Novel compounds and methods for producing the same

Abstract

This invention relates to a novel anoctamin 1 (ANO1) inhibitory compound, and was completed after confirming that the novel compound can suppress brain tumors by inhibiting the expression and activity of ANO1, a calcium-dependent chloride channel. Therefore, this invention provides a novel compound that can be used as an ANO1 inhibitor. Furthermore, since the compound of this invention can simultaneously inhibit ANO1 and EGFR in brain tumor cells, it can be used as a dual-target anticancer agent for ANO1 and EGFR, and can also be used as a combination drug for EGFR-targeted therapy, so it is expected to be widely used in the field of brain tumor prevention and treatment.

Description

[Technical Field]

[0001] This invention relates to a novel compound that can simultaneously inhibit ANO1 (Anoctamin 1) and EGFR (epidermal growth factor receptor), and a method for producing the same.

[0002] This invention claims priority based on Korean Patent Application No. 10-2023-0073989, filed on June 9, 2023, and Korean Patent Application No. 10-2024-0074031, filed on June 5, 2024, and all contents disclosed in the specifications and drawings of the said applications are incorporated herein by reference. [Background technology]

[0003] Glioblastoma is a primary malignant brain tumor that occurs in adults. If left untreated after diagnosis, death occurs within 3 to 6 months, and even with all available treatments, the average survival time is only 12 to 14 months. It is an extremely aggressive disease. In South Korea, approximately 12,000 brain tumors are diagnosed each year. Of these, about 9,000 are benign tumors such as meningiomas or pituitary adenomas, and about 2,000 are diagnosed as malignant tumors. Of the malignant tumor patients each year, approximately 630 are diagnosed with glioblastoma.

[0004] Glioblastomas, which originate from glial cells in brain tissue, grow invasively into surrounding brain tissue, resulting in unclear tumor boundaries and a wider spread than what is visible to the naked eye during radiological examinations or surgery. Due to functional limitations of the brain, glioblastomas are often located in areas that cannot be surgically removed, and complete remission is not achieved with radiation therapy. Furthermore, the presence of the blood-brain barrier makes it difficult for anticancer drugs to penetrate the brain, resulting in a limited number of effective anticancer drugs and making glioblastoma an extremely difficult disease to treat with a poor prognosis. Currently, temozolomide is used as the basic treatment for glioblastoma, but long-term treatment leads to side effects and resistance, making continued treatment difficult. Moreover, despite many advances in modern medicine and the development of new drugs, no new treatments have yet emerged.

[0005] One of the oncogenes that frequently undergoes genetic mutations in glioblastoma patients is EGFR (epidermal growth factor receptor). EGFR has physiological importance, and mutations in EGFR are observed in more than half of patients. When EGF or TNF-α binds to EGFR, various signal transduction processes occur within the cell. However, overexpression or hyperactivation of EGFR induces excessive cell proliferation and other complications, ultimately leading to cancer. Among EGFR mutations, EGFRvIII is characterized by the deletion of amino acids 6 to 273 and is a protein specifically expressed only in cancer cells. EGFRvIII was first discovered in glioblastoma, but has also been confirmed in cancers of other tissues.

[0006] It has been reported that volume regulation of cancer cells by chloride ion channel proteins such as Anoctamin 1 (ANO1) and the CLC (chloride channel) family is important in the migration and metastasis of glioblastoma cells. The ANO1 protein is involved in various physiological functions (pain regulation, blood pressure regulation, brain nerve development, water secretion regulation, etc.), and its antitumor effects have been reported in particular in various cancer cells (breast cancer, head and neck cancer, prostate cancer, pancreatic cancer, lung cancer, etc.). Inhibition of the ANO1 protein in breast cancer produced an anticancer effect via CaMK2 signaling. Furthermore, while the expression level of ANO1 is remarkably low in the normal brain, the ANO1 protein is overexpressed in glioblastoma, and it has been reported that suppressing ANO1 activity inhibits glioblastoma cell migration and metastasis, and suppresses tumor growth in animal models. In addition, it has been reported that suppressing ANO1 in glioblastoma stem cells induces the degradation of epidermal growth factor receptor mutant 3 (EGFRvIII) protein. These findings suggest that inhibiting the ANO1 protein could be a therapeutic strategy for various EGFR mutation-related cancers, including glioblastoma.

[0007] Thus, both the ANO1 protein and EGFR have potential as targets for anti-cancer therapy. However, to date, no substance has been found that can exert a superior antitumor effect through the dual inhibition of both the ANO1 protein and EGFR. [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention relates to a novel anoctamin 1 (ANO1) inhibitory compound and a method for producing the same. The invention was completed after confirming that the compound simultaneously inhibits ANO1, a calcium-dependent chloride channel, and EGFR, a tyrosine kinase, thereby exhibiting excellent anticancer effects.

[0009] Therefore, the object of the present invention is to provide one or more compounds selected from the group consisting of compounds represented by the following chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof.

[0010] [ka] JPEG2026522325000003.jpg121111

[0011] Another object of the present invention is to provide a method for producing the compounds represented by the chemical formulas 1 to 6.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain tumors, comprising as an active ingredient one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0013] A further object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain tumors, comprising (i) one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent against EGFR as an active ingredient.

[0014] Still another object of the present invention is to provide a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent for brain tumors, which contains, as an active ingredient, one or more compounds selected from the group consisting of the compounds represented by the following Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0015] However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those having ordinary knowledge in the technical field to which the present invention pertains from the following description.

Means for Solving the Problems

[0016] To achieve the above object, the present invention provides one or more compounds selected from the group consisting of the compounds represented by the following Chemical Formulas 1 to 6 or a pharmaceutically acceptable salt thereof:

[0017]

Chem.

[0018] Further, the present invention includes (S1A) a step of reacting a compound represented by the following Chemical Formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to produce a compound represented by the following Chemical Formula 8; or

[0019] (S1B) a step of reacting a compound represented by the following Chemical Formula 9 with a compound represented by the following Chemical Formula 10 or Chemical Formula 11 in the presence of a base, and provides a method for producing the compound represented by the above Chemical Formulas 1 to 6:

[0020]

Chem.

[0021] In one embodiment of the present invention, step (S1A) may further include, but is not limited to, the following steps:

[0022] (S1A-1) A step of reducing the compound represented by chemical formula 8 (or its nitro group) by treating it with zinc in the presence of ammonium chloride to produce the compound represented by the following chemical formula 12;

[0023] (S1A-2) A step of reacting the compound represented by chemical formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to produce the compound represented by the following chemical formula 13; and

[0024] (S1A-3) A step of reacting the compound represented by chemical formula 13 with the compound represented by chemical formula 14 or chemical formula 15 in the presence of an acid to produce the compound represented by chemical formula 1 or chemical formula 2.

[0025] [ka] JPEG2026522325000009.jpg49113

[0026] In other embodiments of the present invention, if the compound represented by chemical formula 9 and the compound represented by chemical formula 10 react in the presence of a base in step (S1B), a compound represented by the following chemical formula 16 may be produced, but is not limited thereto.

[0027] [ka]

[0028] In yet another embodiment of the present invention, step (S1B) may further include, but is not limited to, the following steps:

[0029] (S1B-1) A step of reacting the compound represented by chemical formula 16 with trimethylsilyl cyanide in the presence of a base to produce the compound represented by the following chemical formula 17;

[0030] (S1B-2) A step of producing a compound represented by the following chemical formula 18 by hydrogenating the compound represented by chemical formula 17 in the presence of a metal catalyst;

[0031] (S1B-3) A step of reacting the compound represented by the above chemical formula 18 with the compound represented by the following chemical formula 19 in the presence of a reducing agent to produce the compound represented by the following chemical formula 20; and

[0032] (S1B-4) The compound represented by the above chemical formula 20 and NCO - A step of reacting the above to produce a compound represented by chemical formula 6; or a step of reacting the compound represented by chemical formula 20 with the compound represented by the following chemical formula 21 in the presence of a base to produce a compound represented by the following chemical formula 22.

[0033] [ka] JPEG2026522325000012.jpg48118

[0034] In yet another embodiment of the present invention, step (S1B-4) may further include, but is not limited to, a step of hydrolyzing the compound represented by chemical formula 22 in the presence of an acid to produce the compound represented by chemical formula 3.

[0035] In yet another embodiment of the present invention, the metal catalyst may be one or more selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney nickel (Raney-Ni).

[0036] In yet another embodiment of the present invention, if the compound represented by chemical formula 9 and the compound represented by chemical formula 11 react in the presence of a base in step (S1B), a compound represented by the following chemical formula 23 may be produced, but is not limited thereto.

[0037] [ka]

[0038] In yet another embodiment of the present invention, step (S1B) may further include, but is not limited to, the following steps.

[0039] (S1Ba-1) A step of reacting the compound represented by the chemical formula 23 with a peroxide to produce the compound represented by the chemical formula 24 below, and

[0040] (S1Ba-2) A step of reacting the compound represented by the above chemical formula 24 with the compound represented by the following chemical formula 25 in the presence of a base to produce the compound represented by the above chemical formula 4; or

[0041] (S1Bb-1) A step of phosphorylating the compound represented by the above chemical formula 23 in the presence of a base to produce the compound represented by the following chemical formula 26, and

[0042] (S1Bb-2) A step of reacting the compound represented by chemical formula 26 with the compound represented by the following chemical formula 27 in the presence of a base to produce the compound represented by chemical formula 5.

[0043] [ka]

[0044] In yet another embodiment of the present invention, the peroxide may be one or more selected from the group consisting of m-CPBA (meta-chloroperoxybenzoic acid), H2O2, DMDO (dimethyldioxirane), and oxone, but is not limited thereto.

[0045] In yet another embodiment of the present invention, the compound represented by chemical formula 25 can be produced by reacting the compound represented by the following chemical formula 28 with ethylamine in the presence of a reducing agent, but is not limited thereto.

[0046] [ka]

[0047] In yet another embodiment of the present invention, the reducing agent may be one or more selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium hydrogen boride (NaBH4), but is not limited thereto.

[0048] In yet another embodiment of the present invention, the acid may 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).

[0049] In yet another embodiment of the present invention, the base may be one or more selected from the group consisting of potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), cupric 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.

[0050] In yet another embodiment of the present invention, the production method may consist of one or more solvents selected from the group consisting of organic solvents, water, and mixtures thereof, but is not limited thereto.

[0051] In yet another embodiment of the present invention, the organic solvent may 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), hexafluoroisopropanol (HFIP), acetonitrile (ACN), dimethylformamide (DMF), nitromethane, trimethoxymethane (CH(OMe)3), acetic acid, isopropanol (IPA), and chloroform, but is not limited thereto.

[0052] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of brain tumors, comprising one or more compounds selected from the group consisting of compounds represented by the above chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0053] Furthermore, the present invention provides a method for preventing or treating brain tumors, comprising the step of administering to an individual in need one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof, or a composition containing the same as an active ingredient.

[0054] Furthermore, the present invention provides for the prevention or treatment of brain tumors one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6, or pharmaceutically acceptable salts thereof, or compositions containing the same as an active ingredient.

[0055] Furthermore, the present invention provides applications for one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 for the manufacture of drugs for the treatment of brain tumors.

[0056] In one embodiment of the present invention, the brain tumor may be, but is not limited to, a brain tumor associated with one or more mutations selected from the group consisting of EGFR (Epidermal growth factor receptor) and ANO1 (Anoctamin 1).

[0057] In other embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof can suppress EGFR and ANO1 simultaneously (e.g., inhibition of expression and / or activity). In other words, the compound or a pharmaceutically acceptable salt thereof may be a dual inhibitor targeting both EGFR and ANO1.

[0058] In yet another embodiment of the present invention, the compound or a pharmaceutically acceptable salt thereof may satisfy one or more properties selected from the group consisting of: (a) suppressing tumor metastasis; and (b) Suppress cancer resistance to anticancer drugs.

