Application of compound or its salt in preparing preparation for inhibiting WNT7A gene activity

Abstract

This invention belongs to the field of cancer drug technology and specifically relates to the use of a compound or a salt thereof in the preparation of a preparation for inhibiting WNT7A gene activity. The compound can effectively inhibit WNT7A gene activity and tumor growth and spread; it also exhibits good drug safety both in vivo and in vitro, has low toxicity to normal cells, and does not cause significant side effects at either the cellular or in vivo levels.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer drugs, and specifically relates to the use of a compound or a salt thereof in preparing a preparation for inhibiting WNT7A gene activity. Background Art

[0002] WNT7A is a key protein in the WNT family, playing a crucial role in human development, tissue homeostasis, and various physiological and pathological processes. Numerous studies have shown that WNT7A is closely associated with the occurrence, progression, and poor prognosis of various malignancies. For example, studies have found that high WNT7A expression can promote the occurrence and progression of breast cancer, while low WNT7A expression is associated with a better prognosis. In colorectal cancer, high WNT7A expression can promote the proliferation and metastasis of EpCAM-positive cells and affect the drug resistance of tumor cells, leading to poor prognosis. In ovarian cancer, WNT7a promotes the growth and invasion of ovarian cancer cells in a β-catenin / Tcf-MMP7-dependent manner. WNT7a induces the production of fibroblast-like cells associated with ovarian cancer metastasis, resulting in a close association between high WNT7A expression and poor prognosis. Furthermore, Wu et al. found that hypoxia-induced high WNT7A expression may promote cell migration by promoting the transformation of PDAC epithelial cells to mesenchymal cells, thereby promoting tumor metastasis and leading to poor prognosis.

[0003] The poor prognostic impact of WNT7A may involve multiple biological and molecular mechanisms, including promoting tumor cell proliferation, invasion, and metastasis; regulating stem cell characteristics and epithelial-mesenchymal transition; and influencing the tumor microenvironment and oncogenic pathways. Within these mechanisms, WNT7A often interacts with other signaling pathways and proteins, forming a complex signaling network. Existing research has shown that WNT7A is a member of the WNT pathway family, a pathway that plays a crucial role in human development and various physiological and pathological processes and is widely considered to be a key regulator of cell signaling and gene expression in the human body. The WNT / β-catenin pathway, a key WNT pathway, is the primary pathway involved in WNT7A's biological activities. The WNT / β-catenin pathway is a critical signaling pathway involved in a wide range of biological processes, including cell growth, differentiation, and apoptosis. This pathway is initiated through the interaction between the WNT receptor and the Frizzled (FZD) receptor, which form a complex, and exerts its biological effects by regulating the stability and nuclear translocation of β-catenin. Furthermore, overactivation of the WNT / β-catenin pathway promotes cancer cell proliferation and metastasis. In summary, the WNT / β-catenin pathway can influence the proliferation and survival of tumor cells by regulating the expression of multiple cell cycle and apoptosis-related genes. For example, genes such as MYC and CCND1 are among the downstream target genes of the WNT / β-catenin pathway, and their high expression is closely associated with the occurrence and progression of malignant tumors. Furthermore, the WNT / β-catenin pathway can also influence the characteristics of cancer stem cells, which have the ability to promote tumor proliferation, metastasis, and drug resistance. This pathway promotes the self-renewal and proliferation of cancer stem cells by regulating the expression of cytokines such as IL-6, Notch, and Hedgehog.

[0004] In summary, WNT7A is a gene that has been identified as having a negative impact on cancer prognosis. Overexpression of WNT7A can stimulate cell proliferation, metastasis, and invasion in various cancers, thereby promoting tumor progression. However, there is currently a lack of clinical translational research targeting WNT7A, hindering the use of more direct and precise treatments for related cancers. To inhibit WNT7A gene activity, RNA interference (RNAi) technologies such as siRNA and shRNA can be used to specifically suppress WNT7A expression, effectively inhibiting biological processes such as cell proliferation and invasion. However, these approaches have limitations, such as low specificity, limited dose response, and unsuitability for drug development. Alternatively, WNT7A-specific monoclonal antibodies can be used. By targeting the WNT7A protein or its interacting proteins, these antibody drugs can effectively inhibit activation of the WNT signaling pathway, thereby suppressing tumor growth and metastasis. However, the high production cost and the large injection dose required restrict the use of WNT7A-specific monoclonal antibodies.

