KR101510995B1 - Pharmaceutical composition for treating or preventing microRNA-31 related diseases - Google Patents

Abstract

The present invention relates to a novel isoxazolylphenylsulfonamide derivative or a salt thereof and a pharmaceutical composition for treating or preventing microRNA-31-related diseases containing the same as an active ingredient. The isoxazolylphenylsulfonamide derivative or a salt thereof is a microsphere- The present invention can be used for the treatment of cancer diseases such as lung cancer, esophageal squamous cell carcinoma, colon cancer or ovarian cancer by controlling RNA-31 expression.

Description

[0001] The present invention relates to a pharmaceutical composition for treating or preventing a microRNA-31 related disease,

The present invention relates to a novel isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof and a pharmaceutical composition for treating or preventing a microRNA-31 (miRNA-31) related disease containing the same as an effective ingredient.

MicroRNAs (miRNAs) are small non-coding RNAs with a length of 18-25 nucleotides and are known to regulate post-transcriptional expression of target mRNA. miRNAs are involved in a number of cellular processes such as cell development, proliferation, survival and death. In addition, downregulated expression of miRNAs is observed in various types of cancer diseases. For example, the miR-155, miR-21 and miR-17-92 clusters are frequently upregulated in a variety of cancers including lung, breast, gastric, colon and lymphatic cancers, while miR-15a-miR-16-1 And miR-29b-1-miR-29a clusters are observed in leukemia and other cancers. In general, over-expressing miRNAs function as carcinogenic miRNAs by down-regulating tumor suppressor genes, while down-regulation of miRNAs function as tumor suppressor miRNA target carcinogens.

MiRNA-31 is a carcinogenic miRNA that is overexpressed in cancer cells such as lung cancer, esophageal squamous cancer and breast cancer ( J Clin Invest . 120: 1298, 2010; Clin Sci ( Lond ). 121: 437, 2011; Oncogenesis 2: 1, 2013) and also functions as an activator of the RAS signaling system associated with colon cancer growth ( J. Biol . Chem . 288: 9508, 2013).

International Patent Publication No. 2010-119698 discloses a therapeutic agent for head and neck tumors using a microRNA inhibitor.

On the other hand, miRNA-31-specific modulators need to be developed because these cancers can be treated through modulation of miRNA-31 associated with various cancer diseases.

It is an object of the present invention to provide a novel isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof capable of treating various cancer diseases through miRNA-31 regulation and a method for the treatment or prevention of miRNA-31 related diseases And to provide a pharmaceutical composition for administration.

In order to achieve the above object, the present invention provides a method for producing an isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound represented by the formula (1), a compound represented by the formula (2), a compound represented by the formula (3) to provide:

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

The isoxazolyl phenyl sulfonamide derivative or a pharmaceutically acceptable salt thereof may be a microRNA-31 modulator.

The present invention also relates to a pharmaceutical composition comprising an isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound represented by the formula (1), a compound represented by the formula (2), a compound represented by the formula (3) There is provided a pharmaceutical composition for treating or preventing a microRNA-31 related disease:

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

The microRNA-31 related disease may be a cancer disease, but may be a cancer disease selected from the group consisting of lung cancer, esophageal squamous cell cancer, colon cancer and ovarian cancer, but is not limited thereto.

The present invention also provides a method for treating cancer, comprising the steps of: treating a candidate compound in a blood sample obtained from a patient suspected of having cancer; And analyzing the secreted alkaline phosphatase (SEAP) activity in the blood sample treated with the candidate compound.

The isoxazolylphenylsulfonamide derivative according to the present invention or a pharmaceutically acceptable salt thereof can be used for the treatment of microRNA-31-related cancer diseases such as lung cancer, esophageal squamous cell carcinoma, colon cancer or ovarian cancer by controlling microRNA-31 expression It can be useful for the treatment of cancer diseases.

Figure 1 shows a reaction system for screening miRNA-31 modulators according to the present invention,
4 shows the expression of mi-RNA in the isoxazolyl phenyl sulfonamide derivatives according to the present invention by the treatment with the compounds 1, 2 and 6,
FIG. 5 shows the expression level of miRNA-31 when Compound 7 was treated in the isoxazolylphenylsulfonamide derivative according to the present invention.

 The terms, techniques, and the like described in the present invention are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified. In addition, all documents referred to in this specification are included in the present specification as a document for explaining the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention provides an isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound of the formula (1), a compound of the formula (2), a compound of the formula (3) and a compound of the formula (4)

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

The isoxazolylphenylsulfonamide derivative according to the present invention can be prepared by the following reaction formula.

One. Benzenesulfonyl  Chloride and Tetrabutylammonium Hydrogen sulphate  Used Sulphone Amide  Derivative synthesis.

