Application of compound and triple negative breast cancer cell proliferation inhibitor or drug

Abstract

The invention relates to discovery and application of two triple-negative breast cancer cell proliferation inhibitors, in particular to discovery of dauricine compound action targets in rhizoma menispermi, and the targets are triple-negative breast cancer cells (SUM159, HCC1937, MDA-MB-231 and MDA-MB-468). The triple negative breast cancer cell proliferation inhibitor is dauricine and N-demethylated dauricine. In-vitro cell experiments show that the compound dauricine and the N-demethylated dauricine in the invention have different degrees of proliferation inhibition effects on triple negative breast cancer cells (SUM159, HCC1937, MDA-MB-231 and MDA-MB-468). Therefore, the triple-negative breast cancer cell proliferation inhibitor provided by the invention provides a candidate compound with a novel structure for the development of the anti-triple-negative breast cancer drug, and has very important drug research value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to two compounds, piracetamine and N-demethylpiracetamine, which have anti-triple-negative breast cancer activity and their therapeutic uses, including the treatment of various subtypes of triple-negative breast cancer. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a specific subtype of breast cancer that does not express human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER). It accounts for approximately 15-20% of all breast cancer cases and is most common in premenopausal women under 40 years of age. TNBC is characterized by high metastatic potential, high invasiveness, rapid progression, high recurrence rate, poor prognosis, and a high likelihood of developing drug resistance. Compared to other breast cancer subtypes, TNBC lacks a standard treatment regimen; currently, surgery, radiotherapy, and chemotherapy are the most common treatments. Due to specific mutations and abnormally activated signaling pathways in TNBC, targeted therapy and immunotherapy have also become potential clinical treatment options. Therefore, the search for new chemotherapy drugs for breast cancer and the development of new TNBC treatment strategies have become urgent needs. (Wolff AC, Hammond MEH, Allison KH, et al. Humanepidermal growth factor receptor 2 testing in breast cancer: American society of clinical oncology / college of American pathologists clinical practice guideline focused update [J]. Journal of Clinical Oncology, 2018, 36(20): 2105–2122; Wu ZY, Kim HJ, Lee JW, et al. Long-term oncologic outcomes ofimmediate breast reconstruction vs conventional mastectomy alone for breastcancer in the setting of neoadjuvant chemotherapy[J].Jama Surgery,2020,155(12):1142–1150; Moran MSRadiation therapy in the locoregional treatment of triple-negative breast cancer[J].Lancet Oncology.2015,16(3):e113–122; VaidyaJ.S.,Wenz F.,Bulsara M.,et al.An international randomised controlled trial tocompare TARGeted Intraoperative radioTherapy(TARGIT)with conventionalpostoperative radiotherapy after breast-conserving surgery for women withearly-stage breast cancer(the TARGIT-A trial)[J].Health TechnologyAssessment,2016,20(73):1–188;Orecchia R.,Veronesi U.,Maisonneuve P.,etal.Intraoperative irradiation for early breast cancer(ELIOT):long-termrecurrence and survival outcomes from a single-centre,randomised,phase3equivalence trial[J].Lancet Oncology,2021,22(5):597–608;Sharma P.,KimlerB.F.,O'Dea A.,et al.Randomized phase II trial of anthracycline-free andanthracycline-containing neoadjuvant carboplatin chemotherapy regimens instage I-III triple-negative breast cancer(NeoSTOP)[J].Clinical CancerResearch,2021,27(4):975–982;Carey L.A.,Rugo H.S.,Marcom P.K.,et al.TBCRC 001:randomized phase II study of cetuximab in combination with carboplatin instage IV triple-negative breast cancer[J].Journal of Clinical Oncology,2012,30(21):2615–2623;Heeke A.L.,Tan A.R.Checkpoint inhibitor therapy formetastatic triple-negative breast cancer[J].Cancer and Metastasis Reviews,2021,40(2):537–547;Yang F.,Xiao Y.,Ding J.H.,et al.Ferroptosis heterogeneityin triple-negative breast cancer reveals an innovative immunotherapycombination strategy[J].Cell Metabolism,2023,35(1):84–100;Fusco N.,SajjadiE.,Venetis K.,et al.Low-risk triple-negative breast cancers:Clinico-pathological and molecular features[J].Critical Reviews in OncologyHematology,2022,172:103643;Li Y.,Zhang H.,Merkher Y.,et al.Recent advances intherapeutic strategies for triple-negative breast cancer[J].Journal ofHematology&Oncology,2022,15(1):121)。.

