CN111170980A

CN111170980A - Calycosin derivative and synthesis method and application thereof

Info

Publication number: CN111170980A
Application number: CN202010026500.8A
Authority: CN
Inventors: 陈健; 王勇; 李鑫; 任倩瑶; 秦俭; 陈晓宇
Original assignee: Guilin Medical University
Current assignee: Guilin Medical University
Priority date: 2020-01-10
Filing date: 2020-01-10
Publication date: 2020-05-19
Anticipated expiration: 2040-01-10
Also published as: CN111170980B

Abstract

The invention discloses a mullein isoflavone derivative and a synthesis method and application thereof. The method for synthesizing the isoflavone derivatives of the present invention mainly includes the following steps: dissolving compound 1 and compound 2 in an organic solvent, adding an acid binding agent, and then reacting under heating conditions to obtain a crude product of the target product. The applicant's experimental results show that the verisoflavone derivatives of the present invention can simultaneously inhibit the proliferation of ER-positive breast cancer cells and ER-negative breast cancer cells; as the concentration of the derivatives increases, the proliferation rate of breast cancer cells gradually decreases, and At high concentrations, the inhibitory effect was most pronounced and had no effect on normal breast cells MCF-10A.

Description

Calycosin derivative and synthesis method and application thereof

Technical Field

The invention relates to a calycosin derivative and a synthesis method and application thereof, belonging to the technical field of medicines.

Background

Globally, according to research reports, breast cancer is one of the most common malignant cancers in women, and the incidence rate of breast cancer is also the highest in leaders, which accounts for about 29% of new malignant tumors in women, and the fatality rate is the second highest in the fatality rate of malignant tumors, and has become the first risk factor threatening the life safety of women. According to statistics, by 2016, the number of breast cancer patients in China exceeds about 50 ten thousand, the onset age of the breast cancer patients decreases year by year, the breast cancer patients tend to be low in age, the incidence rate and the mortality rate increase year by year, and the age group with the highest incidence rate is 35-49 years old. The study results of the students at home and abroad on the risk factors and the disease factors of the breast cancer are inconsistent because the students put forward different views. The breast cancer is still preferably treated by operation at present, and chemotherapy, radiotherapy or endocrine treatment and the like are assisted after the operation.

TAK1 (transforming growth factor b activated kinase-1) is a member of the mitogen activated protein kinase family (MAP 3K). It also phosphorylates members of the MKK family, and when phosphorylated, it can activate JNK (Jun N-terminal kinase) and p38 kinase. TAK1 has a unique activity requiring binding proteins TAB1, TAB2 and TAB 3. Ubiquitination activates the complex TAK1/TAB2 or TAK1/TAB3 to phosphorylate IKKb at Ser-177 and Ser-181, thereby activating IKK in the activation loop. In addition, the complex of TAK1/TAB1 may also phosphorylate MKK and activate JNK and p38, but not the IKK complex. Thus, these studies indicate that TAK1 complex is activated, while TAK1 of IKK is activated. Activated JNK phosphorylates C-jun, which in turn can affect apoptosis, inflammation and tumorigenesis. Recent studies have reported that TAK1 is abnormally high in expression in a variety of tumors, including breast cancer.

Genomics studies have shown that most of the human genes are transcribed into non-coding RNAs (ncRNAs). ncRNAs include short RNAs represented by miRNA and long non-coding RNAs. Long non-coding RNAs (lncRNAs) are a class of ncRNAs molecules with transcript lengths between 200nt and 100 kb. In recent years, a great deal of research shows that LncRNAs are involved in a plurality of important regulation processes such as X chromosome silencing, genome imprinting, chromatin modification, transcriptional activation, transcriptional interference, intranuclear transportation and the like. One of the incrnas well studied was Hox transcribed antisense intergenic RNA (HOTAIR), which is located in the 12q13 chromosomal region in the HOXC (homeobox C) gene cluster and belongs to the 2158 nucleotide long incrna. Hotair is a modular scaffold of histone modification complexes that silences the expression of a particular gene by binding its 5 'domain to the PRC2 complex, while the 3' domain binds to the LSD1/CoREST complex. Hotair plays a role as an oncogenic molecule in a variety of cancers (e.g., breast, gastric, bladder and lung). Also noteworthy is the increased expression of HOTAIR in breast cancer, a finding that provides a powerful biomarker for tumor metastasis and patient death.

The radix astragali is dried root of Astragalus membranaceus (Fisch.) Bge. belonging to Leguminosae family perennial herbaceous plant Astragalus membranaceus (Fisch.) Bge. According to the pharmacopoeia, astragalus root has sweet taste and mild nature, and has the efficacies of tonifying qi and invigorating yang, benefiting wei and defensive qi, strengthening exterior, improving cardiac function, expanding coronary artery, promoting urination and detumescence, supporting sore and promoting granulation, resisting bacteria, resisting virus, resisting fatigue, resisting aging, promoting hematopoiesis, protecting liver and the like.

