KR102455529B1 - Method for preparing novel benzopyran derivatives and anticancer composition containing the same - Google Patents

Abstract

The present invention relates to novel benzopyran derivatives having ANO1 (Anoctamin 1) inhibitory activity and uses thereof. The novel benzobenzopyran derivative or pharmaceutically acceptable salt thereof according to the present invention exhibits excellent inhibitory activity on ANO1, and ANO1 overexpressed cancer such as prostate cancer, colorectal cancer, esophageal squamous cell carcinoma, pancreatic cancer, head and neck cancer, and In the prevention or treatment of breast cancer, it is expected that target treatment can be approached more fundamentally.

Description

A novel method for preparing a benzopyran derivative and an anticancer composition comprising the same

The present invention relates to a novel method for preparing a benzopyran derivative and an anticancer composition comprising the same.

Anoctamin1 (Anoctamin1, ANO1) is a calcium-activated chloride ion channel (CaCC) that is expressed in a variety of cells and tissues. perform physiological functions. Inhibitors of TMEM16A/ANO1, a calcium-acitivated chloride channel, have been proven to have anticancer effects through many studies. It has been reported that ANO1 is overexpressed in several types of tumor cells and is involved in cancer cell growth, metastasis, cancer cellization, and cancer development.

Anoctamin2 (ANO2) is a chlorine ion channel with 62% structural similarity to ANO1 and is involved in the reduction of spike production in thalamic cortex and hippocampal CA1 neurons. became Therefore, it is necessary to develop a compound that specifically inhibits ANO1 without inhibiting ANO2. Specific inhibition of ANO1 chloride ion channel activity and expression induces apoptosis in cancer cells and can inhibit cancer cell growth and cancer development. For example, it was found that ANO1 protein was overexpressed in prostate cancer cells (PC-3), and cell growth, metastasis, invasion, and tumor development were inhibited when ANO1 expression was suppressed. ANO1, which functions to move chloride ions and regulate cell volume, is recognized as a major marker in androgen-independent prostate cancer. Therefore, ANO1 inhibitors can be good drug targets in ANO1-overexpressing carcinomas, including prostate cancer and esophageal cancer.

Various ANO1 inhibitors have been reported so far, digallic acid, T16A _inh -A01, benzbromarone, tannic acid, CaCC _inh -A01, MONNA, Ani9, etc., but all are in the early stage of new drug development Therefore, there is a need to develop drugs that selectively inhibit the function of ANO1 channels and strongly decrease protein expression.

The present inventors have developed a new low molecular weight compound ANO1 inhibitor with high potential for development as an anticancer agent, analgesic agent, asthma agent, intestinal motility agent, hyperhidrosis agent, and the like. The novel ANO1 inhibitor shows very high selectivity for ANO2, the ion channel most similar to ANO1.

ANO1 overexpressing PC3 cells (prostate cancer cells), HT-29 cells (colon cancer cells), FaDu cells (head and neck cancer cells), MCF7 cells (breast cancer cells), etc. It has been established that there is

Therefore, new ANO1 inhibitors have the potential to be developed as drugs that can inhibit the growth of cancer cells that overexpress ANO1 or as therapeutic agents for ANO1-related diseases.

The present invention has been devised to solve the above problems, and the present inventors have intensively studied to find a new substance that can be used as an ANO1 inhibitor for anticancer purposes. As a result, a novel benzopyran derivative exhibiting ANO1 inhibitory activity was identified and , the present invention was completed on the basis of this.

Accordingly, an object of the present invention is to provide a novel benzopyran derivative having ANO1 inhibitory activity or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a method for preparing a novel benzopyran derivative having ANO1 inhibitory activity.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the benzopyran derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating prostate cancer, colorectal cancer, head and neck cancer and breast cancer, comprising the benzopyran derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to inhibit ANO1 overexpressed in prostate cancer cells, colorectal cancer cells, head and neck cancer cells and breast cancer cells, comprising the benzopyran derivative or a pharmaceutically acceptable salt thereof as an active ingredient. It is to provide an enemy composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention provides a compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

및

이고,R ₁ is

and

ego,

,

및 C_1-4 알킬이고,R ₂ is

,

and C _1-4 alkyl;

,

및

이고,R ₃ is

,

and

ego,

R ₄ , R ₅ and R ₆ are each independently hydrogen, C _1-4 haloalkoxy and C _1-4 alkoxy;

where X is nitrogen, oxygen, carbon and sulfur, and Y is halogen and hydrogen.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical for preventing or treating prostate cancer, colon cancer, esophageal squamous cell cancer, pancreatic cancer, head and neck cancer and breast cancer, comprising the derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. A composition is provided.

In addition, the cancer provides that ANO1 is overexpressed, the pharmaceutical composition.

In one embodiment of the present invention, the composition may inhibit the activity of ANO1.

The novel benzopyran derivative or a pharmaceutically acceptable salt thereof according to the present invention is an excellent target for ANO1 and exhibits inhibitory activity, and the pharmaceutical composition comprising the derivative is ANO1 overexpressed cancer such as prostate cancer, colon cancer, esophagus. It is expected to be usefully used for the prevention and treatment of squamous cell carcinoma, pancreatic cancer, head and neck cancer, and breast cancer.

