阅读说明：本技术 用于预防或治疗非酒精性脂肪肝病的含有gpr119配体作为活性成分的药物组合物 (Pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease comprising GPR119 ligand as active ingredient ) 是由 金美庆 李宝蓝 朴汉洙 李丞镐 蔡洧娜 于 2019-09-11 设计创作，主要内容包括：本发明涉及用于预防或治疗非酒精性脂肪肝病的药物组合物,所述组合物含有GPR119(G蛋白偶联受体119)配体作为活性成分。根据本发明的药物组合物显示出改善脂质代谢、减少肝组织中的脂肪积累以及预防由肝组织中的炎症和纤维化引起的组织学损伤的优异效果,因此可以被用于预防或治疗非酒精性脂肪肝病。(The present invention relates to a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, which contains a GPR119(G protein-coupled receptor 119) ligand as an active ingredient. The pharmaceutical composition according to the present invention shows excellent effects of improving lipid metabolism, reducing fat accumulation in liver tissue, and preventing histological damage caused by inflammation and fibrosis in liver tissue, and thus can be used for preventing or treating non-alcoholic fatty liver disease.)

1. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an effective ingredient:

[ chemical formula 1]

Wherein A is oxadiazole, dihydrooxazole, thiazole or thiadiazole, A is independently unsubstituted or substituted with at least one substituent selected from the group consisting of halogen, C1-C6 straight or branched chain alkyl and C1-C6 alcohol group, said alkyl or alcohol group being independently unsubstituted or substituted with halogen or C1-C6 alkoxy;

b is pyridine, pyrimidine, pyrazine or oxadiazole, B is independently unsubstituted or substituted with at least one substituent selected from the group consisting of halogen, C1-C6 linear or branched alkyl, C1-C6 alcohol, C1-C6 alkoxy and oxadiazolyl, said C1-C6 linear or branched alkyl, C1-C6 alcohol, C1-C6 alkoxy or oxadiazolyl is independently unsubstituted or substituted with halogen, C1-C6 alkyl or C1-C6 alkoxy; and

x is independently F, Cl, Br or I.

2. The pharmaceutical composition according to claim 1, wherein, in the chemical formula 1, a is

And

r1 to R3, R5 and R6 are each independently one or more substituents selected from the group consisting of hydrogen, halogen, C1-C6 straight or branched chain alkyl and C1-C6 alcohol, said alkyl or alcohol independently being unsubstituted or substituted with halogen or C1-C6 alkoxy.

3. The pharmaceutical composition according to claim 1, wherein, in the chemical formula 1, B is

And

r7 to R11 are independently substituted with one or more substituents selected from the group consisting of hydrogen, halogen, C1-C6 straight or branched chain alkyl, C1-C6 alcohol, C1-C6 alkoxy, and oxadiazolyl, which are independently unsubstituted or substituted with halogen, C1-C6 alkyl, or C1-C6 alkoxy.

4. The pharmaceutical composition according to claim 1, wherein, in the chemical formula 1, X is F.

5. The pharmaceutical composition of claim 1, wherein, in the chemical formula 1, a is oxadiazole substituted with C1-C6 linear or branched alkyl group, B is pyrimidine substituted with C1-C6 linear or branched alkyl group, and X is F.

6. The pharmaceutical composition according to claim 1, wherein the compound represented by chemical formula 1 is 3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2, 4-oxadiazole.

7. The pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is selected from simple fatty liver, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits deposition of triglycerides in liver tissue.

9. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits infiltration of inflammatory cells in liver tissue.

10. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition inhibits fibrosis of liver tissue.

11. A method for preventing or treating non-alcoholic fatty liver disease, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a compound represented by the following chemical formula 1, pharmaceutically acceptable salt thereof, optical isomer thereof, hydrate thereof or solvate thereof, or a mixture thereof:

[ chemical formula 1]

Wherein A, B and X are the same as those shown in claim 1.

12. A food composition for preventing or alleviating non-alcoholic fatty liver disease, comprising a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an effective ingredient:

[ chemical formula 1]

Wherein A, B and X are the same as in claim 1.

13. A method for preventing or alleviating non-alcoholic fatty liver disease, the method comprising administering to a subject in need of improvement a food composition comprising a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof:

[ chemical formula 1]

Wherein A, B and X are the same as in claim 1.

14. A feed composition for preventing or alleviating non-alcoholic fatty liver disease, comprising a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an effective ingredient:

[ chemical formula 1]

Wherein A, B and X are the same as in claim 1.

15. Use of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate thereof or a solvate thereof, or a mixture thereof for preventing or treating non-alcoholic fatty liver disease:

[ chemical formula 1]

Wherein A, B and X are the same as in claim 1.

16. Use of a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate thereof or a solvate thereof, or a mixture thereof for the manufacture of a medicament for preventing or treating non-alcoholic fatty liver disease:

[ chemical formula 1]

Wherein A, B and X are the same as in claim 1.

Technical Field

The present invention relates to a pharmaceutical composition, a food composition and a feed composition for preventing or treating non-alcoholic fatty liver disease, which comprise a GPR119(G protein-coupled receptor 119) ligand as an active ingredient; to a method for treating and ameliorating nonalcoholic fatty liver disease; or use of the composition in preventing or treating non-alcoholic fatty liver disease.

