WO2024066540A1 - 一种天冬氨酸蛋白酶靶向识别pet分子探针及应用 - Google Patents

一种天冬氨酸蛋白酶靶向识别pet分子探针及应用 Download PDF

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WO2024066540A1
WO2024066540A1 PCT/CN2023/102803 CN2023102803W WO2024066540A1 WO 2024066540 A1 WO2024066540 A1 WO 2024066540A1 CN 2023102803 W CN2023102803 W CN 2023102803W WO 2024066540 A1 WO2024066540 A1 WO 2024066540A1
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molecular probe
gpn
devd
compound
caspase
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French (fr)
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邱玲
林建国
刘清竹
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江苏省原子医学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo

Definitions

  • the present application relates to a PET molecular probe for targeted recognition of aspartic protease and its application, belonging to the field of chemical technology.
  • Tumor refers to a disease caused by abnormal cell growth in an organism due to a variety of factors. It is divided into malignant tumors and benign tumors according to its characteristics and harm to the organism. Malignant tumors, also known as cancer, have biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. Due to the heterogeneity and individual differences of malignant tumors, different cancer patients need to develop specific treatment plans. Therefore, it is very important for cancer patients to choose the most appropriate clinical treatment plan by evaluating the treatment efficacy. By evaluating early treatment responses and treatment results, side effects during cancer treatment can be reduced, treatment methods can be adjusted in a timely manner, and treatment costs can be reduced.
  • Aspartate proteinase-3 (caspase-3), as a crucial "death execution protease" in the apoptosis cascade reaction, is often used as a biomarker for detecting tumor cell apoptosis. Therefore, by detecting the changes in caspase-3 levels in tumor cells after treatment, immediate feedback on the efficacy of cancer treatment can be achieved.
  • PET Positron emission tomography
  • the present application provides a molecular probe targeting aspartate protease, wherein the molecular probe has the following structure:
  • R is a radionuclide labeling group.
  • the radionuclide labeling group is 68 Ga, [ 18 F]AlF, 64 Cu or 89 Zr.
  • the molecular probe when the radionuclide labeling group is 68 Ga, the molecular probe has the following structure:
  • the molecular probe when the radionuclide labeling group is [ 18 F]A1F, the molecular probe has the following structure:
  • the labeling precursor of the molecular probe has the following structure:
  • the molecular probe targets caspase-3.
  • the present application also provides a method for preparing the above-mentioned molecular probe, which comprises: dissolving the compound GPN-7 and NOTA-NHS in DMF (N,N-dimethylformamide) to obtain a solution; adding DIPEA (N,N-diisopropylethylamine) to the solution and reacting to obtain a reaction solution; purifying the reaction solution to obtain a labeled precursor of the molecular probe GPN-DEVD; and The molecular probe's labeling precursor GPN-DEVD is labeled with radioactive nuclides to obtain the molecular probe;
  • the compound GPN-7 has the structure shown below:
  • the preparation method of the compound GPN-7 is: dissolving the compound GPN-6 in methanol (MeOH) to obtain a solution; adding Tips (triisopropylsilane) and SEt (2-(ethyldisulfanyl)pyridine) to the solution and reacting to obtain a reaction solution; concentrating the reaction solution, precipitating with ether, centrifuging, and drying to obtain the compound GPN-7;
  • the compound GPN-6 has the structure shown below:
  • the preparation method of the compound GPN-6 is as follows: dissolving the compound GPN-5 in CH 2 Cl 2 (dichloromethane) to obtain a solution; adding TFA (trifluoroacetic acid) and TIPS (triisopropylsilane) to the solution for reaction to obtain a reaction solution; concentrating the reaction solution, precipitating with ether, centrifuging, and drying to obtain the compound GPN-6;
  • the compound GPN-5 has the structure shown below:
  • the preparation method of the compound GPN-5 is: dissolving the compound GPN-4, the compound A1 and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in THF (tetrahydrofuran) to obtain a solution; adding DIPEA (N,N-diisopropylethylamine) to the solution, reacting under the protection of nitrogen to obtain a reaction solution; concentrating, purifying and drying the reaction solution to obtain the compound GPN-5;
  • the compound GPN-4 has the structure shown below:
  • the compound A1 has the structure shown below:
  • the preparation method of the compound GPN-4 is: mixing the compound GPN-3 and a piperidine aqueous solution and reacting to obtain a reaction solution; extracting, concentrating, purifying, and drying the reaction solution to obtain the compound GPN-4;
  • the compound GPN-3 has the structure shown below:
  • the preparation method of the compound GPN-3 is: dissolving the compound GPN-2, lysine and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in THF (tetrahydrofuran) to obtain a solution; adding DIPEA (N, N-diisopropylethylamine) to the solution, reacting under the protection of nitrogen to obtain a reaction solution; concentrating, purifying and drying the reaction solution to obtain the compound GPN-3;
  • the compound GPN-2 has the structure shown below:
  • the preparation method of the compound GPN-2 is: dissolving the compound GPN-1 in CH 2 Cl 2 (dichloromethane) to obtain a solution; adding TFA (trifluoroacetic acid) and TIPS (triisopropylsilane) to the solution and reacting to obtain a reaction solution; concentrating the reaction solution, precipitating with ether, centrifuging, and drying to obtain the compound GPN-2;
  • the compound GPN-1 has the structure shown below:
  • the preparation method of the compound GPN-1 is: dissolving BOC-glycine, isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM) in THF (tetrahydrofuran), and reacting under the protection of nitrogen to obtain a reaction solution A; dissolving 2-cyano-6-aminobenzothiazole (CBT) in THF (tetrahydrofuran) to obtain a dissolved solution; mixing the dissolved solution and the reaction solution A, and reacting under the protection of nitrogen to obtain a reaction solution B; extracting, concentrating, purifying and drying the reaction solution B to obtain the compound GPN-1.
  • the present application also provides the use of the above molecular probe in aspartate protease imaging, and the application is not for the purpose of disease diagnosis and treatment.
  • the aspartate protease is aspartate protease-3.
  • the present application also provides an imaging agent targeting aspartate protease, wherein the imaging agent contains the above-mentioned molecular probe.
  • the present application provides a molecular probe, which has the following advantages:
  • a glycine was added between CBT and lysine to construct the intramolecular cyclized backbone GPN.
  • This extended self-assembled backbone can Improve the flexibility of the compound so that CBT and the cysteine in the molecule are more likely to undergo click condensation reaction for intramolecular condensation cyclization, making it less affected by high concentrations of free cysteine, further promoting the condensation cyclization and self-assembly of the probe in the tumor to form aggregates, so as to achieve long-term high-quality visualization of tumor apoptosis levels;
  • this molecular probe can monitor changes in caspase-3 levels in tumor cells through PET imaging, thereby realizing a non-invasive imaging strategy for detecting abnormal levels of tumor cell apoptosis or monitoring treatment-induced tumor cell apoptosis in various disease states, so as to achieve immediate feedback on the efficacy of cancer treatment, and has good application prospects.
  • the radionuclide labeling group is 68 Ga; the 68 Ga nuclide is prepared by a 68 Ge/ 68 Ga generator (ITG), is cheap and readily available, and the incubation temperature required for 68 Ga labeling is low (37° C.), the time is short (10 min), and the labeling yield is high.
  • the radionuclide labeling group is [ 18 F]AlF;
  • the [ 18 F]AlF aluminum fluoride radiolabeling is based on the formation of [ 18 F][AlF] 2+ cations and is complexed with the multidentate bifunctional chelator NOTA, which has good stability, making it difficult for the molecular probe to lose 18 F during imaging, resulting in lower bone uptake.
  • FIG. 8 HPLC spectrum of compound GPN-2.
  • FIG. 10 HPLC spectrum of compound GPN-3.
  • FIG. 12 HPLC spectrum of compound GPN-4.
  • Figure 13 Synthesis route of the caspase-3 targeted molecular probe precursor GPN-DEVD.
  • FIG. 15 HPLC spectrum of compound GPN-5.
  • FIG. 17 HPLC spectrum of compound GPN-6.
  • FIG. 20 ESI-MS analysis results of the caspase-3 targeted molecular probe precursor GPN-DEVD.
  • FIG. 21 HPLC spectrum of the caspase-3 targeted molecular probe precursor GPN-DEVD.
  • Figure 22 HPLC spectrum of the shear-reduction of the caspase-3 targeted molecular probe precursor GPN-DEVD.
  • Figure 23 ESI-MS analysis results of the caspase-3-targeted molecular probe precursor GPN-DEVD-reduced.
  • Figure 24 ESI-MS analysis results of the caspase-3 targeted molecular probe precursor GPN-DEVD-C.
