Pharmaceutical composition for preventing or treating brain tumor

Abstract

The present invention relates to the use of a novel anoctamin 1 (ANO1) inhibiting compound for the prevention and treatment of brain tumors, and is accomplished by confirming that the compound can inhibit brain tumors by inhibiting the expression or activity of the calcium-dependent chlorine channel ANO1. Growth, migration and proliferation of brain tumor cells treated by the compound are remarkably inhibited, and the inhibition effect is proved to be superior to that of a traditional ANO1 inhibitor. Particularly, the compound of the present invention can simultaneously inhibit ANO1 and EGFR in brain tumor cells, thereby exhibiting a strong anti-cancer effect by dual inhibition of the two proteins, and when used in combination with an EGFR-targeted anti-cancer agent, further enhancing the efficacy of the anti-cancer agent and inhibiting cancer resistance. Therefore, the compound can be used as a double-target anticancer agent of ANO1 and EGFR, and can also be used as a combined agent for EGFR targeted therapy, so that the compound is expected to be widely applied to the fields of prevention and treatment of brain tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to a pharmaceutical composition for preventing, ameliorating, or treating a brain tumor, which includes a novel compound capable of simultaneously inhibiting ANO1 (Anoctamin 1) and EGFR (epidermal growth factor receptor) as an active ingredient.

[0002] This application claims priority to and the benefit of Korean Patent Application No. 10-2023-0073989, filed June 9, 2023, and Korean Patent Application No. 10-2024-0074033, filed June 5, 2024, the entire disclosures of which are incorporated herein by reference. BACKGROUND

[0003] Glioblastoma is a primary malignant brain tumor that occurs in adults. It is a highly malignant disease that can lead to death within 3 to 6 months if not treated promptly after diagnosis, and even if treated using various available treatment methods, the average survival period is only 12 to 14 months. In Korea, about 12,000 people are diagnosed with brain tumors each year. Among them, about 9,000 are diagnosed with benign tumors such as meningioma or pituitary adenoma, and about 2,000 are diagnosed with malignant tumors. About 630 malignant tumor patients are diagnosed with glioblastoma each year.

[0004] Since glioblastoma originates from glial cells in brain tissue, it invasively grows into the surrounding brain tissue, so its tumor boundary is not clear and extends further than the actual visual boundary seen by radiological examination or surgery with the naked eye. Glioblastoma is usually located in a region of the brain that cannot be surgically resected due to functional reasons, and radiotherapy also cannot achieve complete remission. In addition, glioblastoma is a poor prognosis incurable disease, and treatment is very difficult due to the lack of effective anticancer agents, as anticancer agents are not easily penetrated into the brain due to the blood-brain barrier. The current standard treatment for glioblastoma is the use of Temozolomide, but long-term treatment can cause side effects and drug resistance, making continuous treatment difficult. Despite many modern medical advances and new drug development, no new therapy has been developed.

[0005] One of the oncogenes that frequently undergoes genetic alteration in glioblastoma patients is the epidermal growth factor receptor (EGFR). EGFR is physiologically important, and mutations are observed in more than half of such patients. Various intracellular signaling events occur when EGFR binds to EGF or TNF-α. However, overexpression or overactive mutations of EGFR lead to excessive cell proliferation, which ultimately results in cancer. Among these EGFR mutations, EGFRvIII is a protein characterized by the deletion of amino acids 6 through 273 and is specifically expressed in cancer cells. Although EGFRvIII was first discovered in glioblastoma, it has also been found in cancers of other tissues.

[0006] It has been reported that chloride channel proteins such as Anoctamin 1 (ANO1) and the chloride channel (CLC) family play important roles in the volume control of cancer cells, particularly in the migration and metastasis of glioblastoma cells. ANO1 protein is involved in various physiological functions (pain control, blood pressure control, brain development, water secretion control, etc.). In particular, their anti-cancer effects in various cancer cells (breast cancer, head and neck cancer, prostate cancer, pancreatic cancer, lung cancer, etc.) have been reported. Inhibition of ANO1 protein in breast cancer exerts its anti-cancer effect through CaMK2 signaling. Furthermore, although ANO1 expression levels are significantly low in the normal brain, it has been reported to be overexpressed in glioblastoma, and inhibition of ANO1 activity suppresses cell migration and metastasis in glioblastoma, thereby suppressing cancer growth in animal models. Additionally, it has been reported that inhibition of ANO1 in glioblastoma stem cells induces the degradation of epidermal growth factor receptor variant 3 (EGFRvIII) protein. These studies suggest that inhibiting ANO1 protein may be a therapeutic strategy for various EGFR mutation-related cancers, including glioblastoma.

[0007] Therefore, both ANO1 and EGFR are potential targets for anti-cancer therapy. However, no therapeutic drugs have yet been found that can exhibit excellent anti-cancer effects through the dual inhibition of ANO1 and EGFR. Summary of the Invention

[0008] [Technical Issues] This invention relates to the use of a novel anoctamin 1 (ANO1) inhibitory compound for the prevention and treatment of brain tumors, and is accomplished by confirming that the compound can simultaneously inhibit calcium-dependent chloride channel ANO1 and tyrosine kinase EGFR, thereby exhibiting excellent anticancer activity.

[0009] Therefore, the object of the present invention is to provide one or more compounds selected from the group consisting of compounds represented by chemical formulas 1 to 6; or pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] .

[0010] Another object of the present application is to provide a method for preparing a compound represented by Chemical Formulae 1 to 6.

[0011] Still another object of the present application is to provide a pharmaceutical composition for preventing or treating a brain tumor, which comprises, as an active ingredient, at least one compound selected from the group consisting of compounds represented by Chemical Formulae 1 to 6 or a pharmaceutically acceptable salt thereof.

[0012] Another object of the present application is to provide a pharmaceutical composition for preventing or treating a brain tumor, which comprises, as an active ingredient, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulae 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anti-cancer agent against EGFR.

[0013] Still another object of the present application is to provide a pharmaceutical composition for enhancing the anti-cancer effect of a brain tumor anti-cancer agent, which comprises, as an active ingredient, at least one compound selected from the group consisting of compounds represented by Chemical Formulae 1 to 6 or a pharmaceutically acceptable salt thereof.

[0014] However, the technical problems to be solved by the present application are not limited to the above-mentioned problems, and other problems not described herein will be sufficiently understood by those of ordinary skill in the art through the following description.

[0015] [Technical Solution] To achieve the above object, the present application provides one or more compounds selected from the group consisting of compounds represented by Chemical Formulae 1 to 6; or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] .

[0016] Further, the present application provides a method for preparing a compound represented by Chemical Formulae 1 to 6, comprising: (S1A) preparing a compound represented by the following Chemical Formula 8 by reacting a compound represented by the following Chemical Formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base; or (S1B) reacting a compound represented by the following Chemical Formula 9 with a compound represented by the following Chemical Formula 10 or 11 in the presence of a base: [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] .

[0017] In one embodiment of the present application, step (S1A) can further include the following steps, but is not limited thereto: (S1A-1) reducing a compound represented by Chemical Formula 8 (nitro) with zinc in the presence of ammonium chloride to prepare a compound represented by Chemical Formula 12; (S1A-2) reacting a compound represented by Chemical Formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to prepare a compound represented by Chemical Formula 13; and (S1A-3) reacting a compound represented by Chemical Formula 13 with a compound represented by Chemical Formula 14 or Chemical Formula 15 in the presence of an acid to prepare a compound represented by Chemical Formula 1 or Chemical Formula 2: [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] .

[0018] In another embodiment of the present application, when the compound represented by Chemical Formula 9 is reacted with the compound represented by Chemical Formula 10 in the presence of a base in step (S1B), a compound represented by the following Chemical Formula 16 is prepared, but is not limited thereto: [Chemical Formula 16] .

[0019] In still another embodiment of the present application, step (S1B) can further include the following steps, but is not limited thereto: (S1B-1) reacting the compound represented by Chemical Formula 16 with trimethylsilyl cyanide in the presence of a base to prepare a compound represented by the following Chemical Formula 17; (S1B-2) hydrogenating the compound represented by Chemical Formula 17 in the presence of a metal catalyst to prepare a compound represented by the following Chemical Formula 18; (S1B-3) reacting the compound represented by Chemical Formula 18 with a compound represented by the following Chemical Formula 19 in the presence of a reducing agent to prepare a compound represented by the following Chemical Formula 20; and (S1B-4) reacting the compound represented by Chemical Formula 20 with NCO− to prepare the compound represented by Chemical Formula 6, or reacting the compound represented by Chemical Formula 20 with a compound represented by the following Chemical Formula 21 in the presence of a base to prepare a compound represented by the following Chemical Formula 22: [Chemical Formula 17] [Chemical Formula 18] [Chemical Formula 19] [Chemical Formula 20] [Chemical Formula 21] [Chemical Formula 22] .

[0020] In another embodiment of the present application, step (S1B-4) can further include hydrolyzing the compound represented by Chemical Formula 22 in the presence of an acid to prepare the compound represented by Chemical Formula 3, but is not limited thereto.

[0021] In still another embodiment of the present application, the metal catalyst can be one or more selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney nickel (Raney-Ni), but is not limited thereto.

[0022] In another embodiment of the present application, when the compound represented by Chemical Formula 9 is reacted with the compound represented by Chemical Formula 11 in the presence of a base in step (S1B), a compound represented by the following Chemical Formula 23 is prepared, but is not limited thereto: [Chemical Formula 23] .

[0023] In still another embodiment of the present application, step (S1B) can further include the following steps, but is not limited thereto: (S1Ba-1) reacting the compound represented by Chemical Formula 23 with a peroxide to prepare a compound represented by the following Chemical Formula 24; and (S1Ba-2) reacting the compound represented by Chemical Formula 24 with a compound represented by the following Chemical Formula 25 in the presence of a base to prepare the compound represented by Chemical Formula 4; or (S1Bb-1) phosphorylating the compound represented by Chemical Formula 23 in the presence of a base to prepare a compound represented by the following Chemical Formula 26; and (S1Bb-2) reacting the compound represented by Chemical Formula 26 with a compound represented by the following Chemical Formula 27 in the presence of a base to prepare the compound represented by Chemical Formula 5: [Chemical Formula 24] [Chemical Formula 25] [Chemical Formula 26] [Chemical Formula 27] .

[0024] In another embodiment of the present application, the peroxide can be one or more selected from the group consisting of meta-chloroperoxybenzoic acid (m-CPBA), H2O2, dimethyldioxirane (DMDO), and potassium peroxymonosulfate, but is not limited thereto.

[0025] In still another embodiment of the present application, the compound represented by Chemical Formula 25 can be prepared by reacting a compound represented by the following Chemical Formula 28 with ethylamine in the presence of a reducing agent, but is not limited thereto: [Chemical Formula 28] .

[0026] In another embodiment of the present application, the reducing agent can be one or more selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium borohydride (NaBH4), but is not limited thereto.

[0027] In still another embodiment of the present application, the acid can be one or more selected from the group consisting of pivalic acid (PivOH), acetic acid (AcOH), trifluoromethanesulfonic acid (TfOH), p-toluenesulfonic acid (TsOH), benzoic acid, zinc bromide (ZnBr2), hydrogen chloride (HCl), and trifluoroacetic acid (TFA), but is not limited thereto.

[0028] In another embodiment of the present application, the base can be one or more selected from the group consisting of potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), copper bromide (CuBr2), cesium carbonate (Cs2CO3), lithium hydroxide (LiOH), sodium hydride (NaH), potassium hydride (KH), potassium hydroxide (KOH), triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), pyridine, and piperidine, but is not limited thereto.

[0029] In still another embodiment of the present application, the method can be performed in one or more solvents selected from the group consisting of organic solvents, water, and a mixture thereof, but is not limited thereto.

[0030] In another embodiment of the present application, the organic solvent can be one or more selected from the group consisting of dichloromethane (DCM), dichloroethane (DCE), 1,4-dioxane, tetrahydrofuran (THF), toluene, hexane, benzene, xylene, chlorobenzene, methanol (MeOH), ethanol (EtOH), t-amyl alcohol (t-AmOH), trifluoroethanol (TFE), hexafluoroisopropyl alcohol (HFIP), acetonitrile (ACN), dimethylformamide (DMF), nitromethane, trimethoxymethane (CH(OMe)3), acetic acid, isopropyl alcohol (IPA), and chloroform, but is not limited thereto.

[0031] Further, the present application provides a pharmaceutical composition for preventing or treating a brain tumor, which comprises at least one compound selected from the group consisting of the compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0032] Further, the present application provides a method for preventing or treating a brain tumor, which comprises administering to a subject in need at least one compound selected from the group consisting of the compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof or a composition comprising the same as an active ingredient.

[0033] Further, the present application provides use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof or a composition comprising the same as an active ingredient for preventing or treating a brain tumor.

[0034] Further, the present application provides use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 for manufacturing a medicament for treating a brain tumor.

[0035] In one embodiment of the present application, the brain tumor can be associated with at least one mutation selected from the group consisting of EGFR (epidermal growth factor receptor) and ANO1 (Anoctamin 1), but is not limited thereto.

[0036] In another embodiment of the present application, the compound or a pharmaceutically acceptable salt thereof can simultaneously inhibit EGFR and ANO1 (for example, by inhibiting expression and / or activity), but is not limited thereto. In other words, the compound or a pharmaceutically acceptable salt thereof can be a dual inhibitor targeting both EGFR and ANO1.