Tetrabutylammonium hydrogen sulfate and NaOH aqueous solution were added to a solution of benzenesulfonyl chloride and amine in benzene as shown in Reaction Scheme 1 below. After vigorous stirring in the atmosphere until no starting material was observed by TLC , The reaction mixture is diluted with EtOAc and water and the resulting organic layer is washed, dried, concentrated in vacuo and the residue purified by flash column chromatography on silica gel to give the desired isoxazolyl phenyl sulfonamide derivative :

[Reaction Scheme 1]

2. ( E ) -4- (4- Methoxyphenyl ) -3- Methyl boot -3-en-2-one Isoxazolyl phenyl sulfonamide  Derivative synthesis

2-1) ( E ) -4- (4- Methoxyphenyl ) -3- Methyl boot -3-en-2-one

An HCl solution was added dropwise to a mixture of p-anisaldehyde and 2-butanone as shown in Reaction Scheme 2-1, and the mixture was neutralized with NaOH, and the aqueous layer was extracted with diethyl ether. The extract was washed with brine, dried over anhydrous MgSO ₄ , concentrated under reduced pressure and purified by column chromatography to give ( E ) -4- (4-methoxyphenyl) -3-methylbut-3-en-2- Can:

[Reaction Scheme 2-1]

2-2) (3 E ) -4- (4- Methoxyphenyl ) -3- Methyl boot -3-en-2-one Oxime  synthesis

( E ) -4- (4-methoxyphenyl) -3-methyl-but-3-en-1-one obtained in Reaction Scheme 2-1 was stirred while stirring in a methanol solution of hydroxyamine hydrochloride containing sodium acetate anhydride. 2-one in methanol was added dropwise and the reaction was carried out. The mixture was poured into water and filtered. The residue was washed with water and dried under reduced pressure to obtain ( 3E ) -4- (4-methoxyphenyl) -3-methylbut- 2-one oxime can be obtained:

[Reaction Scheme 2-2]

2-3) 5- (4- Methoxyphenyl ) -3,4- Dimethyl isoxazole  synthesis

Manganese dioxide was added to a chloroform solution of 3 E ) -4- (4-methoxyphenyl) -3-methylbut-3-en-2-one oxime obtained in the above reaction scheme 2-2 as shown in the following Reaction Scheme 2-3 Was subjected to a reflux reaction. The reaction was terminated by TLC, diluted with chloroform and filtered, and the filtrate was concentrated under reduced pressure. And then purified by column chromatography to give 5- (4-methoxyphenyl) -3,4-dimethylisoxazole:

[Reaction Scheme 2-3]

2-4) 5- (3,4- Dimethyl isoxazole -5-yl) -2- Methoxybenzene -One- Sulfonyl  Chloride synthesis

As shown in Scheme 2-4, sulfonic acid chloride was added to 5- (4-methoxyphenyl) -3,4-dimethylisoxazole obtained in the above Scheme 2-3, followed by dilution with EtOAc, Washed with brine. The washed product is dried over anhydrous MgSO ₄ and filtered, and the filtrate is concentrated under reduced pressure to give the desired isoxazolylphenylsulfonamide derivative:

[Reaction Scheme 2-4]

3. 5- (4- Methoxyphenyl ) Isoxazol  Used Isoxazolyl phenyl sulfonamide  Derivative synthesis

3-1) 5- (4- Methoxyphenyl ) Isoxazol  synthesis

4-methoxyacetophenone was slowly added dropwise to a suspension of tetrahydrofuran with sodium hydride and ethyl formate as shown in Reaction Scheme 3-1, and the mixture was stirred at room temperature. The reaction was then terminated with water and the aqueous layer was washed with ethyl acetate. Hydroxyamine hydrochloride was added to the washed aqueous layer, stirred at room temperature, acidified with 2N HCl, extracted with dichloromethane and washed with brine. The washings are dried over anhydrous MgSO ₄ and the filtrate is concentrated under reduced pressure and purified by column chromatography to give 5- (4-methoxyphenyl) isoxazole:

[Reaction Scheme 3-1]

3-2) 5- (3- Chlorosulfonyl -4- Methoxyphenyl ) Isoxazol  synthesis

As shown in Scheme 3-2, sulfonic acid chloride was added to 5- (4-methoxyphenyl) isoxazole obtained in Reaction Scheme 3-1, and the mixture was stirred at 60 ° C. The residue was diluted with ethyl acetate, Lt; / RTI > The washings are dried over anhydrous MgSO ₄ and the filtrate is concentrated under reduced pressure and purified by column chromatography to give the desired isoxazolylphenylsulfonamide derivative:

[Reaction Scheme 3-2]

The isoxazolylphenylsulfonamide derivatives according to the present invention can be prepared as corresponding salts by known methods. Such salts are preferably water-soluble without toxicity. Preferred salts include salts of alkali metals such as potassium, sodium and the like; Salts of alkaline earth metals such as calcium, magnesium and the like; And amines such as tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris (hydroxymethyl) amine, lysine, arginine, And salts of pharmaceutically acceptable amines such as N-methyl-D-glucamine and the like.