Calycosin (calycosin) is an isoflavone compound extracted and separated from radix astragali (Ma Xiaofeng et al, research on flavone components in Mongolian radix astragali, Chinese herbal medicine, vol. 36, 9 th, 9.2005, p1293-1296), and its structural formula is shown as follows:

the existing research shows that calycosin has the effects of resisting oxidative stress, resisting virus, regulating apoptosis and the like, but has the defects of larger effective concentration and undefined target spot. Low concentrations of calycosin (<16 μ M) promote proliferation of ER-positive breast cancer cells MCF-7; although high concentrations of calycosin (>20 μ M) were able to inhibit proliferation of ER-positive breast cancer cells MCF-7 and T47D, it had no effect on proliferation of ER-negative breast cancer cells (zhouging, chengjian, study of the effect and mechanism of calycosin at different concentrations on ER-positive breast cancer cells, proceedings of the eleventh national chemotherapeutics and pharmacology symposium of chinese pharmacology, 7/1/2012, p 322-323). At present, no report related to calycosin derivatives capable of simultaneously inhibiting ER positive breast cancer cells and ER negative breast cancer cells exists.

Disclosure of Invention

The invention aims to provide a calycosin derivative which has a novel structure and low toxicity to normal mammary gland cells and can inhibit ER positive breast cancer cells and/or ER negative breast cancer cells, a synthesis method and application thereof.

The calycosin derivative disclosed by the invention is a compound shown as the following formula (I) or a pharmaceutically acceptable salt thereof:

the chemical name of the compound represented by the formula (I) is: 3- (4-methoxy-3- (ethoxycarbonylethoxy) phenyl) -7- (ethoxycarbonylethoxy) -4H-benzopyran-4-one, molecular weight 456.4.

The invention also provides a synthesis method of the compound shown in the formula (I), which mainly comprises the following steps: the method mainly comprises the following steps: dissolving a compound 1 and a compound 2 in an organic solvent, adding an acid-binding agent, and then reacting under a heating condition to obtain a target crude product; wherein the structures of the compound 1 and the compound 2 are respectively as follows:

the compound 1,

Compound 2.

In the above synthesis method, the molar ratio of compound 1 to compound 2 is stoichiometric, and in actual operation, compound 2 may be slightly in excess.

In order to accelerate the reaction rate and further improve the yield, it is preferable to add an iodide, which is sodium iodide and/or potassium iodide, before the reaction. The addition of an iodide, which is generally used in an amount of 0.5 to 1.5 times the amount of the compound 1 substance, activates the hydroxyl group on the compound 1 to facilitate the reaction.

In the above synthesis method, the organic solvent is acetone and/or N, N-Dimethylformamide (DMF). The amount of the organic solvent to be used may be determined as required, and it is usually preferable to dissolve the starting materials to be reacted, and specifically, the total amount of the starting materials to be reacted is dissolved in 6 to 20mL of the first organic solvent based on 1mmol of the compound 1.

In the above synthesis method, the acid-binding agent is a conventional choice in the prior art, and specifically may be one or a combination of two or more of common basic substances selected from sodium carbonate, sodium bicarbonate, potassium carbonate, ammonium carbonate, sodium hydroxide, potassium hydroxide, triethylamine, N-diisopropylethylamine, triethanolamine, tetrabutylammonium bromide, 4-dimethylaminopyridine, pyridine and the like, and preferably is triethylamine, pyridine, sodium carbonate or potassium carbonate. The ratio of the dosage of the acid-binding agent to the dosage of the compound 1 substance is preferably 1: 1.

in the above synthesis method, the reaction is preferably carried out at not less than 40 ℃, and more preferably at a temperature ranging from 45 ℃ to the boiling point of the first organic solvent. The completion of the reaction can be detected by TLC tracking, and the developing solvent is preferably chloroform: methanol 60: 1 (volume ratio).

In the above synthesis method, since the solubility of the target product in the organic solvent is high after the reaction is finished, the target product in the reactant is low after the reaction is finished, and in order to separate out more target product, ice water is usually added to the reactant after the reactant is cooled, and the target product is largely separated out in the form of precipitate by changing the polarity of the solution; then filtering and collecting the precipitate, wherein the precipitate is the crude product of the target product. The crude target product can also be dissolved by ethyl acetate and then dried by anhydrous sodium sulfate to remove water in the crude target product.

The above synthesis method produces a crude product of the compound represented by formula (I), and in order to improve the purity of the crude product, the crude product may be purified by conventional purification methods. In the application, the purification step is to recrystallize the crude target product in ethanol. Of course, purification by silica gel column chromatography is also possible.

The invention also comprises the application of the compound shown in the formula (I) or the pharmaceutically acceptable salt thereof in preparing a medicament for treating ER positive breast cancer, or in preparing a medicament for treating ER negative breast cancer, or in preparing a medicament for simultaneously treating ER positive breast cancer and ER negative breast cancer.

