CN110627868B

CN110627868B - A kind of18F-labeled compound and legumain-targeted PET imaging probe

Info

Publication number: CN110627868B
Application number: CN201910796902.3A
Authority: CN
Inventors: 邱玲; 林建国; 吕高超; 刘清竹; 李珂; 黄洪波; 彭莹; 谢敏浩
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2021-05-18
Anticipated expiration: 2039-08-27
Also published as: CN110627868A

Abstract

The invention discloses a radioactive drug and a preparation method thereof in the technical field of nuclear medicine¹⁸F-labeled compound and legumain targeted PET imaging probe. The invention provides a compound shown as a formula (I), which generates the shearing of asparagine sites and the reduction of disulfide bonds in a tumor microenvironment with high legumain expression, and forms a radioactive dimer by utilizing a biocompatible CBT-Cys click condensation reaction¹⁸F-1-dimer, thereby increasing tumor imaging effect. The invention adopts one-step ion exchange "¹⁸The compound with the structure shown in the formula (I) is synthesized by the F-labeling method, the method is simple to operate, and further purification by preparative HPLC is not needed. The invention also provides a legumain targeted PET imaging probe which is a compound shown in the formula (I). The PET imaging probe has the advantages of high stability, high sensitivity, strong specificity, good safety and the like.

Description

A kind of18F-labeled compound and legumain-targeted PET imaging probe

Technical Field

The invention relates to the technical field of radiopharmaceuticals and nuclear medicine, in particular toRelates to a¹⁸F-labeled compound, legumain targeted PET imaging probe, preparation method and application.

Background

Legumain (legumain), also known as asparaginyl endopeptidase, is a lysosomal cysteine protease. Legumain has high selectivity and is the only enzyme known to human to recognize asparagine (Asn) at the P1 site of peptide substrates. Recent studies have shown that legumain enzyme can be involved in a variety of biological events, such as inhibiting osteoclastogenesis, affecting the efficiency and kinetics of Major Histocompatibility Complex (MHC) class II antigen presentation, modulating the role of M2 macrophages in alleviating renal interstitial fibrosis in obstructive renal disease, etc., and is of great importance in maintaining normal renal physiology and homeostasis. In addition, it has been shown that upregulation of legumain expression levels is closely associated with the development of many diseases, including inflammation, atherosclerosis, and tumors. Legumain is highly expressed in many tumors, such as ovarian cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, lung cancer, lymphoma, and central nervous system related cancers. Although the underlying mechanism by which legumain enzymatically induces tumorigenesis has not yet been fully elucidated, the close correlation between the high expression of legumain enzyme and the proliferation, invasion, and metastasis of tumor cells has been demonstrated. Therefore, legumain has become an effective target for tumor diagnosis and treatment. How to effectively monitor legumain enzyme activity in tumors by using the existing molecular imaging technology has important significance for improving the early diagnosis level of tumors.

Although Legumain is closely related to tumor cell proliferation, invasion, metastasis, the exact mechanism by which Legumain promotes tumorigenesis and progression is not clear. Therefore, the method can effectively detect legumain activity in the tumor in time, is favorable for deepening understanding of biological effects of legumain in a tumor occurrence and development microenvironment, and has great significance for improving the early diagnosis level of the tumor.

Current research on legumain activity detection focuses mainly on the strategy of fluorescence imaging, and Positron Emission Tomography (PET) imaging technology has not yet been applied.

Disclosure of Invention

It is therefore an object of the present invention to provide a method of producing¹⁸F-labeled compound, legumain targeted PET imaging probe, preparation method and application. The specific technical scheme is as follows:

a kind of¹⁸A compound labeled F having the structure shown in formula (I) below:

wherein R is R₁C (O) or R₁-C(O)-R₂；

The R is₁Is C₁-C₁₀Alkyl groups of (a);

the R is₂Is a polypeptide sequence, the amino acid number n of the polypeptide sequence is 1-10.

Preferably, said R is₁Is C₁-C₃The number of amino acids n of the polypeptide sequence is 4-10.

The above-mentioned¹⁸The F-labeled compound has the following formula¹⁸F-1:

the above-mentioned¹⁸The F-labeled compound has the following formula¹⁸F-2:

the above-mentioned¹⁸A process for the preparation of an F-labelled compound comprising the radiosynthesis of the compound: reacting a precursor compound having a structure represented by the following formula (II) with¹⁸Incubating the F anion at 75-85 ℃ for 20-40 min;

the preparation method of the precursor compound with the structure shown in the formula (II) comprises the following steps,

carrying out condensation reaction on the compound shown as the formula II-1 and the compound shown as the formula II-2 to obtain a compound shown as a formula II-3;

carrying out deprotection reaction on the compound shown as the formula II-3 to obtain a compound shown as a formula II-4;

carrying out click condensation reaction on the compound shown in the formula II-4, alkyl aminomethyl boron trifluoride, tri (2-benzimidazolylmethyl) amine and copper (I) hexafluorophosphate to obtain a precursor compound shown in the formula II;

in the condensation reaction, the compound shown in the formula II-1, the compound shown in the formula II-2, a coupling agent and a dissolving agent are mixed and dissolved, then an organic synthetic solvent is added, and the reaction is carried out at normal temperature under the protection of nitrogen; preferably, in the condensation reaction, the coupling agent used comprises HBTU, EDCI or DCC, and the dissolving agent used comprises DMF, THF, DMSO, CH₂Cl₂Or H₂O, organic synthetic solvents used include DIPEA or TEA;

preferably, in the deprotection reaction, the compound shown in the formula II-3 is dissolved until the solution turns orange red, and then a peptide synthesis protective agent is added for reaction at normal temperature; preferably, in the deprotection reaction, a solvent comprising a mixture of TFA and DCM and/or MeCN is used for dissolution; preferably, the TFA: DCM: MeCN (volume ratio) ═ 1-5: 1-10: 0 to 3; preferably, the peptide synthesis protective agent is TIPS.

