KR101870968B1 - New Thiophene Compound or Pharmaceutically Acceptable Salt Thereof Having Activation on PPAR, FXR and AMPK, and Medical Use Thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a health functional food comprising a novel thiophene compound and a novel thiophene compound capable of activating PPAR, FXR and AMPK, or a pharmaceutically acceptable salt thereof, and PPAR subtypes of alpha, beta, and gamma, and exhibit FXR or AMPK activation effect. Therefore, it is possible to provide a pharmaceutical composition for preventing or treating fatty liver disease including lipid hepatitis or a pharmaceutical composition for preventing or improving health function It can be usefully used as a food composition.

Description

FIELD OF THE INVENTION The present invention relates to novel thiophene compounds having PPAR, FXR and AMPK activating activity, or pharmaceutically acceptable salts thereof, and medicinal uses thereof,

The present invention is directed to novel thiophene compounds having PPAR, FXR and AMPK activity, or pharmaceutically acceptable salts thereof and their medicinal uses.

Most of the fat accumulated in the liver is triglyceride. The fatty liver is mainly caused by alcoholic fatty liver disease and nonalcoholic fatty liver disease (obesity, diabetes, hyperlipemia, drug) liver disease, NAFLD).

Alcoholic fatty liver disease progresses from the initial simple fatty liver to fatty liver and cirrhosis. Non-alcoholic fatty liver disease is known to remain in simple fatty liver and does not progress. However, recently, it has been found that non-alcoholic fatty liver disease may progress from simple fatty liver to fatty liver or liver cirrhosis.

Recently, as the nutritional status improves and the adult diseases increase, the number of patients with fatty liver is increasing. According to the Korean Hepatology Society, the prevalence of liver disease has declined over the past decade, but the prevalence of fatty liver has tripled over 20 years, among which the ratio of nonalcoholic fatty liver exceeds 50% appear.

Nonalcoholic fatty liver disease is a disease characterized by fatty change (steatosis) and lobular hepatitis (steatohepatitis), which are hallmarks of alcoholic hepatitis, And so on. The pathologic findings of the liver are diverse, ranging from non-alcoholic Fatty Liver (NAFL) to non-alcoholic steatohepatits (NASH), fibrosis associated with fatty liver, liver cirrhosis, It is used to mean all inclusive. Most of these nonalcoholic fatty liver diseases are accompanied by complications such as insulin resistance, obesity, diabetes, and hyperlipidemia. In the presence of such complications, treatment should be performed. The principle of treatment for nonalcoholic fatty liver disease is improvement of lifestyle such as diet and exercise therapy, but it is a reality that it is difficult to perform reliably. In particular, nonalcoholic steatohepatitis is more likely to develop cirrhosis or hepatocellular carcinoma, so more aggressive medication is needed.

To date, polyene phosphatidylcholine has been used clinically as a therapeutic agent for fatty liver. Fibrate-based drugs, such as clofibrate, a therapeutic agent for hyperlipidemia, have been used clinically in the treatment of fatty liver. It is thought that the double fibrate drug improves lipid metabolism through hepatic β - oxidase enzyme in liver. However, the above-mentioned fibrate drugs have a side effect such as liver dysfunction.

Currently, there are currently no drugs licensed as therapeutic agents for fatty liver, especially NASH. The promising target of NASH treatment is peroxisome proliferator activated receptor (PPAR), Farnesoid X Receptor (FXR) ), 5'-AMP-activated protein kinase (AMPK) and thyroid receptor-β (TR-β) are known.

PPAR alpha agonists are known to promote beta-oxidation of free fatty acids and ketone body formation, thereby improving excess fat. PPAR gamma regulates the production and differentiation of fat cells, fat metabolism and insulin sensitization, It is reported that it can be improved. PPAR delta is known to modulate fatty acid oxidation and inflammation to improve the symptoms of NASH (nonalcoholic steatohepatitis), and FXR is known to improve NASH symptoms by affecting bile acid regulation and glucose and lipid metabolism.

AMPK (5'-AMP-activated protein kinase) is an enzyme that plays an important role in the energy homeostasis of cells. In addition, it promotes hepatic fatty acid oxidation and ketogenesis and inhibits cholesterol production, fat production and triglyceride biosynthesis And also plays an important role in improving glucose uptake. Because of this function, many papers report that AMPK plays a crucial role in the treatment of alcoholic or nonalcoholic fatty liver disease.

Currently, a number of compounds with single targets are undergoing clinical trials. However, in order to have a large market share in the pharmaceutical market through clinical trials, the side effects should be very small and at the same time, the efficacy should be superior. Multiple target compounds are known to be ideal for satisfying these conditions. Therefore, it should be a drug capable of simultaneously activating two or more of these targets.

Therefore, there is a need for the development of a composition for the treatment of alcoholic or non-alcoholic fatty liver which is capable of simultaneously activating PPAR and FXR having excellent therapeutic effect without any side effects or activating a target such as AMPK more than a known drug.

1. Korean Patent No. 10-1486312.

It is an object of the present invention to provide novel thiophene compounds.

Another object of the present invention is to provide a novel pharmaceutical composition showing the effect of preventing or treating fatty liver disease.

It is still another object of the present invention to provide a health functional food exhibiting an effect of preventing or ameliorating fatty liver disease.

In order to achieve the above object, the present invention provides a thiophen compound represented by the following formula (I): or a pharmaceutically acceptable salt thereof;

[Chemical Formula 1]

In Formula 1,

W is S or O,

R ¹ and R ² may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl and halogen,

R ³ and R ⁴ may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy and halogen, or R ³ and R ⁴ are connected to each other to form a 5- or 6- And the 5-or 6-membered ring may be substituted or unsubstituted,

m is 0 or 1,

n is any integer selected from 0 to 3,

Ar ¹ and Ar ² may be the same or different and are each selected from the group consisting of aromatic, heteroaromatic, cycloalkyl, and heterocycloalkyl.

To achieve these and other objects, the present invention provides a pharmaceutical composition for prevention or treatment of fatty liver disease comprising the thiophene compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

According to another aspect of the present invention, there is provided a health functional food for preventing or ameliorating fatty liver disease comprising the thiophene compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a pharmaceutical composition or a health functional food comprising a novel thiophene compound and a novel thiophene compound capable of activating PPAR, FXR and AMPK, or a pharmaceutically acceptable salt thereof, the present invention can activate any one or more of the PPAR subtypes consisting of?,?, and?, and also has FXR and AMPK activation effect and triglyceride reduction effect. Thus, it is possible to provide a pharmaceutical composition for preventing or treating fatty liver disease Or as a health functional food for improvement.

Fig. 1 shows the results of confirming the activity of the compounds 1 to 15 according to the present invention against PPAR (Fig. 1A is the result for PPAR alpha, Fig. 1B is the result for PPAR beta, Fig. 1C is the result for PPAR gamma to be.)
Fig. 2 shows the results of confirming the activity of the compounds 1 to 15 (MHY3294 to MHY3304 and MHY3310 to MHY3313) according to the present invention against FXR.
Fig. 3 shows the results of confirming the oxidative stress-removing activity of the compounds 1 to 15 (MHY3294 to MHY3304 and MHY3310 to MHY3313) according to the present invention.
Figure 4 shows the oral administration schedule of Compound 7 (MHY3300) in mice for in vivo experiments.
Fig. 5 shows the oxidative stress-removing activity of Compound 7 (MHY3300) in vivo.
Fig. 6 shows the result of confirming cytotoxicity of Compound 7 (MHY3300).
FIG. 7 shows the result of confirming the activity of Compound 7 (MHY3300) on PPAR in cells.
Figure 8 shows the results of measuring the amount of AMPK phosphorylated over time after treatment with Compound 7 (MHY3300) in cells.
Fig. 9 shows the oral administration schedule of Compound 7 (MHY3300) in mice for in vivo AMPK measurement experiment.
Figures 10 and 11 are the results of confirming the amount of phosphorylated AMPK in Compound 7 (MHY3300) treatment in vivo.
Fig. 12 shows the result of measurement of the amount of triglyceride in the compound 7 (MHY3300) treatment.