[0003] Northern Bean Root (Menispermum dauricum DC., M. dauricum) is the dried rhizome of the Menispermaceae family plant. Also known as Bat Kudzu Root, it is harvested in spring and autumn, and dried after removing fibrous roots and dirt. In my country, Northern Bean Root is mainly produced in Northeast, North, East China, Sichuan, and Hubei provinces. It was first recorded in the *Chinese Medicinal Plants*. According to the *Chinese Pharmacopoeia*, Northern Bean Root is bitter and cold in nature, and has the effects of clearing heat and detoxifying, dispelling wind and relieving pain. It can be used to treat sore throat, dysentery caused by heat toxins, and rheumatic pain. The chemical composition of Northern Bean Root is complex, including alkaloids, phenolic acids, quinones, alcohols, polysaccharides, and volatile oils. Alkaloids are present in high amounts and are diverse, accounting for 1.7%–2.5% of the total composition. The main component is bat kudzu alkaloid. Alkaloids are the main active substances in Northern Bean Root, possessing anti-inflammatory, anti-Alzheimer's disease, cardiovascular protective, and anti-tumor pharmacological activities. There are no reports in the existing technology of the use of basalpinx compounds as inhibitors of triple-negative breast cancer in anti-triple-negative breast cancer drugs. (Wei HL, Han Y., Zhou H., et al. Isoquinoline alkaloid dimers with dopamine D1 receptor activities from Menispermum dauricum DC[J]. Phytochemistry, 2021, 194: 113015; National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. China Medical Science and Technology Press, 2020. 89; Guo Yueshan. Identification and comparative study of Chinese medicinal slices of Sophora flavescens and Sophora tonkinensis[J]. China Medical Guide, 2018, 16(20): 217-218; Lv L., Yin B., You Y., et al. Protective effects of total alkaloids from Menispermum dauricum against airway inflammation in asthmatic mice[J]. Planta Medica, 2020, 86(10): 665–673; Chen CY, Liu P., Wang J., et al. Dauricine attenuates spatial memory impairment and Alzheimer-likepathologies by enhancing mitochondrial function in a mouse model of Alzheimer's disease[J].Frontiers In Cell and Developmental Biology,2020,8:624339; Liu QN, Zhang L., Gong PL, et al.Daurisoline suppressed early afterdepolarizations and inhibited L-type calcium current[J].The American Journal of Chinese Medicine.2010,38(1):37–49;Yang Z.,Li C.,Wang X.,et al.Dauricineinduces Apoptosis,inhibits proliferation and invasion through inhibiting NF-κB signaling pathway in colon cancer cells[J]. Journal of Cellular Physiology, 2010, 225(1):266–275). . Summary of the Invention

[0004] The purpose of this invention is to provide a series of basilicon compounds and their applications. These compounds can be used as inhibitors of triple-negative breast cancer cell proliferation in the preparation of anti-triple-negative breast cancer drugs.

[0005] The application of the puerarin compounds described in this invention in the preparation of materials for the prevention and / or treatment of triple-negative breast cancer, wherein the puerarin compounds are puerarin and N-demethylpuerarin, and their structural formulas are as follows:

[0006]

[0007] This invention relates to the discovery and application of two inhibitors of triple-negative breast cancer cell proliferation, namely the discovery of the target of purslane compounds from Sophora flavescens, specifically triple-negative breast cancer cells (SUM159, HCC1937, MDA-MB-231, and MDA-MB-468), and the inhibitors of triple-negative breast cancer cell proliferation being purslane and N-demethylpurslane.

[0008] The beneficial effects of this invention are:

[0009] In vitro cell experiments showed that the compounds piracetamine and N-demethylpiracetamine in this invention have a proliferation inhibitory effect on triple-negative breast cancer cells (SUM159, HCC1937, MDA-MB-231 and MDA-MB-468) in a dose-dependent manner, and their inhibitory effect is better than that of the positive control drug 5-fluorouracil. Therefore, the research results of this invention can broaden the clinical application scope of such compounds. Attached Figure Description

[0010] Figure 1 The structural formulas of piperine and N-demethylpiperine.