[0059] In yet another embodiment of the present invention, the anticancer agent may be, but is not limited to, a targeted anticancer agent against tyrosine kinase.

[0060] In yet another embodiment of the present invention, the tyrosine kinase may 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.

[0061] In yet another embodiment of the present invention, the pharmaceutical composition may be administered in combination with a targeted anticancer agent against tyrosine kinase, but is not limited thereto.

[0062] In yet another embodiment of the present invention, the pharmaceutical composition may be administered simultaneously with, separately from, or sequentially with the targeted anticancer agent, but is not limited thereto.

[0063] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of brain tumors, comprising (i) one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent against EGFR as an active ingredient.

[0064] Furthermore, the present invention provides a method for preventing or treating brain tumors, comprising the steps of (i) administering one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) administering a targeted anticancer agent against EGFR to an individual in need thereof.

[0065] Furthermore, the present invention provides for the prevention or treatment of brain tumors a composition comprising (i) one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof, and (ii) a targeted anticancer agent against EGFR as an active ingredient.

[0066] Furthermore, the present invention provides for the use of a composition for the manufacture of a drug for the treatment of brain tumors, comprising (i) one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent against EGFR as an active ingredient.

[0067] In one embodiment of the present invention, the compound or a pharmaceutically acceptable salt thereof can suppress the resistance of brain tumor cells to targeted anticancer agents against EGFR, but is not limited to this.

[0068] In other embodiments of the present invention, the composition may be in the form of a mixture comprising the compound or a pharmaceutically acceptable salt thereof, and a targeted anticancer agent against EGFR.

[0069] In yet another embodiment of the present invention, the composition may be formulated in a manner in which the compound or a pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR are administered simultaneously, separately, or sequentially.

[0070] Furthermore, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of a brain tumor anticancer agent, comprising one or more compounds selected from the group consisting of compounds represented by the above chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0071] Furthermore, the present invention provides a method for enhancing the anticancer effect of a brain tumor anticancer agent, comprising the step of administering to an individual requiring the use of one or more compounds selected from the group consisting of compounds represented by the above chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof, or a composition containing the same as an active ingredient.

[0072] Furthermore, the present invention provides a method for enhancing the anticancer effect of brain tumor anticancer agents using one or more compounds selected from the group consisting of compounds represented by the above chemical formulas 1 to 6, or pharmaceutically acceptable salts thereof, or compositions containing the same as an active ingredient.

[0073] Furthermore, the present invention provides uses for one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6, or pharmaceutically acceptable salts thereof, for the production of formulations for enhancing the anticancer effect of brain tumor anticancer agents.

[0074] Furthermore, the present invention provides the use of one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof for the production of resistance inhibitors for brain tumor cells to brain tumor anticancer drugs.

[0075] In one embodiment of the present invention, the brain tumor anticancer agent may be, but is not limited to, a targeted anticancer agent against tyrosine kinase.

[0076] In other embodiments of the present invention, the tyrosine kinase may 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.

[0077] In yet another embodiment of the present invention, the composition may be administered simultaneously with, separately from, or sequentially with the brain tumor anticancer agent, but is not limited thereto. [Effects of the Invention]

[0078] This invention relates to a novel anoctamin 1 (ANO1) inhibitory compound, and was completed after confirming that the novel compound can suppress brain tumors by inhibiting the expression and activity of ANO1, a calcium-dependent chloride channel.

[0079] Therefore, the present invention provides a novel compound that can be used as an ANO1 inhibitor. Furthermore, since the compound of the present invention can simultaneously suppress ANO1 and EGFR in brain tumor cells, it can be used as a dual-target anticancer agent for ANO1 and EGFR, and can also be used as a combination formulation for EGFR-targeted therapy. Thus, it is expected to be widely used in the field of brain tumor prevention and treatment. [Brief explanation of the drawing]

[0080] [Figure 1]Figure 1 shows the names, structural formulas, and basic information of compounds AON-MG23-01 and AON-MG23-02 according to the present invention.

[0081] [Figure 2] Figure 2 shows the results of a CCK assay performed on human glioblastoma cell lines after treating them with compounds AON-MG23-01 or AON-MG23-02 of the present invention at various concentrations to confirm the cell growth inhibitory effect of compounds AON-MG23-01 and AON-MG23-02 on human glioblastoma cells.

[0082] [Figure 3] Figure 3 shows the results of analyzing the degree of cell migration over time after treating human glioblastoma cell lines with compounds AON-MG23-01 and AON-MG23-02 of the present invention at various concentrations, in order to confirm the cell migration inhibitory effect of compounds AON-MG23-01 and AON-MG23-02 of the present invention on human glioblastoma cells.

[0083] [Figure 4] Figure 4 shows the results of analyzing the degree of Matrigel penetration of human glioblastoma cell lines cultured on Matrigel after being treated with AON-MG23-01 or AON-MG23-02 at various concentrations to confirm the metastatic inhibitory effect of the compounds AON-MG23-01 and AON-MG23-02 of the present invention on glioblastoma.

[0084] [Figure 5] Figure 5 shows the results of Western blotting performed on human glioblastoma cell lines after treating them with compounds AON-MG23-01 or AON-MG23-02 at various concentrations to confirm the effects of compounds AON-MG23-01 and AON-MG23-02 on EGFR and ANO1 protein expression in glioblastoma. The left side shows images of protein bands detected by Western blotting, and the right side shows the quantified levels of EGFR and ANO1 protein.

[0085] [Figures 6a-6b] Figures 6a and 6b show the results of a whole-cell patch-clamp experiment conducted on human glioblastoma cell lines treated with either AON-MG23-01 or AON-MG23-02 of the present invention to confirm whether the compounds AON-MG23-01 and AON-MG23-02 of the present invention suppress ANO1 activity in glioblastoma. Figure 6a shows the results of measuring the current of calcium-dependent chloride ion channels from -100mV to +100mV, and Figure 6b shows the current / voltage (I / V) plot at +80mV in Figure 6a.

[0086] [Figures 7a-7b] Figures 7a and 7b show the results of comparing the metastatic inhibitory effects of the compound AON-MG23-02 of the present invention with EGFR inhibitors and ANO1 inhibitors in human glioblastoma.

[0087] [Figures 8a-8b] Figures 8a and 8b show the results of comparing the inhibitory effects of the compound AON-MG23-02 of the present invention with an EGFR inhibitor and an ANO1 inhibitor on ANO1, EGFR, and pEGFR protein expression in glioblastoma cell lines.

[0088] [Figure 9a-d] Figures 9a to 9d show the results of Western blotting after treating human glioblastoma cell lines with AON-MG23-05, AON-MG23-06, AON-MG23-07, or AON-MG23-08, derivatives of the compound AON-MG23-02 of the present invention, at various concentrations, in order to confirm the effects of these derivatives on EGFR and ANO1 protein expression in glioblastoma. Figure 9a shows the results of Western blotting after treating with AON-MG23-05, Figure 9b shows the results of Western blotting after treating with AON-MG23-06, Figure 9c shows the results of Western blotting after treating with AON-MG23-07, and Figure 9d shows the results of Western blotting after treating with AON-MG23-08.

[0089] [Figures 10a-10b] Figures 10a and 10b show the results of analyzing the degree of Matrigel penetration of human glioblastoma cell lines cultured on Matrigel after being treated with AON-MG23-05, AON-MG23-06, AON-MG23-07, or AON-MG23-08, derivatives of the compound AON-MG23-02 of the present invention, at various concentrations, in order to confirm the glioblastoma metastasis inhibitory effect of these derivatives.

[0090] [Figure 11] Figure 11 shows the 1H NMR data of AON-MG23-05, a derivative of the compound AON-MG23-02 of the present invention.

[0091] [Figure 12] Figure 12 shows the 1H NMR data of AON-MG23-06, a derivative of the compound AON-MG23-02 of the present invention.

[0092] [Figure 13] Figure 13 shows the 1H NMR data of AON-MG23-07, a derivative of the compound AON-MG23-02 of the present invention.

[0093] [Figure 14] Figure 14 shows the 1H NMR data of AON-MG23-08, a derivative of the compound AON-MG23-02 of the present invention. [Modes for carrying out the invention]

[0094] This invention relates to a novel compound having an anoctamin 1 (ANO1) inhibitory effect, and was completed after confirming that the compound inhibits the expression and activity of ANO1, a calcium-dependent chloride channel, and suppresses the growth and metastasis of brain tumor cells. In particular, it has been confirmed that the compound of this invention effectively inhibits EGFR expression along with ANO1 in brain tumor cells, and is expected to exhibit an even stronger anticancer effect compared to conventional ANO1 or EGFR mono-inhibitors, and consequently, is expected to suppress the development of resistance to EGFR-targeted therapeutic agents in brain tumor cells.

[0095] Therefore, the object of the present invention is to provide one or more compounds selected from the group consisting of compounds represented by the following chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof.

[0096] [ka] JPEG2026522325000017.jpg121120

[0097] In the present invention, the compound represented by chemical formula 1 may be referred to as "N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazine-1-yl)piperidine-1-yl)phenyl)amino)pyrimidine-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide" or "AON-MG23-01". The compound represented by chemical formula 2 may be referred to as "N-(2-((5-chloro-2-((4-(4-(dimethylamino)piperidine-1-yl)-2-methoxyphenyl)amino)pyrimidine-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide" or "AON-MG23-02".

[0098] As used herein, the term "Ms" refers to methylsulfonyl, i.e., -SO2(CH3).

[0099] Furthermore, the compound represented by the chemical formula 3 may be referred to as "2-(4-(2-((2-hydroxyethyl)(naphthalen-2-ylmethyl)amino)ethyl)benzyl)isoindolin-1-one(2-(4-(2-((2-hydroxyethyl)(naphthalen-2-ylmethyl)amino)ethyl)benzyl)isoindolin-1-one)", "AON-MG23-05", or "Target A".

[0100] The compound represented by the aforementioned chemical formula 4 may be referred to as "2-(4-(2-(ethyl(naphthalen-2-ylmethyl)amino)-1-hydroxyethyl)benzyl)isoindolin-1-one (2-(4-(2-(ethyl(naphthalen-2-ylmethyl)amino)-1-hydroxyethyl)benzyl)isoindolin-1-one)", "AON-MG23-06", or "Target B".

[0101] The compound represented by the aforementioned chemical formula 5 may be referred to as "2-(4-(2-(bis(naphthalen-2-ylmethyl)phosphoryl)ethyl)benzyl)isoindolin-1-one(2-(4-(2-(bis(naphthalen-2-ylmethyl)phosphoryl)ethyl)benzyl)isoindolin-1-one)", "AON-MG23-07", or "Target C".

[0102] The compound represented by the aforementioned chemical formula 6 may be referred to as "1-(naphthalen-2-ylmethyl)-1-(4-((1-oxoisoindolin-2-yl)methyl)phenethyl)urea", "AON-MG23-08", or "Target D".

[0103] Unless otherwise specified, the terms "compounds of the present invention" or "compounds represented by chemical formulas 1 to 28" are used to encompass the compounds represented by chemical formulas 1 to 28 themselves, their salts, and their isomers.

[0104] As used in this invention, the term "pharmaceutically acceptable salt" includes all pharmaceutically acceptable inorganic acids, organic acids, or salts derived from bases.

[0105] As used herein, the term "pharmaceutically acceptable" means a compound or composition that is suitable for use in contact with the tissues of an organism (e.g., human) without excessive toxicity, irritation, allergic reactions, or other problems or complications, has a reasonable gain / risk ratio, is suitable for use in contact with the tissues of an organism (e.g., human), and is within the bounds of sound medical judgment.