1 is a graph showing the results of an experiment to determine the specificity of selectively inhibiting the ANO1 activity of the 3h compound, which is a benzopyran derivative of the present invention (A: 3h dose-dependent activity of ANO1 channel in FRT cells expressing ANO1 and YFP Graph that inhibits ANO1, B: A graph measuring whether 3h inhibits the activity of ANO2, which has a similar protein structure to ANO1, C: 3h , a dose-response curve showing the dose-dependent amount of inhibition of ANO1 and ANO2, D: 3h A graph measuring whether the activity of CFTR, a chlorine ion channel, was inhibited, E: a graph measuring the effect of 3h on the intracellular calcium concentration).
2 is a graph showing the results of experiments to identify the effect of reducing cell viability through ANO1 protein degradation of the 3h compound, which is a benzopyran derivative of the present invention (A, B: PC-3 (prostate cancer cells) and FaDu (esophageal cancer cells) for 3 h Western blot measuring the degree of ANO1 proteolysis by compound and blot showing ANO1 protein levels in PC-3 cells and FaDu cells in which the ANO1 gene was removed, C, D: expressing ANO1 and not expressing ANO1 by removing the ANO1 gene PC-3 and FaDu cells were treated with the compound for 3 h and cell viability was measured).
3 is a graph showing the results of an experiment to identify the anticancer effect through apoptosis of the 3h compound, which is a benzopyran derivative of the present invention (A, B: ANO1-expressing PC-3, FaDu cells for 3 h by concentration Graphs measuring fluorescence indicating activation of Caspase-3 after treatment, C, D: ANO1-expressing PC-3 and FaDu cells treated with compound for 3 h and observed cells in which apoptosis occurred through caspase-3 activation Photographs, E, F: Western blot confirmed whether apoptosis occurred by treating PC-3 and FaDu cells for 3 h at each concentration and measuring PARP protein and cleaved PARP protein).

Hereinafter, the present invention will be described in detail.

The present invention provides a compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1,

및

이고,R ₁ is

and

ego,

,

및 C_1-4 알킬이고,R ₂ is

,

and C _1-4 alkyl;

,

및

이고,R ₃ is

,

and

ego,

R ₄ , R ₅ and R ₆ are each independently hydrogen, C _1-4 haloalkoxy and C _1-4 alkoxy;

where X is nitrogen, oxygen, carbon and sulfur, and Y is halogen and hydrogen.

As used herein, "alkyl" generally refers to linear and branched saturated hydrocarbon groups having the specified number of carbon atoms (eg, from 1 to 12 carbon atoms). Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and n-heptyl, and the like. Alkyl may be attached to a parent group or substrate at any ring atom provided that attachment does not violate valence requirements. Likewise, an alkyl or alkenyl group may contain one or more non-hydrogen substituents provided that attachment does not violate valence requirements.

"Alkoxy" is an alkyl group bonded to oxygen. The range of (R-O)alkoxy groups is very large and includes methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy.

"Haloalkoxy" refers to a compound in which the hydrogen atom of alkoxy is replaced with a halogen atom, and may be attached to a parent group or substrate at any ring atom provided that attachment does not violate valence requirements.

In another embodiment of the present invention, in the above formulas, R ₁ is

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

또는

인 화합물이다.

;

;

or

is a phosphorus compound.

In another embodiment of the present invention, in the above formulas, R ₁ is

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

또는

인 화합물이다.

;

;

or

It is a phosphorus compound.

As another embodiment of the present invention, the benzopyran derivative of Formula 1 is (7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)(phenyl)methanone;

(4-chlorophenyl)(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)methanone;

(4-bromophenyl)(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)methanone;

1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)ethan-1-one;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-phenylprop-2-en-1-one;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2-methoxyphenyl)prop-2-en-1-one ;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(3-methoxyphenyl)prop-2-en-1-one ;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-one ;

(E)-3-(2,4-dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene-1 -On;

(E)-3-(3,5-dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene-1 -On;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(3,4,5-trimethoxy-phenyl)prop-2 -en-1-one;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1 -On;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(furan-2-yl)prop-2-en-1-one ;

(E)-3-(5-bromofuran-2-yl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene -1-one;

(E)-3-(5-bromothiophen-2-yl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene -1-one;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-indol-2-yl)prop-2-en-1 -On;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(thiophen-2-yl)prop-2-en-1- On;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2-(trifluoromethoxy)phenyl)prop-2-ene -1-one;

(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2,4,5-trimethoxyphenyl)prop-2- en-1-one; or

4-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)pyridine.

In addition, as shown in Scheme 1 below, the compound in the present invention is prepared by using compound 1 and several α-bromoketones to prepare compound 2 (step 1);

preparing compound 3 by Kleisen-Schmidt Aldol reaction using compound 2 and arylaldehyde prepared in step 1 (step 2); and

The benzopyran derivative of claim 1 may be prepared by a method comprising the step of preparing compound 5 (step 3) using compound 1 and chloromethyl pyridine, but the present invention is not limited thereto.

[Scheme 1]

Meanwhile, the compound of the present invention may be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt.

