CN115772208A - Granzyme B targeted activation type PET imaging probe and application thereof - Google Patents

Abstract

The invention relates to a granzyme B targeted activation type PET imaging probe and application thereof, belonging to the technical field of chemistry. The invention provides a molecular probe of a targeting granzyme B, which has an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence targeted and identified by granzyme B or an isoleucine-glutamic acid-proline-aspartic acid sequence targeted and identified by granzyme B, can cut asparagine sites and reduce disulfide bonds in a tumor microenvironment with high expression of granzyme B, utilizes a biocompatible CBT-Cys click condensation reaction, and enables naked amino and sulfydryl to be easily condensed with cyano groups on CBT, so that intramolecular condensation cyclization self-assembly is rapidly carried out to form a radioactive macrocyclic product; in addition, when high-concentration L-Cys exists in the reaction environment, intramolecular condensation cyclization of the molecular probe is hardly influenced; meanwhile, the molecular probe has the advantages of high stability, high sensitivity, strong specificity, good safety and the like.

Description

Granzyme B targeted activation type PET imaging probe and application thereof

Technical Field

The invention relates to a granzyme B targeted activation type PET imaging probe and application thereof, belonging to the technical field of chemistry.

Background

Immunotherapy is a treatment method for preventing and treating diseases by regulating the immune state of the body with drugs or biological agents to generate appropriate immune response to the diseases. In recent years, immunotherapy has been widely used for clinical treatment of cancer, but the clinical objective response rate is less than 30% due to the large individual difference between cancer patients. On the other hand, immunotherapy is often associated with severe immune-related adverse reactions, including allergic reactions, inflammatory reactions, and even death of the patient. Therefore, longitudinal monitoring and visualization of immune responses is critical to better stratify patients and select responders during immunotherapy, and is of great significance to accurate assessment of immunotherapy response rates.

Granzyme B is a serine protease released by cytoplasmic granules in cytotoxic T cells and Natural Killer (NK) cells, inducing programmed cell death or apoptosis in target cells. When Cytotoxic T Lymphocytes (CTLs) recognize target cells, granzymes are released from solutes, enter the cytoplasm of the target cells, and effectively induce apoptosis through direct and indirect lamination processes and activation, mitochondrial permeability, or against other nuclear proteins. There are five different granzymes, a, B, H, K and M, in humans, of which granzyme B is the most abundant type, with high cytotoxic efficacy and various apoptosis-inducing mechanisms. Granzyme B functions in one of the two major pathways mediating cell death by T cells, and is one of the most prominent serine proteases involved in T cell cytotoxicity. Therefore, by monitoring the expression level of granzyme B, the activity of cytotoxic T cells in immune action can be identified, and the early curative effect of tumor immunotherapy can be accurately evaluated.

The molecular imaging technology can display specific molecules at the tissue level, the cell level and the subcellular level by using an imaging means, reflect the change of the molecular level in a living body state, and carry out qualitative and quantitative research on the biological behavior in the aspect of imaging. Therefore, the molecular imaging technology is used for effectively monitoring the granzyme B expression level of the tumor part in the immunotherapy process, and has important significance for accurately evaluating the early curative effect of the tumor immunotherapy. However, the current PET molecular probes for detecting granzyme B expression level lack sufficient sensitivity and targeting.

Disclosure of Invention

In order to solve the problems, the invention provides a granzyme B-targeted molecular probe, which has an isoleucine-glutamate-phenylalanine-aspartate sequence targeted and identified by granzyme B or an isoleucine-glutamate-proline-aspartate sequence targeted and identified by granzyme B;

when the molecular probe has an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence targeted and recognized by granzyme B, the molecular probe targeted to granzyme B has the following structure:

when the molecular probe has an isoleucine-glutamate-proline-aspartate sequence targeted and recognized by granzyme B, the molecular probe targeted to granzyme B has the following structure:

wherein R is a radionuclide-labeled group.

In one embodiment of the present invention, the radionuclide-labeling group is ⁶⁸ Ga、[ ¹⁸ F]AlF、[ ¹⁸ F]AmBF ₃ 、 ⁶⁴ Cu or ⁸⁹ Zr。

In one embodiment of the invention, when having the isoleucine-glutamic acid-phenylalanine-aspartic acid sequence recognized as being targeted by granzyme B, and the radionuclide labeling group is set forth in [ 2 ] ¹⁸ F]AmBF ₃ The molecular probe targeting granzyme B has the following structure:

when the gene has a isoleucine-glutamic acid-phenylalanine-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]In AlF, the molecular probe targeting granzyme B has the following structure:

when the gene has a isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]AmBF ₃ The molecular probe targeting granzyme B has the following structure:

when the gene has a isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]In AlF, the molecular probe targeting granzyme B has the following structure:

in one embodiment of the invention, when the plasmid B has an isoleucine-glutamate-phenylalanine-aspartate sequence targeted for recognition, and the radionuclide labeling group is ⁶⁸ Ga, the molecular probe targeting granzyme B has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence which is targeted and recognized by granzyme B, and the radionuclide labeling group is ⁶⁸ Ga, the molecular probe targeting granzyme B has the following structure:

in one embodiment of the invention, when having the isoleucine-glutamic acid-phenylalanine-aspartic acid sequence recognized as being targeted by granzyme B, and the radionuclide labeling group is set forth in [ 2 ] ¹⁸ F]AmBF ₃ When the compound is a precursor of the molecular probe targeting granzyme B, the precursor compound has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is ¹⁸ F]AmBF ₃ When the compound is used, the precursor compound of the molecular probe targeting granzyme B has the following structure:

in one embodiment of the invention, when the plasmid B has an isoleucine-glutamate-phenylalanine-aspartate sequence targeted for recognition, and the radionuclide labeling group is ⁶⁸ Ga, the precursor compound of the molecular probe targeting granzyme B has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence which is targeted and recognized by granzyme B and the radionuclide labeling group is ⁶⁸ Ga, the precursor compound of the molecular probe targeting granzyme B has the following structure:

the invention also provides a method for preparing the molecular probe of the targeted granzyme B, when the molecular probe has an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence which is targeted and recognized by the granzyme B, and the radionuclide labeled group is ¹⁸ F]AmBF ₃ The method comprises the following steps:

the method comprises the following steps: carrying out condensation reaction on a compound IEFD and a compound SF to obtain a compound I-1;

step two: carrying out deprotection reaction on the compound I-1 to obtain a compound I-2;

step three: carrying out click condensation reaction on a compound I-2, alkyl aminomethyl boron trifluoride, tri (2-benzimidazolylmethyl) amine and copper (I) hexafluorophosphate to obtain a precursor compound M-1 of a molecular probe of the targeted granzyme B;

step four: carrying out radionuclide labeling on a precursor compound M-1 of the molecular probe of the targeting granzyme B to obtain the molecular probe of the targeting granzyme B;

the compound IEFD has the structure shown below:

the compound SF has the following structure:

the compound I-1 has the following structure:

the compound I-2 has the following structure:

when the gene has a isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]AmBF ₃ The method comprises the following steps:

the method comprises the following steps: carrying out condensation reaction on a compound IEPD and a compound SF to obtain a compound II-1;

step two: carrying out deprotection reaction on the compound II-1 to obtain a compound II-2;

step three: carrying out click condensation reaction on a compound II-2, alkyl aminomethyl boron trifluoride, tri (2-benzimidazolylmethyl) amine and copper (I) hexafluorophosphate to obtain a precursor compound H-1 of the molecular probe of the targeted granzyme B;

