Application of D724-0491 in the preparation of drugs for treating breast cancer

Abstract

This invention belongs to the field of biopharmaceutical technology, and specifically relates to the application of D724-0491 in the preparation of products for the prevention and treatment of breast cancer. D724-0491 inhibits the binding of HBXIP and KEAP1, reduces the level of NRF2 nuclear translocation, thereby inducing oxidative stress in tumor cells and promoting cancer cell death. It is a novel targeted therapy drug for breast cancer, improving the prognostic survival rate of breast cancer patients.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to the application of D724-0491 in the preparation of products for the prevention and treatment of breast cancer. Background Technology

[0002] Cancer is a malignant disease threatening human life and health, and the distribution of different types of cancer varies significantly across countries and regions worldwide. Breast cancer, in particular, shows a rising incidence rate globally. Furthermore, researchers predict that by 2040, the number of new breast cancer cases will reach 3 million annually, while the number of deaths will exceed 1 million per year. Unfortunately, current clinical diagnostic and treatment methods for breast cancer cannot fully meet the treatment needs of patients; moreover, patients' quality of life is significantly reduced due to traditional treatments such as surgery or chemotherapy. Therefore, in-depth research into the pathogenesis of breast cancer and the exploration and development of targeted therapies for breast cancer are of paramount importance.

[0003] Hepatitis B virus X protein-binding protein (HBXIP) is a membrane protein located on the surface of human lysosomes. It forms a pentameric regulatory complex with P18 (Lamtor1), P14 (Lamtor2), MP1 (Lamtor3), and C7orf59 (Lamtor4) of the Lamtor family. The latter is primarily responsible for activating rapamycin complex 1 (mTORC1); HBXIP-C7orf59 is the core component of this pentamer. Furthermore, HBXIP has been found to play a role in insulin regulation and DNA damage repair. In studies of malignant diseases such as tumors, HBXIP has been found to be highly expressed in various malignant tumors, including cervical cancer, breast cancer, and non-small cell lung cancer, and it often predicts tumor stage, metastasis, and poor prognosis. Currently, research on the carcinogenic mechanisms associated with HBXIP is ongoing. For example, researchers have demonstrated that HBXIP can act as a coactivator of multiple oncogenes, promoting the expression of related oncoproteins. Furthermore, HBXIP has been shown to regulate the expression of related oncoproteins at multiple levels, including transcription, post-transcriptional, and translation, thereby promoting tumorigenesis and development. In addition, recent studies have found that HBXIP may serve as a potential oxidative stress regulator in breast cancer. In summary, HBXIP has been shown to regulate malignant tumor progression through multiple mechanisms, making the search for related targeted drugs particularly important.

[0004] Tumor cells exhibit significantly higher levels of reactive oxygen species (ROS) than normal cells, which can promote malignant tumor progression by regulating signaling pathways and proteins. To adapt to high ROS levels, tumor cells enhance their antioxidant capacity through various mechanisms. Researchers have discovered that interfering with the protein-protein interaction between Kelch-like ECH-associated protein (KEAP1) and the nuclear transcription factor erythroid 2-associated factor 2 (NRF2) is one of the main mechanisms by which tumor cells enhance their antioxidant capacity. Normally, NRF2 is primarily located in the cytoplasm at physiological levels, where it binds to KEAP1 and undergoes ubiquitination and degradation; only a small amount of NRF2 enters the nucleus and activates related antioxidant elements (AREs) to maintain normal physiological ROS levels. However, under pathological conditions such as tumors, NRF2 no longer binds to KEAP1 but instead enters the nucleus in large quantities and acts on downstream AREs, thereby enhancing the antioxidant capacity of tumor cells. Therefore, in previous tumor-related research, searching for small molecule compounds that target the KEAP1-NRF2 protein and its interaction is a logical approach. Recent research has revealed a highly conserved GLNLG motif on HBXIP, enabling it to actively bind to the KEAP1 protein. Furthermore, the affinity of HBXIP-KEAP1 binding is higher than that of NRF2. Therefore, HBXIP, highly expressed in breast cancer, can indirectly activate NRF2 to enter the nucleus and act on AREs (anti-reactive proteins) due to its higher affinity for KEAP1, thereby reducing the accumulation of ROS in breast cancer cells and ultimately promoting malignant progression. Interestingly, further research has shown that NRF2 entering the nucleus can also act on the promoter sequence of HBXIP, creating a positive feedback loop that further promotes breast cancer development. Therefore, identifying targeted inhibitors acting on the HBXIP-KEAP1 binding site to reduce the level of NRF2 entering the nucleus and thus induce oxidative stress in breast cancer cells presents a potential therapeutic possibility for breast cancer. Summary of the Invention

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] This invention provides the application of D724-0491 in the preparation of drugs for treating breast cancer, wherein the structural formula of D724-0491 is:

[0007]

[0008] In the above technical solution, the active ingredient of the breast cancer drug is D724-0491.

