Azobenzene sulfonate compound, preparation method and application thereof, and preparation method of radioactive probe

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an azobenzene sulfonate compound, a preparation method and application thereof, and a preparation method of a radioactive probe. The invention provides an azobenzene sulfonate compound. The azobenzene sulfonate compound has a structure as shown in a formula I. The preparation method for preparing the radioactive probe by using the azobenzene sulfonate compound as the radioactive labeling probe precursor is simple, convenient and rapid, labeling is rapid, the radiochemical yield of the obtained radioactive probe is high, and the azobenzene sulfonate compound can be applied to preparation of a fluorine-18 radioactive probe kit. The obtained radioactive probe targets a protein aggregate with a beta-folded structure, can be applied to imaging agents for diseases related to the beta-folded protein aggregate, and is beneficial to diagnosis and drug development of the diseases related to the beta-folded protein aggregate.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an azobenzene sulfonate compound and a preparation method and application thereof, and a preparation method of a radioactive probe. Background Art

[0002] Proteins are important biological macromolecules of living organisms. When they are misfolded, aggregated and eventually form protein deposits in the body, they can cause a series of diseases. At present, more than 30 diseases have been reported to be associated with misfolded protein aggregates. Among them, protein aggregates with β-folded structures, such as β-amyloid protein aggregates, tau protein aggregates, and α-synuclein aggregates, are closely related to the course of a series of central nervous system degenerative diseases and are typical pathological markers of such diseases, including Alzheimer's disease, cerebral amyloid angiopathy, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick's disease, primary age-related tauopathy, globular glial tauopathy, Parkinson's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure, Alzheimer's disease Lewy body variant, etc. These proteins vary in amino acid sequence, structure, and expression level, but they all undergo a pathological and physiological process in the brains of patients in which they gradually form aggregates rich in β-folded structures from monomeric forms in the physiological environment. Research data show that pathological changes characterized by β-folded protein aggregates occur earlier than the clinical symptoms of the disease, and protein aggregates gradually diffuse in the brain as the disease progresses. They are an important target for early diagnosis, disease course monitoring and therapeutic drug research of related diseases.

[0003] Positron emission tomography (PET) is an advanced molecular imaging technology in the field of nuclear medicine. It uses radioactive imaging agents to perform molecular-level, non-invasive, quantitative three-dimensional tomographic imaging analysis of biological targets in the body, and plays a significant role in clinical imaging diagnosis, disease course monitoring, and drug research for tumors, neurodegenerative diseases, mental illnesses, cardiovascular diseases, etc. The preparation of radioactive imaging agents and radiolabeled precursors is an important prerequisite for achieving PET imaging of related diseases, and is also the focus and difficulty of technical research. Since β-amyloid protein aggregates, tau protein aggregates, and α-synuclein aggregates all have β-folded structures, it is still extremely challenging to design targeting ligands that can specifically recognize protein aggregates and prepare precursor compounds that are easy to label. Summary of the invention

[0004] The purpose of the present invention is to provide an azobenzene sulfonate compound and a preparation method and application thereof, and a method for preparing a radioactive probe. The azobenzene sulfonate compound provided by the present invention can be used as a labeling precursor for efficiently preparing a radiolabeled β-folded protein aggregate targeting probe.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides an azobenzene sulfonate compound having a structure shown in Formula I:

[0007]

[0008] Among them, R 1 is alkyl, substituted alkyl or substituted aryl; R is n is 1 to 3.

[0009] Preferably, the carbon number of the alkyl group is 1 to 3; the carbon number of the substituted alkyl group is 1 to 3; the substituent of the substituted alkyl group is halogen, amino, nitro or hydroxyl; the substituent of the substituted aryl group is alkyl, halogen or amino.

[0010] Preferably, it has a structure as shown in any one of Formulas 1 to 27:

[0011]

[0012]

[0013]

[0014]

[0015] The present invention also provides a method for preparing the azobenzene sulfonate compound described in the above technical solution, comprising the following steps:

[0016] A primary amine compound, sodium nitrite, phenol and water are mixed and subjected to a diazotization-coupling reaction under alkaline conditions to obtain an intermediate 1; the primary amine compound is 6-aminobenzothiazole, 6-aminoquinoline or 6-aminoquinoxaline; the intermediate 1 has a structure shown in Formula II:

[0017]

[0018] The intermediate 1, the PEGylation reagent and the first organic solvent are mixed, and a first nucleophilic substitution reaction is carried out under alkaline conditions to obtain the intermediate 2; -CH 2 CH 2 The number of O- is 1 to 3; the intermediate 2 has a structure shown in formula III:

[0019]

[0020] The intermediate 2, a sulfonylating agent, an organic base and a second organic solvent are mixed to carry out a second nucleophilic substitution reaction to obtain the azobenzene sulfonate compound; the sulfonylating agent is an alkylsulfonyl chloride, a substituted alkylsulfonyl chloride or a substituted arylsulfonyl chloride.

[0021] Preferably, the PEGylation reagent comprises ClCH 2 CH 2 OH, ClCH 2 CH 2 -O-CH 2 CH 2 OH, ClCH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 OH, BrCH 2 CH 2 OH, BrCH 2 CH 2 -O-CH 2 CH 2 OH and BrCH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 One or more of OH;

[0022] The sulfonylating agent includes one or more of methanesulfonyl chloride, trifluoromethanesulfonyl chloride and p-toluenesulfonyl chloride;

[0023] The organic base includes one or more of pyridine, 4-dimethylaminopyridine and triethylamine.

[0024] Preferably, the temperature of the diazotization-coupling reaction is 0 to 4°C, and the reaction time is 1 to 5 hours;

[0025] The temperature of the first nucleophilic substitution reaction is 80-110° C., and the reaction time is 2-8 hours;

[0026] The second nucleophilic substitution reaction includes a first stage reaction and a second stage reaction carried out in sequence; the temperature of the first stage reaction is 0-4°C, and the reaction time is 5-30 minutes; the temperature of the second stage reaction is 20-35°C, and the reaction time is 3-8 hours.

[0027] The present invention also provides the use of the azobenzenesulfonate compound described in the above technical solution in the preparation of a radioactive probe, a radioactive probe kit, a radioactive imaging agent or a radioactive imaging kit.

[0028] Preferably, the radioactive imaging agent is an imaging agent for detecting neurodegenerative diseases associated with abnormal protein aggregates; the protein aggregates include one or more of β-amyloid aggregates, tau aggregates and α-synuclein aggregates; the neurodegenerative diseases include one or more of Alzheimer's disease, cerebral amyloid angiopathy, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick's disease, primary age-related tauopathy, globular cell tauopathy, Parkinson's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure and Alzheimer's disease Lewy body variant;

[0029] The imaging of the radioactive imaging agent is positron emission tomography, positron emission tomography-computed tomography or positron emission tomography-magnetic resonance imaging.

[0030] The present invention also provides a method for preparing a radioactive probe, comprising the following steps:

[0031] In the presence of a phase transfer catalyst, the azobenzenesulfonate compound described in the above technical solution, the fluorine-18 anion ([ 18 F]F - ) is mixed with an ultra-dry solvent to perform a radioactive organic chemical reaction to obtain the radioactive probe; the radioactive probe has a structure shown in Formula IV:

[0032]

[0033] Among them, R is n is 1 to 3.

[0034] Preferably, the phase transfer catalyst is kryptofix 2.2.2. / one or more of potassium carbonate, tetrabutylammonium hydroxide and tetrabutylammonium bicarbonate;

[0035] The radiation organic chemical reaction is carried out in a closed environment; the temperature of the radiation organic chemical reaction is 105-150° C., and the reaction time is 5-15 minutes.