step four: carrying out radionuclide labeling on a precursor compound H-1 of the molecular probe of the targeting granzyme B to obtain the molecular probe of the targeting granzyme B;

the compound IEPD has the structure shown below:

the compound II-1 has the following structure:

the compound II-2 has the following structure:

the invention also provides a method for preparing the molecular probe of the targeted granzyme B, namely isoleucine-glutamic acid-phenylalanine-aspartic acid with granzyme B targeted recognitionSequence and radionuclide-labeling group of ⁶⁸ Ga, the method comprising the steps of:

the method comprises the following steps: carrying out condensation reaction on a compound IEFD-ZL and a compound CBT-1 to obtain a compound III-1;

step two: carrying out deprotection reaction on the compound III-1 to obtain a compound III-2;

step three: carrying out condensation reaction on the compound III-2 and 2- (ethylinsulfanyl) pyridine to obtain a compound III-3;

step four: carrying out condensation reaction on the compound III-3 and hydroxysuccinimide-tetraazacyclododecane tetraacetic acid to obtain a precursor compound M-2 of the molecular probe of the targeted granzyme B;

step five: carrying out radionuclide labeling on a precursor compound M-2 of the molecular probe of the targeting granzyme B to obtain the molecular probe of the targeting granzyme B;

the compound IEFD-ZL has the structure shown below:

the compound CBT-1 has the following structure:

the compound III-1 has the following structure:

the compound III-2 has the following structure:

the compound III-3 has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence which is targeted and recognized by granzyme B, and the radionuclide labeling group is ⁶⁸ Ga, the method comprising the steps of:

the method comprises the following steps: carrying out condensation reaction on a compound IEPD-ZL and a compound CBT-1 to obtain a compound IV-1;

step two: carrying out deprotection reaction on the compound IV-1 to obtain a compound IV-2;

step three: carrying out condensation reaction on the compound IV-2 and 2- (ethidium propyl) pyridine to obtain a compound IV-3;

step four: carrying out condensation reaction on the compound IV-3 and hydroxysuccinimide-tetraazacyclododecane tetraacetic acid to obtain a precursor compound H-2 of the molecular probe of the targeting granzyme B;

step five: carrying out radionuclide labeling on a precursor compound H-2 of the molecular probe of the targeting granzyme B to obtain the molecular probe of the targeting granzyme B;

the compound IEPD-ZL has the structure shown as follows:

the compound IV-1 has the following structure:

the compound IV-2 has the following structure:

the compound IV-3 has the following structure:

in one embodiment of the present invention, the method for labeling with a radionuclide is an isotope exchange method.

The invention also provides application of the molecular probe targeting granzyme B in granzyme B imaging, and the application aims at diagnosis and treatment of diseases.

The invention also provides an imaging agent of the targeted granzyme B, which contains the molecular probe of the targeted granzyme B.

The technical scheme of the invention has the following advantages:

the invention provides a molecular probe of a targeting granzyme B, which has an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence identified by the granzyme B in a targeting manner or an isoleucine-glutamic acid-proline-aspartic acid sequence identified by the granzyme B in a targeting manner, can cut asparagine loci and reduce disulfide bonds in a tumor microenvironment with high granzyme B expression, utilizes a biocompatible CBT-Cys click condensation reaction, and rapidly carries out intramolecular condensation cyclization self-assembly by virtue of easy condensation of naked amino and sulfydryl and cyano groups on the CBT to form a radioactive macrocyclic product; and when high concentration of L-Cys exists in the reaction environment, intramolecular condensation cyclization of the molecular probe is hardly influenced; meanwhile, the molecular probe has the advantages of high stability, high sensitivity, strong specificity, good safety and the like. Therefore, the molecular probe can accurately and dynamically monitor the activity of the granzyme B, and when the molecular probe is used for the early curative effect of tumor immunotherapy, the imaging effect can be enhanced, so that the accuracy of the evaluation of the early curative effect of the tumor immunotherapy is improved.

Furthermore, the molecular probe adopts an isotope exchange method for radionuclide labeling, the method is simple to operate, further purification is not required by preparative HPLC, and the purification time is shortened, so that the possibility of label failure caused by complicated steps is reduced, and the obtained PET tracer has high radiochemical yield and good specific activity, thereby greatly promoting the development of the PET tracer and the application thereof in disease diagnosis.

Drawings

FIG. 1: mass spectrum of precursor compound M-1.

FIG. 2: high performance liquid chromatogram of precursor compound M-1.

FIG. 3: hydrogen spectrum of precursor compound M-1.

FIG. 4 is a schematic view of: carbon spectrum of precursor compound M-1.

FIG. 5 is a schematic view of: molecular probe [ 2 ] ¹⁸ F]HPLC analysis results before and after M-1 radiosynthesis and purification.

FIG. 6: mass spectrum of precursor compound M-2.

FIG. 7: high performance liquid chromatogram of precursor compound M-2.

FIG. 8: molecular probe [ 2 ] ⁶⁸ Ga]HPLC analysis of M-2 radiosynthesis.

FIG. 9: molecular probe [ 2 ] ¹⁸ F]Stability HPLC analysis of M-1 incubated in PBS for 1, 2, 4 hours.

FIG. 10: molecular probe [ 2 ] ¹⁸ F]Stability HPLC analysis of M-1 incubated in mouse serum for 1, 2, 4 hours.

FIG. 11: the enzyme-cutting high-performance liquid chromatogram of the precursor compound M-1.

FIG. 12: results of enzyme kinetic calculations for precursor compound M-1.

FIG. 13: results of the western blot analysis of granzyme B expression levels in different cells.

FIG. 14 is a schematic view of: results of western blot quantitative analysis of granzyme B expression levels in different cells.

FIG. 15: molecular probe [ 2 ] ¹⁸ F]Uptake of M-1 in 4T1 cells co-incubated with T cells and 4T1 cells not co-incubated with T cells.

FIG. 16: molecular probe [ 2 ] ⁶⁸ Ga]Uptake of M-2 in 4T1 cells co-incubated with T cells and 4T1 cells not co-incubated with T cells.

FIG. 17: molecular probe [ 2 ] ¹⁸ F]micro-PET visualization of M-1 in 4T 1-bearing and non-immune-treated 4T 1-bearing miceLike this.

FIG. 18: molecular probe [ 2 ] ¹⁸ F]Results of quantitative analysis of M-1 uptake in tumors and muscles of 4T1 tumor-bearing mice that were not treated with immunotherapy.

FIG. 19 is a schematic view of: molecular probe [ 2 ] ¹⁸ F]Results of quantitative analysis of M-1 uptake in tumors and muscles of 4T1 tumor-bearing mice undergoing immunotherapy.

FIG. 20: molecular probe [ 2 ] ¹⁸ F]M-1 ratio of tumor and muscle uptake in 4T1 tumor-bearing mice that were immunotherapeutically and 4T1 tumor-bearing mice that were not immunotherapeutically.

FIG. 21: molecular probe [ 2 ] ⁶⁸ Ga]micro-PET imaging of M-2 in 4T1 tumor bearing mice that were immune treated and 4T1 tumor bearing mice that were not immune treated.

FIG. 22: molecular probe [ 2 ] ⁶⁸ Ga]Results of quantitative analysis of M-2 uptake in tumors and muscles of 4T1 tumor-bearing mice that were not treated with immunotherapy.