In the present invention, a novel thiophene compound having an effect of activating PPAR, FXR and AMPK was synthesized while studying a compound showing a therapeutic effect on fatty liver, thereby completing the present invention.

Accordingly, the present invention provides a thiophen compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof;

[Chemical Formula 1]

In Formula 1,

W is S or O,

R ¹ and R ² may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl and halogen,

R ³ and R ⁴ may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy and halogen, or R ³ and R ⁴ are connected to each other to form a 5- or 6- And the 5-or 6-membered ring may be substituted or unsubstituted,

m is 0 or 1,

n is any integer selected from 0 to 3,

Ar ¹ and Ar ² may be the same or different and are each selected from the group consisting of aromatic, heteroaromatic, cycloalkyl, and heterocycloalkyl.

The aromatic compound may be any one selected from the group consisting of phenyl, biphenyl, naphthyl, and anthracene, but is not limited thereto.

The thiophene compound represented by the above-mentioned formula (1) can be obtained by reacting 3- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) benzoic acid [3- 3-carboxamido) benzoic acid], 5- (2-4-tert-butylbenzamido) thiophene-3-carboxamide [ 4- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] thiophene-3-carboxamido) benzoic acid, 3- (2- (2-naphthaamido) thiophene-3-carboxamido) benzoic acid [3- (2- (2-Naphthamido) thiophene-3-carboxamido) -2-methylbenzoic acid (2-Naphthamido) thiophene-3-carboxamido) benzoic acid], 3- (2-naphthylamido) thiophene- -Biphenyl-4-ylcarboxamide Midothiophene-3-carboxamido) benzoic acid [3- (2-Biphen 4-ylcarboxamidothiophene-3-carboxamido) benzoic acid, 5- (2-biphenyl-4-ylcarboxamidothiophene- 4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid], 4- (2-biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [4- (2-Biphenyl-4-ylcarboxamidothiophene -3-carboxamido) benzoic acid], 3- (2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxamide [ ) thiophene-3-carboxamido) benzoic acid], 2-methyl-5- (2- (4- (trifluoromethyl) benzamido) thiophene- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid], 4- (2- (4- (2- (4- (tert-butyl) benzamido) thiophene-3-carboxamido) benzoic acid) Phenyl) acetic acid [2- (3- (2- (4- (tert-Butyl) b 3-carboxamido) phenyl) acetic acid], 2- (3- (2- ((1,1'-biphenyl-4-ylcarboxamido) thiophene- ) Acetic acid] and 2- (3- (2- (4- (tri (phenoxycarbonyl) aminomethyl) thiophene-3- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] It is any one selected from the group.

In addition, the present invention provides a pharmaceutical composition for preventing or treating fatty liver disease comprising, as an active ingredient, a thiophene compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

In Formula 1,

W is S or O,

R ¹ and R ² may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl and halogen,

R ³ and R ⁴ may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy and halogen, or R ³ and R ⁴ are connected to each other to form a 5- or 6- And the 5-or 6-membered ring may be substituted or unsubstituted,

m is 0 or 1,

n is any integer selected from 0 to 3,

Ar ¹ and Ar ² may be the same or different and are each selected from the group consisting of aromatic, heteroaromatic, cycloalkyl, and heterocycloalkyl.

The aromatic compound may be any one selected from the group consisting of phenyl, biphenyl, naphthyl, and anthracene, but is not limited thereto.

The thiophene compound represented by the above-mentioned formula (1) can be obtained by reacting 3- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) benzoic acid [3- 3-carboxamido) benzoic acid], 5- (2-4-tert-butylbenzamido) thiophene-3-carboxamide [ 4- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] thiophene-3-carboxamido) benzoic acid, 3- (2- (2-naphthaamido) thiophene-3-carboxamido) benzoic acid [3- (2- (2-Naphthamido) thiophene-3-carboxamido) -2-methylbenzoic acid (2-Naphthamido) thiophene-3-carboxamido) benzoic acid], 3- (2-naphthylamido) thiophene- -Biphenyl-4-ylcarboxamide Midothiophene-3-carboxamido) benzoic acid [3- (2-Biphen 4-ylcarboxamidothiophene-3-carboxamido) benzoic acid, 5- (2-biphenyl-4-ylcarboxamidothiophene- 4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid], 4- (2-biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [4- (2-Biphenyl-4-ylcarboxamidothiophene -3-carboxamido) benzoic acid], 3- (2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxamide [ ) thiophene-3-carboxamido) benzoic acid], 2-methyl-5- (2- (4- (trifluoromethyl) benzamido) thiophene- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid], 4- (2- (4- (2- (4- (tert-butyl) benzamido) thiophene-3-carboxamido) benzoic acid) Phenyl) acetic acid [2- (3- (2- (4- (tert-Butyl) b 3-carboxamido) phenyl) acetic acid], 2- (3- (2- ((1,1'-biphenyl-4-ylcarboxamido) thiophene- ) Acetic acid] and 2- (3- (2- (4- (tri (phenoxycarbonyl) aminomethyl) thiophene-3- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] It is any one selected from the group.

The fatty liver disease may be an alcoholic fatty liver disease or a nonalcoholic fatty liver disease.

The alcoholic fatty liver disease may be selected from the group consisting of alcoholic fatty liver disease, simple alcoholic fatty liver disease, alcoholic liver fibrosis, and alcoholic cirrhosis, preferably alcoholic fatty liver disease, but is not limited thereto . The non-alcoholic fatty liver disease may also be selected from the group consisting of non-alcoholic fatty liver, non-alcoholic simple fatty liver, non-alcoholic liver fibrosis and non-alcoholic cirrhosis, But are not limited thereto.

The thiophene compound represented by Formula 1 may activate at least one selected from the group consisting of FXR, PPAR alpha, PPAR gamma, PPAR beta and AMPK, more preferably having a double activity against PPAR and FXR Can have activity against AMPK.

The pharmaceutical compositions according to the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the production of pharmaceutical compositions.

Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The pharmaceutical composition according to the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method have.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

Further, the dosage of the pharmaceutical composition according to the present invention may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intratracheal, intrauterine or intracerebroventricular injections.

The present invention also provides a health functional food for preventing or ameliorating fatty liver disease comprising a thiophene compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient;

[Chemical Formula 1]

In Formula 1,

W is S or O,

R ¹ and R ² may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl and halogen,

R ³ and R ⁴ may be the same or different and are any one selected from the group consisting of H, C 1 -C 4 alkyl, C 1 -C 4 alkoxy and halogen, or R ³ and R ⁴ are connected to each other to form a 5- or 6- And the 5-or 6-membered ring may be substituted or unsubstituted,

m is 0 or 1,

n is any integer selected from 0 to 3,

Ar ¹ and Ar ² may be the same or different and are each selected from the group consisting of aromatic, heteroaromatic, cycloalkyl, and heterocycloalkyl.

The aromatic compound may be any one selected from the group consisting of phenyl, biphenyl, naphthyl, and anthracene, but is not limited thereto.

The thiophene compound represented by the above-mentioned formula (1) can be obtained by reacting 3- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) benzoic acid [3- 3-carboxamido) benzoic acid], 5- (2-4-tert-butylbenzamido) thiophene-3-carboxamide [ 4- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] thiophene-3-carboxamido) benzoic acid, 3- (2- (2-naphthaamido) thiophene-3-carboxamido) benzoic acid [3- (2- (2-Naphthamido) thiophene-3-carboxamido) -2-methylbenzoic acid (2-Naphthamido) thiophene-3-carboxamido) benzoic acid], 3- (2-naphthylamido) thiophene- -Biphenyl-4-ylcarboxamide Midothiophene-3-carboxamido) benzoic acid [3- (2-Biphen 4-ylcarboxamidothiophene-3-carboxamido) benzoic acid, 5- (2-biphenyl-4-ylcarboxamidothiophene- 4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid], 4- (2-biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [4- (2-Biphenyl-4-ylcarboxamidothiophene -3-carboxamido) benzoic acid], 3- (2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxamide [ ) thiophene-3-carboxamido) benzoic acid], 2-methyl-5- (2- (4- (trifluoromethyl) benzamido) thiophene- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid], 4- (2- (4- (2- (4- (tert-butyl) benzamido) thiophene-3-carboxamido) benzoic acid) Phenyl) acetic acid [2- (3- (2- (4- (tert-Butyl) b 3-carboxamido) phenyl) acetic acid], 2- (3- (2- ((1,1'-biphenyl-4-ylcarboxamido) thiophene- ) Acetic acid] and 2- (3- (2- (4- (tri (phenoxycarbonyl) aminomethyl) thiophene-3- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] It is any one selected from the group.