As used herein, the term “salt” is useful as an acid addition salt formed by a pharmaceutically acceptable free acid. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methylbenzoate Toxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycol late, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the invention is prepared by a conventional method, for example by dissolving the compound in an aqueous solution of an excess of acid and precipitating the salt with a water-miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. can be manufactured. It can also be prepared by evaporating the solvent or excess acid from the mixture and drying, or by suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

In addition, the compound of the present invention includes all salts, isomers, hydrates and solvates that can be prepared by conventional methods as well as pharmaceutically acceptable salts.

Accordingly, the present invention provides a pharmaceutical for preventing or treating ANO1 overexpressed cancers such as prostate cancer, colorectal cancer, head and neck cancer and breast cancer, comprising the benzopyran derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. Composition, use of the imidazole derivative of Formula 1 or a pharmaceutically acceptable salt thereof for the treatment of the above disease, and administering to a subject a therapeutically effective amount of the compound of Formula 1 or a pharmaceutically acceptable salt thereof It provides a method for treating the above disease.

As used herein, the term “prevention” refers to any action that inhibits or delays the onset of ANO1 overexpressed cancer such as prostate cancer, colorectal cancer, head and neck cancer, and breast cancer by administration of the pharmaceutical composition according to the present invention do.

As used herein, the term “treatment” refers to any action in which symptoms for ANO1-overexpressed cancer such as prostate cancer, colon cancer, head and neck cancer, and breast cancer are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention do.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. In this case, pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose. , polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. may be additionally included in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) according to a desired method, and the dosage may vary depending on the condition and weight of the patient, and the disease. Although it varies depending on the degree, drug form, administration route and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition of 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 at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, activity of the drug, and the type of disease in the patient. Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate and excretion rate, disease type, and drugs used in combination, in general 0.0001 to 1000 mg per 1 kg of body weight, preferably 0.001 to 500 mg, may be administered daily or every other day, or divided into 1 to 3 times a day. However, the dosage may be increased or decreased according to the route of administration, the severity of obesity, sex, weight, age, etc., and thus the dosage is not intended to limit the scope of the present invention in any way.

In the present invention, "individual" refers to a subject in need of treatment for a disease, and more specifically, refers to mammals such as human or non-human primates, mice, dogs, cats, horses and cattle. .

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Basic synthesis method of compound No. 2

To a vial filled with 1 (30 mg, 0.15 mmol) in acetonitrile (2 mL) at room temperature was added 2-bromoacetophenone (35.1 mg, 1.2 equiv) and potassium carbonate (60.9 mg, 3 equiv). After stirring at 120 °C for 4 h, the reaction mixture was concentrated under reduced pressure, extracted with CH ₂ Cl ₂ (2 mL) and washed with H ₂ O (5 mL). The aqueous layer was extracted 2 more times with CH ₂ Cl ₂ (1 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-hexane : ethyl acetate : dichloromethane = 30 : 1 : 2) to obtain 2a in a yield of 100%.

The compounds of Examples 2 to 5 were obtained by the method of Example 1.

<Example 2> (7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl) (phenyl) methanone (2a)

yellow oil; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.00 (d, J = 8.0 Hz, 2H), 7.61 (t, J = 7.2 Hz, 1H), 7.52 (t, J = 7.2 Hz, 2H), 7.43-7.40 (m, 2H), 6.90 (d, J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.72 (d, J = 9.6 Hz, 1H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 184.0, 154.0, 152.8, 152.0, 137.8, 132.7, 130.5, 129.4, 128.6, 122.8, 120.8, 118.0, 115.8, 115.0, 106.7, 77.3, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₀ H ₁₆ NaO ₃ 327.0992, found 327.0968.

<Example 3> (4-chlorophenyl) (7,7-dimethyl-7H-furo [2,3-f] chromen-2-yl) methanone (2b)

Yellow solid, mp: 88.3-90.8 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.95 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.42-7.38 (m, 2H), 6.85-6.80 (m, 2H), 5.70 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 182.6, 154.2, 151.9, 139.2, 136.0, 130.9, 130.7, 129.0, 125.8, 122.9, 120.7, 117.9, 115.7, 115.2, 106.7, 77.4, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₀ H ₁₅ ClNaO ₃ 361.0602, found 361.0655.

<Example 4> (4-bromophenyl) (7,7-dimethyl-7H-furo [2,3-f] chromen-2-yl) methanone (2c)

brown solid, mp: 136.1-136.9 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.90 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.8 Hz, 2H), 7.45 (s, 1H), 7.42 (d, J = 8.4 Hz) , 1H), 6.89-6.83 (m, 2H), 5.73 (d, J = 10.0 Hz, 1H), 1.50 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 182.7, 154.2, 152.8, 151.8, 136.4, 131.9, 131.0, 130.6, 127.8, 122.9, 120.6, 117.9, 115.6, 115.2, 106.6, 77.4, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₀ H ₁₅ BrNaO ₃ 405.0097, found 405.0148.