FIG. 23 is a schematic view of: molecular probe [ 2 ] ⁶⁸ Ga]Results of quantitative analysis of M-2 uptake in tumors and muscles of 4T1 tumor-bearing mice undergoing immunotherapy.

FIG. 24: molecular probe [ 2 ] ⁶⁸ Ga]M-2 uptake ratio in tumor and muscle in 4T1 tumor-bearing mice that were immunotherapy and 4T1 tumor-bearing mice that were not immunotherapy.

FIG. 25 is a schematic view of: mass spectrum of precursor compound H-1.

FIG. 26: high performance liquid chromatogram of precursor compound H-1.

FIG. 27 is a schematic view showing: mass spectrum of precursor compound H-2.

FIG. 28: high performance liquid chromatography spectrum of precursor compound H-2.

FIG. 29 is a schematic view of: hydrogen spectra of molecular probe H-1.

FIG. 30: carbon spectrum of precursor compound H-1.

FIG. 31: precursor compound [ 2 ] ¹⁸ F]HPLC analysis before and after the radiosynthesis of H-1.

FIG. 32: molecular probe [ 2 ] ⁶⁸ Ga]HPLC analysis of H-2 radiosynthesis.

FIG. 33 is a schematic view of: molecular probe [ 2 ] ¹⁸ F]Stability HPLC of H-1 incubation in PBS for 1, 2, 4 hoursAnd (6) analyzing.

FIG. 34 is a schematic view of: molecular probe [ 2 ] ¹⁸ F]H-1 was incubated in mouse serum for 1, 2, 4 hours for stability HPLC analysis.

FIG. 35: enzyme digestion high performance liquid chromatogram of precursor compound H-1.

FIG. 36: results of enzyme kinetic calculations for precursor compound H-1.

FIG. 37: molecular probe [ 2 ] ¹⁸ F]Uptake of H-1 in 4T1 cells co-incubated with T cells and 4T1 cells not co-incubated with T cells.

FIG. 38: molecular probe [ 2 ] ⁶⁸ Ga]Uptake of H-2 in 4T1 cells co-incubated with T cells and 4T1 cells not co-incubated with T cells.

FIG. 39: molecular probe [ 2 ] ¹⁸ F]micro-PET imaging of H-1 in 4T 1-primed and 4T 1-naive tumor-bearing mice.

FIG. 40: molecular probe [ 2 ] ¹⁸ F]Results of quantitative analysis of H-1 uptake in tumors and muscles of 4T1 tumor-bearing mice that were not treated with immunotherapy.

FIG. 41: molecular probe [ 2 ] ¹⁸ F]Results of quantitative analysis of H-1 uptake in tumors and muscles of 4T1 tumor-bearing mice undergoing immunotherapy.

FIG. 42: molecular probe [ 2 ] ¹⁸ F]H-1 ratio of uptake in tumor and muscle in 4T1 tumor-bearing mice that were immunotherapeutically and 4T1 tumor-bearing mice that were not immunotherapeutically.

FIG. 43: molecular probe [ 2 ] ⁶⁸ Ga]micro-PET imaging of H-2 in 4T1 tumor bearing mice that were immunotherapy and 4T1 tumor bearing mice that were not immunotherapy.

FIG. 44: molecular probe [ 2 ] ⁶⁸ Ga]Results of quantitative analysis of H-2 uptake in tumors and muscles of 4T1 tumor-bearing mice that were not treated with immunotherapy.

FIG. 45 is a schematic view of: molecular probe [ 2 ] ⁶⁸ Ga]Results of quantitative analysis of H-2 uptake in tumors and muscles of 4T1 tumor-bearing mice undergoing immunotherapy.

FIG. 46: molecular probe [ 2 ] ⁶⁸ Ga]H-2 ratio of uptake in tumor and muscle in 4T1 tumor-bearing mice that were immunotherapeutically and 4T1 tumor-bearing mice that were not immunotherapeutically.

Detailed Description

The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.

The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.

Example 1-1: molecular probe of targeted granzyme B ¹⁸ F]M-1

This example provides a molecular probe of granzyme B ¹⁸ F]M-1, the molecular probe targeting granzyme B ¹⁸ F]M-1 has the structure shown below:

examples 1 to 2: molecular probe for preparing targeting granzyme B ¹⁸ F]Method of M-1

This example provides the molecular probe of targeting granzyme B described in example 1 ¹⁸ F]The preparation method of M-1 comprises the following specific steps:

the method comprises the following steps: according to the literature "LinJ, gaoD, wangs, et. JAmChemSoc.2022;144 (17): 7667-7675. Doi; according to the literature "HeS, liJ, lyuY, huangJ, puK. JAm ChemSoc.2020;142 (15) < 7075-7082. Doi.

Step two: dissolving a compound SF (40mg, 0.053mmol, 1eq), a compound IEFD (1.1 eq) and benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 1.15 eq) in 10mL of anhydrous Tetrahydrofuran (THF) to obtain a solution; adding 1mLN into the solution, and after N-Dimethylformamide (DMF) is completely dissolved, adding N, N-diisopropylethylamine (DIPEA, 4 eq) to adjust the pH of the solution to 8 to obtain a mixture; after the resulting mixture was left to stir (150 rpm) at room temperature (25 ℃) for 3 hours using a nitrogen blanket, the organic solvent was removed using a rotary evaporator to obtain compound I-1 (yield 87%).

Step three: compound I-1 (40mg, 0.031mmol, 1eq) was dissolved in 5mL of Dichloromethane (DCM) to give a solution; 5mL of trifluoroacetic acid (TFA) was added to the solution to obtain a mixture; after the resulting mixture was stirred (150 rpm) at room temperature (25 ℃) for 30min, the organic solvent was removed using a rotary evaporator, followed by washing with ether, precipitation, removal of the supernatant, and drying of the lower layer compound to obtain compound i-2 (yield 23%).

Step four: compound I-2 (20mg, 0.015mmol) was dissolved in DMF/H ₂ O (3ml, v; adding alkyl aminomethyl boron trifluoride (AMBF) to the mixture ₃ 0.053 mmol), tris (2-benzimidazolylmethyl) amine (ligand, 0.0015 mmol), and copper (I) tetrakis (acetate) hexafluorophosphate (Cu (I), 11mg, 0.015mmol) to obtain a reaction system; the reaction system was evacuated, stirred at 45 ℃ for 45min (150 rpm) under nitrogen protection, purified using semi-preparative high performance liquid chromatography, and freeze-dried to give precursor compound M-1 (11 mg, white pale green powder). The mass spectrum, high performance liquid chromatogram and hydrogen spectrum of precursor compound M-1 are respectively shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4, respectively.