Furthermore, the invention also provides a pharmaceutical composition, which consists of a therapeutically effective dose of the compound shown in the formula (I) or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or auxiliary material. The dosage form of the medicine can be any pharmaceutically acceptable dosage form, and specifically can be conventional dosage forms such as granules, tablets, pills, capsules or injections.

the experimental results of the applicant show that the compound shown in the formula (I) can down-regulate the expression level of Hotair and p-TAK1 and can inhibit the phosphorylation level of downstream target C-jun/I kappa B α and simultaneously inhibit the proliferation of ER positive breast cancer cells (such as MCF-7 and T-47D) and ER negative breast cancer cells (such as MDA-MB-231), wherein the MTT experiment shows that the proliferation rate of MCF-7, MDA-MB-231 and T-47D is gradually reduced along with the increase of the concentration of the compound shown in the formula (I), and the inhibition effect is most obvious at high concentration and has no influence on normal breast cells MCF-10A.

Drawings

FIG. 1 is a graph of dose-response curves of 48h of the effect of different concentrations of a compound of formula (I) on breast cancer cells MCF-7, MDA-MB-231, T-47D and normal breast cells MCF-10A, p <0.05vs control; wherein (a) is MCF-7, (b) is T-47D, (c) is MDA-MB-231, and (D) is normal mammary gland cell MCF-10A.

FIG. 2 is a photograph of a single cell clone affecting MCF-7, MDA-MB-231 and T-47D cells with different concentrations of a compound of formula (I); wherein (a) is MCF-7, (b) is MDA-MB-231, and (c) is T-47D.

FIG. 3 shows the effect of different concentrations of a compound of formula (I) on the migratory capacity of MCF-7, MDA-MB-231 and T-47D cells, p <0.05vs control; wherein, (a) is a microscope photograph of the change of the scratched area of the MCF-7 cells at different time, (b) is a histogram of the migration capacity of the MCF-7 cells at 24h, and (c) is a histogram of the migration capacity of the MCF-7 cells at 48 h; (d) the photomicrographs of the MDA-MB-231 cells in the scratch area at different times, (e) the histogram of the migration capacity of the MDA-MB-231 cells at 12h, and (f) the histogram of the migration capacity of the MDA-MB-231 cells at 24 h; (g) the microscopic photographs of the T-47D cells in the different time scratch areas are shown, (h) the histogram of the migration capacity of the T-47D cells at 24h, and (i) the histogram of the migration capacity of the T-47D cells at 36 h.

FIG. 4 shows the effect of different concentrations of a compound of formula (I) on the invasive potential of MCF-7, MDA-MB-231 cells,. p <0.05vs control; wherein, (a) is a staining picture of a compound shown in formula (I) and MCF-7 cells after 48h of incubation at different concentrations, (b) is a staining picture of a compound shown in formula (I) and MDA-MB-231 cells after 48h of incubation at different concentrations, (c) is a bar graph of the invasion capacity of a compound shown in formula (I) and MCF-7 cells after 48h of incubation at different concentrations, and (d) is a bar graph of the invasion capacity of a compound shown in formula (I) and MDA-MB-231 cells after 48h of incubation at different concentrations.

FIG. 5 shows the effect of different concentrations of a compound of formula (I) on the level of mRNA expression of Hotair in MCF-7, MDA-MB-231, T-47D cells,. p <0.05vs control; wherein (a) is MCF-7, (b) is MDA-MB-231, and (c) is T-47D.

FIG. 6 shows the effect of different concentrations of a compound of formula (I) on the level of phosphorylation of proteins of TAK1, C-jun/IkB α in MCF-7, MDA-MB-231, T-47D cells and the downstream pathway of TAK1 was identified as C-jun/IKB α, # p <0.05 vstrol, # p <0.05vs TAK1, wherein (a) is MCF-7, (B) is MDA-MB-231, and (C) is T-47D.

FIG. 7 shows the results of experiments on protein phosphorylation levels of C-jun and IKB α by selecting emoty group (A), TAK1(B), TAK1+16 μ M (C) and empty +16 μ M (D) after successful transfection by up-regulating the phosphorylation expression levels of TAK1 in MCF-7, MDA-MB-231 and T-47D cells, respectively, wherein (a) is MCF-7, (B) is MDA-MB-231, (C) is T-47D, (D) is protein phosphorylation levels of C-jun and IKB α by A, B, C and D group after up-regulating MCF-7, (e) is protein phosphorylation levels of C-jun and IKB α by MDA-MB-231 after up-regulating MDA-MB-231, A, B, C and D group after up-regulating C-jun and IKB α, and (f) is protein phosphorylation levels of C-jun and B α by T-47D after up-regulating T-47D, A, B, C and D group after up-jun and T-47.

