CN113354712A

CN113354712A - Enzyme-targeted control intramolecular condensation molecular probe and preparation method and application thereof

Info

Publication number: CN113354712A
Application number: CN202110656178.1A
Authority: CN
Inventors: 林建国; 邱玲; 刘清竹; 李珂; 吕高超; 彭莹; 张理霞
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-09-07
Anticipated expiration: 2041-06-11
Also published as: CN113354712B

Abstract

The invention relates to an enzyme-targeted control intramolecular condensation molecular probe and a preparation method and application thereof, belonging to the technical field of chemistry. The invention provides a molecular probe, which is obtained by modifying a marker group and a targeting group on the basis of intramolecular condensation of a skeleton GP-13; the intramolecular condensation skeleton GP-13 used by the molecular probe enables the CBT and the intramolecular Cys residue to generate click condensation reaction more quickly by changing the connecting group between the 2-cyano-6-aminobenzothiazole (CBT) and the cysteine (Cys), thereby reducing the competitive combination of the molecular probe and the intracellular free cysteine, and further ensuring that the condensation rate of the molecular probe is not limited by the concentration of the intracellular free cysteine; the molecular probe has extremely high application prospect in target object imaging, such as tumor imaging.

Description

Enzyme-targeted control intramolecular condensation molecular probe and preparation method and application thereof

Technical Field

The invention relates to an enzyme-targeted control intramolecular condensation molecular probe and a preparation method and application thereof, belonging to the technical field of chemistry.

Background

Tumors are diseases caused by abnormal growth of cells in organisms due to various factors, and are classified into malignant tumors and benign tumors according to their characteristics and damage to the organisms. Malignant tumors, also called cancers, are metastatic and invasive and are extremely harmful to the human body.

The tumor targeting molecular probe and the imaging technology are important means for specifically diagnosing tumors, and the wide development prospect is obtained by virtue of the advantages of high sensitivity, whole-body imaging, good safety and the like. The intermolecular condensation type probe is a probe which generates a dimer by utilizing the molecular condensation of CBT and Cys so that a development signal can be kept in a tumor for a long time, and can be used for detecting a tumor marker in a living body. However, it is known from the reported literature (for example, the documents "Chen, Z., et al., expanding the Condensation Reaction between organic compounds and Amino thio To Optimize the molecule In Situ hybridization Formation for the Imaging of proteins and carbohydrates In cells. analytical chemistry 2020,59(8), 3272. sup. In 3279.", "Ye, D., et al., Controlling Intracellular hybridization for the Imaging of protein activity. Angewandle International Edition 2011,50(10), 2275. sup. In 2279". sup. In tumor cells containing a large amount of free cysteine (Cys) at a concentration between 20. mu.M and 100. mu.M, which makes it difficult for these molecules To form a hydrophilic probe or even a dimeric Condensation Reaction between these molecules and the tumor cells. Since the intermolecular condensation rate of the intermolecular condensation-type probe in the tumor cell is affected by the concentration thereof, it may make it more difficult for the intermolecular condensation-type probe to undergo condensation-cyclization in the tumor cell, and may also cause the intermolecular condensation-type probe to be easily pumped out by the tumor cell, thereby causing a decrease in the imaging ability of the intermolecular condensation-type probe.

At present, a proper amount of non-radioactive intermolecular condensation-type probes are injected to consume free cysteine in tumor cells, so that the concentration of the intermolecular condensation-type probes in the tumor cells is prevented from being reduced, the efficiency of the cyclic condensation in the tumor cells is improved, and the purpose of enhancing the imaging capability is finally achieved. However, the co-injection of the non-radioactive intermolecular condensation probe undoubtedly increases the cost of the drug greatly, and the increase of the concentration of the drug in vivo after the co-injection may bring new toxic and side effects, which both greatly hinder the clinical application of the intermolecular condensation probe in the specific diagnosis of tumors. Therefore, it is highly desirable to find intermolecular condensation-type probes that do not competitively condense with free Cys to overcome the drawbacks of using current intermolecular condensation-type probes for specific diagnosis of tumors.

Disclosure of Invention

In order to solve the above problems, the present invention provides a molecular probe having a structure as shown below:

wherein R is₁Is a labeling group, R₂Is a targeting group.

In one embodiment of the invention, the molecular probe is a glutamyl transpeptidase targeted molecular probe; the glutamyl transpeptidase targeted molecular probe has the following structure:

in one embodiment of the present invention, the labeled precursor of the molecular probe targeted by glutamyl transpeptidase has the following structure:

in one embodiment of the invention, the molecular probe is a legumain targeted molecular probe; the legumain targeted molecular probe has the following structure:

in one embodiment of the invention, the labeled precursor of the legumain-targeted molecular probe has the structure shown below:

in one embodiment of the invention, the molecular probe is an aspartate proteolytic enzyme targeted molecular probe; the aspartate proteolytic enzyme targeted molecular probe has the following structure:

in one embodiment of the present invention, the labeled precursor of the aspartic acid proteolytic enzyme targeted molecular probe has the following structure:

the invention also provides an intramolecular condensation skeleton, which has the following structure:

the invention also provides a method for preparing the glutamyl transpeptidase targeted molecular probe, which comprises the following steps: modifying a marking group and a targeting group on the basis of the intramolecular condensation skeleton.

In one embodiment of the present invention, when the molecular probe is a glutamyl transpeptidase targeted molecular probe, the method is: dissolving the intramolecular condensation skeleton, N-BOC-L-glutamic acid-1-tert-butyl ester and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in ultra-dry THF (tetrahydrofuran) to obtain a solution; adding DIPEA (N, N-diisopropylethylamine) into the dissolved solution, and reacting under the protection of nitrogen to obtain a compound GD-14; dissolving a compound GD-14 in a mixed solution of DCM and TFA for reaction to obtain a compound GD-15; dissolving compound GD-15 in DMF/H₂Obtaining mixed solution from the mixed solution of O; adding AMBF to the mixed solution₃Reacting (2-azidoethyl-N, N-dimethylaminomethyl-trifluoroborate), ligand (tris (2-benzimidazolylmethyl) amine) and Cu (I) under the protection of nitrogen to obtain a labeled precursor of the glutamyltranspeptidase targeted molecular probe; carrying out radioactive labeling on a labeled precursor of the glutamyl transpeptidase targeted molecular probe to obtain the glutamyl transpeptidase targeted molecular probe;

the compound GD-14 has the structure shown below:

the compound GD-15 has the structure shown below:

in one embodiment of the present invention, when the molecular probe is a glutamyl transpeptidase targeted molecular probe, the method is: dissolving 35mg of the intramolecular condensation skeleton (0.043mmol), 14mg of N-BOC-L-glutamic acid-1-tert-butyl ester (0.047mmol) and 19mg of HBTU (0.054mmol) in 10mL of ultra-dry THF to obtain a solution; adding 235 mu L of DIPEA (1.35mmol) into the dissolved solution, adjusting the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; washing the crude product with cold ether (4 ℃) for 3 times to remove redundant HBTU in the crude product to obtain a compound GD-14;

dissolving 39.8mg of compound GD-14 in a mixed solution of 2mL of DCM and 2mL of TFA, and stirring at 25 ℃ for 1h to obtain a reaction solution; removing the organic solvent in the reaction solution by using a rotary evaporator to obtain an oily substance; washing the oil 3 times with cold diethyl ether (4 ℃) to remove excess trifluoroacetic acid in the oil to give compound GD-15;

33.7mg of Compound GD-15(0.0357mmol) were dissolved in 3mL of DMF/H₂O(DMF:H₂O is 2:1) to obtain a mixed solution; 199. mu.L of AMBF was added to the mixture₃(0.102mmol), 295. mu.L ligand (0.0029mmol) and 13mg Cu (I) (0.035mmol) were stirred at 45 ℃ for 1h under nitrogen protection to obtain a reaction solution; purifying and drying the reaction solution to obtain a labeled precursor of the glutamyl transpeptidase targeted molecular probe;

and (3) carrying out radioactive labeling on a labeled precursor of the glutamyl transpeptidase targeted molecular probe to obtain the glutamyl transpeptidase targeted molecular probe.

In one embodiment of the present invention, when the molecular probe is a legumain-targeted molecular probe, the method is: dissolving the intramolecular condensation skeleton in ultra-dry THF (tetrahydrofuran) to obtain a solution; adding Ac-AAN-OH (acetyl-alanine-aspartic acid), HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) and DIPEA (N, N-diisopropylethylamine) into the dissolved solution, and reacting under the protection of nitrogen to obtain ｃA compound GP-A; adding TFA and Tips into ｃA mixed solution of ｃA compound GP-A and DCM for reaction to obtain ｃA compound GP-AAN; dissolving a compound GD-15 in DMF to obtain a mixed solution; adding AMBF to the mixed solution₃Reacting (2-azidoethyl-N, N-dimethylaminomethyl-trifluoroborate), ligand (tris (2-benzimidazolylmethyl) amine) and tetrakis (acetonitrile) copper (I) hexafluorophosphate under the protection of nitrogen to obtain the legumain targeted molecular probeA label precursor; carrying out radioactive labeling on a labeled precursor of the legumain targeted molecular probe to obtain the legumain targeted molecular probe;

the compound GP-A has the structure shown below:

the compound GP-AAN has the structure shown below:

in one embodiment of the present invention, when the molecular probe is a legumain-targeted molecular probe, the method is: dissolving 22mg of the intramolecular condensation skeleton GP-13 in 6mL of ultra-dry THF to obtain a dissolved solution; adding 17mg of Ac-AAN-OH, 12mg of HBTU and 20 mu L of DIPEA into the dissolved solution, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain ｃA compound GP-A;

to remove the protected group Trt, 2mL of TFA and 100. mu.L of Tips were added to ｃA mixed solution of 25mg of compound GP-A and 2mL of DCM, and stirred at 25 ℃ for 0.5h to obtain ｃA reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; washing the crude product with cold ether (4 ℃) for 3 times to remove redundant trifluoroacetic acid in the crude product, and then centrifugally purifying the crude product to obtain a compound GP-AAN;

18mg of the compound GP-AAN are dissolved in 2mL DMF and 1mL H₂Obtaining a mixed solution in the step O; adding 212 μ L AMBF to the mixture₃275. mu.L of ligand and 13mg of tetrakis (acetonitrile) copper (I) hexafluorophosphate, and stirring at 45 ℃ for 1 hour under the protection of nitrogen to obtain a reaction solution; purifying the reaction solution to obtain a labeled precursor of the legumain targeted molecular probe;

and carrying out radioactive labeling on the labeled precursor of the legumain targeted molecular probe to obtain the legumain targeted molecular probe.

