New compound and application thereof in preparation of medicine for treating triple negative breast cancer

Abstract

The invention discloses a novel compound and application thereof in preparation of a medicine for treating triple negative breast cancer. The invention provides a compound with a novel structure. Activity studies show that the compound has an excellent treatment effect on triple negative breast cancer, not only can inhibit growth and migration of triple negative breast cancer cells, but also can induce apoptosis of the triple negative breast cancer cells, has no obvious inhibition effect on normal mammary epithelial cells, and is superior to cis-platinum in safety. The invention also provides a method for preparing the compound, which is simple and feasible.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and relates to the development, preparation of compounds and their application in the treatment of triple-negative breast cancer. Specifically, it relates to a new compound and its application in the preparation of drugs for the treatment of triple-negative breast cancer. Background Art

[0002] Breast cancer is a complex disease, which is divided into four categories according to the levels of immunohistochemical indicators, namely Luminal A type, Luminal B type, HER-2 positive type and triple-negative type. Among them, triple-negative breast cancer accounts for about 15%-20% of all breast cancers. Since it does not express estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER-2), the effects of hormone therapy and HER-2 targeted therapy are poor. Coupled with its high risk of deterioration and metastasis, the prognosis is poor. At present, the main treatment methods for triple-negative breast cancer are surgical treatment and chemotherapy. For advanced triple-negative breast cancer with metastasis, chemotherapy is the main treatment method. Taxanes, anthracyclines and platinum drugs are the most commonly used drugs in the chemotherapy regimens for triple-negative breast cancer. However, the toxicity and side effects of chemotherapy drugs often severely limit their application and treatment effects. Therefore, finding highly effective and low-toxic chemotherapy drugs is crucial for improving the overall survival rate of triple-negative breast cancer patients.

[0003] Apoptosis is a way of cell death. Different from necrosis, apoptosis is an active process involving the activation, expression and regulation of a series of genes. Apoptosis is of great significance for clearing senescent, damaged and abnormal cells (such as cancer cells) and even growth and development. Cancer cells can usually escape the recognition and monitoring of the immune system due to gene mutations, and thus continue to grow and divide. Many chemotherapy drugs usually trigger apoptosis by inducing DNA damage in cancer cells or affecting DNA replication (such as platinum drugs, cyclophosphamide, etc.), and these drugs often have strong human toxicity. Therefore, seeking anti-cancer drugs that can induce apoptosis in cancer cells and are low-toxic is also the direction of many scientific research scholars.

[0004] In view of the applicant's discovery of a new compound with excellent therapeutic effect on triple-negative breast cancer, the present invention is hereby proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a compound with a novel structure, the second object is to provide a preparation method of the compound, and the third object is to provide the application of the compound in the preparation of drugs for the treatment of triple-negative breast cancer.

[0006] The above objects of the present invention are achieved by the following technical solutions:

[0007] A compound with a structural formula as shown in formula (I), or a pharmaceutically acceptable salt or solvate thereof:

[0008]

[0009] A method for preparing the compound shown in the above formula (I) includes the following steps:

[0010] (1) Weigh an appropriate amount of 2-methyl-8-hydroxyquinoline and dissolve it in methanol in a reaction vessel. Add an appropriate amount of NaHCO 3 Stir, dissolve liquid bromine in methanol, and drop it into the reaction vessel, then stir and react; after the reaction is complete, add an appropriate amount of solid Na 2 SO 3 to quench the reaction. Filter the mixture by suction, collect the filtrate, add an appropriate amount of distilled water and stir. After mixing, filter by suction again. Vacuum dry the filter cake to obtain the white solid product 5,7-dibromo-2-methyl-8-hydroxyquinoline;

[0011] (2) Weigh an appropriate amount of 5,7-dibromo-2-methyl-8-hydroxyquinoline in a reaction vessel, add an appropriate amount of acetone and dissolve it under ultrasonic conditions. Sequentially add an appropriate amount of K 2 CO 3 , 3-iodo-bromobenzyl, and heat and stir to react; after the reaction is complete, drop an appropriate amount of ammonia water into the solution to react with the remaining benzyl bromide compounds to form ammonium salts, and then perform ultrasonic oscillation; then add the reaction solution and the solids therein to a separatory funnel. First, extract with dichloromethane, then wash the lower layer liquid after extraction with saturated brine, and finally add an appropriate amount of anhydrous sodium sulfate for drying; concentrate and evaporate the dried liquid to dryness, and separate and purify by silica gel column chromatography to obtain the target product 5,7-dibromo-8-(3-iodophenethoxy)-2-methylquinoline.