[0037] In still another embodiment of the present application, the compound or a pharmaceutically acceptable salt thereof can satisfy at least one property selected from the group consisting of: (a) inhibiting tumor metastasis; and (b) cancer resistance to an anticancer agent, but is not limited thereto.

[0038] In another embodiment of the present application, the anticancer agent can be a targeted anticancer agent against tyrosine kinase, but is not limited thereto.

[0039] In still another embodiment of the present application, the tyrosine kinase can be one or more selected from the group consisting of EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (B-Raf proto-oncogene), HER2 (human epidermal growth factor receptor 2), RET (RET proto-oncogene), NTRK1 (neurotrophic receptor tyrosine kinase 1), MET (mesenchymal epithelial transition factor), and NRG1 (neuregulin 1), but is not limited thereto.

[0040] In another embodiment of the present application, the pharmaceutical composition can be administered in combination with a targeted anticancer agent against tyrosine kinase, but is not limited thereto.

[0041] In still another embodiment of the present application, the pharmaceutical composition can be administered simultaneously, separately, or sequentially with the targeted anticancer agent, but is not limited thereto.

[0042] Further, the present application provides a pharmaceutical composition for preventing or treating a brain tumor, comprising, as active ingredients, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anti-cancer agent against EGFR.

[0043] Further, the present application provides a method for preventing or treating a brain tumor, comprising administering, to a subject in need thereof, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anti-cancer agent against EGFR.

[0044] Further, the present application provides a use of a composition for preventing or treating a brain tumor, the composition comprising, as active ingredients, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anti-cancer agent against EGFR.

[0045] Further, the present application provides a use of a composition for manufacturing a medicament for treating a brain tumor, the composition comprising, as active ingredients, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anti-cancer agent against EGFR.

[0046] In one embodiment of the present application, the compound or a pharmaceutically acceptable salt thereof can inhibit drug resistance of a brain tumor cell to a targeted anti-cancer agent against EGFR, but is not limited thereto.

[0047] In another embodiment of the present application, the composition can be in the form of a mixed preparation in which the compound or a pharmaceutically acceptable salt thereof and the targeted anti-cancer agent against EGFR are mixed together, but is not limited thereto.

[0048] In still another embodiment of the present application, the composition can be in the form in which the compound or a pharmaceutically acceptable salt thereof and the targeted anti-cancer agent against EGFR are separately formulated and administered simultaneously, separately or sequentially, but is not limited thereto.

[0049] Further, the present application provides a pharmaceutical composition for enhancing anticancer effects of a brain tumor anticancer agent, comprising, as an active ingredient, at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0050] Further, the present application provides a method for enhancing anticancer effects of a brain tumor anticancer agent, comprising administering, to a subject in need thereof, at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, or a composition comprising the same as an active ingredient.

[0051] In addition, the present application provides use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, or a composition comprising the same as an active ingredient for enhancing anticancer effects of a brain tumor anticancer agent.

[0052] In addition, the present application provides use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof for manufacturing a medicament for enhancing anticancer effects of a brain tumor anticancer agent.

[0053] In addition, the present application provides use of one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof for manufacturing an inhibitor of drug resistance of brain tumor cells to a brain tumor anticancer agent.

[0054] In one embodiment of the present application, the brain tumor anticancer agent can be a targeted anticancer agent against tyrosine kinase, but is not limited thereto.

[0055] In another embodiment of the present application, the tyrosine kinase can be one or more selected from the group consisting of EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (B-Raf proto-oncogene), HER2 (human epidermal growth factor receptor 2), RET (RET proto-oncogene), NTRK1 (neurotrophic receptor tyrosine kinase 1), MET (mesenchymal epithelial transition factor), and NRG1 (neuregulin 1), but is not limited thereto.

[0056] In still another embodiment of the present application, the composition can be administered simultaneously, separately, or sequentially with the brain tumor anticancer agent, but is not limited thereto.

[0057] [Advantageous Effects] The present invention relates to the use of a novel Anoctamin 1 (ANO1) inhibiting compound for the prevention and treatment of brain tumors, which has been completed by confirming that the compound is capable of inhibiting the expression or activity of calcium-dependent chloride channel ANO1, thereby inhibiting brain tumors. Brain tumor cells treated with the compound exhibit a significant growth, migration, and proliferation inhibitory effect, and it was found that this inhibitory effect is superior to that of conventional ANO1 inhibitors. In particular, the compound of the present invention is capable of simultaneously inhibiting ANO1 and EGFR in brain tumor cells, and not only can exhibit a more effective anticancer effect through the dual inhibition of the two proteins, but also enhance the therapeutic efficacy of anticancer agents such as anticancer drugs targeting EGFR, and inhibit drug resistance when used in combination. Therefore, the compound itself can be used as a dual-target anticancer agent against ANO1 and EGFR, and also as a combination agent for EGFR-targeted therapy, and thus is expected to be useful in various applications for the prevention and treatment of brain tumors.

[0058] Accordingly, the present invention provides novel compounds that can be used as ANO1 inhibitors. In addition, the compounds of the present invention can simultaneously inhibit ANO1 and EGFR in brain tumor cells, and thus can be used as a dual-target anticancer agent against ANO1 and EGFR, and also as a combination agent for EGFR-targeted therapy. Therefore, these compounds are expected to be effectively used in various fields for the prevention and treatment of brain tumors. BRIEF DESCRIPTION OF DRAWINGS

[0059] FIG. 1 shows the name, structural formula, and basic information of the compounds AON-MG23-01 and AON-MG23-02 according to the present invention.

[0060] FIG. 2 shows the results of CCK assay after treating human glioblastoma cell lines with different concentrations of the compounds AON-MG23-01 or AON-MG23-02 of the present invention, to confirm the inhibitory effect of the compounds AON-MG23-01 and AON-MG23-02 on the growth of human glioma cells.

[0061] FIG. 3 shows the results obtained by photographing and analyzing the degree of cell migration over time after treating human glioblastoma cell lines with different concentrations of the compounds AON-MG23-01 or AON-MG23-02 of the present invention, to confirm the inhibitory effect of the compounds AON-MG23-001 and AON-MG23-02 of the present invention on cell migration in human glioblastoma cells.

[0062] Figure 4 shows the results obtained by taking and analyzing pictures of the extent of Matrigel invasion of cells after treatment of human glioblastoma cell lines cultured on Matrigel with different concentrations of the compounds of the application AON-MG23-01 or AON-MG23-02 to confirm the inhibitory effect of the compounds of the application AON-MG23-001 and AON-MG23-02 on glioblastoma invasion.

[0063] Figure 5 shows the results of Western Blot analysis performed after treatment of human glioblastoma cell lines with different concentrations of the compounds of the application AON-MG23-01 or AON-MG23-02 to confirm the effect of the compounds AON-MG23-01 and AON-MG23-02 on EGFR and ANOl protein expression in glioblastoma. The left panel shows images of the protein bands detected by Western Blot, while the right panel shows quantification of EGFR and ANOl protein levels.

[0064] Figure 6A and Figure 6B show the results of whole-cell patch-clamp experiments performed after treatment of human glioblastoma cell lines with the compounds of the application AON-MG23-01 or AON-MG23-02 to confirm whether these compounds inhibit ANOl activity in glioblastoma. Figure 6A shows the results of measuring calcium-dependent chloride channel currents from -100 mV to +100 mV, and Figure 6B shows the current-voltage (I / V) plot at +80 mV derived from Figure 6A.

[0065] Figure 7A and Figure 7B show the results comparing the inhibitory effect of the compound of the application AON-MG23-02, an EGFR inhibitor and an ANOl inhibitor on human glioblastoma invasion.

[0066] Figure 8A and Figure 8B show the results comparing the inhibitory effect of the compound of the application AON-MG23-02, an EGFR inhibitor and an ANOl inhibitor on ANOl, EGFR and pEGFR protein expression in glioblastoma cell lines.

[0067] Figures 9A to 9D show the results of Western blot analysis after treatment of human glioblastoma cell lines with different concentrations of derivatives of the compound AON-MG23-02 of the present application (i.e. AON-MG23-05, AON-MG23-06, AON-MG23-07 or AON-MG23-08) to check their effect on the expression of EGFR and ANOl proteins in glioblastoma. Specifically, Figure 9A shows the results for AON-MG23-05, Figure 9B shows the results for AON-MG23-06, Figure 9C shows the results for AON-MG23-07, and Figure 9D shows the results for AON-MG23-08, confirming the expression levels of ANOl and EGFR proteins by Western blot analysis.

[0068] Figures 10A and 10B show the results obtained by taking and analyzing the extent of Matrigel invasion of cells after treatment of human glioblastoma cell line cells cultured on Matrigel with different concentrations of derivatives of the compound AON-MG23-02 of the present application (i.e. AON-MG23-05, AON-MG23-06, AON-MG23-07 or AON-MG23-08) to confirm the inhibitory effect of these derivatives on glioblastoma invasion.

[0069] Figure 11 shows the H NMR data for the derivative AON-MG23-05 of the compound AON-MG23-02 of the present application.

[0070] Figure 12 shows the H NMR data for the derivative AON-MG23-06 of the compound AON-MG23-02 of the present application.

[0071] Figure 13 shows the H NMR data for the derivative AON-MG23-07 of the compound AON-MG23-02 of the present application.

[0072] Figure 14 shows the H NMR data for the derivative AON-MG23-08 of the compound AON-MG23-02 of the present application. DETAILED DESCRIPTION

[0073] The present application relates to novel compounds having an inhibitory effect on Anoctamin 1 (ANO1), and is accomplished by confirming that these compounds can inhibit the expression or activity of calcium-dependent chloride channel ANOl, thereby inhibiting the growth and metastasis of brain tumor cells. In particular, the compounds of the present application have been confirmed to effectively inhibit the expression of EGFR and ANOl in brain tumor cells, and thus are expected to exhibit a stronger anticancer effect than conventional single inhibitors of ANOl or EGFR, and further inhibit the development of drug resistance to EGFR-targeted therapy in brain tumor cells.

[0074] Accordingly, an object of the present application is to provide one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 6, or pharmaceutically acceptable salts thereof: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] .

[0075] In the present application, the compound represented by Chemical Formula 1 can be referred to as “N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide” or “AON-MG23-01”. In addition, the compound represented by Chemical Formula 2 can also be referred to as “N-(2-((5-chloro-2-((4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide” or “AON-MG23-02”.

[0076] As used herein, the term “Ms” refers to a methylsulfonyl group, i.e., -SO2(CH3). In addition, the compound represented by Chemical Formula 3 can be referred to as “2-(4-(2-((2-hydroxyethyl)(naphthalen-2-ylmethyl)amino)ethyl)benzyl)isoindolin-1-one”, “AON-MG23-05”, or “Target A”.

[0077] The compound represented by Chemical Formula 4 can be referred to as “2-(4-(2-(ethyl(naphthalen-2-ylmethyl)amino)-1-hydroxyethyl)benzyl)isoindolin-1-one”, “AON-MG23-06”, or “Target B”.

[0078] The compound represented by Chemical Formula 5 can be referred to as “2-(4-(2-(bis(naphthalen-2-ylmethyl)phosphoryl)ethyl)benzyl)isoindolin-1-one”, “AON-MG23-07”, or “Target C”.

[0079] The compound represented by Chemical Formula 6 can be referred to as “1-(naphthalen-2-ylmethyl)-1-(4-((1-oxoisoindol-2-yl)methyl)phenethyl)urea”, “AON-MG23-08”, or “Target D”.

[0080] Unless otherwise specified herein, the term “compound of the present invention” or “compound represented by Chemical Formulas 1 to 28” and the like are intended to include the compound represented by Chemical Formulas 1 to 28 per se, salts thereof, and isomers thereof.

[0081] In the present invention, the term “pharmaceutically acceptable salt” includes a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base.

[0082] As used herein, the term “pharmaceutically acceptable” means a compound or composition that is suitable for contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0083] Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, hydroiodic acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, (+)-L-tartaric acid, di-L-tartrate, acetic acid, trichloroacetic acid or trifluoroacetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, 4-acetylamino-benzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, hexanoic acid, octanoic acid, cinnamic acid, cyclamic acid, dodecylsulfic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, galacturonic acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, alpha-ketoglutaric acid, hippuric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, (−)-L-malic acid, (±)-DL-mandelic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, benzenesulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, di-oxalic acid, palmitic acid, palmitoleic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, tannic acid, thiocyanic acid, camphoric acid, and undecylenic acid. Acid addition salts can be prepared by conventional methods, e.g., by dissolving the compound in excess aqueous acid, and precipitating the salt using a water soluble organic solvent such as methanol, ethanol, acetone or acetonitrile. Alternatively, the salt can be prepared by heating an equimolar amount of the compound and acid or alcohol in water, followed by evaporation and drying of the mixture, or by filtering the precipitated salt under suction.

[0084] The compounds of the present application can include not only pharmaceutically acceptable salts, but also all isomers, hydrates, and solvates that can be prepared by conventional methods.

[0085] In the present application, the term "isomer" refers to compounds having the same molecular formula but different connectivity or spatial arrangement of constituent atoms within the molecule. Isomers can include, for example, structural isomers and stereoisomers. Stereoisomers can be diastereomers or enantiomers. Enantiomers refer to isomers that are not superimposable on their mirror image, similar to the relationship between left and right hands, and are also known as optical isomers. When a chiral carbon atom has four different substituents, enantiomers are classified as R (Rectus; clockwise) or S (Sinister; counterclockwise). Diastereomers refer to stereoisomers that are not mirror images of each other, which can include cis-trans isomers resulting from differences in atomic spatial arrangement.

