CN109400615B

CN109400615B - Beta-amyloid targeted coumarin compound and preparation and application thereof

Info

Publication number: CN109400615B
Application number: CN201710713030.0A
Authority: CN
Inventors: 王辉; 曹本红; 梁胜
Original assignee: Shanghai Atom Kexing Pharmaceuticals Co ltd; XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Current assignee: Shanghai Atom Kexing Pharmaceuticals Co ltd; XinHua Hospital Affiliated To Shanghai JiaoTong University School of Medicine
Priority date: 2017-08-18
Filing date: 2017-08-18
Publication date: 2021-07-16
Anticipated expiration: 2037-08-18
Also published as: CN109400615A

Abstract

The invention relates to a coumarin compound targeting beta-amyloid and a preparation method and application thereof, wherein the structural formula of the coumarin compound targeting beta-amyloid is as follows:

wherein R is₁Is CH₃Or CF₃；R₂Is C₂H₄Or C₃H₆Linear alkyl group of (1). Said compound using compound B as a labeling precursor, with¹⁸F^‑Compared with the prior art, the invention can fill the blank in the field of early diagnosis and curative effect evaluation of AD by using radiopharmaceuticals in China. And is used as a specific Alzheimer's disease PET imaging probe. The preparation method is simple, is suitable for clinical application of radiopharmaceuticals, and is prepared¹⁸The radiochemical purity of the F-labelled compound is greater than 95%.

Description

Beta-amyloid targeted coumarin compound and preparation and application thereof

Technical Field

The invention relates to coumarin compounds, in particular to a coumarin compound targeting beta-amyloid protein and preparation and application thereof.

Background

Beta-amyloid protein (a β) in brain is derived from Amyloid Precursor Protein (APP), its precursor substance. It has been confirmed that a β is derived from gene-encoded APP, has a molecular weight of about 4kD, is a transmembrane glycoprotein, and is β -sheet in three-dimensional structure, so called "β amyloid". A beta is composed of 28 amino acids outside the cell membrane of APP and 12 amino acids in the transmembrane part, and is positioned in the hydrophobic part of APP. Has strong aggregation and is easy to form precipitates which are extremely difficult to dissolve. A β is the major component of senile plaques, and is a neuropathological marker (Glenner G.Wong C W. Alzheimer's disease: initial report of the publication and characterization of a novel cerebrolytic amyloid protein [ J ]. Biochem Biophys Res Commun,1984,120(3) 855-900. Eille F, touch J. pathogenic and intracellular analysis of the organic form of Alzheimer's disease [ J ]. Prog Neurobiol,2002,66(3):191-203.Lambert M P, Bar A K, clone B A, interactive, soluble bacterial extract, scientific adsorbed protein [ 19-42 ] N. J. (6448. Nature 6448, N. org. A.). J.: 6453. front 6448. A. N. A. J. (N. C. Alzheimer's disease and characterization of A. 4. biological adsorbed protein). There are several theories of the pathogenesis of Alzheimer's Disease (AD), among which Hardy et al put forward the Α β cascade hypothesis of AD most attention, which states that aggregation of Α β plays a central role in the development and progression of AD, and Α β causes neuronal dysfunction and death, ultimately leading to dementia (Hardy J, selko dj. the amyloid hysteric of Alzheimer's disease: progress and dementia on the road to thermal disorders [ J ] Science,2002,297(5580): 356.).

Positron Emission Tomography (PET) using a specific molecular probe targeting Α β is the most appropriate means for early diagnosis of AD. Various major pharmaceutical companies have been developing PET imaging agents that specifically target Α β worldwide, until 2012, products of the company Eli Lilly,¹⁸F-Florbetapir (US7687052/US8506929) received US FDA approval; then, beautyThe national FDA has in turn approved two drugs for marketing, general electric medical company (GE HEALTHCARE)¹⁸F-Flutecetamol (US7270800/US7351401/US8236282/US8691185/US8916131) and PIRAMAL IMAGING¹⁸F-Florbetaben (US 7807135). At present, no PET imaging agent targeting Abeta is approved to be on the market at home, the early diagnosis and curative effect evaluation of AD at home are the situation of no medicine availability, and the research and development of novel PET imaging agents targeting Abeta can fill the blank of the biomedical industry in the field in China.

