KR101418168B1 - Novel tetrahydropyridinol derivative compound and pharmaceutical compositions for prevention or treatment of cancer comprising the same - Google Patents

Abstract

The present invention relates to a novel (novel) tetrahydropyridinol derivative compound and a novel use thereof for treating cancer, and more particularly, to a novel tetrahydropyridinol derivative compound, a pharmaceutical composition for preventing or treating cancer, , And health functional foods. The novel tetrahydropyridinol derivative compounds of the present invention are useful for the down-regulation of β-catenin expression, the upregulation of GSK-3β (glyco-gensynthase kinsase 3-β) cadherin) expression by up-regulating the expression of Wnt / β-catenin signaling inhibits the proliferation and infiltration of cancer cells as well as inhibiting the expression of CDK2 and the expression of p21 and p27, cycle arrest. Therefore, the novel tetrahydropyridinol derivative of the present invention can be used as a health functional food as well as a pharmaceutical such as a pharmacological composition as a substance capable of preventing or treating cancer, and is useful when it is applied to the food industry and the pharmaceutical industry It is expected to be very high. In addition, since the novel tetrahydropyridinol derivative of the present invention does not show cytotoxicity to non-tumorous cells other than cancer cells, the composition of the present invention containing it as an active ingredient has safety advantages even in long-term use.

Description

TECHNICAL FIELD The present invention relates to a novel tetrahydropyridinol derivative compound and a pharmaceutical composition for preventing or treating cancer comprising the same as an effective ingredient.

The present invention relates to a novel (novel) tetrahydropyridinol derivative compound and a novel use thereof for treating cancer, and more particularly, to a novel tetrahydropyridinol derivative compound, a pharmaceutical composition for preventing or treating cancer, , And health functional foods.

In 1982, Roel Nusse and Harold Varmus reported Integrase-1 (Int1), a cancer gene that is activated by breast cancer virus in mice. Several years later, Int1 detected segment polarity (Wg) and the homologue. Wnt is a combination of Wingless and Intergrase-1 names, and many studies have been carried out to date on development and cancer.

Wnt protein is a highly conserved signaling molecule that is secreted in many species and organs during cell fate determination and cell proliferation and embryo development of progenitor cells. Thus, variations in the components of the Wnt gene or the Wnt signal pathway are known to be involved in diseases such as developmental defects or cancer.

The transcription factor that plays a key role in the Wnt signaling pathway is beta-catenin, and beta-catenin is present in the cell membrane in association with cadherin, It is also adjustable. The signal pathway containing beta-catenin is called the canonical pathway, which binds to the lymphocyte-enhancer-binding factor (TCF / LEF) in the canonical pathway and produces cyclin D1 and c- myc, and the like.

The scaffolding proteins APC (adenomatous polyposis coli) and axin (axis inhibition protein), and serine / threosine kinase glycogen synthase kinase 3-beta (hereinafter briefly referred to as Gsk 3-β) And CK1 (casein kinase1), the beta-catenin degrades in the absence of the Wnt signal and maintains a low level of beta-catenin in the cells.

A specific mechanism is that when GSK3-β phosphorylates beta-catenin, beta-transducin-containing protein (Beta TrCP), a ubiquitin ligase, recognizes it and polyubiquitinates beta-catenin, The ubiquitinated beta-catenin is degraded by the proteasome. As a result, beta-catenin fails to enter the nucleus and fails to express carcinogenic genes such as cyclin D1 and c-myc.

Accordingly, various proteins capable of regulating the Wnt signal or genes encoding the Wnt signal can be an important target in the treatment of diseases related to the Wnt signal.

Therefore, the inventors of the present invention synthesized novel tetrahydropyridinol derivative compounds to develop a new therapeutic agent effective for prevention or treatment (improvement) of cancer, and they have been able to down-regulate the expression of β-catenin, Β-catenin signaling through upregulation of expression of β-3β (glyco-gensynthase kinsase 3-β) or upregulation of expression of E-cadherin, thereby inhibiting the proliferation and infiltration of cancer cells The present invention has been completed.

Korean Patent Publication No. 10-2011-0014229 Korean Patent Publication No. 10-2011-0122870

Keana, J. F. W .; Cai, S. X. New reagents for photoaffinity labeling: synthesis and photolysis of functionalized perfluorophenyl azides. J. Org. Chem. 1990, 55, 3640-3647. Kato, S .; Morie, T .; Hino, K .; Kon, T .; Naruto, S .; Yoshida, N .; Karasawa, T .; Matsumoto, J. Novel benzamides as selective and potent gastric prokinetic agents. 1. Synthesis and structure-activity relationships of N - [(2-morpholinyl) alkyl] benzamide. J. Med. Chem., 1990, 33 (5), 1406-1413. Aridoss, G .; Amirthaganesan, S.; Ashok Kumar, N .; Kim, J. T .; Lim, K. T .; Kabilan, S .; Jeong, Y. T. A facile synthesis, antibacterial, and antituberculary studies of some piperidin-4-one and tetrahydropyridine derivatives. Bioorg. Med. Chem. Lett. 2008, 18, 6542-6548. Aridoss, G .; Amirthaganesan, S.; Jeong, Y. T. Synthesis, crystal and antibacterial studies of diversely functionalized tetrahydropyridin-4-ol. Bioorg. Med. Chem. Lett. 2010, 20, 2242-2249.

Accordingly, an object of the present invention is to provide a novel tetrahydropyridinol derivative compound.

Another object of the present invention is to provide a pharmaceutical composition effective for preventing or treating cancer comprising the above-mentioned compound as an active ingredient.

Yet another object of the present invention is to provide a health functional food effective for prevention or improvement of cancer comprising the above-mentioned compound as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a novel compound represented by the following general formula (1).

≪ Formula 1 >

In this formula,

R is CH ₂ Br or CH ₂ Cl.

The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the compound of Chemical Formula 1 as an active ingredient.

In one embodiment of the invention, the cancer is selected from the group consisting of brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial, cytoplasmic, brainstem tumor, head and neck tumor, Nasopharyngeal carcinoma, thymic carcinoma, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, hepatic cancer, nasopharyngeal cancer, nasal carcinoma, nasopharyngeal cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, Cancer of the genital organs, penis (urethral) cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, ovarian cancer, ovarian cancer, Cancer, uterine sarcoma, vaginal cancer, female genital external cancer, and female urethral cancer.

