KR20170024522A - 2-(phenylamino)benzo[d]oxazol-5-ol derivatives, preparation method thereof, and pharmaceutical composition for use in preventing or treating inflammatory diseases containing the same as an active ingredient - Google Patents

Abstract

The present invention relates to a 2-(phenylamino)benzo[d]oxazol-5-ol derivative, a method for preparing the same, and a pharmaceutical composition comprising the same as an active ingredient for preventing or treating inflammatory diseases. According to the present invention, the 2-(phenylamino)benzo[d]oxazol-5-ol derivative, or an optical isomer or pharmaceutically acceptable salt thereof has excellent activities of preventing T cells from producing interferon gamma (IFN), an inflammatory cytokine, by Interleukin-2 (IL-2) known as a T cell proliferation stimulating factor and from being differentiated into T helper cells 1 (Th1 cells) mainly expressing 5-lipoxygenase (5-LO), and of inhibiting expression of Interleukin-6 (IL-6) effectively. Thus, the 2-(phenylamino)benzo[d]oxazol-5-ol derivative is useful for a pharmaceutical composition for preventing or treating inflammatory diseases.

Description

The present invention relates to a 2- (phenylamino) benzo [d] oxazol-5-ol derivative, a process for producing the same, and a pharmaceutical composition for preventing or treating an inflammatory disease containing the same as an active ingredient. 5-ol derivatives, preparation methods thereof, and pharmaceutical compositions for use in preventing or treating inflammatory diseases comprising the same as an active ingredient.

The present invention relates to a 2- (phenylamino) benzo [ d ] oxazol-5-ol derivative, a process for its preparation, and a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases containing it as an active ingredient.

Inflammation is a normal and protective in vivo defense manifestation that is localized to physical trauma, harmful chemicals, microbial infections, or tissue damage caused by stimuli in vivo metabolites. These inflammations are triggered by various chemical mediators produced from damaged tissues and migrating cells, and these chemical mediators are known to exist in various forms depending on the type of inflammation process.

In non-patent reference 1, in vivo inflammatory cells such as macrophages are activated by inducible nitric oxide synthase (iNOS) or cyclo-oxygenase-2 (COX-2) Inflammatory mediators such as nitric oxide (NO), prostaglandin (PG), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha ) And cytokine are produced in large quantities.

In general, inflammatory diseases are caused by the overgrowth of inflammatory cytokines due to the continued growth and proliferation of activated T cells (T lymphocytes). Specifically, T cells are produced by the production of interferon gamma (IFNγ), an inflammatory cytokine, by interleukin-2 (IL-2), which is known as T cell proliferation promoting factor, and 5-lipoxygenase (T helper cells 1) cells characterized by the expression of Lipoxygenase, 5-LO or 5-LOX. In addition, the inflammatory disease is known to be closely related to cytokines such as interleukin-6 (IL-6).

Specifically, interleukin-6 (hereinafter "IL-6") (aka interferon-β2; B-cell differentiation factor; B-cell stimulating factor-2) specifically regulates the acute inflammatory response, including B- Is a multifunctional cytokine that participates in a number of biological processes such as regulation of specific immune responses, inflammation reactions arising from bone metabolism, platelet formation, epithelial proliferation, neuronal differentiation, neuroprotection, and aging.

Non-Patent Document 2-4 discloses that IL-6 is directly related to inflammatory bowel disease such as ulcerative colitis, Crohns disease, and the like, 5 discloses that plasma concentration of IL-6 is directly related to sepsis.

Interferon is a secreted protein in cells that have been infected with viruses. It is a substance that stimulates cells in vivo to resist the invasion of viruses. There are three types of interferon: alpha, beta, and gamma. Among the interferons known so far, the most effective is gamma interferon, produced by activated T cells and macrophages, and abnormal interferon gamma is associated with autoinflammation or autoimmune disease.

Inflammatory bowel disease, typified by Crohn's disease or ulcerative colitis, has a lower incidence and prevalence in Korea than in the western world, but it is increasing recently. Although the pathogenesis and pathogenesis of inflammatory bowel disease has not been clarified yet, it is now known that exposure to environmental causes or inducers of genetic vulnerability causes inflammation and immune responses to the intestinal mucosa, It is known that the reaction continues and amplifies and causes chronic tissue damage. It has been reported that imbalance of Th1 cells (T helper type 1) and Th2 cells (T helper type 2) among CD4 positive helper T cells is associated with inflammatory bowel disease.

