KR20170084712A - HNF4-α antagonist and Use Thereof - Google Patents

Abstract

The present invention relates to HNF4-alpha (hepatocyte nuclear factor 4 alpha) antagonist and its use, and relates to the HNF4-alpha (hepatocyte nuclear factor 4 alpha) antagonist of the present invention and its use. The antagonist specifically binds to the ligand binding domain of HNF4-? To inhibit the activity of HNF4- ?. The HNF4-alpha antagonist of the present invention significantly inhibits the expression of Wnt5a compared to the known HNF4- And can inhibit the growth of stomach cancer cells. Therefore, it can be used not only as a pharmaceutical composition for cancer prevention and treatment or as a health functional food, but also for the treatment or prevention of diseases caused by overexpression of HNF4- Can be applied as a composition.

Description

HNF4-alpha antagonists and their uses {HNF4-alpha antagonist and Use Thereof}

The present invention relates to HNF4-alpha (hepatocyte nuclear factor 4 alpha) antagonist and its use, and more particularly to an antagonist which specifically binds to a ligand binding domain of HNF4-? To inhibit the activity of HNF4-? -A antagonist as an effective ingredient.

Hepatocyte nuclear factor 4 alpha (HNF4-alpha) is a hepatocyte nuclear factor, a DNA-binding protein present in the nucleus, and is known to be a transcription factor that regulates liver-specific gene expression. In addition, HNF4-a is a transcription factor belonging to the steroid receptor family and activates the HNF1-a gene (Sladek, FM, et al., Genes Dev., 4: 2353, 1990). Previous studies have reported that HNF4-? Activates transcription of the target gene in the absence of exogenous ligand, but afterwards specific fatty acid acyl-CoA has been studied to activate HNF4- ?, suggesting that HNF4-? (Hertz, R., et al., Nature , 392: 512, 1998).

In addition, HNF4-α is a gene related to lipid transport such as MTP (microsomal triglyceride transfer protein), apoB (apolipoprotein B) and apoCIII (apolipoprotein CIII), or a peptide such as PEPCK (phosphenolpyruvate carboxykinase) and G6Pase (glucose-6-phosphatase) It is known to control the expression of genes involved in glucose metabolism and it is expressed in the kidney and small intestine as well as in the liver. It is widely used for the metabolism and maintenance of the homeostasis of the body such as the synthesis, transport and secretion of fatty acids, .

The HNF4-α not only regulates the transcription of genes necessary for cholesterol, fatty acid and glucose metabolism and hepatic differentiation (Jian G et al., Molecular and cell biology, 15: 5131, 1995), and the representative drug metabolizing enzyme CYP2D6 it is known to regulate transcription of CYP2B6 (cytochrome P450 2D6), CYP3A4 (cytochrome P450 3A4) and CYP2C9 (cytochrome P450 2D6) (William C et al., The Journal of Biological Chemistry , 271 (41) : 25269, 1996).

In fact, mice lacking liver-specific HNF4-a have been shown to decrease the expression of MTP and apo protein in the liver, decrease serum cholesterol and serum triglyceride, and lose weight (Hayhurst, GP, et al, Mol Cel Biol, 21 :.... 1393, 2001). Therefore, it has been presumed that inhibition of the activity of HNF4-a has lipid metabolism control ability and glucose metabolism control ability, and can be usefully used as a preventive or remedy for hypertriglyceridemia, fatty liver and diabetes.

In the prior art, the transcriptional activity of HNF4-a has been reported to be inhibited by a small heterodimer partner (SHP), one of the target genes of FXR (farnesoid X receptor) (Lee, Y., et al., Mol . Cell Biol . , 20: 187, 2000), long chain fatty acid acyl CoA (palmitoyl CoA, millipyridyl-CoA, dodecanoyl-CoA, stearoyl- CoA, (Nature, 392: 512, 1998) have been reported in the present invention.

Japanese Patent Laid-Open Publication No. 2008-133247 reports a nitrogensistin, which is an inhibitor of HNF4-alpha activity, in order to prevent or ameliorate fatty liver or diabetes. WO2002-024227 discloses that HNF4α protein is overexpressed in colorectal cancer tissues, and HNF4α Inhibition of the expression of the protein has been reported to inhibit tumor cell proliferation. In US Patent Publication No. 2010-0286220, a compound BIM5078, which is an HNF4-alpha antagonist, has been described.

On the other hand, gastric cancer is gastric adenocarcinoma, which is usually found in gastric cancer and submucosal layer of stomach epithelium, muscle sarcoma, and interstitial tumor.