Background

Fatty liver is a pathological state in which triglycerides are excessively accumulated in liver cells, and is medically defined as a state in which triglycerides account for 5% or more of the weight of the liver. Fatty liver is classified into alcoholic fatty liver and non-alcoholic fatty liver according to whether it is caused by excessive alcohol intake. Non-alcoholic fatty liver disease (NAFLD) is a group of diseases that encompasses all aspects of the disease ranging from non-alcoholic fatty liver disease to steatohepatitis and cirrhosis. Simple non-alcoholic fatty liver (non-alcoholic liver) (fatty deposition only in the liver and showing increased fatty deposition in liver tissue due to insulin resistance and the like without any finding of hepatocyte damage and fibrosis) may progress to non-alcoholic steatohepatitis (NASH) (with hepatocyte damage due to inflammatory response caused by oxidative stress and sometimes also fibrosis) and may progress to liver cirrhosis (liver cirrhosis) (with irreversible liver damage if not treated properly).

Recently, the prevalence of nonalcoholic fatty liver disease is rapidly increasing worldwide, the prevalence of simple fatty liver disease is estimated to be 6.3-33%, and the prevalence of non-alcoholic steatohepatitis with inflammation is reported to be 3-5% (Hepatology, 2012 (55): 2005-. Simple fatty liver progresses very slowly to severe liver disease without increasing mortality associated with liver disease, but steatohepatitis may cause cirrhosis and liver cancer and increase mortality associated with liver disease as well as overall mortality. In addition, simple fatty liver may be easily improved if the body weight is reduced by 3 to 5% by changing general lifestyle, but it is recommended to reduce the body weight by at least 10% to reduce inflammation. However, it is not clear whether this weight loss can lead to an improvement in inflammation and fibrosis of the liver (Korean J Gastroenterol, 2012 (60): 64-66).

To date, there is no therapeutic agent for non-alcoholic steatohepatitis. If the disease is worsening, liver transplantation is performed. According to the us statistics, by 2013, hepatitis c was considered the most common cause of liver transplantation, followed by non-alcoholic steatohepatitis. In addition, the growth rate of liver transplantation for hepatitis c was 14% and that for nonalcoholic steatohepatitis was 170% in 10 years from 2004, which is a rapid growth, and NASH was also expected to be the first cause of liver transplantation from 2020, exceeding hepatitis c, so that there is an urgent need to develop an effective therapeutic agent for nonalcoholic fatty liver diseases including nonalcoholic steatohepatitis.

Currently, drug therapy for non-alcoholic steatohepatitis includes methods using insulin resistance enhancers (e.g., pioglitazone) or antioxidants (e.g., vitamin E), where the discovery of improved fat accumulation and inflammation has been confirmed by small-scale clinical trials on non-alcoholic steatohepatitis patients. However, this approach fails to show significant effects in ameliorating inflammation and fibrosis, and it is still impossible to provide these drugs as intensive therapy options due to safety concerns over long-term use.

GPR119(G protein-coupled receptor 119) is distributed in L cells and K cells of the small intestine. GPR119 increases the secretion of incretin hormones such as GLP-1 (glucagon-like peptide-1) or GIP (glucose-dependent insulinotropic polypeptide) if GPR119 is activated by triglyceride metabolites (2-monoacylglycerol) or the like. GPR119 is also distributed in pancreatic beta and alpha cells and helps maintain blood glucose levels by controlling the secretion of insulin and glucagon in a glucose-dependent manner. Furthermore, it has been reported that if GPR119 is overexpressed in a human macrophage line, the expression of GLP-1 receptor is increased, so that ABCA1 (ATP-binding cassette protein a1), which is a transporter for bringing cholesterol out of cells, is increased, thereby increasing ApoA 1-mediated cholesterol reflux, improving Lipid metabolism and reducing the concentration of inflammatory cytokines in blood (J Lipid Res, 2014 (55): 681-.

However, it has not been reported whether GPR119 ligand directly inhibits inflammatory response and thus inhibits inflammatory response as a core of hepatocyte injury in non-alcoholic fatty liver disease. Furthermore, it has not been reported whether GPR119 ligand can improve inflammation and fibrosis after non-alcoholic fatty liver disease is induced.

Disclosure of Invention

Technical problem

An object of the present invention is to provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising GPR119 ligand as an effective ingredient.

Technical scheme

The present invention can provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising a compound represented by the following chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an effective ingredient:

[ chemical formula 1]

The pharmaceutical composition according to the present invention can be used for preventing or treating non-alcoholic fatty liver disease by significantly inhibiting triglyceride deposition, inflammation and fibrosis in liver tissue.

In the chemical formula 1, the first and second,

a is oxadiazole (oxadiazole), dihydrooxazole (dihydrooxazole), thiazole (thiazole) or thiadiazole (thiadiazole), A is independently unsubstituted or substituted with at least one substituent selected from halogen, C1-C6 straight or branched chain alkyl and C1-C6 alcohol groups, said alkyl or alcohol groups independently being unsubstituted or substituted with halogen or C1-C6 alkoxy;

b is pyridine, pyrimidine, pyrazine or oxadiazole, B is independently unsubstituted or substituted with at least one substituent selected from halogen, C1-C6 linear or branched alkyl, C1-C6 alcohol, C1-C6 alkoxy and oxadiazolyl, and C1-C6 linear or branched alkyl, C1-C6 alcohol, C1-C6 alkoxy or oxadiazolyl is independently unsubstituted or substituted with halogen, C1-C6 alkyl or C1-C6 alkoxy; and

each X is independently F, Cl, Br or I, but is not limited thereto.