In one embodiment of the present invention, the compounds may be used to control down-regulation of β-catenin expression, upregulation of GSK-3β (glyco-gensynthase kinsase 3-β) expression, E-cadherin, The up-regulation of expression may inhibit the proliferation and infiltration of cancer cells by the wnt / β-catenin signaling mechanism.

In one embodiment of the present invention, the compound inhibits the proliferation of cancer cells by inhibiting the expression of CDK2 and increasing the expression of p21 and p27 by a cell cycle arrest mechanism of the cancer cells.

In one embodiment of the present invention, the compound may be included in the composition at a concentration of 1 to 1000 μM.

The present invention also provides a health functional food for preventing or ameliorating cancer comprising the compound of formula (1) as an active ingredient.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ram noodles, gums, candy, ice cream, alcoholic beverages, .

The novel tetrahydropyridinol derivative compounds of the present invention are useful for the down-regulation of β-catenin expression, the upregulation of GSK-3β (glyco-gensynthase kinsase 3-β) cadherin expression by inhibiting wnt / β-catenin signaling by inhibiting the proliferation and infiltration of cancer cells as well as inhibiting CDK2 expression and cell cycle arrest of cancer cells by increasing expression of p21 and p27 cycle arrest. Therefore, the novel tetrahydropyridinol derivative of the present invention can be used as a health functional food as well as a pharmaceutical such as a pharmacological composition as a substance capable of preventing or treating cancer, and is useful when it is applied to the food industry and the pharmaceutical industry It is expected to be very high. In addition, since the novel tetrahydropyridinol derivative of the present invention does not show cytotoxicity to non-tumorous cells other than cancer cells, the composition of the present invention containing it as an active ingredient has safety advantages even in long-term use.

Figure 1 shows the cell viability after treatment of each of the novel compounds (5a, 5b, 5c) prepared in Example 1 by concentration in HEK293, THLE-3, Sk-Hep1, Hep3B and MDA- As shown in FIG.
2A is a graph showing changes in expression of tumor suppressor proteins (p53, p16) and cancer genes (beta-catenin: beta-catenin) over time after treatment of the novel compound of Formula 1 with Sk- Respectively.
FIG. 2B is a graph showing the change in expression of beta-catenin over time after Western blot analysis of the novel compound of formula 1 of the present invention to Hep3B cell line.
FIG. 3 is a graph showing the effect of phospho-β-catenin (Tyr 142) and phospho-β-catenin (Ser 33 / 37) was measured by Western blotting.
FIG. 4 is a graph showing the effect of the combination of DCA (5 μM) and a novel compound of the formula (I) of the present invention in liver cancer cell lines (Sk-Hep1 or Hep3B) with other proteins involved in the beta-catenin signal pathway (E-cadherin: , Axin1: tumor suppressor protein, GSK-3beta: beta-catenin phosphorylase) by Western blotting.
FIG. 5 shows the results of treatment of p53, beta-catenin, E-cadherin, GSK-1, and EGFR-2 after treatment of the novel compound of formula 1 of the present invention with breast cancer cell line (MDA- 3β expression was measured by Western blotting.
6A and 6B show changes in expression of cyclin D1 and c-myc, which are the target genes of beta -catenin, after treatment of DCA (5 μM) with the novel compound of formula 1 of the present invention in liver cancer cell lines (Sk-Hep1 or Hep3B) Were measured by Western blotting.
FIG. 6C shows the expression of cyclin D1 and c-myc, the target genes of beta-catenin, over time after treatment of the novel compound of formula 1 with breast cancer cell line (MDA-MB-231) Respectively.
FIG. 7 is a graph showing the cell cycle of SK-Hep1, Hep3B and MDA-MB-231 treated with the novel compound of formula 1 of the present invention for 12 hours, followed by a cell cycle analysis using a flow cytometer.
FIG. 8 is a graph showing Western blot analysis of changes in expression of proteins (p21, p27, cdk2) related to the cell cycle over time after treating the novel compound of formula (I) of the present invention with Hep3B cells.
FIG. 9 is a graph showing the degree of suppression of invasion of cancer cells by a cell invasive assay after treatment of Sk-Hep1 and MDA-MB-231 cells with the novel compounds of formula (I) according to the present invention.
FIG. 10 shows Western blotting of changes in expression of MMP-2 protein (an important role in basement membrane degradation in cancer invasion) over time after treatment of Sk-Hep1 and MDA-MB-231 cells with the novel compounds of formula 1 of the present invention .

The present invention is characterized by providing a novel tetrahydropyridinol derivative represented by the following general formula (1).

≪ Formula 1 >

In this formula,

R is CH ₂ Br or CH ₂ Cl.

The novel compounds of formula (I) of the present invention can be prepared based on the chemical knowledge of those skilled in the art and do not particularly limit the synthesis method thereof.

) 화합물을 제조하는 단계; (b) 테트라하이드로피리딘 유도체 화합물인

(R= CH₂BR 또는 CH₂Cl)을 제조하는 단계; 및 (c) 상기 (b) 단계의 화합물에 DMAP 또는 NEt3를 첨가하여 교반하여 반응 혼합물을 제조한 후, 여기에 상기 (a) 단계에서 제조한 펜타플루오로벤조일 클로라이드(pentafluorobenzoyl chloride)를 디클로로메탄에 녹인 용액을 첨가하여 혼합시키는 단계; (d) 상기 (c) 단계의 반응이 완료된 용액에서 용매를 제거하고 잔여물을 에틸아세테이트로 녹인 후 세척하는 단계; 및 (e) 상기 (d) 단계를 통해 수득한 유기층을 Na₂SO₄로 건조하고, 증발을 통해 얻어진 잔사는 순수한 물질을 얻기 위해 컬럼(0.5:10 to 1:10, ethyl acetate: hexane)을 이용하여 정제하는 단계를 거쳐 제조할 수 있다(하기 실시예1 참조).In one embodiment of the present invention, the novel compound of Formula 1 is prepared by (a) hydrolyzing methylpentafluorobenzoate with sodium hydroxide, methanol and water to prepare a compound in the form of an acid, (SOCl ₂ ) and dry toluene were added to the mixture, and the mixture was refluxed. Then, the solvent was evaporated to obtain pentafluorobenzoyl chloride

) Compound; (b) a tetrahydropyridine derivative compound

(R = CH ₂ BR or CH ₂ Cl); And (c) adding DMAP or NEt3 to the compound of step (b) and stirring to prepare a reaction mixture, and then pentafluorobenzoyl chloride prepared in step (a) is added to dichloromethane Adding and mixing the dissolved solution; (d) removing the solvent from the solution in which the reaction of step (c) has been completed, dissolving the residue in ethyl acetate, and washing; And (e) drying the organic layer obtained in step (d) with Na ₂ SO ₄ and evaporating the residue to obtain a pure substance (0.5: 10 to 1:10, ethyl acetate: hexane) (See Example 1, below). ≪ tb >< TABLE >

The novel compounds of formula (I) of the present invention may be prepared by pharmaceutically acceptable salts and solvates according to conventional methods in the art.

Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration.

As the free acid, organic acids and inorganic acids can be used. As the inorganic acids, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used. Examples of the organic acids include methanesulfonic acid, p- toluenesulfonic acid, acetic acid, trifluoroacetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycollic acid, gluconic acid glutamic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like can be used.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt in particular, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

The pharmaceutically acceptable salts of the novel compounds of formula (I) above include, unless otherwise indicated, salts of acidic or basic groups which may be present in the compounds. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Mesylate) and p-toluenesulfonate (tosylate) salts, which are known in the art and which are known in the art Can be manufactured through a manufacturing process.

The present inventors synthesized a novel tetrahydropyridinol derivative compound of the above formula (1), and found that the compound thus synthesized has a downregulation of β-catenin expression, GSK-3β (glyco- β-catenin signaling by up-regulating the expression of gensynthase kinsase 3-β or by up-regulating expression of E-cadherin, inhibiting the proliferation and infiltration of cancer cells as well as inhibiting the expression of CDK2 Inhibition and induction of cell cycle arrest of cancer cells through increased expression of p21 and p27.

In the following Experimental Example 1 of the present invention, HEK293 (human embryonic kidney cells), THLE-3 (normal human hepatocytes), Sk-Hep1 (human HCC cells ), Hep3B (human HCC cells) and MDA-MB-231 (human breast cancer cells) were treated with the novel compounds of formula 1 and then the cell viability was measured. As a result, in the non-tumor cell lines HEK293 and THLE- The cytotoxicity was not observed, whereas in the cancer cell line, the cell survival rate was remarkably decreased in dependence on the treatment concentration of the compound of the present invention. From these results, it was confirmed that the novel compound of Formula 1 has cytotoxicity specific to liver cancer and breast cancer (see FIG. 1).

In the following Experimental Example 2 of the present invention, the effect of the novel compound of Formula 1 on tumor suppressor protein (p53, p16) or cancer gene (beta-catenin) As a result, it was confirmed that p53 and p16 expression were not changed in the experimental group treated with the novel compound of formula (I) of the present invention in the Sk-Hep1 cell line, while the protein expression of beta-catenin was inhibited in a time dependent manner 2A). However, the expression of beta-catenin was not down-regulated in the Hep3B cell line, and the expression level was not changed with time (FIG. 2B).

In order to further elucidate whether β-catenin protein down regulation appears to be an inhibition of protein activity in Experimental Example 3 of the present invention, the protein of the phosphorylated form of β-catenin was measured. As a result, Phospho-β-catenin (Tyr 142) expression was decreased and phospho-β-catenin (Ser 33/37) expression was increased in the experimental group treated with the novel compound of Formula 1 (see FIG. 3A). However, the above expression pattern was not observed in the Hep3B cell line (see FIG. 3B). From these experimental results, it was determined that the novel compounds of formula (I) of the present invention can inhibit beta-catenin expression specifically in Sk-Hep1 cells in liver cancer cells.

In the following Experimental Example 4 of the present invention, the novel compounds of Formula 1 of the present invention are also useful for the production of other proteins (E-cadherin, Axin1 [tumor suppressor protein] and GSK-3beta [Beta -catenin kinase]). As a result, in the experimental group treated with the novel compound of formula (I) of the present invention, the expression of GSK-3β, a beta -catenin kinase, was markedly elevated in the Sk-Hep1 cell line (Fig. 4A). These results indicate that the novel compounds of formula 1 of the present invention increase GSK-3? Expression in Sk-Hep1 cells and that when the increased GSK-3? Enzyme phosphorylates the Ser 33/37 residue of beta -catenin Beta - catenin was polyubiquitinated by several proteins, and the polyubiquitinated beta - catenin was degraded by proteasomes, which ultimately led to down regulation of beta - catenin protein.

In the following Experimental Example 5 of the present invention, the effect of the novel compound of Formula 1 on the beta-catenin signal pathway in breast cancer cells (MDA-MB-231) It was confirmed that the expression of E-cadherin protein, a binding partner of beta-catenin, was up-regulated while there was no change in beta-catenin and p53 expression (see FIG. 5). These results indicate that the novel compounds of formula 1 of the present invention have an effect on the signal pathway by upregulating GSK-3β expression and blocking β-catenin expression in SK-Hep1 (liver cancer) cells, -231 (breast cancer) cells by up-regulating the expression of E-cadherin.

In the following Experimental Example 6 of the present invention, it was found that the novel compound of Chemical Formula 1 of the present invention can induce cell cycle arrest in cancer cells. As a result, in the experimental group treated with the novel compound of Chemical Formula 1 of the present invention The cell population of G1 group was high, and in particular, the cell population of subG1 group and G1 group was significantly increased in Hep3B cells according to the treatment with the compound of the present invention (see FIG. 7). In addition, it was confirmed that the expression level of p21 and p27 in Hep3B cells was markedly up-regulated, while CDK2 was markedly down-regulated by treatment with the novel compound of formula (I) of the present invention (see FIG. 8). These results suggest that inhibition of growth in Hep3B cells is mediated by cell cycle arrest.

In the following Experimental Example 7 of the present invention, as a result of investigating whether the novel compound of Chemical Formula 1 of the present invention can inhibit the invasion of cancer cells, it was found that Sk-Hep1 and MDA-MB- , The invasiveness of the cancer cells was decreased depending on the compound treatment concentration in the experimental group treated with the new compound of each concentration (see FIG. 9). In addition, Western blot analysis of the protein expression level of MMP-2, which plays an important role in basement membrane degradation in cancer invasion, showed that the Sk-Hep1 or MDA-MB-231 cell line was treated with the novel compound of formula When the time elapsed, it was confirmed that MMP-2 expression was remarkably reduced (see FIG. 10).