[0009] In the above technical solution, the sole active ingredient of the breast cancer drug is D724-0491.

[0010] In the above technical solution, the effective concentration of D724-0491 is further 7.5-60 μmol / L, preferably 7.5-30 μmol / L.

[0011] In the above technical solution, the breast cancer further includes breast cancer cells MCF-M7 and breast cancer cells MDA-MB-231.

[0012] In the above technical solution, the drug is further described as an oral preparation or an injectable preparation.

[0013] In the above technical solution, the drug further inhibits the binding of HBXIP and KEAP1, thereby reducing the level of NRF2 nuclear entry.

[0014] Furthermore, in the above technical solution, the drug inhibits the proliferation of breast cancer cells.

[0015] Furthermore, in the above technical solution, the drug inhibits the invasion of breast cancer cells.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention is the first to propose the application of compound D724-0491 in the preparation of drugs for treating breast cancer. D724-0491 inhibits the binding of HBXIP and KEAP1, reduces the level of NRF2 entering the nucleus, thereby inducing oxidative stress in tumor cells and promoting cancer cell death. It is a new targeted therapy drug for breast cancer and improves the prognosis and survival rate of breast cancer patients. Attached Figure Description

[0018] Figure 1 A schematic diagram of molecular docking shows the interaction site of D724-0491;

[0019] Figure 2 Figure 1 shows the results of the cytotoxicity assay; A. Killing effect of D724-0491 on MDA-MB-231 and MCF-7; B. D724-0491 showed no toxicity to normal mammary epithelial cells and human adrenal epithelial cells; (*P<0.05; **P<0.01; ***P<0.001);

[0020] Figure 3D724-0491 inhibits the proliferation and colony formation of breast cancer cells; A. D724-0491 inhibits the proliferation rate of MDA-MB-231 and MCF-7 breast cancer cells; B. D724-0491 inhibits the size and number of colony formations of MDA-MB-231 and MCF-7 breast cancer cells; C. D724-0491 inhibits the percentage of MDA-MB-231 and MCF-7 breast cancer cell colony formation (*P<0.05; **P<0.01; ***P<0.001);

[0021] Figure 4 D724-0491 inhibits the invasion and metastasis of breast cancer cells; A. D724-0491 inhibits the migration ability of MDA-MB-231 and MCF-7 breast cancer cells; B. D724-0491 weakens the invasive ability of MDA-MB-231 and MCF-7 breast cancer cells; C. The migration rate of MDA-MB-231 and MCF-7 breast cancer cells after treatment with D724-0491; D. The number of MDA-MB-231 and MCF-7 breast cancer cells after invasion after treatment with D724-0491 (*P<0.05; **P<0.01; ***P<0.001);

[0022] Figure 5 D724-0491 can interfere with the HBXIP-Keap1-Nrf2 signaling pathway and affect the nuclear localization of NRF2; A. In MDA-MB-231 and MCF-7 breast cancer cells treated with D724-0491 co-precipitated with KEAP, the binding of NRF2 to KEAP1 increased, while the binding of HBXIP to KEAP1 decreased; B. After treatment with D724-0491, the localization of NRF2 in the cell nucleus decreased (*P<0.05; **P<0.01; ***P<0.001).

[0023] Figure 6 D724-0491 affects the Keap-NRF2-related signaling pathway and the expression of HBXIP; A. D724-0491 inhibits the expression of NRF2 and NQO-1 in MDA-MB-231 and MCF-7 breast cancer cells; B. D724-0491 inhibits the expression of HBXIP in MDA-MB-231 and MCF-7 breast cancer cells; C. D724-0491 inhibits the expression rate of NRF2 and NQO-1 in MDA-MB-231 and MCF-7 breast cancer cells; D. D724-0491 inhibits the expression rate of HBXIP in MDA-MB-231 and MCF-7 breast cancer cells (*P<0.05; **P<0.01; ***P<0.001);

[0024] Figure 7 D724-0491 promotes the accumulation of ROS levels in breast cancer cells; A. The green fluorescence representing ROS increased significantly after treatment with D724-0491; B. The ROS level increased significantly after treatment with D724-0491 (*P<0.05; **P<0.01; ***P<0.001).

[0025] Figure 8 D724-0491 inhibits breast cancer cell proliferation in vivo; A. Nude mouse cadaver photographs treated with Con and D724-0491 groups, the tumor volume in the Con group was significantly larger than that in the D724-0491 group; B. In vitro comparison of tumor tissues from nude mice in the Con and D724-0491 groups, the tumor volume in the Con group was significantly larger than that in the D724-0491 group; C. Changes in body weight of nude mice in the Con and D724-0491 groups during drug administration; D. Changes in tumor tissue volume of nude mice in the Con and D724-0491 groups during drug administration; E. Histochemical results, showing changes in Ki67 and HBXIP expression in the Con and D724-0491 groups, with significantly higher Ki67 and HBXIP expression levels in the Con group (*P<0.05; **P<0.01; ***P<0.001). Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0027] Example 1