The fatty liver disease may be an alcoholic fatty liver disease or a nonalcoholic fatty liver disease.

The alcoholic fatty liver disease may be selected from the group consisting of alcoholic fatty liver disease, simple alcoholic fatty liver disease, alcoholic liver fibrosis, and alcoholic cirrhosis, preferably alcoholic fatty liver disease, but is not limited thereto . The non-alcoholic fatty liver disease may also be selected from the group consisting of non-alcoholic fatty liver, non-alcoholic simple fatty liver, non-alcoholic liver fibrosis and non-alcoholic cirrhosis, But are not limited thereto.

The health functional food may be provided in the form of powder, granules, tablets, capsules, syrups or beverages. The health functional food may be used in combination with other food or food additives other than the thiophene compound represented by the formula (1) , And can be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose, for example, prevention, health or therapeutic treatment.

The thiophene compound represented by the formula (1) contained in the health functional food may be used in accordance with the effective dose of the pharmaceutical composition. However, in the case of long-term ingestion for health and hygiene purposes or for health control purposes And the active ingredient may be used in an amount of more than the above range since there is no problem in terms of safety.

There is no particular limitation on the kind of the above health functional food and examples thereof include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, , Drinks, alcoholic beverages and vitamin complexes.

The thiophene compound represented by formula (1) of the present invention can be used in the form of a pharmaceutically acceptable salt, and the salt can be used in any form of a pharmaceutically acceptable basic salt or acidic salt. The basic salt may be used in the form of an organic base salt or an inorganic base salt and may be in the form of a salt such as a sodium salt, a potassium salt, a calcium salt, a lithium salt, a magnesium salt, a cesium salt, an aminium salt, But are not limited to, aminium salts and pyridinium salts.

Acid salts are also useful as acid addition salts formed by free acids. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, maleic acid, fumaric acid, Benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glyconic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, Etc. may be used. Preferably, hydrochloric acid is used as the inorganic acid, and methanesulfonic acid is used as the organic acid.

The thiophene compound represented by formula (1) of the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

The addition salt according to the present invention can be prepared by a conventional method. For example, the thiophene compound of formula (1) is dissolved in a water-miscible organic solvent such as acetone, methanol, ethanol, acetonitrile or the like, Or by adding an aqueous base solution of an inorganic base, followed by precipitation or crystallization. Or by evaporating a solvent or an excess of a base from the mixture, followed by drying to obtain an additional salt, or by subjecting the precipitated salt to suction filtration.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> thiophene compound synthesis

Compounds 17a-17d, 18a-18d, 19a-19d and 1-15 were synthesized in the same manner as Reaction Scheme 1 below.

[Reaction Scheme 1]

1. Methyl 2-aminothiophene-3-carboxylate (16) Methyl 2-aminothiophene-

A solution of methyl cyanoacetate (10 g, 0.1 mol) and 1,4-dithiane-2,5-diol (10.294 g, 0.067 mol) in methanol mL) was adjusted to 0 ° C and then triethylamine (Et ₃ N, 3.8 mL, 0.027 mol) was slowly added over 30 minutes and stirred at 40 ° C for 2 hours. The resulting solid was filtered and washed with diethyl ether to give compound 16 (7.014 g, 44%) as a white solid.

^{1 H NMR (400 MHz, CDCl} 3) δ 6.95 (d, 1H, J = 5.6Hz, 4-H), 6.17 (d, 1H, J = 5.6 Hz, 5-H), 5.94 (brs, 2H, N H 2), 3.80 (s, 3H, OC H ₃ ); ^{13 C NMR (100 MHz, CDCl} 3) δ 166.1, 163.1, 126.0, 107.4, 107.1, 51.3.

Acyl chloride (1.1 eq.) Corresponding to a solution of compound 16 (100 mg, 0.64 mmol) in anhydrous THF (tetrahydrofuran, 3 mL) was added followed by slow addition of Et ₃ N (1.5 eq.). After reaction at room temperature for 3 hours, the resulting solid was filtered and washed with diethyl ether to give compounds 17a to 17d as a solid.

2. methyl  2- (4- ( Rat - Butyl ) Benzamido ) Thiophene-3- Carboxylate [Methyl 2- (4- ( tert -butyl) benzamido) thiophene-3-carboxylate] (17a)

Beige solid; Yield, 50%; ^{1 H NMR (400 MHz, CDCl} 3) δ 11.96 (s, 1H, N H ), 7.96 (d, 2H, J = 8.4Hz, 2'-H, 6'-H), 7.74 (d, 2H, J = 8.0 Hz, 3'-H, 5 ' -H), 7.24 (d, 1H , J = 5.6Hz), 6.78 (d, 1H, J = 5.6Hz), 3.92 (s, 3H, OC H 3), 1.35 (s, 9H, tert -butyl); ^{13 C NMR (100 MHz, CDCl} 3) δ 166.7, 163.9, 156.7, 149.8, 129.4, 127.6, 126.2, 124.0, 116.4, 113.0, 52.0, 35.4, 31.3.

3. methyl  2- (2- Naphthamide ) Thiophene-3- Carboxylate [Methyl 2- (2- naphthamido ) thiophene -3-carboxylate] (17b)

White solid; Yield, 32%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 11.89 (s, 1H, N H ), 8.55 (s, 1H, 1'-H), 8.13 - 8.12 (m, 2H), 8.02 (d, 1H, J = 8.0 Hz), 7.94 (d, 1H, J = 8.0 Hz), 7.68 (t, 1H, J = 8.8 Hz), 7.63 (t, 1H, J = Hz), 3.89 (s, 3H , OC H 3); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 128.1, 128.4, 123.4, 118.2, 113.7, 52.8.

4. methyl  2 - ([1,1'-biphenyl] -4- Ylcarboxamide ) Thiophene-3- Carboxylate [Methyl 2 - ([1,1'-biphenyl] -4-ylcarboxamido) thiophene-3-carboxylate] (17c)

Beige solid; Yield, 30%; ^{1 H NMR (400 MHz, CDCl} 3) δ 12.05 (s, 1H, N H ), 8.10 (d, 2H, J = 8.4Hz), 7.75 (d, 2H, J = 8.4 Hz), 7.64 (d, 2H, J = 7.2Hz), 7.48 (t, J = 7.2 Hz), 7.41 (t, 1H, J = 7.2 Hz), 7.26 (d, 1H, J = 5.6 Hz), 6.80 OC H ₃ ); ^{13 C NMR (100 MHz, CDCl} 3) δ 166.8, 163.6, 149.7, 145.7, 140.0, 130.9, 129.2, 128.5, 128.3, 127.9, 127.6, 124.1, 116.7, 113.1, 52.2.

5. methyl  2- (4- Trifluoromethyl ) ( Benzamido ) Thiophene-3- Carboxylate [Methyl 2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxylate] (17d)

White solid; Yield, 80%; ^{1 H NMR (400 MHz, CDCl} 3) δ 12.11 (s, 1H, N H ), 8.13 (d, 2H, J = 8.4Hz), 7.80 (d, 2H, J = 8.4Hz), 7.26 (d, 1H, J = 5.6Hz), 6.83 (d, 1H, J = 5.6Hz), 3.94 (s, 3H, OC H 3); ^{13 C NMR (100 MHz, CDCl} 3) δ 166.8, 162.5, 149.1, 135.5, 134.5 (q, J = 32.6 Hz), 128.2, 126.4, 124.2, 123.7 (q, J = 270.8 Hz), 117.0, 113.7, 52.3.

