Novel compound and preparation method therefor

Abstract

The present invention relates to a novel anoctamin 1 (ANO1) inhibitory compound and has been completed by confirming that the novel compound may inhibit brain tumor by inhibiting the expression or activity of ANO1 which is a calcium-dependent chloride channel. Thus, according to the present invention, a novel compound usable as an ANO1 inhibitor may be provided. In addition, the compound of the present invention may simultaneously inhibit ANO1 and EGFR in brain tumor cells, and thus may be used as a dual-target anticancer agent for ANO1 and EGFR, and may also be used as a combined preparation for EGFR-targeted therapy, and thus is expected to be usable in various ways in the field of brain tumor prevention and treatment.

Description

Novel compound and method for preparing the same

[0001] The present invention relates to a novel compound capable of simultaneously inhibiting ANO1 (Anoctamin 1) and EGFR (epidermal growth factor receptor) and a method for preparing the same.

[0002] This invention claims the benefit of priority from 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, the entire contents of which are incorporated herein by reference.

[0003]

[0004] Glioblastoma is a primary malignant brain tumor that occurs in adults. Without treatment, death occurs within three to six months after diagnosis. Even with comprehensive treatment options, the average survival time is only 12 to 14 months. It is a highly malignant disease. Approximately 12,000 people develop brain tumors in South Korea each year. Of these, approximately 9,000 are benign tumors, such as meningiomas or pituitary adenomas, and approximately 2,000 are diagnosed with malignant tumors. Approximately 630 of these malignant tumors are diagnosed with glioblastoma each year.

[0005] Glioblastoma, which originates from glial cells in the brain, grows invasively into surrounding brain tissue, resulting in unclear tumor boundaries and extending farther than the actual visual boundaries seen with the naked eye during radiological examinations or surgery. Glioblastoma is often located in areas of the brain that make surgical removal impossible due to functional reasons, and complete remission is not achieved with radiotherapy. Furthermore, the blood-brain barrier hinders the brain penetration of anticancer drugs, making effective anticancer drugs rare and making treatment very difficult. Temozolomide, the current standard treatment for glioblastoma, is used, but long-term treatment can lead to side effects and resistance, making sustained treatment difficult. Despite numerous advances in modern medicine and the development of new drugs, no new treatment has yet been developed.

[0006] One of the most frequently genetically altered oncogenes in glioblastoma patients is the epidermal growth factor receptor (EGFR). EGFR is physiologically important, and mutations in EGFR are observed in more than half of patients. When EGFR binds to EGF or TNF-alpha, various signaling events occur within the cell. However, mutations that overexpress or overactivate EGFR can lead to excessive cell proliferation and ultimately cancer. Among these EGFR mutations, EGFRvIII is characterized by the deletion of amino acids 6 to 273, resulting in a protein specifically expressed in cancer cells. While first discovered in glioblastoma, EGFRvIII has also been found in other tissue cancers.

[0007] It has been reported that volume control of cancer cells by chloride channel proteins such as Anoctamin 1 (ANO1) and the CLC (chloride channel) family is important in the migration and metastasis of glioblastoma cells. Ano1 proteins are involved in various physiological functions (pain control, blood pressure control, brain nerve development, water secretion control, etc.), and in particular, their anticancer effects have been reported in various cancer cells (breast cancer, head and neck cancer, prostate cancer, pancreatic cancer, lung cancer, etc.). Inhibition of Ano1 proteins in breast cancer exerted anticancer effects through CaMK2 signaling. In addition, although the level of Ano1 expression in normal brain is significantly low, it is overexpressed in glioblastoma, and when Ano1 activity is inhibited, glioblastoma cell migration and metastasis are inhibited, and cancer growth is suppressed in animal models. Furthermore, it has been reported that inhibition of ANOVA in glioblastoma stem cells induces epidermal growth factor receptor mutated 3 (EGFRvIII) protein degradation. These studies suggest that inhibition of ANOVA could be a therapeutic strategy for various EGFR mutation-associated cancers, including glioblastoma.

[0008] Thus, both ANO1 and EGFR have potential as anticancer therapeutic targets. However, no agent capable of exhibiting superior anticancer effects through dual inhibition of ANO1 and EGFR has yet been discovered.

[0009]

[0010] The present invention relates to a novel Anoctamin 1 (ANO1) inhibitory compound and a method for preparing the same, and was completed by confirming that the compound can exhibit an excellent anticancer effect by simultaneously inhibiting ANO1, a calcium-dependent chloride channel, and EGFR, a tyrosine kinase.

[0011] Accordingly, an 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.

[0012] [Chemical Formula 1]

[0013]

[0014] [Chemical Formula 2]

[0015]

[0016] [Chemical Formula 3]

[0017]

[0018] [Chemical Formula 4]

[0019]

[0020] [Chemical Formula 5]

[0021]

[0022] [Chemical Formula 6]

[0023]

[0024] Another object of the present invention is to provide a method for producing compounds represented by the chemical formulas 1 to 6.

[0025] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating brain tumors, comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0026] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating brain tumors, comprising (i) at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) an anticancer agent targeting EGFR as an active ingredient.

[0027] Another object of the present invention is to provide a pharmaceutical composition for enhancing the anticancer effect of a brain tumor anticancer agent, comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0028]

[0029] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0030]

[0031] To achieve the above object, the present invention provides one or more compounds selected from the group consisting of compounds represented by the following chemical formulas 1 to 6 or pharmaceutically acceptable salts thereof:

[0032] [Chemical Formula 1]

[0033]

[0034] [Chemical Formula 2]

[0035]

[0036] [Chemical Formula 3]

[0037]

[0038] [Chemical Formula 4]

[0039]

[0040] [Chemical Formula 5]

[0041]

[0042] [Chemical Formula 6]

[0043]

[0044] In addition, the present invention comprises a step of preparing a compound represented by the following chemical formula 8 by reacting a compound represented by the following chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base (S1A); or

[0045] (S1B) Provided is a method for producing a compound represented by the chemical formulae 1 to 6, comprising a step of reacting a compound represented by the chemical formula 9 below with a compound represented by the chemical formula 10 or 11 below in the presence of a base:

[0046] [Chemical Formula 7]

[0047]

[0048] [Chemical Formula 8]

[0049]

[0050] [Chemical Formula 9]

[0051]

[0052] [Chemical Formula 10]

[0053]

[0054] [Chemical Formula 11]

[0055]

[0056] In one embodiment of the present invention, the step (S1A) may further include, but is not limited to, the following steps:

[0057] (S1A-1) A step of reducing a compound represented by the above chemical formula 8 (the nitro group) with zinc in the presence of ammonium chloride to produce a compound represented by the following chemical formula 12;

[0058] (S1A-2) A step of preparing a compound represented by the following chemical formula 13 by reacting a compound represented by the above chemical formula 12 with 2, 4, 5-trichloropyrimidine in the presence of a base; and

[0059] (S1A-3) A step of producing a compound represented by the chemical formula 1 or 2 by reacting a compound represented by the chemical formula 13 and a compound represented by the chemical formula 14 or 15 below in the presence of an acid.

[0060] [Chemical Formula 12]

[0061]

[0062] [Chemical Formula 13]

[0063]

[0064] [Chemical Formula 14]

[0065]

[0066] [Chemical Formula 15]

[0067]

[0068] In another embodiment of the present invention, when the compound represented by the chemical formula 9 and the compound represented by the chemical formula 10 react in the presence of a base in the step (S1B), a compound represented by the chemical formula 16 below can be produced, but is not limited thereto.

[0069] [Chemical Formula 16]

[0070]

[0071] In another embodiment of the present invention, the step (S1B) may further include, but is not limited to, the following steps:

[0072] (S1B-1) A step of producing a compound represented by the following chemical formula 17 by reacting a compound represented by the above chemical formula 16 with trimethylsilyl cyanide in the presence of a base;

[0073] (S1B-2) A step of producing a compound represented by the following chemical formula 18 by hydrogenating a compound represented by the above chemical formula 17 in the presence of a metal catalyst;

[0074] (S1B-3) A step of preparing a compound represented by the following chemical formula 20 by reacting a compound represented by the above chemical formula 18 with a compound represented by the following chemical formula 19 in the presence of a reducing agent; and

[0075] (S1B-4) Compound represented by the above chemical formula 20 and NCO - A step of preparing a compound represented by the above chemical formula 6 by reacting; or a step of preparing a compound represented by the above chemical formula 22 by reacting a compound represented by the above chemical formula 20 and a compound represented by the following chemical formula 21 in the presence of a base.

[0076] [Chemical Formula 17]

[0077]

[0078] [Chemical Formula 18]

[0079]

[0080] [Chemical Formula 19]

[0081]

[0082] [Chemical Formula 20]

[0083]

[0084] [Chemical Formula 21]

[0085]

[0086] [Chemical Formula 22]

[0087]

[0088] In another embodiment of the present invention, the step (S1B-4) may further include, but is not limited to, a step of preparing a compound represented by the chemical formula 3 by subjecting the compound represented by the chemical formula 22 to a hydrolysis reaction in the presence of an acid.

[0089] In another embodiment of the present invention, the metal catalyst may be at least one selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney-Ni, but is not limited thereto.

[0090] In another embodiment of the present invention, when the compound represented by the chemical formula 9 and the compound represented by the chemical formula 11 react in the presence of a base in the step (S1B), a compound represented by the chemical formula 23 below can be produced, but is not limited thereto.

[0091] [Chemical Formula 23]

[0092]

[0093] In another embodiment of the present invention, the step (S1B) may further include, but is not limited to, the following steps.

[0094] (S1Ba-1) A step of producing a compound represented by the following chemical formula 24 by reacting a compound represented by the above chemical formula 23 with a peroxide, and

[0095] (S1Ba-2) A step of preparing a compound represented by the above chemical formula 4 by reacting a compound represented by the above chemical formula 24 and a compound represented by the following chemical formula 25 in the presence of a base; or

[0096] (S1Bb-1) A step of producing a compound represented by the following chemical formula 26 by phosphorylating the compound represented by the above chemical formula 23 in the presence of a base, and

[0097] (S1Bb-2) A step of producing a compound represented by the above chemical formula 5 by reacting a compound represented by the above chemical formula 26 and a compound represented by the following chemical formula 27 in the presence of a base.

[0098] [Chemical Formula 24]

[0099]

[0100] [Chemical Formula 25]

[0101]

[0102] [Chemical Formula 26]

[0103]

[0104] [Chemical Formula 27]

[0105]

[0106] In another embodiment of the present invention, the peroxide may be at least one selected from the group consisting of m-CPBA (meta-chloroperoxybenzoic acid), H2O2, DMDO (dimethyldioxirane), and oxone, but is not limited thereto.

[0107] In another embodiment of the present invention, the compound represented by the chemical formula 25 can be prepared by reacting the compound represented by the chemical formula 28 below with ethylamine in the presence of a reducing agent, but is not limited thereto.