The inventors of the present invention have found that the novel compounds of formula (I) are cytotoxic only to cancer cells, and that the proliferation and infiltration of cancer cells can be prevented by the wnt / β-catenin signal blocking mechanism in cancer cells having high invasiveness Which can be inhibited.

Therefore, the composition of the present invention comprising a novel compound of the formula (1) as an active ingredient can effectively prevent or treat cancer, and can be particularly effective in the treatment of cancer with high invasiveness.

The composition of the present invention is a pharmaceutical composition comprising the novel compound of formula (1) as an active ingredient, and can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. A disintegrating agent, a sweetener, a binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the novel compounds of formula (I) of the present invention may be included in the composition at a concentration of 1 to 1000 μM, and the compound of the present invention may be contained in an amount of 0.1 to 95% by weight based on the total weight of the composition .

The novel compounds of formula (I) of the present invention may be administered in combination with one or more therapeutic agents in a therapeutically effective amount (pharmaceutical combination). For example, synergistic effects may occur when the novel compounds of formula I of the present invention are used in combination with chemotherapeutic agents. When the compounds of the present invention are administered in combination with other therapies, the dose of the co-administered compound will vary depending on the type of co-drug used, the particular drug employed, the condition being treated, and the like.

In addition, the present invention provides the use of a composition comprising the novel compound of formula (1) as an active ingredient for the manufacture of a medicament for the prevention or treatment of cancer. The composition of the present invention comprising the novel compound of the formula (1) as an active ingredient can be used for the preparation of a medicament for the prevention or treatment of cancer.

The present invention also provides a method of preventing or treating cancer, comprising administering to a mammal a novel compound of Formula 1.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms 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 will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the compound of the present invention at a dose of 0.01 mg / kg to 250 mg / kg once to several times a day.

In the therapeutic method of the present invention, the composition comprising the novel compound of formula (I) of the present invention as an active ingredient may be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, In a conventional manner.

The present invention also provides a health functional food for cancer prevention or improvement comprising the novel compound of formula (1) as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and rings for prevention and improvement of cancer.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in tablet form may be prepared by granulating a mixture of the novel compound of formula (1), which is an active ingredient of the present invention, with an excipient, a binder, a disintegrant and other additives by a conventional method, Or the mixture can be directly compression-molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a mixture of the novel compound of the formula (1), which is an active ingredient of the present invention, with an additive such as an excipient into a normal hard capsule, Of the present invention is mixed with an additive such as an excipient to fill a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of the novel compound of formula (1), the active ingredient of the present invention, excipient, binder, disintegrant and the like by a conventionally known method, It can be applied to other skin preparations, or the surface can be coated with materials such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of the novel compound of formula (1), an active ingredient of the present invention, excipient, binder, disintegrant, and the like into granules by a conventionally known method, , A mating agent, and the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example >

reagent

All solvents and reagents were reagent grade from commercial sources and were used without further purification. TLCon Silica Gel 60 F254 precoated plates were run to evaluate the reaction process and the purification of the manufactured products. Melting points were measured and recorded using an Electrothermal-9100 (Shimadzu, Japan) apparatus. For 1 H NMR and 13 C NMR, ¹ H was performed at 400 MHz, and ¹³ C completely separated by proton was performed using a JNM ECP-400 (JEOL, Japan) device operating at 100.6 MHz. In this case, CDCl ₃ was used as a solvent, and in the case of CDCl ₃ , ppm values were indicated based on TMS as an internal standard (CDCl 3, δ = 7.26 ppm for proton and 77.00 ppm for 11 carbon NMR). The mass spectra were measured using a JMS-700 (JEOL) instrument and the final compounds were purified using silica gel (200-400 mesh-60).

Cell line purchase and cell culture

All cell lines were purchased from the American Type Culture Collection (ATCC). Human HCC Sk-Hep1 and Hep3B cells were cultured in MEM / EBSS containing 10% FBS at 37 ° C and 5% CO ₂ air. Human breast cancer MDA-MB-231 cell line and non-cancerous human embryonic kidney HEK293 cell line were cultured in DMEM medium containing 10% FBS at 37 ° C under 5% CO ₂ air. THLE-3 human normal liver cells were cultured in a CO ₂ incubator at 37 ° C in BEBM medium containing 10% FBS. The stock solutions (10 ml) of the three different compounds were prepared by dissolving them in DMSO. 100μl of complete medium containing 1 × 10 ⁴ cells was dispensed into each well of a 96 well (well) cell culture microtiter teopan (microtiter plate). The plate was incubated in a 37 ° C 5% CO ₂ incubator for 24 hours before testing. After removal of the medium, 100 μl of fresh medium containing different concentrations of compound was added to each well and incubated at 37 ° C for 24 hours. After incubation, 10 [mu] l of EZ-Cytox Cell Viability Assay Solution WST-1 reagents (Daeil Lab Service, Korea) was added to each well, followed by further incubation for 4 hours. Absorbance at 460 nm was measured using a microplate reader (Molecular Devices). IC ₅₀ was defined as the concentration required to inhibit cell proliferation by 50% on the basis of 100% survival in cells treated with DMSO.

statistics

Statistical validity between compound treated values and non-treated groups was determined with GraphPad Prism 5.0. Results were expressed as mean ± standard deviation of three independent experiments. Data were analyzed using one-way analysis of variance and turkey analysis was performed for various comparisons.

< Example  1>

Tetrahydropyridinol ( tetrahydropyridinol ) Preparation of derivative compounds

<1-1> Pentafluorobenzoyl  Chloride ( pentafluorobenzoyl chloride ) Produce

The industrially available methylpentafluorobenzoate (1) was hydrolyzed at 25 [deg.] C by adding sodium hydroxide (NaOH), methanol and water to the acid form (2). 20 ml of thionyl chloride (SOCl ₂ ) and 15 ml of dry toluene were added to 0.005 mol of the compound (2), and the mixture was refluxed for 6 hours. Then, the reaction mixture was cooled, the solvent was evaporated in vacuo, 10 ml of toluene was added again to the reaction solution, and the solvent was completely evaporated. The residue thus obtained was dissolved in dichloromethane (DCM) and used for preparing the objective compound of the present invention.