[0028] Using molecular docking technology, we sought compounds that target and interfere with the protein-protein interactions between Keap1 and NRF2. The results showed that D724-0491 primarily acts on the hydrophobic region of HBXIP, forming strong hydrogen bonds with Gly28 and Asn30. Simultaneously, the long-chain hydroxyl groups of D724-0491 also form hydrogen bonds with Asp39 and Gly43, respectively, suggesting that hydrogen bonding plays a crucial role in maintaining the binding of D724-0491 to HBXIP. A targeted inhibitor, D724-0491(C), was identified at this site. 17 H 19 CIN4O2),

[0029] 2-(2-{[3-(4-chlorophenyl)-5-methylpyrazolo[1,5-a]pyrimidin-7-yl]amino}ethoxy)ethan-1-ol( Figure 1 It inhibits the binding of HBXIP to KEAP1, reduces the level of NRF2 nuclear translocation, and thus induces oxidative stress in tumor cells. The structural formula of D724-0491 is:

[0030]

[0031] The D724-0491 used in the following examples was purchased from Shanghai Taoshu Biotechnology Co., Ltd.

[0032] The security of D724-0491 was verified.

[0033] MTT cytotoxicity assay:

[0034] 1. Plating: MCF-7 and MDA-MB-231 breast cancer cell lines were seeded into 24-well plates, with 1 × 10⁶ cells per well. 5 After mixing the cells using the cross-hatching method, observe them under a microscope and incubate them in an incubator. When the cell density reaches approximately 70%-80%, administer the drug.

[0035] 2. Drug Administration: Calculate the required drug dosage in advance according to the preset drug concentration gradient. Prepare an equal number of 5mL EP tubes according to the drug concentration gradient. Add an appropriate amount of DMEM complete culture medium to each tube, then add the corresponding volume of drug according to the experimental plan, mix well, and set aside. Remove the 24-well plate, remove the original culture medium using a pipette pump, and add 100μL of DMEM complete culture medium containing the drug to each well. After completion, mix thoroughly using the cross-hatching method in a clean bench, observe under a microscope, and incubate overnight in an incubator.

[0036] 3. Processing: The next day, dissolve the MTT reagent in advance. Take out the 24-well plate and add 20 μL of MTT reagent directly to each well. After 4 hours, aspirate the MTT and culture medium mixture from the wells. Add 100 μL of DMSO to each well. Cut an appropriate amount of aluminum foil to wrap the 24-well plate, then incubate at room temperature on a shaker for 5 minutes. After incubation, measure the absorbance at 570 nm using a spectrophotometer and analyze the data.

[0037] The results showed that, at the same treatment concentration, D724-0491 could effectively kill MCF-7 and MDA-MB-231 breast cancer cells. Figure 2 A), D724-0491 showed no cytotoxicity to normal mammary epithelial cells 10A and human adrenal epithelial cells 293T. Figure 2 B). Therefore, we selected D724-0491 as a potential drug for further research.

[0038] Example 2D724-0491 can inhibit the proliferation and colony formation ability of breast cancer cells.

[0039] This study investigated the effect of this inhibitor on the proliferation of breast cancer cells. MCF-7 and MDA-MB-231 breast cancer cells were treated with D724-0491 at concentration gradients of 7.5, 15, and 30 μmol / L, and the cells were counted continuously for four days. The results are as follows:

[0040] 1. Plate preparation: A 24-well plate was selected for the experiment, with 1×10⁻⁶ plates per well. 5 Cells were seeded individually. Each treatment group (different drug concentration treatment groups) was replicated in 3 wells, observed under a microscope, and then incubated overnight in an incubator.

[0041] 2. Treatment: On the second day, after the cell density reaches the requirements of different treatment purposes, the cells are treated (administered) in 24-well plates and then placed in an incubator for culture.

[0042] 3. Cell counting: Digest the cells in the corresponding number of wells each day, resuspend them dozens of times until the cells no longer clump together, and count the cells using a Boehringer-Stokes counting chamber. After rinsing the chamber with an appropriate amount of cell suspension from each group, count the cells under a microscope. This process is repeated for 4 days.

[0043] The results showed that, compared with the control group (untreated cells), the proliferation of breast cancer cells treated with the inhibitor D724-0491 was significantly inhibited in a dose-dependent manner. Figure 3 A).

[0044] In addition, cell clonogenesis experiments were conducted:

[0045] 1. Plating: Select 6-well plates for the experiment. Seed breast cancer cells MCF-7 and MDA-MB-231 at a density of 800 cells per well. After mixing with the cross-hatching method, observe under a microscope and incubate in an incubator for 24-48 hours.

[0046] 2. Treatment: After the culture period, administer the drug to the treated group of cells. After treatment, continue culturing in an incubator for 10-15 days, during which the number and size of breast cancer cell clones are observed daily.

