CN117801062A

CN117801062A - Aspartic proteinase targeting recognition PET molecular probe and application thereof

Info

Publication number: CN117801062A
Application number: CN202211230629.6A
Authority: CN
Inventors: 邱玲; 林建国; 张理霞; 刘清竹
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2024-04-02
Also published as: WO2024066540A1

Abstract

The invention relates to an aspartic proteinase targeting recognition PET molecular probe and application thereof, belonging to the technical field of chemistry. The invention provides a molecular probe, which is constructed by adding glycine between CBT and lysine to obtain an intramolecular cyclic skeleton GPN, wherein the prolonged autonomous skeleton can improve the flexibility of a compound, so that the CBT and intramolecular cysteine are easier to undergo click condensation reaction to undergo intramolecular condensation cyclization, the influence of free high-concentration cysteine on the intramolecular condensation cyclization is smaller, the condensation cyclization and self-assembly of the probe in a tumor are further promoted to form an aggregate, so that the long-time high-quality visual tumor apoptosis level is achieved, the molecular probe has better selectivity for caspase-3 and the capability of specifically monitoring tumor apoptosis in vitro, and the molecular probe has good capability of monitoring immune therapy induced apoptosis in a tumor xenograft tumor model receiving immune therapy.

Description

Aspartic proteinase targeting recognition PET molecular probe and application thereof

Technical Field

The invention relates to an aspartic proteinase targeting recognition PET molecular probe and application thereof, belonging to the technical field of chemistry.

Background

Tumors are diseases in organisms caused by abnormal growth of cells due to various factors, and are classified into malignant tumors and benign tumors according to their characteristics and harm to organisms. Malignant tumors, also known as cancers, have biological characteristics such as abnormal cell differentiation and proliferation, loss of control of growth, infiltration, and metastasis. Since heterogeneity and individual differentiation of malignant tumors lead to the need to formulate specific treatment regimens for different cancer patients, it is important for cancer patients to select the most appropriate clinical treatment regimen by assessing the therapeutic efficacy. By evaluating early treatment response and treatment outcome, side effects during cancer treatment may be reduced, treatment methods may be adjusted in time, and treatment costs may be reduced.

At present, a plurality of treatment methods are often used clinically to induce tumor cell apoptosis so as to achieve the purpose of resisting tumor. Thus, the level of apoptosis of tumor cells can be used as a new indicator for evaluating the efficacy of cancer treatment. Aspartic proteinase-3 (caspase-3) is often used as a biomarker for detecting apoptosis in tumor cells as a "death-performing protease" critical in the apoptotic cascade. Therefore, the immediate feedback of the curative effect of the cancer treatment can be realized by detecting the change of the caspase-3 level in the tumor cells after the treatment.

Positron Emission Tomography (PET) is one of the very advanced noninvasive imaging technologies in the field of molecular imaging, has high sensitivity and deep tissue penetrability, and can be used for molecular target verification and clinical disease diagnosis of living subjects. A PET imaging probe targeting caspase-3 is developed to monitor the change of caspase-3 level in tumor cells, so as to obtain a non-invasive imaging strategy capable of detecting abnormal apoptosis level of tumor cells or monitoring the treatment-induced apoptosis level of tumor cells under various disease states, which is helpful for realizing immediate feedback of the curative effect of cancer treatment.

Disclosure of Invention

In order to solve the problems, the invention provides an aspartic protease targeted molecular probe, which has the following structure:

wherein R is a radionuclide label group.

In one embodiment of the invention, the radionuclide labeling group is ⁶⁸ Ga、[ ¹⁸ F]AlF、 ⁶⁴ Cu or ⁸⁹ Zr。

In one embodiment of the invention, when the radionuclide label group is ⁶⁸ The molecular probe has the following structure when Ga:

in one embodiment of the invention, when the radionuclide label group is [ [ ¹⁸ F]In AlF, the molecular probe has the following structure:

in one embodiment of the invention, the labeling precursor of the molecular probe has the structure shown below:

in one embodiment of the invention, the molecular probe targets aspartic proteinase-3 (caspase-3).

The invention also provides a method for preparing the molecular probe, which comprises the following steps: dissolving compounds GPN-7 and NOTA-NHS in DMF (N, N-dimethylformamide) to obtain a solution; adding DIPEA (N, N-diisopropylethylamine) into the dissolution solution, and then carrying out reaction to obtain a reaction solution; purifying the reaction liquid to obtain a labeling precursor GPN-DEVD of the molecular probe; labeling a radionuclide on a labeling precursor GPN-DEVD of an aspartic protease targeted molecular probe to obtain the molecular probe;

the compound GPN-7 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-7 comprises the following steps: dissolving a compound GPN-6 in methanol (MeOH) to obtain a solution; adding Tips (triisopropylsilane) and SEt (2- (ethylisoulfanyl) pyridine) into the solution, and reacting to obtain a reaction solution; concentrating the reaction solution, precipitating with diethyl ether, centrifuging, and drying to obtain a compound GPN-7;

the compound GPN-6 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-6 comprises the following steps: dissolving the compound GPN-5 in CH ₂ Cl ₂ (dichloromethane) to obtain a solution; adding TFA (trifluoroacetic acid) and TIPS (triisopropylsilane) into the solution, and then carrying out reaction to obtain a reaction solution; concentrating the reaction solution, precipitating with diethyl ether, centrifuging and drying to obtain a compound GPN-6;

the compound GPN-5 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-5 comprises the following steps: dissolving a compound GPN-4, a compound A1 and HBTU (benzotriazole-tetramethylurea hexafluorophosphate) in THF (tetrahydrofuran) to obtain a solution; adding DIPEA (N, N-diisopropylethylamine) into the dissolution solution, and then carrying out reaction under the protection of nitrogen to obtain reaction solution; concentrating, purifying and drying the reaction liquid to obtain a compound GPN-5;

the compound GPN-4 has the following structure:

the compound A1 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-4 comprises the following steps: mixing a compound GPN-3 with a piperidine aqueous solution, and then reacting to obtain a reaction solution; extracting, concentrating, purifying and drying the reaction liquid to obtain a compound GPN-4;