Crohn's disease is associated with Th1 cell-associated interferon-γ, IFN-γ, tumor necrosis factor-α, TNF-α, interleukin-2 and IL-2 (TGF-β), IL-4, and IL-5, which are associated with Th2 cells, and IL-6, IL -21 and IL-23 are known to be associated with both Crohn's disease and ulcerative colitis.

Many inflammatory mediators are involved in the inflammatory response of inflammatory bowel disease, among which leukotriene is a potent inflammatory mediator and plays a major role in the pathophysiology of asthma, arthritis, psoriasis, and inflammatory bowel disease. Rocotrienes are produced in arachidonic acid by 5-lipoxygenase (5-LO), and activated rucotrienes mediate acute inflammatory and hypersensitivity reactions. It is also known that rucotrienes are secreted from leukocytes activated by inflammation or immunological stimulation and increase mucous secretion of endothelial cells, contraction of smooth muscle, and permeability of capillaries. Thus, 5-LO inhibitors that inhibit the production of rucotrienes are expected to play a major role in the treatment of inflammatory bowel disease, but the typical 5-LO inhibitor zileuton has been shown to have side effects and interactions with other drugs And there was a limitation in use.

Accordingly, the present inventors have made efforts to develop a compound capable of effectively inhibiting IL-6 production while preventing T cells from differentiating into Th1 cells by interleukin-2, Amino] benzo [d] oxazol-5-ol derivative, an optical isomer thereof, or a pharmaceutically acceptable salt thereof exhibits the above-mentioned inhibitory activity and thus exhibits an inflammatory disease such as Crohn's disease, inflammatory bowel disease, rheumatoid arthritis, asthma, The present invention has been completed based on this finding.

Journal of Life Science, 2011, Vol. 21, No. 5, 678-683. Toxicology and Applied Pharmacology 279 (2014) 311-321. International Immunopharmacology 23 (2014) 294-303. Seminars in Immunology 26 (2014) 75-79. Clin Infect Dis. (2000) 31 (6): 1338-1342.

It is an object of the present invention to provide a 2- (phenylamino) benzo [ d ] oxazol-5-ol derivative showing an inhibitory effect on interleukin-6 (IL-6) activity.

Another object of the present invention is to provide a process for preparing the 2- (phenylamino) benzo [ d ] oxazol-5-ol derivative.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating an inflammatory disease containing the 2- (phenylamino) benzo [ d ] oxazol-5-ol derivative as an active ingredient.

Another object of the present invention is to provide a health food composition for preventing or ameliorating an inflammatory disease containing the 2- (phenylamino) benzo [ d ] oxazol-5-ol derivative as an active ingredient.

In order to achieve the above object,

The present invention provides a compound represented by the following general formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1,

R ¹ , R ² and R ³ are independently selected from the group consisting of -H, -OH, halogen, C _{1 -10} linear or branched alkyl, C _1-10 linear or branched alkoxy, -SR ⁴ , -NO ₂ , -NH ₂ Or a -N = N-Ph, wherein R ⁴ is a straight or branched alkyl of C _{1 -10.}

The present invention also relates to a process for producing a compound represented by the formula (1)

Reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (4) (step 1);

A step of cyclizing the compound of formula (4) prepared in step 1 to prepare a compound of formula (5) (step 2); And

Converting the methoxy group of the compound represented by the formula (5) to a hydroxy group to prepare a compound represented by the formula (1) (step 3); and reacting the compound represented by the formula to provide.

[Reaction Scheme 1]

In the above Reaction Scheme 1,

R ¹ , R ² And R < ³ > are independently as defined in the above formula (1).

Further, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease containing the compound represented by the formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health food composition for preventing or ameliorating an inflammatory disease containing the compound represented by the formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The 2- (phenylamino) benzo [ d ] oxazol-5-ol derivative, its optical isomer, or pharmaceutically acceptable salt thereof according to the present invention is useful for the treatment and prevention of T-cell proliferation- Interferon gamma (IFNγ), which is an inflammatory cytokine, and 5-Lipoxygenase (5-LO) are mainly expressed by interleukin-2 and IL-2 The present invention is useful as a pharmaceutical composition for preventing or treating an inflammatory disease associated with the above-mentioned diseases, because it has excellent activity to prevent differentiation and effectively inhibit the expression of interleukin-6 (IL-6).