[0012] Preferably, the temperature of the stirring reaction in step (2) is 100 °C.

[0013] Preferably, the eluent for silica gel column chromatography in step (2) is a mixed solvent of petroleum ether / ethyl acetate with a volume ratio of 20:1.

[0014] Use of the above compound or its pharmaceutically acceptable salt or solvate in the preparation of a drug for treating triple-negative breast cancer.

[0015] Furthermore, the treatment includes inhibiting the growth of triple-negative breast cancer.

[0016] Furthermore, the treatment includes inhibiting the metastasis of triple-negative breast cancer.

[0017] Beneficial effects:

[0018] The present invention provides a novel-structured compound; activity studies have shown that this compound has excellent therapeutic effects on triple-negative breast cancer, not only can inhibit the growth and migration of triple-negative breast cancer cells, but also can induce apoptosis of triple-negative breast cancer cells, and has no obvious inhibitory effect on normal breast epithelial cells, and its safety is better than cisplatin.

[0019] The present invention also provides a method for preparing this compound, which is simple and feasible. Description of the Drawings

[0020] Figure 1 It is the synthetic route diagram of compound HQL-9;

[0021] Figure 2 It is the 1H NMR spectrum of compound HQL-9;

[0022] Figure 3 It is the 13C NMR spectrum of compound HQL-9;

[0023] Figure 4 It is the effect of compound HQL-9 on the proliferation of triple-negative breast cancer cell MDA-MB-468 detected by trypan blue staining method. In the figure, *** indicates that the P value < 0.001, which is statistically significant;

[0024] Figure 5 It is the effect of compound HQL-9 on the migration of MDA-MB-468 cells detected by cell scratch assay. In the figure, * indicates that the P value < 0.05, and *** indicates that the P value < 0.001, which is statistically significant;

[0025] Figure 6 It is the effect of compound HQL-9 on the apoptosis of MDA-MB-468 cells detected by flow cytometry. In the figure, ** indicates that the P value < 0.01, and *** indicates that the P value < 0.001, which is statistically significant;

[0026] Figure 7 It is the effect of compound HQL-9 on apoptosis-related proteins in MDA-MB-468 cells detected by Western blot. Detailed Embodiments

[0027] The following specifically introduces the substantial content of the present invention in combination with embodiments, but does not limit the protection scope of the present invention thereby.

[0028] Example 1: Preparation method of 5,7-dibromo-8-(3-iodophenethoxy)-2-methylquinoline (HQL-9)

[0029]

[0030] (1) Add 2-methyl-8-hydroxyquinoline (3.19 g, 20 mmol) to a 100 mL two-necked round-bottom flask, then add 30 mL of methanol to dissolve it. Add NaHCO 3 (3.18 g, 40 mmol) and stir. Dissolve liquid bromine (3.20 mL, 60 mmol) in 10 mL of methanol, and drop it into the two-necked flask. React fully for 4 h under an electromagnetic stirrer. After detecting the completion of the reaction by thin-layer chromatography spotting, add solid Na 2 SO 3 to the reaction solution in batches to quench the reaction. Filter the mixture by suction, collect the filtrate, then add 40 mL of distilled water and stir for 30 min. After mixing evenly, filter by suction. After vacuum drying the filter cake, the white solid product 5,7-dibromo-2-methyl-8-hydroxyquinoline is obtained.