Coumarin (Coumarin), also known as difuran ring and oxanaphthalenone, is an effective component of the unique plants in south China, such as black coumarins, callianthus fasciatus and the like. The invention innovatively carries out F-18 labeling on the coumarin derivative, develops a specific PET imaging probe, and has very important application prospect for early diagnosis and curative effect evaluation of AD.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a high-purity beta-amyloid targeted coumarin compound, and preparation and application thereof.

The purpose of the invention can be realized by the following technical scheme: a coumarin compound targeting beta-amyloid is characterized in that the structural formula is as follows:

wherein R is₁Is CH₃Or CF₃；R₂Is C₂H₄Or C₃H₆Linear alkyl group of (1).

The preparation method of the beta-amyloid targeted coumarin compound is characterized in that the compound B is used as a labeled precursor, and¹⁸F^-performing nucleophilic substitution reaction, wherein the structural formula of the compound B is as follows:

wherein R is₁Is CH₃Or CF₃；R₂Is C₂H₄Or C₃H₆TsO is octadecyl trichlorosilane.

The method specifically comprises the following steps: in organic solvent under the protection of inert gas, in the presence of phase transfer catalyst, potassium salt and¹⁸F^-the obtained mixture and a compound B are subjected to nucleophilic substitution reaction at the reaction temperature of 40-120 ℃ for 5-30min to obtain the beta-amyloid targeted coumarin compound.

The obtained beta-amyloid targeted coumarin compound is subjected to post-treatment, and the post-treatment method comprises the following steps: separating and purifying beta-amyloid targeted coumarin compounds by using radioactive HPLC, then diluting with pure water, enriching by using a silica gel column, eluting with a physiological saline solution containing 45% ethanol, collecting in a physiological saline vial, finally preparing into a physiological saline solution containing 10% ethanol, and filtering by using a sterile filter membrane to obtain the beta-amyloid targeted coumarin compounds¹⁸F, marking coumarin compound preparation. Before separation and purification by radioactive HPLC, the product can also be purified by Sep-Pak C18 column.

The organic solvent comprises one or more of anhydrous acetonitrile, anhydrous tetrahydrofuran, anhydrous DMF (N, N-dimethylformamide) and anhydrous DMSO (dimethyl sulfoxide), and is preferably anhydrous DMF.

The potassium salt is potassium carbonate or potassium bicarbonate;

the phase transfer catalyst is a cyclic crown ether catalyst selected from 4,7,13,16,21, 24-hexaoxy-1, 10-diazabicyclo [8.8.8]]Hexacosane (K)₂₂₂)。

The inert gas is nitrogen and/or argon and/or helium.

Said catalyst containing phase transfer catalyst, potassium salt and¹⁸F^-the ratio of the amounts of the substances of the components in the mixture of (a): phase transfer catalyst: potassium salt 1: 3.5-7.5: 1,¹⁸F^-the activity of (b) is 40 mu Ci-2 Ci. In the reaction solution of said compound BThe concentration is 0.01-2 mol/L; the mass ratio of the phase transfer catalyst to the compound B is 1: 1-7.5: 1 in which¹⁸F^-Selected from bombardment with a cyclotron₂ ¹⁸And (4) obtaining an O target.

Said composition comprises K222, potassium carbonate and¹⁸F^-the mixture of (a) can be prepared by the following method: eluting with K222 solution for enrichment¹⁸F^-The solvent was evaporated to dryness to obtain a QMA column.

The application of the beta-amyloid targeted coumarin compound is characterized in that the coumarin compound is used as a specific Alzheimer's disease PET imaging probe and is used for early diagnosis and curative effect evaluation of AD.

The labeled precursor compound B used in the present invention is commercially available from Shanghai such as Flint biosciences, Inc., and the rest of the reagents used are commercially available.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. obtained by the invention¹⁸The F-labeled coumarin compound can specifically target beta-amyloid in brain, and the aggregation of the beta-amyloid in brain is one of the reasons for AD generation¹⁸The F-labeled coumarin compound can be used for imaging and characterizing the concentration degree of the coumarin compound in the brain through PET, thereby characterizing the aggregation degree of beta-amyloid protein for early diagnosis of AD.

2. The preparation method is simple, is suitable for clinical application of radiopharmaceuticals, and is prepared¹⁸The radiochemical purity of the F-labeled coumarin compound is more than 95 percent.