Step five: produced on demand by a medical cyclotron ¹⁸ F, fluoride ions; after the production is finished, the ¹⁸ F fluoride ion targeting on anion exchange column (QMA) and pyridazine buffer (300. Mu.L, pH = 2.5) ¹⁸ Eluting F anion from QMA column into polypropylene reaction tube (1 mL), adding precursor compound M-1 (25mM, 30 μ L) into the reaction tube, and incubating at 80 deg.C for 30min to obtain reaction solution; the reaction solution was transferred into a centrifugal tube containing 20mL of ultrapure water, and the obtained molecular probe [ 2 ] ¹⁸ F]M-1 was loaded on a C18 purification column (model Sep-Pak plusC 18) activated with ethanol (10 mL) and ultrapure water (10 mL) successively; washing the C18 column with ultrapure water for three times, and then first with ethanol (500. Mu.L) of the purified molecular probe ¹⁸ F]The M-1 is leached into a penicillin bottle, and the molecular probe is leached by the normal saline ¹⁸ F]M-1 was diluted to 1. Mu. Ci/. Mu.L to give a molecular probe 2 ¹⁸ F]M-1 solution. Molecular probe [ 2 ] using Gabinova radioactivity detector ¹⁸ F]The reaction solution before and after M-1 labeling is subjected to radioactive HPLC detection, and the detection result is shown in figure 5. The molecular probe [ 2 ] is calculated from the radioactive product peak area/total peak area ¹⁸ F]Radiochemical purity (RCP) of M-1, calculated as: the radiochemical purity of the product obtained by labeling is higher than 95%. Calculation of molecular Probe by detection of radioactive HPLC ¹⁸ F]Radiochemical yield (RCY) of M-1, calculated as: 56 +/-1.2 percent.

Examples 1 to 3: molecular probe of targeted granzyme B ⁶⁸ Ga]M-2

This example provides a molecular probe of granzyme B ⁶⁸ Ga]M-2, the molecular probe targeting granzyme B ⁶⁸ Ga]M-2 has the structure shown below:

examples 1 to 4: molecular probe for preparing targeting granzyme B ⁶⁸ Ga]Method of M-2

This example provides the molecular probe of targeting granzyme B described in example 3 ⁶⁸ Ga]The preparation method of the M-2 comprises the following specific steps:

the method comprises the following steps: washing the sand core funnel twice by using dichloromethane, draining, adding 2-chlorotrityl chloride resin (the load is 1.106mmol/g and is 271.25 mg) into the drained sand core funnel, adding 10mL of dichloromethane to soak and swell the 2-chlorotrityl chloride resin, soaking and swelling for 10min, and draining;

step two: adding amino acid Fmoc-isoleucine (0.5 mmol) into the sand core funnel obtained in the first step, and dissolving the amino acid Fmoc-isoleucine with 10mL of DMF (N, N-dimethylformamide) to obtain a dissolved solution; adding DIPEA (N, N-diisopropylethylamine) (130 μ L,0.75 mmol) to the solution to adjust pH to 8, and shaking the solution at 25 deg.C for 3 hrAfter the reaction is finished, the solvent is pumped to be dry; 10mL of DMF/CH was added ₃ OH/DIPEA mixed solution (DMF/CH) ₃ OH/DIPEA =7:2:1, v/v/v), oscillating for 10min, filtering, and repeating the operation once to remove unreacted amino acid; washing twice with 10mL DMF (HPLC type), oscillating for 2min, and vacuum filtering; adding 10mL of DMF solution containing 20vt% piperidine into a sand core funnel, oscillating for 10min, carrying out 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 of DMF (HPLC type) to wash off excess piperidine; after washing, draining the solvent, sampling and carrying out Kaiser test, wherein the color of the reagent is dark purple, which indicates that the FOMC group is removed at the moment, and the amino group is exposed and can be connected with the next amino acid;

step three: and on the basis of the second step, sequentially replacing amino acid Fmoc-glutamic acid (0.5 mmol) with amino acid Fmoc-phenylalanine (0.5 mmol), amino acid Fmoc-aspartic acid (0.5 mmol) and amino acid Fmoc-S-trityl-L-cysteine (0.5 mmol), amino acid Fmoc-glycine (0.5 mmol), amino acid Fmoc-p-aminobenzoic acid (0.5 mmol) and amino acid Fmoc-p-aminobenzoic acid (0.5 mmol), and repeating the operation of the second step to obtain a compound IEFD-ZL (the compound IEFD-ZL is an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence with granzyme B targeted recognition).

Step four: synthesis of Compound CBT-1 according to the literature "Lin, J.et.journal of the American Chemical Society, (JACS) 2022, 144 (17), 7667-7675"; dissolving a compound CBT-1 (10mg, 0.023mmol, 1eq), a compound IEFD-ZL (1.1 eq) and benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 1.15 eq) in 10mL of anhydrous Tetrahydrofuran (THF) to obtain a dissolved solution; adding 1mLN into the solution, and after N-Dimethylformamide (DMF) is completely dissolved, adding N, N-diisopropylethylamine (DIPEA, 4 eq) to adjust the pH of the solution to 8 to obtain a mixture; after the resulting mixture was left to stir (150 rpm) at room temperature (25 ℃) for 3 hours using a nitrogen blanket, the organic solvent was removed using a rotary evaporator to obtain Compound III-1 (yield 76%).

Step five: dissolving compound III-1 (20mg, 0.013mmol, 1eq) in 5mL of Dichloromethane (DCM) to obtain a dissolved solution; 5mL of trifluoroacetic acid (TFA) was added to the solution to obtain a mixture; after the resulting mixture was stirred (150 rpm) at room temperature (25 ℃) for 30min, the organic solvent was removed using a rotary evaporator, and then washed with ether, precipitated, and the supernatant was removed, and the lower layer compound was dried to obtain compound iii-2 (yield 61%).

Step six: compound III-2 (9mg, 0.007mmol, 1eq) was dissolved in 10mL of methanol (MeOH) to give a solution; to the solution were added 200. Mu.L of triisopropylsilane (Tips) and 102. Mu.L of 2- (ethidium fluoride) pyridine (SEt, 0.675 mmol) to obtain a mixture; after the mixture was stirred (150 rpm) at room temperature (25 ℃ C.) for 2h, the organic solvent was removed using a rotary evaporator and then washed 3 times with cold (4 ℃ C.) ether to give compound III-3 (85% yield).

Step seven: after mixing compound III-3 (8mg, 0.006mmol) with hydroxysuccinimide-tetraazacyclododecane tetraacetic acid (NOTA-NHS, 0.009 mmol), DIPEA was added to adjust pH to 8, to obtain a mixture; after the resulting mixture was stirred (150 rpm) for 2 hours at room temperature (25 ℃) using a nitrogen blanket, it was purified using semi-preparative high performance liquid phase and freeze-dried to give precursor compound M-2 (5 mg, white powder). The mass spectrum of the precursor compound M-2 is shown in FIG. 6, and the high performance liquid chromatogram is shown in FIG. 7. The molecular weight of precursor compound M-2 was 1621, with a purity of greater than 95%.

Step eight: using 0.05MHCl from ⁶⁸ Ge/ ⁶⁸ Elution in Ga Generator (ITG) ⁶⁸ Ga, and mixed with 1.25m naoac buffer to adjust pH to 4.0, resulting in a mixture; transferring the mixture to a 5ml EP tube containing the precursor compound M-2 (20. Mu.g), mixing well, and incubating at 37 ℃ for 15min to obtain the molecular probe [ 2 ] ⁶⁸ Ga]M-2; the molecular probe is set in a physiological saline solution ⁶⁸ Ga]M-2 was diluted to 1. Mu. Ci/. Mu.L to give a molecular probe 2 ⁶⁸ Ga]M-2 solution. Molecular probe [ 2 ] using a Gabinova radioactivity detector ⁶⁸ Ga]The radioactive HPLC detection is carried out on the reaction solution before and after the M-2 labeling, and the detection result is shown in figure 8. The molecular probe [ 2 ] was calculated from the radioactive product peak area/total peak area ⁶⁸ Ga]Radiochemical purity (RCP) of M-2, calculated as: the radiochemical purity of the product obtained by labeling is higher than 95%. By passingRadioactivity HPLC detection calculation molecular probe ⁶⁸ Ga]The radiochemical yield (RCY) of M-2 was calculated as: 98 percent.