&Lt; 1-2 > Tetrahydropyridinol  Manufacture of derivative compounds

The tetrahydropyridinol derivative compound is obtained by reacting 4a, 4b, 4c compound (4a = CH ₂ Br, 4b = CH (CH _{3) 2} ) in a pentafluorobenzoyl chloride solution prepared in Example <1-1> ₂ Cl, 4c = CHBrCH ₃ The compounds 4a, 4b and 4c were synthesized by the method described in the literature (G. Aridoss, S. Amirthaganesan, N. Ashok Kumar, JT Kim, KT Lim , S. Kabilan, YT Jeong, Bioorg. Med. Chem. Lett. 2008, 18, 6542-6548, G. Aridoss, S. Amirthaganesan, YT Jeong, Bioorg. Med. Chem. Lett. 2010, 20, 2242-2249 Reference). More specifically, 0.005 mol of each of the compounds 4a, 4b and 4c was dissolved in 20 ml of dry dichloromethane, and then 0.015 mol of DMAP or NEt3 was added and stirred. To this reaction mixture, pentafluorobenzoyl chloride solution prepared in Example <1-1> was added and stirred at room temperature for 5 hours. After the reaction was completed, the solvent was removed and the residue was dissolved in ethyl acetate and washed sequentially with bicarbonate, water and brine. Finally, the organic layer was dried over Na ₂ SO ₄ , and the residue obtained through evaporation was purified by column (0.5: 10 to 1:10, ethyl acetate: hexane) to obtain pure material. The tetrahydropyridinol derivative compound .

The process for preparing the tetrahydropyridinol derivative of the present invention is shown below.

In the above reaction scheme,

_{a: NaOH, MeOH, H 2} O, carried out at 25 ℃; b: SOCl ₂ , dry toluene, reflux performed; c: EtOH, heat, HCl, NH3 reaction; d: RCOX (X = Br and Cl), NEt3 or DMAP, Dry DCM, rt reaction; e: DMAP, dry DCM, rt.

&Lt; 1-3 > Tetrahydropyridinol  Identification of derivative compounds

Nuclear magnetic resonance spectroscopy was carried out to confirm the tetrahydropyridinol derivative compounds 5a, 5b and 5c of the present invention prepared in Example <1-2>.

N - ( Bromoacetyl ) -3- carboxyethyl -2,6- 피덴 -4- O - ( pentafluorobenzoyl ) - △ ³ - tetrahydropyridine  (5a)

Yield: 65%, a semi solid. ^{1 H NMR (400 MHz, CDCl} 3) δ ppm: 7.19-7.03 (m, 10H); J = 7.05 Hz, 2H), 6.88 (bs, 1H), 5.51 (bs, 1H), 4.31 (d, J = 12.86 Hz, 1H), 4.16 3.14 (dd, J = 17.84 Hz, J = 4.98 Hz, 1H), 2.98 (dd, J = 17.84 Hz, J = 6.22 Hz, 1H), 0.99 (t, J = 7.05 Hz, 3H). ¹³ C NMR (100 MHz, CDCl3) ppm: 166.57,162.42,157.23,152.79,148.09 (m), 145.47 (m), 139.04 (m), 138.15,138.02,128.51-126.76 (phenyl carbons), 120.17,103.13 (t, JC-F = 11.14), 61.18, 55.34, 53.82, 42.33, 33.20, 13.64; MS (ESI, positive) m / z calcd. for C ₂₉ H ₂₁ BrF ₅ NO ₅ (M + H): 637.05; found 637.3.

N - ( Chloroacetyl ) -3- carboxyethyl -2,6- 피덴 -4- O - ( pentafluorobenzoyl ) -Δ ³ - tetrahydropyridine  (5b)

Yield: 81%. White solid, mp 142-144 [deg.] C. ¹ H NMR (400 MHz, CDCl 3)? Ppm: 7.20-7.02 (m, 10H, phenyl protons); J = 7.05 Hz, 2H), 6.95 (bs, 1H), 5.51 (bs, 1H), 4.31 (d, J = 12.86 Hz, 1H) 3.14 (dd, J = 17.84 Hz, J = 5.39 Hz, 1H), 2.98 (dd, J = 17.43 Hz, J = 6.22 Hz, 1H), 0.99 (t, J = 7.05 Hz, 3H); ¹³ C NMR (100 MHz, CDCl3) ppm: 166.62,162.44,157.24,152.78,148.13 (m), 145.40 (m), 139.06 (m), 138.19,138.03,128.53-126.72 (phenyl carbons), 120.20,103.17 (t, JC-F = 11.19), 61.19, 55.27, 53.83, 42.32, 33.23, 13.65; MS (ESI, positive) m / z calcd. for C ₂₉ H ₂₁ ClF ₅ NO ₅ (M + H): 593.1; found 593.3.

N -(2- Bromopropanoyl ) -3- carboxyethyl -2,6- 피덴 -4- O - ( pentafluorobenzoyl ) -Δ ³ -tetrahydropyridine (5c)

Yield: 61%, a white solid, mp 140-141 [deg.] C. ¹ H NMR (400 MHz, CDCl 3)? Ppm: 7.19-7.03 (m, 10H), 6.98 (s, 1H), 5.51 (s1H), 4.65 2H), 3.15 (s, 2H), 1.87 (d, J = 6.22 Hz, 3H), 0.96 (t, J = 7.05 Hz, 3H); ¹³ C NMR (100 MHz, CDCl 3) ppm: 169.13,162.40,157.29,152.24,148.06 m, 145.60 m, 139.08 m, 138.54,138.43,128.64-126.26 (phenyl carbons), 119.97,103.31 (t, JC-F = 11.15), 61.07, 55.07, 53.11, 39.10, 32.50, 22.01, 13.62; MS (ESI, positive) m / z calcd. _{for C 30 H 23 BrF 5 NO} 5 (M + H): 651.07; found 651.1.

< Experimental Example  1>

The compound of the present invention Cancer cell  About Anti-proliferation  activation( in vitro ) analysis

Human embryonic kidney HEK293 cells, normal human hepatocyte THLE-3, human HCC (Sk-Hep1 and Hep3B), and human embryonic kidney cells were evaluated in order to evaluate the cytotoxicity of the compounds 5a, 5b, Cell viability assay was performed using human breast cancer MDA-MB-231 cell line.

First, HEK293, THLE-3, Sk-Hep1, Hep3B and MDA-MB-231 cells were resuspended in 100 μl medium at a density of 1 × 10 ⁴ cells, and 96-well plates were dispensed and incubated for 24 hours. Then, the cells were treated with various concentrations of the compounds of the present invention 5a, 5b and 5c, respectively, and the cells were cultured for 24 hours, and then replaced with fresh medium. Then, 10 μl of WST-1 solution was added and reacted for 5 hours. Reaction absorbance was measured at 460 nm using an ELISA reader (Molecular Devices, Sunnyvale, Calif., USA). Results were expressed as mean ± standard deviation through three independent experiments.