[0031] (2) Weigh 5,7-dibromo-2-methyl-8-hydroxyquinoline (63.8 mg, 0.2 mmol) into a 10 mL reaction tube, add 6 mL of acetone and dissolve it under ultrasonic conditions. Add K 2 CO 3 (138.2 mg, 1 mmol) and 3-iodobenzyl bromide (59.4 mg, 0.2 mmol) in sequence, and stir magnetically at 100 °C for 3 h for full reaction. After detecting the completion of the reaction by thin-layer chromatography spotting, add two drops of ammonia water to the solution to react with the residual benzyl bromide compounds to form ammonium salts, and then perform ultrasonic oscillation. Then add the reaction solution and the solids in it to a separatory funnel. First, extract with dichloromethane four times, then wash the lower layer liquid after extraction with saturated brine once, and finally add an appropriate amount of anhydrous sodium sulfate for drying. Evaporate the dried liquid to dryness using a rotary evaporator. Select a system of PE:EA = 20:1 as the eluent and use a silica gel column with a mesh size of 300 - 400 for column chromatography separation and purification to obtain the target product 5,7-dibromo-8-(3-iodophenethoxy)-2-methylquinoline (HQL-9). The synthetic route diagram is shown in Figure 1 , and the proton nuclear magnetic resonance and carbon nuclear magnetic resonance spectra are as shown in Figure 2 and Figure 3 .

[0032] 1 H NMR (400 MHz, CDCl 3 ) δ8.33 (d, J = 8.7 Hz, 1H), 8.13 (t, J = 1.7 Hz, 1H), 7.90 (s, 1H), 7.67 (dt, J = 7.9, 1.4 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.12 (t, J = 7.8 Hz, 1H), 5.36 (s, 2H), 2.81 (s, 3H).

[0033] 13 C NMR (101 MHz, CDCl 3 ) δ 160.12, 151.61, 143.25, 139.67, 137.80, 137.22, 136.13, 132.58, 130.13, 127.84, 126.49, 123.54, 116.62, 116.56, 94.32, 75.43, 25.62.

[0034] Example 2: Activity Test

[0035] 1. Cell Culture and Subculture

[0036] The triple-negative breast cancer cell line MDA-MB-468 was cultured in Leibovitz’s L15 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37 °C in 100% air. The normal mammary epithelial cell line MCF-10A was cultured in DMEM / F12 medium supplemented with 5% horse serum, 10 μg / mL insulin, 20 ng / mL epidermal growth factor, and 0.5 μg / mL hydrocortisone at 37 °C in 5% CO 2 . The cell growth status was observed daily, and when the density reached about 90%, cell subculture was performed by 0.25% trypsin digestion method.

[0037] 2. Screening of Compounds with Excellent Activity Against Triple-Negative Breast Cancer by MTT Assay

[0038] The specific steps were as follows:

[0039] (1) Cell Seeding: After trypsin digestion of MDA-MB-468 cells with good growth status, cell counting was performed under a microscope using a hemocytometer. According to the results, a cell suspension with a density of 5 - 8×10 4 cells / mL was prepared. 100 μL of the cell suspension was added to each well of a 96-well plate and cultured in an incubator for 24 h.

[0040] (2) Drug Treatment: Different concentrations of compound solutions were prepared with fresh medium. The medium in the 96-well plate was discarded, and 100 μL of the corresponding concentration of compound solution was added to each well. There were 3 replicates for each concentration, and then it was placed in an incubator for culture.

[0041] (3) After culturing for 72 h, 10 μL of MTT solution (5 mg / mL) was added to each well and cultured in the incubator for another 2 h.

[0042] (4) Gently aspirate the culture medium in each well, add 150 μL of dimethyl sulfoxide to each well, and oscillate at medium speed for 5 min to fully dissolve the blue-violet formazan at the bottom. Then measure the OD value at a wavelength of 490 nm using an ELISA reader.

[0043] (5) Data processing: Cell inhibition rate (%) = (OD value of control group - OD value of drug-treated group) / (OD value of control group - OD value of blank control group) × 100%. Use Graphpad Prism to calculate the half-maximal inhibitory concentration (IC 50 ).

[0044] The results of MTT assay are shown in Table 1. Among the tested compounds, the IC 50 of HQL-9 against MDA-MB-468 cells was < 1 μM, and its anti-cancer activity was stronger than that of the positive control drug cisplatin. In addition, the MTT results for human normal breast epithelial cells MCF-10A showed that the selectivity of HQL-9 was better than that of cisplatin (as shown in Table 2).