[0036] The present invention provides an azobenzenesulfonate compound having a structure shown in Formula I. The method for preparing a radioactive probe using the azobenzenesulfonate compound provided by the present invention as a radiolabeling precursor is simple and fast, and the labeling is fast, and the radiochemical yield of the obtained radioactive probe is high. The obtained radioactive probe can image protein aggregates with a β-folded structure, so the azobenzenesulfonate compound provided by the present invention can be used to prepare a fluorine-18 labeled radioactive probe and a radioactive probe kit, and then used as a radioactive imaging agent for imaging diseases related to β-folded protein aggregates, which is beneficial to the early diagnosis, disease course monitoring and therapeutic drug research of diseases related to β-folded protein aggregates. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 The synthetic route of the azobenzene sulfonate compound in Examples 1 to 3 of the present invention;

[0039] Figure 2 The radiolabeling route for preparing radioactive probes from the azobenzenesulfonate compounds in Examples 4 to 6 of the present invention;

[0040] Figure 3 The chromatograms are the co-injection verification chromatograms of the fluorine-18 radioactive probe and the reference substance obtained in Examples 4 to 6;

[0041] Figure 4 The fluorine-18 radioactive probe obtained in Example 4 [ 18 F]FA plasma stability chromatogram;

[0042] Figure 5 The fluorine-18 radioactive probe obtained in Example 6 [ 18 Plasma stability chromatogram of F]FC;

[0043] Figure 6 These are the radioautographic images and immunostaining images of the patient's brain tissue using the fluorine-18 radioactive probe obtained in Examples 4 to 6. DETAILED DESCRIPTION

[0044] The present invention provides an azobenzene sulfonate compound having a structure shown in Formula I:

[0045]

[0046] Among them, R1 is an alkyl, substituted alkyl or substituted aryl group; the number of carbon atoms of the alkyl group is preferably 1 to 3, more preferably 1; the number of carbon atoms of the substituted alkyl group is preferably 1 to 3, more preferably 1 to 2; the substituent of the substituted alkyl group is halogen, amino, nitro, hydroxyl, preferably halogen; the halogen is preferably F, Cl, Br; the substituent of the substituted aryl group is preferably alkyl, halogen, amino, more preferably alkyl, the number of carbon atoms of the alkyl group is preferably 1 to 3, more preferably 1; the R 1 Preferably it is methyl, trifluoromethyl or p-methylphenyl, more preferably it is methyl or p-methylphenyl, most preferably it is p-methylphenyl; R is n is 1 to 3.

[0047] In the present invention, the azobenzene sulfonate compound preferably has a structure as shown in any one of Formulas 1 to 27:

[0048]

[0049]

[0050]

[0051]

[0052] More preferred are formulas 1 to 9, and most preferred are formulas 1 to 6.

[0053] The method for preparing a radioactive probe using the azobenzenesulfonate compound provided by the present invention as a radiolabeling precursor is simple and fast, labeling is fast, and the radiochemical yield of the obtained radioactive probe is high. The obtained radioactive probe can image protein aggregates with a β-folded structure, so the azobenzenesulfonate compound provided by the present invention can be used to prepare a fluorine-18 labeled radioactive probe and a radioactive probe kit, and then used as a radioactive imaging agent for imaging diseases related to β-folded protein aggregates, which is beneficial to the early diagnosis, disease course monitoring and therapeutic drug research of diseases related to β-folded protein aggregates.

[0054] The present invention also provides a method for preparing the azobenzene sulfonate compound described in the above technical solution, comprising the following steps:

[0055] A primary amine compound, sodium nitrite, phenol and water are mixed and subjected to a diazotization-coupling reaction under alkaline conditions to obtain an intermediate 1; the primary amine compound is 6-aminobenzothiazole, 6-aminoquinoline or 6-aminoquinoxaline; the intermediate 1 has a structure shown in Formula II:

[0056]

[0057] The intermediate 1, the PEGylation reagent and the first organic solvent are mixed, and a first nucleophilic substitution reaction is carried out under alkaline conditions to obtain the intermediate 2; -CH 2 CH 2 The number of O- is 1 to 3; the intermediate 2 has a structure shown in formula III:

[0058]

[0059] The intermediate 2, a sulfonylating agent, an organic base and a second organic solvent are mixed to carry out a second nucleophilic substitution reaction to obtain the azobenzene sulfonate compound; the sulfonylating agent is an alkylsulfonyl chloride, a substituted alkylsulfonyl chloride or a substituted arylsulfonyl chloride.

[0060] In the present invention, the synthesis route of the azobenzene sulfonate compound is as follows:

[0061]

[0062] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0063] The present invention mixes a primary amine compound, sodium nitrite, phenol and water, and performs a diazotization-coupling reaction under alkaline conditions to obtain an intermediate 1.

[0064] In the present invention, the primary amine compound is 6-aminobenzothiazole, 6-aminoquinoline or 6-aminoquinoxaline; the molar ratio of the primary amine compound, sodium nitrite and phenol is 1:1-5:1-2, more preferably 1:2-4:1-1.5, and most preferably 1:2.25:1; the mass solid-to-liquid ratio of the primary amine compound and water is preferably 1:20, more preferably 1:15.

[0065] In the present invention, the feeding form of the primary amine compound is preferably a primary amine compound solution; the solvent of the primary amine compound solution is preferably concentrated hydrochloric acid; the mass fraction of the concentrated hydrochloric acid is preferably 37%; the feeding form of the sodium nitrite is preferably a sodium nitrite solution; the solvent of the sodium nitrite solution is preferably water; the feeding form of the phenol is preferably a phenol solution; the solvent of the phenol solution is preferably a sodium hydroxide aqueous solution; the mass fraction of the sodium hydroxide aqueous solution is preferably 50%; the present invention does not have any special limitation on the amount of the solvent, and the amount well known to those skilled in the art can be used to allow the reaction to proceed smoothly.

[0066] In the present invention, the temperature of the diazotization-coupling reaction is preferably 0-4°C, more preferably 0-2°C, and most preferably 0°C; the reaction time is preferably 1-5h, more preferably 2-4h; the diazotization-coupling reaction preferably includes a diazotization reaction and a coupling reaction performed sequentially; preferably, the primary amine compound solution is added dropwise to a sodium nitrite solution to obtain a diazotization reaction solution; the temperature of the diazotization reaction is preferably 0-4°C, more preferably 0-2°C, and most preferably 0°C; the reaction time is preferably 0.5-2h, more preferably 0.5-1h, and most preferably 0.5h; preferably, the phenol solution is added to the diazotization reaction liquid to obtain the coupling reaction liquid; the pH value of the coupling reaction liquid is preferably 7-9, more preferably 8-9; the reagent for adjusting the pH value is preferably sodium hydroxide, potassium hydroxide or sodium carbonate, more preferably sodium hydroxide or sodium carbonate; the sodium hydroxide is preferably used in the form of a 50% by mass sodium hydroxide aqueous solution; the coupling reaction temperature is preferably 0-4°C, more preferably 0-2°C, most preferably 0°C; the time is preferably 0.5-3h, more preferably 1-2h, most preferably 2h.

[0067] In the present invention, after the coupling reaction is completed, it is preferred that solid-liquid separation is further included, and the solid material obtained by the solid-liquid separation is purified to obtain the intermediate 1; the purification is preferably silica gel column chromatography purification; the eluent used for the silica gel column chromatography purification is preferably ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is preferably 1:1-8, more preferably 1:1-6, and most preferably 1:1.

[0068] After obtaining the intermediate 1, the present invention mixes the intermediate 1, a PEGylation agent and a first organic solvent, and performs a first nucleophilic substitution reaction under alkaline conditions to obtain an intermediate 2.

[0069] In the present invention, the -CH 2 CH 2 The number of O- is 1 to 3; the PEGylation reagent preferably includes ClCH 2 CH 2 OH, ClCH 2 CH 2 -O-CH 2 CH 2 OH, ClCH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 OH, BrCH 2 CH 2 OH, BrCH 2 CH 2-O-CH 2 CH 2 OH and BrCH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 One or more of OH, more preferably ClCH 2 CH 2 OH, ClCH 2 CH 2 -O-CH 2 CH 2 OH, BrCH 2 CH 2 OH and BrCH 2 CH 2 -O-CH 2 CH 2 One or more of OH, most preferably ClCH 2 CH 2 -O-CH 2 CH 2 OH; the molar ratio of the intermediate 1 to the PEGylation agent is preferably 1:1-30, more preferably 1:5-20, and most preferably 1:10.

[0070] In the present invention, the first organic solvent preferably includes one or more of dimethylformamide, dimethyl sulfoxide, acetonitrile and acetone, and more preferably dimethylformamide.