According to an embodiment of the present invention, in chemical formula 1,

there may be provided:

a is And

r1 to R3, R5 and R6 are each independently one or more substituents selected from the group consisting of hydrogen, halogen, C1-C6 straight or branched chain alkyl and C1-C6 alcohol group, said alkyl or alcohol group being independently unsubstituted or substituted with halogen or C1-C6 alkoxy.

According to another embodiment of the present invention, in chemical formula 1,

there may be provided:

b is And

r7 to R11 are independently substituted with at least one substituent selected from the group consisting of hydrogen, halogen, C1-C6 straight or branched chain alkyl, C1-C6 alcohol, C1-C6 alkoxy, and oxadiazolyl, which are independently unsubstituted or substituted with halogen, C1-C6 alkyl, or C1-C6 alkoxy.

According to an embodiment of the present invention, in chemical formula 1, there may be provided a oxadiazole substituted with a C1-C6 linear or branched alkyl group, a pyrimidine substituted with a C1-C6 linear or branched alkyl group, and X is F.

In the present invention, the term "halogen" may refer to fluorine, chlorine, bromine or iodine.

In the present invention, the term "alkyl" may refer to a straight or branched chain hydrocarbon residue, unless otherwise specified. Examples of the C1-C6 alkyl group may include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl and the like.

In the present invention, unless otherwise specified, the term "alkoxy" may include alkyl-oxy having an alkyl group as defined above. Examples of the C1-C6 alkoxy group may include methoxy, ethoxy, propoxy, butoxy, pentoxy and the like.

In the present invention, unless otherwise specified, the term "heterocycle" or "heterocyclic" may refer to a 5 to 13 membered heteroaromatic or non-aromatic compound comprising one to three heteroatoms selected from N, O and S.

In the present invention, the compound represented by chemical formula 1 may be a compound specifically selected from the following compounds:

2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -4, 5-dihydrooxazole,

(R) -2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -4-methyl-4, 5-dihydrooxazole,

(S) -2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -4-methyl-4, 5-dihydrooxazole,

(S) -2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-methyl-4, 5-dihydrooxazole,

(R) -2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-methyl-4, 5-dihydrooxazole,

2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5, 5-dimethyl-4, 5-dihydrooxazole,

(R) - (2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -4, 5-dihydrooxazol-5-yl) methanol,

(S) - (2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -4, 5-dihydrooxazol-5-yl) methanol,

(R) -3- (2- (4- (3- (3, 5-difluoro-4- (5-methyl-4, 5-dihydrooxazol-2-yl) phenoxy) propyl) piperidin-1-yl) pyrimidin-5-yl) -5-isobutyl-1, 2,4-oxadiazole,

(R) -5- (4- (3- (3, 5-difluoro-4- (4-methyl-4, 5-dihydrooxazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-isopropyl-1, 2,4-oxadiazole,

(S) -5- (4- (3- (3, 5-difluoro-4- (5-methyl-4, 5-dihydrooxazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-isopropyl-1, 2,4-oxadiazole,

5- (4- (3- (4- (5, 5-dimethyl-4, 5-dihydrooxazol-2-yl) -3, 5-difluorophenoxy) propyl) piperidin-1-yl) -3-isopropyl-1, 2,4-oxadiazole,

3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-methyl-1, 2,4-oxadiazole,

3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-propyl-1, 2,4-oxadiazole,

3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2,4-oxadiazole,

5- (tert-butyl) -3- (4- (3- (1- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2,4-oxadiazole,

(3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2, 4-oxadiazol-5-yl) methanol,

2- (3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2, 4-oxadiazol-5-yl) ethan-1-ol,

(S) -1- (3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2, 4-oxadiazol-5-yl) propan-1-ol,

(R) -1- (3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2, 4-oxadiazol-5-yl) propan-2-ol,

(S) -1- (3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2, 4-oxadiazol-5-yl) propan-2-ol,

2- (3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,2, 4-oxadiazol-5-yl) -2-methylpropan-1-ol,

3- (2, 6-difluoro-4- (3- (1- (5-propylpyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5-pentylpyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5-methoxypyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5-isopropoxypyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (4- (3- (1- (5-chloropyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (4- (3- (1- (5-bromopyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-methyl-1, 2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-ethyl-1, 2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

5- (sec-butyl) -3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5- (methoxymethyl) -1,2,4-oxadiazole,

(S) -1- (3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -1,2, 4-oxadiazol-5-yl) propan-1-ol,

2- (3- (2, 6-difluoro-4- (3- (1- (5- (5-isobutyl-1, 2, 4-oxadiazol-3-yl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -1,2, 4-oxadiazol-5-yl) -2-methylpropan-1-ol,

3- (4- (3- (1- (5-chloropyrazin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyridin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (3-isopropyl-1, 2, 4-oxadiazol-5-yl) piperidin-4-yl) propoxy) phenyl) -5-methyl-1, 2,4-oxadiazole,

3- (2, 6-difluoro-4- (3- (1- (3-isopropyl-1, 2, 4-oxadiazol-5-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1,2,4-oxadiazole,