[0086] In addition, the present application provides a method for preparing a compound represented by Chemical Formulas 1 to 6, comprising: (S1A) preparing a compound represented by the following Chemical Formula 8 by reacting a compound represented by the following Chemical Formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base (Reaction Scheme 2); or (S1B) reacting a compound represented by the following Chemical Formula 9 with a compound represented by the following Chemical Formula 10 or 11 in the presence of a base (Reaction Scheme 7 or 13): [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] .

[0087] According to one embodiment of the present application, the base used in step (S1A) or (S1B) can be Cs2CO3, but is not limited thereto.

[0088] According to one embodiment of the present application, step (S1A) can further include the following steps, but is not limited thereto: (S1A-1) reducing a compound represented by Chemical Formula 8 (nitro) with zinc in the presence of ammonium chloride to prepare a compound represented by Chemical Formula 12 (Reaction Scheme 3); (S1A-2) reacting a compound represented by Chemical Formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to prepare a compound represented by Chemical Formula 13 (Reaction Scheme 4); and (S1A-3) reacting a compound represented by Chemical Formula 13 with a compound represented by Chemical Formula 14 or Chemical Formula 15 in the presence of an acid to prepare a compound represented by Chemical Formula 1 or Chemical Formula 2 (Reaction Scheme 5 or 6): [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] .

[0089] According to one embodiment of the present application, when a compound represented by Chemical Formula 13 is reacted with a compound represented by Chemical Formula 14 in the presence of an acid in step (S1A-3), a compound represented by Chemical Formula 1 is prepared (Reaction Scheme 5), and when a compound represented by Chemical Formula 13 is reacted with a compound represented by Chemical Formula 15 in the presence of an acid, a compound represented by Chemical Formula 2 can be prepared (Reaction Scheme 6), and specifically, step (S1A-3) can be a reaction of an amino group of a compound represented by Chemical Formula 13 with a compound represented by Chemical Formula 14 or Chemical Formula 15.

[0090] According to one embodiment of the present application, in step (S1A-2), the base is DIPEA (N,N-diisopropylethylamine), and in step (S1A-3), the acid can be TFA (trifluoroacetic acid), but is not limited thereto.

[0091] In the present application, when a compound represented by Chemical Formula 9 is reacted with a compound represented by Chemical Formula 10 in the presence of a base in step (S1B), a compound represented by the following Chemical Formula 16 can be prepared (Reaction Scheme 7 or 19), but is not limited thereto, and according to one embodiment of the present application, the base can be Cs2CO3, but is not limited thereto: [Chemical Formula 16] .

[0092] According to one embodiment of the present application, step (S1B) can further include the following steps, but is not limited thereto: (S1B-1) reacting a compound represented by Chemical Formula 16 with trimethylsilyl cyanide in the presence of a base to prepare a compound represented by the following Chemical Formula 17 (Reaction Scheme 8 or 20); (S1B-2) hydrogenating the compound represented by Chemical Formula 17 in the presence of a metal catalyst to prepare a compound represented by the following Chemical Formula 18 (Reaction Scheme 9 or 21); (S1B-3) reacting the compound represented by Chemical Formula 18 with a compound represented by the following Chemical Formula 19 in the presence of a reducing agent to prepare a compound represented by the following Chemical Formula 20 (Reaction Scheme 10 or 22); and (S1B-4) reacting the compound represented by Chemical Formula 20 with NCO− to prepare the compound represented by Chemical Formula 6 (Reaction Scheme 23), or reacting the compound represented by Chemical Formula 20 with a compound represented by the following Chemical Formula 21 in the presence of a base to prepare a compound represented by the following Chemical Formula 22 (Reaction Scheme 11), and according to one embodiment of the present application, the base in step (S1B-1) is Cs2CO3 and the base in step (S1B-4) can be K2CO3, but is not limited thereto: [Chemical Formula 17] [Chemical Formula 18] [Chemical Formula 19] [Chemical Formula 20] [Chemical Formula 21] [Chemical Formula 22] .

[0093] In the present application, step (S1B-4) can further include hydrolyzing the compound represented by Chemical Formula 22 in the presence of an acid to prepare the compound represented by Chemical Formula 3 (Reaction Scheme 12), but is not limited thereto, and according to one embodiment of the present application, the acid used in step (S1B-4) can be hydrogen chloride (HCl), but is not limited thereto.

[0094] In the present application, the metal catalyst can be one or more selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney nickel (Raney-Ni), and according to one embodiment of the present application, the metal catalyst can be Raney nickel (Raney-Ni), but is not limited thereto.

[0095] In the present application, the reducing agent can be one or more selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium borohydride (NaBH4), and according to one embodiment of the present application, the reducing agent used in step (S1B-3) can be sodium cyanoborohydride (NaBH3CN), but is not limited thereto.

[0096] In the present application, when the compound represented by Chemical Formula 9 is reacted with the compound represented by Chemical Formula 11 in the presence of a base in step (S1B), a compound represented by the following Chemical Formula 23 can be prepared (Reaction Scheme 13), but is not limited thereto: [Chemical Formula 23] .

[0097] In the present application, step (S1B) can further include the following steps, but is not limited thereto: (S1Ba-1) reacting the compound represented by Chemical Formula 23 with a peroxide to prepare a compound represented by the following Chemical Formula 24 (Reaction Scheme 14); and (S1Ba-2) reacting the compound represented by Chemical Formula 24 with a compound represented by the following Chemical Formula 25 in the presence of a base to prepare the compound represented by Chemical Formula 4 (Reaction Scheme 16); or (S1Bb-1) phosphorylating the compound represented by Chemical Formula 23 in the presence of a base to prepare a compound represented by the following Chemical Formula 26, in particular, the compound represented by Chemical Formula 26 by a β-alkylstyrene phosphorylation reaction (Reaction Scheme 17); and (S1Bb-2) reacting the compound represented by Chemical Formula 26 with a compound represented by the following Chemical Formula 27 in the presence of a base to prepare the compound represented by Chemical Formula 5 (Reaction Scheme 18).

[0098] According to one embodiment of the present application, the base in step (S1Ba-2) is DIEA (N,N-diisopropylethylamine), the base in step (S1Bb-1) is KOH, and the base in step (S1Bb-2) can be NaH, but is not limited thereto: [Chemical Formula 24] [Chemical Formula 25] [Chemical Formula 26] [Chemical Formula 27] .

[0099] In the present application, the compound represented by Chemical Formula 25 can be prepared by reacting a compound represented by Chemical Formula 28 with ethylamine in the presence of a reducing agent (Reaction Scheme 15), and according to one embodiment of the present application, the reducing agent can be sodium triacetoxyborohydride (NaBH(OAc)3), but is not limited thereto: [Chemical Formula 28] .

[0100] In the present application, the peroxide can be one or more selected from the group consisting of m-CPBA (meta-chloroperoxybenzoic acid), hydrogen peroxide (H2O2), dimethyldioxirane (DMDO), and potassium peroxymonosulfate, and according to one embodiment of the present application, the peroxide can be m-CPBA (meta-chloroperoxybenzoic acid), but is not limited thereto.

[0101] In the present application, the acid can be one or more selected from the group consisting of pivalic acid (PivOH), acetic acid (AcOH), trifluoromethanesulfonic acid (TfOH), p-toluenesulfonic acid (TsOH), benzoic acid, zinc bromide (ZnBr2), hydrogen chloride (HCl), and trifluoroacetic acid (TFA), but is not limited thereto.

[0102] In the present application, the base can be one or more selected from the group consisting of potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), copper bromide (CuBr2), cesium carbonate (Cs2CO3), lithium hydroxide (LiOH), sodium hydride (NaH), potassium hydride (KH), potassium hydroxide (KOH), triethylamine (TEA), N,N-diisopropylethylamine (DIPEA), pyridine, and piperidine, but is not limited thereto.

[0103] In the present application, the preparation method can be performed in one or more solvents selected from the group consisting of an organic solvent, water, and a mixture thereof, and the organic solvent can be one or more selected from the group consisting of dichloromethane (DCM), dichloroethane (DCE), 1,4-dioxane, tetrahydrofuran (THF), toluene, hexane, benzene, xylene, chlorobenzene, methanol (MeOH), ethanol (EtOH), t-amyl alcohol (t-AmOH), trifluoroethanol (TFE), hexafluoroisopropyl alcohol (HFIP), acetonitrile (ACN), dimethylformamide (DMF), nitromethane, trimethoxymethane (CH(OMe)3), acetic acid (acetic anhydride, acetic acid, or a mixture of acetic anhydride and acetic acid), and chloroform, but is not limited thereto.

[0104] Further, the present application provides a method for preparing a compound represented by Chemical Formula 1, comprising the steps of: (a) reacting a compound represented by Chemical Formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to prepare a compound represented by Chemical Formula 8 (Reaction Scheme 2); (b) reducing the compound represented by Chemical Formula 8 (nitro group) with zinc in the presence of ammonium chloride to prepare a compound represented by Chemical Formula 12 (Reaction Scheme 3); (c) reacting the compound represented by Chemical Formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to prepare a compound represented by the following Chemical Formula 13 (Reaction Scheme 4); and (d) reacting the compound represented by Chemical Formula 13 with a compound represented by the following Chemical Formula 14 in the presence of an acid to prepare a compound represented by Chemical Formula 1 (Reaction Scheme 5).

[0105] Further, the present application provides a method for preparing a compound represented by Chemical Formula 2, comprising the steps of: (a) reacting a compound represented by Chemical Formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to prepare a compound represented by Chemical Formula 8 (Reaction Scheme 2); (b) reducing the compound represented by Chemical Formula 8 (nitro group) with zinc in the presence of ammonium chloride to prepare a compound represented by Chemical Formula 12 (Reaction Scheme 3); (c) reacting the compound represented by Chemical Formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to prepare a compound represented by the following Chemical Formula 13 (Reaction Scheme 4); and (d) reacting the compound represented by Chemical Formula 13 with a compound represented by the following Chemical Formula 15 in the presence of an acid to prepare a compound represented by Chemical Formula 2 (Reaction Scheme 6).

[0106] Further, the present application provides a method for preparing a compound represented by Chemical Formula 3, comprising the steps of: (a) reacting a compound represented by Chemical Formula 9 with a compound represented by Chemical Formula 10 in the presence of a base to prepare a compound represented by Chemical Formula 16 (Reaction Scheme 7); (b) reacting the compound represented by Chemical Formula 16 with trimethylsilyl cyanide in the presence of a base to prepare a compound represented by Chemical Formula 17 (Reaction Scheme 8); (c) hydrogenating the compound represented by Chemical Formula 17 in the presence of a metal catalyst to prepare a compound represented by Chemical Formula 18 (Reaction Scheme 9); (d) reacting the compound represented by Chemical Formula 18 with a compound represented by Chemical Formula 19 in the presence of a reducing agent to prepare a compound represented by Chemical Formula 20 (Reaction Scheme 10); (d) reacting a compound represented by Chemical Formula 20 with a compound represented by Chemical Formula 21 in the presence of a base to prepare a compound represented by Chemical Formula 22 (Reaction Scheme 11); and (e) hydrolyzing the compound represented by Chemical Formula 22 in the presence of an acid to prepare a compound represented by Chemical Formula 3 (Reaction Scheme 12).

[0107] Further, the present application provides a method for preparing a compound represented by Chemical Formula 4, comprising the steps of: (a) reacting a compound represented by Chemical Formula 9 with a compound represented by Chemical Formula 11 in the presence of a base to prepare a compound represented by Chemical Formula 23 (Reaction Scheme 13); (b) reacting the compound represented by Chemical Formula 23 with a peroxide to prepare a compound represented by Chemical Formula 24 (Reaction Scheme 14); and (c) reacting the compound represented by Chemical Formula 24 with a compound represented by Chemical Formula 25 in the presence of a base to prepare a compound represented by Chemical Formula 4 (Reaction Scheme 16).

[0108] Further, the present application provides a method for preparing a compound represented by Chemical Formula 5, comprising the steps of: (a) reacting a compound represented by Chemical Formula 9 with a compound represented by Chemical Formula 11 in the presence of a base to prepare a compound represented by Chemical Formula 23 (Reaction Scheme 13); (b) phosphorylating the compound represented by Chemical Formula 23 in the presence of a base to prepare a compound represented by Chemical Formula 26, specifically the step of preparing the compound represented by Chemical Formula 26 by a β-alkylstyrene phosphorylation reaction (Reaction Scheme 17); and (c) reacting the compound represented by Chemical Formula 26 with a compound represented by Chemical Formula 27 in the presence of a base to prepare a compound represented by Chemical Formula 5 (Reaction Scheme 18).

[0109] Further, the present application provides a method for preparing a compound represented by Chemical Formula 6, comprising the steps of: (a) reacting a compound represented by Chemical Formula 9 with a compound represented by Chemical Formula 10 in the presence of a base to prepare a compound represented by Chemical Formula 16 (Reaction Scheme 19); (b) reacting the compound represented by Chemical Formula 16 with trimethylsilyl cyanide in the presence of a base to prepare a compound represented by Chemical Formula 17 (Reaction Scheme 20); (c) hydrogenating the compound represented by Chemical Formula 17 in the presence of a metal catalyst to prepare a compound represented by Chemical Formula 18 (Reaction Scheme 21); (d) reacting a compound represented by Chemical Formula 18 with a compound represented by Chemical Formula 19 in the presence of a reducing agent to prepare a compound represented by Chemical Formula 20 (Reaction Scheme 22); (d) reacting a compound represented by Chemical Formula 20 with NCO− to prepare a compound represented by Chemical Formula 6 (Reaction Scheme 23).