LiOH (Lithium hydroxide) aqueous solution (2M solution, 2.7 eq.) Was added to a THF solution of the compounds 17a to 17d (using the same volume capacity as the LiOH aqueous solution to be added) and the mixture was stirred at 75 占 폚 for 5 hours. Water was added and the pH was adjusted to 3 with 2 M aqueous HCl, then the resulting solid was filtered and washed with water to give compounds 18a-18d.

6. 2- (4- Rat - Butyl ) Benzamido ) Thiophene-3- Carboxylic acid [2- (4- ( tert -Butyl) benzamido ) thiophene -3-carboxylic acid] (18a)

White solid; Yield, 94%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.8 Hz, 2'-H, 6'-H), 7.62 (d, 2H, J = 8.8 Hz, , 7.20 (d, 1H, J = 5.6 Hz), 7.03 (d, 1H, J = 5.6 Hz), 1.29 (s, 9H, tert -butyl); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.5, 163.4, 156.7, 148.7, 129.8, 127.7, 126.8, 124.9, 117.5, 114.6, 35.6, 31.5.

7. 2- (2-Naphthamido) thiophene-3-carboxylic acid [2- (2-Naphthamido) thiophene-

Yellow solid; Yield, 91%; ^{1 H NMR (500 MHz, DMSO-} d 6) δ 13.40 (brs, 1H), 12.22 (s, 1H), 8.54 (s, 1H, 1'-H), 8.12 - 8.11 (m, 2H), 8.02 ( 7.0 Hz), 7.24 (d, 1H, J = 8.5 Hz), 7.93 (d, 1H, J = 6.0 Hz), 7.07 (d, 1H, J = 6.0 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.5, 163.6, 148.6, 135.4, 132.8, 129.9, 129.7, 129.3, 129.0, 128.5, 128.0, 125.0, 123.7, 117.6, 114.9.

8. 2- (1,1'-biphenyl) -4- Ylcarboxamide ) Thiophene-3- Carboxylic acid [2 - ([1,1'-Biphenyl] -4-ylcarboxamido) thiophene-3-carboxylic acid] (18c)

White solid; Yield, 78%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.0 Hz, 2'-H, 6'-H), 7.90 (d, 2H, J = 8.0 Hz, , 7.74 (d, 2H, J = 8.4 Hz, 2 '' H, 6 '' H), 7.49 (t, 2H, J = 7.6 Hz, , J = 8.0 Hz, 4 " -H), 7.22 (d, 1H, J = 6.0 Hz), 7.05 (d, 1H, J = 6.0 Hz); 13 C NMR (100 MHz, DMSO- d 6) δ 167.6, 163.1, 148.5, 145.0, 139.4, 131.2, 129.8, 129.2, 128.5, 128.1, 127.7, 125.0, 117.5, 115.0.

9. 2- (4- ( Trifluoromethyl ) Benzamido ) Thiophene-3- Carboxylic acid [2- (4- (Trifluoromethyl) benzamido) thiophene-3-carboxylic acid] (18d)

White solid; Yield, 82%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.4 Hz), 7.22 (d, 1H, J = 5.6 Hz), 6.83 (d, = 5.6 Hz).

Acetic anhydride was added to the above compounds 18a to 18d, and the mixture was refluxed and stirred for 3 hours. After cooling, water was added thereto, and the mixture was stirred at room temperature for 30 minutes. The resultant solid was filtered, or filtered, and further purified by silica gel column chromatography (solvent: dichloromethane) to obtain the compounds 19a to 19d as a solid.

10. 2- (4- ( Rat - Butyl ) Phenyl) 4 H - Thieno [2,3- d ] [1,3] oxazine / RTI &gt; [2- (4- ( tert -Butyl) phenyl) -4 H -thieno [2,3- d ] [1,3] oxazin-4-one] (19a)

White solid; Yield, 74%; ^{1 H NMR (500 MHz, CDCl} 3) δ 8.20 (d, 2H, J = 8.5Hz), 7.52 (d, 2H, J = 8.5 Hz), 7.47 (d, 1H, J = 6.0 Hz), 7.26 (d , 1H, J = 6.0 Hz), 1.36 (s, 9H, tert- butyl); ^{13 C NMR (100 MHz, CDCl} 3) δ 164.9, 159.8, 157.1, 155.8, 128.5, 127.0, 126.1, 123.9, 123.3, 118.0, 35.4, 31.3.

11. 2- (Naphthalen-2-yl) -4H- Thieno [2,3- d ] [2,3] jade photo One [2- ( Naphthalen -2- yl )-4 H - thieno [2,3- d ] [1,3] oxazine -4-one] (19b)

White solid; Yield, 60%; ^{1 H NMR (500 MHz, CDCl} 3) δ J = 8.5 Hz), 7.88 (d, IH, J = 7.5 Hz), 7.81 (d, = 8.0 Hz), 7.60 (t, 1H, J = 7.0 Hz), 7.56 (t, 1H, J = 7.0 Hz), 7.50 Hz); ^{13 C NMR (100 MHz, CDCl} 3) δ 164.6, 159.6, 155.6, 135.6, 132.9, 130.0, 129.6, 128.9, 128.7, 128.0, 127.2, 127.0, 124.3, 124.2, 123.3, 118.2.

12. 2- (1,1'-biphenyl] -4-yl) -4 H - Thieno [2,3- d ] [1,3] oxazine One [2 - ([1,1'-Biphenyl] -4- yl )-4 H -thieno [2,3- d ] [1,3] oxazin-4-one] (19c)

Yellow solid; Yield, 61%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 8.20 (d, 2H, J = 8.4Hz), 7.87 (d, 2H, J = 8.4Hz), 7.75 (d, 2H, J = 7.6Hz), 7.71 (d, IH, J = 5.6 Hz), 7.51-7.47 (m, 3H), 7.41 (t, IH, J = 7.2 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 164.2, 159.2, 155.4, 145.0, 139.3, 129.8, 129.2, 129.0, 127.9, 127.6, 126.3, 123.5, 118.5.

13. 2- (4- ( Trifluoromethyl ) Phenyl) -4 H - Thieno [2,3- d ] [1,3] oxazine One [2- (4- (Trifluoromethyl) phenyl) -4 H -thieno [2,3- d ] [1,3] oxazin-4-one] (19d)

Beige solid; Yield, 99%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 8.34 (d, 2H, J = 8.4Hz), 7.95 (d, 2H, J = 8.4Hz), 7.78 (d, 1H, J = 5.6Hz), 7.55 (d, 1H, J = 5.6Hz); ^{13 C NMR (100 MHz, CDCl} 3) δ 163.9, 158.0, 155.1, 134.4 (q, J = 32.6 Hz), 133.2, 129.0, 126.1, 125.3, 123.8 (q, J = 270.6 Hz), 123.5, 118.7.

The compound 19a to 19d (100 mg) and the corresponding aniline derivative (1.0 equivalent) were dissolved in acetic acid (1.0 - 2.0 mL) and refluxed overnight. After addition of water, the resulting solid was filtered and washed with dichloromethane and ethanol to give compounds 1 to 15 as a solid.

14. 3- (2- (4- Rat - Butylbenzamido ) Thiophene-3- Carboxamido ) Benzoic acid [3- (2- (4- tert -Butylbenzamido) thiophene-3-carboxamido) benzoic acid] (Compound 1, MHY3295)

Light purple solid; Yield, 30%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 1H, J = 7.6 Hz), 7.87 (d, 2H, J = 7.2 Hz, 1H), 8.19 (s, 1H, J = 7.6 Hz), 7.78 (d, 1H, J = 5.6 Hz), 7.70 7.14 (d, 1H, J = 5.6 Hz), 1.30 (s, 9H, tert -butyl); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 126.9, 126.1, 125.6, 123.5, 122.7, 117.4, 116.1, 35.6, 31.5.