In the click condensation reaction, dissolving the compound shown in the formula II-4, adding alkyl aminomethyl boron trifluoride and tri (2-benzimidazolylmethyl) amine, quickly weighing, adding copper (I) tetra (acetate) hexafluorophosphate, adding water, and reacting in an oil bath at 40-50 ℃ under the protection of nitrogen; preferably, in the click condensation reaction, the dissolving solvent used is a mixed solution of DMF and water.

As described above¹⁸The application of the F-labeled compound in preparing a legumain-targeted PET imaging probe.

A legumain targeted PET imaging probe comprises the above¹⁸F labeled compound.

The technical scheme of the invention has the following advantages:

1. the compound with the structure shown in the formula (I) has an alanine-asparagine sequence targeted and identified by legumain, the shearing of asparagine sites and the reduction of disulfide bonds occur in a tumor microenvironment with high legumain expression, the biocompatible CBT-Cys click condensation reaction is utilized, and naked amino and sulfhydryl are easy to condense with cyano on CBT, so that a radioactive dimer is formed¹⁸F-1-dimer, the increase in molecular weight will be reduced¹⁸The F-1-dimer is pumped out of the cell, thereby increasing the tumor imaging effect.

2. The invention provides R in a compound with a structure shown as a formula (I)₂When the polypeptide sequence is hydrophilic, namely the hydrophilic histidine-glutamic acid polypeptide sequence is introduced on the basis of the AAN with the structure shown in the formula (I), the water solubility can be obviously increased, the non-specific uptake of the polypeptide sequence in the liver can be reduced, and the specificity of PET imaging can be increased.

3. The invention adopts¹⁸F-¹⁹"one-step ion exchange" for F Isotope Exchange (IEX) "¹⁸The compound with the structure shown in the formula (I) is synthesized by the F labeling method, the method is simple to operate, further purification by preparative HPLC is not needed, and the purification time is reduced, so that the possibility of label failure caused by complicated experimental steps is reduced, and the obtained PET tracer has high radiochemical yield and good specific activity. The labeling method greatly promotes the development of the PET tracer and the application thereof in disease diagnosis.

4. The compound with the structure shown in the formula (I) is used as a PEGylation probe targeted by legumain, has the advantages of high stability, high sensitivity, strong specificity, good safety and the like, and can effectively monitor the activity of the legumain.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a mass spectrum of a precursor compound having a structure represented by formula 1 obtained in example 1 of the present invention;

FIG. 2 is a high performance liquid chromatogram of a precursor compound having a structure represented by formula 1 obtained in example 1 of the present invention;

FIG. 3 is a hydrogen spectrum of a precursor compound having a structure represented by formula 1 obtained in example 1 of the present invention;

FIG. 4 is a carbon spectrum of a precursor compound having a structure represented by formula 1 obtained in example 1 of the present invention;

FIG. 5 is a fluorine spectrum of a precursor compound having a structure represented by formula 1 obtained in example 1 of the present invention;

FIG. 6 is a mass spectrum of a precursor compound having a structure represented by formula 2 obtained in example 2 of the present invention;

FIG. 7 is a high performance liquid chromatogram of a precursor compound of the structure shown in formula 2 obtained in example 2 of the present invention;

FIG. 8 is a hydrogen spectrum of a precursor compound having a structure represented by formula 2 obtained in example 2 of the present invention;

FIG. 9 is a carbon spectrum of a precursor compound having a structure represented by formula 2 obtained in example 2 of the present invention;

FIG. 10 is a fluorine spectrum of a precursor compound having a structure represented by formula 2 obtained in example 2 of the present invention;

FIG. 11 is a mass spectrum of a precursor compound having a structure represented by formula 3 obtained in comparative example 1 of the present invention;

FIG. 12 is a high performance liquid chromatogram of a precursor compound of the structure shown in formula 3 obtained in comparative example 1 of the present invention;

FIG. 13 is a hydrogen spectrum of a precursor compound of the structure shown in formula 3 obtained in comparative example 1 of the present invention;

FIG. 14 is a carbon spectrum of a precursor compound of the structure shown in formula 3 obtained in comparative example 1 of the present invention;

FIG. 15 is a fluorine spectrum of a precursor compound having a structure represented by formula 3 obtained in comparative example 1 of the present invention;

FIG. 16 is a probe of the present invention¹⁸F-1(a)、¹⁸F-2(b) and¹⁸the results of the radiosynthesis and purification of F-3 (c);

FIG. 17 is a probe of the present invention¹⁸F-1(a,b)、¹⁸F-2(c,d)、¹⁸The results of the in vitro stability test of F-3(e, F);

FIG. 18 shows the results of Western Blot analysis of legumain expression levels in different cells according to the present invention;

FIG. 19 is a probe of the present invention¹⁸F-1 precursor (a) and¹⁸toxicity assay of F-3 precursor (b) to HCT116 cells¹⁸Toxicity of F-1 precursor to PC3(c) cells, Probe¹⁸Toxicity assay of F-2 precursor to HCT116(d) and PC3 (e) cells;

FIG. 20 shows a probe according to the present invention¹⁸F-1 and¹⁸f-3 in cells HCT116(a) and probes¹⁸Uptake of F-2 in HCT116 and PC3 cells (b);

FIG. 21 shows a probe of the present invention¹⁸micro-PET imaging of F-1 in PC3(a, b) and HCT116(c, d) tumor-bearing mice and its quantitative analysis result;

FIG. 22 shows a probe according to the invention¹⁸micro-PET imaging of F-3 in HCT116 tumor-bearing mice and quantitative analysis results thereof;

FIG. 23 is a probe of the present invention¹⁸micro-PET imaging of F-2 in HCT116 tumor-bearing mice and quantitative analysis results thereof.

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 examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps 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.