The isoxazolylphenylsulfonamide derivatives used in the present invention can be prepared from the corresponding acid addition salts by known methods. These acid addition salts are preferably water-soluble without toxicity. Preferred acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate and nitrate, or organic acid salts such as acetate, lactate, tartrate, oxalate, fumarate, maleate, citrate, benzoate, methane Organic acid salts such as sulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, isethionate, glucuronate and gluconate.

The isoxazolylphenylsulfonamide derivative or pharmaceutically acceptable salt thereof used in the present invention can be prepared into a hydrate by a known method.

In particular, the SEAP activity is significantly increased by treatment of the isoxazolyl phenyl sulfonamide derivative or a pharmaceutically acceptable salt thereof according to the present invention, and as shown in FIGS. 4 and 5, the isoxazolyl phenyl It can be confirmed that the expression of miRNA-31 is specifically inhibited in miRNA by treatment with a sulfonamide derivative or a pharmaceutically acceptable salt thereof.

The present invention also relates to a pharmaceutical composition comprising an isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound represented by the formula (1), a compound represented by the formula (2), a compound represented by the formula (3) There is provided a pharmaceutical composition for treating or preventing a microRNA-31 related disease:

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

The microRNA-31-related diseases are cancer diseases. In particular, the cancer diseases may be selected from the group consisting of lung cancer, squamous cell carcinoma, colon cancer and ovarian cancer.

In the present invention, "treatment" means, unless otherwise stated, reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder it means.

The pharmaceutical composition may comprise a pharmaceutically effective amount of an isoxazolyl phenyl sulfonamide derivative or a pharmaceutically acceptable salt thereof, alone or in combination with one or more pharmaceutically acceptable carriers, excipients, disintegrants, sweeteners, , Lubricants, lubricants, flavors, antioxidants, buffers, bacteriostats, diluents, dispersants, surfactants, binders or lubricants. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent, ameliorate, and treat the symptoms of autoimmune diseases.

The isoxazolylphenylsulfonamide derivative or its salt according to the present invention may be contained in an amount of 0.01 to 30.0 parts by weight based on 100 parts by weight of the total amount of the pharmaceutical composition. When the content is less than 0.01 part by weight, the macrophage phagocytosis inhibiting effect is insignificant. When the content is more than 30.0 parts by weight, there is a slight problem of improvement in the effect depending on the usage amount.

The pharmaceutically effective amount of the isoxazolyl phenyl sulfonamide derivative or the pharmaceutically acceptable salt thereof according to the present invention is 0.5 to 100 mg / day / kg body weight, preferably 0.5 to 5 mg / day / kg body weight. However, the pharmaceutically effective amount may be appropriately changed depending on the severity of the disease symptoms, the age, body weight, health condition, sex, administration route and treatment period of the patient.

The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal, and the formulations may be granules, powders, It may be in the form of tablets, tablets, pills, capsules, suppositories, gels, syrups, juices, suspensions, emulsions, drops, or liquids and is not limited to a specific formulation.

The pharmaceutical compositions of the present invention may be in various forms and administered via various routes including oral, transdermal, subcutaneous, intravenous, intramuscular, rectal, intratracheal, intracerebral, or intracerebroventricular And the dose of the active ingredient can be appropriately selected according to various factors such as route of administration, age, sex, weight and severity of the patient.

The isoxazolylphenylsulfonamide derivative or its salt according to the present invention is a safe compound having a safety of 2 g / kg or more at 50% lethal dose (LC ₅₀ ).

According to the present invention, as in Figure 1, the SEAP reporter construct contains a miRNA-31 complementary sequence downstream of the SEAP gene, and the presence of mature miRNA-31 causes a decrease in SEAP expression, whereas the miRNA- Inhibition of < RTI ID = 0.0 > 31 < / RTI >

Accordingly, the present invention provides a method for treating cancer, comprising: treating a candidate compound in a blood sample obtained from a patient suspected of having cancer; And analyzing the secreted alkaline phosphatase (SEAP) activity in the blood sample treated with the candidate compound.

In the microRNA-31 modulator screening method according to the present invention, when the SEAP activity is increased by the candidate compound treatment, the candidate compound can be evaluated as having miRNA-31 inhibitory activity.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples. However, the following examples and experimental examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.

