A new compound and application thereof in preparation of a drug for treating triple-negative breast cancer

Abstract

This invention discloses a novel compound and its application in the preparation of drugs for treating triple-negative breast cancer. The invention provides a compound with a novel structure; activity studies show that this compound has excellent therapeutic effects on triple-negative breast cancer, not only inhibiting the growth and migration of triple-negative breast cancer cells but also inducing apoptosis in triple-negative breast cancer cells, while having no significant inhibitory effect on normal breast epithelial cells, and exhibiting a safety profile superior to cisplatin. This invention also provides a simple and feasible method for preparing this compound.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the development, preparation and application of compounds 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 Technology

[0002] Breast cancer is a complex disease, classified into four types based on immunohistochemical marker levels: Luminal A, Luminal B, HER-2 positive, and triple-negative. Triple-negative breast cancer accounts for approximately 15%-20% of all breast cancers. Because it does not express estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER-2), it responds poorly to hormone therapy and HER-2 targeted therapy. Coupled with its high risk of metastasis, its prognosis is poor. Currently, the main treatments for triple-negative breast cancer are surgery and chemotherapy. For advanced triple-negative breast cancer that has metastasized, chemotherapy is the primary treatment. Taxanes, anthracyclines, and platinum-based drugs are the most commonly used chemotherapy regimens for triple-negative breast cancer; however, the toxicity and side effects of chemotherapy drugs often severely limit their application and therapeutic efficacy. Therefore, finding highly effective and low-toxicity chemotherapy drugs is crucial for improving the overall survival rate of patients with triple-negative breast cancer.

[0003] Apoptosis, a form of cell death, is an active process unlike necrosis, involving the activation, expression, and regulation of a series of genes. Apoptosis plays a crucial role in clearing senescent, damaged, and abnormal cells (such as cancer cells) and even in cell growth and development. Cancer cells, due to gene mutations, can often evade the recognition and surveillance of the immune system, thus continuing to grow and divide. Many chemotherapy drugs typically trigger apoptosis by inducing DNA damage in cancer cells or affecting DNA replication (such as platinum-based drugs and cyclophosphamide), and these drugs often have strong toxicity to humans. Therefore, seeking anticancer drugs that can induce apoptosis in cancer cells with low toxicity is a major research focus for many researchers.

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

[0005] The first objective of this invention is to provide a compound with a novel structure, the second objective is to provide a method for preparing the compound, and the third objective is to provide the application of the compound in the preparation of a drug for treating triple-negative breast cancer.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

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

[0008]

[0009] A method for preparing the compound shown in formula (I) above 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 NaHCO3 and stir. Dissolve liquid bromine in methanol and add it dropwise to the reaction vessel. Stir the reaction. After the reaction is complete, add an appropriate amount of solid Na2SO3 to quench the reaction. Filter the mixture and collect the filtrate. Add an appropriate amount of distilled water and stir. After mixing, filter the mixture and dry the filter cake under vacuum 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 into a reaction vessel, add an appropriate amount of acetone to dissolve 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 dropwise to the solution to react with the remaining benzyl bromide compounds 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 liquid after extraction with saturated saline, and finally add an appropriate amount of anhydrous sodium sulfate for drying; concentrate the dried liquid to dryness, and separate and purify it by silica gel column chromatography to obtain the target product 5,7-dibromo-8-(3-iodophenylethoxy)-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 mixture of petroleum ether and ethyl acetate in a volume ratio of 20:1.

[0014] The use of the above-mentioned compounds or their pharmaceutically acceptable salts or solvates in the preparation of drugs 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] This invention provides a novel compound; activity studies show that this compound has excellent therapeutic effects on triple-negative breast cancer, not only inhibiting the growth and migration of triple-negative breast cancer cells, but also inducing apoptosis in triple-negative breast cancer cells, and has no significant inhibitory effect on normal breast epithelial cells, with a safety profile superior to cisplatin.