The abbreviations in this invention state:

and (3) CBT: 2-cyanobenzothiazole; NMM: n-methylmorpholine;

IBCF (IBCF): isopropyl chloroformate; THF: tetrahydrofuran;

TFA: trifluoroacetic acid; DCM: dichloromethane;

DIPEA: n, N-diisopropylethylamine; DMF: n, N-dimethylformamide;

MeOH: methanol; MeCN: acetonitrile;

TIPS: triisopropyl silicon base; trt: a trityl group;

tBu: tetrabutyl urea; rf: a ratio shift value;

HBTU: o-benzotriazol-tetramethylurea hexafluorophosphate;

HEHEHEAAN：His-Glu-His-Glu-His-Glu-Ala-Ala-Asn；AAN：Ala-Ala-Asn；

HCT 116: human colon cancer cells: PC 3: human prostate cancer cells; MDA-MB-231: human breast cancer cells;

MDA-MB-435: human breast cancer cells; LO 2: human normal liver cells.

The solvents used in the following examples, DCM, MeOH, TFA, diethyl ether, DMF, etc., were all analytical grade, with the DMF being extra dry DMF in the condensation reaction; the "Normal temperature reaction" referred to in the following examples is at a temperature in the range of 20-25 deg.C

Example 1

¹⁸The F-labeled compound is a precursor compound with a structure shown in the following formula 1, and the synthetic route is,

synthesis of intermediate compounds 1-2 according to the methods reported in the literature [ Lin, J.et al.chem Commun (Camb)2017,53, (48),6476-6479 ],

weighing compound 1-2(38mg, 0.08mmol), compound 1-1(50mg, 0.09mmol) and HBTU (62mg, 0.16mmol), adding into a reaction flask, ultrasonic dissolving with ultra-dry DMF for 1min (model: KQ2200E, power: 100W), adding DIPEA (88 μ L, 0.53mmol), and N₂Reacting for 2h at normal temperature under protection. After purification by column chromatography (eluent: DCM: MeOH (vol) ═ 35: 1 to 15: 1), the solvent was collected and evaporated, and dried in vacuo for 2h to give compound 1-3(60mg, 72% yield).

The resulting compounds 1-3 were purified with 4mL of DCM: MeCN: TFA ═ 1: 1: the solution 2 (vol/vol) was dissolved, the solution turned orange-red, TIPS (110 μ L, 0.54mmol) was added and the reaction was carried out at room temperature for 0.5h, and the reaction progress was monitored by dot plate (developing solvent DCM: MeOH (vol/vol) ═ 10: 1, Rf ═ 0.4). After the reaction is finished, the solvent is dried by spinning, a little ether is added, a precipitate is generated, the precipitate is quickly transferred to a centrifuge tube, the centrifuge tube is refrigerated for 10min at the temperature of minus 20 ℃ in a refrigerator, the centrifuge tube is centrifuged for 5min at 3000rpm (centrifuge, L7-80), the supernatant is poured off, the rest precipitate is placed in a vacuum drying oven to be dried for 2h, and a crude product (109mg) of the compound 1-4 is obtained, and after the crude product is separated and purified by column chromatography (eluent: DCM: MeOH is 25: 1-10: 1 (volume ratio), the pure product (41.8mg, yield 92%) of the compound 1-.

The obtained compound 1-4(41.8mg, 0.05mmol) was placed in a 25mL round-bottomed flask, dissolved in about 1mL of DMF, and alkylaminomethylboron trifluoride (AmBF) was added₃54mg, 0.28mmol) and tris (2-benzimidazolylmethyl) amine (ligand, 2.2mg, 0.006mmol), copper (I) tetrakis (acetate) hexafluorophosphate (Cu (I), 12.3mg, 0.03mmol) was weighed rapidly, and about 1mL of ultrapure water was added and mixed, N₂Protecting, reacting for 1h under oil bath at 45 ℃, and when the reaction is complete, carrying out semi-preparative HPLC separation and purification (specification of HPLC column is 250X 10 mm; 5 mu m, volume ratio of mobile phase (acetonitrile solution containing TFA with volume fraction of 0.1 percent and water containing TFA with volume fraction of 0.1 percent) on the reaction solution,Flow rate, time of peak emergence, as shown in Table 1), collecting peak, and lyophilizing to obtain AmBF₃-CBT-Cys (StBu) -AAN (23mg, 44% yield). The mass spectrum, high performance liquid chromatogram, hydrogen spectrum, carbon spectrum and fluorine spectrum of the compound are respectively shown in FIGS. 1-5.

TABLE 1 semi-preparative HPLC separation conditions for the compounds of formula 1

Example 2

¹⁸The F-labeled compound is a precursor compound with a structure shown in the following formula 2, and the synthetic route is,

the polypeptide Ac-HEHEHEAAN-OH, namely the compound 2-1, is synthesized by a solid-phase polypeptide synthesis method, wherein the synthetic route is as follows:

by connecting Fmoc-N-trityl-L-asparagine (298mg, 0.5mmol), Fmoc-methoxycarbonyl-L-alanine (156mg, 0.5mmol), Fmoc-L-glutamic acid-5-tert-butyl ester hydrate (213mg, 0.5mmol), Fmoc-trityl-L-histidine (310mg, 0.5mmol), acetic anhydride (1mL, 10mmol), resin removal, rotary evaporation of the solvent, ether precipitation, and vacuum drying to obtain compound 2-1, (634mg, 56% yield).

Compound 2-1(179.7mg, 0.08mmol), compound 1-2(33.6mg, 0.07mmol) and HBTU (55.2mg, 0.15mmol) were placed in a 50mL round bottom flask withDissolving in appropriate amount of ultra-dry DMF, adding DIPEA (65.5 μ L, 0.4mmol) to adjust pH to 8-9, N₂The reaction was carried out at room temperature for 2h under protection, and the progress of the reaction was monitored by TLC plates (developing solvent DCM: MeOH: 10: 1 (vol.%), Rf: 0.6), and when the reaction was completed, the solvent was spun off by an oil pump, and the product was purified by column chromatography (eluent: DCM: MeOH: 50: 1 to 20: 1 (vol.%)) to obtain compound 2-2(170mg, 86.5% yield).