[0047] 3. Photography: Once the number and size of the breast cancer cell clones have reached a certain level, use a pipette pump to remove the old solution and add 1 mL of PBS along the wall of the culture dish to rinse. The number of rinses may vary, but is generally twice. Add 1 mL of methanol along the wall of the culture dish to fix the cells and time the process for 15 minutes. After fixation, discard the methanol. Remove 0.1% crystal violet from the 4°C freezer and add an appropriate amount of crystal violet to the dish for staining. Time the process for 30 minutes, then recover the crystal violet staining solution. Turn on the tap and adjust the water flow to a low setting, placing the wells of the 6-well plate under the water flow to gently buffer and remove any remaining staining solution. (After drying, take photos.)

[0048] Cell cloning experiments showed that the size and number of breast cancer cell clones decreased with increasing D724-0491 concentration. Figure 3 B, C).

[0049] In summary, it was found that D724-0491 can inhibit the proliferation and colony formation ability of breast cancer cells.

[0050] Example 3D724-0491 can inhibit the invasion and metastasis of breast cancer cells.

[0051] The effects of this inhibitor on the invasive and metastatic abilities of breast cancer cells were investigated. Cell scratch assays were performed as follows:

[0052] 1. Plate preparation: First, draw three horizontal lines on the back of the 6-well plate to divide the plate into four equal parts. Then, seed the wells with appropriate densities of breast cancer cells MDA-MB-231 and MCF-7, mix thoroughly using the cross-hatching method, and incubate in an incubator.

[0053] 2. Processing: Once the cells reached a certain density, draw a vertical line in the center of each well of a six-well plate using a 10 μL pipette tip, then aspirate the old culture medium. Wash once with PBS, add serum-free culture medium, and administer the drugs according to the experimental purpose. After completion, photograph the plate under a microscope; this time is recorded as 0 h. Subsequently, photograph the six-well plates at 12, 24, and 48 h of culture time.

[0054] The results showed that the migration ability of breast cancer cells treated with D724-0491 was significantly reduced. Figure 4 A, C).

[0055] Transwell invasion chamber experiment:

[0056] 1. Preparation of Matrigel: Corning brand Matrigel was used to simulate the invasion environment of breast cancer cells. It was removed from the container and allowed to thaw overnight. The next day, the thawed Matrigel and serum-free culture medium were removed from the 4°C freezer. The Matrigel was diluted in a clean bench at a ratio of 8:1 (serum-free culture medium:Matrigel). Depending on the treatment purpose, an appropriate number of chambers (three parallel wells for different concentrations of the drug group) were placed into a 24-well plate using forceps. Then, 100 μL of Matrigel was added to the top layer of each chamber, and the plate was placed in a clean bench overnight to solidify.

[0057] 2. Plating: After cell digestion, centrifuge and discard the supernatant. Add serum-free culture medium to EP tubes to disperse the cell pellet. Seed breast cancer cells MCF-7 and MDA-MB-231 into the chambers at a rate of 1.5 × 10⁶ cells per chamber. 5 -2.5×10 5Seed cells at the appropriate density. Select a suitable well in a 24-well plate and add 750 μL of DMEM complete medium. Then transfer the chamber into the well and place the 24-well plate in an incubator.

[0058] 3. Processing: Remove the 24-well plate and add appropriate amounts of PBS, methanol, and crystal violet to an equal number of blank wells, according to the number of chambers. Then, use tweezers to place the chambers into the wells containing PBS and rinse them several times. Next, place them into the wells containing methanol for fixation for 1 minute. After removal, stain the chambers in the wells containing crystal violet for 30 minutes. After staining, wash away any residual staining solution with PBS and observe the upper layer of the chamber for any unpenetrated cells. If any are found, gently wipe them away with a cotton swab, and then photograph them under a microscope.

[0059] The results also showed that D724-0491 could significantly inhibit the invasive ability of breast cancer cells in a concentration-dependent manner. Figure 4 (B, D)

[0060] In summary, the data above demonstrate that D724-0491 can effectively inhibit the proliferation, invasion, and metastasis of breast cancer cells.

[0061] Example 4D724-0491 can interfere with the HBXIP-Keap1-Nrf2 signaling pathway and affect the nucleus localization of NRF2.

[0062] To investigate whether the alterations in the binding of HBXIP, KEAP1, and NRF2 in breast cancer cells after D724-0491 treatment were as expected, KEAP1 was first used for immunoprecipitation of NRF2 and HBXIP.

[0063] 1. Bead Pretreatment: After obliquely cutting the pipette tip, pipette approximately 100 μL of beads into an EP tube, add 400 μL of ice-cold PBS, and pipette several times. Centrifuge at 1000g for 3 minutes. Discard the supernatant PBS, add 400 μL of PBS again, and repeat the above operation 4 times. After the final wash, add 400 μL of PBS and store at 4°C for later use. Mix well before each use, and add 80 μL of the bead and PBS mixture to every 1 ml of protein sample.

[0064] 2. Plating: Add 8 ml of DMEM complete culture medium and 500 μL of cell suspension to each dish, mix well using the cross method, observe under a microscope, and place in an incubator. When the cell density reaches approximately 70%-80%, administer the drug according to the predetermined concentration.