[0019] The present invention also provides a simple and feasible method for preparing the compound. Attached Figure Description

[0020] Figure 1 This is a synthetic route diagram for compound HQL-9;

[0021] Figure 2 The image shows the 1H NMR spectrum of compound HQL-9.

[0022] Figure 3 The image shows the carbon NMR spectrum of compound HQL-9.

[0023] Figure 4 The effect of compound HQL-9 on the proliferation of triple-negative breast cancer cells MDA-MB-468 was detected by trypan blue staining. *** in the figure indicates P < 0.001, which is statistically significant.

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

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

[0026] Figure 7 To detect the effect of compound HQL-9 on apoptosis-related proteins in MDA-MB-468 cells using Western blot. Detailed Implementation

[0027] The following describes the substantive content of the present invention in detail with reference to embodiments, but this does not limit the scope of protection of the present invention.

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

[0029]

[0030] (1) 2-Methyl-8-hydroxyquinoline (3.19 g, 20 mmol) was added to a 100 mL two-necked round-bottom flask, and then dissolved in 30 mL of methanol. NaHCO3 (3.18 g, 40 mmol) was added and stirred. Liquid bromine (3.20 mL, 60 mmol) was dissolved in 10 mL of methanol and added dropwise to the two-necked flask. The mixture was stirred for 4 h under a magnetic stirrer. After the reaction was confirmed to be complete by thin-layer chromatography, solid Na2SO3 (22 g, 170 mmol) was added in portions to the reaction solution to quench the reaction. The mixture was filtered, and the filtrate was collected. Then, 40 mL of distilled water was added and stirred for 30 min. After mixing, the mixture was filtered again. The filter cake was dried under vacuum to obtain a white solid product, 5,7-dibromo-2-methyl-8-hydroxyquinoline.

[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 under sonication. Then add K₂CO₃ (138.2 mg, 1 mmol) and 3-iodobenzyl bromide (59.4 mg, 0.2 mmol) sequentially. Stir magnetically at 100 °C for 3 h until fully reacted. After the reaction is complete as detected by thin-layer chromatography, add two drops of ammonia to the solution to react with the remaining benzyl bromide compounds to form an ammonium salt. The reaction mixture was subjected to ultrasonic vibration. The reaction solution and solids were then added to a separatory funnel, extracted four times with dichloromethane, and the lower layer was washed once with saturated brine. Finally, an appropriate amount of anhydrous sodium sulfate was added for drying. The dried liquid was evaporated to dryness using a rotary evaporator. A PE:EA ratio of 20:1 was used as the eluent, and the mixture was purified by chromatography using a 300-400 mesh silica gel column to obtain the target product 5,7-dibromo-8-(3-iodophenylethoxy)-2-methylquinoline (HQL-9). The synthetic route is shown in [reference needed]. Figure 1 The proton and carbon NMR spectra are as follows: Figure 2 and Figure 3 As shown.

[0032] 1 H NMR (400MHz, CDCl3) δ8.33(d,J=8.7Hz,1H),8.13(t,J=1.7Hz,1H),7.90(s,1H),7.67(dt,J=7.9,1.4H z,1H),7.60(d,J=8.1Hz,1H),7.39(d,J=8.6Hz,1H),7.12(t,J=7.8Hz,1H),5.36(s,2H),2.81(s,3H).

[0033] 13C NMR (101MHz, CDCl3) δ160.12,151.61,143.25,139.67,137.80,137.22,136.13, 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 passage

[0036] Triple-negative breast cancer cells MDA-MB-468 were cultured in Leibovitz's L15 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 100% aeration. Normal mammary epithelial cells MCF-10A were 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 and 5% CO2. Cell growth was observed daily, and when confluence reached approximately 90%, cells were passaged using 0.25% trypsin digestion.

[0037] 2. MTT assay for screening compounds with excellent activity against triple-negative breast cancer.