(3- (2, 6-difluoro-4- (3- (1- (3-isopropyl-1, 2, 4-oxadiazol-5-yl) piperidin-4-yl) propoxy) phenyl) -1,2, 4-oxadiazol-5-yl) methanol,

2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-methyl-1, 3, 4-oxadiazole,

2-ethyl-5- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -1,3, 4-oxadiazole,

2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1, 3, 4-oxadiazole,

5- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -N-isopropyl-1, 3, 4-oxadiazol-2-amine,

2- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-methyl-1, 3, 4-oxadiazole,

2- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-ethyl-1, 3, 4-oxadiazole,

2- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1, 3, 4-oxadiazole,

2- (4- (3- (1- (5-chloropyrazin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-methyl-1, 3, 4-oxadiazole,

2- (4- (3- (1- (5-chloropyrazin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-ethyl-1, 3, 4-oxadiazole,

2- (4- (3- (1- (5-chloropyrazin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1, 3, 4-oxadiazole,

5- (4- (3- (3, 5-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-propyl-1, 2,4-oxadiazole,

5- (4- (3- (3, 5-difluoro-4- (5-ethyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-propyl-1, 2,4-oxadiazole,

5- (4- (3- (3, 5-difluoro-4- (5-isopropyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-propyl-1, 2,4-oxadiazole,

5- (4- (3- (3, 5-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-isopropyl-1, 2,4-oxadiazole,

5- (4- (3- (4- (5-ethyl-1, 3, 4-oxadiazol-2-yl) -3, 5-difluorophenoxy) propyl) piperidin-1-yl) -3-isopropyl-1, 2,4-oxadiazole,

5- (4- (3- (3, 5-difluoro-4- (5-isopropyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3-isopropyl-1, 2,4-oxadiazole,

5- (4- (3- (3, 5-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -3- (2,2, 2-trifluoroethyl) -1,2,4-oxadiazole,

3- (4- (3- (3, 5-difluoro-4- (5-isopropyl-1, 3, 4-oxadiazol-2-yl) phenoxy) propyl) piperidin-1-yl) -5-isopropyl-1,2,4-oxadiazole,

2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1, 3, 4-thiadiazole,

2- (2, 6-difluoro-4- (3- (1- (5-propylpyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1, 3, 4-thiadiazole,

2- (2, 6-difluoro-4- (3- (1- (5-pentylpyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1, 3, 4-thiadiazole,

2- (2, 6-difluoro-4- (3- (1- (5-fluoropyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1, 3, 4-thiadiazole,

2- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyrimidin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1, 3, 4-thiadiazole,

2- (2, 6-difluoro-4- (3- (1- (5- (trifluoromethyl) pyridin-2-yl) piperidin-4-yl) propoxy) phenyl) -5-isopropyl-1, 3, 4-thiadiazole, and

4-ethyl-2- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) thiazole.

In one embodiment of the present invention, the compound represented by chemical formula 1 of the present invention may be specifically 3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2, 4-oxadiazole.

In the present invention, non-limiting examples of pharmaceutically acceptable salts of the compound represented by chemical formula 1 may include: inorganic acid salts such as hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; organic carboxylates such as acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid or malic acid, or sulfonates such as methanesulfonic acid or p-toluenesulfonic acid; alkali metal salts such as sodium, potassium or lithium; various acid salts known to form other pharmaceutically acceptable salts; and so on.

In a particular embodiment of the invention, it is confirmed by experiments carried out on histological examination, measurement of ALT and AST concentrations in blood, measurement of expression levels of genes and proteins associated with inflammation and fibrosis, and the like: in an ob/ob mouse model in which nonalcoholic fatty liver disease is induced by a special diet, damage to liver tissue, deposition of triglycerides in liver tissue, infiltration of inflammatory cells, and fibrosis are significantly inhibited by administering a compound represented by chemical formula 1 in the course of inducing the disease (example 1).

In one particular embodiment of the invention, it was demonstrated for the first time that: the compound represented by chemical formula 1 inhibits differentiation of human monocytes and activation of differentiated macrophages by inflammatory factors in a concentration-dependent manner through a mechanism of inhibiting inflammatory response (example 2).

In one particular embodiment of the invention, it was demonstrated that: in the C57BL6 mouse model, in which non-alcoholic fatty liver disease was induced by a special diet, damage of liver tissue, deposition of triglyceride in liver tissue, infiltration of inflammatory cells, and fibrosis were significantly improved by administering the compound represented by chemical formula 1 after induction of disease (example 3).

The pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention may be used in the form of a conventional pharmaceutical preparation. The pharmaceutical preparations can be administered in various oral and parenteral dosage forms at the time of administration, and different dosage forms can be determined depending on the method of use.

If the pharmaceutical preparation is formulated into various oral and parenteral dosage forms, commonly used excipients such as fillers, diluents, extenders, binders, wetting agents, disintegrants, surfactants and the like can be used.

Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and may be prepared by mixing at least one excipient such as starch, calcium carbonate (calcium carbonate), sucrose (sucrose), lactose (lactose), gelatin, etc., in a pharmaceutical composition. In addition, in addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may be used.

Further, liquid preparations for oral administration may include suspending agents, liquids for internal administration, emulsions, syrups and the like, but may contain, in addition to water and liquid paraffin (which are commonly used simple diluents), various excipients such as wetting agents, sweeteners, flavoring agents, preservatives and the like.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol (propylene glycol), polyethylene glycol, vegetable oils (e.g., olive oil), injectable esters (e.g., ethyl oleate), and the like. The base of suppositories may include witepsol, polyethylene glycol, tween 61, cocoa butter, bay oil, glycerogelatin, and the like.