  • Figure 25 Synthesis route of the caspase-3 targeting molecular probe [ 68 Ga]GPN-DEVD.
  • FIG. 26 Radio-HPLC spectrum of the caspase-3-targeted molecular probe [ 68 Ga]GPN-DEVD.
  • FIG27 HPLC spectra of the caspase-3-targeted molecular probe [ 68 Ga]GPN-DEVD after incubation in PBS for different time periods.
  • FIG28 HPLC spectra of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD after incubation in mouse serum for different time periods.
  • FIG. 29 Cellular uptake values of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD in apoptotic 4T1 cells and normal 4T1 cells at different times.
  • FIG30 PET imaging of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD in the BMS-1198-induced tumor apoptosis model 4T1.
  • Figure 31 Quantitative analysis results of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD in tumor and muscle in the BMS-1198-induced tumor apoptosis model 4T1.
  • Figure 32 Quantitative analysis results of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD in tumor and muscle in the normal tumor model 4T1.
  • FIG. 33 Ratio of tumor to muscle uptake values of the caspase-3-targeted molecular probe [ 68 Ga]GPN-DEVD in normal and BMS-1198-induced tumor apoptosis 4T1 models.
  • Figure 34 Western Blot analysis of caspase-3 expression levels in tumors of the BMS-1198 treated and untreated groups.
  • FIG. 35 Synthesis route of the caspase-3-targeting molecular probe [ 18 F]GPN-DEVD.
  • FIG. 36 Radio-HPLC spectrum of the caspase-3-targeting molecular probe [ 18 F]GPN-DEVD.
  • FIG. 37 PET imaging of the caspase-3-targeted molecular probe [ 18 F]GPN-DEVD in the BMS-1198-induced tumor apoptosis model 4T1.
  • FIG. 38 Quantitative analysis results of the caspase-3-targeted molecular probe [ 18 F]GPN-DEVD in tumor and muscle in the BMS-1198-induced tumor apoptosis model 4T1.
  • FIG. 39 Quantitative analysis results of the caspase-3-targeted molecular probe [ 18 F]GPN-DEVD in tumor and muscle in the normal tumor model 4T1.
  • FIG. 40 Ratio of tumor to muscle uptake values of the caspase-3-targeted molecular probe [ 18 F]GPN-DEVD in normal and BMS-1198-induced tumor apoptosis 4T1 models.
  • FIG. 41 PET images of the caspase-3-targeted molecular probes [ 18 F]GPN-DEVD and [ 18 F]GP-16 in the 4T1 model.
  • FIG. 42 Quantitative analysis results of the caspase-3-targeted molecular probe [ 18 F]GPN-DEVD in bone in the 4T1 model.
  • Example 1 A caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD
  • This embodiment provides a caspase-3-targeted molecular probe [ 68 Ga]GPN-DEVD.
  • the caspase-3-targeted molecular probe [ 68 Ga]GPN-DEVD has the following structure:
  • Example 2 A method for preparing a molecular probe targeting caspase-3 [ 68 Ga]GPN-DEVD
  • This example provides a method for preparing the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD described in Example 1, and the specific steps are as follows:
  • Step 1 Rinse the sand core funnel twice with dichloromethane, drain it, add 2-chlorotrityl chloride resin (loading amount is 1.106 mmol/g, 361.6 mg) to the drained sand core funnel, and add 10 mL of dichloromethane to soak and swell the 2-chlorotrityl chloride resin, soak and swell for 10 minutes, and then drain it;
  • 2-chlorotrityl chloride resin loading amount is 1.106 mmol/g, 361.6 mg
  • Step 2 Add FMOC-(4-aminomethyl)benzoic acid (186 mg, 0.5 mmol) and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) (219 mg, 1.15 mmol) to the sand core funnel obtained in step 1, and dissolve with 10 mL of ultra-dry DMF (N, N-dimethylformamide) to obtain a solution; add DIPEA (N, N-diisopropylethylamine) (173 ⁇ L, 2 mmol) to the solution to adjust the pH of the solution to 8, and then shake the solution at 25° C. for 3 h;
  • DIPEA N, N-diisopropylethylamine
  • Step 4 Based on step 3, FMOC-(4-aminomethyl)benzoic acid (186 mg, 0.5 mmol) was replaced with FMOC-(4-aminomethyl)benzoic acid (186 mg, 0.5 mmol), glycine (78.5 mg, 0.5 mmol), N-(9-fluorenylmethoxycarbonyl)-S-trityl-L-cysteine (231.8 mg, 0.5 mmol), Fmoc-L-aspartic acid beta-tert-butyl ester (205.6 mg, 0.5 mmol), Fmoc-L-valine (169.7 mg, 0.5 mmol), Fmoc-tert-butyl-L-glutamic acid (221.7 mg, 0.5 mmol), and Fmoc-L-aspartic acid beta-tert-butyl ester (205.6 mg, 0.5 mmol), and the operation of step 2 was repeated to obtain the desired polypeptide chain;
  • Step 5 Add ultra-dry DMF (8 mL), acetic anhydride (1 mL) and DIPEA (N,N-diisopropylethylamine) (1 mL) to the sand core funnel obtained in step 4 to obtain a mixed solution A; shake the mixed solution A at 25°C for 5 h. After the shaking is completed, drain the solvent and rinse three times, add 10 mL of a CH 2 Cl 2 solution containing 1% (v/v) TFA (trifluoroacetic acid) to obtain a mixed solution B; shake the mixed solution B at 25°C for 10 min.
  • a CH 2 Cl 2 solution containing 1% (v/v) TFA (trifluoroacetic acid
  • Step 6 Dissolve N-Boc-D-propargylglycine (157.5 mg, 0.9 mmol) in 7 mL of ultra-dry THF (tetrahydrofuran) to obtain a mixed solution; add isobutyl chloroformate (IBCF, 97.5 ⁇ L, 0.75 mmol) and nitrogen methylmorpholine (NMM, 165 ⁇ L, 1.5 mmol) to the mixed solution, and stir for 2 h in an ice bath at 150 rpm under the protection of nitrogen to obtain a reaction solution A; dissolve CBT (2-cyano-6-aminobenzothiazole) (87.5 mg, 0.5 mmol) in 3 mL of anhydrous THF to obtain a dissolved solution; add the dissolved solution to the reaction solution A using a syringe, and under the protection of nitrogen, stir for 30 min in a light-proof, ice bath, and 150 rpm conditions, and then at 25 ° C.
  • IBCF isobutyl chlor
  • Step 7 Compound GPN-1 (179.5 mg, 0.39 mmol) was dissolved in 4 mL of dichloromethane (CH 2 Cl 2 ) to obtain a solution; 4 mL of TFA (trifluoroacetic acid) was added dropwise to the solution, and the mixture was stirred at 25° C.
  • dichloromethane CH 2 Cl 2
  • TFA trifluoroacetic acid
  • reaction solution was firstly used to remove the organic solvent using a rotary evaporator, and then washed with CH 2 Cl 2 three times, and then precipitated with cold ether (4° C.), and then transferred to a 50 mL centrifuge tube, and the supernatant was removed by centrifugation; the precipitate was taken and dried to obtain compound GPN-2 (120 mg, yield 100%) as a light yellow solid;
  • Step 11 Compound GPN-5 (63 mg, 0.035 mmol) was dissolved in 4 mL CH 2 Cl 2 to obtain a solution; 4 mL TFA (trifluoroacetic acid) was added to the solution, and the mixture was stirred at 25° C. and 150 rpm for 1.5 h to obtain a reaction solution; the reaction solution was used to remove the organic solvent using a rotary evaporator, and after precipitation with cold ether (4° C.), it was transferred to a 50 mL centrifuge tube, and the supernatant was removed by centrifugation; the precipitate was taken and dried to obtain compound GPN-6 (45 mg, yield 100%) as a light yellow solid;
  • Step 12 Dissolve compound GPN-6 (45 mg, 0.035 mmol) in 3 mL of methanol (MeOH) to obtain a solution; add TIPS (triisopropylsilane) (67 ⁇ L) and SEt (2-(ethyldisulfanyl)pyridine) (6.7 mg, 0.039 mmol) to the solution.