[0110] Further, another object of the present application is to provide a pharmaceutical composition for preventing or treating brain tumors, which comprises one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or pharmaceutically acceptable salts thereof as an active ingredient.

[0111] The compound according to the present application can be used as an inhibitor of calcium-dependent chloride channel Anoctamin 1 (ANO1) for preventing, ameliorating, and / or treating brain tumors. In particular, the compound according to the present application is characterized in that it exhibits an excellent anticancer effect by simultaneously inhibiting the expression or activity of EGFR and ANO1. The preventive and / or therapeutic effect of brain tumors includes not only an effect of inhibiting the growth of brain tumor cells, but also an effect of inhibiting the progression or aggravation of brain tumors caused by migration, invasion, and metastasis.

[0112] In the present application, "EGFR (epidermal growth factor receptor)" is a transmembrane glycoprotein belonging to the protein kinase superfamily, which serves as a receptor for epidermal growth factor (EGF) family proteins. EGFR is a key regulator of cell growth, and when its ligand epidermal growth factor (EGF) binds to EGFR, the receptor dimerizes and undergoes tyrosine autophosphorylation, thereby inducing cell proliferation. Overexpression (amplification) or overactivation of EGFR is often observed in various cancers such as colorectal cancer, head and neck cancer, and glioblastoma, and somatic mutations associated with EGFR lead to persistent activation of EGFR, resulting in uncontrolled cell division. In particular, EGFR gene rearrangement and EGFR gene amplification are common patterns in various cancers. Among them, the most common extracellular domain mutation is EGFRvIII. EGFRvIII mutation is characterized by a deletion of about 801 base pairs corresponding to exons 2 to 7 of the EGFR gene, and the EGFR protein expressed therefrom lacks 267 amino acids in the extracellular domain. Unlike normal EGFR, which requires ligand binding to be activated, mutant EGFR maintains constitutive activity (PCT gain-of-function mutation). EGFRvIII mutation is particularly prevalent in glioblastoma.

[0113] In the present application, "Anoctamin 1 (ANO1, also known as Transmembrane Protein 16A (TMEM16A))" is a calcium-activated chloride channel (Cl -ANO1 is expressed in various normal cells including epithelial cells, smooth muscle cells, vascular endothelial cells, and neurons, and performs various physiological functions by regulating fluid secretion, muscle contraction, and nociception. However, ANO1 has also been known to be associated with tumor invasiveness and poor prognosis of brain tumors including glioblastoma. In fact, overexpression of ANO1 facilitates signaling pathways that stimulate cancer cell proliferation and migration, and inhibition of ANO1 has been reported to inhibit cell migration and growth. Overexpression of ANO1 also appears to be associated with overexpression of EGFR and STAT3, and it has been reported that inhibition of ANO1 enhances the response of head and neck squamous cell carcinoma to EGFR / HER2-targeted therapy. Thus, ANO1 has attracted attention as a target for cancer treatment; however, an ANO1 inhibitor exhibiting superior anticancer efficacy has not yet been developed. The compound according to the present application effectively inhibits the expression of ANO1 in brain tumor cells, and has been confirmed to more strongly inhibit the migration, proliferation, and metastasis of brain tumor cells than conventional ANO1 inhibitors, and thus can be used to prevent, treat, and inhibit the metastasis of brain tumors. In addition, previous studies on the improvement of EGFR / HER2-targeted therapy efficacy through ANO1 inhibition suggest that the compound according to the present application can enhance the responsiveness of brain tumor cells to EGFR / HER-targeted therapy and inhibit the development of drug resistance.

[0114] In particular, the compound according to the present application simultaneously inhibits the expression of EGFR and ANO1 in brain tumor cells, and thus can exhibit a stronger anticancer effect by simultaneously inhibiting ANO1 and EGFR. The association between ANO1 and EGFR has been well established, as the upregulation of ANO1 is associated with the signaling pathway of EGFR, and it has been reported that, after epidermal growth factor (EGF) stimulation, remodeling of the phospho-proteome has a significant impact. In addition, in cancer cells, ANO1 can form a functional complex with EGFR to synergistically regulate cell proliferation. Thus, when EGFR and ANO1 are simultaneously inhibited by using the compound according to the present application, the growth of brain tumors can be more effectively inhibited.

[0115] Meanwhile, ANO1 is known to be overexpressed in cancer cells and to contribute to cancer cell migration and tumor metastasis, and it is also known that a tumor microenvironment that promotes tumor metastasis is generated through the signaling of EGFR. Thus, the compound according to the present application can effectively inhibit the metastasis of brain tumors by simultaneously inhibiting ANO1 and EGFR. The term "metastasis" as used herein refers to a condition in which a malignant tumor spreads from an organ of origin to another distant tissue. Thus, the composition according to the present application can inhibit the metastasis of brain tumors, thereby preventing and treating the systemic spread of brain tumors.

[0116] The compound according to the present application or a pharmaceutically acceptable salt thereof can be used to prevent or treat various cancers.

[0117] In the present application, the term "tumor" is synonymous with "cancer" and refers to a condition that is typically characterized by uncontrolled cell growth, migration and proliferation. Preferably, the cancer according to the present application is a brain tumor. The cancer according to the present application includes primary and recurrent cancers and encompasses both benign and malignant tumors. In the present application, brain tumor refers to any tumor that occurs within the cranium, not limited to a particular type, but includes all tumors that occur in the brain and its surrounding structures. In the present application, brain tumor includes primary brain tumors, recurrent brain tumors and metastatic brain tumors. Furthermore, brain tumor includes astrocytoma, malignant astrocytic carcinoma, glioblastoma, meningioma, pituitary adenoma, schwannoma, acoustic schwannoma and craniopharyngioma, not limited to any particular type.

[0118] In particular, the cancer according to the present application can be a brain tumor that is expected to be improved or treated by simultaneous inhibition of ANOl and EGFR. Preferably, the cancer according to the present application can be a brain tumor that is associated with one or more mutations selected from the group consisting of EGFR (epidermal growth factor receptor) and ANOl (Anoctamin 1). That is, the cancer according to the present application can be a brain tumor that has a mutation of EGFR and / or ANOl. The mutation of EGFR and / or ANOl includes amplification of the EGFR and / or ANOl gene, overexpression of the protein, overactivation of the protein and persistent activation of the protein. That is, the cancer according to the present application can be a brain tumor in which the expression or activity of EGFR and / or ANOl is higher than that of normal cells. Alternatively, the cancer according to the present application can be a brain tumor in which the activation state of EGFR and / or ANOl is persistent. Since the compound according to the present application is capable of simultaneously inhibiting the expression and activity of ANOl and EGFR, it can exhibit a particularly excellent anticancer effect on a brain tumor involving a mutation of EGFR and / or ANOl.

[0119] Alternatively, the cancer according to the present application can be a brain tumor that is associated with EGFRvIII (epidermal growth factor receptor variant III) mutation. EGFRvIII mutation is a mutant form in which amino acids 6 to 273 (or exons 2 to 7) of EGFR are deleted, which is characterized in that it remains in an activated state without binding to a ligand (EGF). EGFRvIII is specifically expressed only in cancer cells and is found in about 30% of glioblastomas.

[0120] The compound according to the present application can exhibit one or more effects selected from the group consisting of: (a) inhibiting the proliferation or growth of brain tumor cells; (b) inhibiting the migration of brain tumor cells; (c) inhibiting the metastasis of brain tumors; (d) inhibiting drug resistance of brain tumor to anticancer agents; and (e) enhancing responsiveness of brain tumor cells to anticancer agents.

[0121] The inventors demonstrate by specific examples that brain tumor cells treated with the compound according to the present application exhibit inhibited proliferation, migration, and metastasis (invasiveness), and find that the anticancer effect of the compound is stronger than that of a conventional ANOl inhibitor (CaCCinh-A01).

[0122] Further, since the compound according to the present application can simultaneously inhibit ANOl and EGFR (i.e., dual inhibition), it can exhibit an excellent anticancer effect compared to a single inhibitor of ANOl or EGFR, in particular, by inhibiting ANOl, it can inhibit the development of drug resistance of cancer cells to anticancer agents, and enhance the anticancer efficacy of anticancer agents. For example, the inventors demonstrate by experiments that the ANOl inhibitory effect of the compound according to the present application is superior to that of the conventional ANOl inhibitor CaCCinh-A01, and its anticancer effect is superior to that of the ANOl inhibitor CaCCinh-A01, the EGFR inhibitor Osimertinib, and the combination of Osimertinib and CaCCinh-A01.

[0123] In one embodiment of the present application, the anticancer agent described in (d) and (e) can be a tyrosine kinase-targeted anticancer agent, but is not limited thereto. As used herein, the term "targeted anticancer agent" refers to an agent that exerts an anticancer effect by targeting a specifically altered protein or gene in cancer cells or cancer tissues and interfering with molecular activities involved in cancer growth and development. Preferably, the tyrosine kinase can be one or more selected from the group consisting of: EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (B-Raf proto-oncogene), HER2 (human epidermal growth factor receptor 2), RET (RET proto-oncogene), NTRK1 (neurotrophic tyrosine kinase 1), MET (mesenchymal epithelial transition factor), and NRG1 (neuregulin 1). More preferably, the tyrosine kinase can be EGFR. EGFR includes normal EGFR and mutant EGFR (e.g., EGFRvIII).

[0124] As described in (e), the enhanced responsiveness of cancer cells to anticancer agents means that the inhibition of cancer cell growth and migration by anticancer agents is further enhanced.

[0125] In the present application, the term "enhancing anticancer efficacy" means all actions capable of eventually enhancing the function of an anticancer agent, and includes not only enhancing anticancer effects such as inhibiting tumor growth, inhibiting tumor metastasis, and inhibiting tumor recurrence, but also enhancing anticancer effects by inhibiting drug resistance or tolerance of cancer cells to anticancer agents.

[0126] Accordingly, the compound according to the present application can be used as a compound for combination therapy with a known anticancer agent, preferably a targeted anticancer agent against tyrosine kinase, to enhance anticancer efficacy of a brain tumor anticancer agent.

[0127] For example, the present application provides a pharmaceutical composition for preventing or treating cancer, preferably a brain tumor, comprising, as active ingredients, (i) one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 6, or pharmaceutically acceptable salts thereof; and (ii) a targeted anticancer agent against EGFR.

[0128] In addition, the present application provides a pharmaceutical composition for enhancing anticancer efficacy of an anticancer agent, preferably a brain tumor anticancer agent, comprising, as active ingredients, one or more compounds selected from the group consisting of compounds represented by Chemical Formulas 1 to 6, or pharmaceutically acceptable salts thereof.

[0129] The composition according to the present application can be in the form of a mixture in which the compound or a pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR are mixed, and can be a preparation for simultaneous administration of the compound or a pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR.

[0130] In addition, the composition according to the present application can be in a form in which the compound or a pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR are each formulated and administered simultaneously, separately, or sequentially. In this case, the composition can be a pharmaceutical composition for combination therapy for simultaneous or sequential administration, which comprises a first pharmaceutical composition containing a pharmaceutically effective amount of the compound or a salt thereof as an active ingredient, and a second pharmaceutical composition containing a pharmaceutically effective amount of the targeted anticancer agent against EGFR as an active ingredient. In this case, when administered sequentially, the order of administration is not limited, and the administration schedule can be appropriately adjusted depending on the patient's condition, etc.

[0131] That is, when the pharmaceutical composition is a pharmaceutical composition for combination therapy for sequential administration, the composition can be one in which the compound or a salt thereof ("first component") is administered first, and then the targeted anticancer agent against EGFR ("second component") is administered, or vice versa.

[0132] In addition, when the compound according to the present application is administered in combination with an anticancer agent, the compound can be administered simultaneously, separately or sequentially with the anticancer agent, even in the case of sequential administration, the order of administration is not limited, and the administration schedule can be appropriately adjusted depending on the type of cancer, the type of anticancer agent or the condition of the patient.

[0133] The content of the compound in the composition of the present application can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms and the condition of the patient, for example, can be 0.0001 to 99.9% by weight, or 0.001 to 50% by weight, based on the total weight of the composition, but is not limited thereto. The content is based on the dry weight obtained by removing the solvent.

[0134] The pharmaceutical composition according to the present application can further comprise suitable carriers, excipients and diluents commonly used for the preparation of pharmaceutical compositions. The excipient can be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials and controlled-release additives.

[0135] The pharmaceutical composition according to the present application can be used in the form of a powder, granules, sustained-release granules, enteric-coated granules, a liquid, eye drops, elixir, emulsion, suspension, spirit, tablet, aromatic water, lemonade, tablet, sustained-release tablet, enteric-coated tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric-coated capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, infusion or external preparation (e.g., plaster, emulsion, paste, spray, inhalant, patch, sterile injection solution or aerosol) prepared according to the usual method. The external preparation can have a preparation such as cream, gel, patch, spray, ointment, plaster, emulsion, liniment, paste or cataplasm.

[0136] Lactose, glucose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used as the carriers, excipients and diluents that can be contained in the pharmaceutical composition according to the present application.

[0137] Usual diluents or excipients such as fillers, thickening agents, binders, wetting agents, disintegrants and surfactants are used for the preparation.