Experimental example 1-1: in vitro stability experiments of molecular probes targeting granzyme B

The experimental example provides an in vitro stability experiment of a molecular probe targeting granzyme B, which comprises the following specific processes:

experiment one: the molecular probe targeting granzyme B prepared in example 2 ¹⁸ F]M-1 solution was mixed with PBS buffer (pH 7.4, 0.01M) at a ratio of 1:9 to obtain a mixed solution; incubating the mixture at 37 ℃ for 1, 2 or 4h; after the incubation was completed, the incubation solution was subjected to radioactive HPLC analysis using a Gabinova radioactivity detector, and the analysis result is shown in FIG. 9.

Experiment two: the molecular probe targeting granzyme B prepared in example 2 ¹⁸ F]M-1 solution and mouse serum (obtained from beiga biotechnology, tokyo) were mixed at 1:9 to obtain a mixed solution; incubating the mixture at 37 ℃ for 1, 2 or 4h; after the incubation, 20. Mu.L of the incubation solution was added with an equal volume of acetonitrile, centrifuged at 12000g for 5min to separate the serum from the protein, and the supernatant was aspirated for radioactive HPLC analysis using a Gabinova radioactivity detector, the results of which are shown in FIG. 10.

As can be seen from FIGS. 9 to 10, when the incubation time was increased to 4 hours, the molecular probe [ 2 ] of the target granzyme B ¹⁸ F]The radiochemical purity of M-1 is above 95%, which proves that no other products are generated during the incubation process, indicating that the molecular probe [ 2 ] ¹⁸ F]M-1 has better stability.

Experimental examples 1-2: experiment of lipid-water distribution coefficient of molecular probe of targeting granzyme B

Reference is made to "Lin J, gao D, wang S, et al.J Am Chem Soc.2022;144 (17): 7667-7675 "molecular probe for detecting granzyme B prepared in example 2, [ 2 ] ¹⁸ F]M-1 has a fat water partition coefficient.

The experiment shows that the molecular probe of the target granzyme B ¹⁸ F]The lipid-water partition coefficient of M-1 is 0.113. + -. 0.0018, which indicates that the molecular probe [ 2 ] ¹⁸ F]M-1 is lipophilic and may be predominantly through-expressed in vivoMetabolizing by the hepatic pathway.

Experimental examples 1 to 3: enzyme kinetics experiment of molecular probe of target granzyme B

The experimental example provides an enzyme kinetics experiment of a molecular probe of the target granzyme B, which comprises the following specific processes:

after adding active mouse cathepsin C (0.57 μ L,0.481 mg/mL) and recombinant mouse granzyme B (25 μ L,0.1 mg/mL) to an activation buffer (50 mMMES buffer containing 50mM NaCl, pH = 5.5), the recombinant mouse granzyme B was activated by incubation at 37 ℃ for 4 hours to obtain activated recombinant mouse granzyme B; diluting the activated recombinant mouse granzyme B to 1.0 ng/mu L by using Tris buffer solution (50mM, pH = 7.5) to obtain diluted solution of the activated recombinant mouse granzyme B; mu.L of dilution of activated recombinant mouse granzyme B was added to 1. Mu.L of the molecular probe for targeted granzyme B prepared in example 2 ¹⁸ F]M-1 (10 mM) and incubated at 37 ℃ for more than 4 hours; after the incubation, the incubation solution was subjected to radioactive HPLC analysis using a Gabi Nova radioactivity detector, and the results of the analysis are shown in FIGS. 11 to 12.

As can be seen from FIGS. 11 to 12, the molecular probe [ 2 ] ¹⁸ F]M-1 has excellent ability to target granzyme B. Further, the molecular probes in different concentrations are set ¹⁸ F]M-1 (concentrations of 12.5, 25, 50, 100, 150, 200, 250 and 400. Mu.M, respectively) was incubated with granzyme B and detected by HPLC. Granzyme B as a pair of molecular probes ¹⁸ F]The enzymatic Michaelis-Menten constant (Km) of M-1 was 198.90. Mu.M, and the granzyme B was used as a pair of molecular probes [ 2 ], [ ¹⁸ F]M-1 has a catalytic Rate constant (kcat) of 1.12s ^-1 Particle enzyme B on molecular probe ¹⁸ F]The catalytic efficiency (kcat/Km) of M-1 was 5645M ^-1 s ^-1 . This result further illustrates the molecular probe [ 2 ] ¹⁸ F]M-1 has excellent ability to target granzyme B.

Experimental examples 1 to 4: cell expression assay for granzyme B

The experimental example provides a cell expression experiment of granzyme B, which comprises the following specific processes:

t cells (extracted from spleen of normal Balb/C mice purchased from Kyoto Kavens laboratory animals Co., ltd., heizhou) and 4T1 cells (purchased from cell bank of the Central department) were seeded at cell densities of 300 ten thousand and 30 ten thousand, respectively, into 6-well plates supplemented with 2mL of RPMI1640 medium (purchased from Biological Industries) using 4T1 cells without addition of T cells as a control, and cultured at 37 ℃; after culturing until the cell density reaches 90%, sucking the culture medium in the hole, removing T cells in the hole, sucking the liquid in the hole, and adding 200 mu L of RIPA lysis buffer containing protease inhibitor PMSF (2 mu L,1 mM) into the hole at 4 ℃ to lyse the cells to obtain cell lysate; the cell lysate was centrifuged at 12000rpm for 20 minutes at 4 ℃ and the supernatant was collected; after the protein concentration of the supernatant was determined, western Blot analysis was performed and the blots were detected using a Chemi DOC XRS + gel imaging system, the results of which are shown in FIGS. 13-14.

The results are shown in FIGS. 13-14, where it is evident that granzyme B was highly expressed in 4T1 cells co-incubated with T cells, whereas 4T1 cells not incubated with T cells were less expressed. The 4T1 cells co-incubated with the T cells and the 4T1 cells not incubated with the T cells can be respectively used as cells with high expression and low expression of granzyme B for subsequent targeting experiments of the molecular probe.

Experimental examples 1 to 5: cellular uptake assay of molecular probes targeting granzyme B

The experimental example provides a cell uptake experiment of a molecular probe targeting granzyme B, and the specific process is as follows:

t cells (extracted from spleen of normal Balb/C mice purchased from Kyoto Kavens laboratory animals Co., ltd., heizhou) and 4T1 cells (purchased from cell bank of the Central department) were seeded at cell densities of 300 ten thousand and 30 ten thousand, respectively, into 6-well plates supplemented with 2mL of RPMI1640 medium (purchased from Biological Industries) using 4T1 cells without addition of T cells as a control, and cultured at 37 ℃; after 24h incubation, the wells were freed of T cells and 200. Mu.L of culture medium (containing 1X 10 cells) was taken from the wells ⁶ 4T1 cells) with 100. Mu.L of the molecular probe comprising the targeted granzyme B prepared in example 2 ¹⁸ F]Serum-free medium of M-1 (1. Mu. Ci/100. Mu.L) was co-added to the radioimmunoassay and incubated at 37 ℃ for 0.5, 1, 2 and 4h; after the completion of the incubation, the 4T1 cells in the radioimmunized tube were washed twice with cold (4 ℃ C.) PBS buffer to remove the non-ingested molecular probe 2 ¹⁸ F]M-1; after washing, the amount of radioactive dose taken up by the cells was measured by a gamma counter and the corresponding cell concentration was measured, and three sets of experiments were performed in parallel at each time point, and the results of the measurements are shown in fig. 15. The same method was used to detect the granzyme B-targeting molecular probe prepared in example 4 ⁶⁸ Ga]The cellular uptake of M-2 was determined as shown in FIG. 16.