<Example 5> 1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)ethan-1-one (2d)

Yellow solid, mp: 107.9-109.8 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.42 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 10.0 Hz, 1H), 6.81 (d, J = 8.4 Hz) , 1H), 5.71 (d, J = 10.0 Hz, 1H), 2.57 (s, 3H), 1.48 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 188.0, 153.9, 152.5, 152.4, 130.6, 122.7, 120.7, 115.7, 114.9, 114.3, 106.7, 77.3, 28.0, 26.4; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₁₅ H ₁₄ NaO ₃ 265.0835, found 265.0796.

<Example 6> Basic synthesis method of compound No. 3

To a vial of 2d (30 mg, 0.12 mmol) and benzaldehyde (1.2 equiv) in ethanol (1 mL) at room temperature was added potassium carbonate (1 equiv). After stirring at 60° C. for 3 h, the reaction mixture was concentrated under reduced pressure, extracted with CH ₂ Cl ₂ (2 mL) and washed with H ₂ O (5 mL). The aqueous layer was extracted twice or more with CH ₂ Cl ₂ (1 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-hexane : ethyl acetate : dichloromethane = 50 : 1 : 2) to obtain 38.1 mg of 3a in a yield of 96%.

The compounds of Examples 7 to 19 were obtained by the method of Example 6.

<Example 7> (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-phenylprop-2-en-1-one ( 3a)

Yellow solid, mp: 98.1-99.6 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.92 (d, J = 15.6 Hz, 1H), 7.69-7.67 (m, 2H), 7.58 (s, 1H), 7.53 (d, J = 15.6 Hz, 1H) , 7.44-7.42 (m, 4H), 6.93 (d, J = 9.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d, J = 10.0 Hz, 1H), 1.50 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.1, 154.0, 153.6, 152.6, 144.1, 134.9, 130.8, 130.5, 129.1, 128.7, 122.8, 121.5, 121.0, 115.8, 115.0, 114.5, 106.7, 77.3, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₂ H ₁₈ NaO ₃ 353.1148, found 353.1122.

<Example 8> (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2-methoxyphenyl)prop-2- en-1-one (3b)

Yellow solid, mp: 143.3-144.2 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.24 (d, J = 16.0 Hz, 1H), 7.68 (dd, J = 1.2, 7.6 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.55 (s, 1H), 7.43-7.37 (m, 2H), 7.00 (t, J = 7.6 Hz, 1H), 6.94 (t, J = 10.0 Hz, 2H), 6.82 (d, J = 8.4 Hz, 1H) , 5.73 (d, J = 10.0 Hz, 1H), 3.94 (s, 3H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.7, 159.1, 153.9, 153.8, 152.5, 139.6, 132.0, 130.5, 129.4, 123.9, 122.7, 122.2, 121.1, 120.8, 115.9, 114.8, 114.2, 111.4, 106.7, 77.2 , 55.7, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₃ H ₂₀ NaO ₄ 383.1254, found 383.1251.

<Example 9> ( E )-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)-3-(3-methoxyphenyl)prop-2-en-1-one (3c)

Yellow solid, mp: 103.6-104.8 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.88 (d, J = 16.0 Hz, 1H), 7.58 (s, 1H), 7.50 (d, J = 15.6 Hz, 1H), 7.42 (d, J = 8.4 Hz) , 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 9.2 Hz, 1H), 7.18 (s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.92 (d) , J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d, J = 9.6 Hz, 1H), 3.87 (s, 3H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.0, 160.1, 154.0, 153.6, 152.6, 144.0, 136.3, 130.5, 130.1, 122.8, 121.7, 121.3, 121.0, 116.5, 115.8, 115.0, 114.6, 113.8, 106.7, 77.3 , 55.5, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₃ H ₂₀ NaO ₄ 383.1254, found 383.1235.

<Example 10> (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(4-methoxyphenyl)prop-2- en-1-one (3d)

Yellow solid, mp: 138.8-139.3 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.89 (d, J = 15.6 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.44 (s, 1H), 7.41 (d, J = 5.2 Hz, 1H), 6.94 (t, J = 9.6 Hz, 3H), 6.83 (d, J = 8.8 Hz, 1H), 5.73 (d, J = 9.6 Hz, 1H), 3.87 (s) , 3H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.1, 160.9, 152.8, 151.5, 142.9, 129.5, 129.5, 126.6, 121.6, 120.0, 118.1, 114.8, 113.8, 113.5, 113.0, 105.7, 102.8, 76.2, 54.5, 27.0 ; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₃ H ₂₀ NaO ₄ 383.1254, found 383.1213.

<Example 11> (E)-3-(2,4-dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop- 2-en-1-one (3e)

Yellow solid, mp: 121.4-123.1 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.17 (d, J = 16.0 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.55-7.51 (m, 2H), 7.41 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 9.6 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.54 (dd, J = 2.0, 10.0 Hz, 1H), 6.48-6.48 (m, 1H), 5.72 (d, J = 10.0 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 3H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.8, 163.3, 160.7, 154.1, 153.6, 152.4, 139.7, 131.2, 130.4, 122.6, 121.1, 119.8, 117.2, 115.9, 114.7, 113.7, 106.7, 105.6, 98.5, 77.4 , 55.7, 55.6, 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₄ H ₂₂ NaO ₅ 413.1359, found 413.1295.