3. The invention can fill the blank in the field of early diagnosis and curative effect evaluation of AD by using radiopharmaceuticals in China.

Drawings

FIG. 1 is a HPLC detection chart of the product prepared by the present invention;

FIG. 2 is an analytical HPLC assay of the product prepared according to the present invention;

FIG. 3 shows that the product prepared by the invention is applied to a PET imaging probe to perform AD model mouse experimental micro-PET imaging, wherein a is an APP/PS1 dual-transgenic AD model rat micro-PET 30min static imaging, and b is a normal rat micro-PET 30min static imaging.

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

Compound A (R)₁Is CH₃；R₂Is C₂H₄) Preparation of coumarin compounds

Get K₂₂₂(10-12mg) and potassium carbonate (1-2mg) in an acetonitrile solution to be over-enriched¹⁸F]F^-The QMA column (2mCi) was collected in a reaction flask, the flask was kept at 90 ℃ and nitrogen (1mL/min) was blown to dry, then anhydrous acetonitrile solvent was added to evaporate to dryness, and the process was repeated three times and 0.6mL anhydrous DMF was added. 0.3mL of a reaction flask containing 2mg of precursor compound B (R)₁Is CH₃；R₂Is C₂H₄) The anhydrous DMF solution is sealed and reacted for 30min at the temperature of 120 ℃, and the anhydrous DMF solution is cooled to room temperature after the reaction is finished.

The reaction product was subjected to sep-pak C18 column in advance by adding 5mL of pure water, and then subjected to separation and purification using semipreparative C18 column, as shown in FIG. 1, and the peak at t 8.1min was unreacted¹⁸F^-The peak with t being 25.5min is a product peak, collecting the product for 25-27min, diluting the collected product with 100mL of pure water, enriching the product through a silica gel column, eluting the product with 2mL of physiological saline solution containing 45% ethanol, collecting the product in a vial containing 8mL of physiological saline, finally preparing the physiological saline solution containing 10% ethanol, and filtering the solution through a sterile filter membrane to obtain the product¹⁸F, marking the coumarin compound preparation, obtaining the uncorrected radiochemical yield of 10% by an activity meter, detecting by HPLC, and obtaining the following HPLC conditions: the analytical column is Agilent ZORBAX eclipseXDB C18 column (4.6 mm. times.250 mm); the mobile phase was water (a) and acetonitrile (b) with 0.1% trifluoroacetic acid added, and the gradient conditions were: 0-60min, 30% → 50% b; the flow rate is 1.0 mL/min; UV (220nm) detection and radioactivity detection. To obtain¹⁸Radiochemical purity of F-labelled coumarin compound preparation>98％。

Reference compound structure C is shown below:

wherein R is₁Is CH₃Or CF₃；R₂Is C₂H₄Or C₃H₆Linear alkyl group of (1). The synthesis method of the reference compound C adopts the same method as the invention, and the element F is¹⁸A stable isotope of F.

Collecting preparation A (hot) (R)₁Is CH₃；R₂Is C₂H₄) With its reference compound C (cold) (R)₁Is CH₃；R₂Is C₂H₄) The HPLC check pattern of the co-injection is shown in FIG. 2, and the HPLC conditions are the same as above. The retention time of the above formulation A was 7.51min and that of reference compound C was 7.63 min.

Reference Compound C (R)₁Is CH₃；R₂Is C₂H₄) Warp beam¹The structural identification results of HNMR and MS are shown as follows:

¹HNMR(DMSO-d6):δ7.92(d,3JHH＝8.4Hz,2H,Ar-H),7.86(s,1H,Ar-H),7.75(d,3JHH＝8.4Hz,2H,Ar-H),7.59(s,1H,Ar-H),7.40(d,3JHH＝1.6Hz,8.0Hz,2H,Ar-H),6.72(d,3JHH＝1.6Hz,8.8Hz,2H,Ar-H),6.38(s,1H,Ar-H),4.14-4.11(t,3JHH＝5.2Hz,2H,CH2),3.47-3.45(t,3JHH＝4.4Hz,2H,CH2),2.54(s,CH3),2.34(s,CH3)

TOF-ESI-MS：M(C19H16FN3O2)＝337.12(m/z),338.2(M+1)+

in the preparation method, the precursor compound B is a product of Shanghai Bingsheng Biotech limited, and the other chemical reagents are products of national chemical reagents company, [ 2 ]¹⁸F]F^-Is composed of a C-30 cyclotron¹⁸O-H₂O target generation¹⁸O(p,n)¹⁸F, nuclear reaction.