[0065] 3. Drug administration: Calculate the required drug volume according to the predetermined drug concentration, administer the drug directly into the culture dish after protecting it from light, shake well and continue culturing.

[0066] 4. Collecting cells: Prepare new EP tubes in advance and label them accordingly. Aspirate the cell suspension into the corresponding EP tubes.

[0067] 5. Incubation: Take 40 μL of supernatant from both the control group and the treatment group, add 10 μL of 5× loading buffer, mix thoroughly, and boil for 5 minutes to denature. Store at -20°C. Take the HBXIP antibody from the -20°C freezer, calculate the required volume of antibody for the remaining supernatant according to the antibody instructions, add the antibody, and incubate at 4°C with rotation for 4 hours. After completion, add 80 μL of the prepared beads and PBS mixture and incubate overnight for at least 16 hours.

[0068] 6. Processing: The following day, after incubation, centrifuge at 5000 rpm for 10 minutes in a low-temperature centrifuge. Discard the supernatant, add 1 ml of PBS, and wash by rotation at 4°C for 10 minutes. Then centrifuge again at 5000 rpm for 10 minutes in a low-temperature centrifuge, discarding the supernatant. Repeat twice. After completion, add 8 μL of 5× loading buffer and boil to denature.

[0069] The results showed that, compared with the control group (untreated group), the binding of NRF2 to Keap1 was significantly increased after treatment with 30 μmol / L D724-0491, while the binding of HBXIP to Keap1 was significantly decreased. Figure 5 A).

[0070] Furthermore, previous studies have demonstrated that NRF2 dissociated from Keap1 can translocate from the cytoplasm to the nucleus. Therefore, immunofluorescence was used to investigate the nuclear translocation of NRF2 after inhibitor treatment, and the experimental verification is as follows:

[0071] 1. Slide preparation: Place the slides in a six-well plate one day in advance, add an appropriate amount of alcohol, and sterilize overnight in a laminar flow hood. The next day, discard the alcohol, add PBS to rinse three times, and then use as is.

[0072] 2. Laying the board: 1×10 per hole 6 Calculate the required cell suspension volume to determine the cell density, then use a pipette to transfer the resuspended cell suspension into 6-well plates. Mix thoroughly using the cross-hatching method, observe under a microscope, and incubate for 24 hours.

[0073] 3. Administration: The next day, calculate the required drug volume according to the target concentration, gently mix after administration and place in an incubator for 24 hours.

[0074] 4. Treatment: The next day, aspirate the original culture medium using a pipette. Add PBS along the wall of the culture dish and rinse for 5 minutes. Repeat this operation 3 times, discarding the PBS after the last rinse. Fix the cells with an appropriate amount of 4% paraformaldehyde for 15 minutes, rinsing 3 times with PBS afterward. Add 2 mL of 0.5% permeabilization buffer (PBS + Triton-100 mixture) along the wall of the culture dish and permeabilize the cells for 10 minutes. After permeabilization, rinse with PBS for 5 minutes. Repeat this operation 3 times.

[0075] 5. Blocking: Add an appropriate amount of normal goat serum to the culture dish, just enough to submerge the slide, and block at room temperature for 30 minutes. After completion, rinse with PBS for 5 minutes. Repeat this operation 3 times.

[0076] 6. Incubation: Dissolve the required antibody dilution solution in advance. Add NRF2 antibody dilution solution to the culture dish and incubate overnight on a shaker at 4°C. The next day, discard the antibody dilution solution and rinse with PBS for 5 minutes. Repeat this operation 3 times. Add secondary antibody, wrap the 6-well plate with aluminum foil, and incubate on a shaker for 1 hour. After incubation, rinse with PBS for 5 minutes. Repeat this process three times.

[0077] 7. Staining: Under light-protected conditions, add DAPI dilution buffer and stain for 5 minutes. Then rinse with PBS for 5 minutes. Repeat this process three times.

[0078] 8. Photography: Turn off the lights in the laminar flow hood. Slowly add an appropriate amount of 4% paraformaldehyde along the wall of the culture dish, ensuring it covers the slide, and time for 15 minutes. After completion, rinse with 1 mL of PBS for 3 minutes, repeating this operation three times. Then, add an appropriate amount of diluted DAPI staining to the slide and time for 5 minutes. Then, rinse with PBS for 5 minutes, repeating this operation four times. During this time, prepare an appropriate number of slides and label them accordingly. After rinsing, remove the slides and add an appropriate amount of anti-fluorescence quencher to the slides to be used. Then, mount the slides (with the cell side) onto the cell slide. Wrap the slides in aluminum foil and take them to a dark room for photography using a fluorescence microscope.

[0079] The results showed that after treatment with 30 μmol / L D724-0491, NRF2 localized in the cell nucleus was significantly decreased. Figure 5 B).

[0080] Example 5D724-0491 can affect the Keap-NRF2 related signaling pathway and HBXIP expression.

