KR20150054231A - Pharmaceutical composition comprising imidazopyridine derivative with PI3-kinase inhibitory activity for liver cirrrhosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing an imidazopyridine derivative as an active component which has phosphotidylinostiol-3-kinase (PI-3-kinase; PI3K) inhibitory activity. The imidazopyridine derivative can induce an apoptosis of hepatix stellate cell by effectively blocking signal transduction pathways of PI3K/Akt, and inhibits expression of fibrosis factors, therefore, the imidazopyridine derivative can be used for prevention or treatment of cirrhosis, effectively.

Description

     A pharmaceutical composition for preventing or treating cirrhosis comprising an imidazopyridine derivative having PI3K inhibitory activity as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing or treating cirrhosis, which comprises an imidazopyridine derivative having PI3K inhibitory activity as an active ingredient.

Liver fibrosis is a common consequence of chronic liver injury caused by various pathologic factors and leads to cirrhosis. The pathological process of this process is characterized by the accumulation of extracellular matrix (ECM) proteins. Liver fibrosis is usually caused by the activation of Cooper and HSCs, which leads to an increase of inflammatory cells, which in turn leads to hepatocyte damage. In particular, hepatic stellate cells (HSCs) are the major cells that cause extracellular matrix formation in patients with chronic liver disease and play an important role in the growth and maintenance of hepatic fibrosis. When liver cirrhosis occurs, activated hepatic stellate cells grow exponentially, and these cells secrete profibrogenic cytokines, resulting in alpha-smooth muscle actin (α-SMA), collagen, and metalloproteinases And produce ECM-related molecules such as tissue inhibitors (TIMPs). Until now, liver fibrosis has been known to be irreversible, but recent clinical and experimental studies have reported that liver fibrosis process can reverse fibrosis by reversible reaction. Thus, activated hepatic stellate cells have become a major target to prevent progression of hepatic fibrosis.

Phosphatidylinositol 3-kinase (PI3K) is known to be an important factor controlling many cell functions such as cell proliferation, survival, permeation, and migration. PI3K is composed of 85-kDa and 110-kDa subunits and activates Akt to activate lower signaling molecules such as mTOR and P70S6K.

Activation of the PI3K / AKT signaling pathway has recently been shown to stimulate collagen synthesis in fibroblasts, affecting a variety of fibrotic diseases. In particular, the activation of Akt has been implicated in the proliferation of hepatic stellate cells (HSCs) and in the formation of type 1 collagen transcription and translation. In this regard, the PI3K signal is emerging as an important role in the fibrotic response. Therefore, inhibition of PI3K signal could inhibit the activation of hepatic stellate cells (HSCs), suggesting that treatment of hepatic fibrosis would be possible.

The inventors of the present invention completed the present invention upon confirming that imidazopyridine derivatives that block PI3K signal effectively inhibit fibrosis of liver cirrhosis while studying substances having an inhibitory effect on liver fibrosis among the imidazopyridine derivatives.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cirrhosis, which comprises an imidazopyridine derivative having PI3K inhibitory activity as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating liver cirrhosis comprising an imidazopyridine derivative having PI3K inhibitory activity as an active ingredient.

The imidazopyridine derivative having PI3K inhibitory activity according to the present invention has an excellent effect of suppressing the progression of fibrosis by inhibiting the expression of fibrosis factors such as collagen I and vimentin by inhibiting activation of the PI3K / Akt signaling system, Can be useful for the prevention or treatment of cancer.