The results are shown in Fig. Figures 1A and 1B compare the survival rates of HEK293, THLE-3 and Sk-Hep1 cells in Compounds 5a and 5b of the present invention. Although both compounds exhibited cytotoxicity in a dose-dependent manner, 5b showed significantly higher cytotoxicity in Sk-Hep1 cells than in 5a. On the other hand, both compounds showed very low cytotoxicity in non-tumorous cell lines HEK293 and THLE-3 cells. The IC ₅₀ concentrations of compounds 5a and 5b were 12 μM and 6 μM, respectively, in Sk-Hep1 cells. In addition, the cytotoxicity of 5a compound in Hep3B and MDA-MB-231 cells was measured, and as shown in Fig. 1C, proliferation was inhibited in both cell lines depending on the treatment concentration of 5a compound. The IC ₅₀ concentrations in the Hep3B and DA-MB-231 cell lines of the 5a compound were 24 μM and 48 μM, respectively. Similarly, compound 5b of the present invention also inhibited the growth of Hep3B and MDA-MB-231 cells in a concentration-dependent manner. The IC ₅₀ concentrations in the Hep3B and DA-MB-231 cell lines of the 5b compound were 11 μM and 20 μM, respectively.

On the other hand, as shown in FIG. 1E, the 5c compound of the present invention showed almost no cytotoxicity in all three cell lines (Sk-Hep1, Hep3B, MDA-MB-231).

These results indicate that 5a and 5b of the compounds of the present invention can inhibit proliferation of liver cancer and breast cancer cells, and cytotoxicity of 5c is weak in cancer cells. Therefore, it was concluded that 5a and 5b of the compounds of the present invention had anticancer activity, and further experiments were conducted using compounds 5a and 5b of the present invention in the following experiment.

The IC ₅₀ concentrations of compounds 5a, 5b of the invention for each cancer cell line Cancer cell line IC50 (5a) IC50 (5b) Sk-Hep1 12 μM 6 μM Hep3B 24 μM 11 μM MDA-MB-231 48 μM 20 μM

< Experimental Example  2>

The tumor suppressor protein or the tumor suppressor protein in liver cancer cells of the present invention compound (5a, 5b) Cancer gene  Effect on expression

In Experimental Example 1, 5a and 5b of the compounds of the present invention were found to inhibit cell proliferation of hepatoma and breast cancer cell lines. In this experiment, it was confirmed that the inhibitory activity against cancer cells was increased uptake of tumor suppressor protein (p53, p16) Regulation or down-regulation of the cancer gene (beta-catenin: beta-catenin).

For this experiment, Sk-Hep1 and Hep3B cells of hepatocellular carcinoma cells were first treated with IC ₅₀ (see Table 1) for 3, 6 and 12 hours, respectively, and then centrifuged and washed with cold PBS buffer &Lt; / RTI &gt; The pellet obtained through the above procedure was dissolved in a buffer solution containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM DTT, 0.5% NP-40, 1% Triton X-100, 1% Deoxycholate, Cocktail (Intron biotechnology, Korea)]. Cells were lysed in cold lysis buffer for 1 hour and then centrifuged at 14,000 rpm for 20 minutes to remove insoluble material and collect protein lysates. Equal amounts of protein were dissolved in 12% SDS-PAGE and transferred to nitrocellulose membrane (PALL life science). The membrane was reacted with PBST buffer containing 5% skim milk for 2 hours at room temperature. The membrane was reacted with primary antibody against p53, p16, β-catenin and GAPDH (Cell Signaling Technology) overnight at 4 ° C., and then reacted with HRP-conjugated secondary antibody (cell signal technology) . All membranes were visualized as WESTSAVETM Gold ECL (Ab Frontier) and exposed to Hyperfilm (GE Healthcare). GAPDH was used as a control.

As a result, as shown in Fig. 2A, there was no change in the expression of p53 and p16 in the experimental group treated with the compound (5a or 5b) of the present invention in the Sk-Hep1 cell line, while the expression of the beta -catenin protein was suppressed in a time- . In particular, protein expression of beta-catenin was completely inhibited at 12 hours after treatment with the compound (5a or 5b) of the present invention.

On the other hand, as shown in FIG. 2B, in the experimental group treated with the compound (5a or 5b) of the present invention in the Hep3B cell line, the protein expression of the beta -catenin was not down-regulated and the expression level was not changed with time I could confirm.

< Experimental Example  3>

The beta-glucosidase activity of the compounds (5a, 5b) Catechin  Regulation of expression

In Experimental Example 2, it was confirmed that the compound (5a or 5b) of the present invention inhibited Sk-Hep1 cell proliferation by down-regulating the protein of beta -catenin, which is known as the cancer gene. In order to further elucidate whether regulation appears to be an inhibition of protein activity, we examined the protein measurement of beta-catenin in the phosphorylated form.

Western blotting was used for protein measurement of the phosphorylated form of beta-catenin. Cells were treated with (1) control (DMSO), (2) DCA (5 μM) alone treatment group, (3) DCA (5 μM) and the compound 5a treatment group of the present invention, (4) DCA And treated with Compound 5b of the present invention. Here, the combination of DCA and the compound of the present invention was treated with DCA for 30 minutes and then with the compound of the present invention for 12 hours. Cells prepared as described above were subjected to Western blotting in the same manner as in Experimental Example 2 except that anti-phospho-beta-catenin (Ser 33/37) and anti-phospho-beta-catenin (Tyr 142) Respectively. For reference, deoxycholic acid (DCA) is a substance that can activate the phosphorylation of beta-catenin.

As a result, the expression of phospho-β-catenin (Tyr 142) was increased in the experimental group treated with DCA (5 μM) alone in Sk-Hep1 cells and the expression of DCA and the compound (5a or 5b) And the phospho-β-catenin (Tyr 142) expression was decreased in the combination treatment group. On the other hand, the expression of phospho-beta-catenin (Ser 33/37) was increased in the experimental group treated with DCA and the compound (5a or 5b) of the present invention in comparison with the DCA alone treatment group .

On the other hand, the above-mentioned expression pattern was not observed in the Hep3B cell line (see Fig. 3B).