In one embodiment of the present invention, when the molecular probe is an aspartate proteolytic enzyme targeted molecular probe, the method is: dissolving the intramolecular condensation skeleton, DEVD (aspartic acid-glutamic acid-valine-aspartic acid) and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in THF (tetrahydrofuran) to obtain a solution; adding DIPEA (N, N-diisopropylethylamine) into the dissolved solution, and reacting under the protection of nitrogen to obtain a compound GP-14; dissolving a compound GP-14 in a mixed solution of DCM and TFA for reaction to obtain a compound GP-15; dissolving the compound GP-15 in DMF and H₂Obtaining mixed solution from the mixed solution of O; adding AMBF to the mixed solution₃(2-Azidoethyl-N, N-dimethylaminomethyl-trifluoroborate), THPTA (tris (3-hydroxypropyltriazolemethyl) amine), LAASS (sodium ascorbate) and CUSO₄Then, reacting under the protection of nitrogen to obtain a labeled precursor of the molecular probe targeted by the aspartic acid proteolytic enzyme; carrying out radioactive labeling on a labeled precursor of the molecular probe targeted by the aspartic acid proteolytic enzyme to obtain the molecular probe targeted by the aspartic acid proteolytic enzyme;

the compound GP-14 has the structure shown below:

the compound GP-15 has the structure shown below:

in one embodiment of the present invention, when the molecular probe is an aspartate proteolytic enzyme targeted molecular probe, the method is: dissolving 23mg of the above intramolecular condensation skeleton, 23mg of DEVD (0.033mmol) and 17mg of HBTU (0.045mmol) in 4mL of DMF to obtain a solution; adding 13 mu L of DIPEA into the solution to adjust the pH value of the solution to 8, and stirring for 12h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; carrying out rotary evaporation and vacuum drying on the reaction liquid to obtain a compound GP-14;

adding 50mg of compound GP-14 into a mixed solution of 3mL of TFA and 3mL of DCM, and stirring at 25 ℃ for 0.5h to obtain a reaction solution; removing the solvent in the reaction solution by using a rotary evaporator to obtain a crude product; washing the crude product with DCM for 3 times, adding anhydrous ether, and collecting precipitate; drying the precipitate to obtain a dried substance; purifying the dried product by semi-preparative high performance liquid chromatography to obtain a compound GP-15;

42mg of Compound GP-15 are dissolved in 3mL DMF and 0.6mL H₂Obtaining a mixed solution in the step O; adding 88 μ L AMBF to the mixture₃6.51mg THPTA, 6mg LAASS and 3.8mg CUSO₄Then, reacting for 45min at 45 ℃ under the protection of nitrogen to obtain a reaction solution; purifying the reaction solution by semi-preparative high performance liquid chromatography to obtain a labeled precursor of the molecular probe targeted by the aspartic acid proteolytic enzyme;

and (3) carrying out radioactive labeling on the labeled precursor of the molecular probe targeted by the aspartic acid proteolytic enzyme to obtain the molecular probe targeted by the aspartic acid proteolytic enzyme.

The invention also provides a method for preparing the intramolecular condensation skeleton, which comprises the following steps: dissolving a compound GP-12 in methanol to obtain a mixed solution; adding Tips (triisopropylsilane) and SEt (2- (ethylidenepropyl) pyridine) into the mixed solution for reaction to obtain the intramolecular condensation skeleton;

the compound GP-12 has the structure shown below:

in one embodiment of the present invention, the method is: 295mg of compound GP-12 is dissolved in 15mL of methanol to obtain a mixed solution; after adding 300. mu.L of Tips and 70. mu.L of SEt (0.47mmol) to the mixture, the mixture was stirred at 25 ℃ for 2 hours to obtain a reaction solution; removing methanol in the reaction solution by using a rotary evaporator to obtain a crude product; the crude product was washed with cold ether (4 ℃) 3 times to remove excess SEt from the crude product, and then purified and dried to obtain the intramolecular condensation skeleton described above.

In one embodiment of the present invention, the compound GP-12 is prepared by: dissolving a compound GP-11 in a mixed solution of DCM (dichloromethane) and Tips (triisopropylsilane) to obtain a dissolved solution; adding TFA (trifluoroacetic acid) into the dissolved solution to react to obtain a compound GP-12;

the compound GP-11 has the structure shown below:

in one embodiment of the present invention, the compound GP-12 is prepared by: dissolving 429.6mg of compound GP-11 in a mixed solution of 4mL of DCM and 160 mu L of Tips to obtain a dissolved solution; adding 4mL of TFA into the dissolved solution, and stirring at 25 ℃ for 1h to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain an oily substance; the oil was washed 3 times with cold diethyl ether (4 ℃) to remove excess HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in the oil, yielding compound GP-12.

In one embodiment of the present invention, the compound GP-11 is prepared by: dissolving a compound GP-10, N-tert-butyloxycarbonyl-S-trityl-L-cysteine and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in anhydrous THF (tetrahydrofuran) to obtain a mixed solution; adding DIPEA (N, N-diisopropylethylamine) into the mixed solution, and reacting under the protection of nitrogen to obtain a compound GP-11;

the compound GP-10 has the structure shown below:

in one embodiment of the present invention, the compound GP-11 is prepared by: 287.8mg of the compound GP-10(0.443mmol), 225.6mg of N-t-butoxycarbonyl-S-trityl-L-cysteine (0.487mmol) and 210.5mg of HBTU (0.509mmol) are dissolved in 10mL of anhydrous THF to obtain a mixture; adding 188 mu L DIPEA (1.08mmol) into the mixed solution to adjust the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the oil was washed 3 times with cold ether (4 ℃) to remove excess HBTU in the oil, and the crude product was centrifuged and dried to give compound GP-11.

In one embodiment of the present invention, the compound GP-10 is prepared by: dissolving a compound GP-9 in a mixed solution of DCM (dichloromethane) and TFA (trifluoroacetic acid) for reaction to obtain a compound GP-10;

the compound GP-9 has the structure shown below:

in one embodiment of the present invention, the compound GP-10 is prepared by: dissolving 333.9mg of compound GP-9 in a mixed solution of 4mL of DCM and 4mL of TFA, and stirring for 1h at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after re-dispersing the crude product by adding 20mL of cold diethyl ether (4 ℃ C.), the crude product was centrifuged and dried to obtain the compound GP-10.

In one embodiment of the present invention, the compound GP-9 is prepared by: dissolving a compound GP-8, Boc-glycine and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in ultra-dry THF (tetrahydrofuran) to obtain a mixed solution; adding DIPEA (N, N-diisopropylethylamine) into the mixed solution, and reacting under the protection of nitrogen to obtain a compound GP-9;

the compound GP-8 has the structure shown below:

in one embodiment of the present invention, the compound GP-9 is prepared by: 320.7mg of the compound GP-8(0.541mmol), 104.1mg of Boc-glycine (0.595mmol) and 235.8mg of HBTU (0.622mmol) are dissolved in 10mL of ultra dry THF to obtain a mixture; adding 235 mu LDIPEA (1.35mmol) into the mixed solution, adjusting the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified to give compound GP-9.

In one embodiment of the present invention, the compound GP-8 is prepared by: dissolving a compound GP-7 in a mixed solution of DCM (dichloromethane) and TFA (trifluoroacetic acid) for reaction to obtain a compound GP-8;

the compound GP-7 has the structure shown below:

in one embodiment of the present invention, the compound GP-8 is prepared by: 380.2mg of compound GP-7 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then the mixture is stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the crude product 3 times with cold ether to remove excess trifluoroacetic acid from the oil, the crude product was dried to give compound GP-8.

In one embodiment of the present invention, the compound GP-7 is prepared by: dissolving compounds GP-6, 4- [ (tert-butylcarbonylamino) methyl ] benzoic acid and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in anhydrous THF (tetrahydrofuran) to obtain a mixed solution; adding DIPEA (N, N-diisopropylethylamine) into the mixed solution, and reacting under the protection of nitrogen to obtain a compound GP-7;

the compound GP-6 has the structure shown below:

in one embodiment of the present invention, the compound GP-7 is prepared by: 296.6mg of the compound GP-6(0.650mmol), 179.5mg of 4- [ (tert-butylcarbonylamino) methyl ] benzoic acid (0.715mmol) and 283.3mg of HBTU (0.748mmol) are dissolved in 10mL of anhydrous THF to obtain a mixture; adding 282 mu L DIPEA (1.625mmol) into the mixed solution to adjust the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified to give compound GP-7.

In one embodiment of the present invention, the compound GP-6 is prepared by: dissolving a compound GP-5 in a mixed solution of DCM (dichloromethane) and TFA (trifluoroacetic acid) for reaction to obtain a compound GP-6;

the compound GP-5 has the structure shown below:

in one embodiment of the present invention, the compound GP-6 is prepared by: 367.1mg of compound GP-5 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then the mixture is stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the crude product 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid from the oil, the crude product was centrifuged to give compound GP-6.

In one embodiment of the present invention, the compound GP-5 is prepared by: dissolving compounds GP-4, 4- [ (tert-butylcarbonylamino) methyl ] benzoic acid and HBTU in anhydrous THF (tetrahydrofuran) to obtain a mixed solution; adding DIPEA (N, N-diisopropylethylamine) into the mixed solution, and reacting under the protection of nitrogen to obtain a compound GP-5;

the compound GP-4 has the structure shown below:

in one embodiment of the present invention, the compound GP-5 is prepared by: 235.9mg of the compound GP-4(0.768mmol), 212.3mg of 4- [ (tert-butylcarbonylamino) methyl ] benzoic acid (0.845mmol) and 334.7mg of HBTU (0.883mmol) are dissolved in 10mL of anhydrous THF to obtain a mixed solution; adding 317 mu L DIPEA (1.92mmol) into the mixed solution to adjust the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified to give compound GP-5.