[0005] In addition to the two RNA interference techniques mentioned above, with the development of modern computer technology, researchers can also use a variety of methods such as computational chemistry and molecular docking to screen small molecule compounds that can specifically inhibit target genes. Moreover, small molecule compounds have the advantages of stable efficacy, ease of use, and low cost. Therefore, it is necessary to develop or explore a small molecule compound that can specifically inhibit the WNT7A gene. Summary of the Invention

[0006] To address the above issues, one objective of the present invention is to provide a small molecule compound, 1365-0109 (the compound represented by Formula I below), that can target and inhibit the WNT7A gene. This compound can be used to prepare a formulation that inhibits WNT7A gene activity. This small molecule compound was initially screened using computational chemistry and molecular docking, followed by further screening through in vitro and in vivo assays.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides a compound or a salt thereof for use in preparing a preparation for inhibiting WNT7A gene activity. The compound has a structural formula as shown in Formula I.

[0009]

[0010] Another aspect of the present invention provides a use of a compound or a salt thereof in the preparation of a drug for treating cancer. The compound has a structural formula as shown in Formula I.

[0011]

[0012] The beneficial effects of the present invention include at least:

[0013] (1) The compound represented by formula I can effectively inhibit the activity of the WNT7A gene.

[0014] (2) The compound represented by formula I can effectively inhibit the growth and spread of tumors. Compared with the control group, the tumors grow more slowly, are smaller, and weigh less, and the survival period is significantly prolonged.

[0015] (3) The compound represented by Formula I exhibits good drug safety both in vivo and in vitro, has low toxicity to normal cells, and does not cause obvious side effects at the cellular and in vivo levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram of a human breast cancer tissue sample with high WNT7A expression levels;

[0017] Figure 2 This is a diagram of a human breast cancer tissue sample with low WNT7A expression levels;

[0018] Figure 3 This is a comparison of the overall survival (OS) of breast cancer patients with different WNT7A expression levels in cancer tissue samples;

[0019] Figure 4 This is a photo of tumor growth after subcutaneous orthotopic tumor formation using shWNT7A-EO771;

[0020] Figure 5 This is a time-volume curve of tumor growth after subcutaneous orthotopic tumor formation using shWNT7A-EO771;

[0021] Figure 6 This is a graph showing the tumor volume in the shWNT7A-EO771 group;

[0022] Figure 7 Figure 2 is the tumor weight of mice in the shWNT7A-EO771 group;

[0023] Figure 8 Schematic diagram of the active pocket of WNT7A protein;

[0024] Figure 9 2D diagrams of the effects of 5,286,670 small molecule compounds;

[0025] Figure 10 2D diagram of the interaction of 5,683,035 small molecule compounds;

[0026] Figure 112D diagram of the interaction of 5,180,332 small molecule compounds;

[0027] Figure 12 2D diagrams of the interactions between 5,673,999 small molecule compounds;

[0028] Figure 13 2D diagrams of the interactions of 5,283,766 small molecule compounds;

[0029] Figure 14 2D diagram of the effects of 7954-4074 small molecule compounds;

[0030] Figure 15 2D diagram of the action of the small molecule compound F067-1148;

[0031] Figure 16 2D diagram of the effect of small molecule compound C200-3685;

[0032] Figure 17 2D diagram of the action of small molecule compound 1365-0109;

[0033] Figure 18 2D diagram of the action of small molecule compound 3253-0183;

[0034] Figure 19 Figure 2 shows the inhibition of WNT7A and FZD5 by different small molecule inhibitors in the triple-negative breast cancer cell line MDA-MB-231;

[0035] Figure 20 Figure 2 shows the inhibition of WNT7A and FZD5 by different small molecule inhibitors in triple-negative breast cancer cell line EO771;