As shown in FIG. 15, the molecular probe [ 2 ] in 4T1 cell after cocultivation with T cells ¹⁸ F]The uptake of M-1 was consistently high over 4 hours, reaching a maximum (6.71% ID/mg) in 0.5 hours, and was maintained at 4.03% ID/mg or more for the next two hours. In contrast, the uptake values of 4T1 cells not co-cultured with T cells were always lower than 1.95% ID/mg within 4 hours, much lower than the co-cultured group. Specification molecular Probe [ 2 ] ¹⁸ F]M-1 can well target cells with high granzyme B expression.

As shown in FIG. 16, the molecular probe [ 2 ] in 4T1 cells after cocultivation with T cells ⁶⁸ Ga]The intake of M-2 reached the highest value (4.38% ID/mg) in 0.5 hours, and was maintained at 2.31% or more in the next 2 hours. In contrast, the uptake of 4T1 cells not co-cultured with T cells was consistently less than 1.8% ID/mg over 4 hours, much less than the co-cultured group. Illustrative molecular Probe [ 2 ] ⁶⁸ Ga]M-2 can well target cells with high granzyme B expression.

Experimental examples 1 to 6: mouse PET imaging experiment of molecular probe of targeting granzyme B

The experimental example provides a mouse PET imaging experiment of a molecular probe of a targeting granzyme B, and the specific process is as follows:

4T1 cells were plated at 1X 10 ⁶ Doses of each were implanted subcutaneously in the right axilla of female BALB/C mice (6 weeks old, purchased from haven laboratory animals, inc); one week after tumor growth, mice were divided into two groups, one group was treated group, mice received immunotherapy, treatment regimen was BEC (purchased from MedChemExpress) therapy (injection dose was 20 mg/kg), administered once a day for 3 days continuously, and the other group was untreated group; on day 2 after the end of the treatment, the mice were anesthetized with oxygen containing 2vt% (vt% represents volume ratio) of isoflurane at a flow rate of 2L/min; four limbs of the miceAfter the immobilization with the tail, the molecular probe prepared in example 2 of 150. Mu. Ci dissolved in 100. Mu.L of physiological saline ¹⁸ F]Molecular probe [ 2 ] prepared in M-1 and example 4 ⁶⁸ Ga]M-2 is injected through tail vein; after the probe injection is finished, the dynamic PET scanning is immediately executed for 60min, and the PET imaging result is shown in figures 17 and 21; after scanning is finished, dividing a PET imaging result for 60min into 12 frames of images by using an OSEM3D/MAP algorithm, and carrying out one frame every 5min to realize real-time analysis on in-vivo imaging of the mouse; the distribution of the probe in the tumor site and other organ tissues was sketched and compared by using a region of interest (ROI) technique in ASIPRO software, and the analysis results are shown in FIGS. 18-20 and 22-24, wherein the uptake value of the molecular probe in each tissue in vivo is expressed in% ID/mL (percentage of injected dose per milliliter).

As shown in FIGS. 17 to 20, the molecular probe [ 2 ] ¹⁸ F]M-1 is not obviously taken up at the tumor part before immunotherapy, the taking of the tumor part after immunotherapy is obviously increased, and a good imaging effect is maintained within 1 hour. The uptake at the tumor site was quantitatively analyzed, reaching a maximum value at 10min, about 4% ID/mL, remaining at 3% ID/mL after 1 hour, and the tumor to muscle uptake ratio was at most 4. In the non-immune treated mice, the tumor uptake value was maintained at about 1% by ID/mL within 1 hour, and the tumor-to-muscle uptake ratio was also low. Specification molecular Probe [ 2 ] ¹⁸ F]M-1 can be used for accurately monitoring the curative effect of immunotherapy by PET imaging.

As shown in FIGS. 22 to 24, the molecular probe ⁶⁸ Ga]The M-2 is not obviously absorbed by the tumor part before immunotherapy, the absorption of the tumor part after the immunotherapy is obviously increased, and a good imaging effect is maintained within 1 hour. The uptake at the tumor site was quantitatively analyzed, and the uptake reached a maximum value of about 2.2% at 40min, and remained at 2.2% ID/mL after 1 hour, with a tumor-to-muscle uptake ratio of at most 3. In the non-immune treated mice, the tumor uptake value was maintained at about 1% by ID/mL within 1 hour, and the tumor-to-muscle uptake ratio was also low. Illustrative molecular Probe [ 2 ] ⁶⁸ Ga]M-2 can be used for accurately monitoring the curative effect of immunotherapy by PET imaging.

Example 2-1: molecular probe of targeted granzyme B ¹⁸ F]H-1

This example provides a molecular probe of granzyme B ¹⁸ F]H-1, the molecular probe targeting granzyme B [ 2 ], [ ¹⁸ F]H-1 has the structure shown below:

example 2-2: molecular probe for preparing targeting granzyme B ¹⁸ F]Method for H-1

This example provides the molecular probe of the targeted granzyme B described in example 1 ¹⁸ F]The preparation method of H-1 comprises the following specific steps:

according to the literature "HeS, liJ, lyuY, huangJ, puK.JAmChemSoc.2020;142 (15): 7075-7082. "synthetic compound IEPD; on the basis of example 1-2, the compound IEFD (1.1 eq) was replaced with the compound IEPD (1.1 eq) to give compound II-1 (yield 69%), compound II-2 (yield 82%), precursor compound H-1 (15 mg, white powder) and molecular probe (molecular probe) ¹⁸ F]H-1 solution. The mass spectrum of the precursor compound H-1 is shown in FIG. 25, the high performance liquid chromatogram is shown in FIG. 26, the hydrogen spectrum is shown in FIG. 29, and the carbon spectrum is shown in FIG. 30. Molecular probe [ 2 ] using Gabinova radioactivity detector ¹⁸ F]The reaction solution before and after H-1 labeling was subjected to radioactive HPLC detection, and the detection results are shown in FIG. 31. The molecular probe [ 2 ] is calculated from the radioactive product peak area/total peak area ¹⁸ F]Radiochemical purity (RCP) of H-1, calculated as: the radiochemical purity of the product obtained by labeling is higher than 95%. Calculating the molecular Probe by detection of radioactivity HPLC ¹⁸ F]Radiochemical yield (RCY) of H-1 calculated as: 41 percent.