Gastric adenocarcinoma is the fourth most common cancer in the world, the second most common cause of cancer death in 2000 (700,349 deaths). Is considered a single heterogeneous disorder with several epidemiological and histopathologic features. Gastric cancer treatment is based mainly on clinical parameters such as tumor, node, metastasis (TNM) staging, which determines whether patients should be treated surgically or by surgery and chemotherapy. Unlike breast cancer and colorectal cancer, stomach cancer differs markedly from stage 1 to stage 4 according to the TNM stage system. That is, the 5-year survival rate is more than 90% in case 1 and less than 20% in case 4. Gastric cancer based on the above-described staging system can be divided into Early Gastric Cancer, Locally Advanced Gastric Cancer, Locally Advanced Invasive Gastric Cancer, and Metastatic Gastric Cancer .

The inventors of the present invention confirmed that HNF4-α was overexpressed in early stomach cancer and increased the expression of Wnt5a through a previous study (Hae Ryung Chang et al., Gut. , 0: 1, 2014) It was confirmed that inhibition of HNF4-alpha gene expression or treatment of HNF4-alpha antagonist can inhibit tumor formation by controlling Wnt signal.

Accordingly, the inventors of the present invention have made intensive efforts to select HNF4-alpha antagonists for the prevention and treatment of gastric cancer. As a result, they have found that a compound that specifically binds to the ligand binding domain of HNF4- . It was confirmed that the selected compound specifically reduced the expression of Wnt5a and markedly inhibited the growth of stomach cancer cells, thus completing the present invention.

DISCLOSURE OF THE INVENTION The present invention has been conceived to solve the above-mentioned problems, and it is an object of the present invention to provide an HNF4-alpha antagonist specifically binding to a ligand binding domain of HNF4- And to provide a therapeutic pharmaceutical composition.

In order to solve the above-described problems of the present invention, the present invention provides a compound represented by the following general formula (1) or (2): And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist comprising one or more selected from the group consisting of:

[Chemical Formula 1]

(2)

Each of R ₁ to R ₅ independently represents a hydrogen atom (H), an oxygen atom (O), a nitro group (NO ₂ ), a halogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms An alkyl group or an alkyl group having 1 to 6 carbon atoms.

According to a preferred embodiment of the present invention, the formula (1) may be represented by the following formula (3)

(3)

The formula (2) may be represented by the following formula (4).

[Chemical Formula 4]

According to another preferred embodiment of the present invention, the above-mentioned Formulas (1) and (2) can specifically bind to the ligand binding domain of HNF4- ?.

The present invention also relates to a composition comprising at least one compound selected from the group consisting of the following formulas (5) and (5-1) to (5-7) And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

[Chemical Formula 5]

[Formula 5-1] [Formula 5-2]

[Formula 5-3] [Formula 5-4]

[Formula 5-5] [Formula 5-6]

[Formula 5-7]

The present invention also relates to a composition comprising at least one compound selected from the group consisting of the following formulas (6) and (6-1) to (6-10) as derivatives thereof; And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

[Chemical Formula 6]

[Formula 6-1] [Formula 6-2]

[Formula 6-3] [Formula 6-4]

[Formula 6-5] [Formula 6-6]

[Formula 6-7] [Formula 6-8]

[Formula 6-9] [Chemical Formula 6-10]

The present invention also provides a compound represented by the following formula (7) and derivatives thereof represented by the following formulas (7-1) to (7-5): And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

(7)

[Formula 7-1]

[Formula 7-2]

[Formula 7-3]

[Chemical Formula 7-4]

[Formula 7-5]

The present invention also relates to a composition comprising at least one compound selected from the group consisting of the following formula (8) and derivatives thereof (8-1); And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

[Chemical Formula 8]

[Formula 8-1]

 The present invention also includes a pharmaceutical composition for preventing and treating cancer comprising the HNF4-alpha antagonist as an active ingredient, and includes a health functional food for cancer prevention and improvement comprising the HNF4-alpha antagonist as an active ingredient .

According to a preferred embodiment of the present invention, the HNF4-alpha antagonist may inhibit HNF4-a activity and decrease wnt5a (Wingless-type MMTV integration site family, member 5A) expression.

According to a preferred embodiment of the present invention, the cancer may be at least one selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, cervical cancer, and liver cancer.

The HNF4-alpha antagonist selected in the present invention specifically binds to the ligand binding domain of HNF4- alpha and thus inhibits the activity of HNF4- alpha And the HNF4-alpha antagonist of the present invention can remarkably reduce the expression of Wnt5a significantly and inhibit the growth of stomach cancer cells as compared with the HNF4-alpha antagonist known in the art, Can be applied not only to pharmaceutical compositions or health functional foods, but also to compositions for treating or preventing diseases caused by HNF4-a over-expression.