FIG. 1 is a graph showing the results of evaluating IFN gamma (interferon gamma) production inhibitory effects of the compounds of Examples 4 and 5 according to the present invention.
FIG. 2 is a graph showing the results of evaluating IFN gamma (interferon gamma) production inhibitory effects of the compounds of Examples 4 and 5 according to the present invention.
FIG. 3 is a graph showing the results of evaluating the toxicity of the compounds of Examples 4 and 5 according to the present invention to primary CD4 + T cells. FIG.
4 is an image showing evaluation of the growth inhibitory effect of primary CD4 + T cells of the compound of Example 4 according to the present invention.
5 is an image showing evaluation of the growth inhibitory effect of primary CD4 + T cells of the compound of Example 5 according to the present invention.
FIG. 6 is an image obtained by treating the CD4 + T cells activated by concentration for 72 hours for the compounds of Example 4 and Example 5, and then performing annexin V staining to confirm cell death.
FIG. 7 is an image obtained by treating the CD4 + T cells activated by concentration for 72 hours for the compounds of Example 4 and Example 5, followed by staining of propidium iodide to confirm cell death.
FIG. 8 is a graph showing the results of analysis of proliferation factor IL-2 production in primary CD4 + T cells according to the concentrations of the compounds of Examples 4 and 5 according to the present invention.
FIG. 9 is a graph showing the results of analysis of proliferation factor IL-2 mRNA expression in primary CD4 + T cells according to the concentrations of the compounds of Examples 4 and 5 according to the present invention.
10 is an image showing the results of analysis of intracellular cytokine production in primary CD4 + T cells according to the concentrations of the compounds of Examples 4 and 5 according to the present invention.
11 is a graph showing the results of evaluating the inhibitory effect of Th1 (T helper cells 1) cell differentiation according to the concentration of the compounds of Examples 4 and 5 according to the present invention.
12 is a graph showing the results of evaluating the inhibitory activity against the stool consistency of the compound of Example 4 according to the present invention.
13 is a graph showing the results of evaluating how the compound of Example 4 according to the present invention affects the colon length.
14 is a graph showing the results of evaluating the inflammatory cytokine IFN? Inhibitory activity of the compound of Example 4 according to the present invention in a colitis-induced animal model.
Fig. 15 is an image showing the result of performing colitis-reduced histopathological analysis by the compound of Example according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following general formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In Formula 1,

R ¹ , R ² and R ³ are independently selected from the group consisting of -H, -OH, halogen, C _{1 -10} linear or branched alkyl, C _1-10 linear or branched alkoxy, -SR ⁴ , -NO ₂ , -NH ₂ Or a -N = N-Ph, wherein R ⁴ is a straight or branched alkyl of C _{1 -10.}

Preferably,

R ¹ is -H, -OH, halogen, C _{1 -5} straight or branched chain alkyl or C _{1 -5} straight or branched alkoxy;

R ² is selected from the group consisting of -H, -OH, halogen, C _{1 -5} straight or branched chain alkyl, C _{1 -5} straight or branched alkoxy, -SR ⁴ , -NO ₂ , -NH ₂ Or a -N = N-Ph, wherein R ⁴ is a straight or branched alkyl of C _{1 -5;} And

R ³ is -H, C _{1 -5} straight or branched chain alkyl or C _{1 -5} straight or branched alkoxy.

More preferably,

R ¹ is -H, -OH, -Cl or -Br;

R ² is -H, -OH, -CH ₃ , -CH ₂ CH ₃ , -CH (CH ₃ ) ₂ , - (CH ₂ ) ₃ CH ₃ , -Cl, -I, -SCH ₃ , -NO ₂ , -NH ₂ or -N = N-Ph; And

R ³ is -H, -CH ₂ CH _3, or -OCH ₃ .

Preferable examples of the compound represented by the formula (1) according to the present invention include the following compounds.