In addition, the pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to the present invention may exhibit an effective amount within an administration range of about 1 to about 1000 mg. The administration amount or intake amount can be administered in various administration doses and methods by distributing the composition according to the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity, for example, once or multiple times per day.

In the present invention, non-alcoholic fatty liver disease (NAFLD) may include primary and secondary non-alcoholic fatty liver diseases. Specifically, in the present invention, non-alcoholic fatty liver disease (NAFLD) may include simple steatosis (simple steatosis), non-alcoholic steatohepatitis (NASH) and hepatic fibrosis (liver fibrosis) and cirrhosis (liver cirrhosis) caused by the progression of these diseases, but is not limited thereto.

The pharmaceutical composition of the present invention may comprise a compound represented by chemical formula 1 or one or more active ingredients having similar functions.

The present invention may also provide a method for preventing or treating non-alcoholic fatty liver disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof.

In the present invention, the term "subject in need of treatment" may refer to a mammal including a human, and the term "administering" may refer to providing a predetermined substance to a subject by any suitable method. The term "therapeutically effective amount" may refer to the amount of active ingredient or pharmaceutical composition that induces an animal or human to exhibit a biological or medical response of interest to a researcher, veterinarian, medical doctor or other clinician, and such amount may include the amount of active ingredient or pharmaceutical composition that is used to induce alleviation of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention may be varied depending on the desired effect.

In the present invention, the route of administration of the pharmaceutical composition of the present invention may be administered by any general route as long as it can reach the target tissue.

Administration can be performed orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, endothelially, intranasally, intrapulmonary, rectally, intracavity, intraperitoneally, and intrathecally, but is not limited thereto.

The pharmaceutical compositions of the present invention may be administered once daily or at least twice daily at certain intervals.

For the prevention and treatment of non-alcoholic fatty liver disease, the pharmaceutical composition of the present invention may be used alone or in combination with surgery, endocrine therapy, drug therapy and methods of using biological response modifiers.

The present invention may also provide a food composition for preventing or improving non-alcoholic fatty liver disease, comprising the compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an active ingredient.

In the present invention, the term "alleviating" may refer to all actions that make a disease better or better by administering the composition.

In the present invention, the term "food" may mean meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, instant noodles, other noodles, chewing gum, dairy products (including ice cream), various soups, beverages, tea, health drinks, alcoholic beverages, vitamin complex, health functional foods, health supplements, etc., and includes all foods in the conventional sense.

The term "functional health food" may be the same term as food for special health use (FoSHU) and refers to food having high medical and medical effects, which is processed to effectively exhibit bioregulatory functions in addition to providing nutrients. In this case, the term "functional" may refer to controlling nutrients or obtaining beneficial effects on health such as physiological effects, etc., in terms of the structure and function of the human body.

"health food" may refer to a food having an effect of effectively maintaining or enhancing health as compared to general foods, and "health supplement food" may refer to a food for health supplement purposes. In some cases, the terms health functional food, health food and health supplement may be used interchangeably.

The food of the present invention can be prepared by a method conventionally used in the art, and the food can be prepared by adding raw materials and ingredients conventionally added in the art during the preparation process. Specifically, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavor enhancers may be included, and examples of the carbohydrates may include glucose, fructose, maltose, sucrose, oligosaccharides, dextrin, cyclodextrin, xylitol, sorbitol, erythritol, saccharin, or synthetic flavor enhancers, but are not limited thereto. The food composition of the present invention may be formulated into various dosage forms without limitation as long as the dosage forms are considered as foods.

The present invention may also provide a method for preventing or improving non-alcoholic fatty liver disease, the method comprising administering to a subject in need of improvement a food composition comprising a compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an effective ingredient.

The present invention may also provide a feed composition for preventing or improving non-alcoholic fatty liver disease, comprising the compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof as an effective ingredient.

In the present invention, the term "feed" may refer to any natural or artificial diet, meal or the like, or ingredient of a meal, to be consumed or digested by or suitable for livestock. The feed may comprise feed additives or supplementary feeds.

The kind of the feed is not particularly limited, and a feed conventionally used in the art may be used. Non-limiting examples of feeds may include: plant feeds such as grains, roots, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starch, oil meal, grain by-products, and the like; animal feed such as protein, mineral, oil and fat, mineral, single cell protein, zooplankton, food, etc. The feed may be used alone or by mixing at least two feeds.

The present invention may also provide the use of the compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof for preventing or treating non-alcoholic fatty liver disease.

The present invention may also provide use of the compound represented by chemical formula 1, a pharmaceutically acceptable salt thereof, an optical isomer thereof, a hydrate or solvate thereof, or a mixture thereof for preparing a pharmaceutical preparation for preventing or treating non-alcoholic fatty liver disease.

In the treatment methods, food compositions, mitigation methods, feed compositions, and uses thereof, the compound represented by chemical formula 1 may specifically be 3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2, 4-oxadiazole.

The matters mentioned in the pharmaceutical composition, the method of treatment, the food composition, the method of alleviation, the feed composition and the use thereof according to the invention apply equally if not contradictory to each other.