[0108] [Chemical Formula 28]

[0109]

[0110] In another embodiment of the present invention, the reducing agent may be at least one selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium borohydride (NaBH4), but is not limited thereto.

[0111] In another embodiment of the present invention, the acid may be at least one selected from the group consisting of pivalic acid (PivOH), acetic acid (AcOH), trifluoromethanesulfonic acid (TfOH), paratoluenesulfonic acid (TsOH), benzoic acid, zinc bromide (ZnBr2), hydrogen chloride (HCl), and trifluoroacetic acid (TFA), but is not limited thereto.

[0112] In another embodiment of the present invention, the base may be at least one 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.

[0113] In another embodiment of the present invention, the manufacturing method may be performed in one or more solvents selected from the group consisting of organic solvents, water, and mixtures thereof, but is not limited thereto.

[0114] In another embodiment of the present invention, the organic solvent may be at least one 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.

[0115] In addition, the present invention provides a pharmaceutical composition for preventing or treating brain tumors, comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0116] In addition, the present invention provides a method for preventing or treating a brain tumor, comprising administering to a subject in need thereof at least one compound 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.

[0117] In addition, the present invention provides a use for preventing or treating brain tumors, comprising at least one compound 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 comprising the same as an active ingredient.

[0118] In addition, the present invention provides the use of one or more compounds selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 for the manufacture of a drug for treating brain tumors.

[0119] In one embodiment of the present invention, the brain tumor 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), but is not limited thereto.

[0120] In another embodiment of the present invention, the compound or a pharmaceutically acceptable salt thereof can simultaneously inhibit EGFR and ANO1 (e.g., inhibit expression and / or activity), but is not limited thereto. In other words, the compound or a pharmaceutically acceptable salt thereof can be a dual inhibitor targeting both EGFR and ANO1.

[0121] In 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, but not limited to:

[0122] (a) inhibiting tumor metastasis; and

[0123] (b) Inhibits cancer resistance to anticancer drugs.

[0124] In another embodiment of the present invention, the anticancer agent may be a target anticancer agent for tyrosine kinase, but is not limited thereto.

[0125] In another embodiment of the present invention, the tyrosine kinase may be at least one 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.

[0126] In another embodiment of the present invention, the pharmaceutical composition may be administered in combination with a targeted anticancer agent for tyrosine kinase, but is not limited thereto.

[0127] In 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.

[0128] In addition, the present invention provides a pharmaceutical composition for preventing or treating brain tumors, comprising (i) at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent for EGFR as an active ingredient.

[0129] In addition, the present invention provides a method for preventing or treating brain tumors, comprising administering to a subject in need thereof (i) at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent for EGFR.

[0130] In addition, the present invention provides a composition comprising (i) at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent for EGFR as an active ingredient, for use in the prevention or treatment of brain tumors.

[0131] In addition, the present invention provides the use of a composition comprising (i) at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent for EGFR as an active ingredient for the manufacture of a drug for treating a brain tumor.

[0132] In one embodiment of the present invention, the compound or a pharmaceutically acceptable salt thereof can suppress the resistance of brain tumor cells to the EGFR-targeting anticancer agent, but is not limited thereto.

[0133] In another embodiment of the present invention, the composition may be in the form of a mixture comprising the compound or a pharmaceutically acceptable salt thereof and the EGFR-targeting anticancer agent, but is not limited thereto.

[0134] In another embodiment of the present invention, the composition may be in a form in which the compound or a pharmaceutically acceptable salt thereof and the EGFR-targeting anticancer agent are each formulated and administered simultaneously, separately, or sequentially, but is not limited thereto.

[0135] In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of a brain tumor anticancer agent, comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0136] In addition, the present invention provides a method for enhancing the anticancer effect of a brain tumor anticancer agent, comprising administering to a subject in need thereof at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; or a composition containing the same as an active ingredient.

[0137] In addition, the present invention provides a use of a brain tumor anticancer agent for enhancing the anticancer effect of at least one compound 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.

[0138] In addition, the present invention provides 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, for the manufacture of a preparation for enhancing the anticancer effect of a brain tumor anticancer agent.

[0139] In addition, the present invention provides 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, for the production of an inhibitor of resistance of brain tumor cells to a brain tumor anticancer agent.

[0140] In one embodiment of the present invention, the brain tumor anticancer agent may be a target anticancer agent for tyrosine kinase, but is not limited thereto.

[0141] In another embodiment of the present invention, the tyrosine kinase may be at least one 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.

[0142] In 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.

[0143]

[0144] The present invention relates to a novel Anoctamin 1 (ANO1) inhibitory compound, and was completed by confirming that the novel compound can suppress brain tumors by suppressing the expression or activity of ANO1, a calcium-dependent chloride channel.

[0145] Therefore, according to the present invention, a novel compound that can be used as an ANO1 inhibitor can be provided. Moreover, since the compound of the present 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 utilized as a combination agent for EGFR-targeted therapy, and thus, it is expected to be utilized in various fields of brain tumor prevention and treatment.

[0146]

[0147] Figure 1 shows the names, structural formulas, and basic information of compounds AON-MG23-01 and AON-MG23-02 according to the present invention.

[0148] Figure 2 shows the results of a CCK assay performed after treating a human glioblastoma cell line with various concentrations of the compound AON-MG23-01 or AON-MG23-02 of the present invention to confirm the cell growth inhibitory effect of compounds AON-MG23-01 and AON-MG23-02 on human glioblastoma cells.

[0149] Figure 3 shows the results of photographing and analyzing the degree of cell migration over time after treating a human glioblastoma cell line with various concentrations of the compound AON-MG23-01 or AON-MG23-02 of the present invention to confirm the cell migration inhibitory effect of the compounds AON-MG23-01 and AON-MG23-02 of the present invention on human glioblastoma cells.

[0150] FIG. 4 shows the results of photographing and analyzing the extent of cell penetration into Matrigel after treating human glioblastoma cell lines cultured on Matrigel with various concentrations of the compound AON-MG23-01 or AON-MG23-02 of the present invention to confirm the inhibitory effect of the compounds AON-MG23-01 and AON-MG23-02 of the present invention on glioblastoma metastasis.

[0151] Figure 5 shows the results of Western blotting performed after treating human glioblastoma cell lines with various concentrations of the compound AON-MG23-01 or AON-MG23-02 of the present invention to determine the effects of compounds AON-MG23-01 and AON-MG23-02 on EGFR and ANO1 protein expression in glioblastoma. The left side is an image of protein bands detected by Western blotting, and the right side is the quantification of EGFR and ANO1 protein levels.

[0152] FIGS. 6a and 6b show the results of whole-cell patch clamp experiments performed on human glioblastoma cell lines treated with the compound AON-MG23-01 or AON-MG23-02 of the present invention to confirm whether the compound AON-MG23-01 and AON-MG23-02 of the present invention inhibit the activity of ANO1 in glioblastoma. FIG. 6a shows the results of measuring calcium-dependent chloride ion channel currents from -100 mV to +100 mV, and FIG. 6b shows the current / voltage (I / V) plot at +80 mV of FIG. 6a.

[0153] Figures 7a and 7b show the results of comparing the metastasis inhibition effects of the compound AON-MG23-02 of the present invention and EGFR inhibitors and ANO1 inhibitors in human glioblastoma.

[0154] Figures 8a and 8b show the results of comparing the inhibitory effects of the compound AON-MG23-02 of the present invention and an EGFR inhibitor and an ANO1 inhibitor on the expression of ANO1, EGFR, and pEGFR proteins in glioblastoma cell lines.

[0155] FIGS. 9A to 9D are the results of Western blotting performed after treating human glioblastoma cell lines with various concentrations of AON-MG23-05, AON-MG23-06, AON-MG23-07, or AON-MG23-08, which are derivatives of the compound AON-MG23-02 of the present invention, to determine the effects of AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08 on the expression of EGFR and ANO1 proteins in glioblastoma. FIG. 9A shows the results of Western blotting after treating AON-MG23-05, FIG. 9B shows the results of AON-MG23-06, FIG. 9C shows the results of AON-MG23-07, and FIG. 9D shows the results of Western blotting after treating AON-MG23-08.

[0156] FIG. 10a and FIG. 10b show the results of photographing and analyzing the extent of cell penetration into Matrigel after treating human glioblastoma cell lines cultured on Matrigel with various concentrations of AON-MG23-05, AON-MG23-06, AON-MG23-07, or AON-MG23-08, which are derivatives of the compound AON-MG23-02 of the present invention, to confirm the inhibitory effect of AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08 on glioblastoma metastasis.

[0157] Figure 11 shows H NMR data of AON-MG23-05, a derivative of compound AON-MG23-02 of the present invention.

[0158] Figure 12 shows H NMR data of AON-MG23-06, a derivative of the compound AON-MG23-02 of the present invention.

[0159] Figure 13 shows H NMR data of AON-MG23-07, a derivative of the compound AON-MG23-02 of the present invention.

[0160] Figure 14 shows H NMR data of AON-MG23-08, a derivative of compound AON-MG23-02 of the present invention.

[0161]

[0162] The present invention relates to a novel compound having an anoctamin 1 (ANO1) inhibitory effect, and was completed by confirming that the compound can inhibit the growth and metastasis of brain tumor cells by inhibiting the expression or activity of ANO1, a calcium-dependent chloride channel. In particular, the compound of the present invention was confirmed to effectively inhibit the expression of EGFR together with ANO1 in brain tumor cells, and thus is expected to exhibit a more potent anticancer effect than conventional single inhibitors of ANO1 or EGFR, and further, is expected to be able to inhibit the development of resistance to EGFR targeted therapeutic agents in brain tumor cells.

[0163] Accordingly, an 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.

[0164] [Chemical Formula 1]

[0165]

[0166] [Chemical Formula 2]

[0167]

[0168] [Chemical Formula 3]

[0169]

[0170] [Chemical Formula 4]

[0171]

[0172] [Chemical Formula 5]

[0173]

[0174] [Chemical Formula 6]

[0175]

[0176] In the present invention, the compound represented by the above chemical formula 1 may be referred to as "N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide" or "AON-MG23-01". In addition, the compound represented by the above chemical formula 2 may be referred to as "N-(2-((5-chloro-2-((4-(4-(dimethylamino)piperidin-1-yl)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide" or "AON-MG23-02".

[0177] The term "Ms" as used herein means methylsulfonyl, i.e., -SO2(CH3).

[0178] Additionally, the compound represented by the above 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".

[0179] The compound represented by the above 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”.

[0180] The compound represented by the above 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”.

[0181] The compound represented by the above chemical formula 6 may be referred to as "1-(naphthalen-2-ylmethyl)-1-(4-((1-oxoisoindolin-2-yl)methyl)phenethyl)urea (1-(naphthalen-2-ylmethyl)-1-(4-((1-oxoisoindolin-2-yl)methyl)phenethyl)urea)", "AON-MG23-08", or "Target D".