Brief Description of the Drawings Fig. 1 is a structural diagram (A) of HNF4-a and a schematic diagram (B) showing diseases that can be caused by over-expression of HNF4-a.
Fig. 2 is a schematic diagram showing the binding between the basic structure of naphthofuran and myristic acid and the ligand binding domain of HNF4-a.
FIG. 3 is a data showing simulation of the binding of the compound represented by Formulas 3 and 4, which is HNF4-α antagonists, and BIM5078, which is a positive control, to the ligand binding domain of HNF4-α.
Fig. 4 is data showing the change in energy when the compound represented by formulas 3 and 4, which are HNF4-alpha antagonists, and BIM5078, a positive control, bind to the ligand binding domain of HNF4- ?.
FIG. 5 is data showing the energy change when the HNF4-.alpha. Antagonist represented by the general formula (5) and its derivatives represented by the general formulas (5-1) to (5-7) binds to the ligand binding domain of HNF4- ?.
FIG. 6 is a simulation data showing the binding of the HNF4-.alpha. Antagonist represented by the formula (5) and its derivatives (5-1 to 5-7) to the ligand binding domain of HNF4-.
Fig. 7 is data showing the energy change when binding to the ligand binding domain of HNF4-a represented by the general formula (6) and derivatives thereof represented by the general formulas (6-1) to (6-10).
FIG. 8 is a simulation data showing the binding of the HNF4-alpha antagonist represented by the formula (6) and its derivatives (6-1 to 6-10) to the ligand binding domain of HNF4-a.
9 is data showing energy changes when the HNF4-alpha antagonist represented by the general formula (7) and derivatives thereof represented by the general formulas (7-1) to (7-5) binds to the ligand binding domain of HNF4- ?.
FIG. 10 is a data showing simulation of the binding of the HNF4-alpha antagonist represented by the formula (7) and its derivatives (7-1 to 7-5) to the ligand binding domain of HNF4-a.
Fig. 11 is a graph showing data on the energy change and binding of the HNF4-alpha antagonist of formula (8-1), which is represented by formula (8) and its derivative, when bound to the ligand binding domain of HNF4-a.
12 is a schematic diagram showing HNF4-a and WNT pathways.
FIG. 13 is a graph showing the cancer cell killing effect when the compound represented by the general formula (3) is treated in a gastric cancer cell line.

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

As described above, HNF4-alpha antagonists that inhibit the activity of HNF4-alpha have been studied for the treatment of various diseases caused by HNF4-alpha overexpression in the prior art. (Hae Ryung Chang et al., Gut. , 0: 1, 2014), inhibiting the activity of HNF4-a overexpressed in early stomach cancer can inhibit tumor formation by controlling Wnt signal, HNF4-α antagonist for the prevention and treatment of gastric cancer.

Accordingly, the present inventors sought to solve the above-mentioned problem by providing an HNF4-α antagonist which specifically binds to the ligand binding domain of HNF4-α and inhibits the activity of HNF4-α. HNF4- , The expression of Wnt5a can be markedly reduced and the growth of stomach cancer cells can be suppressed compared with HNF4-alpha antagonist known in the art. Therefore, the pharmaceutical composition for prevention and treatment of cancer including the HNF4-alpha antagonist or the health functional food Can be provided.

Accordingly, the present invention relates to a composition comprising at least one compound selected from the group consisting of the following formulas (1) and (2); And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist comprising one or more selected from the group consisting of:

[Chemical Formula 1]

(2)

Each of R ₁ to R ₅ independently represents a hydrogen atom (H), an oxygen atom (O), a nitro group (NO ₂ ), a halogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms An alkyl group or an alkyl group having 1 to 6 carbon atoms.

The halogen atom may be, for example, at least one atom selected from the group consisting of bromine (Br), chlorine (Cl), fluorine (F) and iodine (I) As saturated straight chain or branched hydrocarbons containing 6 carbon atoms, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec- ≪ / RTI >

The above-mentioned "alkyl group having 1 to 6 carbon atoms which may be substituted with the same or different 1 to 6 halogen atoms" includes, in addition to the above "alkyl group having 1 to 6 carbon atoms", for example, trifluoromethyl group, trichloromethyl group, difluoromethyl group , Dichloromethyl, dibromomethyl, fluoromethyl, chloromethyl, bromomethyl, iodomethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, 2-bromoethyl Fluoroethyl group, 3-fluoroethyl group, 3-chloropropyl group, 3,3,3-trifluoropropyl group, 4-fluorobutyl group, 3-fluoro- 3-trifluoro-2-methylpropyl group, 6,6,6-trichlorohexyl group, and the above-mentioned "alkyl group having 1 to 6 carbon atoms which is substituted by 1 to 3 of the above" halogen atoms " Quot ;, preferably one to three of the above "halogen atoms" Quot; alkyl group having 1 to 3 carbon atoms "which may be substituted, more preferably 1 to 3 " alkyl group having 1 to 3 carbon atoms " which may be substituted by the same or different 1 to 3" fluorine atom or chlorine atom " More preferably a methyl group, an ethyl group, a propyl group, a chloromethyl group or a trifluoromethyl group, particularly preferably a methyl group, an ethyl group or a trifluoromethyl group.

In the above formula (1), NO ₂ may be located in R ₂ or R ₄ , or a halogen atom may be located in R ₃ , and more preferably, it may be represented by the following formula (3).