[0071] In the present invention, the regulating reagent for the alkaline condition is preferably potassium carbonate, more preferably anhydrous potassium carbonate; the intermediate 1, potassium carbonate and the first organic solvent are preferably heated to the temperature required for the nucleophilic substitution reaction, and then a PEGylation reagent is added to obtain a first nucleophilic substitution reaction solution; the molar ratio of the intermediate 1 to potassium carbonate is preferably 1:1-10, more preferably 1:2-6, and most preferably 1:3; the temperature of the first nucleophilic substitution reaction is preferably 80-110°C, more preferably 90-100°C, and most preferably 100°C; the reaction time is preferably 2-8h, more preferably 3-6h, and most preferably 3.5h.

[0072] In the present invention, after the first nucleophilic substitution reaction is completed, it is preferred that the reaction further includes pure water termination reaction, extraction, organic phase drying and purification in sequence; the extraction reagent is preferably ethyl acetate; the reagent used for drying the organic phase is preferably anhydrous sodium sulfate; the purification is preferably silica gel column chromatography purification; the eluent used for silica gel column chromatography purification is preferably ethyl acetate and petroleum ether; the volume ratio of ethyl acetate and petroleum ether is preferably 1-2:1-6, more preferably 1-2:1-3.

[0073] After obtaining the intermediate 2, the present invention mixes the intermediate 2, a sulfonylating agent, an organic base and a second organic solvent to carry out a second nucleophilic substitution reaction to obtain the azobenzene sulfonate compound.

[0074] In the present invention, the sulfonylating agent is preferably an alkylsulfonyl chloride. Substituted alkylsulfonyl chloride or substituted arylsulfonyl chloride The carbon number p of the alkyl sulfonyl chloride is preferably 1 to 3, more preferably 1; the carbon number m of the substituted alkyl sulfonyl chloride is preferably 1 to 3, more preferably 1 to 2; the substituent X of the substituted alkyl sulfonyl chloride includes halogen, amino, nitro, hydroxyl, preferably halogen; the halogen is preferably F, Cl, Br; the substituent Y of the substituted aryl sulfonyl chloride is preferably alkyl, halogen, amino, more preferably alkyl, and the carbon number of the alkyl is preferably 1 to 3, more preferably 1; the sulfonylating agent preferably includes one or more of methanesulfonyl chloride, trifluoromethanesulfonyl chloride and p-toluenesulfonyl chloride, more preferably p-toluenesulfonyl chloride; the organic base preferably includes one or more of pyridine, 4-dimethylaminopyridine and triethylamine, more preferably triethylamine; the molar ratio of the intermediate 2, the sulfonylating agent and the organic base is preferably 1:1 to 5:1 to 5, more preferably 1:1 to 2:1 to 3, and most preferably 1:1.5:2. In the present invention, the organic base acts as a catalyst and an acid-binding agent for the sulfonylation reaction to enhance the reaction activity, absorb the acid generated in the reaction, and improve the reaction performance and reaction yield.

[0075] In the present invention, the second organic solvent includes one or more of dichloromethane, acetonitrile and dimethyl sulfoxide, more preferably dichloromethane.

[0076] In the present invention, the intermediate 2, the organic base and part of the second organic solvent are preferably mixed to obtain solution 1, the sulfonylating agent and the remaining second organic solvent are mixed to obtain solution 2, and solution 2 is added to solution 1 to cause a second nucleophilic substitution reaction; the present invention does not have any special limitation on the amount of the solvent, and the amount well known to those skilled in the art can be used to ensure smooth reaction; the second nucleophilic substitution reaction preferably includes a first stage reaction and a second stage reaction carried out in sequence; the temperature of the first stage reaction is preferably 0 to 4°C, more preferably 0 to 2°C; the reaction time is preferably 5 to 30 min, more preferably 10 to 30 min, and most preferably 10 min; the temperature of the second stage reaction is preferably 20 to 35°C, more preferably 20 to 30°C; the reaction time is preferably 3 to 8 h, more preferably 4 to 6 h.

[0077] In the present invention, the second nucleophilic substitution reaction preferably further includes pure water termination reaction, extraction, organic phase drying and purification in sequence; the extraction reagent is preferably dichloromethane; the reagent used for drying the organic phase is preferably anhydrous sodium sulfate; the purification is preferably preparative thin layer chromatography purification; the developing solution used for the preparative thin layer chromatography purification is preferably ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is preferably 1:1-2, more preferably 1:1.5-2.

[0078] The preparation method of the azobenzene sulfonate compound provided by the invention comprises three-step reactions of azotization, condensation and nucleophilic substitution, has fewer reaction steps and better yield of the azobenzene sulfonate compound.

[0079] The present invention also provides the use of the azobenzenesulfonate compound described in the above technical solution in the preparation of a radioactive probe, a radioactive probe kit, a radioactive imaging agent or a radioactive imaging kit.

[0080] In the present invention, the radioactive probe is preferably a fluorine-18 radioactive probe or a hydrogen-3 radioactive probe.

[0081] In the present invention, the radioactive imaging agent is preferably an imaging agent for detecting neurodegenerative diseases associated with abnormal protein aggregates; the protein aggregates preferably include one or more of β-amyloid protein aggregates, tau protein aggregates and α-synuclein aggregates; the neurodegenerative diseases preferably include one or more of Alzheimer's disease, cerebral amyloid angiopathy, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick's disease, primary age-related tauopathy, globular cell tauopathy, Parkinson's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure and Alzheimer's disease Lewy body variant.

[0082] In the present invention, the imaging of the radioactive imaging agent is preferably positron emission tomography, positron emission tomography-computed tomography or positron emission tomography-magnetic resonance imaging.

[0083] The present invention also provides a method for preparing a radioactive probe, comprising the following steps:

[0084] In the presence of a phase transfer catalyst, the azobenzene sulfonate compound and the fluorine-18 anion ([ 18 F]F - ) is mixed with an ultra-dry solvent, and subjected to a radioactive organic chemical reaction to obtain the protein aggregate targeting probe; the radioactive probe has a structure shown in Formula IV:

[0085]

[0086] Among them, R is n is 1 to 3.

[0087] In the present invention, the radiolabeling route of the azobenzenesulfonate compound as a radiolabeling precursor to prepare a radioactive probe is as follows:

[0088]

[0089] In the present invention, the phase transfer catalyst is preferably kryptofix 2.2.2. / Potassium carbonate (K 2.2.2. / K 2 CO 3 ), one or more of tetrabutylammonium hydroxide and tetrabutylammonium bicarbonate, preferably kryptofix 2.2.2. / K 2 CO 3 or tetrabutylammonium hydroxide, most preferably kryptofix 2.2.2. / K 2 CO 3 ; the kryptofix 2.2.2. / K 2 CO 3 Kryptofix 2.2.2. and K 2 CO 3 The mass ratio of is preferably 1 to 5:1, more preferably 2 to 4:1, and most preferably 4:1; the phase transfer catalyst is preferably dissolved in a certain volume ratio of acetonitrile-water solution to prepare an eluent to play a role; the volume ratio of acetonitrile and water is preferably 1 to 49:1, more preferably 3 to 20:1, and most preferably 4:1; in the present invention, it is preferred that [ 18 F]F - The enriched QMA column is eluted into the reaction tube with 1.0 mL of eluent; the QMA column is preferably wetted with 5 mL of ethanol, 10 mL of 0.5 mol / L sodium carbonate and 20 mL of air; the [ 18 F]F - The activity of is preferably 50 to 200 mCi, more preferably 100 to 180 mCi; the [ 18 F]F - After eluting into the reaction tube, it is also preferred to sequentially include nitrogen blowing and organic solvent azeotropic heating and drying; the organic solvent in the organic solvent azeotropic heating and drying is preferably anhydrous acetonitrile; the temperature of the organic solvent azeotropic heating and drying is preferably 100-120° C., more preferably 105° C., the time is preferably 1-10 min, more preferably 3 min, the number of times is preferably 1-5 times, more preferably 3 times; the nitrogen blowing and organic solvent azeotropic heating and drying have the effect of ensuring that the reaction system is anhydrous and dry.