[0045] Table 1 IC 50 values of compounds against triple-negative breast cancer cells MDA-MB-468

[0046]

[0047] Table 2 IC 50 values of HQL-9 and cisplatin against human normal breast epithelial cells MCF-10A

[0048] Compound HQL-9 Cisplatin <![CDATA[IC 50 (μM)]]> 43.56±4.89 4.55±0.31

[0049] The above experimental results indicate that HQL-9 has excellent inhibitory effects on triple-negative breast cancer cells and has no obvious inhibitory effects on normal cells, and its selectivity and safety are better than those of cisplatin.

[0050] 3. Detection of the effect of HQL-9 on the proliferation of triple-negative breast cancer cells MDA-MB-468 by trypan blue staining method

[0051] Seed MDA-MB-468 cells into a 12-well plate at a density of 1 × 10 5 cells per well and incubate in an incubator for 24 h. Then dilute HQL-9 with culture medium to the preset concentration gradients of 0 μM, 1 μM, 2.5 μM, and 5 μM and perform drug addition treatment, with 3 replicates for each concentration. Then digest the cells at 24 h, 48 h, and 72 h respectively, add 0.4% trypan blue solution for staining, and then count the number of live cells under a microscope. Dead cells are blue and live cells are not stained. Each experiment is independently repeated three times. Finally, use GraphPad Prism software to process the data and plot for analysis.

[0052] The experimental results are as Figure 4As shown in the figure, with the extension of time and the increase of concentration, the difference in the number of viable cells between the HQL-9 drug group and the control group without drug addition became larger and larger, indicating that HQL-9 could effectively inhibit the growth of triple-negative breast cancer cells MDA-MB-468.

[0053] 4. Evaluation of the effect of HQL-9 on the migration of triple-negative breast cancer cells MDA-MB-468 by cell scratch assay

[0054] Inoculate MDA-MB-468 cells in a six-well plate at a density of 8×10 5 cells / well and culture until the density reaches about 90%. Scratch the cells in each well with a 200 μL pipette tip, and then gently rinse the scratched area with PBS to remove the scratched cells. Prepare serum-free media containing HQL-9 at different concentrations (0 μM, 0.5 μM, 1 μM, 2.5 μM) for drug treatment. Take pictures of the scratch status at 0 h under the microscope, and continue to take pictures of the scratch at the same site after 48 h. Use Image J software to calculate the scratch area, and then calculate the cell migration rate.

[0055] Cell migration rate = (scratch area at 0 h - scratch area at 48 h) / scratch area at 0 h × 100%. Each experiment was independently repeated three times, and GraphPad Prism software was used to process the data and plot the analysis.

[0056] The experimental results are as Figure 5 shown, and the compound HQL-9 can significantly inhibit the migration of triple-negative breast cancer cells.

[0057] 5. Detection of the effect of HQL-9 on the apoptosis of triple-negative breast cancer cells MDA-MB-468 by flow cytometry

[0058] Use Annexin V / PI double staining method to stain triple-negative breast cancer cells MDA-MB-468 treated with different concentrations of HQL-9, and then detect them on a flow cytometer.

[0059] Specific steps:

[0060] (1) Cell inoculation: Inoculate triple-negative breast cancer cells MDA-MB-468 with good growth status at 4×10 5 cells per well into a six-well plate and place it in an incubator for 24 h.

[0061] (2) Drug treatment: After the cells adhered for 24 h, prepare media containing HQL-9 at different concentrations (0 μM, 1 μM, 2.5 μM, 5 μM) for drug addition, and place it in an incubator for 72 h.

[0062] (3) Cell collection: Discard the culture medium, add 1 mL of PBS to each well for washing, discard the PBS, add an appropriate amount of trypsin to each well, after digestion is complete, add culture medium to terminate digestion, centrifuge the cell suspension, centrifuge at 1000 g for 5 min, discard the supernatant, resuspend with 1 mL of PBS, centrifuge again at 1000 g for 5 min, and discard the supernatant.

[0063] (4) Staining: Resuspend the cells by adding 195 μL of binding buffer, add 5 μL of AnnexinⅤ-FITC, mix gently, then add 10 μL of propidium iodide staining solution, mix gently, and stain in the dark at room temperature for 20 min.

[0064] (5) Detection by flow cytometry: Complete the detection within 1 h using a flow cytometer.