< Example  1> Isoxazolyl phenyl sulfonamide  Derivative synthesis

< Example  1-1> Benzenesulfonyl  Chloride and Tetrabutylammonium Hydrogen sulphate  Used Sulphone Amide  Derivative synthesis.

3-methyl-lH-indole, 2-methyl-lH-indole, 3,4-dihydroisoquinoline, 0.084 mmol) was added to a mixture of benzenesulfonyl chloride (20 mg, 0.07 mmol) (1.2 mg, 1.2 equiv) in benzene (1 mL) was added tetrabutylammonium hydrogen sulfate (2.4 mg, 0.007 mmol) and 50% aqueous NaOH solution (1 mL), and TLC (EtOAc: n-hexane = 4) was vigorously stirred in the atmosphere until no more starting material was observed. The reaction mixture was diluted with EtOAc and water, and the resultant organic phase was washed with water and brine, dried over MgSO _4, and concentrated in vacuo. The residue was purified by flash column chromatography on silica gel (EtOAc / n -hexane / CH ₂ Cl ₂ = 1: 10: 0.5) to synthesize the desired compound.

[Reaction Scheme 1]

JEOL JNM-LA 300 (300 MHz), JEOL JNM-GCX (400 MHz) or BRUKER AMX-500 (500 MHz) analyzers were used for ¹ H NMR and ¹³ C NMR analyzes. Mass spectrometry and HRMS were performed with VG Trio-2 GC-MS and JEOL JMS-AX, respectively.

1. 5- (3- ( Indole phosphorus -One- IlSilfoil )-4- Methylphenyl ) -3,4- Dimethyl isoxazole  [5- (3- ( Indolin -One- ylsulfonyl ) -4-methylphenyl) -3,4-dimethylisoxazole] (Compound 1)

^{1 H-NMR (500 MHz,} CD 2 Cl 2) δ 8.13 (d, 1H, J = 1.7 Hz), 7.79 (dd, 1H, J = 1.8, 8.0 Hz), 7.44 (d, 1H, J = 8.0 Hz ), 7.38 (d, 1H, J = 8.1 Hz), 7.19-7.18 (m, 1H), 7.13 (t, 1H, J = 7.8 Hz), 6.99 (t, 1H, J = 7.5 Hz), 4.04 (t 2H, J = 8.5 Hz), 3.07 (t, 2H, J = 8.5 Hz), 2.64 (s, 3H), 2.24 (s, 3H), 2.00 (s, 3H);

¹³ C-NMR (CDCl ₃ , 75 MHz) δ 162.2, 161.2, 142.2, 139.1, 138.2, 133.6, 131.0, 130.4, 127.8, 126.9, 126.4, 125.3, 123.6, 114.4, 110.0, 49.9, 27.9, 20.7, ;

LRMS (FAB) m / z 369 (M + H &lt; ⁺ & gt ^; );

_{HRMS (FAB) calcd for C 20} H 21 N 2 O 3 S (M + H +): 369.1267; Found 369.1263.

2. 3,4-Dimethyl-5- (4- methyl -3 - ((3- methyl -One H -Indol-1-yl) Sulfonyl ) Phenyl ) Isoxazol [3,4- Dimethyl -5- (4- methyl -3 - ((3- methyl -One H - indole -One- yl ) ulfonyl ) phen

yl ) isoxazole ] (Compound 2)

^{1 H-NMR (400 MHz,} CD 2 Cl 2) δ 7.78-7.73 (m, 3H), 7.55-7.53 (m, 1H), 7.39-7.37 (m, 2H), 7.28-7.23 (m, 2H), 2.62 (s, 3H), 2.28 (s, 3H), 2.20 (s, 3H), 1.86 (s, 3H);

¹³ C-NMR (CDCl ₃ , 75 MHz) δ 161.8, 161.1, 138.6, 138.6, 135.3, 133.6, 131.3, 131.0, 127.1, 125.5, 124.8, 123.6, 123.2, 119.6, 117.6, 113.4, 110.1, 20.4, 10.2, 9.7 , 7.7;

LRMS (FAB) m / z 381 (M + H &lt; ⁺ & gt ^; );

_{HRMS (FAB) calcd for C 21} H 21 N 2 O 3 S (M + H +): 381.1267; found 381.1270.