(3)

Formula 2 is preferably, in R ₃ NO ₂ is located, or, R may be the same or a C 1 -C 6 alkyl group where different 1 to 6 halogen atoms may be substituted in the ₂ or R _4, and more preferably May be represented by the following formula (4).

[Chemical Formula 4]

In the present invention, the HNF4-alpha antagonist represented by the above-mentioned formulas (1) and (2) or the HNF4-alpha antagonist represented by the above formulas (3) and (4) can specifically bind to the ligand binding domain of HNF4- The activity of HNF4-a can be inhibited.

In the present invention, "HNF4-alpha (Hepatocyte nuclear factor 4 alpha)" is known as a DNA binding protein present in the nucleus. 1A is a schematic diagram showing the structure of HNF4-alpha protein. The HNF4-alpha protein is composed of DNA binding domain, ligand binding domain and transactivation domain, and HNF4- -α antagonist specifically binds to the ligand binding domain of HNF4-a, thereby inhibiting HNF4-a activity.

FIG. 1B is a schematic diagram showing a disease that may be caused by overexpression of HNF4-alpha, which is caused by HNF4-alpha, which is involved in diabetes, atherosclerosis, hemophilia, thrombosis, hypoxia, It is known to cause diseases such as MCAD deficiency (medium chain acyl-CoA dehydrogenase deficiency), OTP deficiency (Ornithine transcarbamylase deficiency), and cancer development due to hepatitis B virus (HBV).

The inventors of the present invention found that 1) inhibition of HNF4-α expression using shRNA, 2) addition of AMPK activating substance (metformin) in a conventional study (Hae Ryung Chang et al., Gut. , 0: Inhibition of HNF4-? Activity and 3) induction of HNF4-? Activity inhibition using BIM5078 (US Patent Publication No. 2010-0286220), which is known as HNF4-α antagonist, And to select more effective HNF4-alpha antagonists in the present invention.

In one embodiment of the present invention, 2648 and 2648 bases, respectively, based on Naphthofuran and Myristic Acid, which are known to specifically bind HNF4-a by binding to the ligand binding domain of HNF4- 9269 compounds were prepared.

Fig. 2 is a schematic diagram showing the basic structure of Naphthofuran and Myristic Acid and binding to the ligand binding domain of HNF4- [alpha], and the ligand of HNF4- alpha of naphthofuran and myristic acid The binding domain binding energies are -6.8 and -5.9, respectively. The larger the "-" value, the stronger the binding energy.

In the present invention, in order to elicit a substance capable of specifically binding to the ligand binding domain of HNF4-? In the compound synthesized using naphthofuran and myristic acid as the basic structures, 1 to 40 compounds, which are HNF4-alpha antagonist candidates that are capable of specifically binding to the ligand binding domain of HNF4-a, were firstly screened using Computer-Aided Drug Design (CADD).

As shown in Tables 2 to 5, all of the first-selected compounds specifically bind to the ligand binding domain of HNF4-? Compared to the basic structures of naphthofuran and myristic acid, and candidate compounds 3 and 4 HNF4-alpha antagonist represented by the above formula (1) was derived based on the candidate substances 11, 17, and 18, and the HNF4-alpha antagonist represented by the above formula (2) Respectively.

In one embodiment of the present invention, the candidate compound 3 represented by Formula 3 and the compound represented by Formula 4, which is the candidate compound 11, are preferentially selected from among the candidates selected first, and BIM5078 (hereinafter referred to as HNF4- U.S. Patent Publication No. 2010-0286220) and a 4IQR (HNF4-α) registered in the Protein Data Bank (PDB, http://www.wwpdb.org/).

FIG. 3 shows a simulation of binding of the compounds represented by formulas (3) and (4) as HNF4-α antagonists and BIM5078 as a positive control to the ligand binding domain of HNF4-α. Compounds represented by formulas (3) and Binding domain of HNF4- [alpha], which means that the compounds can be used as HNF4-alpha antagonists.

Further, as a result of measuring the energy change when the compounds were bound to the ligand binding domain of HNF4-a, it was confirmed that the binding ability was superior to that of the positive control group BIM5078 as shown in Fig.

The present invention also relates to a composition comprising at least one compound selected from the group consisting of the following formulas (5) and (5-1) to (5-7) And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

[Chemical Formula 5]

[Formula 5-1] [Formula 5-2]

[Formula 5-3] [Formula 5-4]

[Formula 5-5] [Formula 5-6]

[Formula 5-7]

In one embodiment of the present invention, the compounds of formulas (5) and (5-1) to (5-7) as derivatives thereof are further selected and synthesized. Of the HNF4-alpha antagonist is bound to the ligand binding domain of HNF4- [alpha], wherein the HNF4-alpha antagonist is represented by the formula (5) and derivatives thereof represented by the following formulas Was specifically bound to the ligand binding domain of HNF4-a.