[0182] Unless otherwise stated herein, the terms “compound of the present invention” or “compound represented by chemical formulae 1 to 28” are used as a concept encompassing all compounds represented by chemical formulae 1 to 28 themselves, salts thereof, and isomers thereof.

[0183] In the present invention, the term “pharmaceutically acceptable salt” includes all salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

[0184] As used herein, the term "pharmaceutically acceptable" means a compound or composition that is suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problems or complications, and with a reasonable benefit / risk ratio, and is within the scope of sound medical judgment.

[0185] Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, hydroiodic acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, (+)-L-tartaric acid, diL-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)-camphorsulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-Hydroxyethanesulfonic acid, galactaric acid, gentisic acid, glucoheptanoic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, a-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, di-oxalic acid, palmitic acid, pamoic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, Examples thereof include stearic acid, tannic acid, thiocyanic acid, camsylic acid, and undecylic acid. Acid addition salts can be prepared by conventional methods, for example, by dissolving the 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, equimolar amounts of the compound and an acid or alcohol in water can be heated, and the mixture is then evaporated to dryness, or the precipitated salt can be filtered with suction.

[0186] The scope of the compounds of the present invention may include not only pharmaceutically acceptable salts, but also all isomers, hydrates and solvates that can be prepared by conventional methods.

[0187] In the present invention, the term "isomer" refers to a compound having the same molecular formula but different connection methods or spatial arrangements of constituent atoms within the molecule. 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 left and right hands, and are also called optical isomers. Enantiomers are distinguished as R (Rectus: clockwise) and S (Sinister: counterclockwise) when four or more substituents are different at the chiral center carbon. Diastereoisomers refer to stereoisomers that are not mirror images, and can be divided into cis-trans isomers due to different spatial arrangements of atoms.

[0188]

[0189] In addition, the present invention comprises a step (S1A) of preparing a compound represented by the following chemical formula 8 by reacting a compound represented by the following chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base (Reaction Scheme 2); or

[0190] (S1B) Provided is a method for producing a compound represented by the chemical formulae 1 to 6, comprising a step (reaction formula 7 or 13) of reacting a compound represented by the chemical formula 9 below with a compound represented by the chemical formula 10 or 11 below in the presence of a base:

[0191] [Chemical Formula 7]

[0192]

[0193] [Chemical Formula 8]

[0194]

[0195] [Chemical Formula 9]

[0196]

[0197] [Chemical Formula 10]

[0198]

[0199] [Chemical Formula 11]

[0200]

[0201] According to one embodiment of the present invention, the base in the step (S1A) or step (S1B) may be, but is not limited to, Cs2CO3.

[0202] According to one embodiment of the present invention, the step (S1A) may further include, but is not limited to, the following steps:

[0203] (S1A-1) A step of reducing a compound represented by the above chemical formula 8 (nitro group) with zinc in the presence of ammonium chloride to produce a compound represented by the following chemical formula 12 (Reaction Formula 3);

[0204] (S1A-2) A step of preparing a compound represented by the following chemical formula 13 by reacting a compound represented by the above chemical formula 12 with 2, 4, 5-trichloropyrimidine in the presence of a base (Reaction Scheme 4); and

[0205] (S1A-3) A step of producing a compound represented by the chemical formula 1 or 2 by reacting a compound represented by the chemical formula 13 and a compound represented by the chemical formula 14 or 15 below in the presence of an acid (reaction formula 5 or 6).

[0206] [Chemical Formula 12]

[0207]

[0208] [Chemical Formula 13]

[0209]

[0210] [Chemical Formula 14]

[0211]

[0212] [Chemical Formula 15]

[0213]

[0214] According to one embodiment of the present invention, when the compound represented by the chemical formula 13 and the compound represented by the chemical formula 14 are reacted in the presence of an acid in the step (S1A-3), the compound represented by the chemical formula 1 is produced (Reaction Scheme 5).

[0215] When the compound represented by the above chemical formula 13 and the compound represented by the above chemical formula 15 are reacted in the presence of an acid, the compound represented by the above chemical formula 2 can be produced (Reaction Scheme 6), and specifically, the step (S1A-3) may be a reaction of the compound represented by the above chemical formula 13 with an amino group of the compound represented by the above chemical formula 14 or 15.

[0216] According to one embodiment of the present invention, in the step (S1A-2), the base may be DIPEA (N,N-diisopropylethylamine), and in the step (S1A-3), the acid may be TFA (trifluoroacetic acid), but is not limited thereto.

[0217] In the present invention, when the compound represented by the chemical formula 9 and the compound represented by the chemical formula 10 react in the presence of a base in the step (S1B), a compound represented by the chemical formula 16 below 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.

[0218] [Chemical Formula 16]

[0219]

[0220] According to one embodiment of the present invention, the step (S1B) may further include, but is not limited to, the following steps.

[0221] (S1B-1) A step of producing a compound represented by the following chemical formula 17 by reacting a compound represented by the above chemical formula 16 with trimethylsilyl cyanide in the presence of a base (Reaction Formula 8 or 20);

[0222] (S1B-2) A step of producing a compound represented by the following chemical formula 18 by hydrogenating a compound represented by the above chemical formula 17 in the presence of a metal catalyst (Reaction Formula 9 or 21);

[0223] (S1B-3) A step of preparing a compound represented by the following chemical formula 20 by reacting a compound represented by the above chemical formula 18 with a compound represented by the following chemical formula 19 in the presence of a reducing agent (reaction formula 10 or 22); and

[0224] (S1B-4) Compound represented by the above chemical formula 20 and NCO - A step of reacting the compound represented by the chemical formula 6 above (Reaction Scheme 23); or a step of reacting the compound represented by the chemical formula 20 above with the compound represented by the chemical formula 21 below in the presence of a base to produce a compound represented by the chemical formula 22 below (Reaction Scheme 11). According to one embodiment of the present invention, in the step (S1B-1), the base may be Cs2CO3, and in the step (S1B-4), the base may be K2CO3, but is not limited thereto.

[0225] [Chemical Formula 17]

[0226]

[0227] [Chemical Formula 18]

[0228]

[0229] [Chemical Formula 19]

[0230]

[0231] [Chemical Formula 20]

[0232]

[0233] [Chemical Formula 21]

[0234]

[0235] [Chemical Formula 22]

[0236]

[0237] In the present invention, the step (S1B-4) may additionally include a step of preparing a compound represented by the chemical formula 3 by subjecting the compound represented by the chemical formula 22 to a hydrolysis reaction in the presence of an acid (Reaction Scheme 12), but is not limited thereto. According to one embodiment of the present invention, the acid in the step (S1B-4) may be hydrogen chloride (HCl), but is not limited thereto.

[0238] In the present invention, the metal catalyst may be at least one selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney-Ni, and according to one embodiment of the present invention, the metal catalyst may be Raney-Ni, but is not limited thereto.

[0239] In the present invention, the reducing agent may be at least one selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium borohydride (NaBH4), and according to one embodiment of the present invention, the reducing agent in the (S1B-3) step may be sodium cyanoborohydride (NaBH3CN), but is not limited thereto.

[0240] In the present invention, when the compound represented by the chemical formula 9 and the compound represented by the chemical formula 11 react in the presence of a base in the step (S1B), a compound represented by the chemical formula 23 below can be produced (reaction formula 13), but is not limited thereto.

[0241] [Chemical Formula 23]

[0242]

[0243] In the present invention, the (S1B) step may further include, but is not limited to, the following steps:

[0244] (S1Ba-1) A step of producing a compound represented by the following chemical formula 24 by reacting a compound represented by the above chemical formula 23 with a peroxide (Reaction Formula 14), and

[0245] (S1Ba-2) A step of preparing a compound represented by the chemical formula 4 by reacting a compound represented by the chemical formula 24 and a compound represented by the chemical formula 25 below in the presence of a base (Reaction Scheme 16); or

[0246] (S1Bb-1) A step for producing a compound represented by the following chemical formula 26 by phosphorylating a compound represented by the above chemical formula 23 in the presence of a base, specifically, a step for producing a compound represented by the following chemical formula 26 through a β-alkylstyrene phosphorylation reaction (Reaction Scheme 17), and

[0247] (S1Bb-2) A step of producing a compound represented by the chemical formula 5 by reacting a compound represented by the chemical formula 26 and a compound represented by the chemical formula 27 below in the presence of a base (Reaction Formula 18).

[0248] According to one embodiment of the present invention, the base in the (S1Ba-2) step may be DIEA (N,N-Diisopropylethylamine), the base in the (S1Bb-1) step may be KOH, and the base in the (S1Bb-2) step may be NaH, but is not limited thereto.

[0249] [Chemical Formula 24]

[0250]

[0251] [Chemical Formula 25]

[0252]

[0253] [Chemical Formula 26]

[0254]

[0255] [Chemical Formula 27]

[0256]

[0257] In the present invention, the compound represented by the chemical formula 25 can be prepared by reacting the compound represented by the chemical formula 28 below 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 triacetoxyborohydride (NaBH(OAc)3), but is not limited thereto.

[0258] [Chemical Formula 28]

[0259]

[0260] In the present invention, the peroxide may be at least one 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.

[0261] In the present invention, the acid may be at least one selected from the group consisting of pivalic acid (PivOH), acetic acid (AcOH), trifluoromethanesulfonic acid (TfOH), paratoluenesulfonic acid (TsOH), benzoic acid, zinc bromide (ZnBr2), hydrogen chloride (HCl), and trifluoroacetic acid (TFA), but is not limited thereto.

[0262] In the present invention, the base may be at least one 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.

[0263] In the present invention, the manufacturing method can be carried out in one or more solvents selected from the group consisting of organic solvents, water, and mixtures thereof, and the organic solvent can be one or more selected from the group consisting of dichloromethane (DCM), dichloroethane (DCE), 1,4-dioxane, tetrahydrofuran (THF), toluene, hexane, benzene, xylene, chlorobenzene, methanol (MeOH), ethanol (EtOH), t-amyl alcohol (t-AmOH), trifluoroethanol (TFE), 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.

[0264] In addition, the present invention provides a method for preparing a compound represented by chemical formula 1, comprising the following steps:

[0265] (a) A step of preparing a compound represented by the chemical formula 8 by reacting the compound represented by the chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base (Reaction Scheme 2);

[0266] (b) a step of reducing the compound represented by the chemical formula 8 (the nitro group) with zinc in the presence of ammonium chloride to produce a compound represented by the chemical formula 12 (reaction formula 3);

[0267] (c) a step of preparing a compound represented by the following chemical formula 13 by reacting the compound represented by the above chemical formula 12 with 2, 4, 5-trichloropyrimidine in the presence of a base (reaction formula 4); and

[0268] (d) A step of producing a compound represented by the chemical formula 1 by reacting a compound represented by the chemical formula 13 and a compound represented by the chemical formula 14 below in the presence of an acid (Reaction Formula 5).