(1) (Z) -2 - ((4- (phenyldiazenyl) phenyl) amino) benzo [d] oxazol-5-ol;

(2) 2- (phenylamino) benzo [d] oxazol-5-ol;

(3) 2 - ((4-ethylphenyl) amino) benzo [d] oxazol-5-ol;

(4) 2 - ((3,4-dichlorophenyl) amino) benzo [d] oxazol-5-ol;

(5) 2 - ((4-hydroxyphenyl) amino) benzo [d] oxazol-5-ol;

(6) 2 - ((2-ethylphenyl) amino) benzo [d] oxazol-5-ol;

(7) 2 - ((4-iodophenyl) amino) benzo [d] oxazol-5-ol;

(8) 2 - ((4-isopropylphenyl) amino) benzo [d] oxazol-5-ol;

(9) 2 - ((4- (methylthio) phenyl) amino) benzo [d] oxazol-5-ol;

(10) 2 - ((3-bromophenyl) amino) benzo [d] oxazol-5-ol;

(11) 2 - ((4-nitrophenyl) amino) benzo [d] oxazol-5-ol;

(12) 2- (p-Tolylamino) benzo [d] oxazol-5-ol;

(13) 2 - ((4-butylphenyl) amino) benzo [d] oxazol-5-ol;

(14) 2 - ((2-methoxy-4-nitrophenyl) amino) benzo [d] oxazol-5-ol;

(15) 2 - ((4-aminophenyl) amino) benzo [d] oxazol-5-ol; And

(16) 2 - ((3-hydroxyphenyl) amino) benzo [d] oxazol-5-ol.

The compound represented by the formula (1) of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid and the like, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. Examples of such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, But are not limited to, but are not limited to, but are not limited to, but are not limited to, but are not limited to, halides, halides, halides, halides, halides, halides, But are not limited to, lactose, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene Hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-carboxylate, benzenesulfonate, Sulfonate, naphthalene-2-sulfonate, mandelate and the like.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a derivative of the formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, Followed by filtration and drying. Alternatively, the solvent and excess acid may be distilled off under reduced pressure, followed by drying and crystallization in an organic solvent.

In addition, the present invention encompasses not only the compound represented by the formula (1) and pharmaceutically acceptable salts thereof, but also solvates, optical isomers and hydrates thereof which can be prepared therefrom.

Further, the present invention relates to a process for preparing a compound represented by the following formula 1,

Reacting a compound represented by the formula (2) with a compound represented by the formula (3) to prepare a compound represented by the formula (4) (step 1);

A step of cyclizing the compound of formula (4) prepared in step 1 to prepare a compound of formula (5) (step 2); And

Converting the methoxy group of the compound represented by the formula (5) to a hydroxy group to prepare a compound represented by the formula (1) (step 3); and reacting the compound represented by the formula to provide.

[Reaction Scheme 1]

(In the above Reaction Scheme 1,

R ¹ , R ² And R < ³ > are independently as defined in the above formula (1)).

Hereinafter, a method for preparing the compound represented by Formula 1 according to the present invention will be described in detail.

In step (1), the compound represented by formula (2) is reacted with the compound represented by formula (3) to produce a compound represented by formula (4).

Solvents usable herein include tetrahydrofuran (THF); Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; Dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, xylene sulfonate, ethyl acetate, phenylacetate, phenyl Propionate, naphthalene-1-sulphonate, naphthalene-2-sulphonate, mandelate, sulphate, Etc. may be used.

The reaction temperature is preferably between 0 ° C and the boiling point of the solvent, and the reaction time is not particularly limited, but it is preferable to carry out the reaction for 12-36 hours.

In the method for preparing the compound represented by the formula (1) according to the present invention, the step (2) is a step of cyclizing the compound represented by the formula (4) prepared in the step (1) to prepare the compound represented by the formula (5).

Solvents usable herein include tetrahydrofuran (THF); Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; Dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, xylene sulfonate, ethyl acetate, phenylacetate, phenyl Propionate, naphthalene-1-sulphonate, naphthalene-2-sulphonate, mandelate, sulphate, , Acetonitrile, etc. may be used.

The reaction temperature is preferably between 0 ° C and the boiling point of the solvent, and the reaction time is not particularly limited, but it is preferable to carry out the reaction for 12-36 hours.

In the method for preparing the compound represented by the formula 1 according to the present invention, the step 3 is a step for converting the methoxy group of the compound represented by the formula 5 prepared in the step 2 into a hydroxy group to prepare a compound represented by the formula 1 And more specifically, a step of reacting a compound represented by the formula (5) in a solvent with a reducing agent to prepare a compound represented by the formula (1).