Phosphorylation in the Ser 33/37 residue of beta-catenin acts as a target for beta-catenin degradation by the ubiquitin-proteasome complex whereas phosphorylation of the beta -catenin in the Tyr142 residue is catalyzed by beta-catenin with axin-APC And functions to suppress the merging of the complex. Therefore, if the increase of Ser 33/37 residue phosphorylation of beta -catenin promotes beta-catenin degradation, and if Tyr142 residue phosphorylation of beta -catenin is enhanced, accumulation of beta -catenin in the cytoplasm and entry into the nucleus Promotes transcription initiation of oncogenic genes such as cyclin D1 and c-myc.

From the above experimental results, it was determined that the compounds 5a and 5b of the present invention can inhibit beta-catenin expression specifically in Sk-Hep1 cells in liver cancer cells.

< Experimental Example  4>

The beta-glucosidase activity of the compounds (5a, 5b) Catechin  Impact on signal

In Experimental Example 2, it was confirmed that the compound (5a or 5b) of the present invention had an inhibitory effect on protein expression of only beta-catenin. In this experiment, it was confirmed that the compounds of the present invention inhibited protein expression of other proteins involved in the beta-catenin signal pathway (E-cadherin [transfer inhibitory protein], Axin1 [tumor suppressor protein] and GSK-3 [beta -catenin kinase]).

E-cadherin, Axin1 and GSK-3β proteins were measured by Western blotting. Cells were treated with (1) control (DMSO), (2) DCA (5 μM) alone treatment group, (3) DCA (5 μM) ) DCA (5 [mu] M) and compound 5b of the present invention. Here, the combination of DCA and the compound of the present invention was treated with DCA for 30 minutes and then with the compound of the present invention for 12 hours. The cells thus prepared were subjected to Western blotting in the same manner as in Experimental Example 2, except that anti-E-cadherin, anti-Axin and anti-GSK-3? Were used as primary antibodies.

As a result, as shown in Fig. 4A, the control group treated with DMSO in Sk-Hep1 cells showed very low protein expression of E-cadherin and Axin1, and no change in expression was observed according to the treatment with the compound of the present invention. On the other hand, it was confirmed that the expression of GSK-3? Was significantly up-regulated in the experimental group in which the compounds of the present invention were co-administered with DCA (5 μM). On the other hand, the above expression pattern was not observed in the Hep3B cell line (see Fig. 4B).

As a result, the compounds 5a and 5b of the present invention increase GSK-3β expression in Sk-Hep1 cells and recognize that the increased GSK-3β enzyme phosphorylates the Ser 33/37 residue of β-catenin Beta-catenin was polyubiquitinated by several proteins, and the multi-ubiquitinated beta-catenin was recognized by the proteasome and was ultimately down-regulated.

< Experimental Example  5>

The beta-glucosidase activity of the compounds (5a, 5b) of the present invention in breast cancer cells, Catechin  Protein expression and beta - Catechin  Effect on signal path

In Experimental Examples 2 to 4, it was confirmed that the compound (5a or 5b) of the present invention down-regulates the beta-catenin protein in liver cancer cells and affects the expression of proteins involved in the beta-catenin signal pathway. To further confirm the above results, the same experiment was carried out using MDA-MB-231, a highly invasive breast cancer cell line.

First, MDA-MB-231 cells were exposed to compound 5a or 5b of the present invention, followed by Western blotting for beta -catenin and p53. As a result, as shown in Fig. 5A, no changes were observed in beta -catenin and p53 expression by treatment with the compound of the present invention (5a or 5b).

In addition, the expression of E-cadherin and GSK-3?, Which are binding partners of beta-catenin, was observed in MDA-MB-231 cells according to the treatment of the compound of the present invention. Compounds 5a and 5b of the present invention were found to up-regulate E-cadherin expression, but did not affect GSK-3 ?.

In conclusion, compounds 5a and 5b of the present invention were found to act as different mechanisms in the wnt / β-cartenin signaling pathway. In other words, in SK-Hep1 cells, upregulation of GSK-3β expression and blockade of β-catenin expression affect this signal pathway. In MDA-MB-231 cells, E-cadherin expression is upregulated, .

< Experimental Example  6>

The induction of cell cycle arrest in the cancer cells of the compound (5a, 5b)

<6-1> Beta- Of catenin  Regulation of target gene expression

In this experiment, in order to examine whether the compounds 5a and 5b regulate the expression of cyclin D1 and c-myc, which are the target genes of beta -catenin, Western blots of proteins expressed from Sk-Hep1, Hep3B and MDA- Gt;

Cells were treated with (1) control (DMSO), (2) DCA (5 μM) alone treatment group, (3) DCA (5 μM) ) DCA (5 [mu] M) and compound 5b of the present invention. At this time, the combination treatment of DCA and the compound of the present invention treated the compound of the present invention for 12 hours after treating DCA for 30 minutes. The cells thus prepared were subjected to Western blotting in the same manner as in Experimental Example 2, except that anti-cyclin D1, anti-c-myc and anti-GSK-3? Were used as primary antibodies.

As a result, as shown in FIG. 6, in the experimental group in which the compounds (5a, 5b) of the present invention were treated together with DCA (5 μM) against the Sk-Hep1 and MDA-MB- The expression was suppressed. However, the expression of these beta-catenin target proteins was not changed in Hep3B cells.

<6-2> induction of cell cycle arrest

In this experiment we examined whether compounds 5a and 5b could induce cell cycle arrest, which was assessed by measuring cell cycle population changes using a flow cytometer.

(SK-Hep1: 5a-12M, 5b-6M; Hep3B: 5a-24M, 5b-11M; MDA-MB -231: 5a-48 [mu] M, 5b-20 [mu] M) for 12 hours. The cells were treated with trypsin, and the cells were collected by centrifugation and fixed overnight at 4 ° C with 70% ethanol. The cells were then resuspended in PBS buffer containing 0.2 mg / ml RNase A and 0 μg / ml propidium iodide (PI). The cells thus treated were kept in the dark for 30 minutes. Cell cycle distribution was analyzed using a FACS Calibur flow cytometer (Becton Dickinson).

As a result, as shown in Fig. 7, the percentage of G1 cell population was higher in all experimental groups treated with the compounds of the present invention compared to the untreated control group. In particular, in Hep3B cells, it was confirmed that the percentage of subG1 and G1 cell populations was significantly increased with the compound treatment of the present invention. These results confirm that inhibition of cell growth in the Sk-Hep1 or MDA-MB-231 cell line is driven by the wnt / β-catenin signaling pathway, while inhibition of growth in Hep3B cells is via cell cycle arrest .