In one embodiment of the present invention, the compound GP-4 is prepared by: dissolving a compound GP-3 in a mixed solution of DCM (dichloromethane) and TFA (trifluoroacetic acid) for reaction to obtain a compound GP-4;

the compound GP-3 has the structure shown below:

in one embodiment of the present invention, the compound GP-4 is prepared by: 309.6mg of compound GP-3 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then the mixture is stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was washed 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid from the oil, and then centrifuged and dried to obtain compound GP-4.

In one embodiment of the present invention, the compound GP-3 is prepared by: dissolving a compound GP-2, tert-butyloxycarbonyl-D-propargyl glycine and HBTU (benzotriazole-tetramethyluronium hexafluorophosphate) in super-dry THF (tetrahydrofuran) to obtain a mixed solution; adding DIPEA (N, N-diisopropylethylamine) into the mixed solution, and reacting under the protection of nitrogen to obtain a compound GP-3;

the compound GP-2 has the structure shown below:

in one embodiment of the present invention, the compound GP-3 is prepared by: 195.3mg of the compound GP-2(0.842mmol), 200mg of tert-butoxycarbonyl-D-propargylglycine (0.939mmol) and 369mg of HBTU (0.974mmol) are dissolved in 10mL of ultra dry THF to obtain a mixture; adding 368 mu L of DIPEA (2.106mmol) into the mixed solution, adjusting the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified to give compound GP-3.

In one embodiment of the present invention, the compound GP-2 is prepared by: dissolving a compound GP-1 in a mixed solution of DCM (dichloromethane) and TFA (trifluoroacetic acid) for reaction to obtain a compound GP-2;

the compound GP-1 has the structure shown below:

in one embodiment of the present invention, the compound GP-2 is prepared by: 281mg of compound GP-1 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain an oily substance; the oil was washed 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid from the oil, and the crude product was centrifuged and dried to give compound GP-2.

In one embodiment of the present invention, the compound GP-1 is prepared by: dissolving Boc-glycine in anhydrous THF (tetrahydrofuran) to obtain a mixed solution; adding IBCF (isobutyl chloroformate) and NMM (N-methylmorpholine) into the mixed solution, and reacting under the protection of nitrogen to obtain a reaction solution; dissolving CBT (2-cyano-6-aminobenzothiazole) in anhydrous THF to obtain a solution; the reaction solution and the dissolution solution are mixed and then reacted to obtain the compound GP-1.

In one embodiment of the present invention, the compound GP-1 is prepared by: 315mg of Boc-glycine (1.8mmol) was dissolved in 10mL of anhydrous THF to obtain a mixed solution; 195. mu.L of IBCF were sequentially added to the mixture(1.5mmol) and 330. mu.L NMM (3.0mmol), stirring for 2h in ice bath under the protection of nitrogen to obtain reaction solution A; 175mg of CBT (2-cyano-6-aminobenzothiazole) (1mmol) was dissolved in 5mL of anhydrous THF to obtain a solution; adding the dissolved solution into the reaction solution A by using an injector, and stirring for 30min in an ice bath under the protection of nitrogen to obtain a reaction solution B; reacting the reaction solution B for 16 hours at 25 ℃ in the absence of light to obtain reaction solution C; after quenching the reaction in reaction solution C with 2mL of hydrochloric acid (concentration: 1mM), THF in reaction solution C was removed using a rotary evaporator to obtain crude product A; after dissolving the crude product A in 20mL EtOA (ethyl acetate), saturated NaHCO was used₃The solution and NaCl solution (with the concentration of 36.6g/100mL) are respectively washed for 3 times to remove the solvent in the crude product, and a crude product B is obtained; and purifying the crude product B to obtain the compound GP-1.

The invention also provides the application of the intramolecular condensation scaffold in the preparation of molecular probes.

In one embodiment of the invention, the molecular probe is a glutamyl transpeptidase targeted molecular probe, a legumain targeted molecular probe or an aspartate proteolytic enzyme targeted molecular probe.

The invention also provides the application of the molecular probe or the intramolecular condensed skeleton in target object imaging.

In one embodiment of the invention, the target is glutamyl transpeptidase, legumain or apoptotic cells.

The invention also provides an imaging agent of the target object, and the imaging agent contains the molecular probe or the intramolecular condensation skeleton.

The technical scheme of the invention has the following advantages:

the invention provides a molecular probe, which is obtained by modifying a marker group and a targeting group on the basis of intramolecular condensation of a skeleton GP-13; the intramolecular condensation skeleton GP-13 used by the molecular probe enables the CBT and the intramolecular cysteine residue to generate click condensation reaction more quickly by changing the connecting group between the CBT and the Cys, thereby reducing the competitive combination of the molecular probe and the intracellular free cysteine and further ensuring that the condensation rate of the molecular probe is not limited by the concentration of the molecular probe; the molecular probe has extremely high application prospect in target object imaging, such as tumor imaging.

Drawings

FIG. 1: synthetic route of compound GP-1.

FIG. 2: synthetic route for compound GP-2.

FIG. 3: synthetic route of compound GP-3.

FIG. 4: synthetic route of compound GP-4.

FIG. 5: synthetic route of compound GP-5.

FIG. 6: synthetic route of compound GP-6.

FIG. 7: synthetic route of compound GP-7.

FIG. 8: synthetic route of compound GP-8.

FIG. 9: synthetic route to compound GP-9.

FIG. 10: synthetic route of compound GP-10.

FIG. 11: synthetic route of compound GP-11.

FIG. 12: synthetic route to compound GP-12.

FIG. 13: a synthetic route of an intramolecular condensation framework GP-13.

FIG. 14: ESI-MS analysis of Compound GP-1.

FIG. 15: HPLC chromatogram of Compound GP-1.

FIG. 16: ESI-MS analysis of Compound GP-2.

FIG. 17: HPLC profile of compound GP-2.

FIG. 18: ESI-MS analysis of Compound GP-3.

FIG. 19: HPLC profile of compound GP-3.

FIG. 20: ESI-MS analysis of Compound GP-4 resulted.

FIG. 21: HPLC profile of compound GP-4.

FIG. 22: ESI-MS analysis of Compound GP-5 resulted.

FIG. 23: HPLC profile of compound GP-5.

FIG. 24: ESI-MS analysis of Compound GP-6 resulted.

FIG. 25: HPLC profile of compound GP-6.

FIG. 26: ESI-MS analysis of Compound GP-7 resulted.

FIG. 27 is a schematic view showing: HPLC profile of compound GP-7.

FIG. 28: ESI-MS analysis of Compound GP-8 resulted.

FIG. 29: HPLC profile of compound GP-8.

FIG. 30: ESI-MS analysis of Compound GP-9.

FIG. 31: HPLC profile of compound GP-9.

FIG. 32: ESI-MS analysis of Compound GP-10.

FIG. 33: HPLC profile of compound GP-10.

FIG. 34: ESI-MS analysis of Compound GP-11 resulted.

FIG. 35: HPLC profile of compound GP-11.

FIG. 36: ESI-MS analysis of Compound GP-12.

FIG. 37: HPLC profile of compound GP-12.

FIG. 38: ESI-MS analysis result of intramolecular condensation skeleton GP-13.

FIG. 39: HPLC chromatogram of intramolecular condensation backbone GP-13.

FIG. 40: intramolecular condensation backbone GP-13 is condensed intramolecular to GP-C under reducing conditions at pH 7.4.

FIG. 41: HPLC profile of intramolecular condensation backbone GP-13 after incubation at 37 ℃ for different times under reducing conditions at pH 7.4.

FIG. 42: and (3) a mass spectrum of the intra-molecular condensation cyclization product GD-C.

FIG. 43: a product possibly generated by incubating an intramolecular condensation skeleton GP-13 and free Cys and a synthetic route thereof.

FIG. 44: HPLC spectrogram of intramolecular condensation skeleton GP-13 after incubation with free Cys of different concentrations for different times.

FIG. 45: a synthetic route to the labelled precursor GD-16 of a glutamyl transpeptidase targeted molecular probe.

FIG. 46: ESI-MS analysis of Compound GD-14.

FIG. 47: HPLC profile of compound GD-14.

FIG. 48: ESI-MS analysis of Compound GD-15.

FIG. 49: HPLC profile of compound GD-15.

FIG. 50: ESI-MS analysis of GD-16, a labeled precursor of a glutamyl transpeptidase-targeted molecular probe.

FIG. 51: HPLC profile of the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe.

FIG. 52: nuclear magnetic resonance hydrogen spectra of the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe.

FIG. 53: nuclear magnetic resonance carbon spectrum of labeled precursor GD-16 of glutamyl transpeptidase targeted molecular probe.

FIG. 54: molecular probe targeted by glutamyl transpeptidase¹⁸F]A synthetic route to GD-16.

FIG. 55: molecular probe targeted by glutamyl transpeptidase¹⁸F]radio-HPLC profiles before and after GD-16 purification.

FIG. 56: molecular probe targeted by glutamyl transpeptidase¹⁸F]radio-HPLC profiles of GD-16 after various incubation times in PBS buffer.

FIG. 57: molecular probe targeted by glutamyl transpeptidase¹⁸F]radio-HPLC profile of GD-16 after 60min of blood circulation in mice.

FIG. 58: cell viability after 12H incubation of the labelled precursor GD-16 of the glutamyl transpeptidase-targeted molecular probe with U87 cells and NCI-H1299 cells, respectively.

FIG. 59: cell viability of the labelled precursor GD-16 of the glutamyltranspeptidase-targeted molecular probe after 24H incubation with U87 cells and NCI-H1299 cells, respectively.

FIG. 60: molecular probe targeted by glutamyl transpeptidase¹⁸F]Cellular uptake of GD-16 in U87 cells and U87 cells pretreated with GGsTop.

FIG. 61: u87 tumor-bearing mice injected with glutamine via tail veinAcyltranspeptidase-targeted molecular probe¹⁸F]PET visualization after GD-16.

FIG. 62: molecular probe for targeting glutamyl transpeptidase taken by tumor and muscle¹⁸F]Quantitative analysis result of GD-16.