<Example 12> ( E )-3-(3,5-dimethoxyphenyl)-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)prop-2-en-1-one (3f)

Yellow solid, mp: 74.5-75.5 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.82 (d, J = 16.0 Hz, 1H), 7.57 (s, 1H), 7.48-7.40 (m, 2H), 6.91 (d, J = 10.0 Hz, 1H) , 6.83-6.81 (m, 3H), 6.53 (s, 1H), 5.72 (d, J = 10.0 Hz, 1H), 3.84 (s, 6H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.9, 161.2, 154.0, 153.5, 152.6, 144.1, 136.7, 130.5, 122.8, 121.9, 121.0, 115.7, 115.0, 114.6, 106.6, 106.6, 102.9, 77.3, 55.6, 28.0 ; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₄ H ₂₂ NaO ₅ 413.1359, found 413.1319.

<Example 13> ( E )-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)-3-(3,4,5-trimethoxy-phenyl)prop-2-en-1-one (3g)

Yellow solid, mp: 137.9-138.4 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.85 (d, J = 16.0 Hz, 1H), 7.59 (s, 1H), 7.44-7.38 (m, 2H), 6.93-6.90 (m, 3H), 6.83 ( d, J = 8.4 Hz, 1H), 5.74 (d, J = 10.0 Hz, 1H), 3.95 (s, 6H), 3.91 (s, 3H), 1.50 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.9, 154.0, 153.6, 152.6, 144.3, 140.7, 130.6, 130.4, 129.0, 122.8, 121.0, 120.7, 115.8, 115.0, 114.5, 106.7, 106.0, 77.3, 61.2, 56.4 , 28.0; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₅ H ₂₄ NaO ₆ 443.1465, found 443.1431.

<Example 14> ( E )-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)-3-(1 H -pyrrol-2-yl) prop-2-en-1-one (3h)

Brown solid, mp: 116.2-117.1 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.49 (s, 1H), 7.88 (d, J = 15.6 Hz, 1H), 7.49 (s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 15.6 Hz, 1H), 7.03 (s, 1H), 6.81-6.76 (m, 3H), 6.34 (s, 1H), 5.66 (d, J = 9.6 Hz, 1H), 1.46 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.3, 153.9, 153.7, 152.4, 134.0, 130.5, 129.5, 123.8, 122.6, 121.1, 116.0, 115.7, 115.1, 114.8, 113.8, 111.7, 106.6, 77.2, 28.0; HRMS (ESI-QTOF) m/z [M+H] ⁺ calcd for C ₂₀ H ₁₈ NO ₃ 320.1281, found 320.1234.

<Example 15> ( E )-3-(5-Bromothiophen-2-yl)-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)prop-2-en-1-one (3i)

Yellow solid, mp: 140.9-141.3 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.86 (d, J = 15.2 Hz, 1H), 7.53 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.17 (d, J = 15.2 Hz) , 1H), 7.09 (s, 1H), 7.04 (s, 1H), 6.88 (d, J = 10.0 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 5.71 (d, J = 10.0 Hz) , 1H), 1.48 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.2, 154.0, 153.4, 152.5, 141.9, 135.3, 132.6, 131.5, 130.5, 122.7, 120.9, 120.5, 116.7, 115.7, 115.0, 114.5, 106.6, 77.3, 28.0; HRMS (ESI-QTOF) m/z [M+H] ⁺ calcd for C ₂₀ H ₁₆ BrO ₃ S 414.9998, found 414.9941.

<Example 16> (E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-indol-2-yl)prop- 2-en-1-one (3j)

Dark yellow solid, mp: 239.9-240.4 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.54 (s, 1H), 7.94 (d, J = 15.6 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 16.0 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.14 (t) , J = 7.4 Hz, 1H), 7.00 (s, 1H), 6.92 (d, J = 9.6 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H), 5.73 (d, J = 1.0 Hz, 1H) ), 1.50 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.4, 153.8, 153.6, 152.4, 138.0, 134.0, 133.5, 130.4, 128.6, 125.1, 122.6, 121.8, 120.9, 120.8, 118.6, 115.6, 114.9, 114.0, 111.2, 110.5 , 106.5, 106.5, 27.8; HRMS (ESI-QTOF) m/z [M+H] ⁺ calcd for C ₂₄ H ₂₀ NO ₃ 370.1438, found 370.1521.

<Example 17> ( E )-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)-3-(thiophen-2-yl)prop-2-en-1-one (3k)

Yellow solid, mp: 121.4-122.1 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.03 (d, J = 15.2 Hz, 1H), 7.56 (s, 1H), 7.37-7.46 (m, 3H), 7.31 (d, J = 15.6 Hz, 1H) , 7.08-7.13 (m, 1H), 6.93 (d, J = 10.0 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d, J = 9.6 Hz, 1H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.5, 153.8, 153.4, 152.4, 140.2, 136.3, 132.3, 130.4, 129.0, 128.4, 122.6, 120.9, 120.2, 115.6, 114.8, 114.2, 106.5, 27.8; HRMS (ESI-QTOF) m/z [M+H] ⁺ calcd for C ₂₀ H ₁₇ O ₃ S 337.0893, found 337.0925.