[0269] In addition, the present invention provides a method for preparing a compound represented by chemical formula 2, comprising the following steps:

[0270] (a) A step of preparing a compound represented by the chemical formula 8 by reacting the compound represented by the chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base (Reaction Scheme 2);

[0271] (b) a step of reducing the compound represented by the chemical formula 8 (the nitro group) with zinc in the presence of ammonium chloride to produce a compound represented by the chemical formula 12 (reaction formula 3);

[0272] (c) a step of preparing a compound represented by the following chemical formula 13 by reacting the compound represented by the above chemical formula 12 with 2, 4, 5-trichloropyrimidine in the presence of a base (reaction formula 4); and

[0273] (d) A step of producing a compound represented by the chemical formula 2 by reacting a compound represented by the chemical formula 13 and a compound represented by the chemical formula 15 below in the presence of an acid (reaction formula 6).

[0274] In addition, the present invention provides a method for preparing a compound represented by chemical formula 3, comprising the following steps:

[0275] (a) a step of producing a compound represented by chemical formula 16 by reacting a compound represented by chemical formula 9 and a compound represented by chemical formula 10 in the presence of a base (reaction formula 7);

[0276] (b) a step of producing a compound represented by the chemical formula 17 by reacting the compound represented by the chemical formula 16 with trimethylsilyl cyanide in the presence of a base (reaction formula 8);

[0277] (c) a step of producing a compound represented by the chemical formula 18 by hydrogenating the compound represented by the chemical formula 17 in the presence of a metal catalyst (reaction formula 9);

[0278] (d) a step of producing a compound represented by the chemical formula 20 by reacting a compound represented by the chemical formula 18 and a compound represented by the chemical formula 19 in the presence of a reducing agent (reaction formula 10);

[0279] (d) a step of preparing a compound represented by the chemical formula 22 by reacting a compound represented by the chemical formula 20 and a compound represented by the chemical formula 21 in the presence of a base (reaction formula 11); and

[0280] (e) A step of producing a compound represented by the chemical formula 3 by subjecting the compound represented by the chemical formula 22 to a hydrolysis reaction in the presence of an acid (reaction formula 12).

[0281] In addition, the present invention provides a method for preparing a compound represented by chemical formula 4, comprising the following steps:

[0282] (a) a step of producing a compound represented by the chemical formula 23 by reacting a compound represented by the chemical formula 9 and a compound represented by the chemical formula 11 in the presence of a base (reaction formula 13);

[0283] (b) a step of reacting a compound represented by the chemical formula 23 with a peroxide to produce a compound represented by the chemical formula 24 (reaction formula 14);

[0284] (c) A step of producing a compound represented by the chemical formula 4 by reacting a compound represented by the chemical formula 24 and a compound represented by the chemical formula 25 in the presence of a base (Reaction Scheme 16).

[0285] In addition, the present invention provides a method for preparing a compound represented by chemical formula 5, comprising the following steps:

[0286] (a) a step of producing a compound represented by the chemical formula 23 by reacting a compound represented by the chemical formula 9 and a compound represented by the chemical formula 11 in the presence of a base (reaction formula 13);

[0287] (b) a step of producing a compound represented by the chemical formula 26 by phosphorylating the compound represented by the chemical formula 23 in the presence of a base, specifically, a step of producing a compound represented by the chemical formula 26 through a β-alkylstyrene phosphorylation reaction (Reaction Scheme 17); and

[0288] (c) A step of producing a compound represented by the chemical formula 5 by reacting a compound represented by the chemical formula 26 and a compound represented by the chemical formula 27 in the presence of a base (Reaction Scheme 18).

[0289] In addition, the present invention provides a method for preparing a compound represented by chemical formula 6, comprising the following steps:

[0290] (a) a step of producing a compound represented by chemical formula 16 by reacting a compound represented by chemical formula 9 and a compound represented by chemical formula 10 in the presence of a base (reaction formula 19);

[0291] (b) a step of producing a compound represented by the chemical formula 17 by reacting the compound represented by the chemical formula 16 with trimethylsilyl cyanide in the presence of a base (reaction formula 20);

[0292] (c) a step of producing a compound represented by the chemical formula 18 by hydrogenating the compound represented by the chemical formula 17 in the presence of a metal catalyst (reaction formula 21);

[0293] (d) a step of producing a compound represented by the chemical formula 20 by reacting a compound represented by the chemical formula 18 and a compound represented by the chemical formula 19 in the presence of a reducing agent (reaction formula 22);

[0294] (d) a compound represented by the above chemical formula 20 and NCO - A step of producing a compound represented by the chemical formula 6 by reacting (reaction formula 23).

[0295]

[0296] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating brain tumors, comprising as an active ingredient at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0297] The compound according to the present invention is an inhibitor of the calcium-dependent chloride channel Anoctamin 1 (ANO1), and can be used for the prevention, improvement, and / or treatment of brain tumors. In particular, the compound according to the present invention is characterized by exhibiting excellent anticancer effects by simultaneously inhibiting the expression or activity of EGFR and ANO1. The effect of preventing and / or treating brain tumors includes not only the effect of inhibiting the growth of brain tumor cells, but also the effect of inhibiting the progression or worsening of brain tumors due to migration, invasion, metastasis, etc.

[0298] In the present invention, "EGFR (epidermal growth factor receptor)" is a transmembrane glycoprotein that is a member of the protein kinase superfamily and is a receptor for epidermal growth factor family proteins. EGFR is a major regulator of cell growth. When the ligand epidermal growth factor (EGF) binds to EGFR, the receptor dimerizes and undergoes tyrosine autophosphorylation, inducing cell proliferation. Overexpression (amplification) or hyperactivity of EGFR is frequently observed in various cancers, including anal cancer, head and neck cancer, and glioblastoma. Somatic mutations related to EGFR cause persistent activation of EGFR, leading to uncontrolled cell division. In particular, EGFR gene rearrangement and EGFR gene amplification are observed in various cancers. Among them, the most common extracellular domain mutation is EGFRvIII. The EGFRvIII mutation is characterized by a deletion of approximately 801 base pairs corresponding to exons 2 to 7 of the EGFR gene, and the resulting EGFR protein lacks 267 amino acids corresponding to the extracellular domain. Unlike normal EGFR, which is activated only upon ligand binding, the mutant EGFR remains continuously activated (PCT gain-of-function mutation). EGFRvIII is particularly common in glioblastoma.

[0299] In the present invention, "Anoctamin 1 (ANO1)" is a calcium-activated chloride ion channel (Cl), also referred to as "transmembrane protein 16A (TMEM16A)." -ANO1 is expressed in various normal cells, including epithelial cells, smooth muscle cells, vascular endothelial cells, and neurons, and performs various physiological roles by regulating fluid secretion, muscle contraction, and pain sensation. However, ANO1 is also known to be associated with cancer invasiveness and poor prognosis in brain tumors, including glioblastoma. In fact, overexpression of ANO1 has been reported to promote signaling pathways that stimulate proliferation and migration in cancer cells, and inhibition of ANO1 inhibits cancer cell migration and growth. High expression of ANO1 appears to be associated with high expression of EGFR and STAT3, and inhibition of ANO1 is also known to 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 discovered. The compound according to the present invention effectively inhibits the expression of ANO1 in brain tumor cells and has been confirmed to inhibit the migration, proliferation, metastasis, etc. of brain tumor cells more potently than conventionally known ANO1 inhibitors, and thus can be used for the prevention, treatment, and metastasis inhibition of brain tumors. Furthermore, previous studies on the improvement in the effect of EGFR / HER2 targeted therapy by ANO1 inhibition suggest that the compound according to the present invention can increase the responsiveness of brain tumor cells to EGFR / HER2 targeted therapy and inhibit the development of resistance through the ANO1 inhibitory effect.

[0300] In particular, the compound according to the present invention simultaneously inhibits the expression of EGFR together with ANO1 in brain tumor cells, and thus can exhibit a more potent anticancer effect through the simultaneous inhibition of ANO1 and EGFR. The relationship between ANO1 and EGFR is well known. The upregulation of ANO1 is related to the EGFR signaling pathway and has been reported to have a significant effect on the remodeling of the phosphorylated proteome after epidermal growth factor (EGF) stimulation. Furthermore, in cancer cells, ANO1 can form a functional complex with EGFR to jointly regulate cell proliferation. Therefore, when the compound of the present invention is used to simultaneously inhibit EGFR and ANO1, the growth of brain tumors can be inhibited more effectively.

[0301] Meanwhile, ANO1 is known to be overexpressed in cancer cells and contribute to cancer cell migration and tumor metastasis, and signaling through EGFR is also known to create a tumor microenvironment that is conducive to tumor metastasis. Therefore, the compound according to the present invention can effectively inhibit brain tumor metastasis through simultaneous inhibition of ANO1 and EGFR. The term "metastasis" refers to a state in which a malignant tumor has spread to other tissues distant from the organ in which it developed. Therefore, the composition according to the present invention can prevent and treat brain tumors from spreading throughout the body by inhibiting brain tumor metastasis.

[0302] The compound according to the present invention or a pharmaceutically acceptable salt thereof can be used for the prevention or treatment of various cancers.

[0303] In the present invention, "tumor" is used with the same meaning as "cancer", and typically refers to a condition characterized by uncontrolled cell growth, migration, and proliferation. Preferably, the cancer according to the present invention is a brain tumor. The cancer according to the present invention includes primary and recurrent cancers, and both benign and malignant tumors. In the present invention, the brain tumor refers to all tumors that occur within the skull, and is not limited to a specific type, and includes all tumors that occur in the brain and structures surrounding the brain. In the present invention, the brain tumor includes primary brain tumors, recurrent brain tumors, and metastatic brain tumors. In addition, the brain tumor includes all types of astrocytomas, malignant astrocytomas, glioblastomas, meningiomas, pituitary adenomas, schwannomas, acoustic neuromas, and craniopharyngiomas, and is not limited to a specific type.

[0304] 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 a mutation of EGFR and / or ANO1. The mutation of EGFR and / or ANO1 includes amplification of the gene of EGFR and / or ANO1, overexpression of the protein, overactivity of the protein, and persistent 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 higher than that of normal cells. Alternatively, the cancer according to the present invention may be a brain tumor in which the activation state of EGFR and / or ANO1 is persistent. Since the compound according to the present invention can simultaneously inhibit the expression and activity of ANO1 and EGFR, it can exhibit particularly excellent anticancer effects on brain tumors carrying mutations in EGFR and / or ANO1.

[0305] Alternatively, the cancer according to the present invention may be a brain tumor with an EGFRvIII (epidermal growth factor receptor variant III) mutation. The EGFRvIII mutation is a mutation in which amino acids 6 to 273 of EGFR are deleted (or exons 2 to 7 are deleted), and is characterized by its continued activation even without binding of a ligand (EGF). EGFRvIII is specifically expressed only in cancer cells and is found in approximately 30% of glioblastomas.