FIG. 63: molecular probe for targeting glutamyl transpeptidase taken by tumor and muscle¹⁸F]Ratio of the intake values of GD-16.

FIG. 64: mark precursor GP-AAN-AMBF of legumain protease targeted molecular probe₃The synthetic route of (1).

FIG. 65: ESI-MS analysis of the GP-A compound.

FIG. 66: HPLC profile of compound GP-A.

FIG. 67: ESI-MS analysis of the GP-AAN compound.

FIG. 68: HPLC profile of compound GP-AAN.

FIG. 69: mark precursor GP-AAN-AMBF of legumain protease targeted molecular probe₃ESI-MS analysis of the results.

FIG. 70: mark precursor GP-AAN-AMBF of legumain protease targeted molecular probe₃HPLC profile of (a).

FIG. 71: legumain targeted molecular probe¹⁸F]GP-AAN-AMBF₃The synthetic route of (1).

FIG. 72: legumain targeted molecular probe¹⁸F]GP-AAN-AMBF₃The HCT116 cells and PC3 cells of (a).

FIG. 73: HCT116 tumor-bearing mouse injected with legumain-targeted molecular probe [ alpha ], [ beta ] -peptide¹⁸F]GP-AAN-AMBF₃The latter PET images.

FIG. 74: a synthetic route of the labeled precursor GP-16 of the aspartate protease targeted molecular probe.

FIG. 75: ESI-MS analysis of Compound GP-14.

FIG. 76: HPLC profile of compound GP-14.

FIG. 77: ESI-MS analysis of Compound GP-15.

FIG. 78: ESI-MS analysis of the labelled precursor GP-16 of the aspartate proteolytic enzyme targeted molecular probe.

FIG. 79: HPLC profile of the labeled precursor GP-16 of the aspartate proteolytic enzyme targeted molecular probe.

FIG. 80: molecular probe [ 2 ] targeted by aspartic acid protease¹⁸F]Synthetic route of GP-16.

FIG. 81: molecular probe [ 2 ] targeted by aspartic acid protease¹⁸F]HPLC chromatogram of GP-16 purified.

FIG. 82: the HeLa tumor-bearing mouse is injected with an aspartic acid protease targeted molecular probe through the tail vein¹⁸F]PET visualization post GP-16. In fig. 82, the dashed line is the HeLa tumor location.

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: molecular internal condensation skeleton

This example provides an intramolecular condensation backbone GP-13, the intramolecular condensation backbone GP-13 having the structure shown below:

example 2: method for preparing molecular internal condensation framework

This example provides a method for preparing an intramolecular condensation backbone GP-13 as described in example 1, comprising the steps of:

the method comprises the following steps: 315mg of Boc-glycine (1.8mmol) was dissolved in 10mL of anhydrous THF to obtain a mixed solution; adding 195 mu L of IBCF (1.5mmol) and 330 mu L of NMM (3.0mmol) in sequence into the mixed solution, and stirring for 2h in an ice bath under the protection of nitrogen to obtain a reaction solution A; dissolve 175mg of CBT (1mmol) in 5mL of anhydrous THF to obtain a solution; adding the dissolved solution into the reaction solution by using an injector, and stirring for 30min in an ice bath under the protection of nitrogen to obtain a reaction solution B; reacting the reaction solution B for 16 hours at 25 ℃ in the absence of light to obtain reaction solution C; after quenching the reaction in reaction solution C with 2mL of hydrochloric acid (concentration: 1mM), THF in reaction solution C was removed using a rotary evaporator to obtain crude product A; after dissolving the crude product A in 20mL EtOA (ethyl acetate), saturated NaHCO was used₃The solution and NaCl solution (with the concentration of 36.6g/100mL) are respectively washed for 3 times to remove the solvent in the crude product, and a crude product B is obtained; the crude product B was purified by chromatography on silica gel (Hex: EtOA ═ 1:1, v/v) to give compound GP-1(281mg, 84.6% yield) (synthetic route for compound GP-1 is shown in fig. 1).

Step two: 281mg of compound GP-1 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain an oily substance; after washing the oil 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid from the oil, the crude product was centrifuged and dried to give compound GP-2(195.3mg, 99.5% yield) (see FIG. 2 for a synthetic route for compound GP-2).

Step three: 195.3mg of the compound GP-2(0.842mmol), 200mg of tert-butoxycarbonyl-D-propargylglycine (0.939mmol) and 369mg of HBTU (0.974mmol) are dissolved in 10mL of ultra dry THF to obtain a mixture; adding 368 mu L of DIPEA (2.106mmol) into the mixed solution, adjusting the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified by chromatography on silica gel (Hex: EtOA ═ 1:1, v/v) to give compound GP-3(309.6mg, yield 86.1%) (the synthetic route for compound GP-3 is shown in fig. 3).

Step four: 309.6mg of compound GP-3 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then the mixture is stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the crude product 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid in the oil, the crude product was centrifuged and dried to give compound GP-4(235.9mg, 99.5% yield) (see FIG. 4 for a synthetic route of compound GP-4).

Step five: 235.9mg of the compound GP-4(0.768mmol), 212.3mg of 4- [ (tert-butylcarbonylamino) methyl ] benzoic acid (0.845mmol) and 334.7mg of HBTU (0.883mmol) are dissolved in 10mL of anhydrous THF to obtain a mixed solution; adding 317 mu L DIPEA (1.92mmol) into the mixed solution to adjust the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified by chromatography on silica gel (DCM: MeOH ═ 15:1, v/v) to give compound GP-5(367.1mg, yield 85.3%) (the synthetic route for compound GP-5 is shown in fig. 5).

Step six: 367.1mg of compound GP-5 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then the mixture is stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the crude product 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid in the oil, the crude product was centrifuged to give compound GP-6(296.6mg, 99.2% yield) (see FIG. 6 for a synthetic route for compound GP-6).

Step seven: 296.6mg of the compound GP-6(0.650mmol), 179.5mg of 4- [ (tert-butylcarbonylamino) methyl ] benzoic acid (0.715mmol) and 283.3mg of HBTU (0.748mmol) are dissolved in 10mL of anhydrous THF to obtain a mixture; adding 282 mu L DIPEA (1.625mmol) into the mixed solution to adjust the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified by chromatography on silica gel (DCM: MeOH ═ 12:1, v/v) to give compound GP-7(380.2mg, 84.4% yield) (synthetic route for compound GP-7 is shown in fig. 7).

Step eight: 380.2mg of compound GP-7 is dissolved in a mixed solution of 2mL of DCM and 2mL of TFA, and then the mixture is stirred for 30min at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the crude product 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid in the oil, the crude product was dried to give compound GP-8(320.7mg, 98.5% yield) (see FIG. 8 for a synthetic route for compound GP-8).

Step nine: 320.7mg of the compound GP-8(0.541mmol), 104.1mg of Boc-glycine (0.595mmol) and 235.8mg of HBTU (0.622mmol) are dissolved in 10mL of ultra dry THF to obtain a mixture; adding 235 mu L of DIPEA (1.35mmol) into the mixed solution, adjusting the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; the crude product was purified by chromatography on silica gel (DCM: MeOH ═ 7:1, v/v) to give compound GP-9(333.9mg, yield 82.3%) (the synthetic route for compound GP-9 is shown in fig. 9).

Step ten: dissolving 333.9mg of compound GP-9 in a mixed solution of 4mL of DCM and 4mL of TFA, and stirring for 1h at 25 ℃ to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after redispersion by addition of 20mL of cold diethyl ether (4 ℃ C.) to the crude product, the crude product was centrifuged and dried to give Compound GP-10(287.8mg, 99.5% yield) (see FIG. 10 for a synthetic route for Compound GP-10).

Step eleven: 287.8mg of the compound GP-10(0.443mmol), 225.6mg of N-t-butoxycarbonyl-S-trityl-L-cysteine (0.487mmol) and 210.5mg of HBTU (0.509mmol) are dissolved in 10mL of anhydrous THF to obtain a mixture; adding 188 mu L DIPEA (1.08mmol) into the mixed solution to adjust the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the oil 3 times with cold ether (4 ℃) to remove excess HBTU in the oil, the crude product was centrifuged and dried to give compound GP-11(429.6mg, 88.5% yield) (see FIG. 11 for a synthetic route for compound GP-11).

Step twelve: dissolving 429.6mg of compound GP-11 in a mixed solution of 4mL of DCM and 160 mu L of Tips to obtain a dissolved solution; adding 4mL of TFA into the dissolved solution, and stirring at 25 ℃ for 1h to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain an oily substance; washing the oil 3 times with cold diethyl ether (4 ℃) removed excess HBTU from the oil to give compound GP-12 (see FIG. 12 for a synthetic route to compound GP-12).

Step thirteen: 295mg of compound GP-12 is dissolved in 15mL of analytical grade methanol to obtain a mixed solution; after adding 300. mu.L of Tips and 70. mu.L of SEt (0.47mmol) to the mixture, the mixture was stirred at 25 ℃ for 2 hours to obtain a reaction solution; removing analytical grade methanol in the reaction solution by using a rotary evaporator to obtain a yellow crude product; after washing the yellow crude product 3 times with cold ether (4 ℃) to remove excess SEt from the yellow crude product, the yellow crude product was purified using semi-preparative HPLC to give a purified product, which was freeze-dried to give the intramolecular condensation backbone GP-13(268.2mg, 87.6% yield) (the synthetic route for the intramolecular condensation backbone GP-13 is shown in FIG. 13);

wherein, the purification conditions of semi-preparative HPLC are shown in Table 1;

the procedure for semi-preparative HPLC purification was: the mobile phase cascade of table 1 was selected and the sample dissolved in DMF was purified by C18 reverse phase chromatography column.

ESI-MS analysis of the intramolecular condensation backbone GP-13 was performed using an electrospray ionization source, and the results are shown in FIG. 38.

HPLC detection is carried out on the intramolecular condensation framework GP-13 by using Waters1525, and the detection result is shown in figure 39.