  • TIPS triisopropylsilane
  • SEt (2-(ethyldisulfanyl)pyridine
  • the mixture was stirred at 25°C and 150 rpm for 2 h to obtain a reaction solution; the reaction solution was used to remove the organic solvent using a rotary evaporator, precipitated with cold ether (4°C), and then transferred to a 50 mL centrifuge tube, and the supernatant was removed by centrifugation; the precipitate was dried to obtain a light yellow powder compound GPN-7 (15 mg, yield 31%);
  • Step 13 Dissolve compound GPN-7 (7 mg, 0.0052 mmol) and NOTA-NHS (5 mg, 0.015 mmol) in 1 mL DMF (N, N-dimethylformamide) to obtain a solution; add DIPEA (N, N-diisopropylethylamine) (26 ⁇ L, 0.015 mmol) to the solution to adjust the pH of the solution to 8, and stir at 25 ° C and 150 rpm for 2 hours under the protection of nitrogen to obtain a reaction solution; purify the reaction solution using semi-preparative HPLC (the purification conditions of semi-preparative HPLC are shown in Table 1; the process of semi-preparative HPLC purification is: select the mobile phase echelon in Table 1, and purify the sample dissolved in DMF through a C18 reverse chromatographic column) to obtain a labeled precursor GPN-DEVD (4 mg, 51.2%) of a caspase-3 targeted molecular probe (the synthetic route of the labeled precursor GPN
  • Step 14 Use 0.05M HCl to elute 68 Ga from the 68 Ge/ 68 Ga generator (ITG) and mix with 1.25M NaOAc buffer to adjust the pH to 4.0; then transfer the mixture directly to a 1 mL plastic tube containing 20 ⁇ g of the labeled precursor GPN-DEVD of the caspase-3 targeted molecular probe, and after mixing, incubate the mixture in an oil bath at 37°C for 10 min to obtain the molecular probe [ 68 Ga]GPN-DEVD; the product is analyzed by radio-HPLC (the synthesis route of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD is shown in Figure 25).
  • Example 3 A caspase-3 targeted molecular probe [ 18 F]GPN-DEVD
  • This embodiment provides a caspase-3-targeted molecular probe [ 18 F]GPN-DEVD.
  • the caspase-3-3-targeted molecular probe [ 18 F]GPN-DEVD has the following structure:
  • Example 4 A method for preparing a molecular probe [ 18 F]GPN-DEVD targeting caspase-3
  • This example provides a method for preparing the caspase-3 targeted molecular probe [ 18 F]GPN-DEVD described in Example 1. The specific steps are as follows:
  • reaction solution was first transferred to a centrifuge tube containing 20 mL of ultrapure water, and then the reaction solution was added to a centrifuge tube containing 20 mL of ultrapure water.
  • the molecular probe [ 18 F]GPN-DEVD in the solution was loaded onto a C 18 purification column (model Sep-Pak plus C 18 ) activated with ethanol (10 mL) and ultrapure water (10 mL) in sequence; the loaded C 18 purification column was washed three times with ultrapure water, and then the molecular probe [ 18 F]GPN-DEVD captured on the C 18 purification column was eluted into a vial with ethanol (500 ⁇ L) to obtain the molecular probe [ 18 F]GPN-DEVD; the product was analyzed by radio-HPLC (the synthetic route of the caspase-3 targeted molecular probe [ 18 F]GPN-DEVD is shown in FIG35 ).
  • This comparative example provides a caspase-3-targeted molecular probe [ 18 F]GP-16, wherein the caspase-3-targeted molecular probe [ 18 F]GP-16 has the following structure (for the molecular structure and preparation method of [ 18 F]GP-16, see the patent application with publication number CN113354712A):
  • This experimental example provides a radiochemical yield (RCY) and radiochemical purity (RCP) experiment of a caspase-3 targeted molecular probe.
  • the specific process is as follows:
  • the labeling time required for the molecular probe [ 68 Ga]GPN-DEVD is shorter, the labeling conditions are mild and suitable for compounds that are unstable at high temperatures, and the labeling yields of the molecular probe [ 18 F]GPN-DEVD and the molecular probe [ 68 Ga]GPN-DEVD are higher than those of the molecular probe [ 18 F]GP-16.
  • This experimental example provides an in vitro reduction and macrocyclization experiment of a caspase-3-targeted molecular probe.
  • the specific process is as follows:
  • the reduction of the disulfide bond in the labeled precursor GPN-DEVD of the caspase-3-targeted molecular probe produced a reduced product (GPN-DEVD-reduced), which produced a cyclized product (GPN-DEVD-C) after incubation with recombinant human caspase-3 in the reaction buffer.
  • This experimental example provides an in vitro stability experiment of a caspase-3 targeted molecular probe.
  • the specific process is as follows:
  • This experimental example provides a lipid-water partition coefficient experiment of a molecular probe targeting caspase-3.
  • the specific process is as follows:
  • the experimental results show that the lipid-water partition coefficient of the caspase-3 targeted molecular probe [ 68 Ga]GPN-DEVD is -2.02 ⁇ 0.03, and the lipid-water partition coefficient of the caspase-3 targeted molecular probe [ 18 F]GPN-DEVD is -1.98 ⁇ 0.09, which indicates that the molecular probes [ 68 Ga]GPN-DEVD and [ 18 F]GPN-DEVD have good hydrophilicity.
  • This experimental example provides a cell uptake experiment of a caspase-3 targeted molecular probe.
  • the specific process is as follows:
  • T cells from healthy BALB/C mice (purchased from Changzhou Cavens Laboratory Animal Company) were separated by density gradient centrifugation using Dayou T cell separation solution (purchased from Dakoway Company) and inoculated in a 6-well plate at a seeding volume of 2 ⁇ 10 5 ; in order to activate T cells, CD3 + antibody (purchased from Dakoway Company) and CD28 + (purchased from Dakoway Company) were simultaneously embedded in the 6-well plate at an addition volume of 5 ⁇ g/mL, and the 6-well plate was incubated at 4°C for 16 hours.
  • the activated T cells were resuspended in 1.5 mL of fresh 1640 medium and inoculated in the 6-well plate at a seeding amount of 1.8 ⁇ 10 5 cells/well, and then co-cultured at 37°C for 24 hours.
  • fresh 1640 medium was added to the 6-well plate at an addition amount of 1.5 mL, and then cultured at 37°C for 24 hours.
  • the 6-well plate was washed with PBS buffer to remove the T cells, and 2 mL of fresh serum-free medium ([ 68 Ga]GPN-DEVD prepared in Example 2) containing [ 68 Ga]GPN-DEVD was added to the 6-well plate.
  • Ga]GPN-DEVD concentration was 1 ⁇ Ci/100 ⁇ L), incubated at 37°C for 0.5, 1, 2 or 3h. After the incubation, the 4T1 cells were centrifuged at 6000rpm for 5min. After the centrifugation, the radioactive dose taken up by the cells was measured by a ⁇ counter, and the cell uptake percentage was calculated. Three groups of parallel experiments were performed at each time point, and the calculation results are shown in Figure 29.
  • the cellular uptake of [ 68Ga ]GPN-DEVD in the positive group was significantly higher than that in the negative group at all time points.
  • the cellular uptake of [ 68Ga ]GPN-DEVD in the positive group was 5.94 ⁇ 0.12%ID/mg at 0.5h, 6.32 ⁇ 0.09%ID/mg at 1h, 5.58 ⁇ 0.15%ID/mg at 2h, and 5.51 ⁇ 0.12%ID/mg at 3h
  • the cellular uptake of [ 68Ga ]GPN-DEVD in the negative group was 1.59 ⁇ 0.14%ID/mg at 0.5h, 1.8 ⁇ 0.02%ID/mg at 1h, 1.86 ⁇ 0.16%ID/mg at 2h, and 1.54 ⁇ 0.06%ID/mg at 3h.
  • the molecular probe [ 68Ga] GPN-DEVD Ga]GPN-DEVD can detect caspase-3 activity in apoptotic 4T1 cells more sensitively and specifically.
  • This experimental example provides a mouse PET imaging experiment using a caspase-3 targeted molecular probe.
  • the specific process is as follows:
  • mice were anesthetized with oxygen containing 2% (v/v) isoflurane at a flow rate of 2L/min; after fixing the limbs and tail of the mice, 150 ⁇ Ci of the molecular probe prepared in Example 2 dissolved in 100 ⁇ L normal saline was injected into the mouse tail vein.