Examples 2 to 3: molecular probe of targeted granzyme B ⁶⁸ Ga]H-2

This example provides a molecular probe of targeting granzyme B ⁶⁸ Ga]H-2, the molecular probe targeting granzyme B ⁶⁸ Ga]H-2 has the structure shown below:

examples 2 to 4: molecular probe [ 2 ] for preparing targeted granzyme B ⁶⁸ Ga]Process for H-2

This example provides the molecular probe of targeting granzyme B described in example 3 ⁶⁸ Ga]The preparation method of H-2 comprises the following specific steps:

on the basis of examples 1 to 4, the amino acid Fmoc-phenylalanine (0.5 mmol) was replaced with the amino acid Fmoc-proline (0.5 mmol) to obtain a compound IEFD-ZL (compound IEFD-ZL is a isoleucine-glutamic acid-proline-aspartic acid sequence recognized for granzyme B targeting), a compound IV-1 (yield 72%), a compound IV-2 (yield 31%), a compound IV-3 (yield 43%), a precursor compound H-2 (11 mg, white powder) and a molecular probe [ 2 ] ⁶⁸ Ga]H-2 solution. The mass spectrum of the precursor compound H-2 is shown in FIG. 27, and the high performance liquid chromatogram is shown in FIG. 28. The precursor compound H-2 has a molecular weight of 1571 and a purity of greater than 90%. Molecular probe [ 2 ] using Gabinova radioactivity detector ⁶⁸ Ga]The reaction solution before and after H-2 labeling was subjected to radioactive HPLC detection, and the detection results are shown in FIG. 32. The molecular probe [ 2 ] was calculated from the radioactive product peak area/total peak area ⁶⁸ Ga]Radiochemical purity (RCP) of H-2, calculated as: the radiochemical purity of the product obtained by labelling was higher than 95%. Calculating the molecular Probe by detection of radioactivity HPLC ⁶⁸ Ga]Radiochemical yield (RCY) of H-2 calculated as: 98 percent.

Experimental example 2-1: in vitro stability experiment of molecular probe of targeting granzyme B

The molecular probe [ 2 ] targeting granzyme B was carried out in the same manner as in Experimental example 1-1 ¹⁸ F]The in vitro stability of H-1 is shown in FIGS. 33-34.

As can be seen from FIGS. 33 to 34, when the incubation time was increased to 4h, the molecular probe of the targeted granzyme B, [ 2 ] ¹⁸ F]The radiochemical purity of H-1 is above 95%, which proves that no other products are generated during the incubation process, indicating that the molecular probe [ 2 ] ¹⁸ F]H-1 has better stability.

Experimental example 2-2: experiment of lipid-water distribution coefficient of molecular probe of targeting granzyme B

Reference is made to "LinJ, gaoD, wangs, et. JAmChemoc.2022; 144 (17): 7667-7675 "molecular probe for detecting granzyme B prepared in example 2, [ 2 ] ¹⁸ F]The lipid water partition coefficient of H-1.

The molecular probe of the target granzyme B is measured by experiments ¹⁸ F]The lipid water partition coefficient of H-1 is-0.803. + -. 0.0046, which indicates that the molecular probe 2 ¹⁸ F]H-1 has hydrophilicity, and has the advantages of being excreted from the kidney and reducing liver metabolism in the aspect of metabolism.

Experimental examples 2 to 3: enzyme kinetics experiment of molecular probe of targeting granzyme B

The molecular probe [ 2 ] targeting granzyme B was carried out in the same manner as in Experimental example 1-3 ¹⁸ F]The results of the enzyme kinetics experiments for H-1 are shown in FIGS. 35 to 36.

As can be seen from FIGS. 35 to 36, the molecular probe [ 2 ] ¹⁸ F]H-1 has excellent ability to target granzyme B. Further, molecular probes of different concentrations are used ¹⁸ F]H-1 (concentrations of 12.5, 25, 50, 100, 150, 200, 250 and 400. Mu.M, respectively) was incubated with granzyme B and detected by HPLC. Granzyme B as a pair of molecular probes ¹⁸ F]The enzymatic Michaelis-Menten constant (Km) of H-1 is 94.11. Mu.M, and the particle enzyme B is [ 2 ] molecular probe ¹⁸ F]H-1 has a catalytic Rate constant (kcat) of 0.60s ^-1 Particle enzyme B on molecular probe ¹⁸ F]The catalytic efficiency (kcat/Km) of H-1 was 6375M ^-1 s ^-1 . This result further illustrates the molecular probe [ 2 ] ¹⁸ F]H-1 has excellent ability to target granzyme B.

Experimental examples 2 to 4: cellular uptake assay of molecular probes targeting granzyme B

The molecular probe [ 2 ] targeting granzyme B was carried out in the same manner as in Experimental examples 1 to 5 ¹⁸ F]Molecular probe [ 2 ] of H-1 and target granzyme B ⁶⁸ Ga]The results of the experiments on the cellular uptake of H-2 are shown in FIGS. 37 to 38.

As shown in FIG. 37, the molecular probe [ 2 ] in 4T1 cells after cocultivation with T cells ¹⁸ F]The uptake value of H-1 is always high within 4 hours, and reaches the maximum value within 1 hour(2.75% by weight, ID/mg) and maintained at 1.57% by weight or more for the next two hours. In contrast, the uptake of 4T1 cells not co-cultured with lymphocytes was consistently less than 1.1% ID/mg over 4 hours, much less than the co-cultured group. Specification molecular Probe [ 2 ] ¹⁸ F]H-1 can well target cells with high granzyme B expression.

As shown in FIG. 38, the molecular probe [ 2 ] in 4T1 cells after cocultivation with T cells ⁶⁸ Ga]The uptake of H-2 was consistently high over 4 hours, reaching a maximum (2.3% ID/mg) in 1 hour. In contrast, the uptake of 4T1 cells not co-cultured with lymphocytes was consistently less than 1.02% ID/mg over 4 hours, much less than the co-cultured group. Illustrative molecular Probe [ 2 ] ⁶⁸ Ga]H-2 can target cells with high granzyme B expression well.

Experimental examples 2 to 5: mouse PET imaging experiment of molecular probe of targeting granzyme B

The molecular probe [ 2 ] targeting granzyme B was carried out in the same manner as in Experimental examples 1 to 6 ¹⁸ F]Molecular probe [ 2 ] of H-1 and target granzyme B ⁶⁸ Ga]The results of the H-2 mouse PET imaging experiments are shown in FIGS. 39-46.

As shown in FIGS. 39 to 42, the molecular probe [ 2 ] ¹⁸ F]H-1 is not obviously taken up at the tumor part before immunotherapy, the taking of the tumor part after immunotherapy is obviously increased, and a good imaging effect is maintained within 1 hour. The uptake at the tumor site was quantitatively analyzed, and the uptake reached a maximum value at 40min, about 3.5% ID/mL, and remained at 3.5% ID/mL after 1 hour, with a tumor-to-muscle uptake ratio of at most 4. In untreated mice, tumor uptake values were maintained at about 1% ID/mL within 1 hour, and tumor-to-muscle uptake ratios were also lower. Specification molecular Probe [ 2 ] ¹⁸ F]The H-1 can be used for accurately monitoring the curative effect of immunotherapy by PET imaging.

As shown in FIGS. 43 to 46, the molecular probe ⁶⁸ Ga]H-2 is not obviously taken up at the tumor part before immunotherapy, the taking of the tumor part after immunotherapy is obviously increased, and a good imaging effect is maintained within 1 hour. The uptake at the tumor site was quantitatively analyzed, and the uptake value reached the highest value at 40min, about 3.5% ID/mL, and remained at 3 after 1 hour.5% ID/mL, tumor to muscle uptake ratio of at most 5. In untreated mice, tumor uptake values were maintained at around 1% id/mL within 1 hour, and tumor to muscle uptake ratios were also low. Specification molecular Probe [ 2 ] ⁶⁸ Ga]The H-2 can be used for accurately monitoring the curative effect of the immunotherapy by PET imaging.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims (10)

1. The molecular probe targeting granzyme B is characterized in that the molecular probe targeting granzyme B has an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence targeted and recognized by granzyme B or an isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B;

when the molecular probe has an isoleucine-glutamic acid-phenylalanine-aspartic acid sequence targeted and recognized by granzyme B, the molecular probe targeted to granzyme B has the following structure:

when the molecular probe has an isoleucine-glutamate-proline-aspartate sequence targeted and recognized by granzyme B, the molecular probe targeted to granzyme B has the following structure:

wherein R is a radionuclide-labeled group.