The present invention also relates to a composition comprising at least one compound selected from the group consisting of the following formulas (6) and (6-1) to (6-10) as derivatives thereof; And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

[Chemical Formula 6]

[Formula 6-1] [Formula 6-2]

[Formula 6-3] [Formula 6-4]

[Formula 6-5] [Formula 6-6]

[Formula 6-7] [Formula 6-8]

[Formula 6-9] [Chemical Formula 6-10]

In one embodiment of the present invention, the compounds of formulas (6) and (6-1) to (6-10) as derivatives thereof are further selected and synthesized, and FIGS. 7 and 8 show Formulas (6-1) to Of the HNF4-alpha antagonist to the ligand binding domain of HNF4- [alpha], wherein the HNF4-alpha antagonist is represented by the formula (6) and derivatives thereof represented by the following formulas (6-1) to Was specifically bound to the ligand binding domain of HNF4-a.

The present invention also provides a compound represented by the following formula (7) and derivatives thereof represented by the following formulas (7-1) to (7-5): And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

(7)

[Formula 7-1]

[Formula 7-2]

[Formula 7-3]

[Chemical Formula 7-4]

[Formula 7-5]

In one embodiment of the present invention, the compound of the formula (7) and the derivatives thereof (7-1 to 7-5), which are derivatives thereof, were further selected and synthesized. Of the HNF4- [alpha] antagonist is bound to the ligand binding domain of HNF4- [alpha], and the binding of the HNF4-alpha antagonist to the ligand binding domain of HNF4- Was specifically bound to the ligand binding domain of HNF4-a.

The present invention also relates to a composition comprising at least one compound selected from the group consisting of the following formula (8) and derivatives thereof (8-1); And salts thereof; (Hepatocyte nuclear factor 4 alpha) antagonist including at least one selected from the group consisting of HNF4-?

[Chemical Formula 8]

[Formula 8-1]

In an embodiment of the present invention, the formula (8) and its derivative (8-1) are further selectively synthesized, and FIG. 11 is a graph showing the relationship between the HNF4-α antagonist of formula (8) Binding domain of the HNF4- [alpha] -adrenergic receptor, the binding of the HNF4-alpha antagonist to the ligand binding domain of HNF4- [alpha] Respectively.

The present invention also includes a pharmaceutical composition for preventing and treating cancer comprising the HNF4-alpha antagonist as an active ingredient, and includes a health functional food for cancer prevention and improvement comprising the HNF4-alpha antagonist as an active ingredient .

The HNF4-alpha antagonist may inhibit HNF4-a activity and decrease wnt5a (Wingless-type MMTV integration site family, member 5A) expression.

As shown in FIG. 12, HNF4-alpha binds to the promoter portion of the wnt5a (Wingless-type MMTV integration site family, member 5A) gene and promotes the expression of wnt5a. HNF4-alpha antagonist binds ligand binding of HNF4- Domain specifically binds to HNF4- [alpha] to inhibit binding of the promoter portion of the wnt5a gene, resulting in a decrease in wnt5a expression, thereby regulating the wnt signal.

In the present invention, the cancer may be at least one selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, cervical cancer, and liver cancer, and preferably gastric cancer.

As shown in FIG. 13, when the HNF4-alpha antagonist of Chemical Formula 3 was treated to the gastric cancer cell line, the growth of gastric cancer cells was effectively inhibited. Induced gastric cancer deaths and killed half of gastric cancer cell lines at 50 μM compared to the control group.

In one embodiment of the present invention, in order to confirm whether the HNF4-alpha antagonist selected in the present invention can be used as a preventive or therapeutic agent for gastric cancer, HNF4-alpha antagonist was treated with various gastric cancer cell lines, , And the expression of wnt5a in each of the cells treated with HNF4-alpha antagonist was markedly decreased.

For in vivo chemotherapy, HNF4-α antagonist was injected orally or directly into tumor tissues after inducing tumor formation by grafting gastric cancer cells to the side of mice lacking immunity. It was confirmed that the size of tumor cells was remarkably reduced as compared with the negative control group not treated with HNF4-alpha antagonist.

Thus, it was confirmed that the HNF4-alpha antagonist selected in the present invention specifically binds to the ligand binding domain of HNF4-alpha and thus inhibits the activity of HNF4- alpha. The HNF4-alpha antagonist of the present invention inhibits HNF4- alpha antagonist, the expression of Wnt5a can be markedly reduced and the growth of stomach cancer cells can be inhibited. Therefore, it has been confirmed that the composition can be used as a pharmaceutical composition or health functional food for cancer prevention and treatment.

In addition, the present invention can be applied as a composition for treating or preventing a disease caused by overexpression of HNF4-alpha antagonist HNF4-alpha selected in the present invention.