The resulting compound 2-2 was purified with DCM: MeCN: TFA ═ 1: 1: 4 (volume ratio) (6mL) was dissolved, the solution turned orange-red, TIPS (60. mu.L, 0.29mmol) was added and the reaction was allowed to proceed for 0.5h at ambient temperature, and the progress of the reaction was monitored by TLC plates. After the reaction is finished, the solvent is dried by spinning, a little diethyl ether is added, a precipitate is generated, the precipitate is quickly transferred to a centrifuge tube, the centrifuge tube is refrigerated for 10min at minus 20 ℃ in a refrigerator, the centrifuge tube is centrifuged for 5min at 3000rpm (the centrifuge model L7-80), the supernatant is poured off, and the rest precipitate is placed in a vacuum drying oven to be dried for 2h, so that the compound 2-3(90mg, the yield is 78.9%) is obtained.

The resulting compound 2-3(50mg, 0.03mmol) was taken up in a 25mL round-bottomed flask, dissolved in about 1mL DMF, and AmBF was added₃(31.4mg, 0.16mmol) and ligand (1.3mg, 0.003mmol), copper (I) tetrakis (acetate) hexafluorophosphate (23.9mg, 0.06mmol) was weighed rapidly, and about 1mL of ultrapure water, N, was added₂Protecting, performing oil bath reaction at 45 ℃ for 1h, performing semi-preparative HPLC separation and purification (specification of HPLC column 250X 10 mm; 5 μm, volume ratio of mobile phase (acetonitrile solution containing 0.1% TFA by volume fraction, water containing 0.1% TFA by volume fraction), flow rate, and peak emergence time (shown in Table 2) when the reaction is complete, performing peak inoculation, collecting, and freeze-drying to obtain AmBF₃-CBT-Cys (StBu) -HEHEHEAAN (14mg, 40.2% yield). The mass spectrum, high performance liquid chromatogram, hydrogen spectrum, carbon spectrum and fluorine spectrum of the compound are respectively shown in FIGS. 6-10.

TABLE 2 semi-preparative HPLC separation conditions for the compounds of formula 2

Example 3

And (3) radioactive synthesis:

probe needle¹⁸The radioactive synthesis route of the F-1 compound is as follows,

produced on demand by a medical cyclotron¹⁸F, fluoride ions. After the production is finished, the¹⁸The F fluoride ion was targeted to an anion exchange column (QMA) and washed with pyridazine buffer (300. mu.L, pH 2.5)¹⁸F anion (150-300mCi) was eluted from QMA column into polypropylene reaction tube (1mL) and AmBF synthesized in example 1 was added₃-CBT-Cys (StBu) -AAN (a precursor compound of the structure shown in formula 1, i.e., Probe precursor 1) (25mM, 30. mu.L) was added to the reaction tube and incubated at 80 ℃ for 30min (80 ℃ is the optimal condition for the radiosynthesis).

And (3) radioactive purification:

after completion of the radiosynthesis, the reaction solution was transferred to a centrifuge tube containing 20mL of ultrapure water, and the resultant radiolabeled probe was then introduced¹⁸F-1 was loaded on a C18 purification column (model Sep-Pak plusC18 of column) activated with ethanol (10mL) and then with ultrapure water (10 mL). The C18 column was washed three times with ultrapure water, and then the purified radiolabeled product was rinsed into penicillin bottles with ethanol (500. mu.L) and diluted to an appropriate concentration with physiological saline for use. The entire radiosynthesis and purification process was completed within 30 min.

Example 4

And (3) radioactive synthesis:

probe needle¹⁸The radioactive synthesis route of the F-2 compound is as follows,

produced on demand by a medical cyclotron¹⁸F, fluoride ions. After the production is finished, the¹⁸The F fluoride ion was targeted to an anion exchange column (QMA) and washed with pyridazine buffer (300. mu.L, pH 2.5)¹⁸F anion (150-300mCi) was eluted from QMA column into polypropylene reaction tube (1mL) and synthesized in example 2 AmB¹⁹F₃-CBT-Cys (StBu) -HEHEHEAAN (precursor compound of the structure shown in formula 2, i.e., Probe precursor 2) (25mM, 30. mu.L) was added to the reaction tube and incubated at 80 ℃ for 30 min.

And (3) radioactive purification:

after completion of the radiosynthesis, the reaction solution was transferred to a centrifuge tube containing 20mL of ultrapure water, and the resultant radiolabeled probe was then introduced¹⁸F-2 was loaded on a C18 purification column (model Sep-Pak plusC18 of column) activated with ethanol (10mL) followed by ultra pure water (10 mL). The C18 column was washed three times with ultrapure water, and then the purified radiolabeled product was rinsed into penicillin bottles with ethanol (500. mu.L) and diluted to an appropriate concentration with physiological saline for use. The entire radiosynthesis and purification process was completed within 30 min.

Comparative example 1

Probe precursor 3, i.e. Compound AmBF₃FAAN, which has a structure shown in the following formula 3, and the synthetic route is,

according to the solid-phase polypeptide synthesis method, Fmoc-N-trityl-L-asparagine, N-fluorenylmethyloxycarbonyl-L-alanine and Fmoc-L-propargyl glycine are sequentially connected, resin is removed, a solvent is evaporated in a rotary mode, and the compound is polypeptide FAAN, namely the compound 3-1(269.2mg, the yield is 32%).