[0011] Figure 2 Inhibition curves of piracetamine, N-demethylpiracetamine and 5-fluorouracil on triple-negative breast cancer cells SUM159(A), HCC1937(B), MDA-MB-231(C) and MDA-MB-231(D).

[0012] Figure 3 Effects of N-demethylhexane on apoptosis in SUM159 cells. Detailed Implementation

[0013] The substantive content of the invention will be further illustrated below with reference to the accompanying drawings and embodiments, but this does not limit the invention in any way. The invention will be further described below by way of specific embodiments.

[0014] Unless otherwise specified, all chemical reagents used in the embodiments of this invention are obtained through conventional commercial means, but this does not limit the scope of the methods to be protected by this invention.

[0015] SUM159, HCC1937, MDA-MB-231, and MDA-MB-468 triple-negative breast cancer cells (TNBC) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Heteroside and N-demethylheteroside (brand: Yuanye) and 5-fluorouracil (brand: Yuanye) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. DMEM / F12, RIMP 1640, and Leibovitz's L-15 culture media were purchased from Darthill Biotechnology Co., Ltd. Fetal bovine serum (FBS) was purchased from Suzhou Qianshe Biotechnology Co., Ltd. (brand: PAN). Phosphate-buffered saline (PBS) was purchased from Sangon Biotech (Shanghai) Co., Ltd. (brand: Sangon). Dimethyl sulfoxide (DMSO) was purchased from Beyotime Biotechnology Co., Ltd. (brand: Beyotime). The cell viability assay platform was the Ensight multi-plate reader from PE Corporation.

[0016] Example 1: Triple-negative breast cancer proliferation inhibition activity experiment

[0017] 1. Cell Culture

[0018] SUM159 cells were cultured in DMEM / F12 medium containing 10% FBS, HCC1937 cells in RIMP 1640 medium containing 10% FBS, and MDA-MB-231 and MDA-MB-468 cells in Leibovitz's L-15 medium containing 10% FBS in an incubator at 37°C with 5% CO2 air. When the cells were in the logarithmic growth phase, they were used for triple-negative breast cancer activity assays.

[0019] 2. Anti-triple-negative breast cancer activity test

[0020] (1) TNBC cells in logarithmic growth phase (SUM159, HCC1937, MDA-MB-231 and MDA-MB-468) were divided into groups of 4 × 10⁻⁶ cells. 3 Inoculate the cells at a density of 1 cell / well into 96-well plates, label the different 96-well plates, and incubate them in an incubator at 37°C with a CO2 concentration of 5% by volume for 24 hours.

[0021] (2) Add 100 μL of different final concentrations of piracetamine and N-demethylpiracetamine (40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM and 0.156 μM) and 5-FU (1000 μM, 333 μM, 111 μM, 37.03 μM, 12.3 μM, 4.12 μM, 1.37 μM, 0.46 μM and 0.15 μM) to different wells of a 96-well plate, and then place the cells in an incubator containing 37°C and 5% CO2 air for 72 h.

[0022] (3) Replace the old medium in the corresponding well with DMEM / F12, RIMP1640 and Leibovitz's L-15 medium containing 0.5% CCK8 detection reagent (brand: biosharp; catalog number: BS350B). Incubate at 37°C and 5% CO2 air content for 2-4 hours (3 hours in this case). Read the OD value of each well at 450 nm wavelength using an ELISA reader and calculate the survival rate.