3. 5- (3 - ((1 H -Indol-1-yl) Sulfonyl )-4- Methylphenyl ) -3,4-dimethylisoxazole [5- (3 - ((1 H - Indol -1-yl) sulfonyl) -4-methylphenyl) -3,4-dimethylisoxazole] (Compound 3)

^{1 H-NMR (400 MHz,} CD 2 Cl 2) δ 7.87 (d, 1H, J = 1.3 Hz), 7.78-7.74 (m, 2H), 7.65 (d, 1H, J = 3.6 Hz), 7.61-7.59 (m, 1H), 7.37, (d, 1H, J = 8.0 Hz), 7.27-7.21 (m, 2H), 6.74 (d, 1H, J = 3.6 Hz), 2.60 (s, 3H), 2.22 (s , &Lt; / RTI &gt; 3H), 1.92 (s, 3H);

¹³ C-NMR (CDCl ₃ , 75 MHz) δ 161.8, 161.2, 138.6, 138.3, 134.8, 133.7, 131.2, 130.4, 127.2, 126.9, 125.8, 124.7, 123.5, 121.6, 113.2, 110.2, 108.1, 20.2, 10.1, 7.7 ;

LRMS (FAB) m / z 367 (M + H &lt; ⁺ & gt ^; );

_{HRMS (FAB) calcd for C 20} H 19 N 2 O 3 S (M + H +): 367.1111; found 367.1114.

4. 3,4-Dimethyl-5- (4- methyl -3 - ((2- methyl -One H -Indol-1-yl) Sulfonyl ) Phenyl ) Isoxazol  [3,4- Dimethyl -5- (4-methyl-3 - ((2-methyl-1 H -indol-1-yl) sulfonyl) phenyl) isoxazole] (Compound 4)

¹ H-NMR (500 MHz, CDCl ₃ )? 8.05-8.03 (m, 1H), 7.80 (dd, 1H, J = 1.6, 7.9 Hz), 7.56-7.54 1H), 7.41 (d, 1H , J = 8.0 Hz), 7.35 (d, 1H, J = 1.4 Hz), 7.33-7.27 (m, 2H), 2.57 (s, 3H), 2.49 (s, 3H), 2.21 (s, 3 H), 1.73 (s, 3 H);

¹³ C-NMR (CDCl ₃ , 75 MHz) δ 161.6, 161.2, 139.4, 138.6, 135.8, 133.8, 132.9, 131.1, 127.3, 127.0, 125.4, 124.4, 124.2, 118.3, 114.6, 112.8, 110.3, , 7.5.

5. 5- (3 - ((3,4- Dihydroquinoline -1 (2 H )-Work) Sulfonyl )-4- Methylphenyl ) -3,4- Dimethyl isoxazole  [5- (3 - ((3,4- Dihydroquinolin -1 (2 H ) - yl ) ulfonyl )-4- 메틸 피닐 ) -3,4

- dimethylisoxazole ] (Compound 5)

^{1 H-NMR (500 MHz,} CD 2 Cl 2) δ 8.14 (d, 1H, J = 1.7 Hz), 7.79 (dd, 1H, J = 1.8, 8.0 Hz), 7.44-7.42 (m, 2H), 7.12 2H, J = 6.8 Hz), 2.45 (s, 3H), 2.25 (s, 3H), 2.06 (s, 1.84-1.76 (m, 2H);

¹³ C-NMR (CDCl ₃ , 75 MHz) δ 162.3, 161.2, 139.7, 138.7, 137.1, 133.6, 130.3, 130.1, 129.3, 127.2, 127.0, 126.4, 124.9, 124.1, 110.1, 46.3, 26.6, 22.2, 20.5, 10.2 , 8.0;

LRMS (FAB) m / z 383 (M + H &lt; ⁺ & gt ^; );

_{HRMS (FAB) calcd for C 21} H 23 N 2 O 3 S (M + H +): 383.1424; found 383.1425.

< Example  1-2> E ) -4- (4- Methoxyphenyl ) -3- methyl - 3 Beaten -2-one Isoxazolyl phenyl sulfonamide  Derivative synthesis

One)( E ) -4- (4- Methoxyphenyl ) -3- Methyl boot -3-en-2-one

A solution of 4.0 M HCl in 1,4-dioxane (10 mL) was added to a mixture of p-anisaldehyde (1217 μL, 10.0 mmol) and 2-butanone (996 μL, 10.0 mmol) The mixture was stirred at room temperature for 12 hours, neutralized with cold 2N-NaOH, and extracted three times with diethyl ether (50 mL). The extract was washed with brine, dried over anhydrous MgSO ₄ , concentrated under reduced pressure, and purified by column chromatography (EtOAc: Hexane = 1:15 → 1:12) to obtain the compound (1341.7 mg, 7.05 mmol).

[Reaction Scheme 2-1]

^{1 H NMR, (300MHz, CDCl} 3): δ 7.48 (s, 1H), 7.42 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 3.85 (s, 3H), 2.45 (s, 3 H), 2.07 (s, 3 H).