[0036] Figure 21 This is a graph showing the effects of different concentrations of 1365-0109 small molecules on MDA-MB-231 cell lines at different action times;

[0037] Figure 22 This is a graph showing the effects of different concentrations of 1365-0109 small molecules on EO771 cell lines at different action times;

[0038] Figure 23 This is a picture showing the actual tumor growth after intervention with different concentrations of the 1365-0109 small molecule;

[0039] Figure 24 This is the time-volume diagram of tumor growth after intervention with different concentrations of 1365-0109 small molecules;

[0040] Figure 25 This is a diagram showing the tumor volume after intervention with different concentrations of 1365-0109 small molecules;

[0041] Figure 26 This is a graph showing the tumor weight after intervention with different concentrations of 1365-0109 small molecules;

[0042] Figure 27 The figure shows the effects of different concentrations of 1365-0109 small molecules on the routine blood test, ASL and ALT of mice; the horizontal axis in the figure is from left to right for the shNC group, 5 mg / kg group, 10 mg / kg group and 15 mg / kg group. DETAILED DESCRIPTION

[0043] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0044] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context clearly has a different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0045] An embodiment of the present invention provides a use of a compound or a salt thereof in preparing a preparation for inhibiting WNT7A gene activity. The compound has a structural formula as shown in Formula I.

[0046]

[0047] Specifically, the full name of the WNT7A gene is Wnt family member 7A, ID: 7476. In addition, the present invention uses the ChemBridge and ChemDiv libraries to screen small molecule compounds, identifying the active site of the molecule by determining the WNT7A active pocket. The resulting small molecules are docked, and the results are ranked by docking score. Those with the highest scores are selected for synthesis. The effectiveness of the small molecule inhibitors in blocking WNT7A and FZD5 is verified through experiments such as COIP, and the efficacy and safety of the drugs are verified through in vitro physical tests.

[0048] In addition, as mentioned above, WNT7A is an important molecular marker that is associated with poor prognosis in various types of tumors. The above-mentioned compounds or salts thereof can effectively inhibit the activity of WNT7A, thereby affecting behaviors related to WNT7A gene activity, such as tumor occurrence, development and poor prognosis.

[0049] In addition, the compound represented by the above formula I is also referred to as 1365-0109 below.

[0050] It should be noted that the salt form of the above compounds is a salt known in the art, generally a hydrochloride.

[0051] In some specific embodiments, the dosage form of the above-mentioned preparation may include tablets, powders, liquids, suspensions or gels.

[0052] It should be noted that the above-mentioned compound or its salt can be prepared into different dosage forms by adding excipients according to different needs, such as tablets, powders, liquids, suspensions or gels. Excipients are all known in the art. For example, the preparation of tablets mainly uses diluents (such as starch, dextrin, sucrose or glycosides), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (povidone, syrup or hypromellose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvinylpyrrolidone, etc.); for example, the preparation of liquids mainly uses expanders, suspending agents, emulsifiers or colorants.

[0053] In addition, the above-mentioned preparations are not specifically drugs, and in addition to drugs, the purpose of inhibiting the activity of the WNT7A gene can also be achieved in other forms.

[0054] Another embodiment of the present invention provides the use of the above-mentioned compound or its salt in the preparation of a drug for treating cancer. It should be noted that the use of the above-mentioned compound in tumor inhibition can significantly reduce the proliferation and invasion ability of cancer cells without causing significant damage to cells, mouse internal organs, blood routine, or liver function.

[0055] In some specific embodiments, the above-mentioned use in preparing a drug for treating cancer includes: use of the above-mentioned compound or its salt in preparing a drug for inhibiting WNT7A gene activity.

[0056] In some specific embodiments, the above-mentioned use in preparing a drug for inhibiting WNT7A gene activity and the use in a drug for inhibiting WNT7A gene activity include: use of the above-mentioned compound or its salt in preparing a drug for inhibiting the WNT / β-catenin signaling pathway.