[0038] The specific steps are as follows:

[0039] (1) Cell seeding: After trypsin digestion, MDA-MB-468 cells in good growth condition were counted under a microscope using a hemocytometer. Based on the results, cells were prepared at a density of 5–8 × 10⁶ cells / year. 4 Add 100 μL of cell suspension per well to a 96-well plate and incubate for 24 h.

[0040] (2) Drug treatment: Prepare compound solutions of different concentrations using fresh culture medium, discard the culture medium in the 96-well plate, add 100 μL of the corresponding concentration of compound solution to each well, make 3 replicates for each concentration, and then put it in an incubator for incubation.

[0041] (3) After culturing for 72 hours, add 10 μL of MTT solution (5 mg / mL) to each well and continue culturing in an incubator for 2 hours.

[0042] (4) Gently aspirate the culture medium from each well, add 150 μL of dimethyl sulfoxide to each well, and shake at medium speed for 5 min to fully dissolve the blue-purple formazan at the bottom. Then measure the OD value at 490 nm wavelength using a microplate 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%. The half-maximal inhibitory concentration (IC50) was calculated using Graphpad Prism. 50 ).

[0044] The MTT assay results are shown in Table 1. Among the tested compounds, HQL-9 showed the highest IC50 value in MDA-MB-468 cells. 50 <1 μM, with stronger anticancer activity than the positive control drug cisplatin. Furthermore, MTT assays on normal human mammary epithelial cells MCF-10A showed that HQL-9 exhibited superior selectivity compared to cisplatin (as shown in Table 2).

[0045] Table 1 shows the IC50 values ​​of the compounds against triple-negative breast cancer cells MDA-MB-468. 50 value

[0046]

[0047] Table 2. IC50 of HQL-9 and cisplatin against normal human mammary epithelial cells MCF-10A 50 value

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

[0049] The above experimental results show that HQL-9 has excellent inhibitory effects on triple-negative breast cancer cells, but no significant inhibitory effect on normal cells, and its selectivity and safety are superior to cisplatin.

[0050] 3. Effect of trypan blue staining on the proliferation of MDA-MB-468 triple-negative breast cancer cells.

[0051] MDA-MB-468 cells were distributed at a rate of 1×10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 12-well plates and incubated for 24 hours. HQL-9 was then diluted with culture medium to a pre-defined concentration gradient of 0 μM, 1 μM, 2.5 μM, and 5 μM, and administered as a treatment, with three replicates for each concentration. Cells were digested at 24, 48, and 72 hours, stained with 0.4% trypan blue solution, and the number of viable cells was counted under a microscope. Dead cells appeared blue, while viable cells were not stained. Each experiment was repeated three times independently. Data were then processed and analyzed using GraphPad Prism software.

[0052] Experimental results are as follows Figure 4 As shown, 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 became increasingly larger, indicating that HQL-9 can effectively inhibit the growth of triple-negative breast cancer cells MDA-MB-468.

[0053] 4. Cell scratch assay to evaluate the effect of HQL-9 on the migration of triple-negative breast cancer cells MDA-MB-468.

[0054] MDA-MB-468 cells were loaded at 8 × 10 5 Cells were seeded at a density of 10 cells / well in six-well plates and cultured until the confluence reached approximately 90%. Each well was scratched with a 200 μL pipette tip, and the scratched area was gently rinsed with PBS to remove the scratched cells. Serum-free culture medium containing HQL-9 at different concentrations (0 μM, 0.5 μM, 1 μM, 2.5 μM) was prepared and applied. The scratched area was photographed under a microscope at 0 h, and again at the same location after 48 h. The scratch area was calculated using ImageJ software, and then the cell migration rate was calculated.

[0055] Cell migration rate = (0h scratch area - 48h scratch area) / 0h scratch area × 100%. Each experiment was independently repeated three times. Data were processed and plotted using GraphPad Prism software.

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

[0057] 5. Flow cytometry analysis of the effect of HQL-9 on apoptosis in triple-negative breast cancer cells MDA-MB-468.