[0081] Studies have shown that NRF2 entering the cell nucleus can activate the expression of downstream antioxidant elements such as NQO-1, thereby enhancing the antioxidant capacity of tumor cells. Furthermore, it is noteworthy that NRF2 in the cell nucleus can also activate the HBXIP promoter, thus promoting HBXIP expression and forming a positive feedback loop. Therefore, to investigate the changes in related proteins after D724-0491 treatment, nucleocytoplasmic separation was performed on breast cancer cells treated with the inhibitor, and the following experimental verification was conducted:

[0082] 1. Plating: MCF-7 and MDA-MB-231 breast cancer cells were seeded at an appropriate density in culture dishes, mixed using the cross method in a clean bench, observed under a microscope, and then cultured in an incubator.

[0083] 2. Drug administration: When the cell density reaches approximately 60%-70%, calculate the required dosage based on the target concentration. Add the drug to the wells of the designated culture dish, gently mix using the cross-hatching method, and place in an incubator for further culture. Harvest the cells.

[0084] 3. Prepare reagents: Prepare an ice box in advance. Take the nucleo-cytoplasmic separation kit out of the -20℃ freezer and place it on the ice box to thaw. Calculate the required total amount of CEA-A and NEB, assuming that one culture dish requires 100μL of CEA-A and 50μL of NEB. Then prepare CEA-A and NEB according to the instructions of the nucleo-cytoplasmic separation kit and place them on ice for later use.

[0085] 4. Processing: Add 100 μL of CEA-A to the EP tube, vortex for 15 seconds, and incubate on ice for 10 minutes. After incubation, add 5.5 μL of pre-chilled CEB-B to the EP tube, vortex for a few seconds, and incubate on ice for 1 minute. After incubation, vortex for a few seconds and centrifuge at 10000 xg for 5 minutes at 4°C. During this time, prepare an appropriate number of new EP tubes labeled "Cytoplasm". After centrifugation, carefully pipette the supernatant into the prepared new EP tubes. Then add 500 μL of NEB to the precipitate, vortex for 15 seconds, and incubate on ice for 10 minutes. Repeat the above steps a total of 4 times. After centrifugation, centrifuge at 16000 rpm for 10 minutes, and prepare an appropriate number of new EP tubes labeled "Nucleus". After centrifugation, only aspirate the supernatant and transfer it to the EP tube labeled "Nucleus".

[0086] Changes in relevant proteins were detected using Western blot technology.

[0087] 1. Cell Processing: Prepare new EP tubes and label them as treatment and control groups, setting them aside for later use. Cell processing is the same as cell passage as before. Aspirate the cell suspension into the corresponding EP tubes, centrifuge at 800 rpm for 3 minutes, and remove the supernatant using a pipette pump. Add 1 mL of PBS to the EP tube to re-disperse the cell pellet, centrifuge at 2000 rpm for 3 minutes, and discard the supernatant. Observe the amount of cell pellet and, based on experience, add an appropriate amount of RIPA protein lysis buffer to the EP tubes, and lyse by rotation at 4°C for 40 minutes. Subsequently, centrifuge at 12000 rpm for 20 minutes in a low-temperature centrifuge, and use a pipette of the appropriate volume to transfer the supernatant into a spare EP tube.

[0088] 2. Protein Quantification: Remove the BCA protein quantification kit, 5× loading buffer, and standards from the 4℃ freezer. Calculate and prepare an appropriate amount of working solution. Select an appropriate number of wells in a 96-well plate, add the specified volume of protein sample and the above-mentioned materials sequentially, and incubate for approximately 15 minutes. Measure the absorbance using a spectrophotometer. Simultaneously, calculate the extracted protein concentration and the required amounts of 5× loading buffer and PBS using the standard curve presented by the standards. After adding the above-mentioned materials to the sample, denature at 98℃, and finally store at -20℃ for later use.

[0089] 3. Electrophoresis: Prepare PAGE gels of the required concentration in advance, load protein samples of equal mass according to the set grouping, and perform electrophoresis at an appropriate voltage for the corresponding time according to the molecular weight of the target protein.

[0090] 4. Transfer: After electrophoresis, cut the target protein into the molecular weight range specified by the Maker, then place it in the transfer tank and transfer it at an appropriate voltage for a certain period of time according to the molecular weight of the target protein.

[0091] 5. Sealing: After the transfer is complete, remove 5% milk sealing solution from the refrigerator and pour it into the container. Transfer PVDF to the sealing solution and place it on a shaker at room temperature for one hour.

[0092] 6. Antibody Incubation: After blocking, recover or discard the blocking solution depending on the number of times it has been used. Pour PBST into the container, shake a few times, then discard the PBST. Pour in an appropriate amount of PBST again and wash the membrane three times on a shaker, 5 minutes each time. During this time, take out the required pre-prepared primary antibody dilution and thaw it in water. After washing, place the PVDF membrane in the corresponding primary antibody dilution according to its molecular weight and incubate overnight on a shaker at 4°C. The next day, wash the membrane three times with PBST, 15 minutes each time, and recover the antibody. Then add the appropriate secondary antibody to the container, place the PVDF membrane inside, incubate at room temperature for 1 hour, and wash the membrane again with PBST for 15 minutes, repeating this process three times.