  • the needle [ 68Ga ]GPN-DEVD, the molecular probe [ 18F ]GPN-DEVD prepared in Example 4 and the molecular probe [ 18F ]GP-16 of Comparative Example 1 were injected through the tail vein respectively; the mice in the control group were administered with the same method and dosage; after the probe injection, a 60-min dynamic PET scan was immediately performed, and the PET imaging results are shown in Figures 30, 37 and 41; after the scan, the 60-min PET imaging results were segmented into 12 frames of images, one frame every 5 minutes, using the OSEM3D/MAP algorithm to achieve real-time analysis of the in vivo imaging of the mice; the region of interest (ROI) technology in the ASIPRO software was used to outline and analyze the distribution of the probe in the tumor site and other organ tissues, and the analysis results are shown in Figures 31-33 and 38-40, and the bone uptake value was obtained by ROI analysis, and the analysis results are shown in Figure 42, wherein the uptake value of the molecular probe in each tissue in viv
  • the tumor/muscle ratio of the BMS-1198-treated group (7.45 ⁇ 0.35 at 10 min and 4.66 ⁇ 0.87 at 60 min) was higher than that of the untreated group (1.86 ⁇ 0.28 at 10 min and 1.61 ⁇ 0.48 at 60 min).
  • the BMS-1198 treatment group had a higher level of caspase-3 expression, while the blank group had almost no caspase-3 expression. Therefore, BMS-1198 can achieve a therapeutic effect by inducing tumor apoptosis.
  • the tumor/muscle ratio was higher in the BMS-1198-treated group (4.28 ⁇ 0.40 at 10 min and 3.13 ⁇ 0.26 at 60 min) relative to the untreated group (1.42 ⁇ 0.65 at 10 min and 2.15 ⁇ 0.26 at 60 min).

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Abstract

本申请涉及一种天冬氨酸蛋白酶靶向识别PET分子探针及应用，属于化学技术领域。本申请提供了一种分子探针，此分子探针在CBT与赖氨酸之间添加一个甘氨酸构建得到分子内环化骨架GPN，此延长的自主装骨架可以改善化合物柔性从而使得CBT和分子内的半胱氨酸更易发生点击缩合反应进行分子内缩合环化，使其受游离高浓度的半胱氨酸的影响更小，进一步促进探针在肿瘤内的缩合环化及自组装形成聚集体，以达到长时间高质量可视化肿瘤凋亡水平，此分子探针对caspase-3有较好选择性并具有体外特异性监测肿瘤细胞凋亡的能力，并且，此分子探针在接受免疫治疗的肿瘤异种移肿瘤模型中显示了良好的监测免疫治疗诱导的细胞凋亡的能力。

Description

一种天冬氨酸蛋白酶靶向识别PET分子探针及应用
相关申请的交叉引用
本申请要求在2022年9月30日提交中国专利局、申请号为202211230629.6、发明名称为“一种天冬氨酸蛋白酶靶向识别PET分子探针及应用”的中国专利申请的优先权，其全部内容通过引用的方式并入本文中。
技术领域
本申请涉及一种天冬氨酸蛋白酶靶向识别PET分子探针及应用，属于化学技术领域。
背景技术
肿瘤是指生物体内由多种因素导致的其细胞异常生长而引发的疾病，按其对生物体的特性及危害又分为恶性肿瘤和良性肿瘤。恶性肿瘤，又称为癌症，具有细胞分化和增殖异常、生长失去控制、浸润性和转移性等生物学特征。由于恶性肿瘤的异质性和个体差异化导致不同的癌症患者需要制定特定的治疗方案，因此，对于癌症患者来说通过评估治疗疗效来选择最合适的临床治疗方案非常重要。通过对早期治疗反应和治疗结果进行评估，可以减少癌症治疗过程中的副作用，可以及时调整治疗方法，并且，可以降低治疗费用。
目前，临床上常采用多种治疗方法诱导肿瘤细胞凋亡来达到抗肿瘤的目的。因此，肿瘤细胞凋亡水平可作为评价癌症治疗疗效的一项新指标。天冬氨酸蛋白水解酶-3(caspase-3)作为凋亡级联反应中至关重要的“死亡执行蛋白酶”，常被用来作为检测肿瘤细胞凋亡的生物标志物。因此，可以通过检测治疗后的肿瘤细胞中caspase-3水平的变化，从而实现癌症治疗疗效的即时反馈。
正电子发射断层扫描(PET)是分子影像领域非常先进的无创成像技术之一，具有高灵敏度和深层组织穿透性，可用于活体受试者的分子靶标验证和临床疾病诊断。开发一种靶向caspase-3的PET成像探针以对肿瘤细胞中caspase-3水平的变化进行监测，进而得到能够检测肿瘤细胞凋亡水平异常或监测各种疾病状态下的治疗诱导的肿瘤细胞凋亡水平的非侵入性成像策略，有助于实现癌症治疗疗效的即时反馈。
发明内容
为解决上述问题，本申请提供了一种天冬氨酸蛋白水解酶靶向的分子探针，所述分子探针具有如下所示结构：
其中，R为放射性核素标记基团。
在本申请的一种实施方式中，所述放射性核素标记基团为68Ga、[18F]AlF、64Cu或89Zr。
在本申请的一种实施方式中，当放射性核素标记基团为68Ga时，所述分子探针具有如下所示结构：
在本申请的一种实施方式中，当放射性核素标记基团为[18F]AlF时，所述分子探针具有如下所示结构：
在本申请的一种实施方式中，所述分子探针的标记前体具有如下所示结构：
在本申请的一种实施方式中，所述分子探针靶向天冬氨酸蛋白水解酶-3(caspase-3)。
本申请还提供了一种制备上述分子探针的方法，所述方法为：将化合物GPN-7和NOTA-NHS溶解于DMF(N,N-二甲基甲酰胺)中，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)后进行反应，得到反应液；对反应液进行纯化，得到分子探针的标记前体GPN-DEVD；对天冬氨酸蛋白水解酶靶向的 分子探针的标记前体GPN-DEVD进行放射性核素标记，得到分子探针；
所述化合物GPN-7具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-7的制备方法为：将化合物GPN-6溶解于甲醇(MeOH)中，得到溶解液；在溶解液中加入Tips(三异丙基硅烷)和SEt(2-(ethyldisulfanyl)pyridine)后进行反应，得到反应液；将反应液浓缩、乙醚沉淀、离心、干燥，得到化合物GPN-7；
所述化合物GPN-6具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-6的制备方法为：将化合物GPN-5溶解于CH2Cl2(二氯甲烷)中，得到溶解液；在溶解液中加入TFA(三氟乙酸)和TIPS(三异丙基硅烷)后进行反应，得到反应液；将反应液浓缩、乙醚沉淀、离心、干燥，得到化合物GPN-6；