The pharmaceutical composition for preventing or treating cancer comprising the HNF4-alpha antagonist according to the present invention as an active ingredient and the health functional food for cancer prevention or improvement may further include other natural substances or compounds having an anticancer effect.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. In addition, the pharmaceutical composition for preventing or treating cancer comprising the HNF4-alpha antagonist according to the present invention as an active ingredient can be formulated into various formulations. In the case of formulation, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, and capsules, which may contain at least one excipient (e.g. starch, sucrose, lactose and gelatin) in an HNF4- Etc. can be prepared. In addition to simple excipients, lubricants can also be used. Examples of liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, glycerol, gelatin and the like may be used.

The dosage of the pharmaceutical composition may be varied depending on the age, sex, condition of the subject, the degree of absorption of the active ingredient in the body, the rate of inactivation and the rate of excretion, and the drugs used in combination.

There is no particular limitation on the kind of the health functional food for cancer prevention and improvement which comprises the HNF4-alpha antagonist of the present invention as an active ingredient, and examples thereof include meat, sausage, bread, chocolate, candy, snack, confection, pizza, Noodles, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes.

 The health food is used together with other food or food additives other than the HNF4-alpha antagonist, and can be suitably used according to a conventional method. For example, in order to prevent and improve cancers containing the HNF4-alpha antagonist as an active ingredient, HNF4-alpha is contained as an active ingredient, and calcium, a thick liquid, fructose, purified water, , Sterilized and then cooled to room temperature to prepare a beverage. In addition, the health supplement for cancer prevention and improvement containing the HNF4-alpha antagonist as an active ingredient may be added to the HNF4-alpha antagonist as a nutritional supplement component (vitamins B1, B2, B5, B6, E and acetic acid ester, nicotinamide) 50% ethanol and purified water were added and mixed to form granules. The granules were dried in a vacuum drier and then passed through 12 to 14 mesh to prepare uniform granules. The granules were uniformly extruded to prepare tablets or powders or hard capsules To form a hard capsule product.

 The effective dose of the HNF4-alpha antagonist contained in the health food can be used in accordance with the effective dose of the pharmaceutical composition, and the amount of the active ingredient to be mixed can be suitably determined according to the purpose of use such as preventive or therapeutic treatment. In the case of long-term consumption intended for health and hygiene purposes or for health control purposes, it may be below the above range.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Synthesis of ligand binding domain-binding substances of HNF4 -a and screening of donor substances after HNF4 -a antagonist

In the present invention, a compound having a chemical structure similar to that of Naphthofuran and Myristic Acid, which specifically binds to the ligand binding domain of HNF4-a and is known to regulate HNF4-a, is called pubchem (46 million compounds were stored), and compounds with a Tanimoto score of greater than 0.4 were selected that select the similarity of the chemical structure. As a result of screening, 2648 and 9269 compounds having a chemical structure similar to that of Naphthofuran and Myristic Acid were respectively selected.

In order to derive a substance capable of specifically binding to the ligand binding domain of HNF4-? In a compound synthesized with Naphthofuran and Myristic Acid as a basic structure, a computer-aided drug (CADD) Design was used and a binding simulation with HNF4-α 3FS1 registered in the Protein Data Bank (PDB, http://www.wwpdb.org/) was performed according to the manual, The docking energy was measured.

As shown in FIG. 2, it is possible to bind to the basic structure of Naphthofuran and Myristic Acid and the ligand binding domain of HNF4-alpha, and to bind the HNF4-alpha of naphthofuran and myristic acid The ligand binding domain binding energies were found to be -6.8 and -5.9, respectively. The lower the energy value, the more stable binding to the ligand binding domain of HNF4- ?.

In the main filtering, the energy value is lower than that of naphthofuran and myristic acid, and the energy value is similar to that of BIM5078 (US Patent Publication No. 2010-0286220), which is known as HNF4-α antagonist. Compounds with similar size to BIM5078 were selected.

In the second filtering, the compounds meeting the conditions in Table 1 were selected.

Selection criteria for HNF4-α antagonist candidates  Condition range Partition coefficient (log P) -0.4 to +5.6 Molar refractivity 40 to 130 Molecular weight 180 ~ 500 Number of atoms 20 to 70 (including explicit H) Polar surface area <= 140 Å 2 rotatable bond (dihedral angle) 10 or fewer

The selected candidate substances 1 to 20 contain naphthofuran as a basic structure, and candidate substances 21 to 40 contain a basic structure of myristic acid.