Advantageous effects

The pharmaceutical composition according to the present invention shows excellent effects of alleviating lipid metabolism, reducing fat accumulation in liver tissue, and preventing histological damage caused by inflammation and fibrosis of liver tissue, and thus can be used for preventing or treating non-alcoholic fatty liver disease.

Drawings

Fig. 1 is a photograph of a tissue sample showing the effects of the compound of the present invention in reducing triglycerides and inhibiting inflammatory cell infiltration in a mouse model induced with non-alcoholic steatohepatitis.

FIG. 2 is an analytical graph showing the effects of the compounds of the present invention on inhibition of inflammatory cell infiltration and inhibition of fibrosis in a mouse model induced with non-alcoholic steatohepatitis (#, p <0.05vs. Normal; &, p <0.05& p <0.01vs. Ob-NASH).

Fig. 3 is a photograph of a tissue sample showing the effect of the compound of the present invention in inhibiting fibrosis in a mouse model induced with non-alcoholic steatohepatitis.

FIG. 4 is a graph showing the effect of the compounds of the invention in reducing AST and ALT in a mouse model induced with non-alcoholic steatohepatitis (#, p <0.05vs. Normal;. p <0.05vs. ob-NASH).

Fig. 5 is a view showing the results of measuring the concentrations of proteins associated with inflammation and fibrosis in a mouse model induced with non-alcoholic steatohepatitis (##, p <0.001vs. normal; &, p <0.01& p <0.001vs. ob-NASH).

Fig. 6 is a view showing the results of measuring the expression levels of genes associated with inflammation and fibrosis in a mouse model induced with non-alcoholic steatohepatitis.

Fig. 7 is a view showing the results of evaluating the gene expression of GPR119 in cells at time points before differentiating human monocytes into macrophages and after differentiating 24 hours and 48 hours.

Fig. 8 is a view showing the results of evaluating the effect on differentiation of human monocytes into macrophages according to treatment with the compound of the present invention.

Fig. 9 is a view showing the results of evaluating the effect on the activation of differentiated macrophages according to treatment with the compound of the present invention.

Fig. 10 is a photograph of a tissue sample showing the effects of the compound of the present invention in reducing triglycerides and inhibiting inflammatory cell infiltration in liver tissue in a mouse model induced with non-alcoholic steatohepatitis.

Fig. 11 is a view showing the results of measuring the content of triglyceride in liver tissue in a mouse model induced with non-alcoholic steatohepatitis (&, p <0.01& p <0.001vs. dio-NASH).

Fig. 12 is a view showing changes in NAFLD Activity Score (NAS) before and after administration to the same individual in a mouse model induced with non-alcoholic steatohepatitis.

Fig. 13 is a photograph of a tissue sample showing the effect of inhibiting fibrosis by the compound of the present invention in a mouse model induced with non-alcoholic steatohepatitis.

Fig. 14 is a view showing the results of measuring the amount of type I collagen in liver tissue in a mouse model induced with non-alcoholic steatohepatitis (&, p <0.05& p <0.001vs. dio-NASH).

Fig. 15 is a view showing the results of measuring the concentration of AST and ALT in blood in a mouse model induced with non-alcoholic steatohepatitis (&, p <0.01& p <0.001vs. dio-NASH).

Fig. 16 is a view showing the results of measuring the gene expression levels of monocyte attractant, macrophage marker and fibrosis marker in liver tissue of a mouse model induced with non-alcoholic steatohepatitis (, &, p <0.05, p <0.01& p <0.001vs. dio-NASH).

Detailed Description

The features and advantages of the present invention, and the methods of accomplishing the same, will be apparent from the following detailed description of exemplary embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be embodied in various forms. Hereinafter, the following examples will be presented to better understand the present invention and are provided only to thoroughly explain the scope of the present invention to those skilled in the art, and thus the present invention will be limited only by the scope of the claims.

< example 1> determination of the efficacy of GPR119 ligand in a mouse model induced steatohepatitis by the supply of a special diet

To determine the effect of GPR119 ligand compounds according to the present invention in the prevention of nonalcoholic fatty liver disease, the following experiments were performed.

Preparation of mouse model with non-alcoholic steatohepatitis

Male ob/ob mice with leptin deficiency, which are six weeks old, were fed with a special diet containing high fat, high fructose and high cholesterol for 10 weeks, thereby inducing their progression to non-alcoholic steatohepatitis. A mixed form of diet was prepared such that the daily dose of 3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isopropyl-1,2,4-oxadiazole (3- (4- (3- (1- (5-ethylpyrimidin-2-yl) piperidin-4-yl) propoxy) -2, 6-difluorophenyl) -5-isoproyl-1, 2,4-oxadiazole) (hereinafter, "compound 1") among the compounds represented by chemical formula 1 reached 100 mg/kg/day, and the mixed form of diet was supplied to mice from a time point of 10 weeks from the start of supplying a special diet.

Histological examination

For histological examination, the prepared mouse model induced with non-alcoholic steatohepatitis was necropsied to fix the separated liver tissue in 10% formalin and prepare paraffin blocks, thereby obtaining tissue sections 2 μm thick. To determine the infiltration of inflammatory cells, Hematoxylin and Eosin (HE) staining was performed using an automated staining machine (autostainer XL, Leica), the results of which are shown in fig. 1 and 2.