[0106] Examples of suitable acids include hydrochloric acid, bromate, sulfuric acid, nitric acid, perchloric acid, hydroiodic acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, (+)-L-tartaric acid, di-L-tartaric acid, acetic acid, trichloroacetic acid or trifluoroacetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camhorsulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, galactaric acid, and glycerin. Examples include anthidic acid, glucoheptanoic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, α-oxo-glutaric 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, disoxalic acid, palmitic acid, palmic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, tannic acid, thiocyanic acid, camsilic acid, and undecylic acid. Acid addition salts can be produced by conventional methods, for example, by dissolving a compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Alternatively, they can be produced by heating an equal molar amount of the compound and an acid or alcohol in water, then evaporating and drying the mixture, or by suction filtration of the precipitated salt.

[0107] The scope of compounds in the present invention may include not only pharmaceutically acceptable salts, but also all isomers, hydrates, and solvates that can be produced by conventional methods.

[0108] In this invention, the term "isomer" refers to compounds that have the same molecular formula but differ in the linkage and spatial arrangement of their constituent atoms. Isomers include, for example, structural isomers and stereoisomers. The stereoisomers may be diastereomers or enantiomers. Enantiomers are isomers that do not overlap with their mirror images, like the relationship between a left hand and a right hand, and are also called optical isomers. Enantiomers are classified into R (rectus: clockwise) and S (sinister: counterclockwise) when there are four or more substituents on the chiral central carbon that are different from each other. Diastereomers are stereoisomers that are not mirror images of each other and are divided into cis and trans, which have different spatial arrangements of atoms.

[0109] Furthermore, the present invention also includes the step of (S1A) reacting a compound represented by the following chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to produce a compound represented by the following chemical formula 8 (reaction formula 2); or (S1B) A method for producing compounds represented by chemical formulas 1 to 6 is provided, comprising the step of 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 formula 7 or 13):

[0110] [ka] JPEG2026522325000019.jpg65110

[0111] According to one embodiment of the present invention, the base in step (S1A) or step (S1B) may be Cs2CO3, but is not limited thereto.

[0112] According to one embodiment of the present invention, step (S1A) may further include, but is not limited to, the following steps: (S1A-1) A step in which the nitro group of the compound represented by chemical formula 8 is treated with zinc in the presence of ammonium chloride to reduce it and produce the compound represented by the following chemical formula 12 (reaction formula 3); (S1A-2) A step of reacting the compound represented by chemical formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to produce the compound represented by the following chemical formula 13 (reaction formula 4); and (S1A-3) A step of reacting the compound represented by chemical formula 13 with the compound represented by chemical formula 14 or chemical formula 15 in the presence of an acid to produce the compound represented by chemical formula 1 or chemical formula 2 (reaction formula 5 or 6).

[0113] [ka] JPEG2026522325000021.jpg48115

[0114] According to one embodiment of the present invention, when the compound represented by chemical formula 13 and the compound represented by chemical formula 14 are reacted in the presence of an acid in step (S1A-3), the compound represented by chemical formula 1 is produced (reaction formula 5), When the compound represented by chemical formula 13 and the compound represented by chemical formula 15 are reacted in the presence of an acid, the compound represented by chemical formula 2 may be produced (reaction formula 6). Specifically, step (S1A-3) may involve the compound represented by chemical formula 13 reacting with the amino group of the compound represented by chemical formula 14 or chemical formula 15.

[0115] According to one embodiment of the present invention, the base in step (S1A-2) may be DIPEA (N,N-diisopropylethylamine), and the acid in step (S1A-3) may be TFA (trifluoroacetic acid), but is not limited thereto.

[0116] In the present invention, when the compound represented by chemical formula 9 and the compound represented by chemical formula 10 react in the presence of a base in step (S1B), a compound represented by the following chemical formula 16 can be produced (reaction formula 7 or 19), but is not limited thereto. According to one embodiment of the present invention, the base may be Cs2CO3, but is not limited thereto.

[0117] [ka]

[0118] According to one embodiment of the present invention, step (S1B) may further include, but is not limited to, the following steps.

[0119] (S1B-1) A step to produce a compound represented by the following chemical formula 17 by reacting the compound represented by chemical formula 16 with trimethylsilyl cyanide in the presence of a base (reaction formula 8 or 20); (S1B-2) A step in which the compound represented by chemical formula 17 is subjected to a hydrogenation reaction in the presence of a metal catalyst to produce the compound represented by the following chemical formula 18 (reaction formula 9 or 21); (S1B-3) A step of reacting the compound represented by chemical formula 18 with the compound represented by the following chemical formula 19 in the presence of a reducing agent to produce the compound represented by the following chemical formula 20 (reaction formula 10 or 22); and (S1B-4) The compound represented by the above chemical formula 20 and NCO - A step of reacting to produce a compound represented by the chemical formula 6 (reaction formula 23); or a step of reacting the compound represented by the chemical formula 20 with the compound represented by the following chemical formula 21 in the presence of a base to produce a compound represented by the following chemical formula 22 (reaction formula 11). According to one embodiment of the present invention, the base in step (S1B-1) may be Cs2CO3, and the base in step (S1B-4) may be K2CO3, but is not limited thereto.

[0120] [ka] JPEG2026522325000024.jpg47115

[0121] In the present invention, step (S1B-4) may further include a step of hydrolyzing the compound represented by chemical formula 22 in the presence of an acid to produce the compound represented by chemical formula 3 (reaction formula 12), but is not limited thereto. According to one embodiment of the present invention, the acid in step (S1B-4) may be hydrogen chloride (HCl), but is not limited thereto.

[0122] In the present invention, the metal catalyst may 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 invention, the metal catalyst may be Raney nickel (Raney-Ni), but is not limited thereto.

[0123] In the present invention, the reducing agent may be one or more selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium hydrogen boride (NaBH4). According to one embodiment of the present invention, the reducing agent in step (S1B-3) may be sodium cyanoborohydride (NaBH3CN), but is not limited thereto.

[0124] In the present invention, when the compound represented by chemical formula 9 and the compound represented by chemical formula 11 react in the presence of a base in step (S1B), a compound represented by the following chemical formula 23 can be produced (reaction formula 13), but is not limited thereto.

[0125] [ka]

[0126] In the present invention, step (S1B) may further include, but is not limited to, the following steps: (S1Ba-1) A step of reacting the compound represented by the above chemical formula 23 with a peroxide to produce the compound represented by the following chemical formula 24 (reaction formula 14), and (S1Ba-2) A step of reacting the compound represented by chemical formula 24 with the compound represented by the following chemical formula 25 in the presence of a base to produce the compound represented by chemical formula 4 (reaction formula 16); or (S1Bb-1) A step of phosphorylating the compound represented by chemical formula 23 in the presence of a base to produce the compound represented by the following chemical formula 26, specifically a step of producing the compound represented by the following chemical formula 26 by a β-alkylstyrene phosphorylation reaction (reaction formula 17), and (S1Bb-2) A step in which the compound represented by chemical formula 26 and the compound represented by chemical formula 27 below are reacted in the presence of a base to produce the compound represented by chemical formula 5 (reaction formula 18).

[0127] According to one embodiment of the present invention, the base in step (S1Ba-2) may be DIEA (N,N-Diisopropylethylamine), the base in step (S1Bb-1) may be KOH, and the base in step (S1Bb-2) may be NaH, but is not limited thereto.

[0128] [ka]

[0129] In the present invention, the compound represented by chemical formula 25 can be produced by reacting the compound represented by the following chemical formula 28 with ethylamine in the presence of a reducing agent (reaction formula 15). According to one embodiment of the present invention, the reducing agent may be sodium triacetoxyburohydride (NaBH(OAc)3), but is not limited thereto.

[0130] [ka]

[0131] In the present invention, the peroxide may be one or more selected from the group consisting of m-CPBA (meta-chloroperoxybenzoic acid), hydrogen peroxide (H2O2), dimethyldioxirane (DMDO), and oxone. According to one embodiment of the present invention, the peroxide may be m-CPBA (meta-chloroperoxybenzoic acid), but is not limited thereto.

[0132] In the present invention, the acid may 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.

[0133] In the present invention, the base may be one or more selected from the group consisting of potassium carbonate (K2CO3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), cupric 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.

[0134] In the present invention, the production method may consist of one or more solvents selected from the group consisting of organic solvents, water, and mixtures thereof, and the organic solvent may 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), hexafluoroisopropanol (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.

[0135] Furthermore, the present invention provides a method for producing a compound represented by chemical formula 1, comprising the following steps: (a) A step of reacting the compound represented by chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to produce the compound represented by chemical formula 8 (reaction formula 2); (b) A step in which the nitro group of the compound represented by chemical formula 8 is reduced by treating it with zinc in the presence of ammonium chloride to produce the compound represented by chemical formula 12 (reaction formula 3); (c) A step of reacting the compound represented by chemical formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to produce the compound represented by the following chemical formula 13 (reaction formula 4); and (d) A step of reacting the compound represented by chemical formula 13 with the compound represented by the following chemical formula 14 in the presence of an acid to produce the compound represented by chemical formula 1 (reaction formula 5).

[0136] Furthermore, the present invention provides a method for producing a compound represented by chemical formula 2, comprising the following steps: (a) A step of reacting the compound represented by chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to produce the compound represented by chemical formula 8 (reaction formula 2); (b) A step in which the nitro group of the compound represented by chemical formula 8 is reduced by treating it with zinc in the presence of ammonium chloride to produce the compound represented by chemical formula 12 (reaction formula 3); (c) A step of reacting the compound represented by chemical formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to produce the compound represented by the following chemical formula 13 (reaction formula 4); and (d) A step of producing the compound represented by chemical formula 2 by reacting the compound represented by chemical formula 13 with the compound represented by chemical formula 15 in the presence of an acid (reaction formula 6).

[0137] Furthermore, the present invention provides a method for producing a compound represented by chemical formula 3, which includes the following steps: (a) A step of reacting the compound represented by chemical formula 9 with the compound represented by chemical formula 10 in the presence of a base to produce the compound represented by chemical formula 16 (reaction formula 7); (b) A step of reacting the compound represented by chemical formula 16 with trimethylsilyl cyanide in the presence of a base to produce the compound represented by chemical formula 17 (reaction formula 8); (c) A step of producing the compound represented by chemical formula 18 by hydrogenating the compound represented by chemical formula 17 in the presence of a metal catalyst (reaction formula 9); (d) A step of reacting the compound represented by chemical formula 18 with the compound represented by chemical formula 19 in the presence of a reducing agent to produce the compound represented by chemical formula 20 (reaction formula 10); (d) A step of reacting the compound represented by chemical formula 20 with the compound represented by chemical formula 21 in the presence of a base to produce the compound represented by chemical formula 22 (reaction formula 11); and (e) A step of producing the compound represented by chemical formula 3 by hydrolyzing the compound represented by chemical formula 22 in the presence of an acid (reaction formula 12).

[0138] Furthermore, the present invention provides a method for producing a compound represented by chemical formula 4, which includes the following steps: (a) A step of reacting the compound represented by chemical formula 9 with the compound represented by chemical formula 11 in the presence of a base to produce the compound represented by chemical formula 23 (reaction formula 13); (b) A step of reacting the compound represented by chemical formula 23 with a peroxide to produce the compound represented by chemical formula 24 (reaction formula 14); (c) A step of reacting the compound represented by chemical formula 24 and the compound represented by chemical formula 25 in the presence of a base to produce the compound represented by chemical formula 4 (reaction formula 16).

[0139] Furthermore, the present invention provides a method for producing a compound represented by chemical formula 5, which includes the following steps: (a) A step of reacting the compound represented by chemical formula 9 with the compound represented by chemical formula 11 in the presence of a base to produce the compound represented by chemical formula 23 (reaction formula 13); (b) A step of phosphorylating the compound represented by chemical formula 23 in the presence of a base to produce the compound represented by chemical formula 26, specifically a step of producing the compound represented by the following chemical formula 26 by a β-alkylstyrene phosphorylation reaction (reaction formula 17); and (c) A step of reacting the compound represented by chemical formula 26 with the compound represented by chemical formula 27 in the presence of a base to produce the compound represented by chemical formula 5 (reaction formula 18).