HNF4-alpha antagonist candidate substances 1 to 10 Structure (CID) Lowest E Structure (CID) Lowest E

Naphthofuran -6.8 One

23055118 -11.00 6

2404390 -10.00 2

23345854 -11.00 7

35728809 -10.00 3

3649084 -10.40 8

66661464 -10.00 4

2943931 -10.30 9

7935304 -10.00 5

3904673 -10.30 10

3282588 -9.90

HNF4-alpha antagonist candidate substance 11 to 20 Structure (CID) Lowest E Structure (CID) Lowest E 11

2089313 -9.80 16

9839993 -9.70 12

2397577 -9.80 17

1323001 -9.60 13

29617456 -9.80 18

2087412 -9.60 14

26796617 -9.70 19

2438477 -9.60 15

7900920 -9.70 20

52368191 -9.60

HNF4-alpha antagonist candidate substances 21 to 30 Structure (CID) Lowest E Structure (CID) Lowest E

MYR (11005) -5.9 21

59048949 -9.60 26

58493861 -9.40 22

22660511 -9.50 27

22660515 -9.30 23

22660512 -9.50 28

14511605 -9.10 24

44625575 -9.50 29

13280700 -9.00 25

57764746 -9.40 30

22445971 -9.00

HNF4-alpha antagonist candidate substances 31 to 40 Structure (CID) Lowest E Structure (CID) Lowest E 31

19593543 -8.90 36

53783087 -8.70 32

21773206 -8.90 37

17764038 -8.60 33

69919679 -8.90 38

20320744 -8.60 34

22383158 -8.80 39

44625744 -8.60 35

22157196 -8.70 40

59582177 -8.60

As shown in Tables 2 to 5, the docking energy of the first-selected compounds was -11.0 to -9.6 kcal / mol, and compared with the basic structures of naphthofuran and myristic acid, the ligands of HNF4-? HNF4-alpha antagonist represented by the following Formula 1 was derived based on the candidate substances 3, 4, 5, 6, 7, 10, 12 and 14, and candidate substances 11 and 17 And 18, an HNF4-alpha antagonist represented by the following formula 2 was derived.

[Chemical Formula 1]

(2)

Each of R ₁ to R ₅ is a C _1-4 alkyl group substituted with H, NO ₂ , a halogen atom or a halogen atom.

HNF4 -α screening for antagonists

In the present invention, in the first candidate substance selected from the above, the compound having the naphthofuran as a basic structure among the substances having the lowest binding energy, which is the candidate substance 3, and the candidate compound 11, The compound was preferentially screened and submitted to MediGen Co. Ltd and synthesized. A protein information bank (PDB, http: http: //www.ibm.com) was used together with BIM5078 (US Patent Publication No. 2010-0286220) known as HNF4- : //www.wwpdb.org/) and 4IQR (HNF4-α).

(3)

[Chemical Formula 4]

[BIM5078]

As a result, as shown in FIG. 3, it was confirmed that the compounds represented by Chemical Formula 3 and Chemical Formula 4 of the present invention were able to bind to the ligand binding domain of HNF4-α. When the compounds were bound to the ligand binding domain of HNF4- As a result, it was confirmed that BIM5078 showed better binding ability than the positive control group as shown in Fig. This means that the compounds can be used as HNF4-alpha antagonists.

* 215

HNF4 -a antagonist screening and HNF4 Measurement of docking energy with -α

In the present invention, HNF4-? Capable of specifically binding to the ligand binding domain of HNF4-? Was further selected to prepare compounds represented by the following formulas (5) to (8). The compounds represented by formulas (5-1) to (5-7), derivatives represented by formula (5), compounds represented by formulas (6-1) to 7-5 and a compound represented by the formula (8-1), which is a derivative of the formula (8), were further prepared, and the docking energy of each compound with HNF4-a was measured.

[Chemical Formula 5]

* 221 [Formula 5-1] [Formula 5-2]

[Formula 5-3] [Formula 5-4]

[Formula 5-5] [Formula 5-6]

[Formula 5-7]

[Chemical Formula 6]

[Formula 6-1] [Formula 6-2]

[Formula 6-3] [Formula 6-4]

[Formula 6-5] [Formula 6-6]

[Formula 6-7] [Formula 6-8]

[Formula 6-9] [Chemical Formula 6-10]

(7)

[Formula 7-1]

[Formula 7-2]

[Formula 7-3]

[Chemical Formula 7-4]

[Formula 7-5]

(8)

[Formula 8-1]

As a result, as shown in FIG. 5 to FIG. 11, the HNF4-α antagonist was bound to the ligand binding domain of HNF4-.alpha. α-antagonist specifically binds to the ligand binding domain of HNF4-α.

HNF4 Synthesis of -α antagonist

Compounds were synthesized to confirm the effect of the compound of formula (3) (Dibenzo [b, d] furan-2-yl 4-chloro-3-nitrobenzoate) The compound Dibenzo [b, d] furan-2-ol was purchased from Aldrich CRP and the compound 4-Chloro-3-nitrobenzoyl chloride was purchased from Medigen. The two compounds were mixed and treated with TEA and DMAP for one day. The reaction formula of the compound represented by the formula (3) is shown in the following reaction formula (1).