1 is a graph showing the effect of the present invention on the production of ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- And the cell proliferation inhibitory activity was measured by MTT assay.
FIG. 2 is a graph showing the cytotoxic effect of HS-173 according to the present invention on hepatic stellate cells, by TUNEL assay, Western blot analysis, and JC-1 fluorescence staining assay (c).
FIG. 3 is a graph showing the effect of fibrosis factors (α-SMA, Vimentin, fibronectin, type 1 collagen and type IV collagen, TIMP-1 and MMP-2) in hepatic stellate cells of HS-173 of the present invention in hepatic stellate cells (A) and Western blot analysis (b), respectively.
FIG. 4 is a graph showing the inhibitory effect of Akt, P70S6K phosphorylase, which is a downstream signaling factor of the PI3K / Akt signaling system of HS-173 of the present invention.
FIG. 5 is a graph showing the inhibition of liver fibrosis production and ALT and AST concentration increase by HS-173 of the present invention in a mouse liver cirrhosis model.
FIG. 6 is a graph showing the efficacy of cirrhosis treatment by reducing the expression of fibrosis markers by HS-173 of the present invention in a mouse liver cirrhosis model.
FIG. 7 is a graph showing the inhibitory effect of HS-173 on Akt, P70S6K phosphorylation, which is a lower signaling factor of the PI3K / Akt signal transduction system in the mouse liver cirrhosis model.
8 is a graph showing the effect of HS-173 according to the present invention on fibrosis factor and PI3K / Akt signal transduction system in the mouse liver cirrhosis model.

The present invention relates to a prophylactic or therapeutic agent for cirrhosis, which contains, as an active ingredient, an imidazopyridine derivative represented by the following general formula (1) having a phosphotidylinostiol-3-kinase (PI3K) A pharmaceutical composition is provided.

In Formula 1, X is carbon or nitrogen,

R1 is hydrogen, an alkyl group of C ₁ ~ C _6; An acetyl group; Pyrazole group; A morpholine group; - (C = O) O- ( C 1 -C 6 alkyl); Cyano; Tetrazole group; An oxadiazole group; Or a C ₆ to C ₃₀ aryl group substituted with a cyano group or a C ₆ to C ₃₀ heteroaryl group,

R 2 is a C ₆ to C ₃₀ aryl group or a C ₆ to C ₃₀ heteroaryl group which is substituted or unsubstituted with at least one member selected from the group consisting of an amine group, an arylsulfonamide group and a morpholinosulfonyl group.

로 이루어진 군으로부터 선택된 1종 이상이고,Preferably, R1 in the formula (1) is

And at least one member selected from the group consisting of

로 이루어진 군으로부터 선택된 1종 이상이다.R2 is

≪ / RTI >

More preferably, the imidazopyridine derivative of Formula 1 is at least one selected from the group consisting of the following compounds:

1) N- (5- (3- (5-methyl-1,2,4-oxadiazol-3-yl) imidazo [1,2- a] pyridin- Sulfonamide;

2) N- (5- (3-Cyanoimidazo [1,2-a] pyridin-6-yl) pyridin-3yl) benzenesulfonamide;

3) N- (5- (3-morpholinoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

4) N- (5- (3-acetylimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

5) N- (5- (3-acetylimidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide;

6) 5- (3-morpholinoimidazo [1,2-a] pyridin-6-yl) -3- (morpholinosulfonyl) pyridin-2-amine;

7) 3,5-Difluoro-N- (5- (3- (5-methyl-1,2,4-oxadiazol- Yl) pyridin-3-yl) benzenesulfonamide;

8) 2,4-Difluoro-N- (5- (3- (5-methyl-1,2,4-oxadiazol-3- yl) imidazo [ Yl) pyridin-3-yl) benzenesulfonamide;

9) N- (5- (3- (3-cyanophenyl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

10) N- (5- (3- (2H-tetrazol-5-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

11) N- (5- (3- (Pyridin-3-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

12) N- (5- (3- (Pyridin-4-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

13) 3- (imidazo [1,2-a] pyridin-6-yl) aniline;

14) 5- (Imidazo [l, 2-a] pyridin-6-yl) pyridin-3-amine;

15) N- (5- (imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

16) ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate;

17) 6- (6-Amino-5- (morpholinosulfonyl) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carbonitrile;

18) N- (5- (3- (1H-pyrazol-4-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;

19) N- (5- (3-Cyanoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) -2,4-difluorobenzenesulfonamide;

20) N- (5- (3- (2H-tetrazol-5-yl) imidazo [1,2- a] pyridin-6-yl) pyridin- Sulfonamide;

21) N- (5- (3-Acetylimidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide;

22) N- (5- (Imidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide; And

23) Ethyl 6- (5- (2,4-difluorophenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate.

Most preferably, the imidazopyridine derivative of Formula 1 is ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate.