[0140] Furthermore, the present invention provides a method for producing a compound represented by chemical formula 6, which includes the following steps: (a) A step of reacting the compound represented by chemical formula 9 with the compound represented by chemical formula 10 in the presence of a base to produce the compound represented by chemical formula 16 (reaction formula 19); (b) A step of reacting the compound represented by chemical formula 16 with trimethylsilyl cyanide in the presence of a base to produce the compound represented by chemical formula 17 (reaction formula 20); (c) A step of producing the compound represented by chemical formula 18 by hydrogenating the compound represented by chemical formula 17 in the presence of a metal catalyst (reaction formula 21); (d) A step of reacting the compound represented by chemical formula 18 and the compound represented by chemical formula 19 in the presence of a reducing agent to produce the compound represented by chemical formula 20 (reaction formula 22); (d) The compound represented by the chemical formula 20 and NCO - A step in which the two are reacted to produce the compound represented by the chemical formula 6 (reaction formula 23).

[0141] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of brain tumors, comprising as an active ingredient one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0142] The compounds according to the present invention are inhibitors of the calcium-dependent chloride channel Anoctamine 1 (ANO1), and can be used for the prevention, improvement, and / or treatment of brain tumors. In particular, the compounds according to the present invention are characterized by exhibiting excellent anticancer effects by simultaneously suppressing the expression or activity of EGFR and ANO1. The aforementioned preventive and / or therapeutic effects on brain tumors include not only the effect of suppressing the growth of brain tumor cells, but also the effect of suppressing the progression and worsening of brain tumors due to migration, invasion, metastasis, etc.

[0143] In this invention, "EGFR (epidermal growth factor receptor)" is a transmembrane glycoprotein that is a member of the protein kinase superfamily and is a receptor for the epidermal growth factor family of proteins. EGFR is a major regulator of cell growth, and when its ligand, epidermal growth factor (EGF), binds to EGFR, the receptor undergoes dimerization and tyrosine autophosphorylation, inducing cell proliferation. Overexpression (amplification) or overactivity of EGFR is frequently observed in various cancers such as anal cancer, head and neck cancer, and glioblastoma, but EGFR-related somatic mutations cause persistent activation of EGFR and induce uncontrolled cell division. In particular, EGFR gene rearrangements and EGFR gene amplification are observed in various cancers. Among these, the most common extracellular domain mutation is EGFRvIII. EGFRvIII mutations are characterized by the deletion of over 801 base pairs in exons 2-7 of the EGFR gene, resulting in the expression of EGFR protein with 267 amino acids missing from the extracellular domain. Unlike normal EGFR, which is activated only when a ligand binds, the mutated EGFR maintains a persistently activated state (PCT gain of function mutation). EGFRvIII is frequently observed, particularly in glioblastoma.

[0144] In this invention, "Anoctamin 1 (ANO1)" is also called "transmembrane protein 16A (TMEM16A)," and is a calcium-activated chloride ion channel (Cl -ANO1 is a neurotransmitter channel. ANO1 is expressed in a variety of normal cells, including epithelial cells, smooth muscle cells, vascular endothelial cells, and neurons, and plays diverse physiological roles by regulating fluid secretion, muscle contraction, and pain sensation. However, ANO1 is known to be associated with cancer invasiveness and poor prognosis in brain tumors, including glioblastoma. In fact, it has been reported that overexpression of ANO1 promotes signaling pathways that stimulate proliferation and migration in cancer cells, and that suppression of ANO1 inhibits the migration and growth of cancer cells. High expression of ANO1 is also thought to be associated with high expression of EGFR and STAT3, and it is known that suppression of ANO1 can improve the response of head and neck squamous cell carcinoma to EGFR / HER2 targeted therapy. Therefore, ANO1 is attracting attention as a target for cancer treatment, but an ANO1 inhibitor that actually exhibits excellent anticancer effects has not yet been found. The compounds according to the present invention have been confirmed to effectively suppress ANO1 expression in brain tumor cells and to suppress the migration, proliferation, and metastasis of brain tumor cells more potently than conventionally known ANO1 inhibitors. Therefore, they can be used for the prevention, treatment, and suppression of metastasis of brain tumors. Furthermore, previous research on improving the efficacy of EGFR / HER2 targeted therapy by ANO1 suppression suggests that the compounds according to the present invention can enhance the responsiveness of brain tumor cells to EGFR / HER targeted therapy and suppress the development of resistance through their ANO1 inhibitory effect.

[0145] In particular, the compounds according to the present invention simultaneously suppress the expression of EGFR along with ANO1 in brain tumor cells, and can exert an even more potent anticancer effect through the simultaneous suppression of ANO1 and EGFR. The relationship between ANO1 and EGFR is well known, and it has been reported that upward regulation of ANO1 is related to the EGFR signaling pathway and has a significant impact on phosphorylated proteome remodeling after epidermal growth factor (EGF) stimulation. Furthermore, in cancer cells, ANO1 can form a functional complex with EGFR to jointly regulate cell proliferation. Therefore, by simultaneously suppressing EGFR and ANO1 using the compounds of the present invention, the growth of brain tumors can be inhibited even more effectively.

[0146] Furthermore, ANO1 is known to be overexpressed in cancer cells and contribute to cancer cell migration and tumor metastasis, and EGFR-mediated signaling is also known to generate a tumor microenvironment that is useful for tumor metastasis. Therefore, the compounds according to the present invention can effectively suppress brain tumor metastasis through the simultaneous suppression of ANO1 and EGFR. The term "metastasis" refers to the state in which a malignant tumor has spread to other tissues away from the organ in which it originated. Therefore, the compositions according to the present invention can suppress brain tumor metastasis and prevent and treat the spread of brain tumors throughout the body.

[0147] The compounds according to the present invention or their pharmaceutically acceptable salts can be used for various cancer prevention or treatment applications.

[0148] In this invention, "tumor" is used synonymously with "cancer" and refers to a condition typically characterized by uncontrolled cell growth, migration, and proliferation. Preferably, the cancer according to this invention is a brain tumor. The cancer according to this invention includes primary and recurrent cancers and encompasses all benign and malignant tumors. In this invention, the brain tumor is all tumors occurring within the skull and is not limited to specific types, but includes all tumors occurring in the brain and surrounding structures. In this invention, the brain tumor includes primary brain tumors, recurrent brain tumors, and metastatic brain tumors. Furthermore, the brain tumor includes, but is not limited to, astrocytomas, malignant astrocytomas, glioblastomas, meningiomas, pituitary adenomas, schwannomas, acoustic neuromas, and craniopharyngiomas.

[0149] In particular, the cancer according to the present invention may be a brain tumor that is expected to be improved or treated by simultaneously inhibiting ANO1 and EGFR. Preferably, the cancer according to the present invention may be a brain tumor associated with one or more mutations selected from the group consisting of EGFR (Epidermal growth factor receptor) and ANO1 (Anoctamin 1). That is, the cancer according to the present invention may be a brain tumor having mutations in EGFR and / or ANO1. The mutations in EGFR and / or ANO1 include amplification of the EGFR and / or ANO1 genes, overexpression of the protein, overactivity of the protein, and sustained activation of the protein. That is, the cancer according to the present invention may be a brain tumor in which the expression or activity of EGFR and / or ANO1 is even higher compared to normal cells. Alternatively, the cancer according to the present invention may be a brain tumor in which the activated state of EGFR and / or ANO1 persists. Since the compounds according to the present invention can simultaneously suppress the expression and activity of ANO1 and EGFR, they can exhibit particularly excellent anticancer effects against brain tumors accompanied by mutations in EGFR and / or ANO1.

[0150] Alternatively, the cancer according to the present invention may be a brain tumor accompanied by an EGFRvIII (epidermal growth factor receptor variant III) mutation. The EGFRvIII mutation is a mutation in which amino acids 6-273 of EGFR are deleted (or exons 2-7 are deleted), and is characterized by its ability to remain activated even without ligand (EGF) binding. EGFRvIII is specifically expressed only in cancer cells and is found in approximately 30% of glioblastomas.

[0151] The compounds according to the present invention can exert one or more effects from the group consisting of the following: (a) Suppress the proliferation or growth of brain tumor cells; (b) Suppress the migration of brain tumor cells; (c) Suppress the metastasis of brain tumors; (d) Suppressing resistance of brain tumors to anticancer drugs; and (e) It enhances the responsiveness of brain tumor cells to anticancer drugs.

[0152] Based on specific examples, the inventors confirmed that brain tumor cells treated with the compound according to the present invention exhibited suppressed proliferation, migration, and metastasis (penetration), demonstrating that the anticancer effect of the compound is more potent than that of conventionally known ANO1 inhibitors (CaCCinh-A01).

[0153] Furthermore, since the compounds according to the present invention can simultaneously inhibit ANO1 and EGFR (i.e., double inhibition), they can exhibit even better anticancer effects compared to ANO1 or EGFR inhibitors alone. In particular, they can suppress the development of resistance in cancer cells to anticancer drugs through ANO1 inhibition, thereby enhancing the anticancer effect of the anticancer drug. For example, the inventors have confirmed through experiments that the ANO1 inhibitory effect of the compounds according to the present invention is not only superior to that of the conventional ANO1 inhibitor CaCCinh-A01, but also that their anticancer effect is superior to that of the ANO1 inhibitor CaCCinh-A01, the EGFR inhibitor Osimertinib, and the combination of Osimertinib and CaCCinh-A01.

[0154] In one embodiment of the present invention, the anticancer agents described in (d) and (e) above may be, but are not limited to, targeted anticancer agents against tyrosine kinase. In the present invention, "targeted anticancer agent" means a formulation that exhibits an anticancer effect by targeting proteins or genes that are specifically altered in cancer cells or cancer tissue and interfering with molecular activities involved in cancer growth and development. Preferably, the tyrosine kinase may 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). More preferably, the tyrosine kinase may be EGFR. The aforementioned EGFR includes normal EGFR and mutant EGFR (e.g., EGFRvIII).

[0155] In (e) above, the statement that the responsiveness of cancer cells to anticancer drugs is enhanced means that the suppression of cancer cell growth and migration by anticancer drugs is further enhanced.

[0156] In this invention, "enhancing the anticancer effect" refers to all effects that can ultimately strengthen the function of the anticancer drug. This includes not only enhancing the anticancer effects of the anticancer drug, such as inhibiting tumor growth, inhibiting tumor metastasis, and inhibiting tumor recurrence, but also enhancing the anticancer effect by suppressing the resistance and tolerance formation of cancer cells to the anticancer drug.

[0157] Therefore, the compounds according to the present invention can be used as compounds for co-administration with known anticancer agents (preferably targeted anticancer agents against tyrosine kinases) for the purpose of enhancing the anticancer effect of brain tumor anticancer agents.

[0158] For example, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer (preferably brain tumors), comprising (i) one or more compounds selected from the group consisting of compounds represented by the chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent against EGFR as an active ingredient.

[0159] Furthermore, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent (preferably a brain tumor anticancer agent), comprising one or more compounds selected from the group consisting of compounds represented by the above chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0160] The composition according to the present invention is in the form of a mixture comprising the compound or a pharmaceutically acceptable salt thereof and a targeted anticancer agent against EGFR, and may also be in the form of a mixture for simultaneous administration of the compound or a pharmaceutically acceptable salt thereof and the targeted anticancer agent against EGFR.

[0161] Furthermore, the composition according to the present invention may be in the form of formulations containing the compound or a pharmaceutically acceptable salt thereof, and a targeted anticancer agent against EGFR, which are administered simultaneously, separately, or sequentially. In this case, the composition may be a combination-administered pharmaceutical composition for simultaneous or sequential administration, comprising 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 the case of sequential administration, the order of administration is not restricted, and the administration therapy can be appropriately adjusted according to the patient's condition, etc.

[0162] In other words, if the pharmaceutical composition is a pharmaceutical composition for combination administration for sequential administration, the composition can be administered such that the compound or a salt thereof ("first component") is administered first, followed by the targeted anticancer agent against EGFR ("second component"), and the reverse order is also possible.