2) (3 E ) -4- (4- Methoxyphenyl ) -3- Methyl boot -3-en-2-one Oxime  synthesis

( E ) -4- (4-aminophenoxy) -benzene obtained in the above Reaction Scheme 2-1 was stirred for 30 minutes while stirring a mixture of sodium carbonate (414 mg, 5.05 mmol) in methanol (3 mL) 3-methylbut-3-en-2-one (800 mg, 4.2 mmol) in methanol (2 mL) was added dropwise and the mixture was reacted at 60 ° C for 3 hours. Water (20 mL) was added to the mixture and the mixture was filtered. The residue was washed with water, and the water was dried under reduced pressure to obtain the compound of the following reaction formula 2-2.

[Reaction Scheme 2-2]

^{1 H NMR, (300MHz, CDCl} 3): δ 8.93 (s, 1H), 7.27 (d, J = 9.0 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 6.87 (s, 1H), 3.83 (s, 3H), 2.17 (s, 3H), 2.08 (s, 3H).

3) 5- (4- Methoxyphenyl ) -3,4- Dimethyl isoxazole  synthesis

As shown in Reaction Scheme 2-3, a solution of 3 E ) -4- (4-methoxyphenyl) -3-methylbut-3-en-2-one oxime (500 mg, 2.44 mmol) Manganese dioxide (423.6 mg, 4.87 mmol) was added to chloroform (50 mL) and the mixture was refluxed for 3 hours. The reaction was terminated by TLC, diluted with chloroform and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (CAN: CHCl3 = ₁ : 40) to obtain the desired compound (320 mg, 1.583 mmol).

[Reaction Scheme 2-3]

^{1 H NMR, (300MHz, CD} 2 Cl 2): δ 7.64 (d, J = 9.0 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H), 3.85 (s, 3H), 2.24 (s, 3H ), 2.13 (s, 3 H).

4) 5- (3,4- Dimethyl isoxazole -5-yl) -2- Methoxybenzene -One- Sulfonyl  Chloride synthesis

As shown in Scheme 2-4, sulfonic acid chloride was added to 5- (4-methoxyphenyl) -3,4-dimethylisoxazole obtained in the above Scheme 2-3, followed by dilution with EtOAc, Washed with brine. The washed product is dried over anhydrous MgSO ₄ and filtered. The filtrate is concentrated under reduced pressure to obtain the desired isoxazolylphenylsulfonamide derivative.

[Reaction Scheme 2-4]

5) 5- (3- (3,4- Dihydroisoquinoline -2 (1 H ) - IlSilfoil )-4- Methoxyphenyl ) -3,4- Dimethyl isoxazole (Compound 6)

5- (3,4-dimethylisoxazol-5-yl) -2-methoxybenzene-1-sulfonyl chloride (30 mg, 0.1 mmol) obtained in the above Reaction Scheme 2-4, (15.0 mg, 0.1 mmol) and 1.5 mL of 50% NaOH in water (v / v) were added to the reaction mixture, followed by the addition of 1,2,3,4-tetrahydroisoquinoline And the mixture was stirred at room temperature for 5 hours. The residue was diluted with EtOAc and washed with water and brine. The washed product was dried over anhydrous MgSO ₄ , concentrated under reduced pressure and purified by column chromatography (EtOAc: Hex: MC = 1: 3: 0.5) to obtain the desired compound (20 mg, 50%) (Compound 6).

[Reaction Scheme 2-5]

^{1 H NMR, (600MHz, CDCl} 3): δ 8.32 (d, J = 2.3 Hz, 1H), 7.87 (dd, J = 8.7 Hz, 2.3 Hz, 1H), 7.13-7.18 (m, 2H), 7.06- 7.08 (m, 2H), 7.02 (d, J = 8.7 Hz, 1H), 4.58 (s, 2H), 3.74 (s, 3H), 3.64 (t, 5.9 Hz, 2H), 2.76 (t, 5.9 Hz, 2H), 2.29 (s, 3H ), 2.18 (s, 3H) 13 C NMR (150 MHz, CDCl 3): δ 162.47, 161.16, 157.23, 133.52, 132.61, 132.59, 129.74, 128.95, 127.98, 126.57, 126.34, 126.06, 121.42, 112.52, 109.24, 55.93, 47.14, 43.31, 28.57, 10.24, 8.15.