TABLE 1 purification conditions of semi-preparative HPLC

Experimental example 1: intramolecular condensation cyclization experiment of intramolecular condensation skeleton

This experimental example provides an intramolecular condensation cyclization experiment of intramolecular condensation backbone GP-13 described in example 1, the specific procedure is as follows:

dissolving an intramolecular condensation skeleton GP-13 in DMF to prepare a GP-13 solution with the concentration of 25 mM; mu.L GP-13 solution was added to 158. mu.L DMF/H₂Mixed solution of O (DMF: H)₂O ═ 1:1, v/v), to give a mixed solution; adding into the mixed solutionAfter 10. mu.L of HCl solution (concentration: 1mM) was adjusted to

pH

3, 20. mu.L of TCEP (concentration: 50mM) was added to the mixture to obtain a reaction system; 10 μ L of saturated NaHCO was added to the reaction system₃After the pH value of the reaction system is 7.4 by the solution, incubating the reaction system at 37 ℃ to obtain a reaction solution; respectively taking out 25 mu L of reaction liquid after incubation for 1, 3 and 5min, and adding 10 mu L of HCl solution (with the concentration of 1mM) into the reaction liquid to quench the reaction to obtain a solution to be detected; the liquid to be tested was detected using high performance liquid chromatography, and the rate of condensation cyclization of intramolecular condensation backbone GP-13 was obtained by analyzing HPLC data. All of the above processes operate at zero degrees celsius. The detection results are shown in FIGS. 40 to 42.

As shown in fig. 40 to 42, after the intramolecular condensation backbone GP-13 was incubated in a reducing environment at pH 7.4 for 1min, it was found that the intramolecular condensation backbone GP-13 had been completely converted into a product with a half-retention time of 15.03min, which was very close to the liquid-phase retention time (15.07min) of the reduction product GP-12, but after the product was collected and subjected to mass spectrometry, it was found that the product was an intramolecular condensation cyclization product GP-C, and no reduction product GP-12 was found, indicating that GP-13 was highly susceptible to click condensation reaction in a reducing environment at pH 7.4 to generate a cyclization product.

Experimental example 2: experiment of competitive reaction between intramolecular condensation skeleton and L-Cys

This experimental example provides a competition reaction experiment of the intramolecular condensation backbone GP-13 and L-Cys described in example 1, the specific procedure is as follows:

dissolving an intramolecular condensation skeleton GP-13 in DMF to prepare a GP-13 solution with the concentration of 25 mM; diluting GP-13 solution with concentration of 25mM with DMF to obtain GP-13 solution with concentration of 0.5 mM; four 1.5mL reaction tubes were first loaded with 35. mu. L H₂O and 10. mu.L HCl (1mM in concentration) to adjust pH to 3, then 10. mu.L of TCEP (50 mM in concentration) and 20. mu.L of GP-13 solution (0.5 mM in concentration) were added to the four reaction tubes, and then 1. mu.L of an aqueous solution of Cys (10 mM in concentration), 10. mu.L of an aqueous solution of Cys (100 mM in concentration) or 10. mu.L of an aqueous solution of Cys (1000 mM in concentration) were added to the four reaction tubes, respectively, so that free Cys and the intramolecular condensation scaffold G in the four reaction tubesThe concentration ratio of P-13 is 1:1, 10:1, 100:1 and 1000:1 respectively, and finally 15 mul of saturated NaHCO is added into four reaction tubes₃Adjusting the pH value of the solution to 7.4 to activate click reaction; incubating the four reaction tubes at 37 ℃ for 10min, and dropwise adding 10 mu L of HCl solution (with the concentration of 1mM) into the four reaction tubes to quench reaction to obtain four solutions to be detected; and detecting the liquid to be detected by using high performance liquid chromatography, and analyzing HPLC data to obtain the generation condition of a competition product between the molecular internal condensation skeleton GP-13 and the L-Cys in the liquid to be detected. The detection results are shown in FIGS. 43 to 44.

As shown in FIGS. 43 to 44, when free cysteine was present in the reaction environment at the same concentration as that of the intramolecular condensation backbone GP-13, only the condensation product GD-C was detected, and no other products were found. It was subsequently continued to explore whether high concentrations of L-Cys would affect the intramolecular condensation cyclization of the intramolecular condensation backbone GP-13. When the concentration of free cysteine was 200 times that of the intramolecular condensation backbone GP-13, no other products were detected. Continuing to increase the concentration of free cysteine, a new product with a retention time of 13.4min was observed when the concentration of free cysteine was 1000 times that of the intramolecular condensation backbone GP-13, which was 5% of the total product, presumably GD-Cys. Although the intramolecular condensation skeleton GP-13 still generates a small amount of competitive products by condensation with cysteine ammonia in an environment with high concentration of free cysteine, the reactivity of the intramolecular condensation skeleton GP-13 with free cysteine is greatly reduced compared with intermolecular condensation.

Example 3: labeling precursor of glutamyl transpeptidase targeted molecular probe

This example provides a labeled precursor GD-16 of a glutamyl transpeptidase targeted molecular probe, which labeled precursor GD-16 has the following structure:

example 4: method for preparing glutamyl transpeptidase targeted molecular probe

This example provides a method for preparing the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe described in example 3, comprising the steps of:

the method comprises the following steps: 35mg of the intramolecular condensation skeleton GP-13(0.043mmol) prepared in example 2, 14mg of N-BOC-L-glutamic acid-1-tert-butyl ester (0.047mmol) and 19mg of HBTU (0.054mmol) were dissolved in 10mL of extra dry THF to obtain a solution; adding 235 mu L of DIPEA (1.35mmol) into the dissolved solution, adjusting the pH value to 8, and stirring for 3h at 25 ℃ under the protection of nitrogen to obtain a reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; washing the crude product 3 times with cold ether (4 ℃) removed excess HBTU from the crude product to give compound GD-14(39.8mg, 84.2% yield);

step two: dissolving 39.8mg of compound GD-14 in a mixed solution of 2mL of DCM and 2mL of TFA, and stirring at 25 ℃ for 1h to obtain a reaction solution; removing the organic solvent in the reaction solution by using a rotary evaporator to obtain an oily substance; washing the oil 3 times with cold ether (4 ℃) removed excess trifluoroacetic acid from the oil to give compound GD-15(33.7mg, 98.6% yield);

step three: 33.7mg of Compound GD-15(0.0357mmol) were dissolved in 3mL of DMF/H₂O(DMF:H₂O is 2:1) to obtain a mixed solution; 199. mu.L of AMBF was added to the mixture₃(0.102mmol), 295. mu.L ligand (0.0029mmol) and 13mg Cu (I) (0.035mmol) were stirred at 45 ℃ for 1h under nitrogen protection to obtain a reaction solution; purifying the reaction solution by semi-preparative HPLC to obtain a purified product, and freeze-drying the purified product to obtain a labeled precursor GD-16(25.7mg, yield 63.2%) of the glutamyl transpeptidase targeted molecular probe (the synthetic route of the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe is shown in FIG. 45);

wherein, the purification conditions of the semi-preparative HPLC are shown in Table 2;

the procedure for semi-preparative HPLC purification was: the mobile phase cascade of table 2 was selected and the sample dissolved in DMF was purified by C18 reverse phase chromatography column.

ESI-MS analysis of compound GD-14 using electrospray mass spectrometry gave the results shown in FIG. 46.

The HPLC detection of the compound GD-14 was performed by using waters high performance liquid, and the detection result is shown in FIG. 47.

ESI-MS analysis of compound GD-15 by electrospray mass spectrometry gave the results shown in FIG. 48.

The HPLC detection of the compound GD-15 was performed by using waters high performance liquid, and the detection result is shown in FIG. 49.

ESI-MS analysis of glutamyl transpeptidase targeted molecular probe GD-16 was performed by electrospray mass spectrometry, and the analysis results are shown in FIG. 50.

HPLC detection is carried out on the molecular probe GD-16 targeted by the glutamyl transpeptidase by using waters high performance liquid, and the detection result is shown in figure 51.

And performing nuclear magnetic resonance on the molecular probe GD-16 targeted by the glutamyltranspeptidase by using a duke 400 nuclear magnetic resonance instrument, wherein the resonance result is shown in figures 52-53.

TABLE 2 purification conditions of semi-preparative HPLC

Example 5: glutamyl transpeptidase targeted molecular probe (radioactivity)

This example provides a molecular probe targeted by glutamyl transpeptidase¹⁸F]GD-16, the glutamyl transpeptidase targeted molecular probe [ 2 ]¹⁸F]GD-16 has the structure shown below:

example 6: method for preparing glutamyl transpeptidase targeted molecular probe

This example provides the molecular probe [ 2 ] targeted by the glutamyltranspeptidase described in example 5¹⁸F]A method of preparing GD-16, said method using a fluorine-18 isotope exchange process, comprising the steps of:

use of a catalyst consisting of 0.5mol/L NaHCO₃(10mL) Capture with pure Water (10mL) activated QMA columnFluorine-18 species (-9.25 GBq) produced by the accelerator. Then, fluorine-18 on the QMA column was eluted into the reaction tube using 300 μ L of pyridazine hydrochloride buffer (pH 2.5), and then 25 μ L of the labeled precursor GD-16(25mM) of the glutamyl transpeptidase-targeted molecular probe prepared in example 4 was added to the reaction tube, shaken and mixed well, and placed in an oil bath at 80 ℃ for reaction for 30 min. mu.L of the reaction solution was diluted with 1000. mu.L of acetonitrile and subjected to radio-HPLC analysis, and the radiolabel yield (RCY) was determined. The remaining reaction solution was then diluted with 20mL of purified water, and the molecular probe targeted by glutamyltranspeptidase in the dilution was captured using a C18 column previously activated with 10mL of ethanol and 10mL of purified water¹⁸F]GD-16. The crude product loaded in the C18 column was then washed three times with 10mL of pure water to remove unreacted fluorine-18 ions, and finally the product was eluted with 500. mu.L of ethanol into a vial to give a glutamyltranspeptidase-targeted molecular probe [ 2 ]¹⁸F]GD-16 (glutamyl transpeptidase targeted molecular probe [, ]¹⁸F]The synthetic route of GD-16 is shown in FIG. 54, a molecular probe for glutamyl transpeptidase targeting¹⁸F]The radio-HPLC spectra before and after GD-16 purification are shown in FIG. 55).