[0306] The compound according to the present invention can exhibit one or more effects from the group consisting of:

[0307] (a) inhibiting the proliferation or growth of brain tumor cells;

[0308] (b) inhibiting the migration of brain tumor cells;

[0309] (c) inhibiting metastasis of brain tumors;

[0310] (d) inhibiting resistance of brain tumors to anticancer drugs; and

[0311] (e) Enhances the responsiveness of brain tumor cells to anticancer drugs.

[0312] The present inventors confirmed through specific examples that brain tumor cells treated with the compound according to the present invention were inhibited from proliferation, migration, and metastasis (invasiveness), and the anticancer effect of the compound was found to be stronger than that of the previously known ANO1 inhibitor (CaCCinh-A01).

[0313] Furthermore, since the compound according to the present invention can simultaneously inhibit ANO1 and EGFR (i.e., dual inhibition), it can exhibit a more excellent anticancer effect than a single inhibitor of ANO1 or EGFR, and in particular, it can suppress the development of resistance of cancer cells to anticancer drugs through ANO1 inhibition and enhance the anticancer effect of the anticancer drug. For example, the inventors of the present invention confirmed through experiments that not only the ANO1 inhibitory effect of the compound of the present invention is superior to the conventional ANO1 inhibitor CaCCinh-A01, but also the anticancer effect is superior to the ANO1 inhibitor CaCCinh-A01, the EGFR inhibitor Osimertinib, and the combination of Osimertinib and CaCCinh-A01.

[0314] In one embodiment of the present invention, the anticancer agent described in (d) and (e) above may be a targeted anticancer agent for tyrosine kinase, but is not limited thereto. In the present invention, the term "targeted anticancer agent" refers to an agent that exhibits an anticancer effect by targeting a protein or gene that is specifically changed in cancer cells or cancer tissues and interfering with molecular activities involved in the growth and development of cancer. Preferably, the tyrosine kinase may be at least one 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 EGFR includes normal EGFR and mutant EGFR (e.g., EGFRvIII).

[0315] In the above (e), the enhanced responsiveness of cancer cells to anticancer drugs means that the inhibition of cancer cell growth and migration by the anticancer drugs is further enhanced.

[0316] In the present invention, “enhancing anticancer effect” refers to all effects that can ultimately strengthen the function of an anticancer agent, and is a concept that includes not only enhancing the anticancer effect of an anticancer agent, such as suppressing tumor growth, suppressing tumor metastasis, and suppressing tumor recurrence, but also suppressing the formation of resistance or tolerance in cancer cells to the anticancer agent, thereby ultimately enhancing the anticancer effect.

[0317] Therefore, the compound according to the present invention can be used as a compound for combined administration with a known anticancer agent (preferably a target anticancer agent for tyrosine kinase) for the purpose of enhancing the anticancer effect of a brain tumor anticancer agent.

[0318] For example, the present invention provides a pharmaceutical composition for preventing or treating cancer (preferably, brain tumor), comprising (i) at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof; and (ii) a targeted anticancer agent for EGFR as an active ingredient.

[0319]

[0320] In addition, the present invention provides a pharmaceutical composition for enhancing the anticancer effect of an anticancer agent (preferably, a brain tumor anticancer agent), which comprises as an active ingredient at least one compound selected from the group consisting of compounds represented by the above chemical formulas 1 to 6 or a pharmaceutically acceptable salt thereof.

[0321] The composition according to the present invention is in the form of a mixture comprising the compound or a pharmaceutically acceptable salt thereof; and the target anticancer agent for EGFR, and may be in the form for simultaneous administration of the compound or a pharmaceutically acceptable salt thereof; and the target anticancer agent for EGFR.

[0322] In addition, the composition according to the present invention may be in a form in which the compound or a pharmaceutically acceptable salt thereof and the EGFR-targeting anticancer agent are each formulated and administered simultaneously, separately, or sequentially. In this case, the composition may be a pharmaceutical composition for combination administration 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 EGFR-targeting anticancer agent as an active ingredient. In this case, in the case of sequential administration, the administration order is not limited, and the administration regimen may be appropriately adjusted depending on the patient's condition, etc.

[0323] That is, when the pharmaceutical composition is a combination-administration pharmaceutical composition for sequential administration, the composition may be administered such that the compound or its salt (“first component”) is administered first and then the EGFR-targeting anticancer agent (“second component”) is administered, and the reverse order is also possible.

[0324] In addition, when the compound according to the present invention is used in combination with an anticancer agent, the compound may be administered simultaneously, separately, or sequentially with the anticancer agent. Even when administered sequentially with the anticancer agent, the administration order is not limited, but the administration regimen may be appropriately adjusted depending on the type of cancer, the type of anticancer agent, the condition of the patient, etc.

[0325] The content of the compound in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9 wt% or 0.001 to 50 wt% based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0326] 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 excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0327] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0328] 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, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0329] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0330] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao 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 ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0331] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0332] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0333] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0334] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0335] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include 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; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0336] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0337] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0338] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0339] 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. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0340] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0341] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0342] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, along with various related factors such as the disease to be 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 may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg, administered daily or every other day, or divided into 1 to 3 times a day. However, since the dosage may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0343] In the present invention, the term "subject" means a subject requiring treatment for a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0344] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any appropriate method.

[0345] In the present invention, “prevention” means any action that suppresses or delays the onset of a target disease, “treatment” means any action that improves or beneficially changes a target disease and its metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0346] In addition, the present invention provides a kit for preventing or treating cancer comprising a pharmaceutical composition according to the present invention.

[0347] The kit according to the present invention may include, without limitation, other components, compositions, solutions, devices, etc. that are typically required for the prevention or treatment of cancer, in addition to the compound and the targeted anticancer agent, and in particular, may include instructions for appropriate use and storage of the compound according to the present invention.

[0348]

[0349] Terms or words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0350]

[0351] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0352]

[0353] [Example]

[0354] Example A. Novel compounds AON-MG23-01 and AON-MG23-02

[0355] 1. Preparation of a compound according to the present invention

[0356] 1-1. Preparation of N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide (AON-MG23-01)

[0357] [Reaction Formula 1]

[0358]

[0359] The AON-MG23-01 compound was prepared through the above reaction scheme 1. The specific preparation method is as follows:

[0360] <Step 1> Synthesis of N-cyclopropyl-N-(2-nitrophenyl)methanesulfonamide

[0361] [Reaction Formula 2]

[0362]

[0363] 1-Fluoro-2-nitrobenzene (1.00 eq) and cyclopropyl amine (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, refluxed, and stirred overnight. After completion of the reaction, the temperature was lowered to room temperature, and extraction was performed using ethyl acetate and water. The organic layer was added with anhydrous sodium sulfate, stirred, 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 light yellow solid, with a yield of 36%.

[0364]

[0365] <Step 2> Synthesis of N-(2-aminophenyl)-N-cyclopropylmethanesulfonamide

[0366] [Reaction Formula 3]

[0367]

[0368] N-Cyclopropyl-N-(2-nitrophenyl)methanesulfonamide (1.00 eq) synthesized in Step 1 was added to 1,4-dioxane and water (3:1) and stirred, then the reactor was cooled to 0°C, and zinc (Zn, 10.0 eq) and ammonium chloride (NH4Cl, 10.0 eq) were added. The mixture was stirred for 4 hours while gradually increasing the temperature. After completion of the reaction, the mixture was filtered using Celite, and the filtrate was extracted with ethyl acetate and water. The organic layer was added with anhydrous sodium sulfate, stirred, filtered using a filter, and the filtrate was concentrated under reduced pressure. The concentrated residue was used in Step 3 without any additional purification.

[0369]

[0370] <Step 3> Synthesis of N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide

[0371] [Reaction Formula 4]

[0372]

[0373] 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 refluxed and stirred overnight. After completion of the reaction, the mixture was evaporated under reduced pressure and extracted with water and dichloromethane. The organic layer was washed with 2 N hydrochloric acid, and anhydrous sodium sulfate was added to the organic layer and stirred. Then, the mixture was filtered using a filter, and the filtrate was concentrated under reduced pressure. The concentrated residue was purified by column chromatography (hexane:ethyl acetate = 3:1) to synthesize N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide as a light yellow solid, with a yield of 58.9%.

[0374]

[0375] <Step 4> Synthesis of N-(2-((5-chloro-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)-N-cyclopropylmethanesulfonamide

[0376] [Reaction Formula 5]

[0377]

[0378] N-Cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide (1.00 eq) synthesized in Step 3 was added to ethanol (EtOH), and 2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline (1.00 eq) and trifluoroacetic acid (TFA, 1.95 eq) were added at room temperature, followed by reflux and stirring overnight. After completion of the reaction, the mixture was neutralized with 1 N sodium hydroxide solution and extracted with water and ethyl acetate. The organic layer was added with anhydrous sodium sulfate, stirred, 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%.

[0379] H NMR (500 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.62 (dd, J = 7.9, 1.6 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.15 (td, J = 7.6, 1.5 Hz, 1H), 6.62 (d, J = 2.5 Hz, 1H), 6.47 (dd, J = 8.7, 2.5 Hz, 1H), 3.75 - 3.71 (m, 5H), 3.25 - 3.22 (m, 4H), 2.67 (td, J = 12.2, 2.4 Hz, 2H), 2.55 - 2.44 (m, 4H), 2.38 - 2.27 (m, 4H), 2.15 (s, 3H), 1.85 (d, J = 11.5 Hz, 2H), 1.52 (qd, J = 12.1, 3.9 Hz, 2H), 1.01 - 0.93 (m, 2H), 0.55 - 0.49 (m, 1H), 0.17 - 0.12 (m, 1H). HRMS: 640.2708, cal: 640.2711

[0380]

[0381] 1-2. Preparation 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)

[0382] [Reaction Formula 6]

[0383]

[0384] Compound AON-MG23-01 and Steps 1, 2, and 3 were performed in the same manner, and in Step 4, 1-(4-amino-3-methoxyphenyl)-N,N-dimethylpiperidin-4-amine was used instead of 2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)aniline.

[0385] Specifically, N-cyclopropyl-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide (1.00 eq) synthesized in Step 3 was added to ethanol (EtOH), 1-(4-amino-3-methoxyphenyl)-N,N-dimethylpiperidin-4-amine (1.00 eq) and trifluoroacetic acid (TFA, 1.95 eq) were added at room temperature, and the mixture was refluxed and stirred overnight. After completion of the reaction, the mixture was neutralized with 1 N sodium hydroxide solution and extracted with water and ethyl acetate. The organic layer was added with anhydrous sodium sulfate, stirred, 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%.