<Example 18> ( E )-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)-3-(2-(trifluoromethoxy)phenyl)prop-2-en-1-one (3l)

light yellow solid, mp: 91.7-92.6 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.11 (d, J = 15.6 Hz, 1H), 7.81 (d, J = 6.8 Hz, 1H), 7.54-7.64 (m, 2H), 7.41-7.49 (m, 2H), 7.32-7.41 (m, 2H), 6.90 (d, J = 9.6 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 5.73 (d, J = 9.6 Hz, 1H), 1.49 ( s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 178.7, 153.9, 153.3, 152.5, 148.1, 148.0, 136.7, 131.5, 130.4, 128.9, 128.1, 127.1, 124.5, 122.7, 121.3, 120.8, 115.5, 114.9, 114.7, 106.5 , 27.9; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₂₃ H ₁₇ F ₃ NaO ₄ 437.0971, found 437.0961.

<Example 19> ( E )-1-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (3m)

Yellow solid, mp: 142.5-143.2 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.22 (d, J = 16.0 Hz, 1H), 7.54 (s, 1H), 7.45 (d, J = 16.4 Hz, 1H), 7.42 (d, J = 8.8 Hz) , 1H), 7.16 (s, 1H), 6.92 (d, J = 10.0 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.73 (d, J = 10.0 Hz) , 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.93 (s, 3H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 179.5, 154.9, 153.9, 153.5, 152.7, 152.2, 143.2, 139.3, 130.3, 122.4, 120.9, 119.5, 117.6, 115.7, 115.4, 114.6, 113.6, 111.6, 106.5, 96.7 , 56.6, 56.3, 56.1, 27.9; HRMS (ESI-QTOF) m/z [M+H] ⁺ calcd for C ₂₅ H ₂₅ O ₆ 421.1646, found 421.1688.

<Example 20> Basic synthesis method of compound No. 5

To a solution of compound 1 (60 mg, 0.30 mmol) in anhydrous N,N-dimethylformamide (1.1 mL) at room temperature 4-(chloromethyl)pyridine hydrochloride (49.6 mg, 1 equiv) and potassium carbonate (125.3 mg, 3 equivalent) was added. After stirring at 50 °C for 11 h, the reaction mixture was quenched with water (5 ml) and extracted with ethyl acetate (2 ml). The aqueous layer was extracted more than 5 times with ethyl acetate (1 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo to give a crude residue. To a vial filled with residue in anhydrous N,N-dimethylformamide (1 mL) at room temperature was added potassium tert-butoxide (43.3 mg, 1.5 equiv). After stirring at 80 °C for 16 h, the reaction mixture was quenched with water (5 ml) and extracted with ethyl acetate (2 ml). The aqueous layer was extracted more than 5 times with ethyl acetate (1 mL). The organic layer was dried over MgSO ₄ , filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-hexane : ethyl acetate : dichloromethane = 10 : 1 : 2) to obtain 5a in a yield of 71%.

The compound of Example 21 was obtained by the method of Example 20.

<Example 21> 4-(7,7-dimethyl-7 H -Puro[2,3- f ]chromen-2-yl)pyridine (5a)

Yellow solid, mp: 98.3-99.1 °C; ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.64 (d, J = 4.8 Hz, 2H), 7.64-7.63 (m, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H) , 6.86 (d, J = 9.6 Hz, 1H), 6.78 (d, J = 8.4 Hz, 1H), 5.74 (d, J = 10.0 Hz, 1H), 1.49 (s, 6H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ 152.2, 151.8, 151.5, 150.4, 137.7, 130.8, 122.1, 120.9, 118.3, 115.9, 113.8, 106.5, 105.5, 76.8, 27.9; HRMS (ESI-QTOF) m/z [M+Na] ⁺ calcd for C ₁₈ H ₁₅ NNaO ₂ 300.0995, found 300.0934.

Experimental Example 1. Measurement of ANO1 inhibitory activity

The inhibitory effect of ANO1 channel activity by treatment with the benzopyran derivatives shown in Table 1 according to the present invention was measured.

Example compound
number R _One IC of ANO1 ₅₀ (μM) Decomposition of ANO1 (%) 2 2a

NA ^b ~60 4 2c

NA ~70 5 2d

NA ~70 9 3c

NA ~80 13 3g

NA ~50 14 3h

1.23 ~50 15 3i

NA ~80 16 3j

NA ~60 17 3k

NA ~80 18 3l

NA ~80 19 3m

NA ~85 21 5a

8.64 ~60 ^a IC ₅₀ values were determined using a YFP fluorescence quenching assay in FRT cells expressing ANO1. (mean ± SE, n = 3). ^b NA if inhibition is less than 20%.

These are the values of IC ₅₀ and ANO1 protein degradation that inhibit ANO1 channel activity of benzopyran derivatives in FRT cells expressing ANO1 and YFP.