the compound GPN-3 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-3 comprises the following steps: the compound GPN-2, lysine and HBTU (benzotriazol-tetramethyluronium hexafluorophosphate) are dissolved in THF (tetrahydrofuran) to obtain a solution; adding DIPEA (N, N-diisopropylethylamine) into the dissolution solution, and then carrying out reaction under the protection of nitrogen to obtain reaction solution; concentrating, purifying and drying the reaction liquid to obtain a compound GPN-3;

the compound GPN-2 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-2 comprises the following steps: dissolving the compound GPN-1 in CH ₂ Cl ₂ (dichloromethane) to obtain a solution; adding TFA (trifluoroacetic acid) and TIPS (triisopropylsilane) into the solution, and then carrying out reaction to obtain a reaction solution; concentrating the reaction solution, precipitating with diethyl ether, centrifuging and drying to obtain a compound GPN-2;

the compound GPN-1 has the following structure:

in one embodiment of the invention, the preparation method of the compound GPN-1 comprises the following steps: BOC-glycine, isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM) are dissolved in THF (tetrahydrofuran) and reacted under the protection of nitrogen to obtain a reaction solution A; dissolving 2-cyano-6-aminobenzothiazole (CBT) in THF (tetrahydrofuran) to obtain a solution; mixing the dissolution solution and the reaction solution A, and then reacting under the protection of nitrogen to obtain a reaction solution B; and extracting, concentrating, purifying and drying the reaction liquid B to obtain the compound GPN-1.

The invention also provides application of the molecular probe in aspartic proteinase imaging, and the application is not for disease diagnosis and treatment.

In one embodiment of the invention, the aspartic protease is aspartic protease-3.

The invention also provides an imaging agent for targeting aspartic proteolytic enzyme, which comprises the molecular probe.

The technical scheme of the invention has the following advantages:

the invention provides a molecular probe, which has the following advantages:

firstly, adding glycine between CBT and lysine to construct a intramolecular cyclic skeleton GPN, wherein the prolonged autonomous skeleton can improve the flexibility of the compound so that the CBT and intramolecular cysteine are easier to undergo click condensation reaction to undergo intramolecular condensation cyclization, the influence of free high-concentration cysteine is smaller, and the condensation cyclization and self-assembly of a probe in a tumor are further promoted to form an aggregate so as to achieve long-time high-quality visual tumor apoptosis level;

secondly, the method can combine a plurality of labeling nuclides and labeling methods to research different nuclides ⁶⁸ Ga、[ ¹⁸ F]AlF、 ⁶⁴ Cu or ⁸⁹ Zr) the effect of molecular probe labeling specific activity and imaging effect to select the optimal labeling mode and imaging nuclide, thereby achieving the purpose of high-quality visualization of tumor apoptosis level in a proper time;

thirdly, the in-vitro stability and the safety are good;

fourth, has better selectivity to caspase-3 and has the ability of in vitro specificity monitoring tumor cell apoptosis;

fifth, the ability to monitor immunotherapy-induced apoptosis is shown well in tumor xenograft tumor models that receive immunotherapy.

In conclusion, the molecular probe can monitor the change of caspase-3 level in tumor cells through PET imaging, so as to realize the noninvasive imaging strategy for detecting abnormal apoptosis level of tumor cells or monitoring the treatment-induced apoptosis level of tumor cells under various disease states, thereby realizing the immediate feedback of the curative effect of cancer treatment and having better application prospect.

Further, the radionuclide labeling group is ⁶⁸ Ga； ⁶⁸ From Ga species ⁶⁸ Ge/ ⁶⁸ Ga generator (ITG) is prepared, is cheap and easy to obtain, and ⁶⁸ the incubation temperature required for Ga labeling is low (37 ℃) for a short period of time(10 min) and the labeling yield was high.

Further, the radionuclide label group is [ ¹⁸ F]AlF；[ ¹⁸ F]AlF aluminum fluoride radiolabelling is based on [ ¹⁸ F][AlF] ²⁺ The formation of cations and the complexation with a polydentate bifunctional chelating agent NOTA have good stability, so that the molecular probe is not easy to fall off in the imaging process ¹⁸ F, lower bone uptake.

Drawings

Fig. 1: synthetic route for compound A1.

Fig. 2: ESI-MS analysis results of Compound A1.

Fig. 3: HPLC profile of compound A1.

Fig. 4: synthetic route for the compound GPN-4.

Fig. 5: ESI-MS analysis results of Compound GPN-1.

Fig. 6: HPLC profile of compound GPN-1.

Fig. 7: ESI-MS analysis results of Compound GPN-2.

Fig. 8: HPLC profile of compound GPN-2.

Fig. 9: ESI-MS analysis results of Compound GPN-3.

Fig. 10: HPLC profile of compound GPN-3.

Fig. 11: ESI-MS analysis results of Compound GPN-4.

Fig. 12: HPLC profile of compound GPN-4.

Fig. 13: a synthesis route of a caspase-3 targeted molecular probe precursor GPN-DEVD.

Fig. 14: ESI-MS analysis results of Compound GPN-5.

Fig. 15: HPLC profile of compound GPN-5.

Fig. 16: ESI-MS analysis results of Compound GPN-6.

Fig. 17: HPLC profile of compound GPN-6.

Fig. 18: ESI-MS analysis results of Compound GPN-7.

Fig. 19: HPLC profile of compound GPN-7.

Fig. 20: ESI-MS analysis results of caspase-3 targeted molecular probe precursor GPN-DEVD.

Fig. 21: HPLC profile of caspase-3 targeted molecular probe precursor GPN-DEVD.

Fig. 22: caspase-3 targeted molecular probe precursor GPN-DEVD shear reduction HPLC profile.

Fig. 23: caspase-3 targeted molecular probe precursor GPN-DEVD-reduced ESI-MS analysis results.

Fig. 24: caspase-3 targeted molecular probe precursor GPN-DEVD-C ESI-MS analysis results.