[0057] Specifically, the aforementioned compound or its salt can inhibit the activity of the WNT7A gene, thereby inhibiting the binding of WNT7A to FZD5 and reducing the activation of the WNT / β-catenin signaling pathway, thereby inhibiting the occurrence and development of tumors. Furthermore, the compound has low toxicity to normal cells and does not cause significant side effects at the cellular or in vivo levels.

[0058] It should be noted that WNT7A is a key signaling molecule that triggers a series of biological responses within the cell by binding to specific receptors. A review of literature and relevant databases (http: / / genemania.org / ) revealed that the primary receptor for WNT7A is FZD5. FZD5 is a membrane-associated cell surface receptor protein, a member of the Frizzled family, and is capable of binding to WNT7A and other WNT family proteins. The FZD5 molecule is composed of approximately 300 amino acid residues, with its N-terminus and C-terminus located both inside and outside the cell membrane. On the inner membrane, the FZD5 domain consists of a seven-transmembrane α-helix and a cytoplasmic C-terminal sequence. Binding of WNT7A to the FZD5 receptor activates the WNT / β-catenin signaling pathway and plays a crucial role in biological processes such as gene expression regulation.

[0059] In some specific embodiments, the cancer may be one or more of breast cancer, colorectal cancer, and ovarian cancer. Specifically, the cancer may be any cancer caused by WNT, such as breast cancer, colorectal cancer, and ovarian cancer.

[0060] In some specific embodiments, the dosage form of the above-mentioned drug may include an injection or an oral agent. Specifically, the above-mentioned drug dosage form can be prepared into an injection or an oral agent according to clinical needs, and the method for preparing the injection or oral agent is known in the art.

[0061] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0062] 1. Validation of WNT7A as a tumor marker for predicting the prognosis of breast cancer patients

[0063] In the examples of the present invention, it was verified that WNT7A can be used as an oncological marker to determine the prognosis of breast cancer patients, as follows:

[0064] (1) Sampling and preparation of paraffin sections

[0065] Cancer tissues from breast cancer patients in the Department of Endocrine Surgery, The First Affiliated Hospital of Chongqing Medical University were collected (this study has been approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University, with ethics number: 2022-K121), and tissue paraffin blocks and sections were routinely prepared.

[0066] (II) Immunohistochemical detection of WNT7A expression in cancer tissues

[0067] (1) Dewax paraffin sections to water; (2) Incubate with 3% H2O2 at room temperature for 10 minutes to eliminate endogenous peroxidase activity; (3) Rinse with distilled water, soak in PBS for 5 minutes, and repeat the soaking twice; (4) Citrate antigen retrieval; (5) Block with 10% normal goat serum (diluted with PBS) and incubate at room temperature for 10 minutes; (6) Add WNT7A primary antibody working solution (Abcam, 100792) and incubate at 37°C for 1.5 hours and 4°C overnight; (7) Rinse with PBS for 5 minutes and repeat the rinse three times; (8) Add appropriate amount of biotinylated secondary antibody Working solution (Maixin Technology Co., Ltd., KIT-9730), incubate at 37°C for 25 minutes; (9) Rinse with PBS for 5 minutes, repeat the rinse three times; (10) Add an appropriate amount of horseradish enzyme (alkaline phosphatase-labeled streptavidin can also be used as an alternative) working solution, incubate at 37°C for 25 minutes; (11) Rinse with PBS for 5 minutes, repeat the rinse three times; (12) Develop with DAB colorant for 10 minutes; (13) Rinse thoroughly with tap water, counterstain, dehydrate, make transparent, and seal the slides; (14) Observe WNT7A expression under an optical microscope and count the number of WNT7A-positive cells.

[0068] (3) Results

[0069] The tissue sections were scored under an optical microscope according to the degree of staining (0, 1, 2, and 3 correspond to negative staining, light yellow, light brown, and dark brown, respectively) and the positive range (1, 2, 3, and 4 correspond to 0-25%, 26%-50%, 51%-75%, and 76%-100%, respectively). The total score (staining degree × positive range, 0-12 points) was finally obtained for evaluation. According to the immunohistochemical scoring results, ① WNT7A immunohistochemical score of less than 6 points was judged as low WNT7A expression; ②

[0070] The immunohistochemical score of WNT7A was greater than or equal to 6 points, which was judged as high expression of WNT7A.