Example 2 PET imaging

Prepared by the method¹⁸F-labeled coumarin compound A for micro-PET imaging

AD model mouse and normal control rat tail intravenous injection prepared by the method¹⁸Marking a coumarin compound A by F, keeping the body temperature of the coumarin compound A at about 37 ℃ by using a heating module after isoflurane is inhaled for anesthesia, dynamically scanning for 2h by using R4micro-PET, framing after data acquisition is finished (the first half hour, 2 minutes/frame; the later every 10 minutes/frame), carrying out image reconstruction by using an OSEM3D method, manually selecting each region of interest (ROI) of a brain area by using Inveon Research workbench 2.2(IRW, Siemens) software, and calculating SUV. The image is shown in FIG. 3. As can be seen from the figure, the AD model rats are paired in the brain¹⁸F marked coumarin compound A has obvious concentration, and has no concentration compared with normal mice.

Example 2

A coumarin compound targeting beta-amyloid has the following structural formula:

wherein R is₁Is CH₃₃；R₂Is C₂H₄Linear alkyl group of (1).

The beta-amyloid targeted coumarin compound is prepared by a method comprising the following steps of taking a compound B as a labeled precursor, and reacting with¹⁸F^-Performing nucleophilic substitution reaction, wherein the structural formula of the compound B is as follows:

wherein R is₁Is CH₃；R₂Is C₂H₄TsO is octadecyl trichlorosilane.

The above method specifically comprises the steps of:

1. to obtain a mixture containing¹⁸F^-H of (A) to (B)₂ ¹⁸Enriching the O solution by passing through a QMA column¹⁸F^-Eluting QMA column with solution containing phase transfer catalyst and potassium salt to obtain solution containing phase transfer catalyst, potassium salt and¹⁸F^-the mixed solution of (1), the phase transfer catalyst-containing solutionAgent, potassium salt and¹⁸F^-the ratio of the amounts of the substances of the components in the mixture of (a): phase transfer catalyst: potassium salt 1: 3.5,¹⁸F^-activity of (4) was 40. mu. Ci. Wherein¹⁸F^-Selected from bombardment with a cyclotron₂ ¹⁸And (4) obtaining an O target.

2. In an organic solvent (anhydrous tetrahydrofuran), under the protection of inert gas (nitrogen), adding a phase transfer catalyst K₂₂₂Potassium carbonate and¹⁸F^-h of (A) to (B)₂ ¹⁸And carrying out nucleophilic substitution reaction on the O solution and the compound B at the reaction temperature of 40 ℃ for 30min to obtain the beta-amyloid targeted coumarin compound. The concentration of the compound B in the reaction solution is 0.01 mol/L; the mass ratio of the phase transfer catalyst to the compound B is 1: 1.

3. separating and purifying beta-amyloid targeted coumarin compounds by using radioactive HPLC, then diluting with pure water, enriching by using a silica gel column, eluting with a physiological saline solution containing 45% ethanol, collecting in a physiological saline vial, finally preparing into a physiological saline solution containing 10% ethanol, and filtering by using a sterile filter membrane to obtain the beta-amyloid targeted coumarin compounds¹⁸F, marking coumarin compound preparation. Before separation and purification by radioactive HPLC, the product can also be purified by Sep-Pak C18 column.

Example 3

A preparation method of a coumarin compound targeting beta-amyloid comprises the following steps:

1. to obtain a mixture containing¹⁸F^-H of (A) to (B)₂ ¹⁸Enriching the O solution by passing through a QMA column¹⁸F^-Eluting QMA column with solution containing phase transfer catalyst and potassium salt to obtain solution containing phase transfer catalyst, potassium salt and¹⁸F^-the mixed solution of (1), the phase transfer catalyst, potassium salt and¹⁸F^-the ratio of the amounts of the substances of the components in the mixture of (a): phase transfer catalyst: potassium salt 7.5: 1,¹⁸F^-activity of 2 Ci. Wherein¹⁸F^-Selected from bombardment with a cyclotron₂ ¹⁸And (4) obtaining an O target.