As can be understood from fig. 1 and fig. 2, Ob/Ob mice (Ob-NASH) fed with the special diet for 10 weeks showed a significant increase in triglyceride lipid droplets in liver tissues, as compared to C57BL/6 mice (normal) fed with the normal diet, and also confirmed infiltration of inflammatory cells. In contrast, it was confirmed that when compound 1 of the present invention (Ob-NASH + compound 1) was supplied simultaneously with a special diet, infiltration of inflammatory cells was significantly inhibited.

Then, to assess fibrosis, specific staining of fibers in liver tissue was performed using masson trichrome stain or Sirius Res stain, the results of which are shown in fig. 2 and 3.

As can be understood from fig. 2 and 3, unlike C57BL/6 mice fed with a normal diet, ob/ob mice fed with a special diet for 10 weeks exhibited a significant progress of fibrosis in purple liver tissue as a whole, but fibrosis in liver tissue was significantly inhibited when compound 1 of the present invention was supplied simultaneously with the special diet.

AST and ALT measurements in blood

A mouse model with induced non-alcoholic steatohepatitis was subjected to autopsy, and then plasma was separated therefrom, so that AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were quantified using an automatic blood analyzer (Konelab 20i), and the results thereof are shown in fig. 4.

As shown in fig. 4, it was confirmed that ob/ob mice fed with a special diet for 10 weeks showed a significant increase in ALT and AST levels in blood due to liver tissue damage (which is a marker of liver damage), unlike C57BL/6 mice fed with a normal diet. On the other hand, it was confirmed that ob/ob mice administered with compound 1 of the present invention together with a special diet exhibited significant inhibition of ALT and AST increase, which means alleviation of liver cell damage.

Determination of the concentration of proteins involved in inflammation and fibrosis

To evaluate the effect on the expression of inflammation-related genes and fibrosis-related proteins in liver tissues, liver tissues obtained from a mouse model were put in RIPA buffer (Cell Signaling), pulverized with TissueLyser IITM (Quiagen), and then the total protein concentration was quantified with Pierce BCA protein assay kit (Thermo Fisher Scientific), and the protein concentrations of mouse Ccl2(═ Mcp 1; R & D Systems, MJE00) and Timp1(R & D Systems, MTM100) in liver tissues were quantified using a commercially available ELISA kit, and the results are shown in fig. 5.

As shown in fig. 5, it was confirmed that mice fed with a special diet exhibited increased protein concentrations of Ccl2(═ Mcp1) and Timp1 in liver tissue, the Ccl2(═ Mcp1) is a factor inducing inflammatory cells to enter liver tissue, the Timp1 is an endogenous inhibitor of enzymes degrading fibers deposited in liver tissue, and when the compound 1 of the present invention was administered with a special diet, the protein levels of Ccl2 and Timp1 were significantly inhibited.

Determination of expression of genes associated with inflammation and fibrosis

To measure the expression levels of inflammation-related genes and fibrosis-related genes in liver tissue, the expression level of the genes was measured(Invitrogen) was added to liver tissue collected from the mouse model, and then total RNA was extracted from the tissue according to the method provided by the manufacturer, followed by cDNA synthesis using reverse transcriptase. Then, SYBR Green I Master Mix (Roche, 04707516001) was used in LightCyclerReal-time polymerase chain reaction (Real-time polymerase chain reaction) was performed in an Instrument II (Roche Life science) apparatus, and the results are shown in FIG. 6.

As shown in fig. 6, it was confirmed that mice fed with a special diet for 10 weeks exhibited increased expression of mouse genes associated with inflammation (Ccl2, Lgals3, Tnfa) and genes associated with fibrosis (Tgfb1, Col1a, Timp1, Timp2, Acta2), while increased expression of genes associated with inflammation and fibrosis was significantly inhibited when compound 1 of the present invention was administered with the special diet.

The above results indicate that the compounds of the present invention exert excellent effects on non-alcoholic fatty liver diseases by inhibiting damage, inflammation and fibrosis of liver tissues.

< example 2> determination of inhibition of macrophage activation upon differentiation and differentiation of human monocytes

In order to evaluate the direct anti-inflammatory effect of the compounds according to the invention, the effect on human monocyte differentiation and activation thereof was determined.

First, as a method for detecting a human monocyte (THP-1,TIB-202^TM) Results of assessing GPR119 gene expression in cells before differentiation into macrophages and at time points 24 hours and 48 hours after differentiation using PMA (phorbol 12-myristate 13-acetate) have demonstrated that GPR119 receptor expression increases with the passage of time for monocyte differentiation into macrophages (fig. 7).

Then, the THP-1 monocytes were treated with PMA (50ng/ml) for 48 hours to differentiate into macrophages, while being co-treated with compound 1, and then replaced with serum-free medium, and then treated with LPS (lipopolysaccharide), 0.5ng/ml) for 4 hours, to quantify IL-1 β (interleukin-1 β, R & D Systems, DY201) secreted into the medium using a commercial ELISKA kit, and to evaluate the effect on monocyte differentiation, the results of which are shown in fig. 8.

As shown in FIG. 8, it was confirmed that IL-1. beta. secretion caused by macrophage activation decreased in a concentration-dependent manner when treated with the compound of the present invention in the process of differentiating monocytes into macrophages.