< Example  1-3> 5- (4- Methoxyphenyl ) Isoxazol  Used Isoxazolyl phenyl sulfonamide  Derivative synthesis

1) 5- (4- Methoxyphenyl ) Isoxazol  synthesis

4-methoxyacetophenone (347.7 [mu] L, 2.5 [mu] L) was added to a suspension of sodium hydride (200 mg, 5.0 mmol) and ethyl formate (804.4 L, 10.0 mmol) in tetrahydrofuran mmol) was slowly added dropwise at 0 ° C, followed by stirring at room temperature for 3 hours. The reaction was then terminated with water and the aqueous layer was washed with ethyl acetate (EtOAc).

Hydroxyamine hydrochloride (173.7 mg, 2.5 mmol) was added to the washed aqueous layer, stirred at room temperature for 24 hours, acidified with 2N HCl, extracted with dichloromethane and washed with brine. The washed product was dried over anhydrous MgSO _{4. The} filtrate was concentrated under reduced pressure and purified by column chromatography (EtOAc: Hex = 1: 5) to obtain the desired compound (286.8 mg, 65%).

[Reaction Scheme 3-1]

^{1 H NMR, (300MHz, CDCl} 3): δ 8.25 (d, J = 1.9 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 6.40 ( d, J = 1.9 Hz, 1 H), 3.86 (s, 3 H)

2) 5- (3- Chlorosulfonyl -4- Methoxyphenyl ) Isoxazol  synthesis

Chlorosulfonic acid (503 μL, 7.34 mmol) was added to 5- (4-methoxyphenyl) isoxazole (257.3 mg, 1.47 mmol) obtained in the above reaction scheme 3-1 as shown in the following reaction scheme 3-2 After stirring at 60 ° C for 1 hour and 20 minutes, the residue was diluted with ethyl acetate (EtOAc) and washed with water and brine. The washed product was dried over anhydrous MgSO _{4. The} filtrate was concentrated under reduced pressure and purified by column chromatography (EtOAc: Hex = 1: 2) to obtain the desired compound (213 mg, 53%).

[Reaction Scheme 3-2]

^{1 H NMR, (300MHz, CDCl} 3): δ8.34 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H), 8.14 (dd, J = 8.8 Hz, 2.3 Hz, 1H ), 7.25 (d, J = 8.7 Hz, 1 H), 6.55 (d, J = 1.9 Hz,

3) 5- (3 - (3, 4-dihydroisoquinoline -2 (1 H) - some accounts) -chroman-4-methoxyphenyl) isoxazole [5- (3- (3, 4, -dihydroisoquinolin- 2 ( 1H ) -ylsulfonyl) -4-methoxyphenyl) isoxazole] (Compound 7)

5- (3-Chlorosulfonyl-4-methoxyphenyl) isoxazole (30 mg, 0.11 mmol) obtained in the above Reaction Scheme 2-2 was added to 1.5 ml of benzene as shown in the following Reaction Scheme 4, (15 mg, 0.10 mmol) and 1.5 mL of 50% NaOH in water (v / v) were added to the solution, and the mixture was stirred at room temperature for 5 hours. The residue was diluted with EtOAc and washed with water and brine and dried over anhydrous MgSO _4. The dried filtrate was concentrated under reduced pressure and purified by column chromatography (EtOAc: Hex: MC = 1: 2: 0.5) to obtain the desired compound (27 mg, 66%) (Compound 7).

[Reaction Scheme 4]

^{1 H NMR, (600MHz, CDCl} 3): δ8.37 (d, J = 2.3 Hz, 1H), 8.30 (d, J = 1.9 Hz, 1H), 7.97 (dd, J = 8.6 Hz, 2.3 Hz, 1H ), 7.14-7.18 (m, 2H), 7.08-7.06 (m, 2H), 7.02 (d, J = 8.7 Hz, (s, 3H), 3.65 (t, 5.9 Hz, 2H), 2.76 (t, 5.9 Hz, 2H);

¹³ C NMR (150 MHz, CDCl ₃ ): δ 167.64, 157.92, 150.97, 133.48, 132.55, 131.66, 129.39, 128.97, 128.38, 126.61, 126.37, 126.04, 120.09, 112.60, 98.58, 56.00,

< Experimental Example  1> microphone RNA -31 inhibition activity review

1. Cell culture

HEK-293T cells and A549 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FCS, 100 mg / ml streptomycin and 100 U / ml penicillin or RPMI 1640 medium supplemented with 5% CO ₂ Lt; RTI ID = 0.0 &gt; 37 C. &lt; / RTI &gt;

2. SEAP Reaction system  Construction and drug screening

HEK-293T cells were co-transfected with SEAP reporter constructs containing the hsa-miR-31 expression vector and the miR-31 target sequence using PEI (poly ethylene immune; Sigma-Aldrich, St. Louis, Mo., USA) . Cells transfected 24 hours after the transfection were collected and dispensed at a concentration of 2 x 10 ⁴ cells / well on a 96-well plate. After 4-5 hours, the compounds according to Example 1 were added to the cells to a final concentration of 5 [mu] M and incubated for 24 hours. Subsequently, the cell supernatant was added with a p-nitrophenylphosphate (Sigma-Aldrich) solution and the absorbance was measured at 405 nm using a microplate reader.