[0386] 1H NMR (500 MHz, DMSO-d6) δ 8.33 (s, 1H), 8.19 (s, 1H), 8.09 (s, 1H), 7.99 (s, 1H), 7.62 (dd, J = 7.8, 1.6 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H),7.22 (d, J = 8.1 Hz, 1H), 7.15 (td, J = 7.6, 1.6 Hz, 1H), 6.63 (d, J = 2.5 Hz, 1H), 6.48(dd, J = 8.7, 2.5 Hz, 1H), 3.75 (s, 3H), 3.72 (d, J = 12.2 Hz, 2H), 3.23 (m, 4H), 2.68 (td, J = 12.1, 2.5 Hz, 2H), 2.22 (s, 6H), 1.86 (d, J = 12.4 Hz, 2H), 1.51 (qd, J = 12.0,3.9 Hz, 2H), 1.04 - 0.92 (m, 2H), 0.55 - 0.49 (m, 1H), 0.17 - 0.14 (m, 1H). HRMS:585.2294, cal: 585.2289.

[0387]

[0388] Compounds according to the present invention were prepared through the above steps, and their structural formulas are shown in Table 1 and Figure 1 below.

[0389]

[0390]

[0391]

[0392] 2. Confirmation of the cell proliferation inhibitory effect of the compound of the present invention on human glioblastoma cells

[0393] 1X10 human glioblastoma cell line U87-MG cells were seeded in a 96-well plate 4After seeding at a density of 10 cells / well and culturing for 24 hours, the cells were treated with DMSO as a control and AON-MG23-01 or AON-MG23-02, a compound of the present invention, respectively, and then cultured for 48 hours. The compound of the present invention was treated at various concentrations of 0, 1, 2, and 4 μM. 10 μl of CCK reagent was added, reacted for 1 hour, and then measured at 450 nm absorbance.

[0394] As a result, as shown in Fig. 2, it was confirmed that the compound AON-MG23-01 or AON-MG23-02 of the present invention inhibits the proliferation of U87-MG cells.

[0395]

[0396] 3. Confirmation of the cell migration inhibitory effect of the compound of the present invention on human glioblastoma cells

[0397] 8Х10 human glioblastoma cell line U87-MG cells were plated on SPLScar Block (SPL) plates 4 After seeding at a density of 10 cells / well and culturing for 24 hours, the block was removed after 24 hours, and the compounds of the present invention, AON-MG23-01 or AON-MG23-02, were respectively treated, and then cultured at 37°C in a CO2 incubator for 24 hours. The compounds of the present invention were treated at concentrations of 0, 1, 2, and 4 μM, respectively. Pictures were taken before and after culturing, and the distance of cell migration was measured using Image J software, and based on the results, whether cell migration was inhibited was analyzed.

[0398] As a result, as shown in Fig. 3, when the compound AON-MG23-01 or AON-MG23-02 of the present invention was treated at a concentration of 4 μM, cell migration was inhibited by more than 65% compared to the untreated control group.

[0399]

[0400] 4. Confirmation of the metastasis-inhibiting effect of the compound of the present invention on human glioblastoma

[0401] The human glioblastoma cell line U87-MG was plated on top of a Matrigel-coated filter (Transwell invasion chambers, Corning) at 8X10 4 After seeding at a density of 10 cells / well, the cells were treated with each compound of the present invention (AON-MG23-01 or AON-MG23-02; 0, 1, 2, or 4 μM) and cultured for 24 hours at 37°C in a CO2 incubator. After completion of the culture, cells were fixed and stained using the Diff-Quick Stain Kit (Sysmex, Kobe, Japan). Images of the stained cells were captured under a microscope, and cells were counted using Image J software, followed by statistical analysis.

[0402] As a result, as shown in Fig. 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 the matrix gel compared to the untreated control group, confirming that the metastatic ability of cancer cells was suppressed.

[0403]

[0404] 5. Confirmation of the effect of the compound of the present invention on ANO1 and EGFR protein expression in glioblastoma cell lines.

[0405] It was confirmed whether the compound of the present invention affects the expression of ANO1 and EGFR proteins in glioblastoma cell lines. The human glioblastoma cell line U87-MG was cultured in a 60 mm dish at a density of 1X10 6After seeding at a cell density of 100 cells and culturing for 24 hours, the cells were treated with each compound of the present invention (AON-MG23-01 or AON-MG23-02; 0, 1, 2, 4 μM) and cultured for 48 hours. After culturing, the cells were harvested and lysed for Western blotting. Antibodies 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 same amount (10 μg) of protein obtained therefrom was subjected to 10% SDS-PAGE to separate the proteins by molecular weight, 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. Then, the PVDF membrane was treated with the primary antibody and reacted at 4°C for 16 hours. After the reaction, the PVDF membrane was washed three times with TBS-T. After that, the PVDF membrane was reacted with the 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 Image J software.

[0406] As a result, as shown in Fig. 5, the protein levels of ANO1 and EGFR were significantly suppressed in cells treated with the compound of the present invention compared to the untreated control group, and this protein expression suppression effect was dependent on the treatment concentration of the compound.

[0407]

[0408] 6. Confirmation of the inhibitory effect of the compound of the present invention on ANO1 activity in glioblastoma cell lines.

[0409] The inhibitory effect on ANO1 ion channel activity was confirmed by measuring calcium-dependent chloride ion channel activity by the compound of the present invention using patch clamp. For this purpose, patch clamp was performed on U87-MG cells expressing ANO1. The pipette solution was composed of (in mM): 146 CsCl, 5 Ca-EGTA-NMDG, 8 HEPES, 2 MgCl2, and 10 sucrose (pH 7.3), and the bath solution was composed of (in mM): 50 NaCl, 10 HEPES, 3 KCl, 2 CaCl2, 2 MgCl2, and 5.5 glucose (pH 7.3). U87-MG cells were cultured on coverslips for 4 hours, and then treated with AON-MG23-01 or AON-MG23-02 for 1 hour, respectively (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 calcium-dependent chloride ion channel currents in the range of -100 mV to +100 mV were measured.

[0410] As a result, as shown in Fig. 6a, the group treated with the compound of the present invention showed a significant decrease in ANO1 activity compared to the untreated control group. In particular, as shown in Fig. 6b, at a membrane potential of +80 mV, the group treated with the compound of the present invention showed a decrease in current density by more than 50% compared to the untreated control group. The above results demonstrate 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.

[0411]

[0412] 7. Comparison of the metastasis-inhibiting effects of the compound of the present invention and EGFR inhibitors and ANO1 inhibitors in human glioblastoma.

[0413] The human glioblastoma cell line U87-MG was plated on top of a Matrigel-coated filter (Transwell invasion chambers, Corning) at 6X10 4 After seeding at a density of 10 cells / well, the 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 of Osimertinib and 100 μM of CaCCinh-A01, and cultured at 37°C in a CO2 incubator for 16 hours. After culture, cells were fixed and stained using the Diff-Quick stain Kit (Sysmex, Kobe, Japan). Images of the stained cells were taken under a microscope, and cells were counted using Image J software, followed by statistical analysis.

[0414] As a result, as shown in FIGS. 7a and 7b, the group treated with the compound AON-MG23-02 of the present invention showed a reduced degree of penetration into the matrix gel compared to the untreated control group, the EGFR inhibitor-treated group, and the ANO1 inhibitor-treated group, and the metastasis inhibition effect was similar to that of the EGFR inhibitor and ANO1 inhibitor combination treatment group.

[0415]

[0416] 8. Comparison of the inhibitory effects of the compound of the present invention and the EGFR inhibitor and ANO1 inhibitor on the expression of ANO1, EGFR, and pEGFR proteins in glioblastoma cell lines.

[0417] The inhibitory effects of the compound AON-MG23-02 of the present invention, the EGFR inhibitor Osimertinib, and the ANO1 inhibitor CaCCinh-A01 on the expression of ANO1, EGFR, and pEGFR proteins in glioblastoma cell lines were compared. The human glioblastoma cell line U87-MG was cultured in a 60 mm dish at a density of 100×10 4After seeding at a density of 10 cells / dish and culturing for 24 h, the cells were treated with 4 μM AON-MG23-02, 2 μM EGFR inhibitor Osimertinib, 100 μM ANO1 inhibitor CaCCinh-A01, or a combination of 2 μM Osimertinib and 100 μM CaCCinh-A01, and cultured for 48 h. After completing the culture, the cells were harvested and lysed for Western blotting. Antibodies 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 cell lysis, the obtained protein was separated by molecular weight using 10% SDS-PAGE with the same amount (10 μg), 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. Then, the PVDF membrane was treated with the primary antibody and reacted at 4°C for 16 hours. After the reaction, the PVDF membrane was washed three times with TBS-T for 10 minutes each. The PVDF membrane was then incubated with horseradish peroxidase-labeled secondary antibody at room temperature for 1 hour, then washed three times with TBS-T for 10 minutes each. After washing, the PVDF membrane was reacted with an ECL kit (Thermo, West Pico Plus) and visualized using a da Vinci-Q (Youngin Lab Plus) instrument.The amount of each protein was quantified and analyzed using Image J software.

[0418] As a result, as shown in FIGS. 8a and 8b, it was confirmed that the compound AON-MG23-02 of the present invention reduces the expression of ANO1, EGFR, and pEGFR proteins compared to the untreated control group, the EGFR inhibitor treatment group, the ANO1 inhibitor treatment group, and the EGFR inhibitor and ANO1 inhibitor combination treatment group.

[0419]

[0420] Example B. Derivatives of the novel compound AON-MG23-02

[0421] 1. Preparation of derivatives of AON-MG23-02

[0422] 1-1. AON-MG23-05

[0423] 1-1-A. Preparation of compound 2

[0424] [Reaction Formula 7]

[0425]

[0426]

[0427] 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 with N2 three times, and then stirred at 80 °C in nitrogen gas (N2) for 2 h. TLC (petroleum ether: ethyl acetate = 1: 1, Rf = 0.49) analysis showed that 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 extracted with 6.00 mL of EtOAc (6.00 mL * 3). The mixed 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). Compound 2 (900 mg, 2.35 mmol, yield 20.6%, purity 82.4%) was obtained as a yellow solid.

[0428]

[0429] 1-1-B. Preparation of compound 3

[0430] [Reaction Formula 8]

[0431]

[0432]

[0433] To a solution of compound 2 (900 mg, 2.85 mmol, 1.00 eq) dissolved in ACN (10.0 mL) were added trimethylsilyl cyanide (TMSCN, 338 mg, 3.42 mmol, 427 μL, 1.20 eq) and Cs2CO3 (1.11 g, 3.42 mmol, 1.20 eq). The mixture was stirred at 25 °C for 12 h. LC-MS analysis showed that compound 2 was completely consumed, and 42.0% of the desired mass was detected. The reaction mixture was concentrated under reduced pressure, and the residue was diluted with 2.00 mL of water and extracted with 3.00 mL of EtOAc (3.00 mL * 3). The combined organic supernatant was washed with 4.00 mL of brine (4.00 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether: ethyl acetate = 10: 1 to 0: 1). Compound 3 (600 mg, 2.22 mmol, yield 77.8%, purity 96.9%) was obtained as a yellow solid.