As can be seen from FIG. 55, the molecular probe [ 2 ] targeted by glutamyltranspeptidase¹⁸F]The GD-16 may be obtained as a glutamyltranspeptidase-targeted molecular probe by one-step isotopic exchange¹⁸F]GD-16, the whole labeling process can be completed within 60min, glutamyl transpeptidase targeted molecular probe [ 2 ]¹⁸F]The RCY of GD-16 can reach 13.32 +/-0.54%, the RCP is 94.61 +/-2.55%, and the molar activity is 9.35 +/-0.55 MBq/nmol.

Experimental example 3: in vitro stability test of glutamyl transpeptidase targeted molecular probes

The present experimental example provides the molecular probe [ 2 ] targeted by the glutamyl transpeptidase described in example 5¹⁸F]The in vitro stability experiment of GD-16 specifically processes as follows:

a molecular probe targeting 20. mu.L of glutamyl transpeptidase [ 2 ]¹⁸F]GD-16(0.74 MBq/. mu.L) was dissolved in 180. mu.L of PBS buffer to obtain a mixed solution; incubating the mixture at 37 ℃ for 0, 1, 2 or 4 h; after the incubation, the incubation solution was directly analyzed by radio-HPLC. The results of the analysis are shown in FIG. 56.

As can be seen from FIG. 56, the molecular probe [ 2 ] targeting glutamyltranspeptidase¹⁸F]The radiochemical purity of the probe did not change significantly after incubation of GD-16 with PBS buffer for 4h at 37 ℃.

Experimental example 4: in vivo stability assay of glutamyl transpeptidase-targeted molecular probes

The present experimental example provides the molecular probe [ 2 ] targeted by the glutamyl transpeptidase described in example 5¹⁸F]The in vivo stability experiment of GD-16, the concrete process is as follows:

a molecular probe targeting glutamyltranspeptidase of 11.1MBq¹⁸F]GD-16 was injected into normal white mice (normal white mice purchased from Calvens, Van. After 30min, 5 mu L of tail vein blood is taken, and equal volume of acetonitrile is added to separate out protein in the blood. Cells and proteins in the blood were sedimented using a high-speed centrifuge, and then 20. mu.L of the supernatant was subjected to radio-HPLC analysis. The analytical results are shown in FIG. 57.

As can be seen from FIG. 57, the molecular probe [ 2 ] targeting glutamyltranspeptidase¹⁸F]The GD-16 still keeps good stability after 1 hour of blood circulation in the mouse.

Experimental example 5: experiment of lipid-water distribution coefficient of glutamyl transpeptidase targeted molecular probe

The present experimental example provides the molecular probe [ 2 ] targeted by the glutamyl transpeptidase described in example 5¹⁸F]The specific process of the lipid-water distribution coefficient experiment of GD-16 is as follows:

a molecular probe targeting 20. mu.L of glutamyl transpeptidase [ 2 ]¹⁸F]GD-16(0.74 MBq/. mu.L) was dissolved in ethanol and placed in a 5mL centrifuge tube, to which 980. mu.L of n-octanol and 1mL of pure water were added. Shaking, mixing and centrifuging. Equal volumes of organic and aqueous phases were taken out and placed in two radioimmunoassay tubes, and then radioactivity in the two phases was measured separately using a gamma counter to detect the distribution of the probes. Then by the formula: log (C)_O/C_W) Molecular probe for calculating glutamyl transpeptidase targeting¹⁸F]GD-16 has a lipid-water partition coefficient (log P) that reflects its hydrophilicity and lipophilicity. Co and Cw each approximately represent a molecular probe for targeting glutamyltranspeptidase¹⁸F]The concentration of GD-16 in the aqueous and organic phases. The aqueous phase was recovered, and a certain amount of pure water was added to return the total volume to 1mL, followed by addition of 1mL of n-octanol. Shaking and mixing evenly, and centrifuging. The water phase and the organic phase with equal volume are put into two radioimmunoassay tubes, the radioactivity in the two phases is measured by a gamma counter, and the log P is calculated by a formula. The above process was repeated until the log P value stabilized.

Measuring the molecular probe of the glutamyl transpeptidase target¹⁸F]Log P of GD-16 is-0.93. + -. 0.09, indicating a molecular probe [ glutamyl transpeptidase ] targeting¹⁸F]GD-16 is hydrophilic.

Experimental example 6: assay for evaluating biocompatibility of labeled precursor of glutamyl transpeptidase-targeted molecular probe

This experimental example provides an experiment for evaluating the biocompatibility of the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe described in example 3, the cytotoxicity of the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe against U87 and NCI-H1299 cell lines was determined by 3- (4, 5-dimethyl-thiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT method) as follows:

firstly 8 is multiplied by 10³U87 cells or NCI-H1299 cells (U87 cells and NCI-H1299 cells from Shanghai cell Bank of Chinese academy) were seeded in 96-well plates (1 blank and 5 experimental groups each set to 3 drug wells) supplemented with 100. mu.L of DMEM high-sugar medium (from BI) and 1640 medium (from BI), respectively. At 37 ℃ with 5% (v/v) CO₂After 12h of incubation in the incubator until the cells were adherent, the original medium was removed, 200 μ L of precursor compound GD-16 containing varying concentrations (0, 12.5, 25, 50, 100 μmol) of the glutamyl transpeptidase targeted molecular probe (solvent is complete medium from BI) was added to each experimental well and incubation continued under the same conditions for 12 and 24 h. After the incubation was complete, 20. mu.L of MTT (5mg/mL) was added to each well and incubated at 37 ℃ for 4 h. After removal of supernatant from the wells, formazan was dissolved by addition of 150 μ L DMSO, and the absorbance at 490nm was recorded using a microplate reader according to the formula: percent cell viability ═ average absorbance value of additive group/blank group x 100%Cytotoxicity was evaluated. The evaluation results are shown in FIGS. 58 to 59.

From FIGS. 58 to 59, it can be seen that the U87 cells and the NCI-H1299 cells still have high cell viability after 24H incubation with the labeled precursor GD-16 (100. mu.M) of the glutamyl transpeptidase-targeted molecular probe (cell viability of U87 > 83.39. + -. 6.74%, cell viability of NCI-1299 > 78.67. + -. 3.31%). When 100 μ M of the labeled precursor GD-16 of the glutamyltranspeptidase-targeted molecular probe was incubated for 12H with both U87 cells and NCI-H1299 cells, no significant cytotoxicity was found (cell viability of U87 > 103.53. + -. 3.91%, cell viability of NCI-H1299 > 88.12. + -. 2.95%). These results indicate that the labeled precursor GD-16 of the glutamyl transpeptidase targeted molecular probe is also safe for cell experiments and animal imaging experiments.

Experimental example 7: cellular uptake assay of glutamyl transpeptidase-targeted molecular probes

The present experimental example provides the molecular probe [ 2 ] targeted by the glutamyl transpeptidase described in example 5¹⁸F]The cell uptake experiment of GD-16 specifically processes as follows:

will be 1 × 10⁶Each U87 cell was dispersed in 200. mu.L of DMEM high-glucose medium (purchased from BI Co.) and added to the radioimmunoassay. Molecular probe for targeting glutamyl transpeptidase¹⁸F]GD-16 was diluted to a concentration of 370KBq/mL using DMEM high-sugar medium, and then 100. mu.L was added to each tube. To verify the targeting of glutamyl transpeptidase¹⁸F]GD-16 is targeted to GGT, and a group of blocking experiments are additionally set. Likewise, 1X 10 of the catalyst is added to each tube⁶U87 cells were then incubated with 0.33. mu.L of 100. mu.M GGT inhibitor (GGsTop) per tube at 37 ℃ for 30min, followed by addition of a glutamyl transpeptidase-targeting molecular probe dissolved in 100. mu.L of medium in an radioactive amount of 37KBq in total¹⁸F]GD-16 was added to treated U87 cells. Shaking, and incubating at 37 deg.C for 15min, 30min, 60min, 120min and 240 min. Each time the set incubation time is over, PBS is added immediately to wash the cells to wash away the free glutamyl transpeptidase targeted molecular probe [ 2 ]¹⁸F]GD-16, the amount of probe taken up by the cells was measured by a gamma counter, and the amount of probe present in the U87 cells was calculated as the resultExpressed as a percentage of the total input dose prior to incubation. The results of the experiment are shown in FIG. 60.

As can be seen from FIG. 60, the molecular probe [ 2 ] targeted by glutamyltranspeptidase¹⁸F]GD-16 was rapidly taken up by U87 cells highly expressing GGT, and its uptake value reached 3.21. + -. 0.15% rapidly after 15min incubation. Followed by extension of the incubation time, a molecular probe targeted by glutamyltranspeptidase¹⁸F]The uptake of GD-16 in U87 cells decreased slightly, reaching a minimum uptake value of 2.79. + -. 0.13% at 2h, followed by an increase to 2.94. + -. 0.08% of 4 h. The molecular probe [ 2 ] targeted by glutamyltranspeptidase when U87 cells were previously treated with GGT inhibitor GGsTop for 30min¹⁸F]The uptake of GD-16 in U87 cells was significantly reduced, as is clear from FIG. 60, and after incubation for 15min, the molecular probe [ U87 ] targeted to glutamyl transpeptidase¹⁸F]The GD-16 uptake was reduced to 1.62. + -. 0.06%. Although the molecular probe [ 2 ] targeted by glutamyltranspeptidase is continued with the lapse of incubation time¹⁸F]The uptake of GD-16 in U87 cells eventually reached a plateau (1.77 ± 0.07%), but the uptake value was still significantly lower than in U87 cells that were not treated with GGsTop. These results indicate that the molecular probe [ 2 ] targeted by glutamyl transpeptidase¹⁸F]GD-16 may be tested in vitro cell experiments for the ability to detect GGT expression levels.