Compound 3-1(20mg, 0.02mmol) was placed in a 25mL round bottom flask and quenched with DCM: MeCN: TFA ═ 1: 1: 2 (volume ratio) (about 2mL) was dissolved and the solution was orange red, TIPS (20. mu.L, 0.10 mmol) was added and the reaction was carried out at room temperature for 1 h. After the reaction is finished, removing the solvent by rotary evaporation, adding anhydrous ether to generate a precipitate, quickly transferring the precipitate into a 50mL centrifuge tube, refrigerating the centrifuge tube for 10min at the temperature of minus 20 ℃, taking out the centrifuge tube and centrifuging the centrifuge tube for 5min, pouring out a supernatant, retaining the precipitate, and drying the precipitate for 2h in vacuum to obtain the compound 3-2(13mg, the yield is 92%).

The resulting compound 3-2 was placed in a 25mL round bottom flask, dissolved in about 1mL DMF, and AmBF was added₃(18 mg，0.09mmol)，ligand(1.2mg, 0.003mmol), copper (I) tetrakis (acetate) hexafluorophosphate (6.8mg, 0.03mmol) was weighed out quickly, and about 1mL of ultrapure water was added thereto and mixed, and N was added₂Protecting, performing oil bath reaction at 45 ℃ for 1h, performing semi-preparative HPLC separation and purification on the reaction solution when the reaction is complete, (the specification of an HPLC column is 250 multiplied by 10 mm; 5 mu m, the volume ratio, the flow rate and the peak emergence time of a mobile phase (an acetonitrile solution containing TFA with the volume fraction of 0.1 percent and water containing TFA with the volume fraction of 0.1 percent, shown in Table 3), performing peak inoculation, collecting and freeze-drying to obtain the AmBF₃FAAN, (14mg, 81% yield). The mass spectrum, high performance liquid chromatogram, hydrogen spectrum, carbon spectrum and fluorine spectrum of the compound are respectively shown in FIGS. 11-15.

TABLE 3 semi-preparative HPLC separation conditions for the compound represented by formula 3

Comparative example 2

And (3) radioactive synthesis:

probe needle¹⁸The radioactive synthesis route for F-3 (control probe) is as follows,

produced on demand by a medical cyclotron¹⁸F, fluoride ions. After the production is finished, the¹⁸The F fluoride ion was targeted to an anion exchange column (QMA) and washed with pyridazine buffer (300. mu.L, pH 2.5)¹⁸F anion (150-300mCi) was eluted from QMA column into polypropylene reaction tube (1mL) and AmBF synthesized in comparative example 1 was added₃FAAN (25mM, 30. mu.L) was added to the reaction tube and incubated at 80 ℃ for 30 min.

And (3) radioactive purification:

after completion of the radiosynthesis, the reaction solution was transferred to a centrifuge tube containing 20mL of ultrapure water, and then the radiolabeled probe was loaded on a C18 purification column (type Sep-Pak plusC18 of column) activated with ethanol (10mL) and ultrapure water (10 mL). The C18 column was washed three times with ultrapure water, and then the purified radiolabeled product was rinsed into penicillin bottles with ethanol (500. mu.L) and diluted to an appropriate concentration with physiological saline for use.

The entire radiosynthesis and purification process was completed within 30 min.

Experimental example 1: radiochemical yield (RCY) and radiochemical purity (RCP) measurements of probes

After completion of the radiosynthesis of the probes in examples 3, 4 and 2, a small amount of the reaction solution was diluted with a mixture (1mL) of deionized water and acetonitrile at a volume ratio of 1:1, and the radiochemical yield (RCY) (radiation dose < 5. mu. Ci) (HPLC specification (250X 4.6 mm; 10 μm)) was monitored by radioactive HPLC, the radiochemical purity (RCP) was evaluated by radioactive HPLC (injection dose < 5. mu. Ci) (HPLC specification (250X 4.6 mm; 10 μm) after completion of the radiosynthesis of the probes in examples 3, 4 and 2.

The results are shown in figure 16 which shows,¹⁸F-1、¹⁸f-2 and¹⁸RCY of F-3 was 75. + -. 2.8%, 62%. + -. 1.5% and 69. + -. 1.2%, respectively.¹⁸F-1、¹⁸F-3 and¹⁸the RCP of F-2 was greater than 99%.

Experimental example 2: in vitro stability assay

To determine the stability of the radiolabeled product, probes are used¹⁸F-1、¹⁸F-2、¹⁸F-3 was incubated in phosphate buffered saline (PBS, pH 7.4) and Fetal Bovine Serum (FBS) at 37 ℃ for 0.5h, 1h, 2h and 4h, respectively, and a small sample of PBS or FBS was taken and tested for its radiochemical purity RCP by radioactive HPLC (the test method was the same as in experimental example 1) to evaluate its stability. For FBS-incubated samples, a small amount of acetonitrile was added to the FBS solution before stability evaluation, and a precipitate was formed, and after centrifugation at 12,000rpm for 5min, the supernatant was removed and subjected to radioactive HPLC for RCP detection (detection method same as in Experimental example 1).

As shown in fig. 17, when the incubation time was increased to 4h,¹⁸F-1、¹⁸F-2、¹⁸the radiochemical purity of F-3 is over 95 percent, which proves that no other products are generated in the incubation process, and shows that the three radioactive probes have better stability. The probe has good stability and is a developing bodyInternal studies probe the necessary prerequisites for legumain enzyme targeting specificity.

Experimental example 3: measurement of fat and water partition coefficient

According to the previously reported method [ Huang, H.et al.contrast Media&Molecular Imaging 2018, 2018,1-9.]Determination of the lipid-water partition coefficient (log P) of the probe, mainly by measurement¹⁸F-1、¹⁸F-2、¹⁸The radioactive dose distribution of F-3 in n-octanol and ultrapure water. First, a radioactive probe, saturated ultrapure water and n-octanol were mixed in a ratio of 1: 9: 10 or 1: 4: a ratio of 5(v/v/v) was added to a polypropylene tube, mixed well, centrifuged at 5,000rpm for 5min to sufficiently separate the two phases, three samples (100. mu.L) were taken in parallel from each phase, respectively, and the radioactive dose in each phase was measured using a gamma counter. Log P is calculated by the following formula:

Log P＝Log(C_o/C_w)

wherein C is_oRepresents the octanol phase¹⁸F-1、¹⁸F-2、¹⁸Radioactive dose of F-3, C_wRepresents the aqueous phase¹⁸F-1、¹⁸F-2、¹⁸Radioactive dose of F-3.