FIG. 8 is a graph showing the effect of compounds of formula (I) on the expression levels of Hotair mRNA in the empty group, <0.05vs control, # p <0.05vs HOTAIR, and empty + 16. mu.M group of three cells, respectively, after successful upregulation of Hotair in the three cells; wherein (a) the expression levels of the compound represented by the formula (I) after Hotair in the blank group and the HOTAIR group of MCF-7 cells are up-regulated, (b) the expression levels of the compound represented by the formula (I) after Hotair in the blank group and the HOTAIR group of MDA-MB-231 cells are up-regulated, (c) the expression levels of the compound represented by the formula (I) after Hotair in the blank group and the HOTAIR group of T-47D cells are up-regulated, (D) the expression levels of the compound represented by the formula (I) after Hotair in each group of MCF-7 cells are up-regulated, (e) the expression levels of the compound represented by the formula (I) after Hotair in each group of MDA-MB-231 cells are up-regulated, and (f) the expression levels of the compound represented by the formula (I) after Hotair in each group of T-47D cells are up-regulated.

FIG. 9 shows the results of experiments on protein phosphorylation levels of C-jun and IKB α, measured by selecting emoty group (A), TAK1(B), TAK1+16 μ M (C) and empty +16 μ M (D) after determining successful transfection by up-regulating the Hotair expression levels in MCF-7, MDA-MB-231 and T-47D cells, respectively, wherein p <0.05vs control, # p <0.05vs HOTAIR, and ^ p <0.05vs HOTAIR, wherein (a) is MCF-7, (B) is MDA-MB-231, and (C) is T-47D.

FIG. 10 shows the effect of the compound of formula (I) on the migratory capacity of cells after Hotair upregulation in three cells,. p <0.05vs control; wherein, (a) is a microscope photograph of the change of the scratch area of MCF-7 cells after Holair is up-regulated in different groups after 48h, (b) is a histogram of the migration capacity of MCF-7 cells after Holair is up-regulated in 48h, (c) is a microscope photograph of the change of the scratch area of MDA-MB-231 cells after Holair is up-regulated in different groups after 24h, (D) is a histogram of the migration capacity of MDA-MB-231 cells after Holair is up-regulated in 24h, (e) is a microscope photograph of the change of the scratch area of T-47D cells after Holair is up-regulated in different groups after 48h, and (f) is a histogram of the migration capacity of T-47D cells after Holair is up-regulated in 48 h.

FIG. 11 shows the effect of compounds of formula (I) on the ability of cells to invade after upregulation of Hotair in MCF-7 and MDA-MB-231 cells, respectively,. p <0.05vs control; the staining pictures of different groups and MCF-7 cells after the Holair is up-regulated after 48 hours of incubation, (b) the staining pictures of different groups and MDA-MB-231 cells after 48 hours of incubation after the Holair is up-regulated, (c) the staining pictures of different groups and MCF-7 cells after 48 hours of incubation after the Holair is up-regulated, and (d) the staining pictures of different groups and MDA-MB-231 cells after 48 hours of incubation after the Holair is up-regulated.

FIG. 12 shows that the compound of formula (I) inhibits the tumor growth curve of tumor-bearing nude mice, p <0.05vs control; wherein (a) is a tumor growth curve of a nude mouse with tumor caused by the compound shown in formula (I) for inhibiting MCF-7, and (b) is a tumor growth curve of the nude mouse with tumor caused by the compound shown in formula (I) for inhibiting MDA-MB-231.

FIG. 13 shows the effect of the compound of formula (I) on the level of phosphorylation of TAK1, IkB α, C-jun proteins in tumor-bearing nude mice, wherein (a) is the effect of the compound of formula (I) on the level of phosphorylation of TAK1, IkB α, C-jun proteins in tumor-bearing nude mice caused by MCF-7, and (B) is the effect of the compound of formula (I) on the level of phosphorylation of TAK1, IkB α, C-jun proteins in tumor-bearing nude mice caused by MDA-MB-231.

Detailed Description

The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.

Example 1: the compound of formula (I) was synthesized according to the following synthetic route

Wherein, the chemical name of the compound 1 is: 7-hydroxy-3- (3-hydroxy-4-methoxyl) -4H-benzopyran-4-ketone, and the compound 2 is ethyl chloroacetate.

The specific synthesis method comprises the following steps:

1g (3.52mmol) of Compound 1 (Calycosin) was dissolved in 40ml of acetone in a 50ml round-bottom flask, and 3g of anhydrous K was added₂CO₃And 0.5g of nai, stirred at room temperature for 1 hour, then 0.91g (7.43mmol) of compound 2 (ethyl chloroacetate) is added dropwise, and then placed in a water bath at 45 ℃ for stirring and refluxing for 4 hours (TCL monitors the reaction, and the developing agent is chloroform: methanol 60: 1 in volume ratio); heating was then stopped and stirring was continued until cooling. Ice water was added to the cooled reaction mixture, which was filtered, and the precipitate was collected, dissolved in ethyl acetate and dried over anhydrous sodium sulfate, the resulting solution was evaporated to remove the solvent, and the residue was dissolved in 10ml of ethanol and recrystallized overnight in a refrigerator at-20 ℃ to give 1082.00mg of pale yellow solid in 70.50% yield.