[0163] Furthermore, when the compound according to the present invention is used in combination with an anticancer agent, the compound can be administered simultaneously, separately, or sequentially with the anticancer agent. Even when administered sequentially with the anticancer agent, the order of administration is not restricted, but the administration therapy can be appropriately adjusted depending on the type of cancer, the type of anticancer agent, the patient's condition, etc.

[0164] The content of the compound in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the progression of the symptoms, the patient's condition, etc. For example, it may 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 percentage of the content is a value based on the dry weight after removing the solvent.

[0165] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film coating substances, and release-controlled additives.

[0166] The pharmaceutical compositions according to the present invention can be formulated by conventional methods into dosage forms such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, elixirs, emulsions, suspensions, alcoholic preparations, lozenges, aromatic preparations, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, softened extracts, dried extracts, liquid extracts, injections, capsules, perfusion solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols for use as external preparations. The external preparations may have dosage forms such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasms.

[0167] Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0168] When formulating the product, it is prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients.

[0169] Excipients for tablets, powders, granules, capsules, pills, and lozenges according to the present invention include corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, monocalcium phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, iodine, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel, etc.; gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose Binders such as methylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropylcellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone can be used, as well as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, Disintegrants such as L-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium powder, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ethers, starch, sodium chloride, sodium acetate, sodium oleate, DL-leucine, and light anhydrous silicic acid can be used.

[0170] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamin, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, and the like can be used.

[0171] The syrup according to the present invention may contain a solution of sucrose, other sugars, or sweeteners, and may also contain fragrances, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc., as needed.

[0172] The emulsion according to the present invention can use purified water, and emulsifiers, preservatives, stabilizers, fragrances, etc., can be used as needed.

[0173] The suspending agent according to the present invention can be acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropyl methylcellulose (HPMC), HPMC1828, HPMC2906, HPMC2910, or other suspending agents, and surfactants, preservatives, stabilizers, colorants, and fragrances can be used as needed.

[0174] The injectable preparations according to the present invention include solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, Ringer's lactate injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, iodine, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nicotinamide, hexamine, and dimethylacetamide; weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate) It may also contain buffering agents such as organic compounds, proteins, albumin, peptones, and gums; isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfurizing agents such as 0.1% sodium bisulfite, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and sodium acetone bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Twin 80, and aluminum monostearate.

[0175] The suppositories according to the present invention include cocoa butter, lanolin, whitepsol, polyethylene glycol, glycerol gelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, twin or span, Imhausen, monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, and Cebes Pharma 16. 16) Hexalide Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote 25, Hydrokote 711, Idropostal, Massaestralium Bases such as estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Maspol, Maspol-15, Neospostal-N, Paramount-B, Sposilo (OSI, OSIX, A, B, C, D, H, L), Suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) can be used.

[0176] Solid formulations for oral administration include tablets, pills, powders, granules, and capsules. Such solid formulations are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose, or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0177] Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0178] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, “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 by factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field.

[0179] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or administered as a single or multiple agent. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person of ordinary skill in the art to which the present invention belongs.

[0180] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All possible methods of administration are conceivable, but may include, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, periarticular (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, etc.

[0181] The effective dose of the pharmaceutical composition of the present invention is determined by the type of active drug, along with various relevant factors such as the disease being treated, the route of administration, the patient's age, sex, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention can vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 doses per day. However, the dose can be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc., so the aforementioned dose does not limit the scope of the present invention in any way.

[0182] In this invention, "individual" means a subject requiring treatment for a disease, and more specifically, it means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0183] In the present invention, "administration" means providing a predetermined composition of the present invention to an individual by any appropriate method.

[0184] In the present invention, "prevention" means all actions that suppress or delay the onset of the target disease; "treatment" means all actions that improve or beneficially alter the target disease and the metabolic disorders caused thereby by administering the pharmaceutical composition according to the present invention; and "improvement" means all actions that reduce parameters related to the target disease, such as the severity of symptoms, by administering the composition according to the present invention.

[0185] Furthermore, the present invention provides a kit for the prevention or treatment of cancer, comprising a pharmaceutical composition according to the present invention.

[0186] The kit according to the present invention may, in addition to the compound and targeted anticancer agent, include, without limitation, other components, compositions, solutions, devices, etc., that are normally necessary for the prevention or treatment of cancer, and may particularly include instructions for the appropriate use and storage of the compound according to the present invention.

[0187] The terms and words used herein and in the claims should not be interpreted in a manner limited to their ordinary or dictionary meanings, but rather in a manner consistent with the technical idea of ​​the present invention, based on the principle that inventors may appropriately define the concepts of terms in order to best describe their invention.

[0188] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided only to facilitate understanding of the present invention and do not limit the scope of the present invention. [Examples]

[0189] [Examples] Example A. Novel compounds AON-MG23-01 and AON-MG23-02 1. Production of the compound according to the present invention 1-1.Manufacture 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)

[0190] [ka] JPEG2026522325000029.jpg175115

[0191] The AON-MG23-01 compound was prepared through the reaction equation 1 described above. The specific preparation method is as follows: <Step 1> Synthesis of N-cyclopropyl-N-(2-nitrophenyl)methanesulfonamide

[0192] [ka]

[0193] 1-Fluoro-2-nitrobenzene (1.00 eq) and cyclopropylamine (1.00 eq) were dissolved in acetonitrile (ACN), and methanesulfonyl chloride (MsCl, 1.0 eq) was slowly added at 0°C, followed by stirring at the same temperature for 1 hour. Then, cesium carbonate (Cs2CO3, 5.00 eq) was added at the same temperature, followed by reflux and stirring overnight. After the reaction was complete, the temperature was lowered to room temperature, and the mixture was extracted with ethyl acetate and water. The organic layer was stirred with anhydrous sodium sulfate, 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-nitrophenyl)methanesulfonamide as a pale yellow solid in 36% yield.

[0194] <Step 2> Synthesis of N-(2-aminophenyl)-N-cyclopropylmethanesulfonamide

[0195] [ka]

[0196] In Step 1, 1.00 eq of N-cyclopropyl-N-(2-nitrophenyl)methanesulfonamide was added to 1,4-dioxane and water (3:1). After stirring, the reactor was cooled to 0°C, and zinc (Zn, 10.0 eq) and ammonium chloride (NH4Cl, 10.0 eq) were added. The mixture was then stirred for 4 hours while gradually increasing the temperature. After the reaction was complete, the mixture was filtered using cellulite, and the filtrate was extracted with ethyl acetate and water. Anhydrous sodium sulfate was added to the organic layer and stirred, then filtered using a filter, and the filtrate was concentrated under reduced pressure. The concentrated residue was used in Step 3 without any further purification process.

[0197] <Step 3> Synthesis of N-cyclopropyl-N-(2-((2,5-dichloropyrimidine-4-yl)amino)phenyl)methanesulfonamide

[0198] [ka]

[0199] N-(2-aminophenyl)-N-cyclopropylmethanesulfonamide (1.00 eq) synthesized in Step 2 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. The mixture was then refluxed and stirred overnight. After the reaction was complete, the mixture was evaporated under reduced pressure and extracted with water and dichloromethane. The organic layer was washed with 2N hydrochloric acid, and the organic layer was stirred with anhydrous sodium sulfate. The mixture was then 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-dichloropyrimidine-4-yl)amino)phenyl)methanesulfonamide as a pale yellow solid in 58.9% yield.

[0200] <Step 4> Synthesis of N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazine-1-yl)piperidine-1-yl)phenyl)amino)pyrimidine-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide

[0201] [ka]

[0202] N-cyclopropyl-N-(2-((2,5-dichloropyrimidine-4-yl)amino)phenyl)methanesulfonamide (1.00 eq) synthesized in Step 3 was added to ethanol (EtOH), and 2-methoxy-4-(4-(4-methylpiperazine-1-yl)piperidine-1-yl)aniline (1.00 eq) and trifluoroacetic acid (TFA, 1.95 eq) were added at room temperature. The mixture was then refluxed and stirred overnight. After the reaction was complete, the mixture was neutralized with 1N sodium hydroxide solution and extracted with water and ethyl acetate. The organic layer was stirred with anhydrous sodium sulfate, 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-(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, with a yield of 14%. H NMR (500MHz, DMSO-d6) δ8.33(s, 1H), 8.19(s, 1H), 8.09(s, 1H), 7.99(s, 1H), 7.62(dd, J=7.9, 1.6Hz, 1H), 7.35(d, J =8.7Hz, 1H), 7.23(t, J=7.8Hz, 1H), 7.15(td, J=7.6, 1.5Hz, 1H), 6.62(d, J=2.5Hz, 1H), 6.47(dd, J=8.7, 2.5Hz, 1H), 3.75-3.71(m, 5H), 3.25-3.22(m, 4H), 2.67(td, J=12.2, 2.4Hz, 2H), 2.55-2.44(m, 4H), 2.38-2.27(m, 4H), 2.15(s, 3H), 1.85(d, J=11.5Hz, 2H), 1.52(qd, J=12.1, 3.9Hz, 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

[0203] 1-2.Manufacture of N-(2-((5-chloro-2-((4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-02)

[0204] [ka]

[0205] Steps 1, 2, and 3 were carried out identically to those for compound AON-MG23-01, but in Step 4, 1-(4-amino-3-methoxyphenyl)-N,N-dimethylpiperidine-4-amine was used instead of 2-methoxy-4-(4-(4-methylpiperazine-1-yl)piperidine-1-yl)aniline for synthesis.

[0206] Specifically, N-cyclopropyl-N-(2-((2,5-dichloropyrimidine-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-dimethylpiperidine-4-amine (1.00 eq) and trifluoroacetic acid (TFA, 1.95 eq) were added at room temperature. The mixture was then refluxed and stirred overnight. After the reaction was complete, the mixture was neutralized with 1N sodium hydroxide solution and extracted with water and ethyl acetate. The organic layer was stirred with anhydrous sodium sulfate, 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-(2-((5-chloro-2-((4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-02) as a yellow solid, with a yield of 25%. 1H NMR (500MHz, DMSO-d6) δ8.33(s, 1H), 8.19(s, 1H), 8.09(s, 1H), 7.99(s, 1H), 7.62(dd, J=7.8, 1.6Hz, 1H), 7.3 5(d, J=8.7Hz, 1H), 7.22(d, J=8.1Hz, 1H), 7.15(td, J=7.6, 1.6Hz, 1H), 6.63(d, J=2.5Hz, 1H), 6.48(dd, J=8.7 , 2.5Hz, 1H), 3.75(s, 3H), 3.72(d, J=12.2Hz, 2H), 3.23(m, 4H), 2.68(td, J=12.1, 2.5Hz, 2H), 2.22(s, 6H), 1. 86(d, J=12.4Hz, 2H), 1.51(qd, J=12.0, 3.9Hz, 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.

[0207] The compounds according to the present invention were produced through the above steps, and their structural formulas are shown in Table 1 and Figure 1 below.

[0208] [Table 1]

[0209] 2. Confirmation of the cell proliferation inhibitory effect of the compound of the present invention on human glioblastoma cells. Human glioblastoma cell line U87-MG cells are placed in a 96-well plate in a 1x10 ratio. 4 Cells were seeded at a density of cells / well and cultured for 24 hours. Afterward, they were treated with DMSO as a control group and with either the compound AON-MG23-01 or AON-MG23-02 of the present invention, respectively, followed by 48 hours of culture. The compounds of the present invention were treated at various concentrations of 0, 1, 2, and 4 μM. 10 μl of CCK reagent was added to each treatment, followed by a 1-hour reaction, and then the absorbance was measured at 450 nm.

[0210] As a result, as shown in Figure 2, we were able to confirm that the compounds AON-MG23-01 or AON-MG23-02 of the present invention suppress the proliferation of U87-MG cells.