Fig. 25: caspase-3 targeted molecular probes ⁶⁸ Ga]Synthetic route for GPN-DEVD.

Fig. 26: caspase-3 targeted molecular probes ⁶⁸ Ga]radioactivity-HPLC profile of GPN-DEVD.

Fig. 27: caspase-3 targeted molecular probes ⁶⁸ Ga]HPLC profiles of GPN-DEVD after incubation in PBS for different times.

Fig. 28: caspase-3 targeted molecular probes ⁶⁸ Ga]HPLC profile after incubation of GPN-DEVD in mouse serum for different times.

Fig. 29: caspase-3 targeted molecular probes ⁶⁸ Ga]Cell uptake values of GPN-DEVD at different times in apoptotic cells 4T1 than in normal cells 4T 1.

Fig. 30: caspase-3 targeted molecular probes ⁶⁸ Ga]PET imaging of GPN-DEVD in BMS-1198 induced tumor apoptosis model.

Fig. 31: caspase-3 targeted molecular probes ⁶⁸ Ga]Quantitative analysis of tumor and muscle in BMS-1198 induced tumor apoptosis model 4T1 by GPN-DEVD.

Fig. 32: caspase-3 targeted molecular probes ⁶⁸ Ga]Quantitative analysis of tumor and muscle in the normal tumor model 4T1 by GPN-DEVD.

Fig. 33: caspase-3 targeted molecular probes ⁶⁸ Ga]GPN-DEVD ratio of tumor to muscle uptake values in BMS-1198 induced tumor apoptosis 4T1 model.

Fig. 34: western Blot analysis of detection of caspase-3 expression levels in BMS-1198 treated and untreated tumors.

Fig. 35: caspase-3 targeted molecular probes ¹⁸ F]Synthetic route for GPN-DEVD.

Fig. 36: caspase-3 targeted molecular probes ¹⁸ F]radioactivity-HPLC profile of GPN-DEVD.

Fig. 37: caspase-3 targeted molecular probes ¹⁸ F]PET imaging of GPN-DEVD in BMS-1198 induced tumor apoptosis model.

Fig. 38: caspase-3 targeted molecular probes ¹⁸ F]Quantitative analysis of tumor and muscle in BMS-1198 induced tumor apoptosis model 4T1 by GPN-DEVD.

Fig. 39: caspase-3 targeted molecular probes ¹⁸ F]Quantitative analysis of tumor and muscle in the normal tumor model 4T1 by GPN-DEVD.

Fig. 40: caspase-3 targeted molecular probes ¹⁸ F]GPN-DEVD ratio of tumor to muscle uptake values in BMS-1198 induced tumor apoptosis 4T1 model.

Fig. 41: caspase-3 targeted molecular probes ¹⁸ F]GPN-DEVD and [ ¹⁸ F]PET imaging of GP-16 in 4T1 model.

Fig. 42: caspase-3 targeted molecular probes ⁶⁸ Ga]Quantitative analysis of bone in the 4T1 model by GPN-DEVD.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1: caspase-3 targeted molecular probe ⁶⁸ Ga]GPN-DEVD

This example provides a caspase-3 targeted fractionSub-probe [ ⁶⁸ Ga]GPN-DEVD, molecular probes targeted by caspase-3 [ ⁶⁸ Ga]GPN-DEVD has the structure shown below:

example 2: preparation of caspase-3 targeting molecular probe ⁶⁸ Ga]GPN-DEVD method

This example provides a caspase-3 targeted molecular probe as described in example 1 [ ⁶⁸ Ga]The preparation method of the GPN-DEVD comprises the following specific steps:

step one: washing the sand core funnel with dichloromethane twice, draining, adding 2-chlorotrityl chloride resin (with the load of 1.106mmol/g and 361.6 mg) into the drained sand core funnel, adding 10mL of dichloromethane, soaking and swelling the 2-chlorotrityl chloride resin, soaking and swelling for 10min, and draining;

step two: to the sand core funnel obtained in step one was added FMOC- (4-aminomethyl) benzoic acid (186 mg,0.5 mmol) and HBTU (benzotriazole-tetramethylurea hexafluorophosphate) (219. Mg,1.15 mmol), and dissolved in 10mL ultra-dry DMF (N, N-dimethylformamide) to give a solution; DIPEA (N, N-diisopropylethylamine) (173. Mu.L, 2 mmol) was added to the solution to adjust the pH of the solution to 8, and then the solution was shaken at 25℃for 3 hours.

Step three: after the oscillation is completed, the solvent in the sand core hopper obtained in the second step is pumped out; 10mL DMF/CH was added ₃ Mixed solution of OH/DIPEA (DMF/CH ₃ OH/dipea=17: 2:1, v/v/v) washing the filter cake, oscillating for 10min, suction filtering, and repeating the operation once to remove unreacted amino acid; washing twice with 10 mM (HPLC), oscillating for 2min, and suction filtering; adding 10mL of DMF solution containing 20% (v/v) piperidine into a sand core funnel, oscillating for 10min, performing suction filtration, and repeating the operation for three times to remove FMOC protecting groups on the amino acid; the filter cake was washed five more times with 10ml dmf (HPLC) to wash off excess piperidine; after washing, the solvent was drained, the sample was taken and tested for Kaiser, the reagent color showed dark purple, indicating that FOMC groups had been removed and amino groups were exposed, and the sample was allowed to attachThe next amino acid;

step four: on the basis of step three, 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), fmoc-L-aspartic acid beta-tert-butyl ester (205.6 mg,0.5 mmol) in this order, and the procedure of step two was repeated to obtain the desired polypeptide chain;