[Reaction Scheme 1]

Thus, it was confirmed that the HNF4-alpha antagonist selected in the present invention specifically binds to the ligand binding domain of HNF4-alpha and thus inhibits the activity of HNF4- alpha. The HNF4-alpha antagonist of the present invention inhibits HNF4- alpha antagonist, the expression of Wnt5a can be markedly reduced and the growth of stomach cancer cells can be inhibited. Therefore, it has been confirmed that the composition can be used as a pharmaceutical composition or health functional food for cancer prevention and treatment.

Gastric cancer cell killing effect

The effect of the compound Dibenzo [b, d] furan-2-yl 4-chloro-3-nitrobenzoate of formula 3 on gastric cancer cell death was confirmed. Three gastric cancer cell lines were used in the experiments: AGS, MKN45, NCI-N87. Each gastric cancer cell line was grown in RPMI medium supplemented with 10% PBS (HyClone, GE Healthcare).

Each of the gastric cancer cell lines was treated with the compound of Formula 3 at concentrations of 0, 20, 40, and 50 μM for 48 hours. The cells were then incubated with the MTS assay solution for 2 hours and then measured. The MTS assay solution was used to analyze cell viability and CellTiter 96 ^® solution (Promega) was used. The experiment of this example was performed three times in a 96 well plate.

The cell growth rate obtained in each experiment was calculated based on the cell survival rate of the negative control group treated with no treatment at the average value of three experiments at 100%. As a result, the results shown in Fig. 13 are shown in Fig. According to the results, the compound represented by the formula (3) was killed in a concentration-dependent manner. At 50 μM, it was found to kill up to half of the gastric cancer cell lines.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, It will be obvious. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (12)

A compound represented by the following formula (2); Or a salt thereof as an active ingredient.
(2)

Each of R ₁ to R ₅ independently represents a hydrogen atom (H), an oxygen atom (O), a nitro group (NO ₂ ), a halogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms An alkyl group or an alkyl group having 1 to 6 carbon atoms.

The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the formula (2) is represented by the following formula (4):
[Chemical Formula 4]

.

2. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the formula (2) specifically binds to a ligand binding domain of HNF4- ?.

At least one compound selected from the group consisting of the following formulas (5) and (5-1) to (5-7) as derivatives thereof; And salts thereof; A pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of:

[Chemical Formula 5]

;


[Formula 5-1] [Formula 5-2]

;

;


[Formula 5-3] [Formula 5-4]

;

;


[Formula 5-5] [Formula 5-6]

;

;


[Formula 5-7]

.

At least one compound selected from the group consisting of the following formulas (6) and (6-1) to (6-10) as derivatives thereof; And salts thereof; A pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of:
[Chemical Formula 6]

;


[Formula 6-1] [Formula 6-2]

;

;


[Formula 6-3] [Formula 6-4]

;

;


[Formula 6-5] [Formula 6-6]

;

;


[Formula 6-7] [Formula 6-8]

;

;


[Chemical Formula 6-9] [Chemical Formula 6-10]

;

.
At least one compound selected from the group consisting of the following formulas (7) and (7-1) to (7-5) as derivatives thereof; And salts thereof; A pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of:
(7)
;

[Formula 7-1]

;

[Formula 7-2]

;

[Formula 7-3]

;

[Chemical Formula 7-4]

;

[Formula 7-5]

.
At least one compound selected from the group consisting of the following formula (8) and derivatives thereof (8-1); And salts thereof; A pharmaceutical composition for preventing or treating cancer comprising at least one selected from the group consisting of:
[Chemical Formula 8]

;

[Formula 8-1]

.
8. The organic electroluminescent device according to any one of claims 1 to 7, wherein at least one of the above-mentioned chemical formula 2, chemical formula 4, chemical formula 5, chemical formula 6, chemical formula 7, chemical formula 8, chemical formula 5-1 to 5-7, chemical formula 6-1 to 6- , The compounds of formulas (7-1) to (7-5) or (8-1) inhibit HNF4-a activity and express wnt5a (Wingless-type MMTV integration site family, member 5A) Composition.
The pharmaceutical composition for preventing or treating cancer according to any one of claims 1 to 7, wherein the cancer is at least one selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, cervical cancer and liver cancer .
7. A compound represented by any of the above formulas (2), (4), (5), (6), (7), (8), (5-1) A health functional food for preventing or ameliorating cancer comprising at least one compound selected from the group consisting of formulas (7-1) to (7-5) and (8-1) as an active ingredient.
11. The method of claim 10,
The compounds represented by Formula 2, Formula 4, Formula 5, Formula 6, Formula 7, Formula 8, Formula 5-1 to 5-7, Formula 6-1 to 6-10, Formula 7-1 to 7-5, Wherein the compound inhibits HNF4-a activity and expresses wnt5a (Wingless-type MMTV integration site family, member 5A).
11. The health functional food for cancer prevention and / or improvement according to claim 10, wherein the cancer is at least one selected from the group consisting of gastric cancer, colon cancer, breast cancer, cervical cancer and liver cancer.

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