[0434]

[0435] 1-1-C. Preparation of compound 4

[0436] [Reaction Formula 9]

[0437]

[0438] 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), degassed and purged with N2, and the suspension was degassed under vacuum and purged with hydrogen. The mixture was stirred several times in a H2 (40 psi) environment at 80°C for 12 h. LC-MS analysis showed that compound 3 was completely consumed, and 54.8% of the desired product was detected. The mixture was filtered and concentrated. The crude product was purified by reverse-phase HPLC (column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water (FA)-ACN]; gradient: 2%-32% B over 15 min). Compound 4 ([[4-(2-aminoethyl) phenyl] methyl] isoindoli-1-one, 320 mg, 1.20 mmol, yield 52.5%, purity 100%) was obtained as a white solid.

[0439]

[0440] 1-1-D. Preparation of compound 5

[0441] [Reaction Formula 10]

[0442]

[0443] A mixture of compound 4 (200 mg, 750 μmol, 1.00 eq) and compound 4a (117 mg, 750 μmol, 1.00 eq) dissolved in CH(OMe)3 (2.00 mL) was degassed and purged with N2 three times, and stirred at 25°C in N2 gas for 1 h. 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. Then, the mixture was stirred at 25°C in N2 gas for 1 h. TLC (dichloromethane: methanol = 10: 1; R f= 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 EtOAc (4.00 mL * 3). The combined organic layers were washed with 5.00 mL of brine (5.00 mL * 1), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, dichloromethane: methanol = 60:1 to 10:1). Compound 5 (34.0 mg, 73.1 μmol, yield 9.73%, purity 87.4%) was obtained as a yellow oil.

[0444]

[0445] 1-1-E. Preparation of compound 6

[0446] [Reaction Formula 11]

[0447]

[0448] Compound 5 (54.0 mg, 132 μmol, 1.00 eq) was dissolved in DMF (Dimethylformamide, 0.600 mL) and K2CO3 (36.7 mg, 265 μmol, 2.00 eq), KI (4.41 mg, 26.5 μmol, 0.200 eq), and compound 5a (38.1 mg, 159 μmol, 1.20 eq) were added to the mixture. The mixture was stirred at 50°C for 12 h. TLC (dichloromethane: methanol = 10:1, R f= 0.70) As a result, 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 EtOAc (2.00 mL * 3). The combined organic layers were washed with 3.00 mL of brine (3.00 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a 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%) was obtained as a yellow oil.

[0449]

[0450] 1-1-F. Manufacturing of AON-MG23-05

[0451] [Reaction Formula 12]

[0452]

[0453] Compound 6 (29.0 mg, 51.3 μmol, 1.00 eq) was dissolved in MeOH (0.500 mL) and HCl / MeOH (2.00 M, 1.54 mL, 60.0 eq) was added. The mixture was stirred at 25°C for 0.5 h. LC-MS revealed that compound 6 was completely consumed and 98.7% of the desired yield was detected. The reaction mixture was concentrated under reduced pressure. NaHCO3 was added to the mixture (PH>7). The residue was extracted with 3.00 mL (3.00 mL * 3) of DCM. The combined organic layers were washed with 2.00 mL (2.00 mL * 1) of brine, 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) (18.2 mg, 40.2 μmol, yield 78.3%, purity 99.6%) was obtained as an off-white solid.

[0454] H NMR data of AON-MG23-05 are shown in Figure 11.

[0455]

[0456] 1-2. Manufacturing of AON-MG23-06

[0457] 1-2-A. Preparation of compound 8

[0458] [Reaction Formula 13]

[0459]

[0460] Compound SM1 (3.00 g, 22.5 mmol, 1.00 eq) and 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 in ACN (30 mL), degassed, and stirred at 25 °C for 16 h. LC-MS results showed that 20% of compound SM1 remained. Several new peaks appeared in LC-MS, and 60% of the desired compound was detected. 30 mL of H2O was added to the reaction mixture to quench the residue, and 30 mL (30 mL * 2) of EA (Ethyl acetate) was extracted. The combined organic layers were washed with 30 mL (30 mL * 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 20:1 to 15:1) and TLC (Petroleum ether: Ethyl acetate = 3:1, R f = 0.43) was purified. Compound 8 (2.10 g, 8.42 mmol, yield 37.3%) was obtained as a white solid, which was confirmed by LC-MS and HNMR.

[0461]

[0462] 1-2-B. Preparation of compound 9

[0463] [Reaction Formula 14]

[0464]

[0465] 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 h. After addition, the mixture was stirred at 25 °C for 2 h. 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. Na2S2O320mL was added to the reaction mixture at 25℃ to quench the residue, and extracted with DCM 30mL (30 mL *2). The combined organic layers were washed with brine 30mL (30 mL *2), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a 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, which was confirmed by HPLC and HNMR. In addition, compound 9 (140 mg, 527 μmol, yield 65.7%) was obtained as a yellow oil, which was confirmed by LC-MS and HNMR.

[0466]

[0467] 1-2-C. Preparation of compound 9A

[0468] [Reaction Formula 15]

[0469]

[0470] A solution of ethanamine (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 h, and compound 9B (1.00 g, 6.40 mmol, 1.00 eq), sodium;triacetoxyborohydride (NaBH(OAc)3, 2.04 g, 9.60 mmol, 1.50 eq), and AcOH (3.85 mg, 64.0 μmol, 3.67 μL, 0.01 eq) were added at 25 °C. The resulting mixture was stirred at 25 °C for 1 h. LC-MS results indicated that compound 9B was completely consumed, and one major peak with the desired m / z value was detected. 10 mL of Na2CO3 was added to the reaction mixture to quench it, diluted with 20 mL of H2O, and extracted with 30 mL of EA (30 mL * 2). The combined organic layers were washed with 30 mL of brine (30 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate = 0: 1 to Dichloromethane: Methanol = 20: 1). Compound 9A (520 mg, 2.81 mmol, yield 43.8%) was obtained as a white solid, which was confirmed by HNMR and LC-MS.

[0471]

[0472] 1-2-D. Manufacturing of AON-MG23-06

[0473] [Reaction Formula 16]

[0474]

[0475] A mixture of 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) dissolved in ethanol (0.1 mL) was degassed and purged three times with N2, and then stirred at 80 °C in N2 gas for 12 h. LC-MS results showed that compound 9 was completely consumed, and one major peak with the desired m / z value was detected. After quenching by adding 10 mL of H2O to the reaction mixture, the mixture was extracted with 5 mL of EA (5 mL * 2), and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (FA condition: Phenomenex luna C18 150*25mm*10um; mobile phase: [water (FA)-ACN]; gradient: 15%-45% B, more than 1 min). Then, it was purified by prep-HPLC (neutral condition: Waters Xbridge 150*25mm*5um; mobile phase: [water (NH4HCO3)-ACN]; gradient: 45%-75% B, more than 15 min). AON-MG23-06 (Target B) (29.7 mg, 65.7μmol, yield 26.8%, purity 99.8%) as a white solid was obtained, which was confirmed by LC-MS, HPLC, HNMR, and special NMR.

[0476] H NMR data of AON-MG23-06 are shown in Figure 12.

[0477]

[0478] 1-3. Manufacturing of AON-MG23-07

[0479] 1-3-A. Preparation of compound 10

[0480] [Reaction Formula 17]

[0481]

[0482] 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 with argon and saturated with P (5.00 g, 147.0 mmol, 36.6 eq). The DMSO solution containing compound 8 (1.00 g, 4.01 mmol, 1.00 eq) was stirred at 70°C for 30 min, and then added dropwise while continuously passing inflammable gas. The reaction mixture was further heated (70°C) for 30 min while maintaining the phosphine flow. 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 a residue. Compound 10 (1.10 g, crude) as a white solid was obtained.

[0483]

[0484] 1-3-B. Preparation of AON-MG23-07

[0485] [Reaction Formula 18]

[0486]

[0487] To a solution of compound 10 (1.10 g, 3.88 mmol, 1.00 eq) in THF (Tetrahydrofuran, 10.0 mL) was added NaH (465.9 mg, 11.6 mmol, purity 60.0%, 3.00 eq) at 0°C under N2 gas atmosphere. The mixture was stirred at 0°C for 30 min. 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 min. 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 N2 gas at 0℃, diluted with 20.0 mL of H2O, and extracted with 40.0 mL of EA (20.0 mL * 2). The combined organic layers were washed with 30.0 mL of brine (30.0 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (FA condition; column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water (FA)-ACN]; gradient: 52%-82% B over 15 min). The residue was further purified by prep-HPLC (FA conditions; column: Welch Xtimate C18 150*25mm*5um; mobile phase: [water (FA)-ACN]; gradient: 58%-78% B for 15 min). Off-white AON-MG23-07 (Target C) (35.0 mg, 58.5 μmol, yield 1.51%, purity 97.0%) was obtained.

[0488] H NMR data of AON-MG23-07 are shown in Figure 13.

[0489]

[0490] 1-4. Manufacturing of AON-MG23-08

[0491] 1-4-A. Preparation of compound 2

[0492] [Reaction Formula 19]

[0493]

[0494] A mixture of compound 1 (5.00 g, 18.9 mmol, 1.00 eq), compound SM1 (1.26 g, 9.47 mmol, 0.50 eq), and Cs2CO3 (4.94 g, 15.1 mmol, 0.80 eq) dissolved in ACN (15.0 mL) was degassed and purged with N2 gas three times, and stirred at 80°C for 2 h in N2 gas. LC-MS (EW47929-30-P1A1) results showed that compound SM1 was completely consumed and the desired mass was detected. The reaction mixture was diluted with 30.0 mL of H2O, and 40.0 mL of EA (20.0 mL * 2) was extracted. The combined organic layer was washed with 30.0 mL of brine (30.0 mL * 1), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=20 / 1 to 5 / 1, TLC,PE:EA=3:1, R f =0.22) was purified. Compound 2 (1.18 g, 3.73 mmol, yield 19.7%) was obtained as a yellow solid.

[0495]

[0496] 1-4-B. Preparation of compound 3

[0497] [Reaction Formula 20]

[0498]

[0499] To a solution of compound 2 (1.1 g, 3.48 mmol, 1.00 eq) in ACN (16.0 mL) were added Cs2CO3 (1.36 g, 4.17 mmol, 1.20 eq) and TMSCN (414.1 mg, 4.17 mmol, 522 μL, 1.20 eq). The mixture was stirred at 25 °C for 12 h. 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 of EA (20.0 mL * 2) was extracted. The combined organic layers were washed with 20.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, Petroleum ether / Ethyl acetate=5 / 1 to 3 / 1, PE: EA=5:1, Rf = 5:1, R f =0.47) was purified. Compound 3 (825 mg, 3.08 mmol, yield 88.6%, purity 98.0%) as a yellow solid was obtained.