[0071] Human breast cancer tissue samples with low WNT7A expression levels Figure 1 As shown, human breast cancer tissue samples with low WNT7A expression levels are Figure 2 shown.

[0072] In addition, the overall survival (OS) of breast cancer patients with different WNT7A expression levels in the above tissue samples is as follows Figure 3 As shown, the results showed that the overall survival rate of breast cancer patients with high WNT7A expression levels was significantly lower than that of breast cancer patients with low WNT7A expression levels.

[0073] The above shows that the expression level of WNT7A is associated with the prognosis of breast cancer patients, and WNT7A can be used as an oncological marker to judge the prognosis of breast cancer patients.

[0074] 2. Blocking WNT7A activity can effectively inhibit tumor growth

[0075] In the examples of the present invention, it was verified that blocking WNT7A activity can effectively inhibit tumor growth, as follows:

[0076] (I) Construction of a mouse model of orthotopic tumor formation and spontaneous lung metastasis

[0077] (1) Mouse triple-negative breast cancer cells EO771 (purchased from American Type Culture Collection) were cultured and the WNT7A gene was knocked out using shRNA technology. The WNT7A gene knockout cells were designated as shWNT7-EO771 cells (also known as shWNT7); the WNT7A gene non-knockout cells were designated as shNC-EO771 cells (also known as shNC group); the cells were cultured in the logarithmic growth phase, digested conventionally, centrifuged, the supernatant removed, and washed twice with PBS.

[0078] (2) Resuspend in PBS, count, and adjust the cell density to 1×10 7 / ml.

[0079] (3) Three days before inoculation, 6-8 week old female C57BC / L mice were ear-tagged (purchased from Enswell Biotechnology Co., Ltd.); the prepared cell suspension was blown evenly, and then shNC-EO771 cells and shWNT7A-EO771 cells were subcutaneously inoculated into the unilateral buttocks, designated as the shNC group and the shWNT7A group, respectively. 50 μl / side, i.e., 40×10 4 cells / mouse, and 5 mice were inoculated in each group.

[0080] (4) After inoculation, mice were housed in an SPF environment, and the length and width of the tumors were measured every other day starting from the fifth day.

[0081] (5) On the 15th day, the mice were killed, the in situ tumors were completely removed and weighed, and the growth curve of the mice and the tumor volume (tumor volume (mm)) were prepared using GraphPad software. 3) = long diameter × wide diameter 2 × 0.5) and tumor weight graph.

[0082] (2) Results

[0083] The tumors of mice in each group were as follows Figure 4 As shown (wherein, the upper row is the shNC group and the lower row is the shWNT7 group), the results showed that the tumor volume of EO771 subcutaneous in situ tumor formation in the shWNT7 group was significantly smaller than that in the shNC group.

[0084] The changes in the volume of the tumors of the mice in each group from day 5 to day 15 are shown in Figure 2. Figure 5 As shown, the results showed that the tumor growth of EO771 in the shWNT7 group was significantly slower than that in the control group after subcutaneous in situ tumor formation.

[0085] The tumor volumes of mice in each group after 15 days are shown in Table 1 and Figure 6 As shown in the figure, the tumor volume of EO771 subcutaneous orthotopic tumors in the shWNT7 group was significantly lower than that in the control group.

[0086] Table 1 Tumor volume of ShNC and ShWNT7A mice

[0087]

[0088] The tumor weights of mice in each group after 15 days are shown in Table 2 and Figure 7 As shown, the results showed that the tumor weight of EO771 subcutaneous orthotopic tumors in the shNC group was significantly smaller than that in the control group.

[0089] Table 2 Tumor weights of ShNC and ShWNT7A mice

[0090]

[0091] 3. Finding small molecule inhibitors through virtual molecular screening

[0092] (1) Selection and preparation of protein structure

[0093] During the docking analysis, the The Prep Wiz module in the software package performs pretreatment operations such as hydrogenation and dehydration on the protein to prepare the test protein.