[0138] As an additive of the tablet, powder, granule, capsule, pill and tablet according to the present application, an excipient such as corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primojel and a binder such as gelatin, acacia, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, carboxymethyl cellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin, hydroxyl cellulose, hydroxypropyl starch, hydroxymethyl cellulose, purified shellac, starch, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl alcohol, and polyvinyl pyrrolidone; and a disintegrant such as hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, carboxymethyl cellulose calcium, calcium citrate, sodium dodecyl sulfate, silicic anhydride, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinyl pyrrolidone, calcium phosphate, gelatinized starch, acacia, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, sorbitol solution, and light anhydrous silicic acid; and a lubricant such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium powder, kaolin, vaseline, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubriwax), aluminum stearate, zinc stearate, sodium dodecyl sulfate, magnesium oxide, polyethylene glycol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid.

[0139] Water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitan fatty acid ester (double ester), polyoxyethylene monoalkyl ether, lanolin ether, lanolin ester, acetic acid, hydrochloric acid, ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, proline, polyvinyl pyrrolidone, ethyl cellulose, and sodium carboxymethyl cellulose can be used as an additive of the liquid according to the present application.

[0140] In the syrup according to the present application, a white sugar solution, other sugars or sweeteners, etc. can be used, and, as needed, a flavoring agent, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifying agent, an adhesive, etc. can be used.

[0141] In the emulsion according to the present application, purified water can be used, and, as needed, an emulsifying agent, a preservative, a stabilizer, a flavoring agent, etc. can be used.

[0142] In the suspension according to the present application, a suspending agent such as gum arabic, gum tragacanth, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methyl cellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. can be used, and, as needed, a surfactant, a preservative, a stabilizer, a coloring agent, and a flavoring agent can be used.

[0143] The injection according to the present application can include a solvent such as distilled water for injection, 0.9% sodium chloride solution, Ringer's solution, glucose solution, glucose + sodium chloride solution, PEG, lactated Ringer's solution, ethanol, propylene glycol, non-volatile oil such as sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzoate; a co-solvent such as sodium benzoate, sodium salicylate, sodium acetate, urea, carbamate, monoacetamide, butazolidine, propylene glycol, Tween series, amide nicotinate, hexamine, and dimethylacetamide; a buffer such as a weak acid and a salt thereof (acetic acid and sodium acetate), a weak base and a salt thereof (ammonia and ammonium acetate), an organic compound, a protein, albumin, proteose peptone, and gum; an isotonic agent such as sodium chloride; a stabilizer such as sodium bisulfite (NaHSO3) carbon dioxide gas, sodium pyrosulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen (N2), and ethylenediaminetetraacetic acid; a sulfating agent such as 0.1% sodium bisulfite, sodium formaldehydesulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; an analgesic such as benzyl alcohol, chlorobutanol, procaine hydrochloride, dextrose, and calcium gluconate; and a suspending agent such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0144] In the suppositories according to the present application, bases such as cocoa butter, lanolin, Witepsol, polyethylene glycol, glycerin gelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lotus wax, glycerin monostearate, Tween or span, imhausen, monoglycerol (propylene glycol monostearate), glycerin, Adeps solidus, buytyrum Tego-G, cebes Pharma 16, hexalactone base 95, cotomar, Hydrokote SP, S-70-XXA, S-70-XX 75 (S-70-XX 95), Hydrokote 25, Hydrokote 711, idropostal, massa estrarium (A, AS, B, C, D, E, I, T), masa-MF, masupol, masupol-15, neosuppostal-N, paramount-B, supposiro OSI, OSIX, A, B, C, D, H, L, suppository base type IV AB, B, A, BC, BBG, E, BGF, C, D, 299, suppostal N, Es, Wecoby W, R, S, M, Fs, and tegester glycerin triester material (TG-95, MA, 57) can be used.

[0145] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc, and the like, are used.

[0146] Examples of liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, syrups, and the like, which, in addition to simple common diluents such as water and liquid paraffin, can contain various types of excipients, such as wetting agents, sweeteners, flavors, preservatives, and the like. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-limiting examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0147] The pharmaceutical composition according to the present application is administered in a pharmaceutically effective amount. In the present application, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined depending on the type of disease, the severity of the disease, the drug activity, the sensitivity to the drug, the administration time, the administration route, the excretion rate, the treatment period, and other drugs used simultaneously, and other factors well known in the medical field.

[0148] The composition according to the present application can be administered as a single therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with the therapeutic agents in the prior art, and can be administered in a single dose or multiple doses. In consideration of all the above factors, it is important to administer the composition in the minimum amount capable of obtaining the maximum effect without any side effects, which can be easily determined by one of ordinary skill in the art.

[0149] The pharmaceutical composition of the present application can be administered to a subject via various routes. All administration methods can be predictable, and the pharmaceutical composition can be administered, for example, orally, subcutaneously, intraperitoneally, intravenously, intramuscularly, intrathecally (into the space surrounding the spinal cord), sublingually, transbuccally, rectally, vaginally, ocularly, aurally, nasally, by inhalation, by oral or nasal spray, transdermally, intradermally, etc.

[0150] The pharmaceutical composition of the present application is determined depending on the type of drug as an active ingredient, and various related factors, such as the disease to be treated, the administration route, the age, sex, and weight of the patient, and the severity of the disease. Specifically, the effective amount of the composition according to the present application can vary depending on the age, sex, and weight of the patient, and generally, 0.001 to 150 mg / kg of body weight, preferably 0.01 to 100 mg / kg of body weight can be administered per day, every other day, or divided into 1 to 3 doses per day. However, since the dose can be increased or decreased depending on the administration route, the severity of the disease, sex, weight, and age, the above-mentioned dose by no means limits the scope of the present application.

[0151] As used herein, "subject" means a subject in need of treatment for a disease, and more specifically, a mammal, such as a human or a non-human primate, a mouse, a rat, a dog, a cat, a horse, and a cow.

[0152] As used herein, "administration" means providing the intended composition of the present application to a subject by using any appropriate method.

[0153] As used herein, the term "prevention" means all actions to inhibit or delay the onset of a target disease. As used herein, the term "treatment" means all actions to alleviate or beneficially change a target disease and abnormal metabolic symptoms caused thereby, via administration of a pharmaceutical composition according to the present application. As used herein, the term "alleviation" means all actions to reduce the degree of a parameter (e.g., a symptom) associated with a target disease, via administration of a composition according to the present application.

[0154] Further, the present application provides a kit for preventing or treating cancer, which comprises a pharmaceutical composition according to the present application.

[0155] The kit according to the present application can include, in addition to the compound and the targeted anticancer agent, other components, compositions, solutions, or devices generally required for the prevention or treatment of cancer, but are not limited thereto, and can particularly contain instructions indicating the correct use and storage of the compound according to the present application.

[0156] The terms and words used in the present specification and claims should not be interpreted as being limited to commonly or dictionary meanings, but should be interpreted based on the meanings and concepts as consistent with the technical spirit of the present application, based on the principle that the inventor can properly define the concepts of the terms to best describe his own application.

[0157] [Mode for Invention] Hereinafter, preferred embodiments are given to help understanding of the present application. However, the following embodiments are provided only to help more easily understand the present application, and the content of the present application is not limited by the following embodiments.

[0158] EMBODIMENT Example A: Novel compounds AON-MG23-01 and AON-MG23-02 1. Preparation of the compound according to the present application 1-1. Preparation of N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-01) [Scheme 1] The AON-MG23-01 compound is prepared by the above-described Scheme 1. The specific preparation method is as follows: <Step 1> Synthesis of N-cyclopropyl-N-(2-nitrophenyl)methanesulfonamide [Scheme 2] N-cyclopropyl-N-(2-nitrophenyl) methanesulfonamide synthesized in Step 1 (1.00 eq) was added to 1,4-dioxane and water (3:1) and stirred, then the reactor was cooled to 0 °C and zinc (Zn, 10.0 eq) and ammonium chloride (NH4Cl, 10.0 eq) were added thereto. Thereafter, the resulting mixture was stirred for 4 hours while gradually increasing the temperature. Upon completion of the reaction, the mixture was filtered through celite and the filtrate was extracted with ethyl acetate and water. The organic layer was stirred after adding anhydrous sodium sulfate, filtered with a filter, and then the filtrate was concentrated under reduced pressure. The concentrated residue was used in Step 3 without any additional purification.

[0159] <Step 2> Synthesis of N-(2-aminophenyl)-N-cyclopropylmethanesulfonamide [Scheme 3] N-cyclopropyl-N-(2-nitrophenyl) methanesulfonamide synthesized in Step 1 (1.00 eq) was added to 1,4-dioxane and water (3:1) and stirred, then the reactor was cooled to 0 °C and zinc (Zn, 10.0 eq) and ammonium chloride (NH4Cl, 10.0 eq) were added thereto. Thereafter, the resulting mixture was stirred for 4 hours while gradually increasing the temperature. Upon completion of the reaction, the mixture was filtered through celite and the filtrate was extracted with ethyl acetate and water. The organic layer was stirred after adding anhydrous sodium sulfate, filtered with a filter, and then the filtrate was concentrated under reduced pressure. The concentrated residue was used in Step 3 without any additional purification.

[0160] <Step 3> Synthesis of N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl) methanesulfonamide [Scheme 4] N-(2-aminophenyl)-N-cyclopropylmethanesulfonamide synthesized in Step 2 (1.00 eq) was added to isopropyl alcohol (IPA) and 2,4,5-trichloropyrimidine (1.1 eq) and N,N- diisopropylethylamine (DIPEA, 2.5 eq) were added at room temperature. Thereafter, the resulting mixture was refluxed and stirred overnight. Upon completion of the reaction, the mixture was evaporated under reduced pressure, extracted with water and dichloromethane. The organic layer was washed with 2 N hydrochloric acid, then stirred after adding anhydrous sodium sulfate. Thereafter, the resulting mixture was filtered using a filter, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified by column chromatography (hexane: ethyl acetate = 3:1) to synthesize N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl) methanesulfonamide as a pale yellow solid in a yield of 58.9%.

[0161] <Step 4> Synthesis of N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1- yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide [Scheme 5] N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide (1.00 eq) synthesized in Step 3 was added to ethanol (EtOH) and 2-methoxy-4-(4-(4- methylpiperazin-1-yl)piperidin-1-yl)aniline (1.00 eq) and trifluoroacetic acid (TFA, 1.95 eq) were added at room temperature. Thereafter, the resulting mixture was refluxed and stirred overnight. After the completion of the reaction, the mixture was neutralized with 1N sodium hydroxide solution, extracted with water and ethyl acetate. After the addition of anhydrous sodium sulfate, the organic layer was stirred, filtered with a filter, and then the filtrate was concentrated under reduced pressure. The concentrated residue was purified by column chromatography (hexane: ethyl acetate = 3:1) to synthesize N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1- yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-01) as a yellow solid in a yield of 14%.

[0162] H NMR (500 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.62 (dd, J = 7.9, 1.6 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.15 (td, J = 7.6, 1.5 Hz, 1H), 6.62 (d, J = 2.5 Hz, 1H), 6.47 (dd, J = 8.7, 2.5 Hz, 1H), 3.75 - 3.71 (m, 5H), 3.25 - 3.22 (m, 4H), 2.67 (td, J = 12.2, 2.4 Hz, 2H), 2.55 - 2.44 (m, 4H), 2.38 - 2.27 (m, 4H), 2.15 (s, 3H), 1.85 (d, J = 11.5 Hz, 2H), 1.52 (qd, J = 12.1, 3.9 Hz, 2H), 1.01 - 0.93 (m, 2H), 0.55 - 0.49 (m, 1H), 0.17 - 0.12 (m, 1H). HRMS: 640.2708, cal: 640.2711 1-2. Preparation of N-(2-((5-chloro-2-((4-(4-(dimethylamino)piperidin-l-yl)-2- methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-02) [Scheme 6] Steps 1, 2 and 3 were performed in the same manner as for the AON-MG23-01 compound, and the synthesis in step 4 was performed using 1-(4-amino-3-methoxyphenyl)-N,N- dimethylpiperidin-4-amine instead of 2-methoxy-4-(4-(4-methylpiperazin-l-yl)piperidin-l- yl)aniline.

[0163] Specifically, N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl) methanesulfonamide (1.00 eq) synthesized in Step 3 was added to ethanol (EtOH), and 1-(4-amino-3-methoxyphenyl)-N,N-dimethylpiperidin-4-amine (1.00 eq) and trifluoroacetic acid (TFA, 1.95 eq) were added at room temperature. Thereafter, the resulting mixture was refluxed and stirred overnight. After completion of the reaction, the mixture was neutralized with a 1N sodium hydroxide solution, extracted with water and ethyl acetate. After adding anhydrous sodium sulfate, the organic layer was filtered with a filter, and then the filtrate was concentrated under reduced pressure. The concentrated residue was purified by column chromatography (hexane: ethyl acetate = 3:1) to synthesize N-(2-((5-chloro-2-((4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-02) as a yellow solid at a yield of 25%.

[0164] 1 H NMR (500 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.19 (s, 1H), 8.09 (s, 1H),7.99 (s, 1H), 7.62 (dd, J = 7.8, 1.6 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.22(d, J = 8.1 Hz, 1H), 7.15 (td, J = 7.6, 1.6 Hz, 1H), 6.63 (d, J = 2.5 Hz,1H), 6.48 (dd, J = 8.7, 2.5 Hz, 1H), 3.75 (s, 3H), 3.72 (d, J = 12.2 Hz, 2H),3.23 (m, 4H), 2.68 (td, J = 12.1, 2.5 Hz, 2H), 2.22 (s, 6H), 1.86 (d, J =12.4 Hz, 2H), 1.51 (qd, J = 12.0, 3.9 Hz, 2H), 1.04 - 0.92 (m, 2H), 0.55 -0.49 (m, 1H), 0.17 - 0.14 (m, 1H). HRMS: 585.2294, cal: 585.2289. The compounds according to the present application were prepared by the above-mentioned steps, and their structural formulas are shown in Table 1 and FIG. 1 below.