The imidazopyridine derivatives according to the present invention can form pharmaceutically acceptable salts. Such pharmaceutically acceptable salts include those acids which form non-toxic acid addition salts containing a pharmaceutically acceptable anion, for example inorganic acids such as sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid and the like; And organic acids such as tartaric acid, formic acid, citric acid, acetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, lactic acid, malonic acid, malic acid, maleic acid, salicylic acid, succinic acid, oxalic acid, propionic acid, ; Sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like, and imidazopyridine derivatives according to the present invention comprising free carboxy substituents May include the above acid addition salts and salts of sodium, calcium and ammonium.

In addition, the pharmaceutically acceptable salts include salts of alkali metals or alkaline earth metals formed by pharmaceutically acceptable base addition salts such as lithium, sodium, potassium, calcium, magnesium and the like; Amino acid salts such as lysine, arginine and guanidine; Organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris (hydroxymethyl) methylamine, diethanolamine, choline, triethylamine and the like.

Hereinafter, the present invention will be described in detail.

The imidazopyridine derivatives of the present invention inhibit the growth of hepatic stellate cells in a concentration-dependent manner, induce apoptotic cell death, block the PI3K / Akt signal transduction system, and inhibit type I collagen, And suppresses the progression of fibrosis by inhibiting the expression of the same fibrosing factors, and thus can be effectively used for preventing or treating cirrhosis.

The composition of the present invention may further contain at least one known active ingredient having an anti-fibrosing effect together with the imidazopyridine derivative of the formula (1).

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient 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, 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 administration 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the daily dose of the imidazopyridine derivative of the present invention may be administered in an amount of 1 mg / kg to 30 mg / kg per day, and may be administered once a day, divided into several times have.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for anti-fibrosis effects.

Hereinafter, preferred embodiments and examples of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples and preparative examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and preparation examples.

Example 1. Inhibition effect of imidazopyridine derivative of the present invention on cell proliferation

In order to examine the effect of the imidazopyridine derivative of the present invention on cell proliferation inhibition, cytotoxicity evaluation of hepatic stellate cells was performed.

The imidazopyridine derivative as an active ingredient of the present invention can be prepared by the method described in the publication of Design and Synthesis of Imidazopyridine Analogues as Phosphoinositide 3-Kinase Signaling and Angiogenesis, 2011 American Chemical Society, dx.doi. org / 10.1021 / jm101582z | J. Med. Chem., 2011, 54, 2455-466, Hong Soon Sun et al., 5). In an embodiment of the present invention, ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate as an imidazopyridine derivative for measuring fibrosis- (Hereinafter referred to as "HS-173") was used and dissolved in DMSO in the experiment.

1. Preparation and culture of hepatic stellate cells

Hepatic stellate cells (HST-T6 cells) were obtained from Professor SL Friedman (Liver Disease Research Center, San Francisco Hospital, CA, USA). The cells were inoculated into a 100-mm cell culture dish containing DMEM medium containing 10% fetal bovine serum (FBS), penicillin and streptomycin. The cultivation was carried out in an incubator (CO ₂ incubator) which provided conditions of 5% CO _2, 37 ° C temperature and humidification.

2. Inhibition effect on cell proliferation

The cell proliferation inhibitory effect of the imidazopyridine derivative of the present invention was evaluated by MTT assay after HS-173 treatment of hepatic stellate cells prepared in 1 above.

Specifically, the hepatic stellate cells were inoculated into a 96-well plate at a concentration of 3-10 × 10 ³ cells / well and cultured for 24 hours. Cells from which the medium was removed were treated with 0.1% (v / v) DMSO or HS-173 or LY294002 (conventional PI3K inhibitor) at concentrations of 1, 5 and 10 μM. After each treated cells were cultured for 24, 48, or 72 hours, 20 占 퐇 of MTT solution (2 mg / ml) was added to each well, and the plate was re-cultured at 37 占 폚 for 4 hours. After incubation, the formazan crystals were dissolved in DMSO (100 or 300 μl / well) with shaking constantly for 5 minutes. The absorbance of each plate was measured at 540 nm using a microplate reader. At this time, each analysis was performed using three replicate wells prepared in the same manner, and the median inhibitory concentration (IC ₅₀ ; indicating the drug concentration that suppresses cell growth to 50%) was measured on the dose-response curve ≪ / RTI > The results are shown in Fig.