[0094] (2) Preparation of small molecule database

[0095] The ChemBridge library and the ChemDiv library were used in the virtual screening of the present invention. All compounds were screened for drug-likeness by the Lipinski's rule of five and Veber rule modules in DS4.0, and molecules with poor drug-likeness were eliminated. The remaining molecules were screened by The LigPrep module in the software package was used for preparation, the OPLS_2005 force field was used, and the protonation of the molecule was operated using the Epik module at pH 7.4.

[0096] (3) Finding active sites

[0097] use The SiteMap module in the software package finds the active site of the WNT7A protein. The WNT7A active site is as follows Figure 8 As shown, the active site is defined as the docking pocket.

[0098] (IV) Filtration of compounds with PAINS properties

[0099] To identify whether the screened compounds are PAINS structures, the present invention employed the Canvas 1.1 program for filtering. It should be noted that pan-assay interference compounds (PAINS) refer to compounds in the new drug screening process that exhibit "activity" that is not based on the specific interaction between the compound molecule and the protein, resulting in a false positive result.

[0100] (V) ADME property prediction and cluster analysis

[0101] In order to predict the pharmacokinetic properties of the compounds screened in the present invention, the present invention used The QikProp 3.2 program in the software package calculated the corresponding properties. It should be noted that the QikProp 3.2 program primarily calculates whether a compound violates Lipinski's five rules and Jorgensen's three rules. These two properties provide a preliminary assessment of a compound's ADME properties. Screened compounds must meet at least four of Lipinski's five rules to demonstrate good oral bioavailability. During this process, molecules obtained through different pharmacophore screening and docking were merged, identical molecules were eliminated, and the remaining small molecules were subjected to structural cluster analysis using FCFP_6 fingerprints.

[0102] (6) Molecular docking

[0103] Select the optimal docking condition Glide force field, and use the WNT7A protein file (for the receptor docking study. Before docking, the WNT7A protein receptor needs to prepare and define a size of After all molecules and receptor proteins were prepared, docking was performed using the Glide algorithm at SP docking accuracy, and other parameters remained default.

[0104] (VII) Analysis of docking results

[0105] The small molecules obtained by screening were docked through the ChemBridge library and the ChemDiv library. The results were sorted by docking score, and the ones with high scores were selected to synthesize the five drugs with the highest scores in each database (as shown in Tables 3 and 4).

[0106] Table 3 2D diagram of small molecule interactions obtained by protein screening of the ChemBridge library

[0107]

[0108]

[0109] Table 4 2D diagram of small molecule interactions obtained by protein screening of the ChemDiV library

[0110] No. ID Dockingscore Structure 1 7954-4074 -7.513 Figure 14 2 F067-1148 -7.461 Figure 15 3 C200-3685 -7.451 Figure 16 4 1365-0109 -7.390 Figure 17 5 3253-0183 -7.383 Figure 18

[0111] IV. Screening and efficacy verification of small molecule inhibitor 1365-0109

[0112] (I) Screening of optimal drugs that inhibit the binding of WNT7A to FZD5

[0113] In the examples of the present invention, the above ten small molecule compounds (synthesized by Taoshu Biotechnology Co., Ltd.) were tested using the co-immunoprecipitation (COIP) assay, and the method for determining the best drug for preventing the binding of WNT7A to FZD5 was as follows:

[0114] (1) Cells were seeded in a 10 cm cell culture dish. When the cell density was approximately 90%, the culture medium was discarded. The cells were washed three times with pre-cooled PBS + PMSF (1 ml + 10 ul). 1 ml of PSB + PMSF was added to each dish. The cells were transferred to a 1.5 ml EP tube using a cell scraper and centrifuged at 3000 rpm for 1 minute.

[0115] (2) Add 1 ml of IP buffer to each EP tube and lyse the supernatant for 30 min at 4°C on a Ferris wheel rotator.