IC ₅₀ How to measure

ANO1 and YFP-expressing rat thyroid cell (FRT) cells were cultured in a 96-well micro plate with DMEM/Ham's F12 medium for 48 hours, then the culture medium was removed and then a regular solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 10 mM HEPES, 1 mM CaCl ₂ , 10 mM Glucose D(+), pH7.4) was added to 100 μl per well and the compounds were treated by concentration. After incubation at 37 °C for 20 minutes, NaI solution (140 mM NaI, 5 mM KCl, 1 mM MgCl ₂ , 10 mM HEPES, 1 mM CaCl ₂ , 10 mM Glucose D(+), pH7.4) and ATP (100 μM ), the decrease in YFP fluorescence caused by iodine ions entering the cells was measured at 0.4 second intervals for 5 seconds.

ANO1 protein degradation measurement method

PC-3 cells expressing a high level of ANO1 were cultured in a 6-well plate, treated with the compound at a concentration of 3 μM for 24 hours, and the amount of ANO1 protein was measured using a Western blot technique.

As a result of measuring the inhibitory effect on ANO1 channel activity of the compounds shown in Table 2, compounds having a 2,2-dimethyl-2 H -chromene motif ( 3h , 5a ) strongly inhibited ANO1 channel activity with IC ₅₀ < 10.2 μM, and With an IC ₅₀ value of 1.23 μM, ANO1 channel activity was most strongly inhibited. In addition, compounds with a 2,2-dimethyl-2 H -chromene motif that significantly reduce ANO1 protein are ( 2a , 2c , 2d , 3c , 3g , 3h , 3i , 3j , 3k , 3l , 3m , 5a ), and ANO1 More than 50% of the protein was degraded.

Experimental Example 2. Identification of the specificity of selectively inhibiting the ANO1 activity of the 3h compound

In order to determine the specificity of the 3h compound to selectively inhibit ANO1 activity, the following experiment was performed and shown as a graph in FIG. 1 .

(A) Rat thyroid cells (FRT) expressing ANO1 and YFP were cultured in a 96-well micro plate with DMEM/Ham's F12 medium for 48 hours, then the culture medium was removed and then a regular solution (140 mM NaCl, 5 mM KCl, 1 mM) MgCl ₂ , 10 mM HEPES, 1 mM CaCl ₂ , 10 mM Glucose D(+), pH7.4) was added to 100 μl per well and treated for 3 h by concentration. After incubation at 37 °C for 20 minutes, NaI solution (140 mM NaI, 5 mM KCl, 1 mM MgCl ₂ , 10 mM HEPES, 1 mM CaCl ₂ , 10 mM Glucose D(+), pH7.4) and ATP (100 μM ), the decrease in YFP fluorescence caused by iodine ions entering the cells was measured at 0.4 second intervals for 5 seconds.

(B) In a manner similar to FIG. 3A, FRT cells expressing mouse ANO2 and YFP were treated for 3 h at each concentration, and after 20 minutes, ATP and iodide were treated to increase intracellular calcium, thereby decreasing YFP fluorescence with time through ANO2 activity. was measured.

(C) The dose response curve shows the magnitude of inhibition of YFP fluorescence reduction through ANO1 and ANO2 activity for 3 h by concentration.

(D) FRT cells expressing CFTR and YFP were treated for 3 h at each concentration and 20 minutes later, forskolin and iodide were treated to increase intracellular cAMP, and YFP decreased with time through CFTR activity. Fluorescence was measured. Treatment with 10 μM CFTR _inh -172 completely inhibited CFTR.

(E) PC-3 cells expressing a high level of ANO1 were cultured in a 96-well micro plate with RPMI1640 culture medium for 48 hours, the culture medium was removed, and then calcium assay buffer containing Fluo-4 (Ca ²⁺ dye) was added per well. 100 μl was added and reacted in a light-shielded state for 1 hour, and then the compound was treated with each concentration (0, 1, 3, 10 μM) for 3h . After 20 minutes, the plate was mounted on a micro plate reader and treated with ATP, a P2Y receptor activator, to measure the intracellular calcium concentration.

3h compound inhibited the channel activity of ANO1 by concentration, but did not inhibit intracellular calcium increased by ATP in prostate cancer cells (PC-3). In addition, 3h inhibited the channel activity of ANO1 more than 144 times stronger than ANO2, which has a similar protein structure to ANO1, and did not inhibit the activity of CFTR activated by Forskolin at all up to a concentration of 30 μM. Therefore, it was confirmed that 3h is a novel inhibitor of ANO1 channel activity that specifically inhibits the activity of ANO1.

Experimental Example 3. Identification of the effect of reducing cell viability through ANO1 protein degradation for 3h

In order to investigate the effect of 3h compound on reducing cell viability through ANO1 proteolysis, the following experiment was performed and shown as a graph in FIG. 2 .

(A, B) PC-3 and FaDu cells expressing high levels of ANO1 were treated for 3 h at each concentration as indicated in the figure for 24 hours, and the change in ANO1 protein expression level was measured by Western blot. In addition, the expression level of ANO1 protein in PC-3 and FaDu cells from which the ANO1 gene was removed using CRISPR-Cas9 (CRISPR gene scissors) was measured as a control.

(C, D) PC-3 and FaDu cells expressing high levels of ANO1 and PC-3 and FaDu cells from which the ANO1 gene has been removed were cultured in 96-well plates, approximately 2*103 cells, and incubated for 3 h at each concentration 1 in 24 hours. times, for a total of 48 hours. Then, 20 μl of MTS solution was added to each well and incubated at 37° C. for 30 minutes in 5% CO ₂ , and then absorbance at 490 nm was measured with a micro plate reader (mean ± SE, n = 3).