step five: adding super-dry DMF (8 mL), acetic anhydride (1 mL) and DIPEA (N, N-diisopropylethylamine) (1 mL) into the sand core funnel obtained in the step four to obtain a mixed solution A; shaking the mixture A at 25deg.C for 5 hr, draining the solvent, washing three times, adding 10mL of CH containing 1% (v/v) TFA (trifluoroacetic acid) ₂ Cl ₂ Obtaining a mixed solution B; oscillating the mixed solution B at 25 ℃ for 10min, filtering out filtrate with the compound A1 after the oscillation is completed, and repeating the operation until the 2-chlorotrityl chloride resin has reddish wine and does not fade; the solvent was removed from the collected filtrate by rotary evaporator, and after precipitation with cold diethyl ether (4 ℃), the filtrate was transferred to a 50mL centrifuge tube, and the supernatant was removed by centrifugation; the precipitate was taken and dried to give compound A1 (160 mg, yield 29.5%) as a yellow powder (the synthetic route for compound A1 is shown in fig. 1);

step six: N-Boc-D-propargylglycine (157.5 mg,0.9 mmol) was dissolved in 7mL of ultra-dry THF (tetrahydrofuran) to obtain a mixture; isobutyl chloroformate (IBCF, 97.5. Mu.L, 0.75 mmol) and methylmorpholine azote (NMM, 165. Mu.L, 1.5 mmol) were added to the mixture, followed by stirring under nitrogen at 150rpm in an ice bath for 2 hours to obtain a reaction solution A; CBT (2-cyano-6-aminobenzothiazole) (87.5 mg,0.5 mmol) was dissolved in 3mL dry THF to give a solution; adding the dissolved solution into the reaction solution A by using a syringe, stirring for 30min under the protection of nitrogen under the conditions of light shielding, ice bath and 150rpm, and then reacting at 25 ℃ for 16h to obtain a reaction solution B; adding hydrochloric acid (2 mL, concentration of 1 mol/L) into the reaction liquid B by using a syringe to quench the reaction, and removing the organic solvent in the reaction liquid B by using a rotary evaporator to obtain a crude product A; after the compound GPN-1 in the crude product A is extracted by an ethyl acetate-water system, the crude product A is washed three times by using saturated sodium bicarbonate water solution, excessive hydrochloric acid is neutralized, and an organic phase is collected; drying the organic phase by using anhydrous sodium sulfate, and removing ethyl acetate by using a rotary evaporator to obtain a crude product B; the crude product B was dissolved in 1mL of dichloromethane and purified by chromatography on a silica gel column (n-hexane: ethyl acetate=1:1, v/v) to give a purified product; the purified product was spin-dried using a rotary evaporator to give compound GPN-1 (132 mg, 79.5% yield) as a white solid;

step seven: compound GPN-1 (179.5 mg,0.39 mmol) was dissolved in 4mL dichloromethane (CH) ₂ Cl ₂ ) Obtaining a dissolving solution; after 4mL of TFA (trifluoroacetic acid) was added dropwise to the solution, the mixture was stirred at 25℃and 150rpm for 30 minutes to obtain a reaction solution; the reaction solution was subjected to rotary evaporation to remove the organic solvent, followed by CH ₂ Cl ₂ Washing for three times, precipitating with cold diethyl ether (4 ℃) and transferring to a 50mL centrifuge tube, and centrifuging to remove supernatant; the precipitate was taken and dried to give the compound GPN-2 (120 mg, 100% yield) as a pale yellow solid;

step eight: compound GPN-2 (120 mg,0.39 mmol), lysine (206 mg,0.44 mmol) and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) (174 mg,0.46 mmol) were dissolved in 8mL ultra-dry THF (tetrahydrofuran) to give a solution; DIPEA (N, N-diisopropylethylamine) (174. Mu.L, 1.0 mmol) was added to the solution to adjust the pH of the solution to 8, and the solution was stirred in an oil bath at 25℃and 150rpm under the protection of nitrogen for 16 hours to obtain a reaction solution; the reaction solution was purified by silica gel column chromatography (n-hexane: ethyl acetate=1:1, v/v), and then dried by spin-drying using a rotary evaporator to give compound GPN-3 (145 mg, yield 65%) as a white solid;

step nine: GPN-3 (145 mg,0.28 mmol) was added to 3mL of a 5% (v/v) aqueous piperidine solution to give a mixed solution; stirring the mixed solution in an ice bath at 150rpm for 15min to obtain a reaction solution; hydrochloric acid (3 mL,1 mol/L) was added to the reaction mixture using a syringe to quench the reaction, followed by extraction with a methylene chloride-water systemRemoving excessive hydrochloric acid from the reaction solution, and evaporating to dryness by using a rotary evaporator to obtain a crude product; the crude product was chromatographed using a silica gel column (CH ₂ Cl ₂ :CH ₃ After purification with oh=10:1, v/v) and spin-drying using a rotary evaporator, compound GPN-4 (81 mg, 80.2% yield) was obtained as a white solid (see fig. 4 for the synthetic route for compound GPN-4);

step ten: compound GPN-4 (25 mg,0.052 mmol), compound A1 (80 mg,0.057 mmol) and HBTU (benzotriazol-tetramethyluronium hexafluorophosphate) (23.5 mg,0.06 mmol) were dissolved in 4mL of ultra-dry THF (tetrahydrofuran) to give a solution; DIPEA (N, N-diisopropylethylamine) (23.5. Mu.L, 0.13 mmol) was added to the solution to adjust the pH of the solution to 8, and the solution was stirred in an oil bath at 25℃and 150rpm under the protection of nitrogen for 16 hours to obtain a reaction solution; the reaction solution was purified by silica gel column chromatography (dichloromethane: methanol=20:1, v/v) and dried by rotary evaporation to give GPN-5 (63 mg, yield 58%) as a white solid;

step eleven: compound GPN-5 (63 mg,0.035 mmol) was dissolved in 4mL CH ₂ Cl ₂ Obtaining a dissolving solution; adding 4mLTFA (trifluoroacetic acid) into the solution, and stirring at 25 ℃ and 150rpm for 1.5 hours to obtain a reaction solution; removing the organic solvent from the reaction solution by using a rotary evaporator, precipitating by using cold diethyl ether (4 ℃), transferring to a 50mL centrifuge tube, and centrifuging to remove the supernatant; the precipitate was taken and dried to give the compound GPN-6 (45 mg, yield 100%) as a pale yellow solid;