[0093] 7. Development: Turn on the gel imaging system 30 minutes in advance to preheat it and enter working condition. At the same time, prepare the luminescent solution according to the 1:1 ratio and wrap the EP tube with aluminum foil to protect it from light. After the gel imaging system is ready, place it on the PVDF membrane and add an appropriate amount of ECL developer to perform the development operation.

[0094] The results showed that after treatment with D724-0491, the content of NRF2 in the cell nucleus decreased significantly; in addition, the expression of downstream NRF2-related proteins such as NQO-1 and HBXIP also decreased. Figure 6 AD)

[0095] Example 6D724-0491 can promote the accumulation of intracellular ROS levels in breast cancer cells.

[0096] Based on the above experimental data, it is hypothesized that D724-0491 can promote changes in ROS levels within breast cancer cells. To verify this hypothesis, flow cytometry was used for further analysis.

[0097] 1. Plate seeding: MCF-7 and MDA-MB-231 breast cancer cells were seeded into six-well plates, 2 × 10⁶ cells per well. 5 The number of cells was determined, the mixture was thoroughly mixed, observed under a microscope, and then incubated in an incubator.

[0098] 2. Treatment: Once the cell density is suitable (around 60%-70%), administer the drug. After completion, place the cells in an incubator for further culture.

[0099] 3. Staining: Prepare new EP tubes in advance. Follow the same cell digestion procedure as above, aspirate the cell suspension into the prepared EP tubes, centrifuge, and discard the supernatant. Resuspend the cells in DMEM medium (serum-free and antibiotic-free), turn off the lights in the laminar flow hood, and add 500 μL of DCFH-DA dye to each EP tube. Wrap the EP tubes with aluminum foil and place them in an incubator for 35 minutes, inverting them every 5 minutes. After the reaction, centrifuge, discard the supernatant, resuspend the cells in PBS, wash away any unattached dye, and centrifuge again. Repeat this process three times.

[0100] 4. Detection: During the 35-minute reaction period described above, turn on the flow cytometer in advance and run the self-test program to get the machine ready. Place the EP tube on the flow cytometer, resuspend the cells with a 1 mL pipette, filter, and add the solution to the flow cytometer detection tube to detect and record the ROS concentration.

[0101] Immunofluorescence analysis of changes in intracellular ROS levels in breast cancer cells after treatment with D724-0491:

[0102] 1. Slide preparation: Place the slides in a six-well plate one day in advance, add an appropriate amount of alcohol, and sterilize overnight in a laminar flow hood. The next day, discard the alcohol, add PBS to rinse three times, and then use as is.

[0103] 2. Plate seeding: MCF-7 and MDA-MB-231 breast cancer cells were seeded into prepared six-well plates at a density of (1.2-1.5) × 10⁶ cells per well. 6 Cell density was determined by mixing the cells using the cross method, observing them under a microscope, and then incubating them in an incubator.

[0104] 3. Treatment: Once the cell density reaches the required level, administer the drug to the treatment group. After treatment, place the cells in an incubator for further culture.

[0105] 4. Staining: Discard the original culture medium. Rinse with 1 mL of PBS along the wall of the culture dish and discard the PBS. Repeat this operation twice. Take out the DMEM incomplete culture medium and add 1 mL to each well along the wall of the dish. Turn off the lights in the laminar flow hood, add 1 μL of DFHDA to each well, gently shake, and place in the incubator to react for 35 minutes. After the reaction, rinse with 1 mL of PBS for 3 minutes, repeating 3 times. Discard the PBS from the last rinse.

[0106] 5. Photography: Turn off the lights in the laminar flow hood. Slowly add an appropriate amount of 4% paraformaldehyde along the wall of the culture dish, ensuring the slide is completely submerged, and time for 15 minutes. After completion, rinse with 1 mL of PBS for 3 minutes, repeating this operation three times. Then, add an appropriate amount of diluted DAPI staining to the slide and time for 5 minutes. Next, rinse with PBS for 5 minutes, repeating this operation four times. During this time, prepare an appropriate number of slides and label them accordingly. After rinsing, remove the slides, add an appropriate amount of anti-fluorescence quencher to the slides to be used, and then mount the slides (cell-side) onto the culture dish. Wrap the slides in aluminum foil and take them to a dark room for photography using a fluorescence microscope.

[0107] The results showed that D724-0491 treatment significantly promoted the increase of ROS levels in breast cancer cells. Figure 7 AB).

[0108] Based on the above data, it can be concluded that D724-0491 can interfere with the Keap1-NRF2 signaling pathway by targeting and inhibiting the function of HBXIP protein, thereby increasing the ROS level of breast cancer cells and achieving a killing effect.