The light yellow solid product obtained in this example was characterized by nuclear magnetic resonance, and its hydrogen spectrum and carbon spectrum were as follows:

¹H NMR(400MHz,DMSO)δ8.44(s,1H),8.05(d,J＝8.9Hz,1H),7.37–7.08(m,5H),7.06(d,J＝8.5Hz,1H),5.00(s,2H),4.78(s,2H),4.19(dq,J＝14.2,7.1Hz,4H),3.82(s,3H),3.34(s,1H),1.22(d,J＝7.2Hz,6H).

¹³C NMR(101MHz,DMSO)δ174.94,169.18,168.54,162.42,157.57,154.31,149.34,147.01,127.54,124.62,123.60,122.83,118.55,115.39,115.01,112.58,102.08,65.98,65.56,61.35,61.06,56.15,40.60,40.39,40.19,39.98,39.77,39.56,39.35,14.51.

therefore, the light yellow solid product obtained in this example can be determined to be the compound shown in formula (I), and the structural formula of the compound is shown in formula (I) below:

example 2: synthesis of a Compound of formula (I)

Example 1 is repeated, except thatAnhydrous Na₂CO₃Instead of anhydrous K₂CO₃The reaction was carried out at 55 ℃ by substituting KI for NaI.

Finally, a pale yellow solid, 70.21%, was obtained.

The product obtained in this example was characterized by hydrogen and carbon nuclear magnetic resonance spectroscopy and was identified as the compound represented by formula (I).

Example 3: synthesis of a Compound of formula (I)

Example 1 was repeated, except that DMF was used instead of acetone and triethylamine was used instead of anhydrous K₂CO₃The reaction was carried out at 40 ℃.

Finally, a pale yellow solid, 23.18%, was obtained.

Example 4: synthesis of a Compound of formula (I)

Example 1 was repeated except that no NaI was added.

Finally, a pale yellow solid was obtained with a yield of 16.88%.

The following experiments are combined to further illustrate the application of the compound shown in the formula (I) in preparing the medicine for treating and inhibiting the breast cancer.

All data in the following experimental examples are expressed as mean + -SEM, statistical comparisons were made using one-way analysis of variance and Tukey's test for group-to-group comparisons.

Test drugs: a compound represented by the formula (I) (hereinafter, CAG002 will be referred to simply as "CAG") prepared by the method described in example 1 of the present invention was dissolved in DMSO to prepare a stock solution having a concentration of 100mM, and stored at 4 ℃ for use.

DMEM, calf serum (FBS), Phosphate Buffered Saline (PBS), penicillin-streptomycin (P/S) and 0.25% (W/V) Trypsin/1 mM EDTA (Trypsin-EDTA) were purchased from Invitrogen (USA), and culture medium of MCF-10A was purchased from Shanghai academy of sciences Cell banks PCR reverse transcription kit was purchased from Sigma (St Louis, MO.) MTT detection kit was provided by Roche (Mennheim, Germany.) antibodies to TAK1, C-jun and IkappaB alpha, phosphorylated TAK1, C-jun and IkappaB α antibodies and horseradish peroxidase (HRP) -labeled anti-rabbit IgG secondary antibody were all purchased from Cell Signaling Technology (Beverly, MA).

D) Cell culture: humanized Breast cancer (MCF-7, MDA-MB-231, T-47D (T47D), ATCC, Manassas) was cultured in DMEM with 100U/ml penicillin, 100. mu.g/ml streptomycin and 10% FBS at 37 ℃ with 5% CO₂Culturing in an incubator.

Experimental example 1: MTT method for detecting breast cancer cell proliferation

And (3) taking 90% fused MCF-7, MDA-MB-231, T-47D and MCF-10A, washing and digesting to prepare a cell suspension. After counting, press 5x10³And inoculating the cells in a 96-well plate, and culturing for 24 hours until the cells are completely attached to the wall. The experimental group is provided with CAG002 (0.1-32 μ M) dosing groups with different concentrations, each group has 6 multiple holes, and the culture solution containing the medicine is added into each hole according to 200 μ l. A control was also set, and a blank control was a 0.1% DMSO culture.

MTT results show that CAG002 can inhibit the proliferation of breast cancer cells (MCF-7, MDA-MB-231 and T-47D) in a dose-dependent manner, and the inhibition effect is most obvious when the drug concentration is maximum. Wherein, when the drug concentration is 32 MuM, the inhibition effect is the most obvious, and the inhibition rate is 34 percent (p is less than 0.05) compared with the blank control. But had little inhibitory effect on MCF-10A. The effect of different concentrations of CAG002 on the proliferation of breast cancer cells (MCF-7, MDA-MB-231, T-47D) is shown in FIG. 1.