[0090] In the present invention, the mass of the azobenzene sulfonate compound is preferably 1 to 5 mg, more preferably 2 to 4 mg, and most preferably 3 mg; the ultra-dry solvent preferably includes one or more of dimethyl sulfoxide, anhydrous acetonitrile and dimethylformamide, more preferably dimethyl sulfoxide or anhydrous acetonitrile, and most preferably dimethyl sulfoxide; the volume of the ultra-dry solvent is preferably 0.2 to 1 mL, more preferably 0.4 to 0.8 mL, and most preferably 0.5 mL.

[0091] In the present invention, the azobenzene sulfonate compound is preferably dissolved in an ultra-dry solvent to obtain a precursor solution, and the precursor solution is added to a reaction tube to perform a radioactive organic chemical reaction. The radioactive organic chemical reaction is preferably performed in a closed environment; the temperature of the radioactive organic chemical reaction is preferably 105 to 150° C., more preferably 120 to 140° C., and most preferably 130° C.; the reaction time is preferably 5 to 15 min, more preferably 5 to 10 min, and most preferably 5 min.

[0092] In the present invention, the radiation organic chemical reaction preferably further includes a pure water termination reaction, solid phase extraction column enrichment, elution, purification and collection in sequence; the solid phase extraction column is preferably Sep-Pak C 18 Small column; Sep-PakC 18 The small column is preferably moistened with 5 mL of ethanol, 5 mL of purified water and 10 mL of air; the eluting reagent is preferably ethanol; the purification is preferably liquid chromatography separation and purification; the mobile phase of the liquid chromatography separation and purification is preferably acetonitrile, water and trifluoroacetic acid, the volume ratio of acetonitrile, water and trifluoroacetic acid is preferably 50-90:10-50:0-1%, more preferably 60:40:0.1%, and the flow rate is preferably 2.0-4.0 mL / min, more preferably 3.0 mL / min.

[0093] The preparation method of the radioactive probe provided by the invention is simple and fast, labeling is rapid, and the radiochemical yield of the obtained target is high.

[0094] The present invention also provides the use of the radioactive probe described in the above technical solution in the preparation of imaging agents.

[0095] In the present invention, the imaging agent is preferably an imaging agent for detecting neurodegenerative diseases, more preferably an imaging agent for detecting neurodegenerative diseases associated with abnormal protein aggregates; the protein aggregates are one or more of β-amyloid protein aggregates, tau protein aggregates and α-synuclein aggregates; the neurodegenerative diseases include Alzheimer's disease, cerebral amyloid angiopathy, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick's disease, primary age-related tauopathy, globular glial tauopathy, Parkinson's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure and Lewy body variants of Alzheimer's disease. The present invention does not have any special restrictions on the application process of the radioactive probe in the preparation of the imaging agent, and the method well known to those skilled in the art can be used.

[0096] To further illustrate the present invention, the azobenzenesulfonate compound provided by the present invention, its preparation method and application, and the preparation method of the radioactive probe are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0097] The synthetic route of the azobenzene sulfonate compound in Examples 1 to 3 of the present invention is as follows: Figure 1 As shown, the reaction reagents and conditions involved are: a. sodium nitrite, hydrochloric acid, ice bath for 0.5h; phenol, sodium hydroxide, ice bath for 2h; b. 2-(2-chloroethoxy)ethanol, dimethylformamide, 100°C, 3.5h; c. p-Toluenesulfonyl chloride, triethylamine, dichloromethane, ice bath for 10min and then room temperature for 4-6h. The specific synthesis steps are described in Examples 1-3.

[0098] The radiolabeling route for preparing the radioactive probe by using the azobenzenesulfonate compounds of Examples 1 to 3 in Examples 4 to 6 of the present invention is as follows: Figure 2 As shown, the reaction reagents and conditions involved are a.[ 18 F]F - , K 2.2.2. / K 2 CO 3 , anhydrous dimethyl sulfoxide, 130°C, 5 min. The specific labeling steps are as described in Examples 4 to 6.

[0099] Example 1

[0100] Synthesis of (E)-2-(2-(4-(benzo[d]thiazol-6-yldiazene))phenoxy)ethoxy)ethyl-4-methylbenzenesulfonate (denoted as TsO-A), the structure of the compound is shown below:

[0101]

[0102] Under ice bath conditions, sodium nitrite (155.2 mg, 2.25 mmol) was dissolved in deionized water (1.24 mL) to obtain a sodium nitrite solution; 6-aminobenzothiazole (150.2 mg, 1.0 mmoL) was dissolved in 37% concentrated hydrochloric acid (1.0 mL) to obtain a concentrated hydrochloric acid solution of 6-aminobenzothiazole; phenol (94.1 mg, 1.0 mmoL) was dissolved in 50% sodium hydroxide aqueous solution (0.2 mL) to obtain a sodium hydroxide aqueous solution of phenol; the concentrated hydrochloric acid solution of 6-aminobenzothiazole was added dropwise to the sodium nitrite solution, the obtained feed solution was stirred and reacted under ice bath conditions for 0.5 h, and then the sodium hydroxide aqueous solution of phenol was added dropwise, the pH value of the system was adjusted to 7-9 with a 50% sodium hydroxide aqueous solution, and the stirring reaction was continued for 2 h while maintaining the ice bath conditions. After the reaction is completed, the obtained liquid is filtered, and the filter cake is separated by column chromatography using ethyl acetate and petroleum ether as eluents (the volume ratio of ethyl acetate and petroleum ether in the eluent is 1:2). After elution, a brown-yellow solid is obtained (referred to as intermediate 1-A, 109.8 mg, yield 43.0%).

[0103] The structure of intermediate 1-A is as follows:

[0104]

[0105] The NMR data of intermediate 1-A are as follows: 1 H NMR (400 MHz, DMSO-d 6 )δ10.37(s,1H),9.50(s,1H),8.64(d,J=1.9Hz,1H),8.21(d,J=8.8Hz,1H),8.01(dd,J=8.7,2.0Hz,1H),7.91-7.82(m,2H),7.03-6.93(m,2H).

[0106] In a round-bottom flask, intermediate 1-A (383.0 mg, 1.5 mmol), dimethylformamide (8 mL) and anhydrous potassium carbonate (652.0 mg, 4.5 mmol) were added, and the resulting solution was heated to 100°C, and then 2-(2-chloroethoxy)ethanol (1.6 mL, 15.0 mmol) was added dropwise to the solution, and the temperature was maintained at 100°C for 3.5 h. After the reaction was completed, an appropriate amount of pure water was used to terminate the reaction, and the resulting solution was extracted with ethyl acetate, and the extracted organic phase was dried with anhydrous sodium sulfate, and then column chromatography was performed with ethyl acetate and petroleum ether as eluents (the volume ratio of ethyl acetate to petroleum ether in the eluent was 1:1), to obtain an orange-yellow solid (referred to as intermediate 2-A, 257.6 mg, yield 50.0%).

[0107] The structure of intermediate 2-A is as follows:

[0108]

[0109] The NMR data of intermediate 2-A are as follows: 1 HNMR (400MHz, CDCl 3 )δ9.06(s,1H),8.45(s,1H),8.21-8.19(d,J=8.3Hz,1H),8.09-8.07(d,J=8.7Hz,1H),7.94-7.92(d,J=8.2Hz,2H), 7.03-7.01(d,J=8.2Hz,2H),4.22-4.19(m,2H),3.90-3.88(m,2H),3.80-3.77(m,2H),3.70-3.67(d,J=4.8Hz,2H).

[0110] Under ice bath conditions, the intermediate 2-A (439.6 mg, 1.28 mmol) was dissolved in dichloromethane (11 mL), and triethylamine (0.42 mL, 3.0 mmol) was added. p-Toluenesulfonyl chloride (356.5 mg, 1.87 mmol) was dissolved in dichloromethane (3 mL), added dropwise to the above mixture, stirred in an ice bath for 10 min, and then transferred to room temperature for stirring and reacting for 4 h. After the reaction was completed, an appropriate amount of water was used to terminate the reaction, and the solution after the reaction was extracted with dichloromethane. The obtained organic phase was dried and concentrated, and then purified by preparative thin layer chromatography with a developing solution of ethyl acetate and petroleum ether in a volume ratio of 1:2. An orange-yellow solid (TsO-A, 190 mg, yield 29.9%) was obtained.