As a result, the expression of miR-31 was significantly inhibited by the treatment of Compound 1, Compound 2 and Compound 6, and the miR-31 expression-suppressing activity, which is superior to that of Compound 3 to Compound 5, Respectively.

3. Real time RT - PCR  analysis

6. The wall-A549 cells on the plate at a concentration of 3 x 10 ⁵ cells / well dispensed, and a compound of 5 μM for 24 hours. The cells were then harvested and resuspended in 1 mL of TRI reagent (Molecular Research Center, Cincinnati, OH, USA) to isolate total RNA. One μg of total RNA was transcribed with cDNA using miScript reverse kits (QIAGEN, Valencia, CA, USA). 0.5 μl of the synthesized cDNA was used in a 20 μl PCR reaction system and 2x miScript SYBR Green PCR Kit (QIAGEN) was used. At this time, amplification was carried out under the following thermal cycling conditions. The amplification was carried out using iCycler (Bio-Rad, Hercules, Calif., USA) at 95 ° C for 15 minutes, then at 94 ° C for 15 seconds, at 55 ° C for 30 seconds and at 70 ° C for 30 seconds. Gene expression was normalized to RNU6B expression and miRNA-specific primers for miR-16, miR-21, miR-31 and miR-155 detection were purchased from QIAGEN (miScript primer assay).

As a result, since the expression of miR-31 is specifically inhibited by the treatment of Compound 1, Compound 2 and Compound 6 as compared with other miRNA species as shown in Fig. 4, these compounds specifically inhibit the expression of miR-31 Respectively.

As shown in FIG. 5, when the compound 7 was treated, the expression level of miR-31 was 56%, indicating that Compound 7 specifically inhibited the expression of miR-31.

< Experimental Example  2> Toxicity experiment

Compound 1 was suspended in 0.5% methylcellulose solution in male Balb / c mice and single oral doses of 0.5 g / kg, 1 g / kg and 2 g / kg were administered once, and the survival rate and body weight of mice were examined for 7 days.

After this administration, we observed the mortality of the animal, clinical symptoms, weight change, hematologic test and blood biochemical test, and autopsied the visceral organs and thoracic organs were observed.

As a result, no clinical symptoms or dead animals were found in all animals, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

As a result, Compound 1 of the present invention did not show any toxic change up to 2 g / kg in mouse, and thus it was judged that the oral LD 50 was 2 g / kg or more.

Hereinafter, formulation examples of the composition containing Compound 1 according to the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

&Lt; Formulation Example 1 > Preparation of powders

5 mg of Compound 1, 100 mg of lactose, and 10 mg of talc were mixed and filled in airtight bags to prepare powders.

&Lt; Formulation Example 2 > Preparation of tablet

Compound 1 (5 mg), corn starch (100 mg), lactose (100 mg) and magnesium stearate (2 mg) were mixed and tableted according to a conventional preparation method.

&Lt; Formulation Example 3 > Preparation of capsule agent

Compound 1 (5 mg), corn starch (100 mg), lactose (100 mg) and magnesium stearate (2 mg) were mixed, and the above components were mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

&Lt; Formulation Example 4 > Preparation of injection

Compound 2 (2 mg), sterile distilled water suitable amount for injection, and pH adjuster were mixed, and the contents were adjusted to the above contents in the amount of 2 ml per ampoule according to the usual injection preparation method.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

An isoxazolylphenylsulfonamide derivative selected from the group consisting of the compounds represented by formulas (1), (2), (3) and (4): or a pharmaceutically acceptable salt thereof,
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

2. The isoxazolyl phenyl sulfonamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the isoxazolyl phenyl sulfonamide derivative or a pharmaceutically acceptable salt thereof is a microRNA-31 modulator. Treatment or prevention of cancer diseases containing, as an active ingredient, an isoxazolylphenylsulfonamide derivative or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound represented by the formula (1), a compound represented by the formula (2), a compound represented by the formula (3) Pharmaceutical composition:
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

delete [Claim 5] The pharmaceutical composition according to claim 3, wherein the cancer disease is selected from the group consisting of lung cancer, esophageal squamous cell carcinoma, colon cancer and ovarian cancer. Treating the candidate compound in a blood sample obtained from a patient suspected of having cancer; And analyzing the secreted alkaline phosphatase (SEAP) activity in a blood sample to which the candidate compound has been treated.

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