step twelve: compound GPN-6 (45 mg,0.035 mmol) was dissolved in 3mL of methanol (MeOH) to obtain a solution; TIPS (triisopropylsilane) (67 μl) and set2 (2- (ethyldulfanyl) pyridine) (6.7 mg,0.039 mmol) were added to the solution, followed by stirring at 25 ℃ for 2 hours at 150rpm to obtain a reaction solution; removing the organic solvent from the reaction solution by using a rotary evaporator, precipitating by using cold diethyl ether (4 ℃), transferring to a 50mL centrifuge tube, and centrifuging to remove the supernatant; the precipitate was taken and dried to give compound GPN-7 (15 mg, 31% yield) as a pale yellow powder;

step thirteen: the compound GPN-7 (7 mg,0.0052 mmol) and NOTA-NHS (5 mg,0.015 mmol) were dissolved in 1ml of LDMF (N, N-dimethylformamide) to give a solution; DIPEA (N, N-diisopropylethylamine) (26. Mu.L, 0.015 mmol) was added to the solution to adjust the pH of the solution to 8, followed by stirring at 25℃and 150rpm under nitrogen protection for 2 hours to give a reaction solution; the reaction solution was purified using semi-preparative HPLC (purification conditions of semi-preparative HPLC are shown in Table 1; semi-preparative HPLC purification process comprises selecting mobile phase ladder of Table 1, purifying sample dissolved in DMF by C18 reverse chromatography column) to obtain labeled precursor GPN-DEVD (4 mg, 51.2%) of caspase-3 targeted molecular probe (synthesis route of labeled precursor GPN-DEVD of caspase-3 targeted molecular probe is shown in FIG. 13);

step fourteen: from using 0.05M HCl ⁶⁸ Ge/ ⁶⁸ Elution in Ga Generator (ITG) ⁶⁸ Ga and mixed with 1.25M NaOAc buffer to adjust the pH to 4.0; the mixture was then transferred directly into a 1mL plastic tube containing 20. Mu.g of the labeled precursor GPN-DEVD of the caspase-3 targeted molecular probe, and after mixing, the mixture was incubated in an oil bath at 37℃for 10min to give the molecular probe [ ⁶⁸ Ga]GPN-DEVD; analysis of the product by radiation-HPLC (caspase-3 targeted molecular probes [ ⁶⁸ Ga]The synthetic route for GPN-DEVD is shown in FIG. 25).

ESI-MS analysis is carried out on the compounds A1, GPN-2, GPN-3, GPN-4, GPN-5, GPN-6, GPN-7 and GPN-DEVD by adopting an electrospray ionization source, and HPLC detection is carried out on the compounds A1, GPN-2, GPN-3, GPN-4, GPN-5, GPN-6, GPN-7 and GPN-DEVD by adopting Waters1525, wherein the analysis results are shown in figures 2-3, 5-11 and 14-21.

TABLE 1 purification conditions for semi-preparative HPLC

Example 3: caspase-3 targeted molecular probe ¹⁸ F]GPN-DEVD

This example provides a caspase-3 targeted molecular probe ¹⁸ F]GPN-DEVD, molecular probes targeted by caspase-3-3 [ ¹⁸ F]GPN-DEVD has the structure shown below：

Example 4: preparation of caspase-3 targeting molecular probe ¹⁸ F]GPN-DEVD method

This example provides a caspase-3 targeted molecular probe as described in example 1 [ ¹⁸ F]The preparation method of the GPN-DEVD comprises the following specific steps:

aluminum chloride (6. Mu.L, 2 mM), glacial acetic acid (5. Mu.L, 2 mM) and acetonitrile (384. Mu.L, 2 mM) were mixed to obtain a mixed solution; adding 40 mug of the labeling precursor GPN-DEVD of the caspase-3 targeted molecular probe prepared in the example 2 into the mixed solution to obtain a mixed solution; the mixture was enriched in 98% in 100. Mu.L of target water (target silver cyclotron by 30MeV proton bombardment ^l8 O]Target water generated from water), heating at 100 ℃ for 15min to obtain a reaction solution; the reaction solution was transferred to a centrifuge tube containing 20mL of ultrapure water, and then molecular probes in the reaction solution were detected [ ¹⁸ F]GPN-DEVD is carried in sequence on C activated with ethanol (10 mL) followed by ultra pure water (10 mL) ₁₈ Purification column (model Sep-Pakpplus C) ₁₈ ) Applying; c after loading ₁₈ After the purification column was washed three times with ultrapure water, the purified water was trapped in C by ethanol (500. Mu.L) ₁₈ Molecular probes on purification columns [ ¹⁸ F]Leaching GPN-DEVD into penicillin bottle to obtain molecular probe ¹⁸ F]GPN-DEVD; analysis of the product by radiation-HPLC (caspase-3 targeted molecular probes [ ¹⁸ F]The synthetic route for GPN-DEVD is shown in FIG. 35).

Comparative example 1: caspase-3 targeted molecular probe ¹⁸ F]GP-16

This comparative example provides a caspase-3 targeted molecular probe ¹⁸ F]GP-16, the caspase-3 targeted molecular probe ¹⁸ F]GP-16 has the structure shown below ([ [ V ]) ¹⁸ F]The molecular structure and preparation method of GP-16 are disclosed in patent application document with publication number CN 113354712A):

experimental example 1: radiochemical yield (RCY) and radiochemical purity (RCP) experiments on caspase-3 targeted molecular probes

This experimental example provides a radiochemical yield (RCY) and radiochemical purity (RCP) experiment of a caspase-3 targeted molecular probe, and the specific procedures are as follows:

20. Mu. Ci of the molecular probe prepared in example 2 was taken ⁶⁸ Ga]GPN-DEVD, molecular Probe obtained in example 4 [ ¹⁸ F]GPN-DEVD and molecular probes of comparative example 1 ¹⁸ F]GP-16 is respectively mixed with 1mL of acetonitrile to obtain mixed solution 1-3; the mixture 1 to 3 was subjected to radioactive HPLC detection using Gabi Nova radioactivity detector (available from Elysia-Raytest Co., germany) to obtain molecular probes [ ⁶⁸ Ga]GPN-DEVD, molecular probes ¹⁸ F]GPN-DEVD and molecular probes ¹⁸ F]The radiochemical yield (RCY) and radiochemical purity (RCP) of GP-16 are shown in FIGS. 26 and 36, and the calculation results are shown in Table 2.