[0109] Example 7D724-0491 can inhibit the proliferation of breast cancer cells in vivo.

[0110] To further refine the research, a nude mouse model was used to verify whether D724-0491 also has an inhibitory effect on breast cancer in vivo. Ten SPF-grade nude mice (weighing 17-20g, 4-5 weeks old) were used. After being housed in the corresponding animal laboratory for one week, the ten mice were randomly and equally divided into two groups, labeled Con and D724 on their frames. Cell culture: The cell resuscitation procedure was the same as above. MCF-7 cells with the most recent cryopreservation time were selected, and four tubes were thawed at a time. Approximately 30 culture trays were passaged for cell culture. Inoculation: Each nude mouse was inoculated with 1×10⁻⁶ cells. 7 Cells. The cell digestion procedure is the same as above. Digest and centrifuge five trays of cells as a group, resuspend the cells in DMEM several times to ensure individual cell arrangement in the cell suspension. Aspirate 5-10 μL of cell suspension into a cell counting chamber and calculate the required cell suspension volume. Use a pipette to transfer the calculated cell suspension volume into a new EP tube, centrifuge, discard the supernatant, and re-disperse the cell pellet with serum-free culture medium. Add 50 μL of matrix gel to every 150 μL of cell suspension, mix well, aspirate into a syringe, remove air bubbles, and inoculate between the right neck and armpit of nude mice. Observation: After tumor inoculation, observe the nude mice daily in the animal laboratory. Prepare for drug administration when the tumor reaches the size of a soybean. Change the bedding, water, and food every few days. Drug administration: Calculate the dosage per nude mouse according to the animal drug dosage conversion table as 50 mg / kg, and administer the drug every two days. At the time of drug administration, the weights of nude mice in both the Con and D724 groups were weighed and recorded. The required dosage per mouse was calculated based on the weight of the mice in the D724 group, and the medication was administered intraperitoneally. The control group (untreated group) received a fixed amount of physiological saline. Simultaneously, the long and short diameters of the tumors in the nude mice of both the Con and D724 groups were measured and recorded. Tissue collection: Nude mice were euthanized by cervical dislocation, and the mice were arranged in sequence on a piece of white paper for photographing. The tumor tissue was removed, and the mice were placed back in their original positions, with a ruler placed on top for photographing. After completion, the tumor tissue was weighed and recorded. Finally, cut off a portion of the tissue with scissors and place it in tissue fixation solution for subsequent HE staining (1. Dewaxing: After removing the paraffin sections, place them in a dewaxing chamber for about 40 minutes. After completion, place them at room temperature for 10 minutes. Open the fume hood and then proceed with the dewaxing process in the correct order. 2. Processing: Add appropriate amounts of hematoxylin staining solution (3 minutes), hematoxylin differentiation solution (several seconds), and hematoxylin blue solution (several seconds) to the tissue sites on the sections in sequence. Before each change of staining solution, rinse thoroughly from top to bottom under a small amount of running water. Then, place the sections in 85% ethanol (5 minutes) and 95% ethanol (5 minutes) in sequence to complete dehydration. Then, add eosin staining to the sections for 5 minutes, rinse, and then dewax again in the reverse order of the above process. Mount with neutral resin. 3. Photography: Take photos and analyze); the remaining tissue is placed in appropriately labeled bags and stored in a -80℃ refrigerator.

[0111] The results showed that the tumor volume in nude mice treated with D724-0491 was significantly smaller than that in the Con group. Furthermore, there was no significant difference in body weight changes between the Con and D724-0491 groups, and the changes were minimal. Figure 8 Immunohistochemical analysis of tumor tissues from both the Con and D724-0491 groups revealed a significant decrease in the expression of the tumor marker Ki67 in the D724-0491 group. Figure 8 E); In addition, the expression of HBXIP in the two groups of animals was investigated, and the results were as predicted, with a significant decrease in HBXIP expression in the treatment group (E); Figure 8 E). In conclusion, it can be shown that D724-0491 can effectively inhibit the progression of breast cancer in vivo.

Claims

1. The application of D724-0491 in the preparation of drugs for treating breast cancer, wherein the structural formula of D724-0491 is: .

2. Use according to claim 1, characterized in that, The active ingredient of the breast cancer drug is D724-0491.

3. Use according to claim 1, characterized in that, The only active ingredient of the breast cancer drug is D724-0491。 4. Use according to claim 1, characterized in that, The breast cancer includes breast cancer cells MCF-M7 and breast cancer cells MDA-MB-231.

5. The use according to claim 1, characterized in that, The drug is an oral preparation or an injection preparation.

6. Use according to claim 1, characterized in that, The drug inhibits the binding of HBXIP to KEAP1, reduces the nuclear level of NRF2.

7. Use according to claim 1, characterized in that, The drug inhibits the proliferation of breast cancer cells.

8. The use according to claim 1, characterized in that, The drug inhibits the invasion of breast cancer cells.

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