In addition, after treating differentiated macrophages with compound 1 for 48 hours, secreted IL-1 β was quantified by the same method as described above for activation of immune cells by LPS, to thereby evaluate the effect on activation of differentiated macrophages. Thus, the results are shown in fig. 9.

As shown in fig. 9, it was confirmed that even when differentiated macrophages were treated with the compound of the present invention, IL-1 β secretion caused by macrophage activation decreased in a concentration-dependent manner.

The above results indicate that the compounds of the present invention produce excellent effects in the prevention and treatment of non-alcoholic fatty liver diseases by directly inhibiting the differentiation and activation of immune cells.

< example 3> determination of the efficacy of GPR119 ligand in a mouse model inducing steatohepatitis by supplying a special diet

To determine the effect of GPR119 ligand compounds according to the present invention in the treatment of non-alcoholic fatty liver disease, the following experiments were performed.

Preparation and research design of mouse model with non-alcoholic steatohepatitis

Six-week old male C57BL/6J mice were fed for at least 26 weeks with a special diet containing high fat, high fructose and high cholesterol, inducing their development of non-alcoholic steatohepatitis. Three weeks prior to drug supply, induction of fatty liver and inflammatory fibrosis was confirmed by biopsy of liver tissue, and mice were evenly distributed into each group based on the area of collagen stained in liver tissue. The feed was prepared by mixing the compound according to the invention in a special diet and was supplied for an additional eight weeks. According to the diet supplied to the mice, the components were: control group fed on normal diet (normal); a positive control group (DIO-NASH) in which the mice induced non-alcoholic steatohepatitis due to the special diet are not supplied with the drug; and groups in which compound 1(L) and compound 1(H), respectively) was supplied at 30 mg/kg/day (L) and 100 mg/mg/day (H) to non-alcoholic steatohepatitis-induced mice.

Histological examination

For histological examination, the prepared C57BL/6J mouse model was autopsied to fix the separated liver tissue in 10% formalin and prepare paraffin blocks, thereby obtaining tissue sections 2 μm thick. Hematoxylin and Eosin (HE) staining was then performed using an automatic staining machine (autostainer XL, Leica), the results of which are shown in fig. 10. In addition, the content of triglyceride in the liver tissue was measured by using a triglyceride reagent (Roche diagnostics, #22-045-795) using the liver tissue isolated from the C57BL/6J mouse model, and the results thereof are shown in FIG. 11.

As can be understood from fig. 10 and 11, the mice fed with the special diet alone (DIO-NASH) showed a significant increase in the deposition of fat and infiltration of inflammatory cells in liver tissues, compared to the mice fed with the normal diet (normal), while the group of DIO-NASH compound 1(H) fed with compound 1 of the present invention together with the special diet showed a significant decrease in the deposition of fat and infiltration of inflammatory cells.

In addition, as a result of evaluating NAS (NAFLD activity fraction) before and after administration, which reflects the proportion of adipocytes, infiltration of inflammatory cells, and damage to hepatocytes, based on the above histological examination, the disease was worsened in the DIO-NASH group of 50% and no individual was worsened in the case of administration of the compound, and the disease was improved in the DIO-NASH _ compound 1 group of about 50% (fig. 12).

Then, in order to evaluate fibrosis, the fibers of the liver tissue were specifically stained with Sirius Res stain in each group of mice, and the content of type I collagen in the liver tissue was measured by an image analysis method after immunostaining, and the results thereof are shown in fig. 13 and 14.

As shown in fig. 13 and fig. 14, the mouse fed with only the special diet (DIO-NASH) showed significant progress of fibrosis in the liver tissue, compared to the mouse fed with the normal diet (normal), while the group fed with the compound 1 of the present invention together with the special diet (DIO-NASH compound 1) showed significant reduction of crosslink formation in the fiber of the liver tissue and also showed significant reduction of type I collagen.

AST and ALT measurements in blood

Each group of mice was subjected to autopsy, and then plasma was separated therefrom, thereby quantifying AST (aspartate aminotransferase) and ALT (alanine aminotransferase) using an automatic hematology analyzer (Konelab 20 i). Thus, the results are shown in fig. 15.

As shown in fig. 15, the group of mice fed with the special diet alone (DIO-NASH) showed a significant increase in the concentrations of ALT and AST in the blood, while the group of mice fed with compound 1 of the present invention together with the special diet (DIO-NASH compound 1) showed a significant inhibition in the increase of ALT and AST, as compared to the group of mice fed with the normal diet.

Determination of changes in Gene expression

In order to analyze changes in total gene expression in liver tissues isolated from the above mouse model within eight weeks after drug administration, isolated RNA fractions were used to generate libraries using neoprep (Illumina), and RNA sequencing analysis was performed by NexSeq 500(Illumina), followed by bioinformatics analysis and classification of changes in major genomes for each function, and the results thereof are shown in fig. 16.

As shown in fig. 16, the mice fed with the special diet alone (DIO-NASH) showed increased expression of monocyte attractants, macrophage markers and fibrosis markers in liver tissues compared to the mice fed with the normal diet (normal), while the group of mouse DIO-NASH compounds 1 fed with the compound 1 of the present invention together with the special diet showed a significant decrease of the above markers.

The above results indicate that the compounds of the present invention exert effects on non-alcoholic fatty liver diseases by inhibiting damage, inflammation and fibrosis of liver tissues, and exert more excellent therapeutic effects by co-administration of DPPIV inhibitors.