15. 5- (2-4- Rat - Butylbenzamido ) Thiophene-3- Carboxamido )-2- Methylbenzoic acid [5- (2- (4- tert -Butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] (Compound 2, MHY3310)

Light pink solid; Yield, 56%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 8.09 (s, 1H), 8.15 (d, 1H, J = 2.0 Hz), 7.90 (dd, 1H, J = 8.4, 2.0 Hz), 7.86 1H), 7.76 (d, 1H, J = 6.0 Hz), 7.65 (d, 2H, J = 8.0 Hz), 7.29 2.49 (s, 3H, C H ₃ ), 1.30 (s, 9H, tert -butyl); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 169.1, 164.7, 163.4, 156.5, 147.8, 136.7, 135.5, 132.5, 131.2, 130.0, 127.6, 126.8, 125.3, 123.7, 123.5, 117.4, 116.1, 35.6, 31.5, 21.5.

16. 4- (2- (4- Rat - Butylbenzamido ) Thiophene-3- Carboxamido ) Benzoic acid [4- (2- (4- tert -Butylbenzamido) thiophene-3-carboxamido) benzoic acid] (Compound 3, MHY3294)

White solid; Yield, 39%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H), 7.87 (d, 2H, J = 8.8 Hz), 7.87 (d, 2H, J = , J = 8.0 Hz), 7.78 (d, 1H, J = 6.0 Hz), 7.65 (d, 2H, J = 8.0 Hz), 7.14 (d, 1H, J = 6.0 Hz), 1.31 (s, 9H, tert -butyl); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.6, 165.0, 163.4, 156.6, 148.3, 143.1, 130.9, 129.9, 127.6, 126.8, 126.6, 123.6, 121.1, 117.4, 116.1, 35.6, 31.5.

17. 3- (2- (2- Naphthamide ) Thiophene-3- Carboxamido ) Benzoic acid [3- (2- (2- Naphthamido ) thiophene -3-carboxamido) benzoic acid] (Compound 4, MHY3296)

White solid; Yield, 50%; ¹ H NMR (400 MHz, DMSO- d ₆ )? 13.10 (br s, 1H), 13.05 (s, 1H), 10.26 8.05 (d, 1H, J = 8.8 Hz), 7.81 (d, 1H, J = 1H, J = 7.6 Hz), 7.72 (d, 1H, J = 7.6 Hz), 7.68 Hz), 7.18 (d, 1 H, J = 5.6 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.8, 164.9, 163.6, 147.9, 139.1, 135.4, 132.9, 131.9, 130.1, 130.0, 129.7, 129.3, 129.1, 128.5, 128.0, 126.3, 125.7, 123.6, 122.8, 117.6, 116.4.

18. 5- (2- (2- Naphthamide ) Thiophene-3- Carboxamido )-2- Methylbenzoic acid [5- (2- (2- Naphthamido ) thiophene -3-carboxamido) -2-methylbenzoic acid] (Compound 5, MHY3297)

White solid; Yield, 52%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H), 8.14 (s, IH), 8.03 (d, IH), 8.17 (s, J = 7.2 Hz), 7.95-7.92 (m, 2H), 7.79 (d, 1H, J = 5.6 Hz), 7.68 , 7.31 (d, 1H, J = 8.4Hz), 7.16 (d, 1H, J = 5.6Hz), 2.50 (s, 3H, C H 3).

19. 4- (2- (2- Naphthamide ) Thiophene-3- Carboxamido ) Benzoic acid [4- (2- (2- Naphthamido ) thiophene -3-carboxamido) benzoic acid] (Compound 6, MHY3298)

Light blue solid; Yield, 37%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H), 8.04 (d, IH, J = 8.0 Hz), 7.98 (s, IH) 7.90 (m, 6H), 7.68 (t, 1H, J = 7.2 Hz), 7.64 (t, 1H, J = 7.2 Hz), 7.17 (d, 1H, J = 5.6 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.6, 165.0, 163.6, 148.2, 143.2, 135.4, 132.9, 130.8, 130.0, 129.8, 129.3, 129.0, 128.5, 128.0, 126.6, 124.0, 123.6, 121.2, 117.5, 116.4.

20. 3- (2-Biphenyl-4- Ylcarboxamidothiophene -3- Carboxamido (2-Biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid] (Compound 7, MHY3300)

Beige solid; Yield, 78%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.4 Hz, 1H), 8.07 (d, 2H), 8.07 (d, J = 8.0 Hz), 7.72 (m, 1H), 7.53-7.48 (m, 1H), 7.94 (d, 2H, J = 8.4 Hz) , 3H), 7.42 (t, IH, J = 7.6 Hz), 7.16 (d, IH, J = 6.0 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.8, 164.9, 163.1, 147.9, 144.9, 139.4, 139.1, 131.9, 131.4, 129.8, 129.7, 129.1, 128.5, 128.2, 127.7, 126.3, 125.7, 123.7, 122.8, 117.6, 116.2.

21. 5- (2-Biphenyl-4- Ylcarboxamidothiophene -3- Carboxamido )-2- Methylbenzoic acid [5- (2-Biphenyl-4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid] (Compound 8, MHY3301)

Beige solid; Yield, 92%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.8 Hz), 7.93 (d, 2H), 8.04 (d, 2H, J = J = 8.8 Hz), 7.91 (dd, 1H, J = 8.4, 2.4 Hz), 7.79-7.75 (m, 3H), 7.50 (t, 2H, J = 7.6 Hz), 7.42 7.6 Hz), 7.31 (d, 1H, J = 8.4Hz), 7.15 (d, 1H, J = 5.6Hz), 2.49 (s, 3H, C H 3); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 129.1, 128.8, 128.2, 127.7, 125.3, 123.8, 123.6, 117.5, 116.3, 21.5.

22. 4- (2-Biphenyl-4- Ylcarboxamidothiophene -3- Carboxamido (2-Biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid] (Compound 9, MHY3299)

Beige solid; Yield, 49%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ (D, 2H, J = 8.4 Hz), 7.96 (d, 2H, J = 8.4 Hz) (D, 2H, J = 7.6 Hz), 7.91 (d, 2H, J = 8.4 Hz), 7.80 = 7.6 Hz), 7.42 (t, 1H, J = 7.6 Hz), 7.17 (d, 1H, J = 5.6 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 167.6, 165.0, 163.1, 148.2, 145.0, 143.1, 139.4, 131.3, 130.9, 129.8, 129.1, 128.5, 128.2, 127.7, 126.7, 123.7, 121.1, 117.6, 116.3.

23. 3- (2- (4- ( Trifluoromethyl ) Benzamido ) Thiophene-3- Carboxamido (2- (4- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid (Compound 10, MHY3303)

White solid; Yield, 57%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 8.01 (d, 2H), 8.01 (d, 2H, J = 8.0 Hz), 8.01 (d, J = 8.0 Hz), 7.79 (d, IH, J = 6.0 Hz), 7.71 (d, IH, J = 8.0 Hz), 7.50 = 6.0 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ (Q, J = 32.2 Hz), 131.9, 129.6, 128.8, 127.0, 126.2, 125.8, 124.4 (q, J = 270.5 Hz), 123.7, 122.8, 118.0, 116.8.

24. 2- methyl -5- (2- (4- ( Trifluoromethyl ) Benzamido ) Thiophene-3- Carboxamido 3-carboxamido) benzoic acid] (Compound 11, MHY3304) [0143] &lt; EMI ID =

White solid; Yield, 97%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.0Hz), 8.00 (d, 2H), 8.15 (d, 2H, J = (D, 1H, J = 8.0 Hz), 7.89 (dd, 1H, J = 8.0, 2.4 Hz), 7.78 , J = 5.6 Hz), 2.49 (s, 3 H, C H ₃ ); ^{13 C NMR (100 MHz, DMSO-} d 6) δ (Q, d = 31.9 Hz), 132.5, 131.1, 128.8, 127.0, 125.4, 124.4 (q, J = 271.6 Hz) 123.8, 123.6, 117.9, 116.9, 21.5 .