[0165] [Table 1] 2. Demonstration of the inhibitory effect of the compounds of the present invention on the proliferation of human glioblastoma cells U87-MG cells, a human glioblastoma cell line, were seeded in 96-well plates at a density of 1 X 10 4 The cells were treated with DMSO as a control and the compounds of the present invention, AON-MG23-01 or AON-MG23-02, after 24 hours of incubation. The compounds of the present invention were treated at different concentrations of 0, 1, 2 and 4 μΜ. 10 μΐ^of CCK reagent was added and allowed to react for one hour, after which the absorbance was measured at 450 nm.

[0166] The results, as shown in Figure 2, show that the compounds of the present invention, AON-MG23-01 or AON-MG23-02, inhibit the proliferation of U87-MG cells.

[0167] 3. Demonstration of the inhibitory effect of the compounds of the present invention on the cell migration of human glioblastoma cells U87-MG cells, a human glioblastoma cell line, were seeded on SPL ScarBlock (SPL) plates at a density of 8 X 10 4 The cells were treated with DMSO as a control and the compounds of the present invention, AON-MG23-01 or AON-MG23-02, after 24 hours of incubation. The compounds of the present invention were treated at different concentrations of 0, 1, 2 and 4 μΜ. 10 μΐ^of CCK reagent was added and allowed to react for one hour, after which the absorbance was measured at 450 nm.

[0168] The results, as shown in Figure 3, demonstrate that when the compounds of the present invention, AON-MG23-01 or AON-MG23-02, were treated at a concentration of 4 μΜ, the cell migration was inhibited by more than 65% compared to the untreated control.

[0169] 4. Demonstration of the inhibitory effect of the compounds of the present invention on the metastasis of human glioblastoma U87-MG cells, a human glioblastoma cell line, were seeded on SPL ScarBlock (SPL) plates at a density of 8 X 10 4The cells were seeded at a density of 1 cell / well on Matrigel-coated filters (Transwell invasion chamber, Corning), then the cells were treated with the compounds of the present application (AON-MG23-01 or AON-MG23-02; each at 0, 1, 2 and 4 mM) and incubated in a CO2incubator at 37 °C for 24 hours. After completion of the incubation, the cells were fixed and stained using Diff-Quick Stain kit (Sysmex, Kobe, Japan). Images of the stained cells were taken under a microscope, the cells were counted using Image J software, and then statistical analysis was performed.

[0170] Results, as shown in Figure 4, it can be seen that the groups treated with the compounds of the present application AON-MG23-01 or AON-MG23-02 showed a decrease in the degree of penetration of Matrigel compared to the untreated control, indicating that the metastatic ability of the cancer cells was inhibited.

[0171] 5. Demonstration of the effect of the compounds of the present application on the expression of ANOl and EGFR proteins in glioblastoma cell lines It was determined whether the compounds of the present application affect the expression of ANOl and EGFR proteins in glioblastoma cell lines. The human glioblastoma cell line U87-MG was seeded at a density of 1 X 10 6Cells were seeded at a density of one cell in a 60 mm dish, cultured for 24 hours, and then treated with the compounds of the present application (AON-MG23-01 or AON-MG23-02; each at 0, 1, 2, and 4 mM) and cultured for 48 hours. The cultured cells were harvested, and the harvested cells were lysed for Western blotting. Anti-ANO1 antibody (Abeam, ab53212), anti-pEGFR antibody (Cell Signaling, #4407S), anti-EGFR antibody (Cell Signaling, #4267S), and anti-a-tubulin antibody (Santacruz, #SC-5286) were used as antibodies for Western blotting. The protein concentration was quantified using the BSA method (Protein Assay, Bovine Serum Albumin Assay) (Pierce, Cat. 23225). After cell lysis, an equal amount (10 pg) of protein obtained from the lysed cells was subjected to 10% SDS-PAGE, the proteins were separated according to their molecular weights, transferred to a PVDF membrane (Bio-Rad), and then treated with a blocking buffer (5% skim milk in Tris-buffered saline (TBS) buffer containing 0.1% Tween 20; TBS-T) at room temperature for one hour. Thereafter, the PVDF membrane was treated with a primary antibody and reacted at 4°C for 16 hours. The PVDF membrane after the reaction was washed with TBS-T three times. Then, the PVDF membrane was reacted with a horseradish peroxidase-labeled secondary antibody at room temperature for one hour, and then visualized using an ECL kit (Bio-rad). The amount of each protein was quantified and analyzed using Image J software.

[0172] As a result, as shown in FIG. 5, the protein levels of ANO1 and EGFR were significantly inhibited in cells treated with the compounds of the present application compared to the untreated control, and this protein expression inhibition was dependent on the treatment concentration of the compounds.

[0173] 6. Confirmation of the inhibitory effect of the compounds of the present application on ANO1 activity in a glioblastoma cell line The inhibitory effect on ANO1 ion channel activity was confirmed by measuring the calcium-dependent chloride ion channel activity of the compounds of the present application using patch clamp. To this end, U87-MG cells expressing ANO1 were subjected to patch clamp. The pipette solution consisted of 146CsCl, 5 Ca-EGTA-NMDG, 8 HEPES, 2MgCl2, and 10 sucrose (pH 7.3), and the bath solution consisted of 50NaCl, 10 HEPES, 3KCl, 2CaCl2, 2MgCl2, and 5.5 glucose (pH 7.3). U87-MG cells were cultured on a coverslip for 4 hours, and then treated with AON-MG23-01 or AON-MG23-02 for one hour (at a concentration of 0, 4, and 8 μM). At this time, the calcium-dependent chloride ion channel was activated using 400 mM ATP and a high concentration of calcium, and the calcium-dependent chloride ion channel current was measured in the range of -100 mV to +100 mV.

[0174] As a result, as shown in FIG. 6A, it was confirmed that the group treated with the compound of the present application showed a significant decrease in ANO1 activity compared to the untreated control. In particular, as shown in FIG. 6B, it was confirmed that the group treated with the compound of the present application showed a current density reduction of more than 50% at a membrane potential of +80 mV compared to the untreated control. The results indicate that the compound of the present application can inhibit the activity of the calcium-dependent chloride ion channel ANO1 in the glioblastoma cell line U87-MG cells.

[0175] 7. Comparison of the metastasis inhibitory effect of the compound of the present application with EGFR inhibitors and ANO1 inhibitors in human glioblastoma The human glioblastoma cell line U87-MG was seeded at a density of 6X10 4 cells / well on the top of Matrigel-coated filters (Transwell invasion chamber, Corning) and treated with 4 μM of the compound of the present application AON-MG23-02, 4 μM of the EGFR inhibitor Osimertinib, 100 μM of the ANO1 inhibitor CaCCinh-A01, or a combination of 4 μM of Osimertinib and 100 μM of CaCCinh-A01, and then incubated in a CO2incubator at 37°C for 16 hours. After completion of the incubation, the cells were fixed and stained using a Diff-Quick staining kit (Sysmex, Kobe, Japan). Images of the stained cells were taken under a microscope, the cells were counted using Image J software, and then statistical analysis was performed.

[0176] Results, as shown in Figures 7A and 7B, the group treated with the compound of the application AON-MG23-02 showed a lower degree of Matrigel penetration and had a similar inhibitory effect on metastasis as the EGFR inhibitor and AN01 inhibitor combination treatment group, compared to the untreated control, EGFR inhibitor treatment group and AN01 inhibitor treatment group.

[0177] 8. Comparison of the inhibitory effect of the compound of the application, EGFR inhibitor and AN01 inhibitor on the expression of AN01, EGFR and pEGFR proteins in glioblastoma cell lines The inhibitory effect of the compound of the application AON-MG23-02, EGFR inhibitor Osimertinib and AN01 inhibitor CaCCinh-A01 on the expression of AN01, EGFR and pEGFR proteins in glioblastoma cell lines was compared. Human glioblastoma cell line U87-MG was seeded at 100X10 4Cells were plated at a density of 1 cell / dish in 60 mm dishes and cultured for 24 hours. Thereafter, cells were treated with 4 mM of AON-MG23-02, 2 mM of EGFR inhibitor Osimertinib, 100 mM of ANO1 inhibitor CaCCinh-A01, or a combination of 2 mM of Osimertinib and 100 mM of CaCCinh-A01, and cultured for 48 hours. The cultured cells were harvested, and the harvested cells were lysed for Western blotting. Anti-ANO1 antibody (Abeam, ab53212), anti-pEGFR antibody (Cell Signaling, #4407S), anti-EGFR antibody (Cell Signaling, #4267S), and anti-ACTIN antibody (sigma, #A2066) were used as antibodies for Western blotting. The protein concentration was quantified using a BSA method (Protein Assay, Bovine Serum Albumin Assay) (Pierce, Cat. 23225). After cell lysis, an equal amount (10 pg) of protein obtained from the lysed cells was subjected to 10% SDS-PAGE, the proteins were separated according to their molecular weights, transferred to a PVDF membrane (Bio-rad), and then treated with a blocking buffer (5% skim milk in Tris-buffered saline (TBS) buffer containing 0.1% Tween 20; TBS-T) at room temperature for one hour. Next, the PVDF membrane was treated with a primary antibody and reacted at 4°C for 16 hours. The reacted PVDF membrane was washed with TBS-T three times for 10 minutes. Then, the PVDF membrane was reacted with a horseradish peroxidase-labeled secondary antibody at room temperature for one hour, and then washed with TBS-T three times for 10 minutes. The washed PVDF membrane was treated with an ECL kit (Thermo, West Pico Plus), reacted, and visualized using a da Vinci-Q (YounginLabPlus) device. The amount of each protein was quantified and analyzed using Image J software.

[0178] As a result, as shown in FIGS. 8A and 8B, it was confirmed that the compound AON-MG23-02 of the present application reduced the expression of ANO1, EGFR, and pEGFR proteins compared to the untreated control, the EGFR inhibitor-treated group, the ANO1 inhibitor-treated group, and the EGFR inhibitor and ANO1 inhibitor combination-treated group.

[0179] Example B. Derivatives of the novel compound AON-MG23-02 1. Preparation of AON-MG23-02 derivatives 1-1. AON-MG23-05 1-1-A. Preparation of Compound 2 [Scheme 7] Compound 1 (3.00 g, 11.3 mmol, 1.00 eq), compound SM1 (756 mg, 5.68 mmol, 0.500 eq) and Cs2CO3(2.96 g, 9.09 mmol, 0.800 eq) were mixed in acetonitrile (ACN) (10.0 mL). The resulting mixture was degassed, purged with N2three times, then stirred at 80 °C for 2 hours under nitrogen (N2). TLC (petroleum ether: ethyl acetate = 1:1, Rf=0.49) analysis showed that compound SM1 was completely consumed and a large number of new spots were generated. The reaction mixture was concentrated under reduced pressure. The residue was diluted with 6.00 mL of water, then extracted with 6.00 mL of EtOAc (6.00 mL*3). The combined organic supernatant was washed with 9.00 mL of brine (9.00 mL*1), dried over Na2SO4, filtered, then concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 20:1 to 0:1). Compound 2 (900 mg, 2.35 mmol, yield: 20.6%, and purity: 82.4%) was obtained as a yellow solid. f =0.49) analysis showed that compound SM1 was completely consumed and a large number of new spots were generated. The reaction mixture was concentrated under reduced pressure. The residue was diluted with 6.00 mL of water, then extracted with 6.00 mL of EtOAc (6.00 mL*3). The combined organic supernatant was washed with 9.00 mL of brine (9.00 mL*1), dried over Na2SO4, filtered, then concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 20:1 to 0:1). Compound 2 (900 mg, 2.35 mmol, yield: 20.6%, and purity: 82.4%) was obtained as a yellow solid.

[0180] 1-1-B. Preparation of compound 3 [Scheme 8] Trimethylsilyl cyanide (TMSCN, 338 mg, 3.42 mmol, 427 μL, 1.20 eq) and Cs2CO3(1.11 g, 3.42 mmol, 1.20 eq) were added to a solution of compound 2 (900 mg, 2.85 mmol, 1.00 eq) dissolved in ACN (10.0 mL). The mixture was stirred at 25 °C for 12 hours. LC-MS analysis showed that compound 2 was completely consumed, and 42.0% of the desired mass was detected. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with 2.00 mL of water, then extracted with 3.00 mL of EtOAc (3.00 mL*3). The combined organic supernatant was washed with 4.00 mL of brine (4.00 mL*1), dried over Na2SO4, filtered, then concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 10:1 to 0:1). Compound 3 (600 mg, 2.22 mmol, yield: 77.8%, purity: 96.9%) was obtained as a yellow solid.