As shown in FIG. 1, HS-173 of the present invention was found to inhibit the growth of hepatic stellate cells in a dose- and time-dependent manner, and inhibited the growth of hepatic stellate cells compared to the conventional hepatic stellate cell growth inhibitor LY294002 The effect of the present invention was confirmed.

Thus, it can be seen that the imidazopyridine derivative of the present invention is excellent in the effect of inhibiting the growth of hepatic stellate cells, and thus is excellent in inhibiting cell proliferation.

3. Statistical Analysis

Data were expressed as means ± SD and statistically analyzed by ANOVA and Student's t-test. A P-value ≤0.05 is considered statistically significant. Statistical values were obtained using SPSS software for Windows operating system (SPSS, Chicago, IL version 10.0).

Example 2. Effect of the imidazopyridine derivative of the present invention on apoptosis

In order to examine the cytotoxic effect of the imidazopyridine derivative of the present invention, the following procedure was performed.

1. Measurement of apoptotic effect of ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-

To investigate the hepatocyte apoptotic effect of the imidazopyridine derivatives of the present invention, TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) analysis was performed on hepatic stellate cells.

Specifically, the hepatic stellate cells (HSC-T6) of Example 1 were placed in an 18-mm cover glass and cultured for 24 hours or more in a DMEM medium until the concentration of cells in the medium reached about 70% Lt; / RTI > The cells were fixed with 5 μM HS-173 in ice-cold 1% paraformaldehyde solution for 4 hours, washed with PBS, and subjected to TUNEL staining. The stained cells were observed under a fluorescence microscope to confirm nuclear fragmentation. The results are shown in Fig.

As shown in FIG. 2A, the morphology of apoptotic cells such as nuclear fusion and nucleation of apoptotic bodies in the cells treated with HS-173 of the present invention at a concentration of 5 μM , And HS-173-dependent TUNEL-positive cells.

Thus, it can be seen that the imidazopyridine derivative of the present invention induces apoptosis of hepatic stellate cells, thereby exerting an effect of inhibiting proliferation of hepatic stellate cells.

2. Measurement of cell death effect of ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [l, 2-a] pyridine-3-carboxylate by Westin blot analysis

Western blot analysis was performed on hepatic stellate cells in order to examine the hepatocyte apoptotic effect of the imidazopyridine derivatives of the present invention.

Specifically, hepatic stellate cells (HSC-T6) prepared in Example 1, 1 were repeatedly washed three times with ice-cold PBS, and then washed with 1% Triton X-100, 1% Nonidet P- A buffer solution containing protease and phosphorylase inhibitor was added and dissolved; (10 mg / ml) (ICN Biomedicals, Asse-Relegem, Belgium), PMSF (phenylmethy lsulfonyl fluoride) (1.72 mM), sodium fluoride (NaF) _{(100 mM), NaVO 3 (} 500 mM) and _{Na 4 P 2 O 7 (500} mg / ml; Sigma-Aldrich). The same amount of protein was separated by electrophoresis on 8 or 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to nitrocellulose membranes. Protein transfer was confirmed using a Ponceau S staining solution. The blot was immunostained using an appropriate primary antibody according to a secondary antibody conjugated with horseradish peroxidase. Primary antibodies are used specifically for the following factors; Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), Cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA), and β-actin (Abcam, Cambridge, UK). All secondary antibodies were purchased from Amersham Biosciences and the band intensities were quantified with Image J software. The results are shown in Figure 2b.

As shown in FIG. 2B, the concentration of cleaved caspase-3, which is a marker of apoptosis in a dose-dependent manner in HS-173-treated hepatic stellate cells (HSC-T6), increases and apoptosis And the concentration of Bcl-2, which is known to inhibit the progression, decreases.

Thus, it can be seen that the imidazopyridine derivative of the present invention induces apoptosis of hepatic stellate cells, so that the effect of inhibiting proliferation of hepatic stellate cells is excellent.

3. Measurement of cytotoxic effect of ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate by JC-

In order to examine the effect of the imidazopyridine derivative of the present invention on the membrane potential of hepatic stellate cells, JC-1 fluorescence staining analysis was performed on hepatic stellate cells.