所述化合物GPN-5具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-5的制备方法为：将化合物GPN-4、化合物A1及HBTU(苯并三氮唑-四甲基脲六氟磷酸盐)溶解于THF(四氢呋喃)中，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)后，在氮气的保护下进行反应，得到反应液；将反应液浓缩、纯化、干燥，得到化合物GPN-5；
所述化合物GPN-4具有如下所示结构：
所述化合物A1具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-4的制备方法为：将化合物GPN-3和哌啶水溶液混合后进行反应，得到反应液；将反应液进行萃取、浓缩、纯化、干燥，得到化合物GPN-4；
所述化合物GPN-3具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-3的制备方法为：将化合物GPN-2、赖氨酸及HBTU(苯并三氮唑-四甲基脲六氟磷酸盐)溶解于THF(四氢呋喃)中，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)后，在氮气的保护下进行反应，得到反应液；将反应液浓缩、纯化、干燥，得到化合物GPN-3；
所述化合物GPN-2具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-2的制备方法为：将化合物GPN-1溶解于CH2Cl2(二氯甲烷)中，得到溶解液；在溶解液中加入TFA(三氟乙酸)和TIPS(三异丙基硅烷)后进行反应，得到反应液；将反应液浓缩、乙醚沉淀、离心、干燥，得到化合物GPN-2；
所述化合物GPN-1具有如下所示结构：
在本申请的一种实施方式中，所述化合物GPN-1的制备方法为：将BOC-甘氨酸、氯甲酸异丁酯(IBCF)和N-甲基吗啉(NMM)溶解于THF(四氢呋喃)中后，在氮气的保护下进行反应，得到反应液A；将2-氰基-6-氨基苯并噻唑(CBT)溶解于THF(四氢呋喃)中，得到溶解液；将溶解液和反应液A混合后，在氮气的保护下进行反应，得到反应液B；将反应液B萃取、浓缩、纯化、干燥，得到的化合物GPN-1。
本申请还提供了上述分子探针在天冬氨酸蛋白水解酶显像中的应用，所述应用非疾病的诊断和治疗目的。
在本申请的一种实施方式中，所述天冬氨酸蛋白水解酶为天冬氨酸蛋白水解酶-3。
本申请还提供了一种靶向天冬氨酸蛋白水解酶的显像剂，所述显像剂含有上述分子探针。
本申请技术方案，具有如下优点：
本申请提供了一种分子探针，此分子探针具有以下优势：
第一，在CBT与赖氨酸之间添加一个甘氨酸构建得到分子内环化骨架GPN，此延长的自主装骨架可 以改善化合物柔性从而使得CBT和分子内的半胱氨酸更易发生点击缩合反应进行分子内缩合环化，使其受游离高浓度的半胱氨酸的影响更小，进一步促进探针在肿瘤内的缩合环化及自组装形成聚集体，以达到长时间高质量可视化肿瘤凋亡水平；
第二，能够结合多种标记核素和标记方法，研究不同核素(68Ga、[18F]AlF、64Cu或89Zr)对分子探针标记比活度以及成像效果的影响，以选择最佳的标记方式以及成像核素，进而达到在合适的时间内高质量可视化肿瘤凋亡水平的目的；
第三，具有较好的体外稳定性以及安全性；
第四，对caspase-3有较好选择性并具有体外特异性监测肿瘤细胞凋亡的能力；
第五，在接受免疫治疗的肿瘤异种移肿瘤模型中显示了良好的监测免疫治疗诱导的细胞凋亡的能力。
综上，此分子探针可以通过PET成像对肿瘤细胞中caspase-3水平的变化进行监测，进而实现检测肿瘤细胞凋亡水平异常或监测各种疾病状态下的治疗诱导的肿瘤细胞凋亡水平的非侵入性成像策略，以实现癌症治疗疗效的即时反馈，具有较好的应用前景。
进一步地，所述放射性核素标记基团为68Ga；68Ga核素由68Ge/68Ga发生器(ITG)制备，廉价易得，而且68Ga标记所需的孵育温度低(37℃)时间短(10min)且标记产率高。
进一步地，所述放射性核素标记基团为[18F]AlF；[18F]AlF氟化铝放射性标记基于[18F][AlF]2+阳离子的形成，并与多齿双功能螯合剂NOTA络合，具有很好的稳定性，使分子探针在成像过程中不易脱18F，使骨摄取较低。
附图说明
图1：化合物A1的合成路线。
图2：化合物A1的ESI-MS分析结果。
图3：化合物A1的HPLC谱图。
图4：化合物GPN-4的合成路线。
图5：化合物GPN-1的ESI-MS分析结果。
图6：化合物GPN-1的HPLC谱图。
图7：化合物GPN-2的ESI-MS分析结果。
图8：化合物GPN-2的HPLC谱图。
图9：化合物GPN-3的ESI-MS分析结果。
图10：化合物GPN-3的HPLC谱图。
图11：化合物GPN-4的ESI-MS分析结果。
图12：化合物GPN-4的HPLC谱图。
图13：caspase-3靶向的分子探针前体GPN-DEVD的合成路线。
图14：化合物GPN-5的ESI-MS分析结果。
图15：化合物GPN-5的HPLC谱图。
图16：化合物GPN-6的ESI-MS分析结果。
图17：化合物GPN-6的HPLC谱图。
图18：化合物GPN-7的ESI-MS分析结果。
图19：化合物GPN-7的HPLC谱图。
图20：caspase-3靶向的分子探针前体GPN-DEVD的ESI-MS分析结果。
图21：caspase-3靶向的分子探针前体GPN-DEVD的HPLC谱图。
图22：caspase-3靶向的分子探针前体GPN-DEVD剪切还原HPLC谱图。
图23：caspase-3靶向的分子探针前体GPN-DEVD-reduced ESI-MS分析结果。
图24：caspase-3靶向的分子探针前体GPN-DEVD-C ESI-MS分析结果。
图25：caspase-3靶向的分子探针[68Ga]GPN-DEVD的合成路线。
图26：caspase-3靶向的分子探针[68Ga]GPN-DEVD的放射性-HPLC谱图。
图27：caspase-3靶向的分子探针[68Ga]GPN-DEVD在PBS中孵育不同时间后的HPLC谱图。
图28：caspase-3靶向的分子探针[68Ga]GPN-DEVD在小鼠血清中孵育不同时间后的HPLC谱图。
图29：caspase-3靶向的分子探针[68Ga]GPN-DEVD在凋亡的细胞4T1与正常细胞4T1中的不同时间的细胞摄取值。
图30：caspase-3靶向的分子探针[68Ga]GPN-DEVD在BMS-1198诱导肿瘤凋亡模型4T1中的PET显像图。
图31：caspase-3靶向的分子探针[68Ga]GPN-DEVD在BMS-1198诱导肿瘤凋亡模型4T1中肿瘤与肌肉的定量分析结果。
图32：caspase-3靶向的分子探针[68Ga]GPN-DEVD在正常肿瘤模型4T1中肿瘤与肌肉的定量分析结果。
图33：caspase-3靶向的分子探针[68Ga]GPN-DEVD在正常和BMS-1198诱导肿瘤凋亡4T1模型中肿瘤与肌肉摄取值之比。
图34：Western Blot分析BMS-1198治疗组和未治疗组肿瘤中caspase-3表达水平的检测结果。
图35：caspase-3靶向的分子探针[18F]GPN-DEVD的合成路线。
图36：caspase-3靶向的分子探针[18F]GPN-DEVD的放射性-HPLC谱图。
图37：caspase-3靶向的分子探针[18F]GPN-DEVD在BMS-1198诱导肿瘤凋亡模型4T1中的PET显像图。
图38：caspase-3靶向的分子探针[18F]GPN-DEVD在BMS-1198诱导肿瘤凋亡模型4T1中肿瘤与肌肉的定量分析结果。
图39：caspase-3靶向的分子探针[18F]GPN-DEVD在正常肿瘤模型4T1中肿瘤与肌肉的定量分析结果。
图40：caspase-3靶向的分子探针[18F]GPN-DEVD在正常和BMS-1198诱导肿瘤凋亡4T1模型中肿瘤与肌肉摄取值之比。
图41：caspase-3靶向的分子探针[18F]GPN-DEVD和[18F]GP-16在4T1模型中的PET显像图。
图42：caspase-3靶向的分子探针[18F]GPN-DEVD在4T1模型中骨的定量分析结果。
具体实施方式
提供下述实施例是为了更好地进一步理解本申请，并不局限于所述最佳实施方式，不对本申请的内容和保护范围构成限制，任何人在本申请的启示下或是将本申请与其他现有技术的特征进行组合而得出的任何与本申请相同或相近似的产品，均落在本申请的保护范围之内。
下述实施例中未注明具体实验步骤或条件者，按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规试剂产品。
实施例1：一种caspase-3靶向的分子探针[68Ga]GPN-DEVD