[0211] 3. Confirmation of the inhibitory effect of the compound of the present invention on cell migration in human glioblastoma cells. Human glioblastoma cell line U87-MG cells are placed on SPLScar Block (SPL) plates in an 8x10 ratio. 4 Cells were seeded at a density of cells / well and cultured for 24 hours. After 24 hours, blocks were removed, and cells were treated with either AON-MG23-01 or AON-MG23-02 of the present invention, respectively, and then cultured in a CO2 incubator at 37°C for 24 hours. The compounds of the present invention were administered at concentrations of 0, 1, 2, and 4 μM, respectively. Photographs were taken before and after culture, and the distance of cell migration was measured using ImageJ software. Based on these results, the suppression of cell migration was analyzed.

[0212] As a result, as shown in Figure 3, when the cells were treated with the compound AON-MG23-01 or AON-MG23-02 of the present invention at a concentration of 4 μM, cell migration was suppressed by more than 65% compared to the untreated control group.

[0213] 4. Confirmation of the effect of the compound of the present invention on inhibiting metastasis of human glioblastoma. Human glioblastoma cell line U87-MG is placed on top of a Matrigel-coated filter (Transwell invasion chambers, Corning) at 8x10. 4 After seeding at a density of cells / well, the cells were treated with the compound of the present invention (AON-MG23-01 or AON-MG23-02; 0, 1, 2, 4 μM), respectively, and cultured for 24 hours in a CO2 incubator at 37°C. After culturing, cells were fixed and stained using the Diff-Quick Stain Kit (Sysmex, Kobe, Japan). Stained cells were imaged under a microscope, counted using ImageJ software, and then statistically analyzed.

[0214] As a result, as shown in Figure 4, the group treated with the compound AON-MG23-01 or AON-MG23-02 of the present invention showed a reduced degree of penetration into Matrigel compared to the untreated control group, confirming that the metastatic potential of cancer cells was suppressed.

[0215] 5. Confirmation of the effects of the compounds of the present invention on ANO1 and EGFR protein expression in glioblastoma cell lines.

[0216] We investigated whether the compound of the present invention affects the expression of ANO1 and EGFR proteins in glioblastoma cell lines. Human glioblastoma cell line U87-MG was placed in a 60mm dish (1x10). 6Cells were seeded at a given density and cultured for 24 hours. Afterward, each cell was treated with the compound of the present invention (AON-MG23-01 or AON-MG23-02; 0, 1, 2, or 4 μM), and cultured for 48 hours. The cultured cells were harvested and lysed for Western blotting. Antibodies used for Western blotting included anti-ANO1 antibody (Abcam, ab53212), anti-pEGFR antibody (Cell Signaling, #4407S), anti-EGFR antibody (Cell Signaling, #4267S), and anti-αTubulin antibody (Santacruz, #SC-5286). Protein concentration was quantified using the BSA method (Protein Quantitation Assay, Bovine serum albumin assay) (Pierce, Cat.23225). After lysing the cells, the obtained proteins were subjected to 10% SDS-PAGE with the same amount (10 μg) to separate them by molecular weight. These proteins were then transferred to a PVDF membrane (Bio-rad) and treated with blocking buffer (5% Skim milk in Tris-buffered saline (TBS) buffer containing 0.1% Tween 20, TBS-T) at room temperature for 1 hour. Next, the PVDF membrane was treated with the primary antibody and reacted at 4°C for 16 hours. The PVDF membrane was washed three times with TBS-T after the reaction. Then, the PVDF membrane was reacted with a secondary antibody labeled with horseradish peroxidase at room temperature for 1 hour, and the membrane was visualized using an ECL kit (Bio-rad). The amount of each protein was quantified and analyzed using ImageJ software.

[0217] As a result, as shown in Figure 5, cells treated with the compound of the present invention showed significantly suppressed levels of ANO1 and EGFR compared to the untreated control group, and this protein expression suppression effect was dependent on the treatment concentration of the compound.

[0218] 6. Confirmation of the ANO1 activity inhibitory effect of the compound of the present invention in glioblastoma cell lines. Using patch clamp, the activity of calcium-dependent chloride ion channels by the compounds of the present invention was measured to confirm the inhibitory effect on ANO1 ion channel activity. For this purpose, patch clamp was performed on U87-MG cells expressing ANO1. The pipette solution was 146 CsCl, 5 Ca-EGTA-NMDG, 8 HEPES, 2 MgCl2, 10 sucrose (pH 7.3), and the bath solution was 50 NaCl, 10 HEPES, 3 KCl, 2 CaCl2, 2 MgCl2, 5.5 glucose (pH 7.3). After culturing U87-MG cells on a coverslip for 4 hours, they were treated with AON-MG23-01 or AON-MG23-02 for 1 hour (treated at concentrations of 0, 4, and 8 μM respectively). At this time, chloride ion channels were activated using 400 mM ATP and high-concentration calcium, and the current of calcium-dependent chloride ion channels in the range of -100 mV to +100 mV was measured.

[0219] As a result, as shown in Figure 6a, it was shown that the ANO1 activity in the group treated with the compound of the present invention was clearly decreased compared to the untreated control group. In particular, as shown in Figure 6b, it was shown that the current density in the group treated with the compound of the present invention at a membrane potential of +80 mV decreased by 50% or more compared to the untreated control group. The above results indicate that the compound of the present invention can inhibit the activity of ANO1, a calcium-dependent chloride ion channel, in glioblastoma cell line U87-MG cells.

[0220] 7. Comparison of the effect of inhibiting metastasis of the compound of the present invention, EGFR inhibitor, and ANO1 inhibitor in human glioblastoma Human glioblastoma cell line U87-MG was placed on the upper part of a filter coated with Matrigel (Transwell invasion chambers, Corning) at 6×10 4After seeding at a cell / well density, cells were treated with 4 μM of the compound AON-MG23-02 of the present invention, 4 μM of the EGFR inhibitor Osimertinib, 100 μM of the ANO1 inhibitor CaCCinh-A01, and a combination of 4 μM Osimertinib and 100 μM CaCCinh-A01. The cells were then cultured for 16 hours at 37°C in a CO2 incubator. After culturing, cells were fixed and stained using a Diff-Quick staining kit (Sysmex, Kobe, Japan). Stained cells were imaged under a microscope, counted using ImageJ software, and then statistically analyzed.

[0221] As a result, as shown in Figures 7a and 7b, the group treated with the compound AON-MG23-02 of the present invention showed a reduced degree of penetration into Matrigel compared to the untreated control group, the EGFR inhibitor-treated group, and the ANO1 inhibitor-treated group, and exhibited a similar translocation inhibitory effect to the group treated with a combination of the EGFR inhibitor and the ANO1 inhibitor.

[0222] 8. Comparison of the inhibitory effects of the compounds of the present invention, EGFR inhibitors, and ANO1 inhibitors on ANO1, EGFR, and pEGFR protein expression in glioblastoma cell lines. The inhibitory effects of the compound AON-MG23-02 of the present invention, the EGFR inhibitor Osimertinib, and the ANO1 inhibitor CaCCinh-A01 on ANO1, EGFR, and pEGFR protein expression in glioblastoma cell lines were compared. Human glioblastoma cell line U87-MG was placed in a 60mm dish at a ratio of 100x10. 4Cells were seeded at a cell / dish density and cultured for 24 hours. Then, they were treated with 4 μM AON-MG23-02, 2 μM EGFR inhibitor Osimertinib, 100 μM ANO1 inhibitor CaCCinh-A01, and a combination of 2 μM Osimertinib and 100 μM CaCCinh-A01, followed by 48 hours of culture. After culturing, the cells were harvested and lysed for Western blotting. Antibodies used for Western blotting included anti-ANO1 antibody (Abcam, ab53212), anti-pEGFR antibody (Cell Signaling, #4407S), anti-EGFR antibody (Cell Signaling, #4267S), and anti-ACTIN antibody (sigma, #A2066). Protein concentrations were quantified using the BSA method (Protein Quantitation Assay, Bovine serum albumin assay) (Pierce, Cat.23225). After lysing the cells, the obtained proteins were subjected to 10% SDS-PAGE with an equal amount (10 μg) to separate the proteins by molecular weight. These proteins were then transferred to a PVDF membrane (Bio-rad) and treated with blocking buffer (5% Skim milk in Tris-buffered saline (TBS) buffer containing 0.1% Tween 20, TBS-T) at room temperature for 1 hour. Next, the PVDF membrane was treated with the primary antibody and reacted at 4°C for 16 hours. The PVDF membrane was then washed three times with TBS-T for 10 minutes each. Next, the PVDF membrane was reacted with a horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour, followed by three more washes with TBS-T for 10 minutes each. The washed PVDF membrane was treated with an ECL kit (Thermo, West Pico Plus) and the reaction was visualized using a da Vinci-Q instrument (YOUNGIN lapplus). We used ImageJ software to quantify and analyze the amount of each protein.

[0223] As a result, as shown in FIGS. 8a and 8b, it was confirmed that the compound AON-MG23-02 of the present invention decreased ANO1, EGFR, and pEGFR protein expression compared to the untreated control group, the EGFR inhibitor treatment group, the ANO1 inhibitor treatment group, and the combined treatment group of the EGFR inhibitor and the ANO1 inhibitor.

[0224] Example B. Derivatives of the novel compound AON-MG23-02 1. Preparation of derivatives of AON-MG23-02 1-1. AON-MG23-05 1-1-A. Preparation of Compound 2

[0225]

Chemical formula

[0226] 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 mixture was degassed and purged three times with N2, and then stirred at 80 °C in nitrogen gas (N2) for 2 hours. According to TLC (petroleum ether:ethyl acetate = 1:1, Rf = 0.49) analysis, Compound SM1 was completely consumed and a large amount of new spots were generated. The reaction mixture was concentrated under reduced pressure. The residue was diluted with 6.00 mL of water and 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, and 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). Yellow solid Compound 2 (900 mg, 2.35 mmol, yield 20.6%, purity 82.4%) was obtained.

[0227] 1-1-B. Preparation of Compound 3

[0228]

Chemical formula

[0229] To a solution of compound 2 (900 mg, 2.85 mmol, 1.00 eq) dissolved in ACN (10.0 mL), TMSCN (trimethylsilyl cyanide, 338 mg, 3.42 mmol, 427 μL, 1.20 eq) and Cs2CO3 (1.11 g, 3.42 mmol, 1.20 eq) were added. The mixture was stirred at 25°C for 12 hours. LC-MS analysis revealed that compound 2 was completely consumed, with 42.0% of the desired mass detected. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with 2.00 mL of water and extracted with 3.00 mL (3.00 mL x 3) of toluene. The bound organic supernatant was washed with 4.00 mL (4.00 mL x 1) of brine, dried over Na2SO4, filtered, and then concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, petroleum ether:ethyl acetate = 10:1, 0:1). Compound 3 (600 mg, 2.22 mmol, yield 77.8%, purity 96.9%) was obtained as a yellow solid.

[0230] 1-1-C. Preparation of Compound 4

[0231] [ka]

[0232] Compound 3 (600 mg, 2.29 mmol, 1.00 eq) and Raney-Ni (19.6 mg, 228 μmol, 0.100 eq) were dissolved in toluene (6.00 mL). After degassing and purging with N2, the suspension was degassed under vacuum and purged with hydrogen. The mixture was stirred several times for 12 hours in an H2 (40 psi) environment at 80°C. LC-MS analysis showed that compound 3 was completely consumed, and 54.8% of the desired substance was detected. The mixture was filtered and concentrated. The unpurified product (crude product) was purified by reverse-phase HPLC (column: Phenomenex luna C18 150*40 mm*15 μm; mobile phase: [water (FA)-ACN]; gradient: 2%~32% B for ≥15 minutes). A white solid compound 4 ([[4-(2-aminoethyl)phenyl]methyl]isoindoli-1-one, 320 mg, 1.20 mmol, yield 52.5%, purity 100%) was obtained.