Is measured by experiments¹⁸The lipid-water partition coefficient of F-1 is 0.57 +/-0.03, which indicates that¹⁸F-1 is lipophilic; while the control probe¹⁸The fat-water partition coefficient of F-3 was-1.48. + -. 0.02, indicating that it has hydrophilicity. The results show that¹⁸Compared with the F-3, the method has the advantages that,¹⁸f-1 has better cell membrane permeability and will have higher uptake in tumor cells.

Is measured by experiments¹⁸The Log P of F-2 is-2.48 + -0.03, which indicates that the F-2 has better water solubility, and indicates that the better water solubility is related to the introduction of HEHEHEHE polypeptide chain. Because histidine and glutamic acid both have hydrophilicity, the HEHEHEHEHE polypeptide is introduced on the basis of AAN, so that the water solubility of the probe can be obviously increased, the non-specific uptake of the probe in liver is reduced, and the specificity of PET imaging is increased.

Experimental example 4: western Blot assay for cell expression of legumain

Western Blotting standard techniques were used to determine legumain expression levels in different cells (HCT116, PC3, MDA-MB-231, MDA-MB-435 and LO 2).

The same amount of protein from cell lysates (50. mu.g) was separated on 10 w/v% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the protein bands were transferred to polyvinylidene fluoride (PVDF) transfer membranes, and after blocking the membranes with 5 w/v% skim milk for 1h, the membranes were incubated overnight at 4 ℃ with anti-Legumain antibody (1: 5000 (vol.), Abcam) or with anti-beta-Actin (1: 1000 (vol.)). The bands were washed and incubated with goat anti-mouse or rabbit IgG-HRP (1: 1000 (vol.), Santa Cruz, California, USA) for 1h at room temperature (the choice of goat anti-mouse or rabbit IgG-HRP corresponds to the primary antibody used), and finally the protein bands were visualized using ECL Western blotting kit (ABXBio) and the optical density analysis of the membranes was performed using ImageJ software.

As a result, as shown in FIG. 18, it was apparent that HCT116 cells had higher level of legumain expression, whereas PC3 cells had almost no legumain expression. Thus, HCT116 cells were selected as positive cells and PC3 cells were selected as negative cells for the control.

Experimental example 5: cytotoxicity test

According to the Western Blotting result, HCT116 with high legumain enzyme expression and PC-3 cells with low legumain enzyme expression are selected for carrying out a probe toxicity test.

HCT116 cells or PC-3 cells were cultured at 5X 10⁴The density per well was plated in 96-well plates and cultured for 24 h. After the cells were attached to the wall, the medium was aspirated, and 200. mu.L of high-glucose DMEM medium containing different concentrations (12.5, 25, 50 and 100. mu.M) of the

probe precursors

1, 2, 3 and 10% fetal bovine serum was added to each well at 37 ℃ with 5% CO₂The culture was carried out for 6, 12 and 24h respectively under the environment. After incubation for the indicated time, 20 μ L of MTT working solution (concentration 5 mg/mL) was added to each well, and the cells were further incubated at 37 ℃ for 4h, followed by addition of 150 μ L of dimethyl sulfoxide (DMSO, 150 μ L, analytical purity) to dissolve formazan precipitate produced in each well. Finally, the absorbance value of each well was measured at 490nm using a microplate reader, and the experiments were performed in parallel in three groups.

The cell viability was calculated by the following formula to evaluate the toxicity of the probe to the cells: survival%

The results are shown in FIG. 19. As can be seen from FIG. 19(a), the cell viability for HCT116 cells treated with probe precursor 1 at concentrations of 12.5, 25, 50 and 100. mu.M at 6h was 101.28. + -. 1.97%, 100.00. + -. 3.00%, 95.88. + -. 2.01% and 96.35. + -. 1.14%, respectively; when the culture time was prolonged to 12 and 24 hours, the cell survival rate did not decrease, indicating that probe 1 was non-toxic. Also, as can be seen from fig. 19(b), probe precursor 3 also has lower toxicity to HCT116 cells; as can be seen from FIG. 19(c), probe 1 also has lower toxicity to PC3 cells. As can be seen from FIG. 19(d), the survival rates of the HCT116 cells treated with the probe precursor 2 at different concentrations were 97.74. + -. 1.49%, 92.89. + -. 0.45%, 90.63. + -. 2.09% and 90.19. + -. 1.93% at 6h, respectively, and did not decrease when the time was prolonged to 12 and 24h, indicating that the probe 2 was not toxic to the HCT116 cells. Similarly, as can be seen in FIG. 19(e), PC3 cells treated with different concentrations (12.5, 25, 50 and 100. mu.M) of probe precursor 2 exhibited cell viability rates of 107.10. + -. 7.16%, 101.86. + -. 1.87%, 97.79. + -. 1.96% and 93.19. + -. 4.20% when the time of application was extended to 24h, indicating that probe 2 was also non-toxic to PC3 cells. Therefore, the probe of the present invention can be used for subsequent biological property studies.