[0111] The NMR data of TsO-A are as follows:

[0112] 1 H NMR (400 MHz, CDCl 3 )δ9.08(s,1H),8.49(d,J=1.9Hz,1H),8.23(d,J=8.8Hz,1H),8.11(dd,J=8.7,1.9Hz,1H),7.95(d,J=8.9Hz,2H),7.80(d,J=8.3Hz,2H), 7.31(d,J=8.0Hz,2H),7.02(d,J=9.0Hz,2H),4.24-4.19(m,2H),4.17-4.12(m,2H),3.87-3.82(m,2H),3.81-3.77(m,2H),2.41(s,3H).

[0113] Example 2

[0114] Synthesis of (E)-2-(2-(4-(quinolin-6-yldiazene))phenoxy)ethoxy)ethyl-4-methylbenzenesulfonate (denoted as TsO-B), the structure of the compound is shown below:

[0115]

[0116] Under ice bath conditions, sodium nitrite (155.2 mg, 2.25 mmol) was dissolved in deionized water (1.24 mL) to obtain a sodium nitrite solution; 6-aminoquinoline (144.2 mg, 1.0 mmoL) was dissolved in 37% concentrated hydrochloric acid (1 mL) to obtain a concentrated hydrochloric acid solution of 6-aminoquinoline; phenol (94.1 mg, 1.0 mmoL) was dissolved in 50% sodium hydroxide aqueous solution (0.2 mL) to obtain a phenol sodium hydroxide aqueous solution; the 6-aminoquinoline concentrated hydrochloric acid solution was added dropwise to the sodium nitrite solution, the obtained feed solution was stirred and reacted under ice bath conditions for 0.5 h, and then the phenol sodium hydroxide aqueous solution was added dropwise, the pH value of the system was adjusted to 7-9 with 50% sodium hydroxide, and the stirring reaction was continued for 2 h while maintaining the ice bath conditions. After the reaction, the obtained liquid was filtered, and the filter cake was separated by column chromatography using ethyl acetate and petroleum ether as eluents (the volume ratio of ethyl acetate and petroleum ether in the eluent was 1:2) to obtain an orange solid (referred to as intermediate 1-B, 97.5 mg, yield 40.1%).

[0117] The structure of intermediate 1-B is as follows:

[0118]

[0119] The NMR data of intermediate 1-B are as follows: 1 H NMR (400 MHz, DMSO-d 6 )δ10.40(s,1H),8.96(d,J=2.5Hz,1H),8.57(d,J=8.3Hz,1H),8.49(s,1H),8.21(d,J=9.0Hz,1H) ,8.12(d,J=9.1Hz,1H),7.88(d,J=8.5Hz,2H),7.62(dd,J=8.3,4.2Hz,1H),6.98(d,J=8.6Hz,2H).

[0120] The above intermediate 1-B (249.0 mg, 1.0 mmol), dimethylformamide (8 mL) and anhydrous potassium carbonate (414.0 mg, 3.0 mmol) were added to a round-bottom flask, and the resulting solution was heated to 100°C, and then 2-(2-chloroethoxy)ethanol (1.0 mL, 10.0 mmol) was added dropwise to the solution, and the temperature was maintained at 100°C for 3.5 hours. After the reaction was completed, an appropriate amount of pure water was used to terminate the reaction, and the resulting solution was extracted with ethyl acetate, and the extracted organic phase was dried with anhydrous sodium sulfate, and then column chromatography was performed with ethyl acetate and petroleum ether as eluents (the volume ratio of ethyl acetate to petroleum ether in the eluent was 1:1), and an orange-yellow solid (referred to as intermediate 2-B, 146.2 mg, yield 43.3%) was obtained.

[0121] The structure of intermediate 2-B is as follows:

[0122]

[0123] The NMR data of intermediate 2-B are as follows: 1 H NMR (400 MHz, CDCl 3 )δ8.95(dd,J=4.3,1.7Hz,1H),8.31(ddd,J=18.6,10.8,2.2Hz,3H),8.17(d,J=9.1Hz,1H),7.98(dd,J=9.4,2.8Hz,2H),7.46(dd,J =8.3,4.3Hz,1H),7.06(dd,J=9.4,2.8Hz,2H),4.23(t,J=4.7Hz,2H),3.91(t,J=4.6Hz,2H),3.85-3.76(m,2H),3.76-3.67(m,2H).

[0124] Under ice bath conditions, the intermediate 2-B (168.7 mg, 0.5 mmol) was dissolved in dichloromethane (5 mL), and triethylamine (0.14 mL, 1.0 mmol) was added. p-Toluenesulfonyl chloride (143 mg, 0.75 mmol) was dissolved in dichloromethane (1 mL), added dropwise to the above mixture, stirred in an ice bath for 10 min, and then transferred to room temperature for stirring and reacting for 6 h. After the reaction was completed, an appropriate amount of water was used to terminate the reaction, and the solution after the reaction was extracted with dichloromethane. The obtained organic phase was dried and concentrated, and then purified by preparative thin layer chromatography with a developing solution of ethyl acetate and petroleum ether in a volume ratio of 1:1. An orange-yellow solid (TsO-B, 142.7 mg, yield 58.1%) was obtained.

[0125] The NMR data of TsO-B are as follows:

[0126] 1 H NMR (400 MHz, CDCl 3 )δ8.96(dd,J=4.3,1.7Hz,1H),8.36(d,J=2.1Hz,1H),8.34-8.27(m,2H),8. 18(d,J=9.1Hz,1H),7.98(d,J=8.9Hz,2H),7.80(d,J=8.3Hz,2H),7.48(dd,J =8.2,4.3Hz,1H),7.31(d,J=8.1Hz,2H),7.03(d,J=9.0Hz,2H),4.26-4.19(m ,2H),4.19-4.12(m,2H),3.88-3.82(m,2H),3.82-3.77(m,2H),2.41(s,3H).

[0127] Example 3

[0128] Synthesis of (E)-2-(2-(4-(quinoxaline-6-yldiazene))phenoxy)ethoxy)ethyl-4-methylbenzenesulfonate (denoted as TsO-C), the structure of the compound is shown below:

[0129]

[0130] Under ice bath conditions, sodium nitrite (155.2 mg, 2.25 mmol) was dissolved in deionized water (1.24 mL) to obtain a sodium nitrite solution; 6-aminoquinoxaline (145.2 mg, 1.0 mmoL) was dissolved in 37% concentrated hydrochloric acid (1.0 mL) to obtain a concentrated hydrochloric acid solution of 6-aminoquinoxaline; phenol (94.1 mg, 1.0 mmoL) was dissolved in 50% sodium hydroxide aqueous solution (0.2 mL) to obtain a phenol sodium hydroxide aqueous solution; the 6-aminoquinoxaline concentrated hydrochloric acid solution was added dropwise to the sodium nitrite solution, the obtained feed solution was stirred and reacted under ice bath conditions for 0.5 h, and then the phenol sodium hydroxide aqueous solution was added dropwise, the pH value of the system was adjusted to 7-9 with 50% sodium hydroxide, and the stirring reaction was continued for 2 h while maintaining the ice bath conditions. After the reaction is completed, the obtained liquid is filtered, and the filter cake is separated by column chromatography using ethyl acetate and petroleum ether as eluents (the volume ratio of ethyl acetate and petroleum ether in the eluent is 1:1). After elution, an orange-yellow solid is obtained (referred to as intermediate 1-C, 45.4 mg, yield 18.1%).

[0131] The structure of intermediate 1-C is as follows:

[0132]

[0133] The NMR data of intermediate 1-C are as follows:1 H NMR (400 MHz, DMSO-d 6 )δ10.46(s,1H),9.00(d,J=1.9Hz,1H),8.96(d,J=1.8Hz,1H),8.43(d,J=2.1Hz,1H ),8.25(dd,J=9.0,2.1Hz,1H),8.19(s,1H),7.91-7.85(m,2H),6.98-6.92(m,2H).

[0134] In a round-bottom flask, add intermediate 1-C (250.0 mg, 1.0 mmol), dimethylformamide (8 mL) and anhydrous potassium carbonate (414.0 mg, 3.0 mmol), heat the resulting solution to 100°C, then add 2-(2-chloroethoxy)ethanol (1.0 mL, 10.0 mmol) dropwise to the solution, and maintain the temperature at 100°C for 3.5 h. After the reaction, terminate the reaction with an appropriate amount of pure water, extract the resulting solution with ethyl acetate, dry the extracted organic phase with anhydrous sodium sulfate, and then perform column chromatography separation with ethyl acetate and petroleum ether as eluents (the volume ratio of ethyl acetate to petroleum ether in the eluent is 1:1), and obtain an orange-yellow solid (referred to as intermediate 2-C, 204.0 mg, yield 60.3%).