<6-3> Regulation of cell cycle regulatory protein expression

In Experimental Example <6-2>, it was confirmed that compounds 5a and 5b of the present invention can induce cell cycle arrest in Hep3B cells. In this experiment, Hep3B cells were treated with the compounds (5a, 5b) P21, p27, and cdk2 protein expression, respectively.

For this purpose, (see Table 1) Hep3B cells IC for compound 5a or 5b, respectively of the present invention to the ₅₀ concentration of 3, 6, cells prepared after treatment 12 hours have been carried out the Western blot analysis in the same manner as in Experimental Example 2, However, anti-CDK2, anti-p21 and anti-p27 were used as primary antibodies.

As a result, as shown in FIG. 8, the expression of p21 and p27 in Hep3B cells was markedly up-regulated by the treatment with the compound of the present invention, while the CDK2 was markedly down-regulated.

For reference, in mammalian cells, cyclins D, E and A are continuously synthesized during the G1 period of the cell cycle. The major catalytic partners of these cyclins are CDK2 and CDK4. These CDKs are known to be negatively regulated by CDK-inhibitors such as p21, p27 and p53.

In conclusion, it was determined that the compounds (5a, 5b) of the present invention could inhibit the growth of Hep3B cells through down-regulation of CDK2 expression and induction of expression of p21 and p27.

< Experimental Example  7>

Inhibitory effect of the compound (5a, 5b) of the present invention on cancer cells

<7-1> Cellular invasion essay ( cell invasive assay )

Compounds 5a and 5b were able to effectively inhibit the proliferation of liver cancer and breast cancer cells. Experimental Examples 1 to 6 showed that compounds 5a and 5b effectively suppressed proliferation of liver cancer and breast cancer cells, Respectively. Therefore, in this experiment, it was evaluated through matrigel invasive assay whether compounds 5a and 5b inhibited the invasion of cancer cells in Sk-Hep1 and MDA-MB-231 cell lines with high invasiveness.

The cell invasive assay was performed as previously described using matrigel-coated transwell chambers (Corning life sciences) with an 8 μm hole size 6.5 mm polyvinyl / pyrrolidone-free polycarbonate filter (W. -C. Hung, H.-C. Chang, J. Agricultural Food Chem. 2008, 57, 76-82). Briefly, 5 × 10 ⁴ cells (Sk-Hep1, Hep3B or MDA-MB-231) were suspended with the compound (5a or 5n) of the present invention in an upper layer transwell chamber containing 100 μl of the sample- Lt; 0 &gt; C for 24 hours. The cells placed on the upper surface of the filter were then thoroughly wiped with a cotton swab and the lower layer of the filter was fixed with 4% formaldehyde stained with crystal violet. After staining, the lower surface cells were lysed with 2% SDS for 1 hour, and the lysates were measured at 570 nm using a microplate reader.

As a result, as shown in Fig. 9, Sk-Hep1 and MDA-MB-231 cells were treated with compound 5a or 5b of the present invention in Sk-Hep1 and MDA- , But it did not affect Hep3B cell line.

<7-2> MMP -2 expression inhibitory effect

In this experiment, the expression of MMP-2 protein, known to be involved in the invasion process of cancer cells, was examined by Western blotting. Expression of MMPs containing MMP-2 is known to play an important role in basement membrane degradation in cancer invasion and is associated with cancer metastasis.

To this end, cells prepared Sk-Hep1 or after the MDA-MB-231 cells of this process the compound 5a or 5b, respectively of the invention as IC ₅₀ concentration of 3, 6 and 12 hours are Western blotting in the same manner as in Experimental Example 2 , But anti-MMP-2 was used as primary antibody.

As a result, as shown in FIG. 10, it was confirmed that the expression of MMP-2 was markedly reduced after 12 hours of treatment with each of the compounds of the present invention in Sk-Hep1 or MDA-MB-231 cell lines.

Therefore, compounds 5a and 5b of the present invention have cell proliferation (growth) inhibitory activity as well as invasive inhibitory activity on cell lines (Sk-Hep1, MDA-MB-231) It could be used as an anticancer material.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

HCC: hepatocellular carcinoma
EMEM: Eagle's minimal essential medium
BEBM: bronchial epithelial cell basal medium
PBS: phosphate-buffered saline
DCA: deoxycholic acid
DMSO: Dimethyl sulfoxide
BSA: bovine serum albumin
CDK2: cyclin-dependent kinase 2
WST-1: 2- (4-iodophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt

Claims (8)

A novel compound represented by the following formula (1);
&Lt; Formula 1 >

In this formula,
R is CH ₂ Br or CH ₂ Cl. A pharmaceutical composition for preventing or treating cancer comprising the compound of claim 1 as an active ingredient. 3. The method of claim 2,
Wherein the cancer is selected from the group consisting of a brain tumor, a benign astrocytoma, a malignant astrocytoma, a pituitary adenoma, a neuroendocrine tumor, a brain lymphoma, an oliguria, a cervicitis, Breast cancer, gastric cancer, hepatic cancer, gallbladder cancer, biliary cancer, pancreatic cancer, small bowel cancer, colon cancer, breast cancer, breast cancer, breast cancer, breast cancer, hypopharynx, thyroid cancer, oral cancer, thoracic tumor, small cell lung cancer, non-small cell lung cancer, , Female genital tumors, female genital tumors, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female genital external cancer, and female urethral cancer &Lt; / RTI &gt; is selected. 3. The method of claim 2,
The compounds are useful in the treatment of wnt / beta through upregulation of β-catenin expression, upregulation of GSK-3β (glyco-gensynthase kinsase 3-β) expression or upregulation of E-cadherin expression -catenin signal blocking mechanism to inhibit the proliferation and penetration of cancer cells. 3. The method of claim 2,
Wherein said compound inhibits the proliferation of cancer cells by a mechanism of cell cycle arrest of cancer cells through inhibition of CDK2 expression and expression of p21 and p27. 6. The method according to any one of claims 2 to 5,
Wherein the compound is contained in the composition at a concentration of 1 to 1000 μM. A health functional food for preventing or ameliorating cancer comprising the compound of claim 1 as an active ingredient. 8. The method of claim 7,
Wherein said food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods.

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