25. 4- (2- (4- ( Trifluoromethyl ) Benzamido ) Thiophene-3- Carboxamido (2- (4- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid (Compound 12, MHY3302)

White solid; Yield, 68%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 2H, J = 8.4 Hz), 7.95 (d, 2H), 8.19 (d, 2H, J = J = 8.8 Hz), 7.89 (d, 2H, J = 8.8 Hz), 7.79 (d, 1H, J = 6.0 Hz), 7.20 (d, 1H, J = 6.0 Hz); ^{13 C NMR (100 MHz, DMSO-} d 6) δ J = 32.1 Hz), 130.9, 128.8, 127.0, 126.7, 124.4 (q, J = 270.8 Hz), 123.8, 121.1, 118.0, 116.8, 167.6, 164.8, 162.4, 147.7, 143.0, 136.5, 132.9 (q,

26. 2- (3- (2- (4- ( Rat - Butyl ) Benzamido ) Thiophene-3- Carboxamido ) Phenyl) acetic acid [2- (3- (2- (4- ( tert -Butyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] (Compound 13, MHY3313)

Beige solid; Yield, 54%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 1H), 7.67 (d, 1H, J = 8.0 Hz), 7.77 (d, 1H, J = 8.0 Hz), 7.64 (d, 2H, J = 8.0 Hz), 7.58 (s, 7.04 (d, 1H, J = 8.0 Hz), 3.58 (s, 2H, C H 2), 1.30 (s, 9H, tert -butyl); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 173.3, 164.8, 163.3, 156.5, 147.8, 138.7, 136.2, 130.0, 129.2, 127.6, 126.8, 126.1, 123.6, 123.0, 120.7, 117.3, 116.2, 41.5, 35.5, 31.5.

27. 2- (3- (2 - ([1,1'-biphenyl] -4- Ylcarboxamide ) Thiophene-3- Carboxamido 3-carboxamido) phenyl) acetic acid] (Compound 14, MHY3311) was obtained in the same manner as in [2- (3-

Beige solid; Yield, 33%; ^{1 H NMR (500 MHz, DMSO-} d 6) δ 2H), 7.79 (d, 2H, J = 7.5 Hz), 7.79 (d, 1H, 2H, J = 7.5 Hz), 7.76 (d, 2H, J = 8.0 Hz), 7.69 (D, 1H, J = 7.5 Hz), 7.33 (t, 1H, J = 8.0 Hz), 7.45 s, 2H, C H ₂ ); ^{13 C NMR (100 MHz, DMSO-} d 6) δ 173.3, 164.8, 163.0, 147.7, 144.9, 139.4, 138.7, 136.2, 131.4, 129.8, 129.2, 128.4, 128.2, 127.7, 126.2, 123.6, 123.1, 120.8, 117.5, 116.4, 41.5.

28. 2- (3- (2- (4- ( Trifluoromethyl ) Benzamido ) Thiophene-3- Carboxamido 3-carboxamido) phenyl) acetic acid] (Compound 15, MHY3312) was dissolved in tetrahydrofuran

Beige solid; Yield, 61%; ^{1 H NMR (400 MHz, DMSO-} d 6) δ 8.0 (d, 2H, J = 8.0 Hz), 7.79 (d, 1H, J = 1H, J = 6.0 Hz), 7.66 (d, 1H, J = 8.0 Hz), 7.57 (s, 7.04 (d, 1 H, J = 7.6 Hz), 3.57 (s, 2H, C H ₂ ); ^{13 C NMR (100 MHz, DMSO-} d 6) δ (Q, J = 31.9 Hz), 129.2, 128.8, 127.0, 126.3, 124.4 (q, J = 271.6Hz), 123.6, 123.2, 120.7, 117.9, 117.0, 41.4.

Example 2: PPAR luciferase assay

(1) PPRE-PPAR alpha Luciferase assay

Peroxisome proliferator-activated receptor (PPAR) is a subunit of PPAR α / β / γ, which plays an important role in fatty liver improvement because it is an important factor controlling the homeostasis of glucose and lipid.

In order to confirm the activation of the PPAR, a PPRE (Peroxisome Proliferator Response Element) sequence, which is a promoter region to which the activated PPAR binds, is ligated to the PPRE Luciferase assays were performed using -X3-TK luc plasmids. HepG2 cells (a product of the supplier), which is a kind of human liver cancer cells, were divided into 2 × 10 ⁴ cells on a 96-well cell culture plate and cultured overnight. 0.1 μg of the PPRE-X3-TK luc plasmid and 0.01 μg of the PPARα expression vector were transfected into the cells using lipofectamine. After 24 hours, Compound 1 to Compound 15 and WY14643, well known as a PPARa agonist as a positive control, were treated at a concentration of 10 uM. After 5 hours, the luciferase activity was measured with a luminometer using the One-Glo Luciferase Assay System kit containing luciferin, a luciferase substrate.

(2) PPRE-PPAR? /? Luciferase assay

In order to confirm the activation of PPAR, luciferase analysis was carried out using the PPRE-X3-TK luc plasmid in which the PPRE sequence, the promoter region to which the activated PPAR binds, was ligated to the front of the luciferase gene, a reporter gene. HepG2 cells, a kind of human liver cancer cells, were divided into 2 × 10 ⁴ cells on a 96-well cell culture plate and cultured overnight. 0.1 μg of the PPRE-X3-TK luc plasmid and 0.01 μg of the PPARβ / δ expression vector were transfected into the cells using lipofectamine. After 24 hours, Compound 1 to Compound 15 and GW501516, well known as a PPAR [beta] / [delta] agonist as a positive control, were treated at a concentration of 10 [mu] M. Five hours later, one-glo luciferase assay kit containing luciferin, a luciferase substrate, was used to determine the luciferase activity And measured with a luminometer.

(3) Analysis of PPRE-PPARγ luciferase

In order to confirm the activation of PPAR, luciferase analysis was carried out using the PPRE-X3-TK luc plasmid in which the PPRE sequence, the promoter region to which the activated PPAR binds, was ligated to the front of the luciferase gene, a reporter gene. HepG2 cells, a kind of human liver cancer cells, were divided into 2 × 10 ⁴ cells on a 96-well cell culture plate and cultured overnight. A 0.1μg PPRE-X3-TK luc plasmid and 0.01μg of the PPAR γ expression vector of the injected transfected using Lipofectamine into the cells. After 24 hours, rosiglitazone, known as PPAR gamma agonist, was treated with Compound 1 to Compound 15 and a positive control at a concentration of 10 μM. Five hours later, one-glo luciferase assay kit containing luciferin, a luciferase substrate, was used to determine the luciferase activity And measured with a luminometer.

(4) Experimental results

As a result, as shown in Fig. 1, the PPAR gamma activity was improved when Compound No. 7 (MHY3300) was treated with Compound 1 to Compound 15, and Compound 9 (MHY3299), Compound 10 (MHY3303) Showed improved activity in all of the subtypes α, β and γ.

&Lt; Example 3 > FXR luciferase assay

FXR (Farnesoid X receptor) is also called BAR (Bile acid receptor), and nuclear receptor, Chenodeoxycholic acid and other bile acids act as ligands, and FXR is expressed in liver and small intestine. That is, since FXR is a receptor found in the nucleus, it acts as a key regulator associated with the pathway of bile acid metabolism. In addition, FXR plays an important role in maintaining the homeostasis of triglyceride in the liver. Because of this function, many papers recently report that FXR is an important factor in treating fatty liver. Thus, the FXR activity effects of the compounds 1 to 15 synthesized in Example 1 were confirmed in order to use metabolic diseases such as fatty liver for treatment.