Experimental example 2: plate clone detection of cell monoclonal proliferation

MCF-7, MDA-MB-231 and T47D cells were seeded at low density (500 cells/well, three replicates) into 6-well plates, followed by the application of various concentrations of CAG002(0, 4, 8, 16. mu.M) and incubation for 5-10 days. Then, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and stained with gram stain for 20 minutes. The results are shown in FIG. 2.

The plate clone experiment result shows that CAG002 inhibits the single cell cloning capacity of breast cancer (MCF-7, MDA-MB-231 and T-47D) cells, and the inhibition effect is obvious along with the increase of the concentration.

Experimental example 3: scratch test for detecting migration capacity of breast cancer cells

Cell migration ability was assessed by scratch test assay. An equal number of cells MCF-7, MDA-MB-231, T47D were cultured in 6-well plates until 90% confluence was reached. Wounds were created by scraping the cell sheet with a sterile 10 μ Ι _ pipette tip. Floating cells were removed by gently washing the wells with Phosphate Buffered Saline (PBS). Different concentrations of CAG002 (dissolved in low serum DMEM) were added and the change in the scratched area was observed daily with an inverted microscope (Leica, germany). Wound width was measured to calculate cell migration capacity. The results are shown in FIG. 3.

The result of the scratch experiment shows that the CAG002 has the effect of inhibiting the migration capacity of the breast cancer cells, and the inhibition effect is more obvious along with the increase of the medicine concentration.

Experimental example 4: transwell experiment for detecting invasion capacity of breast cancer cells

Cell invasion was assessed using a 24-well Millicell suspension cell culture insert with an 8 μm polyethylene terephthalate (PET) membrane (Millipore, Millipore of bedford, massachusetts, usa). 5X10 of each group⁴The individual cells were suspended in 200. mu.l of serum-free medium and seeded into the upper chamber. Then, different concentrations of CAG002 were added while 500 μ l of complete medium containing 10% FBS was added to the lower chamber. After 48 hours of incubation at 37 ℃, non-invasive cells were carefully removed from the upper surface of the filter. The invaded cells in the lower chamber (below the filter surface) were fixed in 100% methanol, stained with 0.1mg/mL crystal violet solution (Beyotime Biotechnology, shanghai, china) and counted under the microscope. Five random fields were counted for each well and the average was determined. The results are shown in FIG. 4.

From the results of the Transwell experiment, it can be seen that CAG002 can inhibit the invasive ability of breast cancer cells and show a drug dose-dependent increase.

Experimental example 5: real-time quantitative PCR detection of Hotair gene expression

Total miRNA were isolated using TRIzol reagent (Gibco-BRL) and 1 was used0ng of RNA and a reversible helper first strand cDNA Synthesis kit (Fermentas, Life sciences, USA) were used to prepare cDNA. Relative miRNA expression determination using contrast CT (2)^-△△ct) A method. Next, quantitation of HOTAIR was measured by qPCR using specific primers for SYBR Green qPCR Master Mix (Fermentas, life sciences, usa) and HOTAIR and GAPDH. GAPDH was used as an internal reference gene to calculate the relative expression level of Hotair.

Histograms of the effect of CAG002 on the amount of Hotair gene expression in breast cancer cells are shown in fig. 5, respectively. The result shows that CAG002 can down-regulate the Hotair gene level in MCF-7, MDA-MB-231 and T47D, and the high-concentration inhibition effect is more obvious.

EXAMPLE 6 immunoblot hybridization (Western Blot) to detect phosphorylation levels of TAK1, C-jun and I κ B α

Total protein was obtained from breast cancer cells using immunoprecipitation assay lysate (RIPA; Sigma-Aldrich, St. Louis, Mo., USA) and protein concentration was determined by BCA protein assay kit (Pierce, Waltham, MA, USA.) the protein mass loaded on each lane was 30 μ g. cell lysate was separated on analytical 10% SDSPAGE gel and transferred to polyvinylidene fluoride (PVDF) membrane. nonspecific binding was prevented by incubation with 5% skim milk for 2 hours at room temperature P-TAK1 (1: 1000-10000), TAK1 (1: 1000-10000), PC-JUN (1: 1000), C-JUN (1: 1000), P-I κ B α (1: 1000), I κ B α (1: 1000) overnight at 4 deg.C. then horseradish peroxidase (HRP) conjugated goat anti-rabbit was used as secondary antibody and the results were observed using Electrochemiluminescence (ECL) reagent (Pierce, Waltham, USA) using Western blotting software (Biotech) and NIK density was observed as shown in the U.S.S.S.A. Pat. No. 6.