[0023] The experimental results showed that both piracetamine and N-demethylpiracetamine exhibited inhibitory activity against the proliferation of four types of TNBC cells in a dose-dependent manner. Figure 2 As shown. The IC50 value of piperine inhibiting the proliferation of SUM159, HCC1937, MDA-MB-231 and MDA-MB-468 cells. 50The values ​​were 6.47 μM, 11.3 μL, 6.34 μM, and 5.01 μM, respectively; the IC50 values ​​of N-demethylpiperazine inhibited the proliferation of SUM159, HCC1937, MDA-MB-231, and MDA-MB-468 cells. 50 The values ​​were 5.93 μM, 13.61 μM, 7.55 μM, and 5.73 μM, respectively; the IC50 values ​​of 5-FU inhibited the proliferation of SUM159, HCC1937, MDA-MB-231, and MDA-MB-468 cells. 50 The values ​​were 8.66 μM, 37.67 μM, 9.66 μM, and 131.32 μM, respectively. The IC50 values ​​of piracetamine and N-demethylpiracetamine inhibited the proliferation of each type of TNBC cell. 50 The values ​​were relatively close, indicating that piracetamine and N-demethylpiracetamine had similar inhibitory activity against TNBC cell proliferation. Compared with the positive control 5-FU, piracetamine and N-demethylpiracetamine had stronger anti-TNBC cell proliferation ability, especially against HCC1937 and MDA-MB-468 cells.

[0024] Table 1. Inhibition IC50 values ​​of baicalein, N-demethylbaicalein, and 5-FU on TNBC cells. 50 value.

[0025]

[0026]

[0027] Example 2: Cell cycle detection for triple-negative breast cancer

[0028] 1. Cell Culture

[0029] SUM159 cells were cultured in DMEM / F12 medium containing 10% FBS at 37°C in an incubator with 5% CO2 air. When the cells were in the logarithmic growth phase, they were used for triple-negative breast cancer activity assays.

[0030] 2. Cell cycle detection

[0031] (1) After processing SUM159 cells according to the passage method, they were seeded into 6-well plates at a cell culture incubator at a cell number of 200,000 cells / well and cultured for 24 hours.

[0032] (2) Remove the old culture medium and add 2 mL of DMEM / F12 culture medium to each well and culture for 24 h to synchronize the cell cycle of each group of cells.

[0033] (3) Add 2 mL of N-demethyl baicale at different final concentrations (0 μM, 10 μM, 20 μM, 30 μM) to each well, and then place the cells back into an incubator at 37°C with a CO2 air content of 5% by volume for 24 h.

[0034] (4) Digest the cells with trypsin (brand: Beyotime; catalog number: C0205) (0.2 mL, 3 min), and transfer the cell suspension to a 15 mL centrifuge tube. Turn on the power and set the centrifuge parameters to 1000 rpm for 5 min. Centrifuge the cells under these conditions. Discard the supernatant after centrifugation, resuspend the cells in PBS, and centrifuge again under the same conditions (1000 rpm, 5 min). Discard the supernatant after centrifugation. To prevent cell clumping after centrifugation, gently tap the cell pellet at the bottom of the centrifuge tube.

[0035] (5) Add 0.5 mL of pre-cooled 70% ethanol (ice-water bath) to the cell pellet. Fix the cell sample in a 4°C refrigerator for 30 min. Centrifuge at 1000 g for 5 min. Discard the supernatant after centrifugation, resuspend the cells in PBS, and centrifuge again under the same conditions (1000 rpm, 5 min). Discard the supernatant after centrifugation. To prevent cell clumping after centrifugation, gently tap the cell pellet at the bottom of the centrifuge tube to remove the supernatant.

[0036] (6) Add 0.5 mL of PI staining solution (brand: Beyotime; catalog number: C1052) to each tube of cells, slowly and thoroughly resuspend the cells, and incubate the cell samples in a cell culture incubator (37℃, 5% CO2 air content) for 30 min. Before flow cytometry, vortex each tube of cell samples to mix them again, and transfer the cell samples to flow cytometry tubes with filters. Then, use a Sony SH800S flow cytometer to detect the red fluorescence intensity (PI channel) at 488 nm, and analyze the cell cycle distribution results using FlowJo.

[0037] To investigate the effect of N-demethylpiperine on apoptosis in SUM159 cells, SUM159 cells were treated with different concentrations of N-demethylpiperine for 24 h. The PI staining and flow cytometry results are as follows: Figure 3 As shown in Figure A, the horizontal and vertical axes represent DNA content and effective cell number, respectively. The figure shows that treatment with 0 μM, 20 μM, and 30 μM N-demethylhexamethonium salts increased the number of cells in the G0 / G1 phase and decreased the number of cells in the G2 phase. Statistical analysis of the cell cycle distribution results is as follows: Figure 3As shown in Figure B, treatment with 0 μM, 20 μM, and 30 μM N-demethylpiperine significantly increased the proportion of cells in the G0 / G1 phase, reaching 56.22%, 67.14%, and 79.8%, respectively, while significantly decreasing the proportion of cells in the G2 phase, reaching 30.45%, 13.52%, and 8.89%, respectively. This indicates that N-demethylpiperine induced G0 / G1 phase arrest in TNBC cells.