Specifically, the hepatic stellate cells (HSC-T6) prepared in Example 1, 1 were placed in an 18-mm cover glass to reach a concentration of approximately 70% (density of differentiated cells in the medium) And cultured in DEME medium for 24 hours. Cells were treated or untreated with 5 μM HS-173 for 2 h and then treated with 5 μM JC-1 fluorescent dye (MitoPT, Immunohistochemistry Technologies, Bloomington, MN) and incubated for 20 min in a CO ₂ incubator. The slides were washed twice with PBS and then covered with Dabco (Sigma-Aldrich). Then, confocal laser scanning microscope (CLSM) (Olympus, Tokyo, Japan) was observed. The results are shown in Figure 2c.

As shown in FIG. 2C, the control group showed heterotypic cytoplasmic dysplasia of both red and green in the hepatic stellate cells, whereas in the HS-173 treated group of the present invention, red fluorescence And clusters of mitochondria were increased.

Therefore, it can be seen that the imidazopyridine derivative of the present invention is superior in the effect of inhibiting hepatic cell proliferation by inducing apoptosis by autophoresis due to loss of mitochondrial membrane potential (ψm) of hepatic stellate cells.

Example 3 Measurement of Inhibitory Effect of Imidazopyridine Derivative of the Present Invention on Expression of Fibrogen Factor

In order to examine the effect of the imidazopyridine derivatives of the present invention on the expression of fibrogen factors, the following procedure was carried out.

1. Measurement of inhibitory effect of ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate on the expression of fibrosis factor by immunofluorescence analysis

Immunofluorescence analysis was performed to confirm the inhibitory effect of the imidazopyridine derivatives of the present invention on the expression of fibrosis factors.

Specifically, hepatic stellate cells (HSC-T6) prepared in Example 1, 1 were plated on DEME medium in an 18-mm cover glass at a density of 1-1.5 × 10 ⁵ cells / well, Lt; / RTI > Then, the cells were cultured for 4 hours without adding 1 [mu] M of HS-173 (experimental group) (control group). Then, the cells were washed with PBS and fixed with a mixed solution of acetic acid: ethanol (2: 1) at a temperature of -20 DEG C for 5 minutes. Non-specific binding was blocked at room temperature with 5% chlorine and horse serum / PBS for 1 hour at room temperature before α-smooth muscle actin α-SMA (Sigma-Aldrich), vimentin, p The cells were incubated in a humidified chamber with the addition of a primary antibody against -cdc2 (Cell Signaling Technology), cyclin B1, matrix metalloproteinase (MMP) -2 (Santa Cruz Biotechnology) or fibronectin (Abcam). After completion of the incubation, the cells were incubated in a dark room at room temperature for 1 hour with a fluorescence-labeled second antibody (diluted in a ratio of 1:50; Dinova) in an antibody dilution solution. The nuclei were stained with 4,6-diamino-2-phenylindole (DAPI) in the dark at room temperature for 30 minutes. The slides were washed twice with PBS and then covered with Dabco (Sigma-Aldrich) and observed at 488 and 568 nm using a confocal laser scanning microscope (CLSM) (Olympus, Tokyo, Japan). The results are shown in Fig.

As shown in FIG. 3A, expression of vitamins and fibronectin, which are fibrotic factors, in HS-173-treated hepatic stellate cells of the present invention was decreased as compared with the control group, and the expression of type IV collagen and MMP-2 was also decreased, -1, respectively.

Therefore, the imidazopyridine derivative of the present invention is excellent in the effect of inhibiting the expression of fibrosis factor in hepatic stellate cells, so that the effect of anti-fibrosis is excellent.

2. Measurement of inhibitory effect of ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate on the expression of fibrosis factor by Westin blot analysis

Westin blot analysis was performed to confirm the effect of the imidazopyridine derivatives of the present invention on inhibiting the expression of fibrosing factors.