from FIGS. 6(a) to 6(C), it is known that CAG002 can inhibit the expression levels of TAK1, C-jun and IkappaB α protein phosphorylation in breast cancer cells, and that the expression of protein phosphorylation gradually decreases with increasing concentration, and the expression level is the lowest when the concentration reaches 16 μ M. from the phosphorylation expression of TAK1 in FIGS. 7(a) to 7(C), TAK1 was transfected successfully in three breast cancer cells, further experiments can be performed, and from FIGS. 7(d) to 7(f), it is known that not only the phosphorylation expression of TAK1 increases, but also the phosphorylation of C-jun and IkappaB α proteins increases, and that p <0.05vs emptyy, whereas TAK1+16 μ M (C) is significantly lower than TAK1(B), and that the phosphorylation of TAvKp <0.05 × pty and the phosphorylation of TAK +16 μ M + is significantly lower than that of TAK 3, TAK # p <0.05 × 1. and Ikappak +16 μ M <16 μ M > K <16 μ M > and TAK # p < 7, and TAK < 21 μ M < 7 μ M > and the phosphorylation of TAK < 21 μ M < TAK < 21 μ M > is significantly lower than that of TAK < 7.

FIG. 8 shows the results of detecting the expression of the Hotair gene after the Hotair gene was successfully upregulated in the three breast cancer cells. As can be seen from FIGS. 8(a) to 8(c), the expression of Hotair after upregulation was significantly increased. After the HOTAIR is added with the CAG002, the expression of the Hotair is obviously reduced, and as can be seen from the experiments of selecting emoty group (A), TAK1(B), TAK1+16 μ M (C) and empty +16 μ M (D) respectively from the graphs of 8(D) to 8(f), the CAG002 can inhibit the expression of the gene of the Hotair in the breast cancer cells.

FIG. 9 shows the results of experiments with emoty group (A), TAK1(B), TAK1+ 16. mu.M (C) and empty + 16. mu.M (D) selected after Hotair is up-regulated, and the results of experiments with TAK1, C-jun and IkappaB α protein phosphorylation are up-regulated after Hotair is up-regulated, and the levels of expression of TAK1, C-jun and IkappaB α protein phosphorylation are reduced after CAG002 is added, which shows that CAG002 can regulate the levels of protein phosphorylation of TAK1, C-jun/IkappaB α to affect the growth of mammary cells, and that HotaIKir is the upstream target of TAK1 and C-jun and Balpha.

After up-regulating Hotair, the ability of migration and invasion of breast cancer cells was examined as described in Experimental examples 3 and 4, and the results are shown in FIGS. 10 and 11, respectively. Experimental results show that after the Hotair is up-regulated, the invasion and migration capacity of the breast cancer is obviously improved, which shows that the up-regulation of the Hotair influences the invasion and migration capacity of cells, and meanwhile, after the CAG002 is added, the invasion and migration capacity is obviously inhibited.

Experimental example 7: tumor-bearing nude mouse experiment

Human breast cancer cell strain MCF-7 and MDA-MB-231 cells are cultured, subcultured, amplified, digested by pancreatin, centrifuged and precipitated to prepare about cell suspension. The BALB/c nude mice were collected, the axillary parts of the nude mice were punctured with a syringe containing the cell suspension, and the cell suspension was injected into the subcutaneous parts of the nude mice, each approximately 0.1ml (1X 10 ml)⁷Individual cell), slowly withdrawing the needle, and preparing the human breast cancer tumor-bearing nude mouse model. After surgery, the tumors were measured with a vernier caliper and grouped 10d after tumor implantation. Each group of the tumor-forming nude mice comprises 6 mice, which are divided into a medication group (30mg/kg, 60mg/kg) and a negative control group. The medicine is injected into the abdominal cavity, 1 time and 20 times daily, the same route of the medicine-free group is used for administering the PBS with equal volume, and the length and the width of the tumor are measured by a vernier caliper. After 20 days the animals were sacrificed and the skin and non-tumor tissue surrounding the tumor were separated and weighed on an electronic balance.

CAG002 inhibits tumor growth in tumor-bearing nude mice as shown in FIG. 12. Compared with the model group, 30mg/kg, 60mg/kg CAG002 inhibited the tumor volume of tumor-bearing nude mice, which is consistent with the in vitro results.

EXAMPLE 8 immunohistochemical detection of in vivo expression of p-TAK1, p-C-jun, p-I.kappa.B.alpha.

tumors were fixed overnight in 4% paraformaldehyde, dehydrated with a series of ethanol, carefully embedded in paraffin, and cut into 5 μm thick sections, after deparaffinization in xylene and hydration with a series of ethanol, tissue sections were incubated with 3% H2O2 for 10 minutes, followed by 3 PBS washes, antigens were recovered from the samples by microwave treatment in citrate buffer (pH 6.8), then, the sections were incubated with primary antibodies, anti-p-TAK 1 antibody (1: 200), p-C-jun (1: 150), p-I κ B α antibody (1: 150), respectively, at a constant temperature of 4 ℃ overnight, after 3 washes with PBS, the sections were probed with the corresponding secondary antibodies using a PV-9000 polymer detection kit, and observed for immunoreactivity using 3, 3-Diaminobenzidine (DAB), after counterstaining with hematoxylin a light microscope, the results of DAB are shown in FIG. 13.