Experimental example 8: mouse PET imaging experiment of glutamyl transpeptidase targeted molecular probe

The present experimental example provides the molecular probe [ 2 ] targeted by the glutamyl transpeptidase described in example 5¹⁸F]The mouse PET imaging experiment of GD-16 specifically processes as follows:

u87 tumor-bearing mice (purchased from Calvens laboratory animals, Van. Changzhou) with good viability and appropriate tumor size were anesthetized with oxygen containing 2 vt% isoflurane at a flow rate of 1.5L/min. After the four limbs and the tail of the mouse are fixed, the molecular probe [ 2 ] targeting glutamyl transpeptidase dissolved in about 5.55MBq of physiological saline of 150. mu.L¹⁸F]GD-16 was injected via the tail vein. Control group NCI-H1299 tumor-bearing mice were administered in the same manner and at the same dose. A 60min dynamic PET scan was performed immediately after the probe injection. Tissue drug uptake in the region of interest (ROI) was quantitatively analyzed by ASIProVM software. Swelling and swelling treating medicineTumor to muscle quantification results are expressed as the ratio of tumor uptake per mL muscle to total dose (% ID/mL). The experimental results are shown in FIGS. 61-63.

As shown in FIGS. 61 to 63, the probe 2¹⁸F]After GD-16 is injected for 10min, a bright PET signal can be found at the tumor part, and the probe [ 2 ] can be detected along with the extension of the injection time¹⁸F]The GD-16 can still clearly image the tumor, and the injection probe¹⁸F]After GD-1660min, a relatively bright PET signal was still observed at the tumor site. Quantitative analysis is carried out on PET imaging, and the injection probe in the tail vein can be visually seen¹⁸F]After GD-165min, the probe¹⁸F]GD-16 had a higher uptake at the tumor site (uptake value of 4.12. + -. 0.53% ID/mL). When the probe is used for injection¹⁸F]After GD-1615min, tumor uptake reached a maximum (maximum of 8.77. + -. 0.59% ID/mL) and then tumor uptake slowly declined to 2.84. + -. 0.27% ID/mL 60min after injection. Further, the probe 2¹⁸F]The uptake of GD-16 in muscle is much lower than that of tumors. The ratio of tumor to muscle uptake values during the last 55min of the dynamic PET scan was between 3.64 ± 0.31 and 4.18 ± 0.37, with a maximum value being reached 15min after injection. Probe [ 2 ]¹⁸F]GD-16 clearly visualises GGT expression levels in tumor-bearing mice and also provides good tumour imaging at the end of imaging. This is probably because of the probe [18F ]]The condensation cyclization process of GD-16 after being hydrolyzed by GGT in the tumor and entering into the cell is less influenced by endogenous cysteine, so that more probes form a structure with stronger hydrophobicity and rigidity and larger size, the retention time of the probes in the cell and even the tumor is prolonged, and the uptake of the probes by the tumor is further increased. All the data above indicate probe [18F ]]GD-16 has good visualization ability to the GGT expression level in tumor-bearing mice, and can monitor the GGT expression level in living mice more sensitively.

Example 7: labeling precursor of legumain targeted molecular probe

This example provides a labeled precursor GP-AAN-AMBF of legumain-targeted molecular probes₃The labeled precursor GP-AAN-A of the legumain targeted molecular probeMBF₃Has the following structure:

example 8: method for preparing legumain targeted molecular probe

This example provides the labeled precursor GP-AAN-AMBF of the legumain-targeted molecular probe described in example 7₃The method comprises the following steps:

the method comprises the following steps: dissolving 22mg of the intramolecular condensation skeleton GP-13 prepared in example 2 in 6mL of ultra dry THF to obtain a solution; adding 17mg of Ac-AAN-OH, 12mg of HBTU and 20 mu of LDIPEA into the dissolved solution, and stirring for 3 hours at 25 ℃ under the protection of nitrogen to obtain a reaction solution; the solvent in the reaction mixture was removed by evaporation under reduced pressure to give compound GP-A (25 mg);

step two: to remove the protected group Trt, 2mL of TFA and 100. mu.L of Tips were added to ｃA mixed solution of 25mg of compound GP-A and 2mL of DCM, and stirred at 25 ℃ for 0.5h to obtain ｃA reaction solution; removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; after washing the crude product 3 times with cold ether (4 ℃) to remove excess trifluoroacetic acid from the crude product, the crude product was purified by centrifugation to give compound GP-AAN (18 mg);

step three: 18mg of the compound GP-AAN are dissolved in 2mL DMF and 1mL H₂Obtaining a mixed solution in the step O; adding 212 μ L AMBF to the mixture₃275. mu.L of ligand and 13mg of tetrakis (acetonitrile) copper (I) hexafluorophosphate were stirred at 45 ℃ for 1 hour under the protection of nitrogen to obtain a reaction solution; purifying the reaction solution by high performance liquid chromatography to obtain a labeled precursor GP-AAN-AMBF of the legumain protease targeted molecular probe₃(8mg, yield 63.2%) (labeled precursor GP-AAN-AMBF of legumain-targeted molecular probes₃See fig. 64).

ESI-MS analysis of compound GP-A by electrospray mass spectrometry was performed, and the results are shown in FIG. 65.

The HPLC detection of the compound GP-A is shown in FIG. 66.

ESI-MS analysis of compound GP-AAN by electrospray mass spectrometry gave the results shown in FIG. 67.

HPLC detection of the GP-AAN compound is performed using waters HPLC, and the detection results are shown in FIG. 68.

Labeled precursor GP-AAN-AMBF of legumain protease targeted molecular probe by electrospray mass spectrometry₃ESI-MS analysis was performed, and the analysis results are shown in FIG. 69.

Marking precursor GP-AAN-AMBF of legumain protease targeted molecular probe by using waters high performance liquid₃HPLC detection was carried out, and the detection results are shown in FIG. 70.

Example 9: pepsin targeted molecular probe (radioactivity)

This example provides a legumain-targeted molecular probe [ 2 ]¹⁸F]GP-AAN-AMBF₃The legumain-targeted molecular probe [ 2 ]¹⁸F]GP-AAN-AMBF₃Has the following structure:

example 10: method for preparing legumain targeted molecular probe

This example provides the legumain-targeted molecular probe of example 9¹⁸F]A method for preparing GP-AAN-AMBF3, said method using a fluorine-18 isotope exchange process, comprising the steps of:

referring to example 6, the labeled precursor GP-AAN-AMBF of the legumain protease targeted molecular probe prepared in example 8 was prepared by using a fluorine-18 isotope exchange method₃Molecular probe substituted into legumain targeting¹⁸F]GP-AAN-AMBF₃(legumain-targeting molecular Probe [ 2 ]¹⁸F]GP-AAN-AMBF₃See fig. 71).

Experimental example 9: cellular uptake assay of legumain-targeted molecular probes

This experimental example provides the pods described in example 9Protease-targeted molecular probe [ 2 ]¹⁸F]GP-AAN-AMBF₃The specific process of the cell uptake experiment of (1) is as follows:

will be 1 × 10⁶Each of HCT116 cells and PC3 cells was dispersed in 200. mu.L of DMEM high-glucose medium (purchased from BI Co.) and then added to the disposable tube. Molecular probe for targeting glutamyl transpeptidase¹⁸F]GD-16 was diluted to a concentration of 370KBq/mL using DMEM high-sugar medium, and then 100. mu.L was added to each tube. Shaking, and incubating at 37 deg.C for 15min, 30min, 60min, 120min and 240 min. Each time the set incubation time is over, PBS is added immediately to wash the cells and wash the free legumain-targeted molecular probe [ 2 ]¹⁸F]GP-AAN-AMBF₃The amount of probe taken up by the cells was measured using a gamma counter and expressed as the amount of probe present in HCT116 cells and PC3 cells as a percentage of the total input dose prior to incubation. The results of the experiment are shown in FIG. 72.

As can be seen in FIG. 72, the molecular probe for legumain targeting¹⁸F]GP-AAN-AMBF₃Can be rapidly taken up by HCT116 cells with high legumain expression, and the taking value of the HCT116 cells rapidly reaches 2.83 +/-0.19 percent after 15min of incubation. Followed by extension of the incubation time, a legumain-targeted molecular probe [ 2 ]¹⁸F]GP-AAN-AMBF₃Uptake in HCT116 cells remained in a stable state. A Legumain-targeting molecular probe for PC3 cells with low Legumain expression¹⁸F]GP-AAN-AMBF₃The intake balance is reached at 15min, and the intake value is about 1.853 +/-0.17 percent and is far lower than that of HCT116 cells with high legumain expression. These results indicate that the legumain-targeted molecular probe [ 2 ]¹⁸F]GP-AAN-AMBF₃Has the capability of sensitively detecting the Legumain expression level in vitro.

Experimental example 10: pepsin-targeted mouse PET imaging experiment with molecular probe

The present Experimental example provides the legumain-targeted molecular probe described in example 9¹⁸F]The mouse PET imaging experiment of GP-AAN-AMBF3 comprises the following specific processes:

well-viable HCT116 tumor-bearing mice of appropriate tumor size (purchased from Calvens laboratory animals, Changzhou)) Anesthesia was performed with oxygen containing 2 vt% isoflurane at a flow rate of 1.5L/min. After the four limbs and the tail of the mouse are fixed, the molecular probe [ 2 ] targeting glutamyl transpeptidase dissolved in about 5.55MBq of physiological saline of 150. mu.L¹⁸F]GP-AAN-AMBF₃By tail vein injection. A 60min dynamic PET scan was performed immediately after the probe injection. Tissue drug uptake in the region of interest (ROI) was quantitatively analyzed by ASIProVM software. Tumor to muscle quantification results are expressed as the ratio of tumor uptake per mL muscle to total dose (% ID/mL). The results of the experiment are shown in FIG. 73.

As can be seen from FIG. 73, the probe 2¹⁸F]GP-AAN-AMBF₃The tumor uptake value is 3.2 +/-0.13% ID/mL in a Legumain high-expression HCT116 model at 15min, and the tumor uptake value is lower in muscles and other organs. The PET imaging result indicates the probe¹⁸F]GP-AAN-AMBF₃Can rapidly, sensitively and specifically image Legumain expression level in vivo.