Specifically, hepatic stellate cells (HSC-T6) prepared in Example 1, 1 were repeatedly washed three times with ice-cold PBS, and then washed with 1% Triton X-100, 1% Nonidet P- A buffer solution containing protease and phosphorylase inhibitor was added and dissolved; (10 mg / ml) (ICN Biomedicals, Asse-Relegem, Belgium), PMSF (phenylmethy lsulfonyl fluoride) (1.72 mM), sodium fluoride (NaF) _{(100 mM), NaVO 3 (} 500 mM) and _{Na 4 P 2 O 7 (500} mg / ml; Sigma-Aldrich). The same amount of protein was separated by electrophoresis on 8 or 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to nitrocellulose membranes. Protein transfer was confirmed using a Ponceau S staining solution. The blot was immunostained using an appropriate primary antibody according to a secondary antibody conjugated with horseradish peroxidase. Primary antibodies are used specifically for the following factors; collagen I, TIMP-1 and beta-actin (Abcam, Cambridge, UK). All secondary antibodies were purchased from Amersham Biosciences and the band intensities were quantified with Image J software. The results are shown in FIG. 3B.

As shown in FIG. 3B, it was confirmed that the HS-173 of the present invention reduced the expression of type 1 collagen, TIMP-1, which is a fibrosing factor. Since activated hepatic stellate cells are involved in the synthesis of collagen and accumulation in the liver, a decrease in the expression of type I collagen means that HS-173 of the present invention inhibits hepatic stellate cell activation. In addition, HS-173 strongly inhibited the expression of TIMP-1 in order to inhibit ECM degradation in hepatic stellate cells.

Therefore, the imidazopyridine derivative of the present invention is excellent in the effect of inhibiting the expression of fibrosis factor in hepatic stellate cells, so that it has excellent anti-fibrosing effect.

Example 4. Inhibition of activation of the PI3K / Akt signaling system of the imidazopyridine derivative of the present invention

Western blot analysis was performed to determine whether the anti-fibrotic effect of the imidazopyridine derivatives of the present invention was mediated through the PI3K / Akt signaling pathway.

Specifically, hepatic stellate cells (HSC-T6) prepared in Example 1, 1 were repeatedly washed three times with ice-cold PBS, and then washed with 1% Triton X-100, 1% Nonidet P- A buffer solution containing protease and phosphorylase inhibitor was added and dissolved; (10 mg / ml) (ICN Biomedicals, Asse-Relegem, Belgium), PMSF (phenylmethy lsulfonyl fluoride) (1.72 mM), sodium fluoride (NaF) _{(100 mM), NaVO 3 (} 500 mM) and _{Na 4 P 2 O 7 (500} mg / ml; Sigma-Aldrich). The same amount of protein was separated by electrophoresis on 8 or 12% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to nitrocellulose membranes. Protein transfer was confirmed using a Ponceau S staining solution. The blot was immunostained using an appropriate primary antibody according to a secondary antibody conjugated with horseradish peroxidase. Primary antibodies are used specifically for the following factors; p-Akt, Akt, p-P70S6K, P70S6K (Cell Signaling Technology, Beverly, MA, USA), beta-actin (Abcam, Cambridge, UK). All secondary antibodies were purchased from Amersham Biosciences and the band intensities were quantified with Image J software. The results are shown in FIG.

As shown in FIG. 4, HS-173 of the present invention inhibits the phosphorylation of Akt and P70S6K, which are lower signaling factors of the PI3K / Akt signaling system, in a concentration-dependent manner.

Thus, it can be seen that the antifibrinolytic effect of the imidazopyridine derivative of the present invention is due to inhibition of the activation of the PI3K / Akt signal transduction system by inhibiting Akt, P70S6K phosphorylation, a downstream signaling factor of the PI3K / Akt signaling system .

Example 5: Effect of imidazopyridine of the present invention on cirrhosis treatment, which was verified by animal experiments

Animal experiments were carried out as follows to confirm whether the effect of the imidazopyridine derivative of the present invention on the anti-fibrosis is effective in vivo and is effective for the treatment or prevention of cirrhosis.