本实施例提供了一种caspase-3靶向的分子探针[68Ga]GPN-DEVD，所述caspase-3靶向的分子探针[68Ga]GPN-DEVD具有如下所示结构：
实施例2：一种制备caspase-3靶向的分子探针[68Ga]GPN-DEVD的方法
本实施例提供了实施例1所述caspase-3靶向的分子探针[68Ga]GPN-DEVD的制备方法，具体步骤如下：
步骤一：用二氯甲烷冲洗砂芯漏斗两次，抽干，在抽干的砂芯漏斗中加入2-氯三苯甲基氯树脂(负载量为1.106mmol/g，361.6mg)，并加入10mL的二氯甲烷浸泡溶胀2-氯三苯甲基氯树脂，浸泡溶胀10min后，抽干；
步骤二：向步骤一获得的砂芯漏斗中加入FMOC-(4-氨甲基)苯甲酸(186mg，0.5mmol)和HBTU(苯并三氮唑-四甲基脲六氟磷酸盐)(219mg，1.15mmol)，用10mL超干DMF(N,N-二甲基甲酰胺)溶解，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)(173μL，2mmol)调节溶解液的pH至8后，将溶解液在25℃下振荡3h；
步骤三：振荡完成后，抽干步骤二获得的砂芯漏斗中的溶剂；加入10mL DMF/CH3OH/DIPEA的混合溶液(DMF/CH3OH/DIPEA＝17：2：1，v/v/v)清洗滤饼，振荡10min后抽滤，重复操作一次，以除去未 反应的氨基酸；再用10mL DMF(HPLC型)洗涤两次，振荡2min后抽滤；向砂芯漏斗中加入10mL含20％(v/v)哌啶的DMF溶液，振荡10min后抽滤，重复操作三次，以脱掉氨基酸上的FMOC保护基团；再用10mL DMF(HPLC型)清洗滤饼五次，以洗去多余的哌啶；洗涤结束后，抽干溶剂，取样进行Kaiser测试，试剂颜色显示为深紫色，说明此时FOMC基团已脱除，裸露出氨基，可连接下一个氨基酸；
步骤四：在步骤三的基础上，将FMOC-(4-氨甲基)苯甲酸(186mg，0.5mmol)依次替换为FMOC-(4-氨甲基)苯甲酸(186mg，0.5mmol)、甘氨酸(78.5mg，0.5mmol)、N-(9-芴甲氧羰基)-S-三苯甲基-L-半胱氨酸(231.8mg，0.5mmol)、Fmoc-L-天冬氨酸beta-叔丁酯(205.6mg，0.5mmol)、Fmoc-L-缬氨酸(169.7mg，0.5mmol)、Fmoc-叔丁基-L-谷氨酸(221.7mg，0.5mmol)、Fmoc-L-天冬氨酸beta-叔丁酯(205.6mg，0.5mmol)，并重复步骤二的操作，得到所需多肽链；
步骤五：向步骤四获得的砂芯漏斗中加入超干DMF(8mL)、乙酸酐(1mL)和DIPEA(N,N-二异丙基乙胺)(1mL)，得到混合液A；将混合液A在25℃下振荡5h，振荡完成后，抽干溶剂并荡洗三次，加入10mL含1％(v/v)TFA(三氟乙酸)的CH2Cl2溶液，得到混合液B；将混合液B在25℃下振荡10min，振荡完成后，滤出带有化合物A1的滤液，重复该操作，直至2-氯三苯甲基氯树脂出现酒红色且不褪色时停止；将收集到的滤液用旋转蒸发仪除去溶剂，用冷乙醚(4℃)沉淀后，转移至50mL离心管，离心吸除上清液；取沉淀干燥，得到黄色粉末状的化合物A1(160mg，产率为29.5％)(化合物A1的合成路线见图1)；
步骤六：将N-Boc-D-炔丙基甘氨酸(157.5mg，0.9mmol)溶解于7mL超干THF(四氢呋喃)中，得到混合液；在混合液中添加氯甲酸异丁酯(IBCF，97.5μL，0.75mmol)和氮甲基吗啉(NMM，165μL，1.5mmol)后，在氮气的保护下，于冰浴、150rpm下搅拌2h，得到反应液A；将CBT(2-氰基-6-氨基苯并噻唑)(87.5mg，0.5mmol)溶于3mL无水THF中，得到溶解液；将溶解液使用注射器加入反应液A中后，在氮气的保护下，先于避光、冰浴、150rpm的条件下搅拌30min，然后于25℃下反应16h，得到反应液B；将盐酸(2mL，浓度1mol/L)使用注射器加入反应液B中淬灭反应后，使用旋转蒸发仪除去反应液B中的有机溶剂，得到粗产物A；通过乙酸乙酯-水体系萃取粗产物A中化合物GPN-1后，使用饱和碳酸氢钠水溶液洗涤三次，中和多余的盐酸，收集有机相；取有机相用无水硫酸钠干燥后使用旋转蒸发仪除去乙酸乙酯，得到到粗产物B；将粗产物B用1mL二氯甲烷溶解后使用硅胶柱进行层析(正己烷：乙酸乙酯＝1：1，v/v)纯化，得到纯化产物；将纯化产物用旋转蒸发仪旋干，得到白色固体状的化合物GPN-1(132mg，产率为79.5％)；
步骤七：将化合物GPN-1(179.5mg，0.39mmol)溶解于4mL二氯甲烷(CH2Cl2)中，得到溶解液；在溶解液中滴加4mL TFA(三氟乙酸)后，于25℃、150rpm下搅拌30min，得到反应液；将反应液先用旋转蒸发仪除去有机溶剂，然后用CH2Cl2洗涤三次，再用冷乙醚(4℃)沉淀后，转移至50mL离心管，离心吸除上清液；取沉淀干燥，得到淡黄色固体状的化合物GPN-2(120mg，产率为100％)；
步骤八：将化合物GPN-2(120mg，0.39mmol)、赖氨酸(206mg，0.44mmol)及HBTU(苯并三氮唑-四甲基脲六氟磷酸盐)(174mg，0.46mmol)溶解于8mL超干THF(四氢呋喃)中，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)(174μL，1.0mmol)调节溶解液的pH至8后，在氮气的保护下，于25℃、150rpm下油浴搅拌16h，得到反应液；将反应液使用硅胶柱进行层析(正己烷：乙酸乙酯＝1：1，v/v)纯化后，使用旋转蒸发仪旋干，得到白色固体状的化合物GPN-3(145mg，产率为65％)；
步骤九：将化合物GPN-3(145mg，0.28mmol)加入3mL 5％(v/v)的哌啶水溶液中，得到混合液；将混合液于冰浴、150rpm下搅拌15min，得到反应液；将盐酸(3mL，1mol/L)使用注射器加入反应液中淬灭反应后，先通过二氯甲烷-水体系萃取反应液除去过量的盐酸，然后使用旋转蒸发仪蒸干，得到粗产品；将粗产物使用硅胶柱进行层析(CH2Cl2:CH3OH＝10:1，v/v)纯化后，使用旋转蒸发仪旋干，得到白色固体状的化合物GPN-4(81mg，产率为80.2％)(化合物GPN-4的合成路线见图4)；
步骤十：将化合物GPN-4(25mg，0.052mmol)、化合物A1(80mg，0.057mmol)和HBTU(苯并三氮唑-四甲基脲六氟磷酸盐)(23.5mg，0.06mmol)溶解于4mL超干THF(四氢呋喃)中，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)(23.5μL，0.13mmol)调节溶解液的pH至8后，在氮气的保护下，于25℃、150rpm下油浴搅拌16h，得到反应液；将反应液使用硅胶柱进行层析(二氯甲烷：甲醇＝20：1，v/v)纯化后，使用旋转蒸发仪旋干，得到白色固体状的化合物GPN-5(63mg，产率为58％)；
步骤十一：将化合物GPN-5(63mg，0.035mmol)溶解于4mL CH2Cl2中，得到溶解液；在溶解液中加入4mL TFA(三氟乙酸)后，于25℃、150rpm下搅拌1.5h，得到反应液；将反应液用旋转蒸发仪除去有机溶剂，用冷乙醚(4℃)沉淀后，转移至50mL离心管，离心吸除上清液；取沉淀干燥，得到淡黄色固体状的化合物GPN-6(45mg，产率为100％)；
步骤十二：将化合物GPN-6(45mg，0.035mmol)溶解于3mL甲醇(MeOH)中，得到溶解液；在溶解液中加入TIPS(三异丙基硅烷)(67μL)和SEt(2-(ethyldisulfanyl)pyridine)(6.7mg，0.039mmol) 后，于25℃、150rpm下搅拌2h，得到反应液；将反应液用旋转蒸发仪除去有机溶剂，用冷乙醚(4℃)沉淀后，转移至50mL离心管，离心吸除上清液；取沉淀干燥，得到淡黄色粉末状的化合物GPN-7(15mg，产率31％)；
步骤十三：将化合物GPN-7(7mg，0.0052mmol)和NOTA-NHS(5mg，0.015mmol)溶解于1mL DMF(N,N-二甲基甲酰胺)中，得到溶解液；在溶解液中加入DIPEA(N,N-二异丙基乙胺)(26μL，0.015mmol)调节溶解液的pH至8后，在氮气的保护下，于25℃、150rpm下搅拌2h，得到反应液；使用半制备型HPLC对反应液进行纯化(半制备型HPLC的纯化条件见表1；半制备型HPLC纯化的过程为：选择表1的流动相梯队，将溶解在DMF中的样品通过C18反向色谱柱纯化)，得到caspase-3靶向的分子探针的标记前体GPN-DEVD(4mg，51.2％)(caspase-3靶向的分子探针的标记前体GPN-DEVD的合成路线见图13)；