[0058] The triple-negative breast cancer cells MDA-MB-468 treated with different concentrations of HQL-9 were stained using Annexin V / PI double staining and then detected by flow cytometry.

[0059] Specific steps:

[0060] (1) Cell seeding: MDA-MB-468 triple-negative breast cancer cells in good growth condition were seeded at a rate of 4 × 10⁶ cells per well. 5 One cell was seeded into a six-well plate and incubated in an incubator for 24 hours.

[0061] (2) Drug treatment: After the cells adhered to the wall for 24 hours, different concentrations (0 μM, 1 μM, 2.5 μM, 5 μM) of HQL-9-containing culture medium were prepared, the drug was added, and the cells were incubated in an incubator for 72 hours.

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

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

[0064] (5) On-machine detection: The detection is completed within 1 hour using a flow cytometer.

[0065] (6) Data processing and analysis: Each set of data was independently repeated three times. The results were plotted and statistically analyzed using GraphPadPrism software.

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

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

[0068] MDA-MB-468 breast cancer cells were seeded into six-well plates and cultured for 24 h. They were then treated with different concentrations of HQL-9 for 48 h. Total protein was extracted from the cells using SDS lysis buffer, and equal volumes of protein were loaded into the wells. Separation was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was transferred to a nitrocellulose membrane. The membrane was blocked in 5% skim milk for 1 h. Subsequently, the membrane was incubated overnight at 4°C with the corresponding primary antibody. After washing four times with TBST, the membrane was incubated with a secondary antibody conjugated with horseradish peroxidase for 2 h at room temperature. After washing four more times with TBST, the membrane was developed using an enhanced chemiluminescence detection kit to obtain protein bands.

[0069] Caspase proteases are key participants in the initiation and execution of apoptosis, and PARP is also one of their substrates. Results are as follows: Figure 7 As shown, after treatment with compound HQL-9, MDA-MB-468 cells showed significant cleavage of the apoptosis marker protein PARP, and the increased expression of Cleaved-Caspase 3, Cleaved-Caspase 8, and Cleaved-Caspase 9 further confirmed the occurrence of apoptosis.

[0070] In summary:

[0071] The compound provided by this 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. It has no significant inhibitory effect on normal breast epithelial cells and its safety is superior to that of cisplatin.

[0072] The purpose of the above embodiments is to specifically illustrate the substantive content of the present invention, but those skilled in the art should know that the scope of protection of the present invention should not be limited to the specific embodiments.

Claims

1. A compound with the structural formula shown in formula (I), or a pharmaceutically acceptable salt thereof: （Ⅰ）。 2. A method for preparing the compound of formula (Ⅰ) in claim 1, characterized in that, Includes the following steps: (1) Weigh 2-methyl-8-hydroxyquinoline and dissolve it in methanol in a reaction vessel. Add NaHCO3 and stir. Dissolve liquid bromine in methanol and add it dropwise to the reaction vessel. Stir the reaction. After the reaction is complete, add solid Na2SO3 to quench the reaction. Filter the mixture and collect the filtrate. Add distilled water and stir. After mixing, filter the mixture and dry the filter cake under vacuum to obtain the white solid product 5,7-dibromo-2-methyl-8-hydroxyquinoline. (2) Weigh 5,7-dibromo-2-methyl-8-hydroxyquinoline into a reaction vessel, add acetone and dissolve under ultrasonic conditions, add K2CO3 and 3-iodo-benzyl bromide in sequence, heat and stir to react; after the reaction is complete, add ammonia water dropwise to the solution to react with the remaining benzyl bromide compounds to form ammonium salt, and then sonicate; then add the reaction solution and the solid therein to a separatory funnel, extract with dichloromethane first, wash the lower layer of the extracted liquid with saturated brine, and finally add anhydrous sodium sulfate to dry; concentrate the dried liquid to dryness, and separate and purify it by silica gel column chromatography to obtain the target product 5,7-dibromo-8-(3-iodophenylethoxy)-2-methylquinoline.

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

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

1.

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

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

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

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