[0233] 1-1-D. Preparation of Compound 5

[0234] [ka]

[0235] A mixture prepared by dissolving compound 4 (200 mg, 750 μmol, 1.00 eq) and compound 4a (117 mg, 750 μmol, 1.00 eq) in CH(OMe)3 (2.00 mL) was subjected to three degassing and N2 purging processes, and stirred at 25°C for 1 hour in N2 gas. AcOH (45.0 mg, 750 μmol, 42.9 μL, 1.00 eq) and NaBH3CN (14.1 mg, 225 μmol, 0.300 eq) were added to the mixture. Next, the mixture was stirred at 25°C for 1 hour in N2 gas. TLC (dichloromethane:methanol = 10:1; R fAccording to a ratio of 0.7), 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 HCl (4.00 mL x 3). The bound organic layer was washed with 5.00 mL of brine (5.00 mL x 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by column chromatography (SiO2, dichloromethane:methanol = 60:1 and 10:1). Compound 5 (34.0 mg, 73.1 μmol, yield 9.73%, purity 87.4%) was obtained as a yellow oil.

[0236] 1-1-E. Preparation of Compound 6

[0237] [ka]

[0238] Compound 5 (54.0 mg, 132 μmol, 1.00 eq) was dissolved in DMF (Dimethylformamide, 0.600 mL) to which 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. The mixture was stirred at 50°C for 12 hours. TLC (dichloromethane:methanol = 10:1, Rf = 0.70) results showed that compound 5 remained and one major new spot was detected. The residue was diluted with 2.00 mL of water and extracted with 2.00 mL of pharmaceutically acceptable ethyl acetate (2.00 mL x 3). The bound organic layer was washed with 3.00 mL of brine (3.00 mL x 1), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-TLC (SiO2, dichloromethane:methanol = 10:1). Compound 6 (32.0 mg, 56.6 μmol, yield 42.6%, purity 100%), a yellow oil, was obtained.

[0239] Manufacturing of 1-1-F.AON-MG23-05

[0240] [Chemical formula]

[0241] HCl / MeOH (2.00 M, 1.54 mL, 60.0 eq) was added to a mixture in which Compound 6 (29.0 mg, 51.3 μmol, 1.00 eq) was dissolved in MeOH (0.500 mL). The mixture was stirred at 25 °C for 0.5 h. As a result of LC-MS, Compound 6 was completely consumed, and 98.7% of the desired amount of the substance was detected. The reaction mixture was concentrated under reduced pressure. NaHCO3 was added to the mixture (pH > 7). The residue was extracted with DCM 3.00 mL (3.00 mL * 3). The combined organic layers were washed with brine 2.00 mL (2.00 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-TLC (SiO2, Ethyl acetate:Methanol = 10:1). Target A (AON-MG23-05), an off-white solid (18.2 mg, 40.2 μmol, yield 78.3%, purity 99.6%) was obtained.

[0242] The 1H NMR data of AON-MG23-05 are shown in Figure 11.

[0243] 1-2. Preparation of AON-MG23-06 1-2-A. Preparation of Compound 8

[0244] [Chemical formula]

[0245] Compound SM1 (3.00 g, 22.5 mmol, 1.00 eq), 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) were mixed and degassed in ACN (30 mL) and stirred at 25°C for 16 hours. LC-MS results showed that 20% of compound SM1 remained. Multiple new peaks appeared on LC-MS, and 60% of the desired compounds were detected. 30 mL of H2O was added to the reaction mixture to eliminate it, and 30 mL (30 mL x 2) of EA (Ethyl acetate) was extracted. The bound organic layer was washed with 30 mL (30 mL x 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the 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 and confirmed by LC-MS and HNMR.

[0246] 1-2-B. Preparation of Compound 9

[0247] [ka]

[0248] Compound 8 (200 mg, 802 μmol, 1.00 eq) was dissolved in DCM (Dichloromethane, 0.200 mL), and m-CPBA (meta-Chloroperoxybenzoic acid, 651 mg, 3.21 mmol, 85.0% purity, 4.00 eq) was added dropwise at 0°C for 0.5 hours. After addition, the mixture was stirred at 25°C for 2 hours. TLC (Petroleum ether:Ethyl acetate = 2:1, R) f(=0.33) As a result, compound 8 was completely consumed, and one new spot was formed. 20 mL of Na2S2O3 was added to the reaction mixture at 25°C to eliminate it, and the mixture was extracted with 30 mL (30 mL * 2) of DCM. The bound organic layer was washed with 30 mL (30 mL * 2) of brine, dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-TLC (SiO2, Petroleum ether:Ethyl acetate = 2:1). Compound 9 (580 mg, 1.65 mmol, yield 62.4%) was obtained as a brown solid and confirmed by HPLC and HNMR. Compound 9 (140 mg, 527 μmol, yield 65.7%) was also obtained as a yellow oil and confirmed by LC-MS and HNMR.

[0249] 1-2-C. Preparation of Compound 9A

[0250] [ka]

[0251] A solution of ethaneamine (626 mg, 7.68 mmol, 909 μL, 1.20 eq, HCl) and TEA (Triethylamine, 777 mg, 7.68 mmol, 1.07 mL, 1.2 eq) dissolved in MeOH (10 mL) was stirred at 25°C for 0.5 hours. 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 1 hour. LC-MS results showed that compound 9B was completely consumed and one major peak with the desired m / z value was detected. After adding 10 mL of Na2CO3 to the reaction mixture and allowing it to dissipate, the mixture was diluted with 20 mL of H2O, and 30 mL (30 mL x 2) of EA was extracted. The bound organic layer was washed with 30 mL (30 mL x 1) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the 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 and confirmed by HNMR and LC-MS.

[0252] Manufacturing of 1-2-D.AON-MG23-06

[0253] [ka]

[0254] A mixture prepared by dissolving compound 9 (65.0 mg, 245 μmol, 1.00 eq), compound 9A (54.4 mg, 294 μmol, 1.20 eq), and DIPEA (N,N-Diisopropylethylamine, 94.9 mg, 735 μmol, 128 μL, 3.00 eq) in ethanol (0.1 mL) was degassed, purged three times with N2, and stirred for 12 hours at 80°C in N2 gas. LC-MS results showed that compound 9 was completely consumed, and one major peak with the desired m / z value was detected. After adding 10 mL of H2O to the reaction mixture to eliminate it, it was extracted with 5 mL (5 mL x 2) of EA, concentrated under reduced pressure, and the residue was obtained. The residue was purified by prep-HPLC (FA conditions: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 15%~45% B, 1 minute or more). Subsequently, it was purified by prep-HPLC (neutral conditions: Waters Xbridge 150*25mm*5um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 45%~75% B, 15 minutes or more). A white solid AON-MG23-06 (Target B) (29.7 mg, 65.7 μmol, yield 26.8%, purity 99.8%) was obtained and confirmed by LC-MS, HPLC, HNMR and special NMR.

[0255] The 1H NMR data for AON-MG23-06 is shown in Figure 12.

[0256] 1-3. Manufacturing of AON-MG23-07 1-3-A. Preparation of Compound 10

[0257] [ka]

[0258] A suspension of KOH (675.1 mg, 12.0 mmol, 3.00 eq), DMSO (Dimethyl sulfoxide, 10.0 mL), toluene (7.50 mL), and H2O (2.00 mL) was blown into argon and saturated with P (5.00 g, 147.0 mmol, 36.6 eq). Compound 8 (1.00 g, 4.01 mmol, 1.00 eq) was added to the DMSO solution, which was stirred at 70°C for 30 minutes, and added dropwise to ensure continuous passage of the flammable gas. The reaction mixture was further heated for 30 minutes (70°C) while maintaining the flow of phosphine. TLC (PE:EA = 3:1, R f (=0.38) As a result, compound 8 was completely consumed and one new spot was formed. The mixture was passed through argon, cooled, diluted with 20 mL of water, and extracted with toluene (10 mL * 2). The toluene extract was washed with brine (10 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound 10 (1.10 g, starting material (crude)) was obtained as a white solid.

[0259] Manufacturing of 1-3-B.AON-MG23-07

[0260] [ka]

[0261] Compound 10 (1.10 g, 3.88 mmol, 1.00 eq) was dissolved in THF (Tetrahydrofuran, 10.0 mL), and NaH (465.9 mg, 11.6 mmol, purity 60.0%, 3.00 eq) was added to the solution in a 0°C N2 gas environment. The mixture was stirred at 0°C for 30 minutes. Then, 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 in a 0°C N2 gas environment, then diluted with 20.0 mL of H2O, and 40.0 mL (20.0 mL x 2) of EA was extracted. The bound 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 the residue. The residue was purified by prep-HPLC (FA conditions; column: Phenomenex luna C18 150*40 mm*15 μm; mobile phase: [water(FA)-ACN]; gradient: 52%~82% B 15 min or more). The residue was further purified by prep-HPLC (FA conditions; column: Welch Xtimate C18 150*25 mm*5 μm; mobile phase: [water(FA)-ACN]; gradient: 58%~78% B 15 min). A grayish-white AON-MG23-07 (Target C) (35.0 mg, 58.5 μmol, yield 1.51%, purity 97.0%) was obtained.

[0262] The 1H NMR data for AON-MG23-07 is shown in Figure 13.

[0263] 1-4. Manufacturing of AON-MG23-08 1-4-A. Preparation of Compound 2

[0264] [ka]

[0265] 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 subjected to degassing and N2 gas treatment three times, followed by stirring at 80°C for 2 hours in N2 gas. LC-MS (EW47929-30-P1A1) results showed that compound SM1 was completely consumed and the desired mass was sensed. The reaction mixture was diluted with 30.0 mL of H2O, and 40.0 mL (20.0 mL x 2) of EA was extracted. The bound organic layer was washed with 30.0 mL (30.0 mL x 1) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was analyzed by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 20 / 1 to 5 / 1, TLC, PE:EA = 3:1, R f The compound was purified using a solution with a pH of 0.22. Compound 2 (1.18 g, 3.73 mmol, yield 19.7%) was obtained as a yellow solid.

[0266] 1-4-B. Preparation of Compound 3

[0267] [ka]

[0268] Compound 2 (1.1 g, 3.48 mmol, 1.00 eq) was dissolved in ACN (16.0 mL), and Cs2CO3 (1.36 g, 4.17 mmol, 1.20 eq) and TMSCN (414.1 mg, 4.17 mmol, 522 μL, 1.20 eq) were added to the solution. The mixture was stirred at 25°C for 12 hours. LC-MS analysis showed that compound 2 was completely consumed 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 40.0 mL (20.0 mL x 2) of EA was extracted. The bound organic layer was washed with 20.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was analyzed by column chromatography (SiO2, Petroleum ether / Ethyl acetate = 5 / 1 to 3 / 1, PE:EA = 5:1, Rf = 5:1, R f The compound was purified using a solution with a pH of 0.47. Compound 3 (825 mg, 3.08 mmol, yield 88.6%, purity 98.0%) was obtained as a yellow solid.

[0269] 1-4-C. Preparation of Compound 4

[0270] [ka]

[0271] Compound 3 (825 mg, 3.15 mmol, 1.00 eq) and Raney-Ni (26.9 mg, 314 μmol, 0.10 eq) were mixed in toluene (8.00 mL). The mixture was then vacuum-treated and removed three times with N2, followed by several treatments with H2 under vacuum to remove the mixture. The mixture was stirred at 80°C in an H2 (40 psi) atmosphere for 12 hours. LC-MS results showed that compound 3 was completely consumed and the desired substance was detected. The reaction mixture was filtered and concentrated under low pressure to obtain the residue. The residue was then purified by prep-HPLC (FA conditions; column: Phenomenex luna C18 150*40 mm*15 μm; mobile phase: [water(FA)-ACN]; gradient: 8%~38% B, 15 minutes). Compound 4 (300 mg, 1.10 mmol, 34.9% yield, 97.5% purity) was obtained as a white solid and confirmed by HNMR and LC-MS.