As is clear from Table 2, the molecular probe was compared with the comparative probe [ ¹⁸ F]GP-16, molecular probe ⁶⁸ Ga]GPN-DEVD labeling requires shorter time, and the labeling conditions are mild for compounds unstable at high temperature, and molecular probes [ ¹⁸ F]GPN-DEVD and molecular probes ⁶⁸ Ga]The labeling yields of GPN-DEVD were all higher than those of the molecular probe [18F]GP-16 is high.

TABLE 2 radiochemical yields and radiochemical purity of different molecular probes

Probe name	Incubation time (min)	Incubation temperature (. Degree. C.)	RCY	RCP
					[ ⁶⁸ Ga]GPN-DEVD	10min	37℃	94％	96％
[ ¹⁸ F]GPN-DEVD	15min	100℃	41％	96％
					[ ¹⁸ F]GP-16	30min	80℃	6.8％	97％

Experimental example 2: in vitro reduction and macrocyclization experiments of caspase-3 targeted molecular probes

The experimental example provides an in vitro reduction and macrocyclization experiment of a caspase-3 targeted molecular probe, and the specific process is as follows:

the labeling precursor GPN-DEVD of the caspase-3 targeted molecular probe prepared in example 2 was mixed with PBS buffer (pH 7.4,0.01M) containing 50mM TCEP (tris (2-carboxyethyl) phosphine) at TCEP: GPN-devd=20: 1, mixing the mixture to obtain a mixed solution A; incubating the mixed solution A at 37 ℃ for 1h to obtain an incubation solution A; incubation a was taken with reaction buffer (100 mM NaCl, 50mM HEPES, 1mM EDTA, 10% glycerol, 0.1% chaps and water, ph=7.4, 10% glycerol% means v/v,0.1% chaps% means m/m) at 12:84 to obtain a mixed solution B; after 0.04. Mu.g (4. Mu.L) of recombinant human caspase-3 enzyme (purchased from Biyun Tian Co.) was added to 96. Mu.L of the mixture B, the mixture B was incubated at 37℃for 1 hour to obtain an incubation liquid B; HPLC and LC-MS characterization of incubation B using Waters1525 and electrospray mass spectrometry was performed and the characterization results are shown in FIGS. 22-24.

As shown in fig. 22 to 24: reduction of disulfide bonds in the labeled precursor GPN-DEVD of the caspase-3 targeted molecular probe yields a reduced product (GPN-DEVD-reduced), which after incubation with recombinant human caspase-3 in reaction buffer yields a circularized product (GPN-DEVD-C).

Experimental example 3: in vitro stability test of caspase-3 targeted molecular probes

The experimental example provides an in vitro stability experiment of a caspase-3 targeted molecular probe, and the specific process is as follows:

experiment II: targeting caspase-3 prepared in example 2 ⁶⁸ Ga]GPN-DEVD with PBS buffer (pH 7.4,0.01M) at 1:1, mixing the materials in a volume ratio to obtain a mixed solution; incubating the mixture at 37 ℃ for 1, 2 or 3 hours; after the incubation, the incubation was taken for radioactive HPLC analysis using a Gabi Nova radioactive detector (purchased from Elysia-Raytest Corp., germany), and the radiochemical purity (RCP) was calculated from the product peak area/total peak area, and the analysis and calculation results are shown in FIG. 27.

Experiment one: targeting caspase-3 prepared in example 2 ⁶⁸ Ga]GPN-DEVD was used with mouse serum (from Nanjsen Bei Ga Biotech Co.) at 1:1, mixing the materials in a volume ratio to obtain a mixed solution; incubating the mixture at 37 ℃ for 1, 2 or 3 hours; after the incubation was completed, 20. Mu.L of the incubation liquid was taken, an equal volume of acetonitrile was added, and the serum was separated from the protein by high-speed centrifugation at 12000g for 5min, and the supernatant was aspirated for radioactive HPLC analysis using a Gabi Nova radioactive detector (available from Elysia-Raytest Corp., germany), and the radiochemical purity (RCP) was calculated from the product peak area/total peak area, and the analysis and calculation results are shown in FIG. 28.

As can be seen from FIGS. 27-28, caspase-3 targeted molecular probes [ when the incubation time was increased to 3h ⁶⁸ Ga]The radiochemical purity of GPN-DEVD is above 95%, which proves that the GPN-DEVD has the following characteristics in the incubation processNo other products are generated, the stability is good, and the good stability of the probe is a necessary prerequisite for developing in vivo research on the targeting specificity of the probe to the caspase-3 enzyme.

Experimental example 4: lipid-water distribution coefficient experiment of caspase-3 targeted molecular probe

The experimental example provides a lipid-water distribution coefficient experiment of a caspase-3 targeted molecular probe, and the specific process is as follows:

a separate tube was taken, 1mL of deionized water and 1mL of n-octanol were added, followed by the molecular probes prepared in example 2, respectively ⁶⁸ Ga]GPN-DEVD, molecular Probe obtained in example 4 [ ¹⁸ F]GPN-DEVD is carried out until the concentration is 30 KBq/mu L, so that a mixed solution is obtained; oscillating the mixed solution for 5min, and centrifuging at 5000rpm for 5min to break emulsion to separate two phases; 500. Mu.L each of the n-octanol phase and the aqueous phase was taken into an EP tube, and then the radioactivity of each of the n-octanol phase and the aqueous phase was measured using a gamma counter and LogP was calculated (LogP=LogC _o /C _w ) Wherein C _o Represents octanol phase [ ⁶⁸ Ga]GPN-DEVD and [ ¹⁸ F]Radiation dose of GPN-DEVD, C _w Representing the aqueous phase [ ⁶⁸ Ga]GPN-DEVD and [ ¹⁸ F]Radiation dose of GPN-DEVD; after the first test is finished, 500 mu L of n-octanol and 500 mu L of water are added into the original centrifuge tube again, and the mixture is oscillated again, sampled and tested for LogP by centrifugation; the experiment was repeated until three groups of log p values were consecutively measured to be close, the average value of the three groups of data was taken as the lipid water distribution coefficient value, and the result was expressed as the average value ± standard deviation.