[0116] (3) Centrifuge at 16000g for 15 min at 4°C. Take 30 μl of the supernatant as input. Take 400 μl of the remaining supernatant and transfer them to new 1.5 ml EP tubes as IP samples and IgG samples.

[0117] (4) Add 30ul protein A / G magnetic beads to the IP sample and IgG sample respectively, and rotate on a Ferris wheel at 4°C for 30 minutes to remove non-specific proteins.

[0118] (5) 10000 rpm, 30 s, collect the supernatant, add 4 μg WNT7A antibody and small molecule compound or DMSO or None to the supernatant of the IP group, add 2 μl normal IgG antibody to the IgG sample, and incubate at 4°C on a Ferris wheel rotator overnight.

[0119] (6) Add 30 μl of protein A / G magnetic beads and incubate at 4°C for 2 hours on a Ferris wheel rotator.

[0120] (7) Centrifuge at 1000 rpm for 30 s at 4°C, place 5 ml of the solution on a magnetic rack, and remove the supernatant.

[0121] (8) Wash three times with 1 ml IP buffer, rotating on a Ferris wheel at 4°C for 5 min each time, centrifuging at 4000 rpm at 4°C for 1 min, and remove the supernatant.

[0122] (9) Add 20ul 5× loading buffer to the IP sample and IgG sample, and add 30ul 5× loading buffer to the input sample, and cook at 100℃ for 5 minutes.

[0123] (10) Load the sample according to the western blot steps (separate the sample by SDS-PAGE electrophoresis; transfer the separated protein to polyacrylamide (PVDF) or nitrocellulose membrane; block and wash the membrane, label with primary antibody, wash, label with secondary antibody, wash, and detect the signal. For details, refer to "Wu, Y., et al., FGFR blockade boosts T cell infiltration into triple-negative breast cancer by regulating cancer-associated fibroblasts. Theranostics, 2022.12(10): p.4564-4580").

[0124] The 10 compounds shown in Table 3 and Table 4 were tested for their ability to inhibit the binding of WNT7A to FZD5 in triple-negative breast cancer cell lines MDA-MB-231 and EO771, respectively. The results are shown in Table 3. Figure 19 and Figure 20 As shown, the results showed that the small molecule compound 1365-0109 had the most significant effect in inhibiting the binding of WNT7A and FZD5, whether in the cell line MDA-MB-231 or the cell line EO771, and was superior to other small molecule compounds.

[0125] (II) Cellular activity assay of small molecule compound 1365-0109

[0126] In the examples of the present invention, the method for testing the cytotoxicity of the small molecule compound 1365-0109 is as follows:

[0127] (1) Cells were seeded in a 96-well plate, 3000 cells / well, 100 μl / well, with 3 replicates per group, set as the blank control group.

[0128] (2) On the second day, different concentration gradients of the small molecule compound 1365-0109 (0, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM) were added and incubated in a 37°C, 5% CO2 incubator for 24 h. 10 μL of CCK8 reagent was added to each well of the 96-well plate, protected from light and to avoid bubbles.

[0129] (3) The 96-well plate was returned to the incubator and incubated for another 2 h. The absorbance at λ = 450 nm was detected using a full-wavelength microplate reader (TECAN).

[0130] (4) Cell viability (%) = [(test well - blank well) / (control well - blank well)] × 100%, where: test well: culture medium containing cells and test compound; control well: culture medium containing cells without test compound; blank well: culture medium without cells and test compound.

[0131] (5) Use GraphPad software to create columns.

[0132] The effects of the small molecule compound 1365-0109 on the cell activity of the MDA-MB-231 cell line and the EO771 cell line were tested according to the above method. The results are as follows Figure 21 and 22 As shown in the figure, the results showed that the cell activities of MDA-MB-231 cell line and EO771 cell line were not significantly changed when different concentrations of 1365-0109 were used to treat the cell line.

[0133] 5. Tumor Growth Inhibition and Toxic Side Effects of the Small Molecule Inhibitor 1365-0109

[0134] (I) Construction of a mouse model of orthotopic tumor formation and spontaneous lung metastasis

[0135] (1) Take the mouse triple-negative breast cancer cells EO771 in the logarithmic growth phase, digest them conventionally, centrifuge, remove the supernatant, and wash them twice with PBS. (2) Resuspend them in PBS, count them, and adjust the cell density to 1×10 7 / ml.