[0272] 1-4-D. Preparation of Compound 5

[0273] [ka]

[0274] Compound 4a (175 mg, 1.13 mmol, 1.00 eq) was added to a solution of compound 4 (300 mg, 1.13 mmol, 1.00 eq) dissolved in 5.00 mL of ethanol (EtOH). The mixture was stirred at 25°C under an N2 atmosphere for 1 hour. Then, NaBH3CN (42.4 mg, 675 μmol, 0.60 eq) and AcOH (67.6 mg, 1.13 mmol, 64.4 μL, 1.00 eq) were added at 25°C, and the mixture was stirred under an N2 atmosphere for 1 hour. LC-MS results showed that 2% of compound 4 was completely consumed and the desired substance was detected. The residue was diluted with 10 mL of H2O and extracted with 20 mL (10 mL x 2) of EA. After binding the organic phase, the mixture was washed with 10 mL of brine (10 mL * 1), dried over [Na2SO4], filtered, and concentrated under low pressure to obtain the residue. The residue was then purified by column chromatography (SiO2, DCM / MeOH = 20 / 1 to 15 / 1), TLC, DCM:MeOH = 10:1, Rf = 0.46). Compound 5 (293 mg, 647 μmol, 57.4% yield, 100% purity, FA) was obtained as a white solid and confirmed by LC-MS and HNMR.

[0275] Manufacturing of 1-4-E.AON-MG23-08

[0276] [ka]

[0277] Compound 5b (99.7 mg, 1.23 mmol, 48.5 μL, 5.00 eq) and H2O (1.50 mL) were added to a solution of compound 5 (100 mg, 245 μmol, 1.00 eq) dissolved in AcOH (0.30 mL). The mixture was stirred at 25°C for 1 hour. LC-MS analysis showed that approximately 10% of compound 5 remained, indicating that the desired mass was detected. The reaction was terminated by slowly adding a saturated sodium hydroxide solution until the pH reached 8, and then extracted with 40 mL (20 mL x 2) of DCM. The bound organic layer was washed with 40.0 mL (20.0 mL x 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified by prep-TLC (SiO2, DCM:MeOH = 15:1). The residue was purified by prep-HPLC (basic conditions; column: Waters Xbridge 150*25mm*5um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 25%~55% B, 10 minutes or more). A white solid AON-MG23-08 (Target D) (11.0 mg, 24.4 μmol, 95% yield, 100% purity) was obtained.

[0278] The 1H NMR data for AON-MG23-08 is shown in Figure 14. Derivatives of AON-MG23-02 were prepared using the method described above, and their structural formulas are shown in Table 2 below.

[0279] [Table 2]

[0280] 2. Comparison of the inhibitory effects of four derivatives of AON-MG23-02 on ANO1 and EGFR protein expression in glioblastoma cell lines. The inhibitory effects of AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08, derivatives of AON-MG23-02, on ANO1 and EGFR protein expression were compared in glioblastoma cell lines. Human glioblastoma cell line U87-MG was placed in a 60mm dish at a rate of 100x10 4Cells were seeded at a cell / dish density and cultured for 24 hours. Afterward, they were treated with 0, 8, 16, and 32 μM of AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08 derivatives, respectively, and cultured for 48 hours. After culturing, the cells were harvested and lysed for Western blotting. Antibodies used for Western blotting included anti-ANO1 antibody (Abcam, ab53212), anti-pEGFR antibody (Cell Signaling, #4407S), anti-EGFR antibody (Cell Signaling, #4267S), and anti-ACTIN antibody (sigma, #A2066). Protein concentration was quantified using the BSA method (Protein Quantitation Assay, Bovine serum albumin assay) (Pierce, Cat.23225). After lysing the cells, the proteins obtained were separated by molecular weight by 10% SDS-PAGE using the same amount (10 μg). These proteins were then transferred to a PVDF membrane (Bio-rad) and treated with blocking buffer (5% Skim milk in Tris-buffered saline (TBS) buffer containing 0.1% Tween 20, TBS-T) at room temperature for 1 hour. Next, the PVDF membrane was treated with the primary antibody and reacted at 4°C for 16 hours. The PVDF membrane was then washed three times with TBS-T for 10 minutes each. Next, the PVDF membrane was reacted with a secondary antibody labeled with horseradish peroxidase at room temperature for 1 hour, followed by three more washes with TBS-T for 10 minutes each. The washed PVDF membrane was then treated with an ECL kit (Thermo, West Pico Plus) and the reaction was visualized using a da Vinci-Q instrument (YOUNGIN lapplus). We used ImageJ software to quantify and analyze the amount of each protein.

[0281] As a result, as shown in Figures 9a to 9d, we were able to confirm that the derivatives AON-MG23-05 and AON-MG23-08 reduced the expression of ANO1 and EGFR proteins compared to the untreated control group.

[0282] 3. Comparison of the metastasis-inhibiting effects of four derivatives of AON-MG23-02 in human glioblastoma. Human glioblastoma cell line U87-MG was seeded at a density of 8x10⁴ cells / well onto a Matrigel-coated filter (Transwell invasion chambers, Corning). The cells were then treated with 0, 8, 16, and 32 μM of the compound derivatives of the present invention, AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08, respectively, and cultured for 24 hours at 37°C in a CO₂ incubator. After culturing, cells were fixed and stained using a Diff-Quick staining kit (Sysmex, Kobe, Japan). Stained cells were imaged under a microscope, counted using ImageJ software, and then statistically analyzed.

[0283] As a result, as shown in Figures 10a and 10b, the groups treated with derivatives AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08 showed a reduced degree of penetration into Matrigel compared to the untreated control group, confirming the suppression of metastasis in human glioblastoma. The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects. [Industrial applicability]

[0284] The novel compound of the present invention can not only be used as an ANO1 inhibitor, but can also simultaneously suppress ANO1 and EGFR in brain tumor cells. Therefore, it can be used as a dual-target anticancer agent for ANO1 and EGFR, and can also be utilized as a combination formulation for EGFR-targeted therapy. As such, it can be used in a variety of ways in the field of brain tumor prevention and treatment, and has industrial applicability.

Claims

1. A compound selected from the group consisting of compounds represented by the following chemical formulas 1 to 6, or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 【change】

2. (S1A) A step of reacting a compound represented by the following chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base to produce a compound represented by the following chemical formula 8; or (S1B) A method for producing the compound according to claim 1, comprising the step of reacting a compound represented by the following chemical formula 9 with a compound represented by the following chemical formula 10 or chemical formula 11 in the presence of a base. 【Chemistry 2】 【change】

3. The method for producing the compound according to claim 2 is characterized in that the (S1A) step further comprises the following steps: (S1A-1) A step of reducing the compound represented by chemical formula 8 with zinc in the presence of ammonium chloride to produce the compound represented by the following chemical formula 12; (S1A-2) A step of reacting the compound represented by chemical formula 12 with 2,4,5-trichloropyrimidine in the presence of a base to produce the compound represented by the following chemical formula 13; and (S1A-3) A step of reacting the compound represented by chemical formula 13 with the compound represented by chemical formula 14 or chemical formula 15 in the presence of an acid to produce the compound represented by chemical formula 1 or chemical formula 2. 【Transformation 3】 【change】

4. The method for producing a compound according to claim 2, characterized in that when the compound represented by chemical formula 9 and the compound represented by chemical formula 10 react in the presence of a base in the (S1B) stage, a compound represented by the following chemical formula 16 is produced. 【Chemistry 4】

5. The method for producing the compound according to claim 4 is characterized in that the (S1B) step further comprises the following steps: (S1B-1) A step of reacting the compound represented by chemical formula 16 with trimethylsilyl cyanide in the presence of a base to produce the compound represented by the following chemical formula 17; (S1B-2) A step of producing a compound represented by the following chemical formula 18 by hydrogenating the compound represented by chemical formula 17 in the presence of a metal catalyst; (S1B-3) A step of reacting the compound represented by the chemical formula 18 with the compound represented by the following chemical formula 19 in the presence of a reducing agent to produce the compound represented by the following chemical formula 20; and (S1B-4) The compound represented by the above chemical formula 20 and NCO － A step of reacting the above to produce a compound represented by chemical formula 6; or a step of reacting the compound represented by chemical formula 20 with the compound represented by the following chemical formula 21 in the presence of a base to produce a compound represented by the following chemical formula 22. 【Transformation 5】 【change】

6. The method for producing a compound according to claim 5, further comprising step (S1B-4) of hydrolyzing the compound represented by chemical formula 22 in the presence of an acid to produce the compound represented by chemical formula 3.

7. The method for producing the compound according to claim 5, characterized in that the metal catalyst is one or more selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney nickel (Raney-Ni).

8. The method for producing a compound according to claim 2, characterized in that when the compound represented by chemical formula 9 and the compound represented by chemical formula 11 react in the presence of a base in step (S1B), a compound represented by the following chemical formula 23 is produced. 【Transformation 6】

9. The method for producing the compound according to claim 8 is characterized in that the (S1B) step further comprises the following steps: (S1Ba-1) A step of reacting the compound represented by the chemical formula 23 with a peroxide to produce the compound represented by the chemical formula 24 below, and (S1Ba-2) A step of reacting the compound represented by chemical formula 24 with the compound represented by the following chemical formula 25 in the presence of a base to produce the compound represented by chemical formula 4; or (S1Bb-1) A step of phosphorylating the compound represented by chemical formula 23 in the presence of a base to produce the compound represented by the following chemical formula 26, and (S1Bb-2) A step of reacting the compound represented by chemical formula 26 with the compound represented by the following chemical formula 27 in the presence of a base to produce the compound represented by chemical formula 5. 【Transformation 7】

10. The aforementioned peroxide is m-CPBA (meta-chloroperoxybenzoic acid), H ２ O ２ A method for producing the compound according to claim 9, characterized in that it is one or more selected from the group consisting of DMDO (dimethyldioxirane) and oxone.

11. The method for producing the compound according to claim 9, characterized in that the compound represented by the chemical formula 25 is produced by reacting the compound represented by the chemical formula 28 below with ethylamine in the presence of a reducing agent. 【Transformation 8】

12. The reducing agent is sodium cyanobolohydride (NaBH ３ CN), Triacetoxyborohydride sodium (NaBH(OAc) ３ ), and sodium borohydride (NaBH ４ A method for producing the compound according to claim 5 or 11, characterized in that it is one or more selected from the group consisting of ).

13. The aforementioned acids include pivalic acid (PivOH), acetic acid (AcOH), trifluoromethanesulfonic acid (TfOH), p-toluenesulfonic acid (TsOH), benzoic acid (Benzoic acid), and zinc bromide (ZnBr). ２ A method for producing the compound according to claim 3 or 6, characterized in that it is one or more selected from the group consisting of ), hydrogen chloride (HCl), and trifluoroacetic acid (TFA).

14. The base is potassium carbonate (K ２ CO ３ ), sodium carbonate (Na ２ CO ３ ), sodium bicarbonate (NaHCO ３ ), potassium bicarbonate (KHCO ３ ), cupric bromide (CuBr ２ ), cesium carbonate (Cs ２ CO ３ ), lithium hydroxide (LiOH), sodium hydride (NaH), potassium hydride (KH), potassium hydroxide (KOH), triethylamine (TEA; triethylamine), N,N - diisopropylethylamine (DIPEA; N,N - diisopropylethylamine), pyridine, and piperidine, and is one or more selected from the group consisting of, and is characterized in that it is a method for producing the compound according to any one of claims 2 to 11.

15. The method for producing a compound according to any one of claims 2 to 11, characterized in that the production method comprises one or more solvents selected from the group consisting of organic solvents, water, and mixtures thereof.

16. The aforementioned organic solvents are 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), hexafluoroisopropanol (HFIP), acetonitrile (ACN), dimethylformamide (DMF), nitromethane, and trimethoxymethane (CH(OMe)). ３ A method for producing the compound according to claim 15, characterized in that it is one or more selected from the group consisting of ), acetic acid, isopropanol (IPA), and chloroform.

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