Experimental measurement of caspase-3 targeting molecular probes ⁶⁸ Ga]GPN-DEVD molecular probe with lipid water distribution coefficient of-2.02+/-0.03 and caspase-3 targeting ¹⁸ F]The lipid partition coefficient of GPN-DEVD is-1.98.+ -. 0.09, which indicates a molecular probe [ ⁶⁸ Ga]GPN-DEVD and [ ¹⁸ F]GPN-DEVD has better hydrophilicity.

Experimental example 5: cell uptake assay for caspase-3 targeted molecular probes

The experimental example provides a cell uptake experiment of a caspase-3 targeted molecular probe, and the specific process is as follows:

small BALB/C from healthT cells from mice (available from Kwandsi laboratories, inc.) were subjected to density gradient centrifugation using Dayou T cell isolate (available from Davidae, inc.), followed by centrifugation at 2X 10 ⁵ Individual inoculum sizes were inoculated in 6-well plates; to activate T cells, CD3 will be used ⁺ Antibodies (available from daceae as company) and CD28 ⁺ After simultaneous embedding in 6-well plates at an addition level of 5 μg/mL (available from daceae), the 6-well plates were incubated at 4 ℃ for 16h, after which the activated T cells were obtained by washing three times with PBS buffer (pH 7.4,0.01M); 4T1 cells (from Shanghai Proc. Natl. Acad. Cell Bank) were used at 1.8X10 ⁴ Cell/well inoculum size was inoculated into 6-well plates with 0.4mL 1640 medium (available from BI Corp.) and the 6-well plates were incubated at 37℃for 24 hours, after the incubation was completed, the supernatant was discarded, and 6-well plates containing 4T1 cells (at this time, the concentration of 4T1 cells in the 6-well plates was 2.5X10) ⁴ Cells/well) into two groups, positive groups re-suspend activated T cells in 1.5mL fresh 1640 medium followed by 1.8x10 ⁵ The inoculum size of cells/well was inoculated into 6-well plates, co-cultured at 37℃for 24 hours, and the negative group was cultured at 37℃for 24 hours after adding fresh 1640 medium to the 6-well plates in an addition amount of 1.5mL, and after completion of the culture, the 6-well plates were washed with PBS buffer to remove T cells, and 2mL of the medium containing the medium prepared in example 2 was added to the 6-well plates ⁶⁸ Ga]Fresh serum-free medium of GPN-DEVD (e.g. [ ⁶⁸ Ga]GPN-DEVD concentration of 1 μCi/100 μL), incubating at 37 ℃ for 0.5, 1, 2 or 3h, centrifuging 4T1 cells at 6000rpm for 5min after incubation, measuring the radioactive dose absorbed by the cells by a gamma counter after centrifugation, and calculating the cell uptake percentage, wherein three groups of experiments are performed at each time point, and the calculation results are shown in figure 29.

As shown in FIG. 29, positive group [ ⁶⁸ Ga]Cell uptake by GPN-DEVD was significantly higher at all time points than in the negative and positive group [ ⁶⁸ Ga]Cell uptake of GPN-DEVD 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, 5.51.+ -. 0.12% ID/mg at 3h, and negative group [ ⁶⁸ Ga]Cell uptake of GPN-DEVD 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, 1.54.+ -. 0.06% ID/mg at 3h, as seen in the fractionSub-probe [ ⁶⁸ Ga]GPN-DEVD can detect caspase-3 activity in apoptotic cell 4T1 more sensitively and specifically.

Experimental example 6: mouse PET imaging experiment of caspase-3 targeted molecular probe

The experimental example provides a mouse PET imaging experiment of a caspase-3 targeted molecular probe, and the specific process is as follows:

4T1 tumor cells were prepared according to 1X 10 ⁶ The individual doses (resuspended in 100 μlpbs buffer) were subcutaneously implanted into the right shoulder of female BALB/C mice (6 weeks old, purchased from karussian laboratory animal company, usa); when the tumor diameter reached 0.6mm, the mice were randomly divided into two groups (n=3), the control group (untreated group) mice were not treated, the experimental group (BMS-1198 treated group) mice were injected with BMS-1198 (dose 5mg/kg, 100 μl of physiological saline as solvent once daily, three times in succession, BMS-1198 see PCT patent application text No. WO 2015/160641 A2) for induction of apoptosis at tumor sites; three days after injection, mice were anesthetized with oxygen containing 2% (v/v) isoflurane at a flow rate of 2L/min; after fixing the limbs and tails of mice, 150. Mu. Ci of the molecular probe prepared in example 2 was dissolved in 100. Mu.L of physiological saline [ ⁶⁸ Ga]GPN-DEVD, molecular Probe obtained in example 4 [ ¹⁸ F]GPN-DEVD and molecular probes of comparative example 1 ¹⁸ F]GP-16 was injected by tail vein respectively; control mice were dosed in the same manner; immediately after the probe injection is completed, a dynamic PET scan is performed for 60min, and PET imaging results are shown in FIGS. 30, 37 and 41; after the scanning is finished, dividing a 60-min PET imaging result into 12 frames of images by using an OSEM3D/MAP algorithm, wherein each 5min is one frame, so that real-time analysis of in-vivo imaging of the mice is realized; the distribution situation of the probe in tumor parts and other organ tissues is sketched, analyzed and compared by adopting a region of interest (ROI) technology in ASIPRO software, analysis results are shown in figures 31-33 and 38-40, bone uptake values are obtained through ROI analysis, and analysis results are shown in figure 42, wherein the uptake values of the molecular probe in each tissue in a living body are expressed in terms of% ID/mL (percent injection dose per gram); after the scan was completed, mice were sacrificed and soluble tumor lysates were obtained by RIPA lysate (purchased from bi yun tian corporation) to allowProtein content in the soluble tumor lysate was measured with BCA protein assay kit (purchased from bi cloud) and 50 μg of the protein in the soluble tumor lysate was separated using a 12% (g/100 mL) SDS-PAGE gel; after adsorbing the membrane onto PVDF membrane, the membrane was blocked with TBS buffer containing 5% (g/100 mL) of skim milk (purchased from Biyun Tian Co.) at room temperature (25 ℃) for 60min, then incubated with TBS buffer containing anti-pro-caspase-3 (purchased from Biyun Tian Co.) at 4℃for 16h (anti-pro-caspase-3: TBS buffer=1:1000), primary antibodies were detected with HRP-conjugated secondary antibodies, immunoreactive bands were displayed with immunoblotting luminol reagent, and finally density analysis was performed with imageJ software, as shown in FIG. 34.