[0135] The structure of intermediate 2-C is as follows:

[0136]

[0137] The NMR data of intermediate 2-C are as follows: 1 H NMR (400 MHz, CDCl 3 )δ8.90(dd,J=18.5,1.9Hz,2H),8.61(d,J=2.2Hz,1H),8.35(dd,J=9.0,2.2Hz,1H),8.18(d,J=9.0Hz,1H),8.02(d ,J=8.9Hz,2H),7.08(d,J=9.0Hz,2H),4.30-4.22(m,2H),3.97-3.90(m,2H),3.83-3.76(m,2H),3.74-3.68(m,2H).

[0138] Under ice bath conditions, the intermediate 2-C (101.6 mg, 0.3 mmol) was dissolved in dichloromethane (3 mL), and triethylamine (0.082 mL, 0.6 mmol) was added. p-Toluenesulfonyl chloride (86 mg, 0.45 mmol) was dissolved in dichloromethane (0.5 mL), added dropwise to the above mixture, stirred in an ice bath for 10 min, and then transferred to room temperature for stirring and reacting for 5.0 h. After the reaction was completed, an appropriate amount of water was used to terminate the reaction, and the solution after the reaction was extracted with dichloromethane. The obtained organic phase was dried and concentrated, and then purified by preparative thin layer chromatography with a developing solution of ethyl acetate and petroleum ether in a volume ratio of 1:1. An orange-yellow solid (TsO-C, 38.1 mg, yield 25.8%) was obtained.

[0139] The NMR data of TsO-C are as follows:

[0140] 1 H NMR (400 MHz, CDCl 3 )δ8.89(dd,J=18.3,1.9Hz,2H),8.60(d,J=2.1Hz,1H),8.34(dd,J=9.0,2.2Hz,1H),8.18(d,J=9.0Hz,1H),8.01(d,J=8.9Hz,2H),7.80(d,J=8.0 Hz,2H),7.31(d,J=8.0Hz,2H),7.03(d,J=9.0Hz,2H),4.24-4.19(m,2H) ,4.18-4.14(m,2H),3.87-3.82(m,2H),3.81-3.77(m,2H),2.41(s,3H).

[0141] Example 4

[0142] The azobenzenesulfonate compound TsO-A was used to prepare the fluorine-18 radioactive probe (denoted as [ 18 F]FA), the preparation chemical formula of the compound is as follows:

[0143]

[0144] The azobenzenesulfonate compound TsO-A is radiolabeled to prepare a fluorine-18 radioactive probe [ 18 The method of F]FA is:

[0145] Take about 150mCi[ 18 F]F - The enrichment was carried out on the QMA column and eluted with 1.0 mL of eluent (containing K 2.2.2 2.6mg, K 2 CO 30.7 mg, 80% acetonitrile, 20% water) 18 F]F - Slowly elute from the QMA column into a 10 mL glass reaction tube, heat at 105 °C, and blow dry with nitrogen. Then add 1 mL of anhydrous acetonitrile and azeotropically dry three times to fully remove the water in the system. Accurately weigh 3.22 mg of TsO-A, dissolve in 0.5 mL of anhydrous dimethyl sulfoxide, and transfer the solution to a container containing [ 18 F]F - The reaction tube was sealed and heated at 130°C for 5 min. After the reaction, the reaction tube was transferred to room temperature and purified water was added to terminate the reaction. An appropriate amount of the reaction solution was mixed with the reference substance and co-injected for high performance liquid chromatography detection. The chromatograph was Agilent 1260 and the chromatographic column was Agilent ZORBAX StableBond C 18 (4.6×250mm, 5μm), the mobile phase was acetonitrile: water (volume ratio of 70:30), the flow rate was 1mL / min, and the detector was a dual detector: γ-detector (Bioscan flow-count FC3200) and UV detector. The obtained chromatogram is shown in Figure 3 (A), the retention time difference between the main component peak in the reaction solution radiogram and the main component peak in the UV spectrum of the reference substance is small (the difference is less than 0.1min, caused by the γ-detector and UV detector connected in series), confirming that the main component peak in the reaction solution radiogram is [ 18 F]FA, indicating that [ 18 F]FA.

[0146] The reaction solution was passed through a pre-treated Sep-PakPlus C 18 The free fluoride ions and inorganic salts in the column were eluted with 40 mL of purified water. Then, 0.8 mL of anhydrous ethanol was used to slowly elute the labeled compounds enriched on the column. The eluent was separated by preparative liquid chromatography. The preparative liquid chromatography conditions were: the chromatographic column was Alltech ChromBDS C 18 column (10×250mm, 10μm), the mobile phase was acetonitrile: water: trifluoroacetic acid (volume ratio of 60:40:0.1%), the flow rate was 3mL / min, and the γ-detector (Bioscan flow-count FC3200) was used to measure the radioactive signal. The separated liquid was purified and collected to obtain a high-purity fluorine-18 radioactive probe [ 18 F]FA, the radiochemical yield was 80%, the radiochemical purity was greater than 99%, the specific activity was 19.1 GBq / μmol, and the total radiolabeling time was about 60 min.

[0147] Example 5

[0148] The azobenzenesulfonate compound TsO-B was used to prepare the fluorine-18 radioactive probe (denoted as [ 18 F]FB), the preparation chemical formula of the compound is as follows:

[0149]

[0150] The azobenzenesulfonate compound TsO-B is radiolabeled to prepare a fluorine-18 radioactive probe [ 18 F]FB method is:

[0151] Take about 120mCi[ 18 F]F - The enrichment was carried out on the QMA column and eluted with 1.0 mL of eluent (containing K 2.2.2 2.6mg, K 2 CO 3 0.7 mg, 80% acetonitrile, 20% water) 18 F]F - Slowly elute from the QMA column into a 10 mL glass reaction tube, heat at 105 °C, and blow dry with nitrogen. Then add 1 mL of anhydrous acetonitrile and azeotropically dry three times to fully remove the water in the system. Accurately weigh 2.8 mg of the precursor TsO-B, dissolve it in 0.5 mL of anhydrous dimethyl sulfoxide, and transfer the solution to a container containing [ 18 F]F - The reaction tube was sealed and heated at 130°C for 5 min. After the reaction, the reaction tube was transferred to room temperature and purified water was added to terminate the reaction. An appropriate amount of the reaction solution was mixed with the reference substance and co-injected for high performance liquid chromatography detection. The chromatograph was Agilent 1260 and the chromatographic column was Agilent ZORBAX StableBond C 18 (4.6×250mm, 5μm), the mobile phase was acetonitrile: water (volume ratio of 70:30), the flow rate was 1mL / min, and the detector was a dual detector: γ-detector (Bioscan flow-count FC3200) and UV detector. The obtained chromatogram is shown in Figure 3 (B), the retention time difference between the main component peak in the reaction solution radiogram and the main component peak in the UV spectrum of the reference substance is small (the difference is less than 0.1min, caused by the tandem connection of the γ-detector and the UV-visible detector), confirming that the main component peak in the reaction solution radiogram is [ 18 F]FB, indicating that [ 18 F]FB.

[0152] The reaction solution was passed through a pre-treated Sep-PakPlus C 18The free fluoride ions and inorganic salts in the column were eluted with 40 mL of purified water. Then, 0.6 mL of anhydrous ethanol was used to slowly elute the labeled compounds enriched on the column. The eluent was separated by preparative liquid chromatography. The preparative liquid chromatography conditions were: the chromatographic column was Alltech Chrom BDS C 18 column (10×250mm, 10μm), the mobile phase was acetonitrile: water: trifluoroacetic acid (volume ratio of 60:40:0.1%), the flow rate was 3mL / min, and the γ-detector (Bioscan flow-count FC3200) was used to measure the radioactive signal. The separated liquid was purified and collected to obtain a high-purity fluorine-18 radioactive probe [ 18 F]FB, the radiochemical yield was 70%, the radiochemical purity was greater than 99%, the specific activity was 6.6 GBq / μmol, and the total radiolabeling time was about 60 min.