[0038] Cyclin D1 and CDK2 proteins play crucial roles in the cell cycle transition from G0 / G1 to S phase; their overexpression can lead to cell cycle dysregulation and subsequently, cancer cell development. SUM159 cells were treated with N-desmethylhexine, and the expression of cyclin D1 and CDK2 proteins was analyzed by Western blot. Figure 3 As shown in Figure C, the expression level of calnexin protein was almost the same in each group of cells after treatment with different concentrations of N-demethylhexane, using calnexin protein as an internal control, indicating that the total protein content of each sample was consistent. Statistical analysis of the Western blot results is as follows: Figure 3 As shown in Figure DE, treatment with N-demethylcycline at concentrations of 0 μM, 10 μM, 20 μM, 40 μM, and 60 μM resulted in the following reductions in Cyclin D1 protein expression: 100.00%, 97.67%, 69.00%, 39.00%, and 20.00%, respectively; and CDK2 protein expression: 100%, 65.33%, 58.00%, 40.34%, and 14.33%, respectively. This indicates that N-demethylcycline significantly inhibited the expression of Cyclin D1 and CDK2 proteins in a concentration-dependent manner. Based on the combined results of flow cytometry and Western blot analysis, it is hypothesized that N-demethylcycline exerts its anti-TNBC cell proliferation effect by downregulating the expression of cyclin D1 and CDK2 proteins, thereby arresting the TNBC cell cycle at the G0 / G1 phase.

Claims

1. The use of the compound as, or in the preparation of, an inhibitor of triple-negative breast cancer cell proliferation, characterized in that: The triple-negative breast cancer cell proliferation inhibitor contains one or two of the following compounds, or derivatives of the compounds, and pharmaceutically acceptable salts of the corresponding compounds as active ingredients: the compounds are piracetamine and / or N-demethylpiracetamine.

2. The use of the compound in the preparation of drugs for the prevention and / or treatment of triple-negative breast cancer, characterized in that: The compound is piracetamine and / or N-demethylpiracetamine; the triple-negative breast cancer drug uses one or two of the compound, or its derivatives, and its corresponding pharmaceutically acceptable salt as the active ingredient.

3. The application according to claim 1 or 2, characterized in that: The structural formula of the compound t-N-demethyl t-nitrofurantoin is shown below:

4. The application according to claim 1 or 2, characterized in that: The triple-negative breast cancer cells include one or more of SUM159, HCC1937, MDA-MB-231 and MDA-MB-468; The triple-negative breast cancer includes one or more of the following cell types: SUM159, HCC1937, MDA-MB-231, and MDA-MB-468.

5. The application according to claim 1, 2, 3, or 4, characterized in that: The inhibitor or drug may also include a pharmaceutically acceptable carrier and / or excipient.

6. A triple-negative breast cancer cell proliferation inhibitor, characterized in that: Its active ingredients are one or two of the following compounds: piracetamine and / or N-demethylpiracetamine, or derivatives of piracetamine and / or N-demethylpiracetamine, and pharmaceutically acceptable salts of piracetamine and / or N-demethylpiracetamine.

7. A drug for the prevention and / or treatment of triple-negative breast cancer, characterized in that: Its active ingredients are one or two of the following compounds: piracetamine and / or N-demethylpiracetamine, or derivatives of piracetamine and / or N-demethylpiracetamine, and pharmaceutically acceptable salts of piracetamine and / or N-demethylpiracetamine.

8. The inhibitor according to claim 6 or the drug according to claim 7, characterized in that: The triple-negative breast cancer cells include one or more of SUM159, HCC1937, MDA-MB-231 and MDA-MB-468; The triple-negative breast cancer includes one or more of the following cell types: SUM159, HCC1937, MDA-MB-231, and MDA-MB-468.

9. The inhibitor according to claim 6 or the medicament according to claim 7, characterized in that: The inhibitor or drug may also include a pharmaceutically acceptable carrier and / or excipient.

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