步骤十四：使用0.05M HCl从68Ge/68Ga发生器(ITG)中洗脱68Ga，并与1.25M NaOAc缓冲液混合以将pH值调节至4.0；然后将混合物直接转移到含有20μg的caspase-3靶向的分子探针的标记前体GPN-DEVD的1mL塑料管中，混匀后，将混合物在油浴锅中于37℃孵育10min，得到分子探针[68Ga]GPN-DEVD；通过放射-HPLC对产物进行分析(caspase-3靶向的分子探针[68Ga]GPN-DEVD的合成路线见图25)。
采用电喷雾电离源对化合物A1、GPN-1、GPN-2、GPN-3、GPN-4、GPN-5、GPN-6、GPN-7、GPN-DEVD进行ESI-MS分析，并采用Waters1525对化合物A1、GPN-1、GPN-2、GPN-3、GPN-4、GPN-5、GPN-6、GPN-7、GPN-DEVD进行HPLC检测，分析及检测结果见图2～3、5～11、14～21。
表1半制备型HPLC的纯化条件
实施例3：一种caspase-3靶向的分子探针[18F]GPN-DEVD
本实施例提供了一种caspase-3靶向的分子探针[18F]GPN-DEVD，所述caspase-3-3靶向的分子探针[18F]GPN-DEVD具有如下所示结构：
实施例4：一种制备caspase-3靶向的分子探针[18F]GPN-DEVD的方法
本实施例提供了实施例1所述caspase-3靶向的分子探针[18F]GPN-DEVD的制备方法，具体步骤如下：
将氯化铝(6μL，2mM)、冰醋酸(5μL，2mM)和乙腈(384μL，2mM)混合，得到混合溶液；向混合溶液中添加40μg实施例2制得的caspase-3靶向的分子探针的标记前体GPN-DEVD，得到混合液；将混合液在100μL靶水(通过30MeV质子轰击银回旋加速器靶中富含98％的[l8O]水产生的靶水)中，于100℃下加热15min，得到反应溶液；先将反应溶液转移到含有20mL超纯水的离心管中，然后将反应溶 液中的分子探针[18F]GPN-DEVD先后负载于用乙醇(10mL)和超纯水(10mL)活化的C18纯化柱(型号Sep-Pak plusC18)上；将负载后的C18纯化柱用超纯水洗涤三次后，用乙醇(500μL)将捕获在C18纯化柱上的分子探针[18F]GPN-DEVD淋洗至西林瓶中，得到分子探针[18F]GPN-DEVD；通过放射-HPLC对产物进行分析(caspase-3靶向的分子探针[18F]GPN-DEVD的合成路线见图35)。
对比例1：一种caspase-3靶向的分子探针[18F]GP-16
本对比例提供了一种caspase-3靶向的分子探针[18F]GP-16，所述caspase-3靶向的分子探针[18F]GP-16具有如下所示结构([18F]GP-16的分子结构和制备方法参见公开号为CN113354712A的专利申请文本)：
实验例1：caspase-3靶向的分子探针的放射化学产率(RCY)和放射化学纯度(RCP)实验
本实验例提供了caspase-3靶向的分子探针的放射化学产率(RCY)和放射化学纯度(RCP)实验，具体过程如下：
取20μCi实施例2制得的分子探针[68Ga]GPN-DEVD、实施例4制得的分子探针[18F]GPN-DEVD和对比例1的分子探针[18F]GP-16分别与1mL乙腈混合，得到混合液1～3；使用Gabi Nova放射性检测器(购自德国Elysia-Raytest公司)对混合液1～3进行放射性HPLC检测，获得分子探针[68Ga]GPN-DEVD、分子探针[18F]GPN-DEVD和分子探针[18F]GP-16的放射化学产率(RCY)和放射化学纯度(RCP)，检测结果见图26、36，计算结果见表2。
由表2可知，相对于对比分子探针[18F]GP-16，分子探针[68Ga]GPN-DEVD标记所需时间较短，标记条件温和适合于在高温下不稳定的化合物，而且分子探针[18F]GPN-DEVD和分子探针[68Ga]GPN-DEVD的标记产率都比分子探针[18F]GP-16高。
表2不同分子探针的放射化学产率和放射化学纯度
实验例2：caspase-3靶向的分子探针的体外还原和大环化实验
本实验例提供了caspase-3靶向的分子探针的体外还原和大环化实验，具体过程如下：
将实施例2制得的caspase-3靶向的分子探针的标记前体GPN-DEVD与含50mM TCEP(三(2-羧乙基)膦)的PBS缓冲液(pH 7.4，0.01M)以TCEP：GPN-DEVD＝20：1的浓度比混合，得到混合液A；将混合液A在37℃下孵育1h，得到孵育液A；取孵育液A与反应缓冲液(100mM NaCl、50mM HEPES、1mM EDTA、10％甘油、0.1％CHAPS和水，pH＝7.4，10％甘油的％是指v/v，0.1％CHAPS的％是指m/m)以12：84的体积比混合，得到混合液B；在96μL混合液B中加入0.04μg(4μL)的重组人caspase-3酶(购自碧云天公司)后，将混合液B在37℃下孵育1h，得到孵育液B；使用Waters 1525和电喷雾质谱对孵育液B进行HPLC和LC-MS表征，表征结果见图22～24。
如图22～24显示：caspase-3靶向的分子探针的标记前体GPN-DEVD中二硫键的还原产生了还原产物(GPN-DEVD-reduced)，与重组人caspase-3在反应缓冲液中孵育后，产生了环化产物(GPN-DEVD-C)。
实验例3：caspase-3靶向的分子探针的体外稳定性实验
本实验例提供了caspase-3靶向的分子探针的体外稳定性实验，具体过程如下：
实验一：将实施例2制得的caspase-3靶向的分子探针[68Ga]GPN-DEVD与PBS缓冲液(pH 7.4，0.01M) 以1：1的体积比混合，得到混合液；将混合液在37℃下孵育1、2或3h；孵育结束后，取孵育液使用Gabi Nova放射性检测器(购自德国Elysia-Raytest公司)进行放射性HPLC分析，并通过产物峰面积/总峰面积计算放射化学纯度(RCP)，分析及计算结果见图27。
实验二：将实施例2制得的caspase-3靶向的分子探针[68Ga]GPN-DEVD与小鼠血清(取自购自南京森贝伽生物科技有限公司)以1：1的体积比混合，得到混合液；将混合液在37℃下孵育1、2或3h；孵育结束后，取20μL孵育液，加入等体积乙腈，在12000g条件下高速离心5min使血清与蛋白分离，吸取上清液使用Gabi Nova放射性检测器(购自德国Elysia-Raytest公司)进行放射性HPLC分析，并通过产物峰面积/总峰面积计算放射化学纯度(RCP)，分析及计算结果见图28。
由图27～28可知，当孵育时间增加至3h，caspase-3靶向的分子探针[68Ga]GPN-DEVD的放射化学纯度都在95％以上，证明其在孵育过程中无其他产物生成，具有较好的稳定性，探针具有良好的稳定性是开展体内研究探针对caspase-3酶靶向特异性的必要先决条件。
实验例4：caspase-3靶向的分子探针的脂水分配系数实验
本实验例提供了caspase-3靶向的分子探针的脂水分配系数实验，具体过程如下：
取一支离心管，先加入1mL去离子水和1mL正辛醇，然后分别加入实施例2制得的分子探针[68Ga]GPN-DEVD、实施例4制得的分子探针[18F]GPN-DEVD至浓度为30KBq/μL，得到混合液；将混合液振荡5min后，5000rpm高速离心5min破乳使得两相分离；取正辛醇相、水相各500μL到EP管中，随后使用γ计数器检测正辛醇相、水相各自的放射性活度并计算Log P(Log P＝LogCo/Cw)，其中，Co代表辛醇相中[68Ga]GPN-DEVD和[18F]GPN-DEVD的放射性剂量，Cw代表水相中[68Ga]GPN-DEVD和[18F]GPN-DEVD的放射性剂量；第一次测试结束后，重新添加500μL正辛醇和500μL的水到原来的离心管中，再次振荡、离心取样测Log P；重复多次实验，直到连续测出三组Log P值接近，取三组数据平均值为脂水分配系数值，结果表示为平均值±标准差。
实验测得caspase-3靶向的分子探针[68Ga]GPN-DEVD的脂水分配系数为-2.02±0.03，caspase-3靶向的分子探针[18F]GPN-DEVD的脂水分配系数为-1.98±0.09，这说明分子探针[68Ga]GPN-DEVD和[18F]GPN-DEVD亲水性较好。
实验例5：caspase-3靶向的分子探针的细胞摄取实验
本实验例提供了caspase-3靶向的分子探针的细胞摄取实验，具体过程如下：