2. As claimed in claimThe granzyme B-targeting molecular probe of claim 1, wherein the radionuclide labeling group is ⁶⁸ Ga、[ ¹⁸ F]AlF、[ ¹⁸ F]AmBF ₃ 、 ⁶⁴ Cu or ⁸⁹ Zr。

3. The molecular probe targeting granzyme B as claimed in claim 2, which has an isoleucine-glutamate-phenylalanine-aspartate sequence which is targeted for recognition by granzyme B and the radionuclide tag group is [ seq ] ¹⁸ F]AmBF ₃ The molecular probe targeting granzyme B has the following structure:

when the gene has a isoleucine-glutamic acid-phenylalanine-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]In AlF, the molecular probe targeting granzyme B has the following structure:

when the gene has a isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]AmBF ₃ When the molecular probe targeting granzyme B is used, the molecular probe has the following structure:

when the gene has a isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is [ 2 ] ¹⁸ F]In AlF, the molecular probe targeting granzyme B has the following structure:

4. the granzyme B-targeting molecular probe of claim 2, wherein said probe has an isoleucine-glutamate-phenylalanine-aspartate sequence which is targetedly recognized by granzyme B, and wherein said radionuclide labeling group is ⁶⁸ Ga, the molecular probe targeting granzyme B has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence which is targeted and recognized by granzyme B, and the radionuclide labeling group is ⁶⁸ Ga, the molecular probe targeting granzyme B has the following structure: i plan number

5. The molecular probe targeting granzyme B as claimed in claim 3, wherein the molecular probe has an isoleucine-glutamate-phenylalanine-aspartate sequence which is targeted and recognized by granzyme B, and the radionuclide tag group is [ seq ] ¹⁸ F]AmBF ₃ When the compound is a precursor of the molecular probe targeting granzyme B, the precursor compound has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is ¹⁸ F]AmBF ₃ When the precursor compound of the molecular probe targeting granzyme B has the structure asThe structure shown below:

6. the granzyme B-targeting molecular probe of claim 4, wherein said probe has an isoleucine-glutamate-phenylalanine-aspartate sequence which is targetedly recognized by granzyme B, and the radionuclide labeling group is ⁶⁸ Ga, the precursor compound of the molecular probe targeting granzyme B has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence which is targeted and recognized by granzyme B and the radionuclide labeling group is ⁶⁸ Ga, the precursor compound of the molecular probe targeting granzyme B has the following structure:

7. a method for preparing the molecular probe targeting granzyme B as described in claim 3 or 5, wherein the molecular probe has an isoleucine-glutamate-phenylalanine-aspartate sequence which is targeted and recognized by granzyme B, and the radionuclide labeling group is set to ¹⁸ F]AmBF ₃ The method comprises the following steps:

the method comprises the following steps: carrying out condensation reaction on a compound IEFD and a compound SF to obtain a compound I-1;

step two: carrying out deprotection reaction on the compound I-1 to obtain a compound I-2;

step three: carrying out click condensation reaction on a compound I-2, alkyl aminomethyl boron trifluoride, tri (2-benzimidazolylmethyl) amine and copper (I) hexafluorophosphate to obtain a precursor compound M-1 of a molecular probe of the targeted granzyme B;

step four: carrying out radionuclide labeling on a precursor compound M-1 of the molecular probe of the targeting granzyme B to obtain the molecular probe of the targeting granzyme B;

the compound IEFD has the structure shown below:

the compound SF has the following structure:

the compound I-1 has the following structure:

the compound I-2 has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence targeted and recognized by granzyme B, and the radionuclide labeling group is ¹⁸ F]AmBF ₃ The method comprises the following steps:

the method comprises the following steps: carrying out condensation reaction on a compound IEPD and a compound SF to obtain a compound II-1;

step two: carrying out deprotection reaction on the compound II-1 to obtain a compound II-2;

step three: carrying out click condensation reaction on a compound II-2, alkyl aminomethyl boron trifluoride, tri (2-benzimidazolylmethyl) amine and copper (I) hexafluorophosphate to obtain a precursor compound H-1 of the molecular probe of the targeted granzyme B;

step four: carrying out radionuclide labeling on a precursor compound H-1 of the molecular probe of the targeted granzyme B to obtain the molecular probe of the targeted granzyme B;

the compound IEPD has the structure shown below:

the compound II-1 has the following structure:

the compound II-2 has the following structure:

8. a method for preparing the granzyme B-targeted molecular probe of claim 4 or 6, wherein the granzyme B-targeted molecular probe has an isoleucine-glutamate-phenylalanine-aspartate sequence and a radionuclide labeling group ⁶⁸ Ga, the method comprising the steps of:

the method comprises the following steps: carrying out condensation reaction on a compound IEFD-ZL and a compound CBT-1 to obtain a compound III-1;

step two: carrying out deprotection reaction on the compound III-1 to obtain a compound III-2;

step three: carrying out condensation reaction on the compound III-2 and 2- (ethidium phenyl) pyridine to obtain a compound III-3;

step four: carrying out condensation reaction on the compound III-3 and hydroxysuccinimide-tetraazacyclododecane tetraacetic acid to obtain a precursor compound M-2 of the molecular probe of the target granzyme B;

step five: carrying out radionuclide labeling on a precursor compound M-2 of the molecular probe of the targeted granzyme B to obtain the molecular probe of the targeted granzyme B;

the compound IEFD-ZL has the structure shown below:

the compound CBT-1 has the following structure:

the compound III-1 has the following structure:

the compound III-2 has the following structure:

the compound III-3 has the following structure:

when the gene has an isoleucine-glutamic acid-proline-aspartic acid sequence which is targeted and recognized by granzyme B, and the radionuclide labeling group is ⁶⁸ Ga, the method comprising the steps of:

the method comprises the following steps: carrying out condensation reaction on a compound IEPD-ZL and a compound CBT-1 to obtain a compound IV-1;

step two: carrying out deprotection reaction on the compound IV-1 to obtain a compound IV-2;

step three: carrying out condensation reaction on the compound IV-2 and 2- (ethidium phenyl) pyridine to obtain a compound IV-3;

step four: carrying out condensation reaction on the compound IV-3 and hydroxysuccinimide-tetraazacyclododecane tetraacetic acid to obtain a precursor compound H-2 of the molecular probe of the targeting granzyme B;

step five: carrying out radionuclide labeling on a precursor compound H-2 of the molecular probe of the targeting granzyme B to obtain the molecular probe of the targeting granzyme B;

the compound IEPD-ZL has the structure shown as follows:

the compound IV-1 has the following structure:

the compound IV-2 has the following structure:

the compound IV-3 has the following structure:

9. use of a granzyme B-targeted molecular probe according to any one of claims 1 to 6 for granzyme B imaging, wherein said use is for non-disease diagnostic and therapeutic purposes.

10. An imaging agent targeting granzyme B, comprising the granzyme B-targeting molecular probe of any one of claims 1 to 6.

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