As a result of the experiment, CAG002 decreased the expression of p-TAK1, p-C-jun, p-I κ B α in vivo, also with the decrease of the dose dependence, which is consistent with the results of the in vivo experiment.

And (4) conclusion:

CAG002 produces a Hotair inhibitor effect. Hotair is a molecule that has been shown to function as an oncogenic molecule in a variety of cancers (e.g., breast, gastric, bladder and lung). Also noteworthy is the abnormal increase in HOTAIR expression in breast cancer, a finding that provides a powerful biomarker for tumor metastasis and patient death. The experimental results show that CAG002 can inhibit the proliferation of breast cancer cells better than calycosin by down-regulating the expression level of Hotair, compared with the results of previous researches in the subject group, therefore, CAG002 is more promising to be an anti-tumor drug than calycosin.

MTT experiments show that CAG002 can inhibit the proliferation of MCF-7, MDA-MB-231 and T47D, and the effect is more obvious along with the increase of the addition amount of the medicament; compared with the previous experimental results, the CAG002 inhibition effect is obviously better than that of calycosin. The results of the scarification and transwell experiments suggest that CAG002 may influence the ability of breast cancer cells to migrate and invade, but the regulation through that pathway in particular is unclear.

CAG002 can down-regulate the level of MCF-7, MDA-MB-231 and T47D cell lncRNAs (Hotair). The real-time quantitative PCR and immunoblot hybridization results show that CAG002 can down-regulate the Hotair gene level of MCF-7, MDA-MB-231 and T47D cells and the phosphorylation expression level of p-TAK1, p-C-jun and p-I kappa B α. meanwhile, TAK1 is up-regulated in three breast cancer cells respectively, C-jun and I kappa B α are found to be correspondingly increased, after CAG002 is added, the p-TAK1, p-C-jun and p-I kappa B α expression levels are obviously reduced, which shows that the downstream target point of TAK 2 is C-jun, I kappa B α. then, the Hotair expression in the three cells is up-regulated, after CAG002 is up-regulated, the Hotair expression of the Hotair is obviously reduced, and the growth of the cells is further shown to be influenced by the phosphorylation of the CAG 387-TAK-1, the downstream target point of the growth of the CAK-TAK-JUN is detected by the phosphorylation of the CAG002, and the CAG-JUN-C-JUN is further detected by the change of the CAG.

After the Hotair is up-regulated, the research on the function experiment of the cells is continued, and the result shows that after the Hotair is up-regulated, compared with a control group, the invasion and migration capacity of the cells are obviously improved, but after the CAG002 is added, the invasion and migration capacity is obviously inhibited.

Animal experiment results show that CAG002 has strong proliferation inhibiting effect on tumor bearing nude mice, the tumor volume curve of the drug group is obviously slower than that of the control group, and the immunohistochemical results show that in vivo and in vitro results are consistent.

in conclusion, CAG002 can affect the proliferation, invasion and migration capacity of breast cancer cells by regulating the expression of protein phosphorylation of downstream TAK1, C-jun/I kappa B alpha pathway by Hotair.

Claims

1. A compound of the following formula (I) or a pharmaceutically acceptable salt thereof:

2. a method of synthesizing the compound of claim 1, wherein: the method mainly comprises the following steps: dissolving a compound 1 and a compound 2 in an organic solvent, adding an acid-binding agent, and then reacting under a heating condition to obtain a target crude product; wherein,

the structures of the compound 1 and the compound 2 are respectively as follows:

3. the method of synthesis according to claim 2, characterized in that: and adding an iodide before the reaction, wherein the iodide is sodium iodide and/or potassium iodide.

4. A synthesis method according to claim 2 or 3, characterized in that: the organic solvent is selected from acetone and/or N, N-dimethylformamide.

5. A synthesis method according to claim 2 or 3, characterized in that: the reaction is carried out at a temperature of more than or equal to 40 ℃.

6. A synthesis method according to claim 2 or 3, characterized in that: the reaction is carried out at a temperature ranging from 45 ℃ to the boiling point of the first organic solvent.

7. A synthesis method according to claim 2 or 3, characterized in that: also comprises a step of purifying the crude product of the target product.

8. Use of a compound of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of ER-positive breast cancer and/or for the treatment of ER-negative breast cancer.

9. A pharmaceutical composition characterized by: consisting of a therapeutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or adjuvant.

10. The medicament of claim 9, wherein: the dosage form of the medicine is a pharmaceutically acceptable dosage form.