As shown in FIGS. 30-33, BMS-1198 treatment group injected with molecular probes [ ⁶⁸ Ga]After 20min (5.04.+ -. 0.67% ID/mL) and after 60min (4.52%.+ -. 0.68% ID/mL) GPN-DEVD, we observed [ [ ⁶⁸ Ga]High uptake of GPN-DEVD at tumor sites, in contrast to PET imaging of untreated mice, showed little uptake at tumor sites at 20min (2.29.+ -. 0.65% ID/mL) and 60min (2.40.+ -. 0.75% ID/mL) of intravenous administration; the radioactive uptake (1.17.+ -. 0.42% ID/mL at 10min, 1.48.+ -. 0.53% ID/mL at 60 min) of muscle tissue was similar in the treated and untreated groups; BMS-1198 had a higher tumor/muscle ratio (7.45.+ -. 0.35% ID/mL at 10min, 4.66.+ -. 0.87% ID/mL at 60 min) than untreated (1.86.+ -. 0.28% ID/mL at 10min, 1.61.+ -. 0.48% ID/mL at 60 min).

As shown in FIG. 34, BMS-1198 treated groups had higher levels of caspase-3 expression, while the empty groups had little caspase-3 expression, and thus BMS-1198 could achieve therapeutic effects by inducing apoptosis of tumors.

As shown in FIGS. 37-41, BMS-1198 treatment group was injected with molecular probes [ ¹⁸ F]After 20min (6.4.+ -. 0.66% ID/mL) and after 60min (5.2.+ -. 0.82% ID/mL) GPN-DEVD, we observed [ [ ¹⁸ F]High tumor uptake by GPN-DEVD, in contrast, PET imaging of untreated mice showed little uptake 20min (2.11.+ -. 0.32% ID/mL) and 60min (1.15.+ -. 0.96% ID/mL) after intravenous administration; the radioactivity of the muscle tissue of the treated group and the untreated group was 1.46.+ -. 0.33% at 20minID/mL,60min 1.67+ -0.46% ID/mL; BMS-1198 had a higher tumor/muscle ratio (4.28.+ -. 0.40% ID/mL at 10min, 3.13.+ -. 0.26% ID/mL at 60 min) than untreated (1.42.+ -. 0.65% ID/mL at 10min and 2.15.+ -. 0.26% ID/mL at 60 min).

As shown in FIG. 42, BABL/C mice were injected with molecular probes [ ¹⁸ F]GP-1660min bone uptake was 8.97.+ -. 1.23% ID/mL, observed [ ¹⁸ F]High uptake of GP-16 at bone sites, in contrast [ ¹⁸ F]GPN-DEVD is ingested relatively little for 60min (3.38+ -0.87% ID/mL).

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims

1. An aspartic protease targeted molecular probe, wherein the molecular probe has the structure shown as follows:

wherein R is a radionuclide label group.

2. The molecular probe of claim 1, wherein the radionuclide labeling group is ⁶⁸ Ga、[ ¹⁸ F]AlF、 ⁶⁴ Cu or ⁸⁹ Zr。

3. The molecular probe of claim 2, wherein when the radionuclide labeling group is ⁶⁸ The molecular probe has the following structure when Ga:

4. the molecular probe of claim 2, wherein when the radionuclide label is [ the compound ¹⁸ F]In AlF, the molecular probe has the following structure:

5. the molecular probe according to any one of claims 1 to 4, wherein the labeling precursor of the molecular probe has the structure shown below:

6. the molecular probe of any one of claims 1 to 5, wherein the molecular probe targets aspartic proteinase-3.

7. A method for preparing the molecular probe according to any one of claims 1 to 6, characterized in that the method comprises: dissolving compounds GPN-7 and NOTA-NHS in N, N-dimethylformamide to obtain a dissolving solution; adding N, N-diisopropylethylamine into the solution, and then carrying out a reaction to obtain a reaction solution; purifying the reaction liquid to obtain a labeling precursor GPN-DEVD of the molecular probe; labeling a radionuclide on a labeling precursor GPN-DEVD of an aspartic protease targeted molecular probe to obtain the molecular probe;

the compound GPN-7 has the following structure:

8. the method of claim 7, wherein the compound GPN-7 is prepared by: dissolving a compound GPN-6 in methanol to obtain a solution; adding triisopropylsilane and 2- (ethyldithiopyridine) into the solution to react to obtain a reaction solution; concentrating the reaction solution, precipitating with diethyl ether, centrifuging, and drying to obtain a compound GPN-7;

the compound GPN-6 has the following structure:

9. use of a molecular probe according to any one of claims 1 to 6 for aspartic protease imaging, wherein said use is for diagnostic and therapeutic purposes other than disease.

10. An imaging agent targeting aspartic proteolytic enzyme, characterized in that said imaging agent comprises a molecular probe according to any one of claims 1 to 5.