将来自健康BALB/C小鼠(购自常州卡文斯实验动物公司)的T细胞通过使用达优T细胞分离液(购自达科为公司)进行密度梯度离心分离后，以2×105个的接种量接种在6孔板中；为了激活T细胞，将将CD3+抗体(购自达科为公司)和CD28+(购自达科为公司)同时以5μg/mL的添加量嵌入6孔板中后，将6孔板在4℃下孵育16h，孵育结束后，用PBS缓冲液(pH 7.4，0.01M)洗涤三次，得到已激活的T细胞；将4T1细胞(购自上海中科院细胞库)以1.8×104细胞/孔的接种量接种到添加有0.4mL 1640培养基(购自BI公司)的6孔板中后，将6孔板在37℃孵育24h，孵育结束后，弃上清，将含有4T1细胞的6孔板(此时6孔板中4T1细胞的浓度为2.5×104细胞/孔)分为两组，阳性组将已激活的T细胞重悬于1.5mL新鲜1640培养基后以1.8×105细胞/孔的接种量接种于6孔板中后，于37℃共培养24h，阴性组将新鲜的1640培养基以1.5mL的添加量添加至6孔板中后，于37℃培养24h，培养结束后，用PBS缓冲液洗涤6孔板去除T细胞，并在6孔板中加入2mL含有实施例2制得的[68Ga]GPN-DEVD的新鲜无血清培养基([68Ga]GPN-DEVD浓度为1μCi/100μL)，在37℃下孵育0.5、1、2或3h，孵育结束后，取4T1细胞于6000rpm离心5min，离心结束后，通过γ计数仪测定细胞摄取的放射性剂量，并计算出细胞摄取百分比，每个时间点平行实验三组，计算结果见图29。
如图29所示，阳性组[68Ga]GPN-DEVD的细胞摄取在所有时间点均明显高于阴性组，阳性组[68Ga]GPN-DEVD的细胞摄取在0.5h为5.94±0.12％ID/mg，在1h为6.32±0.09％ID/mg，在2h为5.58±0.15％ID/mg，在3h为5.51±0.12％ID/mg，而阴性组[68Ga]GPN-DEVD的细胞摄取在0.5h为1.59±0.14％ID/mg，在1h为1.8±0.02％ID/mg，在2h为1.86±0.16％ID/mg，在3h为1.54±0.06％ID/mg，可见，分子探针[68Ga]GPN-DEVD可对凋亡细胞细胞4T1中的caspase-3活性进行更加灵敏特异地检测。
实验例6：caspase-3靶向的分子探针的小鼠PET成像实验
本实验例提供了caspase-3靶向的分子探针的小鼠PET成像实验，具体过程如下：
将4T1肿瘤细胞按照1×106个的剂量(重悬于100μL PBS缓冲液)以皮下注射的方式植入雌性BALB/C小鼠(6周龄，购自常州卡文斯实验动物公司)的右侧肩部；当肿瘤直径达到0.6cm时，将小鼠随机分为两组(n＝3)，对照组(未治疗组)小鼠不做处理，实验组(BMS-1198治疗组)小鼠尾静脉注射BMS-1198(剂量5mg/kg，溶剂100μL生理盐水，每天一次，连续三次，BMS-1198参见PCT专利号为WO 2015/160641A2的专利申请文本)诱导肿瘤部位凋亡；注射三天后，用含有2％(v/v)异氟烷的氧气以2L/min的流速麻醉小鼠；将小鼠的四肢与尾巴固定后，将溶解在100μL生理盐水中的150μCi的实施例2制得的分子探 针[68Ga]GPN-DEVD、实施例4制得的分子探针[18F]GPN-DEVD和对比例1的分子探针[18F]GP-16分别通过尾静脉注射；对照组小鼠以同样的方式和剂量给药；探针注射完毕后，立即执行60min的动态PET扫描，PET显像结果见图30、37和41；扫描完成后，使用OSEM3D/MAP算法将60min的PET成像结果分割成12帧图像，每5min一帧，以实现对小鼠体内成像的实时分析；采用ASIPRO软件中感兴趣区(ROI)技术对探针在肿瘤部位及其他器官组织中的分布情况进行勾画和分析比较，分析结果见图31～33和38～40，通过ROI分析获得骨摄取值，分析结果见图42，其中，分子探针在活体内各个组织中的摄取值以％ID/mL(每克注射剂量百分比)表示；扫描完成后，处死小鼠，通过RIPA裂解液(购自碧云天公司)获得可溶性肿瘤裂解物，使用BCA蛋白质分析试剂盒(购自碧云天公司)测量可溶性肿瘤裂解物中的蛋白质含量，并使用12％(g/100mL)SDS-PAGE凝胶分离50μg可溶性肿瘤裂解物中的蛋白质；将膜吸附到PVDF膜上后，先用含5％(g/100mL)脱脂乳的TBS缓冲液(购自碧云天公司)在室温(25℃)下封闭膜60min，然后用含anti-pro-caspase-3(购自碧云天公司)的TBS缓冲液(anti-pro-caspase-3：TBS缓冲液＝1：1000)在4℃下培养16h，再用HRP结合的二级抗体检测一级抗体，接着用免疫印迹鲁米诺试剂显示免疫反应带，最后用ImageJ软件进行密度分析，分析结果见图34。
如图30～33所示，BMS-1198治疗组注射分子探针[68Ga]GPN-DEVD 20min(5.04±0.67％ID/mL)后和60min(4.52％±0.68％ID/mL)后，观察到[68Ga]GPN-DEVD的在肿瘤部位的高摄取，在相比之下，未治疗组的小鼠的PET成像显示，在静脉给药20min(2.29±0.65％ID/mL)和60min(2.40±0.75％ID/mL)肿瘤部位的摄取很少；治疗组和未治疗组肌肉组织的放射性摄取(10min时为1.17±0.42％ID/mL，60min时为1.48±0.53％ID/mL)相似；BMS-1198治疗组的肿瘤/肌肉比率(10min时为7.45±0.35，60min时为4.66±0.87)相对于未治疗组(10min时为1.86±0.28，60min时为1.61±0.48)较高。
如图34所示，BMS-1198治疗组具有较高水平的caspase-3表达，而空白组几乎没有caspase-3的表达，因此，BMS-1198可通过诱导肿瘤凋亡达到治疗效果。
如图37～41所示，BMS-1198治疗组注射分子探针[18F]GPN-DEVD 20min(6.4±0.66％ID/mL)后和60min(5.2±0.82％ID/mL)后，观察到[18F]GPN-DEVD的高肿瘤摄取，在相比之下，未治疗组的小鼠的PET成像显示，在静脉给药后20min(2.11±0.32％ID/mL)和60min(1.15±0.96％ID/mL)的摄取很少；治疗组和未治疗组肌肉组织的放射性在20min时为1.46±0.33％ID/mL，60min时为1.67±0.46％ID/mL；BMS-1198治疗组的肿瘤/肌肉比率(10min时的4.28±0.40，60min时的3.13±0.26)相对于未治疗组(10min时为1.42±0.65和60min时为2.15±0.26)较高。
如图42所示，BABL/C小鼠注射分子探针[18F]GP-16 60min骨摄取为8.97±1.23％ID/mL，观察到[18F]GP-16的骨部位高摄取，在相比之下，[18F]GPN-DEVD 60min(3.38±0.87％ID/mL)的摄取相对较少。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种天冬氨酸蛋白水解酶靶向的分子探针，其特征在于，所述分子探针具有如下所示结构：
    其中，R为放射性核素标记基团。
  2. 如权利要求1所述的分子探针，其特征在于，所述放射性核素标记基团为68Ga、[18F]AlF、64Cu或89Zr。
  3. 如权利要求2所述的分子探针，其特征在于，当放射性核素标记基团为68Ga时，所述分子探针具有如下所示结构：
  4. 如权利要求2所述的分子探针，其特征在于，当放射性核素标记基团为[18F]AlF时，所述分子探针具有如下所示结构：
  5. 如权利要求1～4任一项所述的分子探针，其特征在于，所述分子探针的标记前体具有如下所示结构：
  6. 如权利要求1～5任一项所述的分子探针，其特征在于，所述分子探针靶向天冬氨酸蛋白水解酶-3。
  7. 一种制备权利要求1～6任一项所述的分子探针的方法，其特征在于，所述方法为：将化合物GPN-7和NOTA-NHS溶解于N,N-二甲基甲酰胺中，得到溶解液；在溶解液中加入N,N-二异丙基乙胺后进行反应，得到反应液；对反应液进行纯化，得到分子探针的标记前体GPN-DEVD；对天冬氨酸蛋白水解酶靶向的分子探针的标记前体GPN-DEVD进行放射性核素标记，得到分子探针；
    所述化合物GPN-7具有如下所示结构：
  8. 如权利要求7所述的方法，其特征在于，所述化合物GPN-7的制备方法为：将化合物GPN-6溶解于甲醇中，得到溶解液；在溶解液中加入三异丙基硅烷和2-(乙基二硫基吡啶)后进行反应，得到反应液；将反应液浓缩、乙醚沉淀、离心、干燥，得到化合物GPN-7；
    所述化合物GPN-6具有如下所示结构：
  9. 权利要求1～6任一项所述分子探针在天冬氨酸蛋白水解酶显像中的应用，其特征在于，所述应用非疾病的诊断和治疗目的。
  10. 一种靶向天冬氨酸蛋白水解酶的显像剂，其特征在于，所述显像剂含有权利要求1～5任一项所述分子探针。
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