[0136] (3) Three days before inoculation, 6-8 week old female C57BC / L mice were ear-tagged. The prepared cell suspension was blown evenly and then inoculated subcutaneously on one side of the buttocks at 50 μl / side, i.e., 40×10 4 / mouse, a total of 20 mice were vaccinated.

[0137] (4) After inoculation, the mice were housed in an SPF environment, and the length and width of the tumor were measured every other day starting from the third day. (5) Starting from the fifth day, the mice were randomly divided into four groups. The first group was given only saline and set as the control group. The second group was given a 5 mg / kg dose, the third group was given a 10 mg / kg dose, and the fourth group was given a 15 mg / kg dose. The drug was given once every three days.

[0138] (5) On day 15, the mice were killed.

[0139] (6) The blood of the mice was taken out and divided into two parts, one for routine blood test and the other for AST and ALT test (preparation of working reagents: single reagent method is used directly; double reagent method uses R1 and R2 respectively; set the corresponding parameters on the automatic biochemical enzyme marker; load the sample, and the automatic biochemical analyzer automatically measures; the experimental results are exported after detection by the automatic biochemical analyzer, and graphed using GraphPad); the in situ tumor was completely removed and weighed, and the mouse growth curve and tumor volume (tumor volume (mm)) were prepared using GraphPad software. 3 ) = long diameter × wide diameter 2 × 0.5) and tumor weight graph.

[0140] (2) Results

[0141] The tumors of mice in each group were as follows Figure 23 As shown, the results showed that the tumor volume of EO771 subcutaneous in situ tumor formation in the 1365-0109 group was significantly smaller than that in the control group, and as the concentration of 1365-0109 continued to increase, the tumor volume continued to decrease.

[0142] The changes in tumor volume of mice in each group from day 5 to day 15 are shown in Figure 2. Figure 24 As shown, the results showed that the tumor growth of EO771 subcutaneous in situ tumors in the 1365-0109 group was significantly slower than that in the control group; and as the concentration of 1365-0109 continued to increase, the tumor growth rate gradually decreased.

[0143] The tumor volumes of mice in each group after 15 days are shown in Table 5 and Figure 25 As shown, the results showed that the tumor volume of EO771 subcutaneous in situ tumor formation in the 1365-0109 group was significantly lower than that in the control group, and as the concentration of 1365-0109 continued to increase, the tumor volume gradually decreased.

[0144] Table 5 Tumor volume of mice in different groups

[0145]

[0146] The tumor weights of mice in each group after 15 days are shown in Table 6 and Figure 26As shown, the results showed that the tumor weight of EO771 subcutaneous in situ tumor in the 1365-0109 group was significantly lower than that in the control group, and as the concentration of 1365-0109 continued to increase, the tumor weight gradually decreased.

[0147] Table 6 Tumor weights of mice in different groups

[0148]

[0149] The blood routine indexes (RBC, WBC, HCG and PLT) and AST and ALT indexes of each group of mice were as follows: Figure 27 As shown, the results showed that at different dosage concentrations, there were no obvious abnormalities in the blood routine and liver function of mice, that is, different concentrations of the small molecule compound 1365-0109 had no obvious toxic side effects on the blood routine and ASL, ALT of mice.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. Use of a compound or a salt thereof in the preparation of a drug for treating breast cancer, wherein the compound has the structural formula shown in Formula I:

2. The use according to claim 1, characterized in that Applications include: Use of the compound or its salt in preparing a drug for inhibiting WNT7A gene activity.

3. The use according to claim 1 or 2, characterized in that Applications include: use of the compound or its salt in preparing a drug for inhibiting the WNT / β-catenin signaling pathway.

4. The use according to claim 1 or 2, characterized in that The drug is available in injection or oral form.

5. The use according to claim 3, characterized in that The drug is available in injection or oral form.

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