Specifically, in order to confirm whether or not FXR is activated, the FXR-luc plasmid in which the Hormone response element (HRE) sequence, which is a promoter region to which the activated FXR binds, is ligated to the front of the luciferase gene Luciferase assay was performed. HepG2 cells, a kind of human liver cancer cells, were divided into 2 × 10 ⁴ cells on a 96-well cell culture plate and cultured overnight. 0.1 μg of FXR-luc plasmid and 0.01 μg of FXR expression vector were transfected into cells using lipofectamine. After 24 hours, the cells were treated with Compound 1 to Compound 15 and CDEA (Chenodeoxycholic acid), well known as an FXR agonist, as a positive control at a concentration of 10 μM. After 5 hours, the luciferase activity was measured with a luminometer using the One-Glo Luciferase Assay System Kit containing luciferin, a luciferase substrate.

As a result, Compound 7 (MHY3300), which showed an excellent PPAR gamma activity improvement as shown in Fig. 2, also showed excellent FXR activity.

Example 4 Confirmation of Oxidative Stress Erasing Effect

The compounds prepared in Example 1 were examined for their scavenging activity against oxidative stress such as reactive oxygen species (ROS) and peroxynitrite (ONOO ^- ). First, SIN-1 (3-morpholinosydnonimine) was treated as an oxidative stress inducer in vitro, and then the compounds 1 to 15 were treated at a concentration of 50 μM. The amount of ROS was confirmed by DCF-2DDA, and the amount of ONOO ^- was determined by measuring the amount of fluorescence for 30 minutes using DHR123.

As a result, as shown in Fig. 3, when both MHY3299 (Compound 9), MHY3300 (Compound 7), MHY3301 (Compound 8) and MHY3311 (Compound 14) were treated, both ROS and ONOO ^- were decreased.

Example 5 Identification of Oxidative Stress Erasing Effect in vivo

Based on the results of Examples 3 and 4, Compound 7 (MYH3300), which exhibits the highest FXR activating ability and also has an active oxygen scavenging ability, was selected to confirm the oxidative stress erasing effect in vivo. As shown in FIG. 4, MHY3300 was dissolved in 5% DMSO for 5 days, and the mice were treated with C57BL / 6 mice at a total dose of 5 or 6 mg / kg / day for 4 days Orally. After the liver tissue of the mouse was disrupted, ROS and ONOO ^- were measured in the cytosol fraction according to the method of Example 4 above.

As a result, as shown in FIG. 5, the amount of ROS was not significantly changed when Compound 7 (MYH3300) was treated, but the amount of ONOO ^- was lower than that of the control.

<Example 6> Measurement of PPAR activation ability

As shown in Example 5, compound 7 (MHY3300) having the highest FXR activity was selected and the following experiment was conducted again.

(1) Cytotoxicity measurement

First, in order to determine whether MHY3300 shows intracellular toxicity, MTT assay was performed as a cell viability assay to measure cell viability. Specifically, MHY3300 was treated with HepG2 cells at a concentration for 24 hours to confirm the survival rate of the cells.

As a result, as shown in Fig. 6, it was confirmed that MHY3300 did not show toxicity to the cells.

(2) Measurement of PPAR activation ability

In order to measure the PPAR activation ability of MHY3300 again, a reporter gene (luciferase) assay was performed for each PPAR subtype according to the method described in Example 2 above.

As a result, as shown in FIG. 7, MHY3300 showed high activation activity on PPAR? And?, And especially PPAR? Showed higher activity than GW501516, which is a positive control.

Example 7 Measurement of AMPK Activation Activity

Based on the results of Examples 5 and 6, the following experiment was carried out to confirm whether MHY3300, which exhibited the ability to activate PPAR? And? As well as FXR, was also capable of activating AMPK.

(1) In vitro experiments

In order to confirm AMPK activated by MHY3300, MHY3300 was treated with ACYF by time, and proteins were extracted and subjected to Western blotting. First, MHY3300 cells treated with time were lysed to extract proteins. This protein was quantified and reacted at 100 ° C to denature the protein. The protein samples thus prepared were loaded on SDS-polyacrylamide gel in equal amounts and electrophoresis was carried out to separate them according to their molecular weights. The proteins separated according to the molecular weight were electrostatically transferred to the membrane and reacted by treating the protein transferred to the membrane with an antibody having specificity and ease of detection of the desired protein. More specifically, phosphorylated AMPK (phosphatase-AMPK) protein, and reacted for more than 12 hours. After that, the secondary antibody that specifically binds to the primary antibody was treated and reacted for 1 hour, and the developer containing the substrate of the horse radish peroxidase (HRP) present in the secondary antibody was treated To detect the protein.

As a result, as shown in Fig. 8, when MHY3300 was treated for 1, 2 and 5 hours, the amount of phosphorylated AMPK protein was increased more than that of the control group.

(2) in vivo  Experiment

Based on the in vitro test results, MHY3300 dissolved in 5% DMSO was added to the stabilized C57BL / 6 mouse at 8 mg / day for 2 days to confirm that the AMPK activation ability was maintained in vivo as well, kg / day for a total of 2 times. Thereafter, liver tissue was disrupted and the amount of phosphorylated AMPK protein in the cell substrate fraction was measured via Western blot as in the method performed above.

As a result, as shown in Fig. 10, the amount of phosphorylated AMPK protein in the group treated with MHY3300 was increased.

Based on the above results, one more in vivo experiment was performed. This time, C57BL / 6 mice were treated with high fat diet for 4 weeks. Then, as shown in FIG. 4, MHY3300 was dissolved in 5% DMSO for 5 days and orally administered at a total dose of 5 or 6 mg / kg / day. Thereafter, liver tissue was disrupted and the amount of phosphorylated AMPK protein in the cell substrate fraction was measured via Western blot as in the method performed above.

As a result, as shown in Fig. 11, both MHY3300-treated groups at two concentrations increased the amount of phosphorylated AMPK protein than the control group. That is, these results show that MHY3300 can act as an AMPK agonist in vivo.

&Lt; Example 8 > Measurement of triglyceride in liver

Triglyceride is an ester consisting of three fatty acids and glycerol, which is also a major component of body fat. This can lead to atherosclerosis or even heart disease if the blood circulates too much. Triglycerides also affect the liver, which can cause fat accumulation when accumulated in the liver. Thus, the following experiment was carried out to confirm how MHY3300 changes the amount of triglyceride.

First, the experiment proceeded in vivo. After 4 weeks of high fat diet as shown in FIG. 4, C57BL / 6 mice were dissolved in 5% DMSO for 5 days, 5 was orally administered. Then, about 50 mg of liver was quantified, and the resultant was disrupted in cold PBS (or 0.9% NaCl), treated with a solvent mixture of chloroform and methanol (MeOH) at a ratio of 2: 1, and reacted for 2 hours. After 2 hours, the reacted sample was filtered using filter paper, and the filtered liquid was allowed to dry in a dry oven so that the liquid of the sample flushed. After drying, the amount of triglyceride was measured by absorbance using a triglyceride measurement kit (Stanbio laboratory, TX USA), and the value was divided by the liver weight of 50 mg to measure the amount of triglyceride in the samples.

As a result, as shown in Fig. 12, triglyceride tended to decrease in the treatment time of MHY3300.

In summary, the results of all of the above experiments suggest that compound 7 (MHY3300) activates both AMPK in cell culture and in vivo experiments and also inhibits fat accumulation in the liver by reducing the amount of triglycerides in the liver model animal Could. Therefore, Compound 7 is considered to have the effect of improving the fatty liver and improving the alcoholic or non-alcoholic fatty liver and improving metabolic diseases.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various modifications and changes may be made without departing from the scope of the appended claims.