3 h strongly degraded ANO1 protein in a dose-dependent manner. In addition, 3 h in PC-3 cells and FaDu cells expressing high levels of ANO1 decreased the cell viability according to the concentration, but the cell viability was weakly decreased in PC-3 cells and FaDu cells in which the ANO1 gene was removed. This is the result that 3h selectively degrades the ANO1 protein, causing a significant decrease in the viability of PC-3 and FaDu cells.

Experimental Example 4. Identification of anticancer effect through 3h of apoptosis

In order to elucidate the anticancer effect of the 3h compound through programmed cell death, the following experiment was performed and shown as a graph in FIG. 3 .

(A, B) PC-3 and FaDu cells cultured in a 96-well plate were treated for 3 h at each concentration for 24 hours, washed twice with PBS, and 200 μL of PBS was left, followed by treatment with 1 μM Caspase-3 substrate. and reacted at room temperature for 30 minutes. Then, the plate was mounted on a micro plate reader and fluorescence was measured using ex/em: 485/515 nm filter. 10 μM Ac-DEVD-CHO is a caspase-3 activity-specific inhibitor, and it was co-treated with cells treated with 10 μM 3 h (mean ± SE, n = 3, *P < 0.05 **P < 0.01, *** P < 0.001).

(C, D) PC-3, FaDu cells expressing high levels of ANO1 were cultured in 96-well plates and treated with 10 μM 3h for 24 hours. After washing twice with PBS and leaving 200 μL of PBS, 1 μM Caspase-3 substrate was treated and reacted at room temperature for 30 minutes. Then, cells were mounted on a Lionheart™ FX Automated Microscope, and cells fluorescing by caspasep-3 were observed using an ex/em: 485/515 nm filter. In addition, to stain the nucleus of the cell, 1 μM Hoechst 33342 was applied to the cell, reacted at room temperature for 15 minutes, and the cell with the nucleus stained was observed using ex/em: 346/460 nm filter. By merging the two photos, it was observed how many cells out of the total number of cells undergone apoptosis.

(E, F) PC-3 and FaDu cells expressing high levels of ANO1 were cultured in a 6-well plate. And the cells were treated for 3 h at each concentration for 24 hours, and the amount of PARP protein and cleaved PARP protein was measured using Western blot technique.

When PC-3 and FaDu cells expressing high levels of ANO1 were treated for 3 h at different concentrations, Caspase-3 activity was significantly increased by concentration, and was significantly inhibited by Ac-DEVD-CHO, a caspase-3 activity inhibitor. In addition, the concentration-dependent cleavage of PARP protein in the two cells was performed for 3 h at each concentration. Therefore, it was confirmed that 3h activates Caspase-3 and causes cleavage of the PARP protein, resulting in apoptosis.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (7)

A compound of Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

In Formula 1,
R ₁ is

or

ego,
R ₂ is

,

or C _1-4 alkyl;
R ₃ is

,

or

ego,
R ₄ , R ₅ and R ₆ are each independently a hydrogen atom, C _1-4 haloalkoxy or C _1-4 alkoxy;
Here, Y is a halogen atom or a hydrogen atom.
The method of claim 1,
R ₁ is

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

;

or

phosphorus compound.
The method of claim 1,
R ₁ is

;

;

;

;

;

;

;

;

;

;

or

phosphorus compound.
The method of claim 1,
The compound of Formula 1 is,
(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)(phenyl)methanone;
(4-chlorophenyl)(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)methanone;
(4-bromophenyl)(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)methanone;
1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)ethan-1-one;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-phenylprop-2-en-1-one;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2-methoxyphenyl)prop-2-en-1-one ;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(3-methoxyphenyl)prop-2-en-1-one ;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-one ;
(E)-3-(2,4-dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene-1 -On;
(E)-3-(3,5-dimethoxyphenyl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene-1 -On;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(3,4,5-trimethoxy-phenyl)prop-2 -en-1-one;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1 -On;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(furan-2-yl)prop-2-en-1-one ;
(E)-3-(5-bromofuran-2-yl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene -1-one;
(E)-3-(5-bromothiophen-2-yl)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)prop-2-ene -1-one;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(1H-indol-2-yl)prop-2-en-1 -On;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(thiophen-2-yl)prop-2-en-1- On;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2-(trifluoromethoxy)phenyl)prop-2-ene -1-one;
(E)-1-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)-3-(2,4,5-trimethoxyphenyl)prop-2- en-1-one; or
A compound that is 4-(7,7-dimethyl-7H-furo[2,3-f]chromen-2-yl)pyridine
Claims 1 to 4, comprising the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient, prostate cancer, colon cancer, esophageal squamous cell cancer, pancreatic cancer, head and neck cancer, breast cancer, glioma , Lung cancer, gastric cancer, and a pharmaceutical composition for the prevention or treatment of cancer diseases selected from the group consisting of oral squamous cell cancer.
6. The method of claim 5
The cancer disease is due to ANO1 overexpression, the pharmaceutical composition. delete

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