Experimental example 6: cell uptake assay

HCT116 or PC3 cells were plated in 10cm dishes using 10% fetal bovine serum in high glucose DMEM at 37 deg.C with 5% CO₂The culture was carried out for 4 days in the environment. When the number of cells reaches about 3X 10⁸When necessary, the medium was aspirated and replaced with serum-free medium. The cells (about 10)⁶Cells/200. mu.L) and probe-containing¹⁸F-1、¹⁸F-2、¹⁸Adding F-3 serum-free medium (about 1 μ Ci/100 μ L) into the radioimmunoassay, shaking, mixing, incubating in water bath at 37 deg.C for 0.5, 1, 2 and 4 hr, and adding the culture medium containing the same dosage¹⁸F-1、¹⁸F-2、¹⁸The F-3 radioimmunoassay (no cells) was set as a control. At the indicated time points, cells were washed twice with cold PBS to remove non-ingested¹⁸F-1、¹⁸F-2、¹⁸F-3. And finally, measuring the radioactive dose taken by the cells through a gamma counter, and calculating the cell uptake rate (AD%), wherein the cell uptake rate (AD%) is the ratio of the cell uptake dose to the control group dose. Three sets were run in parallel for each time point.

The results are shown in FIG. 20. As can be seen from FIG. 20(a), after 0.5, 1, 2 and 4h of action, the target probe was passed¹⁸The uptake (AD%) of HCT116 cells by F-1 treatment was 4.19. + -. 0.38, 4.02. + -. 0.14, 4.15. + -. 0.14 and 4.23. + -. 0.03, respectively, as compared to the control probe¹⁸The uptake (AD%) of HCT116 cells treated with F-3 was 0.70. + -. 0.08, 0.70. + -. 0.16, 0.75. + -. 0.10 and 0.89. + -. 0.22, respectively. As can be seen from the comparison of the results of the two,¹⁸HCT116 cellular uptake of F-1 was significantly higher than¹⁸F-3, and the uptake was consistently higher within 4 h. The reason can be summarized into two points: first, with a control probe¹⁸F-3 phase comparison, target Probe¹⁸F-1 is lipophilic; second, a target probe¹⁸After the F-1 is taken up by cells in a targeted way, the shearing of Asn sites and the reduction of disulfide bonds occur in a tumor microenvironment with high legumain expression, and then the Asn sites and the disulfide bonds are condensed with cyano groups on CBT to form dimers¹⁸F-1-dimer (formula below), the increase in molecular size will decrease¹⁸Pumping out of the F-1-dimer, thereby increasing cellular uptake. In contrast, control probes¹⁸F-3 is unable to undergo CBT-Cys condensation and cellular uptake is relatively low. Thus, the target probe¹⁸F-1 can sensitively and specifically detect legumain activity in HCT116 cells.

As can be seen from FIG. 20(b),¹⁸f-2 was incubated in HCT116 cells at 37 ℃ for 0.5, 1, 2 and 4h at cell uptake (AD%) of 3.18. + -. 0.08, 3.64. + -. 0.07, 3.23. + -. 0.06 and 3.03. + -. 0.01, respectively¹⁸When F-2 is incubated in PC3 cells at 37 ℃ for 0.5, 1, 2 and 4h, the cell uptake value (AD%) is 0.61 + -0.01, 0.65 + -0.08, 0.67 + -0.15 and 0.89 + -0.07 respectively. From the point of view of the results,¹⁸the uptake value of F-2 in HCT116 cells is obviously higher than that of PC3 cells, which indicates that the probe has better targeting specificity to HCT116 cells with legumain high expression. When in use¹⁸Upon entry of F-2 into HCT116 cells, legumain-controlled Asn site cleavage and reduction can occurReduction of the disulfide bond under environmental control, followed by CBT-Cys click condensation to form a radioactive dimer¹⁸The increase in the size of the F-2-dimer, as well as the hydrophobic nature of the radioactive dimer itself, results in a decrease in its pumping out of the cell and an increase in the residence time, thereby causing an increase in the uptake of the probe into the cell. Thus, the probe¹⁸F-2 can be used for more sensitively and specifically detecting legumain activity in HCT116 cells.

Experimental example 7: PET imaging of small animals

The present invention relates to animals female BALB/c nude mice 4-8 weeks old were purchased from Shanghai Slek laboratory animals company and injected subcutaneously with HCT116 or PC3 tumor cells (about 3X 10)⁶Cells/100 μ LPBS) were implanted in the right shoulder of the mice. When the diameter of the tumor reaches 0.4-0.6cm, the mouse can be used for micro-PET imaging experiments. For PET imaging, mice were left under anesthesia using isoflurane (2% isoflurane in oxygen at a flow rate of 2L/min). By means of tail vein injection, (1) 100-200. mu. Ci¹⁸F-1 is injected into HCT116 and PC3 tumor-bearing mice to carry out micro-PET dynamic scanning for 60min, and the targeting of the probe in different model animals is determined; (2) will be 100-¹⁸F-2 is injected into an HCT116 tumor-bearing mouse to carry out micro-PET dynamic scanning for 60min, and the influence of introducing a polypeptide sequence HEHEHEHE on the structure of the probe 1 on the PET imaging result is analyzed; (3) 100-200. mu. Ci of control probes¹⁸F-3 is injected into HCT116 tumor-bearing mice to carry out micro-PET dynamic scanning for 60min so as to analyze and compare the probes¹⁸F-1 and¹⁸the aggregation effect of F-2 has an influence on the visualization effect. After the scanning is finished, the PET imaging result of 60min is segmented into 12 frames of images by using the OSEM3D/MAP algorithm, and one frame is processed every 5min, so that the real-time analysis of the in-vivo imaging of the mouse is realized. The region of interest (ROI) technique in ASIPRO software is adopted to outline and analyze and compare the distribution of the probe in the tumor site and other organ tissues. Uptake of the probe in each tissue in vivo was measured as% ID/g (per gram of injection)Amount percent).