Claims (12)

A thiophen compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof;
[Chemical Formula 1]

In Formula 1,
W is S,
R ¹ to R ⁴ are each hydrogen,
m is 0,
n is any integer selected from 0 to 3,
Wherein Ar ¹ is a phenyl group substituted with at least one substituent selected from the group consisting of C ¹ -C 4 alkyl, a trihalomethyl group, and a phenyl group, or a naphthyl group,
Ar ² is phenyl which is unsubstituted or substituted by C 1 -C 4 alkyl. The method according to claim 1,
The thiophene compound represented by the above-mentioned formula (1) can be obtained by reacting 3- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) benzoic acid [3- 3-carboxamido) benzoic acid], 5- (2-4-tert-butylbenzamido) thiophene-3-carboxamide [ 4- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] thiophene-3-carboxamido) benzoic acid, 3- (2- (2-naphthaamido) thiophene-3-carboxamido) benzoic acid [3- (2- (2-Naphthamido) thiophene-3-carboxamido) -2-methylbenzoic acid (2-Naphthamido) thiophene-3-carboxamido) benzoic acid], 3- (2-naphthylamido) thiophene- -Biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [3- (2-Bipheny 4-ylcarboxamidothiophene-3-carboxamido) benzoic acid], 5- (2-biphenyl-4-ylcarboxamidothiophene- 4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid], 4- (2-biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [4- (2-Biphenyl-4-ylcarboxamidothiophene -3-carboxamido) benzoic acid], 3- (2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxamide [ ) thiophene-3-carboxamido) benzoic acid], 2-methyl-5- (2- (4- (trifluoromethyl) benzamido) thiophene- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid], 4- (2- (4- (2- (4- (tert-butyl) benzamido) thiophene-3-carboxamido) benzoic acid) Phenyl) acetic acid [2- (3- (2- (4- (tert-Butyl) be thiophene-3-carboxamido) phenyl) acetic acid], 2- (3- (2- ) Acetic acid] and 2- (3- (2- (4- (tri (phenoxycarbonyl) aminomethyl) thiophene-3- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] Thiophene or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for preventing or treating fatty liver disease comprising, as an active ingredient, a thiophene compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof;
[Chemical Formula 1]

In Formula 1,
W is S,
R ¹ to R ⁴ are each hydrogen,
m is 0,
n is any integer selected from 0 to 3,
Wherein Ar ¹ is a phenyl group substituted with at least one substituent selected from the group consisting of C ¹ -C 4 alkyl, a trihalomethyl group, and a phenyl group, or a naphthyl group,
Ar ² is phenyl which is unsubstituted or substituted by C 1 -C 4 alkyl. The method of claim 3,
The thiophene compound represented by the above-mentioned formula (1) can be obtained by reacting 3- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) benzoic acid [3- 3-carboxamido) benzoic acid], 5- (2-4-tert-butylbenzamido) thiophene-3-carboxamide [ 4- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] thiophene-3-carboxamido) benzoic acid, 3- (2- (2-naphthaamido) thiophene-3-carboxamido) benzoic acid [3- (2- (2-Naphthamido) thiophene-3-carboxamido) -2-methylbenzoic acid (2-Naphthamido) thiophene-3-carboxamido) benzoic acid], 3- (2-naphthylamido) thiophene- -Biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [3- (2-Bipheny 4-ylcarboxamidothiophene-3-carboxamido) benzoic acid], 5- (2-biphenyl-4-ylcarboxamidothiophene- 4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid], 4- (2-biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [4- (2-Biphenyl-4-ylcarboxamidothiophene -3-carboxamido) benzoic acid], 3- (2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxamide [ ) thiophene-3-carboxamido) benzoic acid], 2-methyl-5- (2- (4- (trifluoromethyl) benzamido) thiophene- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid], 4- (2- (4- (2- (4- (tert-butyl) benzamido) thiophene-3-carboxamido) benzoic acid) Phenyl) acetic acid [2- (3- (2- (4- (tert-Butyl) be thiophene-3-carboxamido) phenyl) acetic acid], 2- (3- (2- ) Acetic acid] and 2- (3- (2- (4- (tri (phenoxycarbonyl) aminomethyl) thiophene-3- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] Or a pharmaceutically acceptable salt thereof. The method of claim 3,
Wherein the fatty liver disease is an alcoholic fatty liver disease or a nonalcoholic fatty liver disease. 6. The method of claim 5,
Wherein the alcoholic fatty liver disease is selected from the group consisting of alcoholic fatty liver hepatitis, alcoholic simple fatty liver, alcoholic liver fibrosis and alcoholic liver cirrhosis, wherein the nonalcoholic fatty liver disease is selected from the group consisting of nonalcoholic fatty liver disease, Non-alcoholic liver fibrosis, non-alcoholic liver fibrosis and non-alcoholic liver cirrhosis. The method of claim 3,
Wherein the thiophene compound represented by Formula 1 activates at least one selected from the group consisting of FXR, PPAR alpha, PPAR gamma, PPAR beta and AMPK. The method of claim 3,
Wherein the thiophene compound represented by Formula 1 has a dual activity against PPAR and FXR or has activity against AMPK. 1. A health functional food for preventing or ameliorating fatty liver disease comprising, as an active ingredient, a thiophene compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof;
[Chemical Formula 1]

In Formula 1,
W is S,
R ¹ to R ⁴ are each hydrogen,
m is 0,
n is any integer selected from 0 to 3,
Wherein Ar ¹ is a phenyl group substituted with at least one substituent selected from the group consisting of C ¹ -C 4 alkyl, a trihalomethyl group, and a phenyl group, or a naphthyl group,
Ar ² is phenyl which is unsubstituted or substituted by C 1 -C 4 alkyl. 10. The method of claim 9,
The thiophene compound represented by the above-mentioned formula (1) can be obtained by reacting 3- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) benzoic acid [3- 3-carboxamido) benzoic acid], 5- (2-4-tert-butylbenzamido) thiophene-3-carboxamide [ 4- (2- (4-tert-butylbenzamido) thiophene-3-carboxamido) -2-methylbenzoic acid] thiophene-3-carboxamido) benzoic acid, 3- (2- (2-naphthaamido) thiophene-3-carboxamido) benzoic acid [3- (2- (2-Naphthamido) thiophene-3-carboxamido) -2-methylbenzoic acid (2-Naphthamido) thiophene-3-carboxamido) benzoic acid], 3- (2-naphthylamido) thiophene- -Biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [3- (2-Bipheny 4-ylcarboxamidothiophene-3-carboxamido) benzoic acid], 5- (2-biphenyl-4-ylcarboxamidothiophene- 4-ylcarboxamidothiophene-3-carboxamido) -2-methylbenzoic acid], 4- (2-biphenyl-4-ylcarboxamidothiophene-3-carboxamido) benzoic acid [4- (2-Biphenyl-4-ylcarboxamidothiophene -3-carboxamido) benzoic acid], 3- (2- (4- (trifluoromethyl) benzamido) thiophene-3-carboxamide [ ) thiophene-3-carboxamido) benzoic acid], 2-methyl-5- (2- (4- (trifluoromethyl) benzamido) thiophene- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) benzoic acid], 4- (2- (4- (2- (4- (tert-butyl) benzamido) thiophene-3-carboxamido) benzoic acid) Phenyl) acetic acid [2- (3- (2- (4- (tert-Butyl) be thiophene-3-carboxamido) phenyl) acetic acid], 2- (3- (2- ) Acetic acid] and 2- (3- (2- (4- (tri (phenoxycarbonyl) aminomethyl) thiophene-3- (Trifluoromethyl) benzamido) thiophene-3-carboxamido) phenyl) acetic acid] Or a pharmaceutically acceptable salt, solvate or prodrug thereof. 10. The method of claim 9,
Wherein the fatty liver disease is an alcoholic fatty liver disease or a nonalcoholic fatty liver disease. 12. The method of claim 11,
Wherein the alcoholic fatty liver disease is selected from the group consisting of alcoholic fatty liver hepatitis, alcoholic simple fatty liver, alcoholic liver fibrosis and alcoholic liver cirrhosis, wherein the nonalcoholic fatty liver disease is selected from the group consisting of nonalcoholic fatty liver disease, Non-alcoholic liver fibrosis, non-alcoholic liver fibrosis, and non-alcoholic liver cirrhosis.

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