1. Pretreatment of experimental animals

Male BALB / c mice (6 wk, weight 22-26 g) were purchased from Orient Bio. Animal Inc. Seoul, Republic of Korea. All animal experiments were carried out with the approval of the committee in accordance with the provisions of the Animal Experimental Ethics Committee of the Life Science Laboratory, Inha University School of Medicine. All animals used in the experiment were randomly selected to receive standard rat chow and tap water, and maintained at 21 ° C for 12 hours in a dark and bright state. Male mice used in the experiment were randomly divided into 4 groups (control group, CCl ₄ treated group, (CCl ₄ + HS-173 10 mg / Kg) treated group, (CCl ₄ + HS-173 20 mg / Kg treated group) Divided into nine groups for each group. To induce acute liver injury, 150 μl of 40% CCl ₄ was dissolved in corn oil and injected intraperitoneally twice a week for 4 weeks. Control animals were injected with the same amount of corn oil only. HS-173 was orally administered to animals of each group for 6 days a week at a dose of 10 or 20 mg / Kg for one month when CCl ₄ was administered as above. For oral administration, HS-173 was dissolved in DMSO (DMSA: PEG 400: DW = 1: 5: 4). Liver samples obtained from the above procedure were immediately fixed in 10% neutral buffered formalin to perform histochemical studies, and blood samples for biochemical analysis were collected from the hearts of the above experimental animals.

2. Treatment or preventive effect of liver cirrhosis

In order to confirm the therapeutic effect of the imidazopyridine derivative of the present invention on liver cirrhosis, an liver sample was taken from the animal model treated as described above, and H & E staining and MT staining were performed. Histopathological evaluation was performed, And ALT and AST concentrations were measured. The results are shown in Fig.

As shown in Fig. 5, in the animal model inducing hepatic fibrosis with carbon tetrachloride, broad and severe hemorrhagic cell necrosis, collapse of cellular tissue structure and infiltration of inflammatory cells were observed unlike in the control group. In contrast, HS-173 (10 mg / kg, 20 mg / Kg) treated liver samples showed decreased fibrosis through collagen MT staining. In particular, the group treated with 20 mg / Kg HS-173 showed a marked reduction in the area of fibrosis. In addition, the animals with acute hepatic injury showed an increase in ALT and AST levels, but all of them were decreased due to administration of HS-173.

Therefore, it can be seen that the imidazopyridine derivative of the present invention is excellent in the anti-fibrosing effect, and thus is effective for the treatment or prevention of cirrhosis.

Example 6. Measurement of Inhibitory Effect of Imidazopyridine Derivatives of the Present Invention on In Vitro Extracellular Matrix (ECM) Protein Accumulation and PI3K / Akt Signaling System

Immunohistochemical staining analysis was performed to confirm the inhibitory effect of the imazapyridine derivative of the present invention on the extracellular matrix (ECM) protein accumulation inhibition and the PI3K / Akt signaling system.

Specifically, immunohistochemical staining was performed on liver cell samples collected from each group of the animal models prepared in Example 5-1. (Sigma-Aldrich), α-SMA, vimentin (Sigma-Aldrich), MMP-2 (Santa Cruz Biotechnology), p-Akt, and p-P70S6K (Cell Signaling Technology) in TGF-β, collagen I and TIMP-1 Specific primary antibodies were used and after washing the unbound primary antibody, the hepatic sample fractions were incubated in a 1.5% horse serum PBS for 20 min at 37 ° C in the dark with fluorescently labeled secondary antibody. DAPI was used for nuclear staining. Slides were washed twice with PBS, covered with DABCO (Sigma-Aldrich) and observed with confocal laser scanning microscopy. The results are shown in Figs. 6, 7, and 8. Fig.

As shown in FIG. 6, the expression of α-SMA and type 1 collagen was increased in the animal model inducing acute liver injury as in Example 5, 1. However, HS-173 treatment In animal models, the expression of α-SMA and type 1 collagen was significantly decreased. In addition, in the HS-173-treated animal model, the regulation of metabolic turnover of the extracellular matrix (ECM) mediates the proliferation of hepatic stellate cells, resulting in the expression of MMP-2, And the expression of TIMP-1, which is a marker of TIMP-1, is reduced. FIG. 7 shows inhibition of phosphorylation of Akt and P70S6K by the HS-173 of the present invention and inhibits the activity of the PI3K / Akt signaling system in vivo. In FIG. 8, HS- The expression of p-Akt, p-P70S6K, which promotes hepatic fibrosis, was decreased and the expression of type 1 collagen and visentin was also decreased.