[0153] Example 6

[0154] The azobenzenesulfonate compound TsO-C was used to prepare the fluorine-18 radioactive probe (denoted as [ 18 F]FC), the preparation chemical formula of the compound is as follows:

[0155]

[0156] The azobenzenesulfonate compound TsO-C is radiolabeled to prepare a fluorine-18 radioactive probe [ 18 The method of F]FC is:

[0157] Take about 200mCi[ 18 F]F - The enrichment was carried out on the QMA column and eluted with 1.0 mL of eluent (containing K 2.2.2 2.6mg, K 2 CO 3 0.7 mg, 80% acetonitrile, 20% water) 18 F]F - Slowly elute from the QMA column into a 10 mL glass reaction tube, heat at 105 °C, and blow dry with nitrogen. Then add 1 mL of anhydrous acetonitrile and azeotropically dry three times to fully remove the water in the system. Accurately weigh 3.0 mg of the precursor TsO-C, dissolve it in 0.5 mL of anhydrous dimethyl sulfoxide, and transfer the solution to a container containing [ 18 F]F -The reaction tube was sealed and heated at 130°C for 5 min. After the reaction, the reaction tube was transferred to room temperature and purified water was added to terminate the reaction. An appropriate amount of the reaction solution was mixed with the reference substance and co-injected for high performance liquid chromatography detection. The chromatograph was Agilent 1260 and the chromatographic column was Agilent ZORBAX StableBond C 18 (4.6×250mm, 5μm), the mobile phase was acetonitrile: water (volume ratio of 70:30), the flow rate was 1mL / min, and the detector was a dual detector: γ-detector (Bioscan flow-count FC3200) and UV detector. The obtained chromatogram is shown in Figure 3 (C) The retention time difference between the main component peak in the radiogram of the reaction solution and the main component peak in the UV spectrum of the reference substance is small (the difference is less than 0.1min, caused by the tandem connection of the γ-detector and the UV-visible detector). It is confirmed that the main component peak in the radiogram of the reaction solution is [ 18 F]FC, indicating that [ 18 F]FC.

[0158] The reaction solution was passed through a pre-treated Sep-PakPlus C 18 The free fluoride ions and inorganic salts in the column were eluted with 40 mL of purified water. Then, 0.8 mL of anhydrous ethanol was used to slowly elute the labeled compounds enriched on the column. The eluent was separated by preparative liquid chromatography. The preparative liquid chromatography conditions were: the chromatographic column was Alltech ChromBDS C 18 column (10×250mm, 10μm), the mobile phase was acetonitrile: water: trifluoroacetic acid (volume ratio of 60:40:0.1%), the flow rate was 3mL / min, and the γ-detector (Bioscan flow-count FC3200) was used to measure the radioactive signal. The separated liquid was purified and collected to obtain a high-purity fluorine-18 radioactive probe [ 18 F]FC, the radiochemical yield was 80%, the radiochemical purity was greater than 99%, the specific activity was 41.3 GBq / μmol, and the total radiolabeling time was about 60 min.

[0159] The above examples demonstrate that the method for preparing radioactive probes using the azobenzenesulfonate compounds provided by the present invention as radiolabeling precursors is simple, fast, and has a rapid labeling process. The radiochemical yield of the obtained radioactive probes is high.

[0160] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and test examples, but the present invention is not limited to the test examples.

[0161] Test Example 1

[0162] Fluorine-18 radioactive probe 18 Stability of F]FA in normal mouse plasma

[0163] The fluorine-18 radioactive probe ([ 18 F]FA) (500 μCi / 150 μL) was added to two normal mouse plasmas (150 μL) and mixed at room temperature in the dark. Chromatographic grade acetonitrile (300 μL) was added at 0.5 h and 1 h after mixing. The resulting mixture was then centrifuged at 4000 rpm for 5 min in a centrifuge (Heraeus Biofuge 13), and the supernatant was filtered through a 0.25 μm filter membrane and analyzed by HPLC. Chromatographic conditions: Agilent 1260 chromatograph; Agilent ZORBAX StableBond C column 18 , (4.6×250mm, 5μm); the mobile phase was acetonitrile: water (volume ratio of 70:30); flow rate: 1mL / min, and the radiation signal was measured by a γ-detector (Bioscan flow-countFC3200).

[0164] Depend on Figure 4 It is known that the fluorine-18 radioactive probe [ 18 When F]FA was mixed with normal mouse plasma for 0.5h or 1.0h, its chromatogram was the same as that when it was mixed with plasma for 0h. Figure 1 No new radioactive chromatographic peak appeared, indicating that the fluorine-18 radioactive probe prepared by the azobenzenesulfonate compound provided by the present invention did not degrade or produce new substances within 1 hour of mixing with plasma, and can be further used for in vivo analysis.

[0165] Test Example 2

[0166] Fluorine-18 radioactive probe 18 Stability experiment of F]FC in normal mouse plasma

[0167] The fluorine-18 radioactive probe ([ 18 F]FC) (300 μCi / 150 μL) was added to two normal mouse plasmas (150 μL) and mixed at room temperature in the dark. Chromatographic grade acetonitrile (300 μL) was added at 0.5 h and 1 h after mixing. The resulting mixture was then centrifuged at 4000 rpm for 5 min in a centrifuge (Heraeus Biofuge 13), and the supernatant was filtered through a 0.25 μm filter membrane and analyzed by HPLC. Chromatographic conditions: chromatograph Agilent 1260; chromatographic column Agilent ZORBAX StableBond C 18(4.6×250mm, 5μm); the mobile phase was acetonitrile:water (volume ratio of 70:30); the flow rate was 1mL / min, and the radiation signal was measured by a γ-detector (Bioscan flow-count FC3200).

[0168] Depend on Figure 5 It is known that the fluorine-18 radioactive probe [ 18 When F]FC was mixed with normal mouse plasma for 0.5h or 1.0h, its chromatogram was the same as that when it was mixed with plasma for 0h. Figure 1 No new radioactive chromatographic peak appeared, indicating that the fluorine-18 radioactive probe prepared by the azobenzenesulfonate compound provided by the present invention did not degrade or produce new substances within 1 hour of mixing with plasma, and can be further used for in vivo analysis.

[0169] Test Example 3

[0170] Autoradiography of fluorine-18 radioactive probe in brain tissue of patients with Alzheimer's disease (AD)

[0171] The fluorine-18 radioactive probe (30-50 μCi / 50 μL) obtained in Examples 4-6 was added dropwise to completely cover the brain tissue slice of an AD patient (male, temporal lobe, 85 years old, 6 μm). After incubation at room temperature in the dark for 1 h, the slice was washed twice with 30% ethanol (3 min each time), rinsed once with pure water (30 s each time), and the slice was naturally air-dried and exposed to a phosphor screen overnight. The phosphor screen was removed the next day and developed and analyzed using an imaging system (dual-mode multispectral laser imaging system, Azure Sapphire, USA).

[0172] After development, the same slice was subjected to immunofluorescence staining. The operation was as follows: the same slice was permeabilized with 0.01% Triton-100X solution at room temperature for 5 minutes, and then washed with PBS three times (5 minutes each time). Then, sheep serum working solution was added dropwise until the brain tissue was completely covered, and the slice was blocked at room temperature in the dark for 1.5 hours. Then, an appropriate amount of 6E10 antibody (dilution ratio of 1:500) was added dropwise until the brain tissue was completely covered, and the slice was incubated at 4°C in the dark overnight (15-20 hours). After the primary antibody incubation was completed, the slice was warmed up at room temperature for 1.0 hour. Then, freshly prepared PBST (phosphate buffer, pH 7.4, containing 0.3% Tween20) was used to wash three times (5 minutes each time). After washing, an appropriate amount of secondary antibody Aflex Fluor 647-goat anti-mouse solution (dilution ratio of 1:1000) was added dropwise until the brain tissue was completely covered, and the slice was incubated at room temperature in the dark for 2.0 hours. After incubation, the slice was washed three times (5 minutes each time) with freshly prepared PBST solution. After washing, an appropriate amount of anti-fluorescence quencher was added and the slices were sealed. The sealed slices were photographed using a fully automatic pathological imaging analysis system (VS200, Olympus, Japan). The imaging data was analyzed using OlyVIA. The results of the autoradiography and immunostaining of the fluorine-18 labeled probes obtained in Examples 4 to 6 and the patient's brain tissue slices are shown in Figure 6 As shown, AC: The autoradiography results of fluorine-18 radioactive probe in brain tissue sections of AD patients, (A) [ 18 F]FA,(B)[ 18 F]FB,(C)[ 18 F] FC; DF: 6E10 antibody immunostaining results of the same section as the autoradiography, D corresponds to A, E corresponds to B, and F corresponds to C. The radioactive signal corresponds to the location of Aβ plaques (indicated by the arrow).