Example 11: labeling precursor of aspartate proteolytic enzyme targeted molecular probe

The present example provides a labeled precursor GP-16 of an aspartase-targeted molecular probe, wherein the labeled precursor GP-16 of the aspartase-targeted molecular probe has the following structure:

example 12: method for preparing aspartate proteolytic enzyme targeted molecular probe

This example provides a method for preparing the labeled precursor GP-16 of the aspartic proteolytic enzyme targeted molecular probe described in example 11, comprising the steps of:

the method comprises the following steps: 23mg of intramolecular condensation skeleton GP-13 prepared in example 2, 23mg of DEVD (0.033mmol) and 17mg of HBTU (0.045mmol) were dissolved in 4mL of DMF to obtain a solution; adding 13 mu L of DIPEA into the solution to adjust the pH value of the solution to 8, and stirring for 12h at 24 ℃ under the protection of nitrogen to obtain a reaction solution; carrying out rotary evaporation and vacuum drying on the reaction liquid to obtain a compound GP-14;

step two: adding 50mg of compound GP-14 into a mixed solution of 3mL of TFA and 3mL of DCM, and stirring at 25 ℃ for 0.5h to obtain a reaction solution; removing the solvent in the reaction solution by using a rotary evaporator to obtain a crude product; washing the crude product with DCM for 3 times, adding anhydrous ether, and collecting precipitate; drying the precipitate to obtain a dried substance; purifying the dried product by semi-preparative high performance liquid chromatography to obtain compound GP-15(40 mg);

step three: 42mg of Compound GP-15 are dissolved in 3mL DMF and 0.6mL H₂Obtaining a mixed solution in the step O; adding 88 μ L AMBF to the mixture₃6.51mg THPTA, 6mg LAASS and 3.8mg CUSO₄Then, the reaction is carried out for 45min at 45 ℃ under the protection of nitrogen, and a labeled precursor GP-16 of the molecular probe targeted by the aspartic acid proteolytic enzyme is obtained (the synthetic route of the labeled precursor GP-16 of the molecular probe targeted by the aspartic acid proteolytic enzyme is shown in figure 74).

ESI-MS analysis of compound GP-14 by electrospray mass spectrometry gave the results shown in FIG. 75.

The HPLC detection of the compound GP-14 is performed by waters HPLC, and the detection result is shown in FIG. 76.

ESI-MS analysis of GP-15 by electrospray mass spectrometry was performed and the results are shown in FIG. 77.

ESI-MS analysis of the GP-16 label precursor of the molecular probe targeted by aspartic acid proteolytic enzyme was performed by electrospray mass spectrometry, and the results are shown in FIG. 78.

HPLC detection is carried out on the labeled precursor GP-16 of the molecular probe targeted by the aspartic acid proteolytic enzyme by using waters high performance liquid, and the detection result is shown in figure 79.

Example 13: aspartic acid proteolytic enzyme targeted molecular probe (radioactivity)

This example provides an aspartic acid proteolytic enzyme-targeted molecular probe¹⁸F]GP-16, the molecular probe targeted by the aspartate protease¹⁸F]GP-16 has the structure shown below:

example 14: method for preparing aspartate proteolytic enzyme targeted molecular probe

This example provides the aspartic protease targeted molecular probe of example 13¹⁸F]A method for the preparation of GP-16, said method making use of a fluorine-18 isotope exchange process, comprising the steps of:

referring to example 6, the labeled precursor of the aspartic acid protease targeted molecular probe prepared in example 12 was prepared by the fluorine-18 isotope exchange method¹⁸F]Molecular probe with GP-16 replaced by aspartic acid proteolytic enzyme as target¹⁸F]GP-16 (aspartic acid proteolytic enzyme targeted molecular probe [, ]¹⁸F]The synthetic route of GP-16 is shown in FIG. 80, and a molecular probe targeting an aspartic acid protease¹⁸F]The HPLC chromatogram after GP-16 purification is shown in FIG. 81).

As can be seen in FIG. 81, the molecular probe [ 2 ] targeted by an aspartic acid protease¹⁸F]GP-16 has high radioactive chemical purity, which can reach more than 90%, and the peak time of radioactive HPLC is 15.8 min.

Experimental example 11: mouse PET imaging experiment of aspartate proteolytic enzyme targeted molecular probe

The present example provides the legumain-targeted molecular probe described in example 13¹⁸F]The mouse PET imaging experiment of GP-AAN-AMBF3 comprises the following specific processes:

HeLa tumor-bearing mice (purchased from Calvens laboratory animals, Hezhou) with good viability and appropriate tumor size were treated for 5 days with 5mg/kg of doxorubicin by intratumoral injection and then anesthetized with oxygen containing 2 vt% isoflurane at a flow rate of 1.5L/min. The HeLa untreated group was a negative control. After the four limbs and the tail of the mouse are fixed, a molecular probe [ 2 ] targeting an aspartic acid protease enzyme of about 5.55MBq dissolved in 150. mu.L of physiological saline¹⁸F]GP-16 was injected via the tail vein. A 60min dynamic PET scan was performed immediately after the probe injection. Region of interestTissue drug uptake of the domain (ROI) was quantitatively analyzed by ASIProVM software. Tumor to muscle quantification results are expressed as the ratio of tumor uptake per mL muscle to total dose (% ID/mL). The results are shown in FIG. 82.

As can be seen in FIG. 82, the aspartic acid protease targeting molecular probe [ 2 ]¹⁸F]GP-16 has significant uptake in doxorubicin-induced apoptotic HeLa tumors, whereas in the untreated group the tumor uptake cross was low, dividing into 4.8 + -0.11% ID/mL and 1.3 + -0.08% ID/mL, indicating that the probe can sensitively distinguish apoptotic tumors from normal tumors. The PET imaging result indicates the probe¹⁸F]GP-16 has good caspase apoptosis protein targeting property and can sensitively detect the tumor apoptosis level in vivo.

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. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A molecular probe, characterized in that it has the structure shown below:

wherein R is₁Is a labeling group, R₂Is a targeting group.

2. The molecular probe of claim 1, wherein the molecular probe is a glutamyl transpeptidase targeted molecular probe; the glutamyl transpeptidase targeted molecular probe has the following structure:

3. the molecular probe of claim 1, wherein said molecular probe is a legumain-targeted molecular probe; the legumain targeted molecular probe has the following structure:

4. the molecular probe of claim 1, wherein the molecular probe is an aspartate proteolytic enzyme-targeted molecular probe; the aspartate proteolytic enzyme targeted molecular probe has the following structure:

5. an intramolecular condensation scaffold, characterized in that it has the structure shown below:

6. a method for preparing the molecular probe of any one of claims 1 to 4, wherein the method comprises: modifying the labeling group and the targeting group on the basis of the intramolecular condensation skeleton according to claim 5.

7. The method of claim 6, wherein when the molecular probe is a glutamyl transpeptidase targeted molecular probe, the method is: dissolving an intramolecular condensation skeleton, N-BOC-L-glutamic acid-1-tert-butyl ester and HBTU in ultra-dry THF to obtain a solution; in thatAdding DIPEA into the dissolved solution, and reacting under the protection of nitrogen to obtain a compound GD-14; dissolving a compound GD-14 in a mixed solution of DCM and TFA for reaction to obtain a compound GD-15; dissolving compound GD-15 in DMF/H₂Obtaining mixed solution from the mixed solution of O; adding AMBF to the mixed solution₃Reacting ligand and Cu (I) under the protection of nitrogen to obtain a labeled precursor of the glutamyl transpeptidase targeted molecular probe; carrying out radioactive labeling on a labeled precursor of the glutamyl transpeptidase targeted molecular probe to obtain the glutamyl transpeptidase targeted molecular probe;

the compound GD-14 has the structure shown below:

the compound GD-15 has the structure shown below:

8. the method of claim 6, wherein when the molecular probe is a legumain-targeted molecular probe, the method is: dissolving the intramolecular condensation skeleton according to claim 1 in ultra-dry THF to obtain a solution; adding Ac-AAN-OH, HBTU and DIPEA into the dissolved solution, and reacting under the protection of nitrogen to obtain ｃA compound GP-A; adding TFA and Tips into ｃA mixed solution of ｃA compound GP-A and DCM for reaction to obtain ｃA compound GP-AAN; dissolving a compound GD-15 in DMF to obtain a mixed solution; adding AMBF to the mixed solution₃Reacting the ligand with tetra (acetonitrile) copper (I) hexafluorophosphate under the protection of nitrogen to obtain a labeled precursor of the legumain targeted molecular probe; carrying out radioactive labeling on a labeled precursor of the legumain targeted molecular probe to obtain the legumain targeted molecular probe;

the compound GP-A has the structure shown below:

the compound GP-AAN has the structure shown below:

9. the method of claim 6, wherein when the molecular probe is an aspartate proteolytic enzyme targeted molecular probe, the method is: dissolving the intramolecular condensation skeleton of claim 1, DEVD and HBTU in THF to obtain a solution; adding DIPEA into the dissolved solution, and reacting under the protection of nitrogen to obtain a compound GP-14; dissolving a compound GP-14 in a mixed solution of DCM and TFA for reaction to obtain a compound GP-15; dissolving the compound GP-15 in DMF and H₂Obtaining mixed solution from the mixed solution of O; adding AMBF to the mixed solution₃THPTA, LAASS and CUSO₄Then, reacting under the protection of nitrogen to obtain a labeled precursor of the aspartic acid proteolytic enzyme targeted molecular probe; carrying out radioactive labeling on a labeled precursor of the aspartic acid proteolytic enzyme targeted molecular probe to obtain the aspartic acid proteolytic enzyme targeted molecular probe;

the compound GP-14 has the structure shown below:

the compound GP-15 has the structure shown below:

10. a method for preparing an intramolecular condensation scaffold according to claim 5, wherein the method comprises: dissolving a compound GP-12 in methanol to obtain a mixed solution; adding Tips and SEt into the mixed solution to react to obtain the intramolecular condensation skeleton in the claim 1;

the compound GP-12 has the structure shown below:

11. use of the intramolecular condensed scaffold of claim 5 in the preparation of molecular probes.

12. Use of the molecular probe of any one of claims 1 to 4 or the intramolecular condensed scaffold of claim 5 for imaging of a target.

13. An imaging agent targeted to a target, comprising the molecular probe of any one of claims 1 to 4 or the intramolecular condensation scaffold of claim 5.