[0181] 1-1-C. Preparation of compound 4 [Scheme 9] Compound 3 (600 mg, 2.29 mmol, 1.00 eq) and Raney-Ni (19.6 mg, 228 pmol, 0.100 eq) were dissolved in toluene (6.00 mL), degassed, purged with N2, and then the suspension was degassed under vacuum, purged with hydrogen. The resulting mixture was stirred at 80 °C under H2(40 psi) for 12 hours. LC-MS analysis showed that compound 3 was completely consumed and 54.8% of the desired product was detected. The mixture was filtered and concentrated. The crude product was purified by reverse phase HPLC (column: Phenomenex luna C18 150*40 mm*15 um; mobile phase: [water (FA)-ACN]; gradient: from 2% to 32% B in 15 minutes). Compound 4 ([[4-(2-aminoethyl)phenyl]methyl]isoindolin-1-one, 320 mg, 1.20 mmol, yield: 52.5%, purity: 100%) was obtained as a white solid.

[0182] 1-1-D. Preparation of compound 5 [Scheme 10] A mixture obtained by dissolving compound 4 (200 mg, 750 pmol, 1.00 eq) and compound 4a (117 mg, 750 pmol, 1.00 eq) in CH(Ome)3 (2.00 mL) was degassed, purged with N2 three times, and stirred under N2gas at 25 °C for one hour. AcOH (45.0 mg, 750 pmol, 42.9 pL, 1.00 eq) and NaBH3CN (14.1 mg, 225 pmol, 0.300 eq) were added to the resulting mixture. Thereafter, the mixture was stirred under N2at 25 °C for one hour. TLC (dichloromethane:methanol = 10:1; Rf=0.7) showed that compound 4 was completely consumed and two new spots were formed. The reaction mixture was concentrated under reduced pressure. The residue was diluted with 5.00 mL of water and extracted with 4.00 mL of EtOAc (4.00 mL*3). The combined organic layers were washed with 5.00 mL of brine (5.00 mL*1), dried over Na2S04, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (Si02, dichloromethane:methanol = 60:1 to 10:1). Compound 5 (34.0 mg, 73.1 pmol, yield: 9.73%, purity: 87.4%) was obtained as a yellow oil. f

[0183] 1-1-E. Preparation of compound 6​ [Scheme 11] K2CO3 (36.7 mg, 265 μmol, 2.00 eq), KI (4.41 mg, 26.5 μmol, 0.200 eq) and compound 5a (38.1 mg, 159 μmol, 1.20 eq) were added to a mixture obtained by dissolving compound 5 (54.0 mg, 132 μmol, 1.00 eq) in dimethylformamide (DMF, 0.600 mL). The mixture was stirred at 50 °C for 12 h. TLC (dichloromethane:methanol=10:1, Rf=0.70) results showed compound 5 remained and one new spot was detected. The residue was diluted with 2.00 mL of water and then extracted with 2.00 mL of EtOAc (2.00 mL*3). The combined organic layers were washed with 3.00 mL of brine (3.00 mL*1), dried over Na2SO4, filtered, and then concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, dichloromethane:methanol=10:1). Compound 6 (32.0 mg, 56.6 μmol, yield: 42.6%, purity: 100%) was obtained as a yellow oil. f

[0184] 1-1-F. Preparation of AON-MG23-05 [Scheme 12] HCl / MeOH (2.00 M, 1.54 mL, 60.0 eq) was added to a mixture obtained by dissolving compound 6 (29.0 mg, 51.3 μmol, 1.00 eq) in MeOH (0.500 mL). The mixture was stirred at 25 °C for 0.5 h. LC-MS results showed compound 6 was completely consumed and 98.7% of the desired mass was detected. The reaction mixture was concentrated under reduced pressure. NaHCO3 (pH>7) was added to the mixture. The residue was extracted with 3.00 mL of DCM (3.00 mL*3). The combined organic layers were washed with 2.00 mL of brine (2.00 mL*1), dried over Na2SO4, filtered, and then concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (SiO2, ethyl acetate:methanol=10:1). The target A (AON-MG23-05) was obtained as an off-white solid (18.2 mg, 40.2 μmol, yield: 78.3%, purity: 99.6%).

[0185] The H NMR data of AON-MG23-05 is shown in Figure 11.​

[0186] 1-2. Preparation of AON-MG23-06 1-2-A. Preparation of compound 8 [Scheme 13] Compound SM1 (3.00 g, 22.5 mmol, 1.00 eq) was mixed with compound 7 (4.13 g, 27.0 mmol, 3.81 mL, 1.20 eq) and Cs2CO3 (11.0 g, 33.8 mmol, 1.50 eq) in ACN (30 mL), the resulting mixture was degassed and stirred at 25 °C for 16 hours. LC-MS result showed 20% of compound SM1 remained. Several new peaks appeared in LC-MS, 60% of desired compound was detected. The reaction mixture was quenched by adding 30 mL of H2O and extracted with 30 mL ethyl acetate (EA) (30 mL*2). The combined organic layers were washed with 30 mL brine (30 mL*2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 20:1 to 15:1) and TLC (petroleum ether: ethyl acetate = 3:1, Rf = 0.43). Compound 8 (2.10 g, 8.42 mmol, yield: 37.3%) was obtained as a white solid, which was confirmed by LC-MS and HNMR. f =0.43) purification. Compound 8 (2.10 g, 8.42 mmol, yield: 37.3%) was obtained as a white solid, which was confirmed by LC-MS and HNMR.

[0187] 1-2B. Preparation of compound 9 [Scheme 14] To a mixture obtained by dissolving compound 8 (200 mg, 802 µmol, 1.00 eq) in dichloromethane (DCM, 0.200 mL) was added dropwise m-chloroperoxybenzoic acid (m-CPBA, 651 mg, 3.21 mmol, purity: 85.0%, 4.00 eq) at 0 °C over 0.5 hours. After this addition, the mixture was stirred at 25 °C for 2 hours. TLC (petroleum ether: ethyl acetate = 2:1, Rf = 0.43) showed that the reaction was completed. The reaction mixture was quenched by adding 30 mL of H2O and extracted with 30 mL ethyl acetate (EA) (30 mL*2). The combined organic layers were washed with 30 mL brine (30 mL*2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 20:1 to 15:1) and TLC (petroleum ether: ethyl acetate = 3:1, Rf = 0.43). Compound 9 (200 mg, 802 µmol, yield: 100%) was obtained as a white solid, which was confirmed by LC-MS and HNMR. f= 0.33) showed that compound 8 was completely consumed and a new spot was formed. 20 mL of Na2S2O3was added to quench the reaction mixture at 25 °C, and extracted with 30 mL of DCM (30 mL*2). The combined organic layers were washed with 30 mL of brine (30 mL*2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative TLC (SiO2, petroleum ether: ethyl acetate = 2: 1). Compound 9 (580 mg, 1.65 mmol, yield: 62.4%) was obtained as a brown solid, which was confirmed by HPLC and HNMR. In addition, compound 9 (140 mg, 527 μmol, yield: 65.7%) was obtained as a yellow oil, which was confirmed by LC-MS and HNMR.

[0188] 1-2C. Preparation of compound 9A [Scheme 15] A solution obtained by dissolving ethylamine (626 mg, 7.68 mmol, 909 μL, 1.20 eq, HC1) and triethylamine (TEA, 777 mg, 7.68 mmol, 1.07 mL, 1.2 eq) in MeOH (10 mL) was stirred at 25 °C for 0.5 hours, and compound 9B (1.00 g, 6.40 mmol, 1.00 eq), sodium triacetoxyborohydride (NaBH(OAc)3, 2.04 g, 9.60 mmol, 1.50 eq) and AcOH (3.85 mg, 64.0 μmol, 3.67 μL, 0.01 eq) were added at 25 °C. The resulting mixture was stirred at 25 °C for one hour. LC-MS results showed that compound 9B was completely consumed, and a major peak with the desired m / z value was detected. The reaction mixture was quenched by adding 10 mL of Na2CO3, diluted with 20 mL of H2O, and then extracted with 30 mL of EA (30 mL*2). The combined organic layers were washed with 30 mL of brine (30 mL*1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 0: 1 to dichloromethane:methanol = 20: 1). Compound 9A (520 mg, 2.81 mmol, yield 43.8%) was obtained as a white solid, which was confirmed by HNMR and LC-MS.

[0189] 1-2D. Preparation of AON-MG23-06 [Scheme 16] To a mixture of compound 9 (65.0 mg, 245 mΐ, 1.00 eq), compound 9A (54.4 mg, 294 mΐ, 1.20 eq) and N,N-diisopropylethylamine (DIPEA, 94.9 mg, 735 mΐ, 128 pL, 3.00 eq) dissolved in ethanol (0.1 mL) was degassed, purged with N2 three times, then stirred in N2 gas at 80 °C for 12 hours. LC-MS result showed compound 9 was completely consumed and one major peak with desired m / z value was detected. The reaction mixture was quenched by adding 10 mL of H2O, extracted with 5 mL of EA (5 mL*2), then concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (FA condition: Phenomenex luna C18 150*25 mm*10 pm; mobile phase: [water(FA)-ACN]; gradient: 15%-45% B, >1 min). Subsequently, it was purified by prep-HPLC (neutral condition: Waters Xbridge 150*25 mm*5 pm; mobile phase: [water(NH4HCO3)-ACN]; gradient: 45% to 75% B, >15 min). White solid AON-MG23-06 (target B) (29.7 mg, 65.7 mΐ, 26.8% yield, 99.8% purity) was obtained, which was confirmed by LC-MS, HPLC, H NMR and special NMR.

[0190] H NMR data of AON-MG23-06 is shown in Figure 12.

[0191] 1-3. Preparation of AON-MG23-07 1-3-A. Preparation of compound 10 [Scheme 17] A suspension of KOH (675.1 mg, 12.0 mmol, 3.00 eq), dimethyl sulfoxide (DMSO, 10.0 mL), toluene (7.50 mL) and H2O (2.00 mL) was purged with argon and saturated with P (5.00 g, 147.0 mmol, 36.6 eq). A DMSO solution containing compound 8 (1.00 g, 4.01 mmol, 1.00 eq) was stirred at 70 °C for 30 minutes and added dropwise continuously to ignite the gas flow. The reaction mixture was further heated (70 °C) for 30 minutes while maintaining the flow of phosphine hydrogen. TLC (PE:EA=3:1, Rf=0.6, compound 8; Rf=0.2, compound 9) showed complete consumption of compound 8 and one major peak with desired m / z value was detected by LC-MS. The reaction mixture was quenched by adding 10 mL of H2O, extracted with 5 mL of EA (5 mL*2), then concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (FA condition: Phenomenex luna C18 150*25 mm*10 pm; mobile phase: [water(FA)-ACN]; gradient: 15%-45% B, >1 min). Subsequently, it was purified by prep-HPLC (neutral condition: Waters Xbridge 150*25 mm*5 pm; mobile phase: [water(NH4HCO3)-ACN]; gradient: 45% to 75% B, >15 min). White solid AON-MG23-07 (target B) (29.7 mg, 65.7 mΐ, 26.8% yield, 99.8% purity) was obtained, which was confirmed by LC-MS, HPLC, H NMR and special NMR. f= 0.38) The results showed that compound 8 was completely consumed and new spots were formed. The mixture was passed through argon gas, cooled, diluted with 20 mL of water, and then extracted with toluene (10 mL*2). The toluene extract was washed with brine (10 mL*1), dried over Na2SO4, filtered, and then concentrated under reduced pressure to obtain a residue. Compound 10 (1.10 g, crude) was obtained as a white solid.

[0192] 1-3-B. Preparation of AON-MG23-07 [Scheme 18] To a solution obtained by dissolving compound 10 (1.10 g, 3.88 mmol, 1.00 eq) in tetrahydrofuran (THF, 10.0 mL) was added NaH (465.9 mg, 11.6 mmol, purity: 60.0%, 3.00 eq) at 0 °C under an N2gas atmosphere. The mixture was stirred at 0 °C for 30 minutes. Thereafter, a THF solution containing compound 10a (858.4 mg, 3.88 mmol, 1.00 eq) was added dropwise at 0 °C. The mixture was stirred at 0 °C for 10 minutes. LC-MS results showed that compound 10 was completely consumed, and the desired mass was detected. The reaction mixture was quenched by adding 20 mL of NH4Cl under N2gas at 0 °C, diluted with 20.0 mL of H2O, and extracted with 40.0 mL of EA (20.0 mL*2). The combined organic layer was washed with 30.0 mL of brine (30.0 mL*1), dried over Na2SO4, filtered, and then concentrated under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (FA condition; column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 52% to 82% B in 15 minutes). The residue was further purified by preparative HPLC (FA condition; column: WelchXtimate C18 150*25mm*5um; mobile phase: [water(FA)-ACN]; gradient: 58% to 78% B in 15 minutes). Off-white AON-MG23-07 (target C) (35.0 mg, 58.5 pmol, yield: 1.51%, purity: 97.0%) was obtained.

[0193] The H NMR data of AON-MG23-07 is shown in Figure 13.

[0194] 1-4. Preparation of AON-MG23-08 1-4-A. Preparation of compound 2 [Scheme 19] A mixture of compound 1 (5.00 g, 18.9 mmol, 1.00 eq), compound SM1 (1.26 g, 9.47 mmol, 0.50 eq) and Cs2CO3 (4.94 g, 15.1 mmol, 0.80 eq) dissolved in ACN (15.0 mL) was degassed, treated with N2 gas for three times and stirred in N2 gas at 80 °C for 2 hours. LC-MS (EW47929-30-P1A1) result showed compound SM1 was consumed completely and the desired mass was detected. The reaction mixture was diluted with 30.0 mL of H2O and extracted with 40.0 mL of EA (20.0 mL*2). The combined organic layer was washed with 30.0 mL of brine (30.0 mL*1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 20 / 1 to 5 / 1, TLC, PE:EA = 3:1, Rf = 0.22). Compound 2 (1.18 g, 3.73 mmol, yield: 19.7%) was obtained as a yellow solid. f =0.22). Compound 2 (1.18 g, 3.73 mmol, yield: 19.7%) was obtained as a yellow solid.

