of the PET / MR dual-modality molecular probe according to any one of claims 1 to 2 in preparing a tumor imaging kit.

Images