[0065] (6) Data processing and analysis: Each group of data was independently repeated three times, and GraphPad Prism software was used to plot the results and perform statistical analysis.

[0066] The results are as Figure 6 shown. As the concentration of compound HQL-9 treatment increased, the proportion of apoptotic cells also increased. Among them, the apoptosis rate of cells was as high as 62.79% after treatment with 5 μM HQL-9 for 72 h.

[0067] 6. Detection of the effect of HQL-9 on apoptosis-related proteins in MDA-MB-468 cells by Western blot

[0068] Inoculate breast cancer cells MDA-MB-468 into a six-well plate and culture for 24 h, treat with different concentrations of compound HQL-9 for 48 h, extract total proteins in the cells using SDS lysis buffer, load equal amounts of proteins into the gel wells, separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transfer to a nitrocellulose membrane. Block the membrane in 5% skim milk for 1 h. Subsequently, incubate the membrane with the corresponding primary antibody overnight at 4 °C. After washing four times with TBST, incubate the membrane with the secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. Wash four times again with TBST, and develop using an enhanced chemiluminescence detection kit to obtain protein bands.

[0069] Caspase proteases are important participants in the initiation and execution of apoptosis, and at the same time, PARP is also one of its substrates. The results are as Figure 7 shown. After treatment of MDA-MB-468 cells with compound HQL-9, obvious cleavage of the apoptosis marker protein PARP occurred, and the increased expression of Cleaved-Caspase 3, Cleaved-Caspase 8, and Cleaved-Caspase 9 further proved the occurrence of apoptosis.

[0070] In summary:

[0071] The compound provided by the present invention has excellent therapeutic effects on triple-negative breast cancer. It can not only inhibit the growth and migration of triple-negative breast cancer, but also induce apoptosis of triple-negative breast cancer cells, and has no obvious inhibitory effect on normal breast epithelial cells, with better safety than cisplatin.

[0072] The role of the above embodiments is to specifically introduce the substantial content of the present invention. However, those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A compound having the structural formula (I), or a pharmaceutically acceptable salt or solvate thereof:

2. A method for preparing the compound represented by formula (I) in claim 1, characterized in that: The following steps are involved: (1) Weighing an appropriate amount of 2-methyl-8-hydroxyquinoline and dissolving it in methanol in a reaction container, adding an appropriate amount of NaHCO3 and stirring, dissolving liquid bromine in methanol and adding it dropwise to the reaction container, stirring for reaction; after the reaction is complete, adding an appropriate amount of solid Na2SO3 to quench the reaction, filtering the mixture, collecting the filtrate, adding an appropriate amount of distilled water and stirring, filtering after mixing, and vacuum drying the filter cake to obtain a white solid product 5,7-dibromo-2-methyl-8-hydroxyquinoline; (2) Weigh an appropriate amount of 5,7-dibromo-2-methyl-8-hydroxyquinoline into a reaction container, add an appropriate amount of acetone to dissolve it under ultrasonic conditions, add an appropriate amount of K2CO3 and 3-iodo-benzyl bromide in sequence, heat and stir to react; after the reaction is complete, add an appropriate amount of ammonia water to the solution to react with the residual benzyl bromide compound to form ammonium salt, and then perform ultrasonic vibration; then add the reaction solution and the solid therein to a separatory funnel, first extract with dichloromethane, then wash the lower layer of the extracted liquid with saturated brine, and finally add an appropriate amount of anhydrous sodium sulfate to dry it; the dried liquid is concentrated and evaporated to dryness, and separated and purified by silica gel column chromatography to obtain the target product 5,7-dibromo-8-(3-iodophenethoxy)-2-methylquinoline.

3. The method according to claim 2, characterized in that: The stirring reaction temperature in step (2) is 100°C.

4. The method according to claim 2, characterized in that: The eluent for silica gel column chromatography in step (2) is a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 20:

1.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a drug for treating triple-negative breast cancer.

6. The use according to claim 5, characterized in that: The treatment includes inhibiting the growth of triple-negative breast cancer.

7. The use according to claim 5, characterized in that: The treatment includes inhibiting metastasis of triple-negative breast cancer.

Images