¹⁸The results of micro-PET imaging of F-1 in PC3(a, b) and HCT116(c, d) tumor-bearing mice and its quantitative analysis are shown in FIG. 21. As shown in FIG. 21(a, b), a probe¹⁸The tumor uptake of F-1 in PC3 tumor-bearing mice is low, and the maximum uptake value reaches 1.87 +/-0.08 percent ID/g at 10-15 min. On the contrary, the present invention is not limited to the above-described embodiments,¹⁸the tumor uptake value of F-1 in HCT116 tumor-bearing mice is relatively high, the tumor uptake value reaches 3.08 +/-0.09% ID/g at 10-15min, but the subsequent uptake value shows a rapid descending trend, as shown in fig. 21(c, d). The PET imaging result of the tumor bearing mouse shows that the probe¹⁸F-1 has better imaging effect on HCT116 tumor bearing mice mainly because HCT116 has higher legumain expression and PC3 has lower legumain expression, so¹⁸F-1 has better tumor targeting selectivity in HCT116 tumor-bearing mice and better tumor part imaging effect. Therefore, the micro-PET imaging experiments were carried out below using the HCT116 tumor-bearing murine model.

Control probe¹⁸The micro-PET imaging of F-3 in HCT116 tumor-bearing mice and the quantitative analysis thereof are shown in fig. 22(a, b), the maximum tumor uptake value reaches 1.79 +/-0.15% ID/g in 10-15min, the tumor uptake value is reduced rapidly in 10-20 min, the retention effect is not ideal, the tumor/muscle uptake ratio is low, and the tumor imaging effect is poor.

Compared with¹⁸F-3, PET Probe¹⁸F-1 has better imaging effect in HCT116 tumor-bearing mice because,¹⁸f-1 can generate shearing of Asn site and reduction of disulfide bond in tumor microenvironment and generate CBT-Cys click condensation reaction in radioactive probe molecule, thereby generating radioactive dimer¹⁸F-1-dimer, and form nano-aggregation to generate aggregation effect;¹⁸the increased size and the hydrophobic property of the F-1-dimer and the nano-aggregate thereof cause the pumping-out of cells to be reduced, thereby enhancing the PET imaging result to a certain extent. While the control probe¹⁸F-3, although containing legumain targeting recognition sequence AAN, does not satisfy the conditions of CBT-Cys click condensation in the tumor microenvironment, and cannot generate radioactive dimers toAnd nano aggregates, and the probe itself is hydrophilic, so the tumor imaging effect is poor.

¹⁸The micro-PET imaging results of F-2 in HCT116 tumor-bearing mice and the quantitative analysis results thereof are shown in FIG. 23 (a) and FIG. 23(b), respectively. The results show that¹⁸F-2 tumor-bearing mice show the best imaging effect at 10-15min, the tumor uptake reaches 2.67 +/-0.06% ID/g, and then shows a descending trend, the tumor/muscle uptake ratio of the mice shows an increasing trend from the beginning to the end of scanning, and the tumor/muscle uptake ratio is increased to 2.86 +/-0.10 at 60 min. This result is illustrative of the fact that,¹⁸f-2 ratio¹⁸F-1 has better tumor targeting property and can quickly and effectively detect legumain activity.

And injection¹⁸Compared with the PET imaging result of the F-1 administration group,¹⁸the F-2 injection mode obviously reduces the liver uptake of a tumor-bearing mouse, which indicates that the introduction of the hydrophilic polypeptide chain HEHEHEHEHEHEHE can actually reduce the nonspecific uptake of the probe in the liver, thereby effectively enhancing the targeting specificity of the PET probe on legumain activity detection, improving the biological distribution of the probe in a living body and enhancing the PET imaging effect of the tumor.

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 obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A kind of¹⁸F-labelled compound, characterized in that said¹⁸The F-labeled compound has the following formula¹⁸F-1:

；

or, the said¹⁸The F-labeled compound has the following formula¹⁸F-2:

。

2. the method of claim 1¹⁸A process for the preparation of an F-labelled compound, comprising the radiosynthesis of a compound: reacting a precursor compound having a structure represented by the following formula (II) with¹⁸Incubating the F anion at 75-85 ℃ for 20-40 min;

；

wherein R is R₁C (O) or R₁-C(O)-R₂；

The R is₁is-CH₃；

The R is₂Is a polypeptide sequence; the polypeptide sequence is HEHEHEHE.

3. The method according to claim 2, wherein the precursor compound of formula (II) is prepared by a process comprising,

carrying out click condensation reaction on the compound shown as the formula II-4, alkyl aminomethyl boron trifluoride, tri (2-benzimidazolylmethyl) amine and copper (I) hexafluorophosphate to obtain the compound shown as the formula

A precursor compound as shown;

。

4. the preparation method according to claim 3, wherein in the condensation reaction, the compound represented by the formula II-1, the compound represented by the formula II-2, the coupling agent and the dissolving agent are mixed and dissolved, and then an organic synthetic solvent is added to react at normal temperature under the protection of nitrogen; in the condensation reaction, a coupling agent comprises HBTU, a dissolving agent comprises DMF, and an organic synthetic solvent comprises DIPE;

in the deprotection reaction, dissolving the compound shown as the formula II-3 until the solution turns orange red, then adding a peptide synthesis protective agent, and reacting at normal temperature; in the deprotection reaction, a solvent comprising a mixture of TFA, DCM and MeCN is used for dissolution; the TFA: DCM: MeCN (volume ratio) = 4: 1: 1; the peptide synthesis protective agent is TIPS.

5. The preparation method according to claim 3, wherein in the click condensation reaction, the compound shown in the formula II-4 is dissolved, then alkyl aminomethyl boron trifluoride and tri (2-benzimidazolylmethyl) amine are added, copper (I) tetra (acetate) hexafluorophosphate is rapidly weighed and added, water is added, and then the reaction is carried out under the protection of nitrogen and in an oil bath at 40-50 ℃; in the click condensation reaction, the used dissolving solvent is a mixed solution of DMF and water.

6. The method of claim 1¹⁸The application of the F-labeled compound in preparing a legumain-targeted PET imaging probe.

7. A legumain-targeted PET imaging probe comprising the peptide of claim 1¹⁸F labeled compound.