[0500]

[0501] 1-4-C. Preparation of compound 4

[0502] [Reaction Formula 21]

[0503]

[0504] 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), and the mixture was evacuated and purged three times with N2, and then purged several times with H2 under vacuum. The mixture was stirred at 80°C under H2 (40 psi) for 12 h. LC-MS results showed that compound 3 was completely consumed and the desired product was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was then purified by prep-HPLC (FA condition; column: Phenomenex luna C18 15040 mm 15 μm; mobile phase: [water(FA)-ACN]; gradient: 8%-38% B, for 15 min). Compound 4 (300 mg, 1.10 mmol, 34.9% yield, 97.5% purity) was obtained as a white solid, which was confirmed by HNMR and LC-MS.

[0505]

[0506] 1-4-D. Preparation of compound 5

[0507] [Reaction Formula 22]

[0508]

[0509] 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) in 5.00 mL of ethanol (EtOH). The mixture was stirred at 25°C for 1 h under a N2 atmosphere. 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 stirred for 1 h under a N2 atmosphere. 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 of EA (10 mL * 2). After combining the organic phases, washing with 10 mL of brine (10 mL * 1), drying with [Na2SO4], filtering, and concentrating under reduced pressure to obtain a 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) as a white solid was obtained, which was confirmed by LC-MS and HNMR.

[0510]

[0511] 1-4-E. Manufacturing of AON-MG23-08

[0512] [Reaction Formula 23]

[0513]

[0514] Compound 5 (100 mg, 245 μmol, 1.00 eq) was dissolved in AcOH (0.30 mL) to which compound 5b (99.7 mg, 1.23 mmol, 48.5 μL, 5.00 eq) and H2O (1.50 mL) were added. The mixture was stirred at 25 °C for 1 h. LC-MS analysis showed that approximately 10% of compound 5 remained and the desired mass was detected. The reaction was stopped by slowly adding saturated sodium hydroxide solution until the pH reached 8, and then extracted with 40 mL (20 mL * 2) of DCM. The combined organic layers were washed with 40.0 mL (20.0 mL * 2) of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-TLC (SiO2, DCM: MeOH = 15:1). The residue was purified by prep-HPLC (default conditions; column: Waters Xbridge 150*25mm*5um; mobile phase: [water (ammonia hydroxide v / v)-ACN]; gradient: 25%-55% B, over 10 min). AON-MG23-08 (Target D) (11.0 mg, 24.4 μmol, 95% yield, 100% purity) was obtained as a white solid.

[0515] H NMR data of AON-MG23-08 are shown in Figure 14.

[0516]

[0517] Derivatives of AON-MG23-02 were prepared using the above method, and their structural formulas are shown in Table 2 below.

[0518]

[0519]

[0520] 2. Comparison of the inhibitory effects of AON-MG23-02 derivatives on ANO1 and EGFR protein expression in four glioblastoma cell lines.

[0521] The inhibitory effects of AON-MG23-02 derivatives AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08 on the expression of ANO1 and EGFR proteins in glioblastoma cell lines were compared. The human glioblastoma cell line U87-MG was cultured in a 60 mm dish at a density of 100×10 4Cells were seeded at a density of 10 cells / dish and cultured for 24 h, and then 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 h. After culture, the cells were harvested and lysed for Western blotting. Antibodies 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 same amount (10 μg) of protein obtained therefrom was subjected to 10% SDS-PAGE to separate the proteins by molecular weight, 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. Then, the PVDF membrane was treated with the primary antibody and reacted at 4°C for 16 hours. After the reaction, the PVDF membrane was washed three times for 10 minutes each with TBS-T. After that, the PVDF membrane was reacted with the secondary antibody labeled with horseradish peroxidase at room temperature for 1 hour, and then washed three times for 10 minutes each with TBS-T. After washing, the PVDF membrane was treated with an ECL kit (Thermo, West Pico Plus) and visualized using a da Vinci-Q (Youngin Lab Plus) device. The amount of each protein was quantified and analyzed using Image J software.

[0522] As a result, as shown in FIGS. 9a to 9d, it was confirmed that the derivatives AON-MG23-05 and AON-MG23-08 reduced the expression of ANO1 and EGFR proteins compared to the untreated control group.

[0523]

[0524] 3. Comparison of the metastasis-inhibiting effects of four derivatives of AON-MG23-02 in human glioblastoma cells.

[0525] The human glioblastoma cell line U87-MG was plated on top of a Matrigel-coated filter (Transwell invasion chambers, Corning) at 8X10 4 After seeding at a density of 10 cells / well, the cells were treated with 0, 8, 16, and 32 μM of each of the compound derivatives of the present invention, AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08, and cultured at 37°C in a CO2 incubator for 24 hours. After culture, cells were fixed and stained using a Diff-Quick stain Kit (Sysmex, Kobe, Japan). Images of the stained cells were taken under a microscope, and cells were counted using Image J software, followed by statistical analysis.

[0526] As a result, as shown in Figures 10a and 10b, the groups treated with the derivatives AON-MG23-05, AON-MG23-06, AON-MG23-07, and AON-MG23-08 showed a reduced degree of penetration into the matrix gel compared to the untreated control group, confirming that they inhibit metastasis in human glioblastoma.

[0527]

[0528] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0529] The novel compound of the present invention can be used not only as an ANO1 inhibitor, but also as a dual-target anticancer agent for ANO1 and EGFR, as it can simultaneously inhibit ANO1 and EGFR in brain tumor cells, and can also be used as a combination agent for EGFR-targeted therapy, and thus can be utilized in various ways in the field of prevention and treatment of brain tumors, and thus 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. [Chemical Formula 1] [Chemical Formula 2] [Chemical Formula 3] [Chemical Formula 4] [Chemical Formula 5] [Chemical Formula 6] 2. (S1A) A step of preparing a compound represented by the following chemical formula 8 by reacting a compound represented by the following chemical formula 7 with methanesulfonyl chloride (MsCl) and cyclopropylamine in the presence of a base; or (S1B) A method for producing the compound of claim 1, comprising a 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. [Chemical Formula 7] [Chemical Formula 8] [Chemical Formula 9] [Chemical Formula 10] [Chemical Formula 11] 3. In paragraph 2, A method for producing a compound, characterized in that the above (S1A) step further comprises the following steps: (S1A-1) A step of reducing a compound represented by the above chemical formula 8 with zinc in the presence of ammonium chloride to produce a compound represented by the following chemical formula 12; (S1A-2) A step of preparing a compound represented by the following chemical formula 13 by reacting a compound represented by the above chemical formula 12 with 2, 4, 5-trichloropyrimidine in the presence of a base; and (S1A-3) A step of producing a compound represented by the chemical formula 1 or 2 by reacting a compound represented by the chemical formula 13 and a compound represented by the chemical formula 14 or 15 below in the presence of an acid. [Chemical Formula 12] [Chemical Formula 13] [Chemical Formula 14] [Chemical Formula 15] 4. In paragraph 2, A method for producing a compound, characterized in that when a compound represented by the chemical formula 9 and a compound represented by the chemical formula 10 react in the presence of a base in the step (S1B), a compound represented by the chemical formula 16 is produced. [Chemical Formula 16] 5. In paragraph 4, A method for producing a compound, characterized in that the above (S1B) step further comprises the following steps: (S1B-1) A step of producing a compound represented by the following chemical formula 17 by reacting a compound represented by the above chemical formula 16 with trimethylsilyl cyanide in the presence of a base; (S1B-2) A step of producing a compound represented by the following chemical formula 18 by hydrogenating a compound represented by the above chemical formula 17 in the presence of a metal catalyst; (S1B-3) A step of preparing a compound represented by the following chemical formula 20 by reacting a compound represented by the above chemical formula 18 with a compound represented by the following chemical formula 19 in the presence of a reducing agent; and (S1B-4) Compound represented by the above chemical formula 20 and NCO - A step of preparing a compound represented by the above chemical formula 6 by reacting; or a step of preparing a compound represented by the above chemical formula 22 by reacting a compound represented by the above chemical formula 20 and a compound represented by the following chemical formula 21 in the presence of a base. [Chemical Formula 17] [Chemical Formula 18] [Chemical Formula 19] [Chemical Formula 20] [Chemical Formula 21] [Chemical Formula 22] 6. In paragraph 5, A method for producing a compound, characterized in that the above step (S1B-4) further comprises a step of producing a compound represented by the above chemical formula 3 by subjecting the compound represented by the above chemical formula 22 to a hydrolysis reaction in the presence of an acid.

7. In paragraph 5, A method for producing a compound, characterized in that the metal catalyst is at least one selected from the group consisting of platinum black, rhodium, palladium carbon, and Raney-Ni.

8. In paragraph 2, A method for producing a compound, characterized in that when the compound represented by the chemical formula 9 and the compound represented by the chemical formula 11 react in the presence of a base in the step (S1B), a compound represented by the chemical formula 23 is produced. [Chemical Formula 23] 9. In paragraph 8, A method for producing a compound, characterized in that the above (S1B) step further comprises the following steps: (S1Ba-1) A step of producing a compound represented by the following chemical formula 24 by reacting a compound represented by the above chemical formula 23 with a peroxide, and (S1Ba-2) A step of preparing a compound represented by the above chemical formula 4 by reacting a compound represented by the above chemical formula 24 and a compound represented by the following chemical formula 25 in the presence of a base; or (S1Bb-1) A step of producing a compound represented by the following chemical formula 26 by phosphorylating the compound represented by the above chemical formula 23 in the presence of a base, and (S1Bb-2) A step of producing a compound represented by the above chemical formula 5 by reacting a compound represented by the above chemical formula 26 and a compound represented by the following chemical formula 27 in the presence of a base. [Chemical Formula 24] [Chemical Formula 25] [Chemical Formula 26] [Chemical Formula 27] 10. In paragraph 9, A method for producing a compound, characterized in that the peroxide is at least one selected from the group consisting of m-CPBA (meta-chloroperoxybenzoic acid), H2O2, DMDO (dimethyldioxirane), and oxone.

11. In paragraph 9, A method for producing a compound, characterized in that the compound represented by the above chemical formula 25 is produced by reacting a compound represented by the following chemical formula 28 with ethylamine in the presence of a reducing agent. [Chemical Formula 28] 12. In paragraph 5 or paragraph 11, A method for producing a compound, characterized in that the reducing agent is at least one selected from the group consisting of sodium cyanoborohydride (NaBH3CN), sodium triacetoxyborohydride (NaBH(OAc)3), and sodium borohydride (NaBH4).

13. In paragraph 3 or paragraph 6, A method for producing a compound, characterized in that the acid is at least one selected from the group consisting of pivalic acid (PivOH), acetic acid (AcOH), trifluoromethanesulfonic acid (TfOH), paratoluenesulfonic acid (TsOH), benzoic acid, zinc bromide (ZnBr2), hydrogen chloride (HCl), and trifluoroacetic acid (TFA).

14. In any one of paragraphs 2 to 11, A method for producing a compound, characterized in that the base is at least one 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.

15. In any one of paragraphs 2 to 11, A method for producing a compound, characterized in that the above manufacturing method is performed in one or more solvents selected from the group consisting of organic solvents, water, and mixtures thereof.

16. In paragraph 15, A method for producing a compound, characterized in that the organic solvent is at least one 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.

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