[0195] 1-4-B. Preparation of compound 3 [Scheme 20] Cs2CO3 (1.36 g, 4.17 mmol, 1.20 eq) and TMSCN (414.1 mg, 4.17 mol, 522 μL, 1.20 eq) were added to a solution obtained by dissolving compound 2 (1.1 g, 3.48 mmol, 1.00 eq) in CAN (16.0 mL). The mixture was stirred at 25 °C for 12 hours. LC-MS analysis result showed compound 2 was consumed completely and the desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was diluted with 20.0 mL of H2O and extracted with 40.0 mL of EA (20.0 mL*2). The combined organic layer was washed with 20.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 5 / 1 to 3 / 1, PE:EA = 5:1, Rf = 0.47). Compound 3 (825 mg, 3.08 mmol, yield: 88.6%, purity: 98.0%) was obtained as a yellow solid. f =0.47). Compound 3 (825 mg, 3.08 mmol, yield: 88.6%, purity: 98.0%) was obtained as a yellow solid. f =0.47). Compound 3 (825 mg, 3.08 mmol, yield: 88.6%, purity: 98.0%) was obtained as a yellow solid.

[0196] 1-4-C. Preparation of compound 4 [Scheme 21] Compound 3 (825 mg, 3.15 mmol, 1.00 eq) and Raney Ni (26.9 mg, 314 pmol, 0.10 eq) were mixed in toluene (8.00 mL), the resulting mixture was treated under vacuum, purged with N2three times, then purged with H2several times under vacuum. The mixture was stirred at 80 °C under H2(40 psi) atmosphere for 12 hours. LC-MS results showed that compound 3 was completely consumed and the desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. Thereafter, the residue was purified by preparative HPLC (FA condition; column: Phenomenex luna C18 150*40 mm*15 pm; mobile phase: [water (FA)-ACN]; gradient: 8% to 38% B in 15 minutes). Compound 4 (300 mg, 1.10 mmol, yield: 34.9%, purity: 97.5%) was obtained as a white solid, which was confirmed by HNMR and LC-MS.

[0197] 1-4-D. Preparation of compound 5 [Scheme 22] Compound 4a (175 mg, 1.13 mmol, 1.00 eq) was added to a solution obtained by dissolving compound 4 (300 mg, 1.13 mol, 1.00 mL) in 5.00 mL of ethanol (EtOH). The mixture was stirred at 25 °C for one hour under N2atmosphere. Thereafter, NaBH3CN (42.4 mg, 675 pmol, 0.60 eq) and AcOH (67.6 mg, 1.13 mmol, 64.4 pL, 1.00 eq) were added at 25 °C and stirred for one hour under N2atmosphere. LC-MS results showed that 2% of compound 4 was completely consumed and the desired mass was detected. The residue was diluted with 10 mL of H2O and extracted with 20 mL of EA (10 mL*2). The organic phase was combined, washed with 10 mL of brine (10 mL*1), dried over Na2S04, filtered, and then concentrated under reduced pressure to obtain a residue. Thereafter, the residue was purified by column chromatography (Si02, DCM / MeOH=20 / 1 to 15 / 1), TLC, DCM:MeOH=10:1, Rf=0.46). Compound 5 (293 mg, 647 pmol, yield: 57.4%, purity: 100%, FA) was obtained as a white solid, which was confirmed by LC-MS and HNMR. f =0.46). Compound 5 (293 mg, 647 pmol, yield: 57.4%, purity: 100%, FA) was obtained as a white solid, which was confirmed by LC-MS and HNMR.

[0198] 1-4-E. Preparation of AON-MG23-08 [Scheme 23] Compound 5b (99.7 mg, 1.23 mmol, 48.5 μL, 5.00 eq) and H2O (1.50 mL) were added to a solution obtained by dissolving compound 5 (100 mg, 245 μmol, 1.00 eq) in AcOH (0.30 mL). The mixture was stirred at 25 °C for one hour. LC-MS analysis showed about 10% of compound 5 remained and the desired mass was detected. The reaction was stopped by slow addition of saturated sodium hydroxide solution until the pH reached 8, then the mixture was extracted with 40 mL of DCM (20 mL*2). The combined organic layers were washed with 40.0 mL of brine (20.0 mL*2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by preparative TLC (SiO2, DCM:MeOH=15:1). The residue was purified by preparative HPLC (basic condition; column: Waters Xbridge 150*25 mm*5 um; mobile phase: [water (ammonia v / v)-ACN]; gradient: 25% to 55% B in 10 minutes). AON-MG23-08 (target D) was obtained as a white solid (11.0 mg, 24.4 μmol, yield: 95%, purity: 100%).

[0199] The H NMR data of AON-MG23-08 is shown in Figure 14.

[0200] Derivatives of AON-MG23-02 were prepared in the same way as described above, and their structural formulas are shown in Table 2 below.

[0201] [Table 2] 2. Comparison of the inhibitory effects of four derivatives of AON-MG23-02 on ANO1 and EGFR protein expression in a glioblastoma cell line The inhibitory effects of AON-MG23-02 derivatives AON-MG23-05, AON-MG23-06, AON-MG23-07 and AON-MG23-08 on ANO1 and EGFR protein expression in a glioblastoma cell line were compared. Human glioblastoma cell line U87-MG was treated with 100X10 4Cells were seeded at a density of one cell per dish in 60 mm dishes and cultured for 24 hours. Thereafter, cells were treated with 0, 8, 16 and 32 mM of AON-MG23-05, AON-MG23-06, AON-MG23-07 and AON-MG23-08 derivatives, respectively, and cultured for 48 hours. The cultured cells were harvested and lysed for Western blotting. Anti-ANO1 antibody (Abeam, ab53212), anti-pEGFR antibody (Cell Signaling, #4407S), anti-EGFR antibody (Cell Signaling, #4267S) and anti-ACTIN antibody (sigma, #A2066) were used as antibodies for Western blotting. The protein concentration was quantified using the BSA method (Protein Quantitation Assay, Bovine Serum Albumin Assay) (Pierce, Cat. 23225). After cell lysis, an equal amount (10 pg) of protein obtained from the lysed cells was subjected to 10% SDS-PAGE, the proteins were separated according to their molecular weight, transferred to a PVDF membrane (Bio-rad), and treated with a blocking buffer (5% skim milk in Tris-buffered saline (TBS) buffer containing 0.1% Tween 20, TBS-T) at room temperature for one hour. Then, the PVDF membrane was treated with a primary antibody and reacted at 4°C for 16 hours. The reacted PVDF membrane was washed with TBS-T three times for 10 minutes. Then, the PVDF membrane was reacted with a horseradish peroxidase-labeled secondary antibody at room temperature for one hour, and then washed with TBS-T three times for 10 minutes. The washed PVDF membrane was treated with an ECL kit (Thermo, West PicoPlus), reacted, and visualized using a da Vinci-Q (Youngin Lab Plus) device. The amount of each protein was quantified and analyzed using Image J software.

[0202] As a result, as shown in FIGS. 9A to 9D, it was confirmed that the derivatives AON-MG23-05 and AON-MG23-08 reduced the expression of ANO1 and EGFR proteins compared to the untreated control.

[0203] 3. Comparison of the metastasis inhibitory effects of four derivatives of AON-MG23-02 on human glioblastoma The human glioblastoma cell line U87-MG was seeded at 8 Х 10 4The cells were seeded at a density of 1 cell / well on the top of Matrigel-coated filters (Transwell invasion chamber, Corning), then treated with 0, 8, 16 and 32 μM of the inventive compound derivatives AON-MG23-05, AON-MG23-06, AON-MG23-07 and AON-MG23-08, respectively, and incubated in a CO2incubator at 37°C for 24 hours. After completion of the incubation, the cells were fixed and stained using a Diff-Quick staining kit (Sysmex, Kobe, Japan). Images of the stained cells were taken under a microscope, the cells were counted using Image J software, and then statistical analysis was performed.

[0204] As shown in FIGS. 10A and 10B, it can be seen that the groups treated with the derivatives AON-MG23-05, AON-MG23-06, AON-MK23-07 and AON-MG23-08 showed a reduced degree of penetration of Matrigel compared to the untreated control, indicating that the derivatives inhibited the metastasis of human glioblastoma.

[0205] The above description of the present application is for illustrative purposes only, and those of ordinary skill in the art to which the present application pertains will understand that various modifications can be made in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the above-described embodiments should be considered illustrative in all aspects and not restrictive.

[0206] [Industrial applicability] The present application relates to the use of novel Anoctamin 1 (ANO1) inhibiting compounds for the prevention and treatment of brain tumors. The compounds of the present application are capable of simultaneously inhibiting ANO1 and EGFR in brain tumor cells, and not only exhibit a more effective anticancer effect through the dual inhibition of these two proteins, but also enhance the efficacy of anticancer agents such as anticancer drugs targeting EGFR and inhibit drug resistance when used in combination. Therefore, these compounds can be used as dual-target anticancer agents against ANO1 and EGFR on their own, and as combination agents for EGFR-targeted therapy, and thus are expected to be useful for various applications for the prevention and treatment of brain tumors and have industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating a brain tumor, comprising at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6, or a pharmaceutically acceptable salt thereof, as an active ingredient: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 2.The pharmaceutical composition according to claim 1, wherein the brain tumor is associated with at least one mutation selected from the group consisting of EGFR (epidermal growth factor receptor) and ANO1 (Anoctamin 1). 3.The pharmaceutical composition according to claim 1, wherein the compound or the pharmaceutically acceptable salt thereof simultaneously inhibits EGFR and ANO1. 4.The pharmaceutical composition according to claim 1, wherein the compound or the pharmaceutically acceptable salt thereof satisfies at least one property selected from the group consisting of: (a) inhibiting tumor metastasis; and (b) reducing drug resistance of cancer to an anticancer agent. 5.The pharmaceutical composition according to claim 4, wherein the anticancer agent is a targeted anticancer agent against tyrosine kinase. 6.The pharmaceutical composition according to claim 5, wherein the tyrosine kinase is one or more selected from the group consisting of EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (B-Raf proto-oncogene), HER2 (human epidermal growth factor receptor 2), RET (RET proto-oncogene), NTRK1 (neurotrophic receptor tyrosine kinase 1), MET (mesenchymal epithelial transition factor), and NRG1 (neuregulin 1). 7.The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered in combination with a targeted anticancer agent against tyrosine kinase. 。 8.The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is administered simultaneously, separately or sequentially with the targeted anticancer agent. 9.A pharmaceutical composition for preventing or treating a brain tumor, comprising the following as active ingredients: (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6, or a pharmaceutically acceptable salt thereof, and (ii) a targeted anticancer agent against EGFR [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 10.The pharmaceutical composition according to claim 9, wherein the compound or the pharmaceutically acceptable salt thereof inhibits drug resistance of a brain tumor cell to the targeted anticancer agent against EGFR. 11.The pharmaceutical composition according to claim 9, wherein the composition is in the form of a mixed preparation in which the compound or the pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR are mixed together. 12.The pharmaceutical composition according to claim 9, wherein the composition is in a form in which the compound or the pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR are separately formulated and administered simultaneously, separately or sequentially. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 。 ​ ​ ​ 13.A pharmaceutical composition for enhancing anticancer effect of a brain tumor anticancer agent, comprising at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof as an active ingredient: [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 。 14.The pharmaceutical composition of claim 13, wherein the brain tumor anticancer agent is a targeted anticancer agent against tyrosine kinase. 15.The pharmaceutical composition of claim 14, wherein the tyrosine kinase is one or more selected from the group consisting of EGFR (epidermal growth factor receptor), ALK (anaplastic lymphoma kinase), ROS1 (ROS proto-oncogene 1), BRAF (B-Raf proto-oncogene), HER2 (human epidermal growth factor receptor 2), RET (RET proto-oncogene), NTRK1 (neurotrophic receptor tyrosine kinase 1), MET (mesenchymal epithelial transition factor), and NRG1 (neuregulin 1). 16.The pharmaceutical composition of claim 13, wherein the composition is administered simultaneously, separately or sequentially with the brain tumor anticancer agent. 17.A method for preventing or treating a brain tumor, comprising administering to a subject in need at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, or a composition comprising the same as an active ingredient. 18.Use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, or a composition comprising the same as an active ingredient for preventing or treating a brain tumor. 19.Use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 for manufacturing a medicament for treating a brain tumor. 20.A method for preventing or treating a brain tumor, comprising administering to a subject in need (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anticancer agent against EGFR. 21.Use of a composition comprising, as active ingredients, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anticancer agent against EGFR for preventing or treating a brain tumor. 22.Use of a composition comprising, as active ingredients, (i) at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anticancer agent against EGFR for manufacturing a medicament for treating a brain tumor. 23.A method for enhancing anticancer effect of a brain tumor anticancer agent, comprising administering to a subject in need at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, or a composition comprising the same as an active ingredient.

24. Use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof, or a composition comprising the same as an active ingredient for enhancing anticancer effects of a brain tumor anticancer agent.

25. Use of at least one compound selected from the group consisting of compounds represented by Chemical Formulas 1 to 6 for manufacturing a medicament for enhancing anticancer effects of a brain tumor anticancer agent.

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