2. In the organic fieldIn a solvent (anhydrous tetrahydrofuran), under the protection of inert gas (nitrogen), adding a phase transfer catalyst K₂₂₂Potassium carbonate and¹⁸F^-h of (A) to (B)₂ ¹⁸And carrying out nucleophilic substitution reaction on the O solution and the compound B at the reaction temperature of 120 ℃ for 5min to obtain the beta-amyloid targeted coumarin compound. The concentration of the compound B in the reaction solution is 2 mol/L; the mass ratio of the phase transfer catalyst to the compound B was 7.5: 1.

The structural formula of the coumarin compound targeting the beta-amyloid protein is as follows:

wherein R is₁Is CF₃；R₂Is C₃H₆Linear alkyl group of (1).

wherein R is₁Is CF₃；R₂Is C₃H₆TsO is octadecyl trichlorosilane.

Claims

1. A coumarin compound targeting beta-amyloid is characterized in that the structural formula is as follows:

2. The method for preparing the coumarin compound targeting beta-amyloid according to claim 1, wherein compound B is a labeled precursor, and¹⁸F^-performing nucleophilic substitution reaction, wherein the structural formula of the compound B is as follows:

3. The preparation method of the coumarin compound targeted to the beta-amyloid protein according to claim 2, characterized in that the method specifically comprises the following steps: to obtain a mixture containing¹⁸F^-H of (A) to (B)₂ ¹⁸Enriching the O solution by passing through a QMA column¹⁸F^-Eluting QMA column with solution containing phase transfer catalyst and potassium salt to obtain solution containing phase transfer catalyst, potassium salt and¹⁸F^-under the protection of protective gas in organic solvent, the mixed solution containing phase transfer catalyst, potassium salt and¹⁸F^-the mixture and the compound B are subjected to nucleophilic substitution reaction at the reaction temperature of 100-120 ℃ for 30-60min, and the beta-amyloid targeted coumarin compound is obtained.

4. The preparation method of the coumarin compound targeted to the beta-amyloid protein according to claim 3, wherein the obtained coumarin compound targeted to the beta-amyloid protein is subjected to post-treatment, and the post-treatment method comprises the following steps: separating and purifying beta-amyloid targeted coumarin compounds by using radioactive HPLC, then diluting with pure water, enriching by using a silica gel column, eluting with a physiological saline solution containing 45% ethanol, collecting in a physiological saline vial, finally preparing into a physiological saline solution containing 10% ethanol, and filtering by using a sterile filter membrane to obtain the beta-amyloid targeted coumarin compounds¹⁸F, marking coumarin compound preparation.

5. The method for preparing the coumarin compound targeting beta-amyloid according to claim 3, wherein the organic solvent is one or more of anhydrous acetonitrile, anhydrous tetrahydrofuran, anhydrous DMF and anhydrous DMSO.

6. The method for preparing the coumarin compound targeted to the beta-amyloid protein according to claim 5, wherein the organic solvent is anhydrous DMF.

7. The preparation method of the coumarin compound targeted to the beta-amyloid protein according to claim 3, wherein the potassium salt is potassium carbonate or potassium bicarbonate.

8. The preparation method of the coumarin compound targeting beta-amyloid according to claim 3, wherein the phase transfer catalyst is a cyclic crown ether catalyst selected from the group consisting of 4,7,13,16,21, 24-hexaoxy-1, 10-diazabicyclo [8.8.8] hexacosane.

9. The method for preparing the coumarin compound targeted to the beta-amyloid protein according to claim 3, wherein the protective gas is nitrogen and/or argon and/or helium.

10. The method for preparing beta-amyloid-targeted coumarin compound according to claim 3, wherein the coumarin compound comprises a phase transfer catalyst, a potassium salt and¹⁸F^-the ratio of the amounts of the substances of the components in the mixture of (a): phase transfer catalyst: potassium salt 1: 3.5-7.5: 1,¹⁸F^-the activity of (A) is 40 mu Ci-2 Ci; the concentration of the compound B in the reaction solution is 0.01-2 mol/L; the mass ratio of the phase transfer catalyst to the compound B is 1: 1-7.5: 1, wherein¹⁸F^-Selected from bombardment with a cyclotron₂ ¹⁸And (4) obtaining an O target.

11. The use of a coumarin compound targeting β -amyloid according to claim 1 as a specific PET imaging probe for alzheimer's disease.