Therefore, it can be seen that the antifibrinolytic effect of the imidazopyridine derivative of the present invention is effective in vivo and is effective for the treatment or prevention of cirrhosis.

Formulation Example 1. Preparation of pharmaceutical preparations

1. Manufacturing of powder

20 mg of an imidazopyridine derivative of the formula (1)

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

10 mg of an imidazopyridine derivative of the formula (1)

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

10 mg of an imidazopyridine derivative of the formula (1)

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

10 mg of an imidazopyridine derivative of the formula (1)

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

20 mg of an imidazopyridine derivative of the formula (1)

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Claims (4)

A pharmaceutical composition for preventing or treating liver cirrhosis, which comprises, as an active ingredient, an imidazopyridine derivative represented by the following formula (1) having phosphotidylinostiol-3-kinase (PI3K) inhibitory activity;
[Chemical Formula 1]

In Formula 1, X is carbon or nitrogen,
R1 is hydrogen or a C1-C6 alkyl group; An acetyl group; Pyrazole group; A morpholine group; - (C = O) O- (C1-C6 alkyl); Cyano; Tetrazole group; An oxadiazole group; Or a C6-C30 aryl group or a C6-C30 heteroaryl group substituted or unsubstituted with a cyano group,
R2 is a C6-C30 aryl group or a C6-C30 heteroaryl group which is substituted or unsubstituted with at least one member selected from the group consisting of an amine group, an arylsulfonamide group and a morpholinosulfonyl group. 2. The compound according to claim 1, wherein R < 1 >

And at least one member selected from the group consisting of
R2 is

, Wherein the pharmaceutical composition is at least one selected from the group consisting of:
2. The process according to claim 1, wherein the imidazopyridine derivative of formula (1)
1) N- (5- (3- (5-methyl-1,2,4-oxadiazol-3-yl) imidazo [1,2- a] pyridin- Sulfonamide;
2) N- (5- (3-Cyanoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
3) N- (5- (3-morpholinoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
4) N- (5- (3-acetylimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
5) N- (5- (3-acetylimidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide;
6) 5- (3-morpholinoimidazo [1,2-a] pyridin-6-yl) -3- (morpholinosulfonyl) pyridin-2-amine;
7) 3,5-Difluoro-N- (5- (3- (5-methyl-1,2,4-oxadiazol- Yl) pyridin-3-yl) benzenesulfonamide;
8) 2,4-Difluoro-N- (5- (3- (5-methyl-1,2,4-oxadiazol-3- yl) imidazo [ Yl) pyridin-3-yl) benzenesulfonamide;
9) N- (5- (3- (3-cyanophenyl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
10) N- (5- (3- (2H-tetrazol-5-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
11) N- (5- (3- (Pyridin-3-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
12) N- (5- (3- (Pyridin-4-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
13) 3- (imidazo [1,2-a] pyridin-6-yl) aniline;
14) 5- (Imidazo [l, 2-a] pyridin-6-yl) pyridin-3-amine;
15) N- (5- (imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
16) ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine-3-carboxylate;
17) 6- (6-Amino-5- (morpholinosulfonyl) pyridin-3- yl) imidazo [1,2- a] pyridine-
3-carbonitrile;
18) N- (5- (3- (1H-pyrazol-4-yl) imidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) benzenesulfonamide;
19) N- (5- (3-Cyanoimidazo [1,2-a] pyridin-6-yl) pyridin-3-yl) -2,4-difluorobenzenesulfonamide;
20) N- (5- (3- (2H-tetrazol-5-yl) imidazo [1,2- a] pyridin-6-yl) pyridin- Sulfonamide;
21) N- (5- (3-Acetylimidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide;
22) N- (5- (Imidazo [1,2-a] pyrimidin-6-yl) pyridin-3-yl) benzenesulfonamide; And
23) A compound selected from the group consisting of ethyl 6- (5- (2,4-difluorophenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- Or more of an imidazopyridine derivative as an active ingredient in the preparation of a medicament for preventing or treating cirrhosis.
4. The compound according to claim 3, wherein the imidazopyridine derivative is ethyl 6- (5- (phenylsulfonamido) pyridin-3-yl) imidazo [1,2-a] pyridine- ≪ / RTI > for the prevention or treatment of cirrhosis.

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