[0173] Depend on Figure 6 It can be seen that the fluorine-18 radioactive probe ([ 18 F]FA,[ 18 F]FB,[ 18 A large number of radioactive signal points of F]-FC) accumulate on the patient's brain tissue slices, which can bind to and mark the pathological deposits in the AD patient's brain tissue slices. The radioactive signal deposition points are consistent with the immunostaining location of the β-amyloid protein antibody 6E10, indicating that the fluorine-18 radioactive probe prepared by the azobenzenesulfonate compound of the present invention can effectively image the pathological deposition of β-folded protein aggregates in the brain tissue of Alzheimer's patients and provide high-resolution images.

[0174] Test Example 4

[0175] Biodistribution of Fluorine-18 Radioactive Probe in Normal Mice

[0176] The fluorine-18 radioactive probes obtained in Examples 4 to 6 (10 to 20 μCi / 100 μL, physiological saline solution, containing 10% ethanol) were injected into normal Kunming mice (male, 25 to 28 g) via the tail vein. The mice were killed at 2 min, 10 min, 30 min, and 60 min after injection (3 mice at each time point), and the relevant organs were dissected. The radioactivity was counted using a γ-counter, and the wet weight was recorded. The data are expressed as the percentage dose of radioactivity per gram of organ (%ID / g). The biodistribution of the fluorine-18 radioactive probes obtained in Examples 4 to 6 in normal mice is shown in Tables 1 to 3.

[0177] Table 1 18 Biodistribution of F]FA in normal mice

[0178]

[0179] Table 2 Biodistribution of [18F]FB in normal mice

[0180]

[0181] Table 3 Biodistribution of [18F]FC in normal mice

[0182]

[0183] As can be seen from Tables 1 to 3, the fluorine-18 radioactive probe ([ 18 F]FA,[ 18 F]FB,[ 18 F]FC) After injection into mice, bone uptake in mice remained at a low level within 1 hour, indicating that no obvious defluorination of the probe occurred within 1 hour. At the same time, brain uptake reached a maximum at 2 minutes after injection, which were 6.96, 9.01 and 5.62% ID / g, respectively; the ratios of brain uptake values ​​at 2 minutes after injection and 60 minutes after injection were 2.36, 2.28 and 3.31, respectively. The above shows that the fluorine-18 radioactive probe prepared by the azobenzene sulfonate compound of the present invention can quickly enter the brain, and is effectively cleared from the brain over time, and has good nucleoside stability in vivo.

[0184] The above test examples once again prove that the fluorine-18 radioactive probe prepared by the azobenzene sulfonate compound provided by the present invention has good in vitro and in vivo stability, no obvious mouse toxicity reaction, can quickly enter the brain and effectively target the β-folded protein aggregates in the brain tissue of the imaging patient and provide high-resolution images. The azobenzene sulfonate compound provided by the present invention and the fluorine-18 radioactive probe prepared therefrom, as well as the radioactive probe kit, radioactive imaging agent or radioactive imaging kit based on the azobenzene sulfonate compound provided by the present invention provide a material basis for imaging for the early diagnosis, disease course monitoring and therapeutic drug research of diseases related to β-folded protein aggregates.

[0185] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An azobenzenesulfonate compound, characterized in that It has the structure shown in formula I: Wherein, R1 is an alkyl group, a substituted alkyl group or a substituted aryl group; R is n is 1 to 3.

2. The azobenzenesulfonate compound according to claim 1, characterized in that The carbon number of the alkyl group is 1 to 3; the carbon number of the substituted alkyl group is 1 to 3; the substituent of the substituted alkyl group is halogen, amino, nitro or hydroxyl; the substituent of the substituted aryl group is alkyl, halogen or amino.

3. The azobenzenesulfonate compound according to claim 1, characterized in that Having a structure as shown in any one of Formulas 1 to 27:

4. The method for preparing the azobenzenesulfonate compound according to any one of claims 1 to 3, characterized in that: The following steps are involved: A primary amine compound, sodium nitrite, phenol and water are mixed and subjected to a diazotization-coupling reaction under alkaline conditions to obtain an intermediate 1; the primary amine compound is 6-aminobenzothiazole, 6-aminoquinoline or 6-aminoquinoxaline; the intermediate 1 has a structure shown in Formula II: The intermediate 1, the PEGylation reagent and the first organic solvent are mixed, and a first nucleophilic substitution reaction is carried out under alkaline conditions to obtain the intermediate 2; the number of -CH2CH2O- in the PEGylation reagent is 1 to 3; the intermediate 2 has a structure shown in formula III: The intermediate 2, a sulfonylating agent, an organic base and a second organic solvent are mixed to carry out a second nucleophilic substitution reaction to obtain the azobenzene sulfonate compound; the sulfonylating agent is an alkylsulfonyl chloride, a substituted alkylsulfonyl chloride or a substituted arylsulfonyl chloride.

5. The preparation method according to claim 4, characterized in that: The PEGylation reagent includes one or more of ClCH2CH2OH, ClCH2CH2-O-CH2CH2OH, ClCH2CH2-O-CH2CH2-O-CH2CH2OH, BrCH2CH2OH, BrCH2CH2-O-CH2CH2OH and BrCH2CH2-O-CH2CH2-O-CH2CH2OH; The sulfonylating agent includes one or more of methanesulfonyl chloride, trifluoromethanesulfonyl chloride and p-toluenesulfonyl chloride; The organic base includes one or more of pyridine, 4-dimethylaminopyridine and triethylamine.

6. The preparation method according to claim 4, characterized in that: The temperature of the diazotization-coupling reaction is 0-4°C, and the reaction time is 1-5h; The temperature of the first nucleophilic substitution reaction is 80-110° C., and the reaction time is 2-8 hours; The second nucleophilic substitution reaction includes a first stage reaction and a second stage reaction carried out in sequence; the temperature of the first stage reaction is 0-4°C, and the reaction time is 5-30 minutes; the temperature of the second stage reaction is 20-35°C, and the reaction time is 3-8 hours.

7. Use of the azobenzenesulfonate compound according to any one of claims 1 to 3 in the preparation of a radioactive probe, a radioactive probe kit, a radioactive imaging agent or a radioactive imaging kit.

8. The use according to claim 7, characterized in that: The radioactive imaging agent is an imaging agent for detecting neurodegenerative diseases associated with abnormal protein aggregates; the protein aggregates include one or more of β-amyloid protein aggregates, tau protein aggregates and α-synuclein protein aggregates; the neurodegenerative diseases include one or more of Alzheimer's disease, cerebral amyloid angiopathy, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick's disease, primary age-related tauopathy, globular cell tauopathy, Parkinson's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure and Lewy body variant of Alzheimer's disease; The imaging of the radioactive imaging agent is positron emission tomography, positron emission tomography-computed tomography or positron emission tomography-magnetic resonance imaging.

9. A method for preparing a radioactive probe, characterized in that: The following steps are involved: In the presence of a phase transfer catalyst, the azobenzene sulfonate compound according to any one of claims 1 to 3, fluorine-18 anions and an ultra-dry solvent are mixed to perform a radioactive organic chemical reaction to obtain the radioactive probe; the radioactive probe has a structure shown in Formula IV: Among them, R is n is 1 to 3.

10. The preparation method according to claim 9, characterized in that:The phase transfer catalyst is kryptofix 2.2.

2. / one or more of potassium carbonate, tetrabutylammonium hydroxide and tetrabutylammonium bicarbonate; The radiation organic chemical reaction is carried out in a closed environment; the temperature of the radiation organic chemical reaction is 105-150° C., and the reaction time is 5-15 minutes.

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