As the solvent, tetrahydrofuran (THF); Dioxane; Ether solvents including ethyl ether, 1,2-dimethoxyethane and the like; Lower alcohols including methanol, ethanol, propanol and butanol; Dimethylformamide (DMF), dimethylsulfoxide (DMSO), dichloromethane (DCM), dichloroethane, water, acetonazenesulfonate, toluene sulfonate, chlorobenzene sulfonate, xylene sulfonate, ethyl acetate, phenylacetate, phenyl Propionate, naphthalene-1-sulphonate, naphthalene-2-sulphonate, mandelate, sulphate, Etc., and it is preferable to use dichloromethane (DCM).

In addition, as the reducing agent, boron triborohydride (BBr ₃ ) is preferably used.

Further, the reaction is preferably carried out at a temperature of 0 ° C between the boiling point of the solvent, and the reaction time is not particularly limited, but the reaction is preferably carried out for 12 to 36 hours.

The present invention also provides a pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases containing the compound represented by the formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the pharmaceutical composition comprises IFN gamma (interferon gamma) production which is an inflammatory cytokine by interleukin-2 (IL-2), which is known as a T cell proliferation promoting factor, and 5-lipoxygenase (Interleukin-6, IL-6), and inhibits the differentiation into Th1 (T helper cells 1) cells in which the expression of Lipoxygenase, 5-LO is mainly expressed. The inflammatory diseases include Crohn's disease, inflammatory bowel disease, rheumatoid arthritis, asthma, atopy and the like.

As a result, the compounds of Examples 3, 4, 9, and 15 according to the present invention were found to have a LTC4 release rate of 5-Lipoxygenase The 5-LO IC ₅₀ (㎍ / mL) values showing inhibitory effects were all below 20 μg / mL. In particular, the compounds of Examples 4, 9 and 15 appeared to have a 5-LO IC ₅₀ (μg / mL) value of less than or equal to 7 μg / mL (see Table 2 of Experimental Example 1) .

In order to evaluate the inhibitory effect of the compound of the example according to the present invention on the inhibition of interleukin-6 (IL-6), the STAT3 reporter gene was assayed for the degree of inhibition of activity. As a result, , 5, 6, 13 and 14 showed that IL-6 IC ₅₀ (㎍ / mL), which shows interleukin-6 inhibitory effect, is less than 10 μg / mL. In particular, the compounds of Examples 5, 6 and 13 showed an IL-6 inhibiting effect remarkably better than that of IL-6 IC ₅₀ (㎍ / mL) of less than 5 μg / mL (see Table 3 of Experimental Example 2) .

Further, in order to evaluate the inhibitory effect of the compound of Example according to the present invention on the production of IFNγ (interferon gamma) by splenic T cells, IFNγ (interferon gamma) cytosine produced from splenic T cells (See FIG. 1 of Experimental Example 3) in a concentration-dependent manner by the treatment of the compounds of Examples 4 and 5 according to the present invention.

In addition, the ability of the compounds of the example according to the invention to inhibit As a result of experiments conducted to evaluate the inhibitory effect of IFNγ (interferon gamma) production, CD4 + T cell growth and IFN-γ cytokine production were markedly decreased by treatment with the concentrations of the compounds of Examples 4 and 5 (See FIG. 2 of Experimental Example 4).

Further, in order to evaluate the toxicity of the compound of the example according to the present invention to primary CD4 + T cells, the degree of cytotoxicity was analyzed using a cytotoxicity kit (EZ-cytox). As a result, The cytotoxicity of the compounds of Example 4 and Example 5 to primary CD4 + T cells was not observed to any significant degree, but a significant reduction of the cells was observed at 48 hours after the cell proliferation was actively observed, indicating cytotoxicity (See FIG. 3 of Experimental Example 5).

As a result of experiments to evaluate the growth inhibitory effect on the primary CD4 + T cells of the compounds of the present invention, the compounds of Examples 4 and 5 were found to be primary, CD4 < + > T cells (see FIG. 4 and FIG. 5 of Experimental Example 6).

In order to explain the cause of the decrease of activated CD4 + T cells by the compound of the example according to the present invention, the effect on cell death was analyzed. As a result, the apoptotic cell death marker (Apoptotic cell death marker, annexin V, and at the same time, an increased number of cells stained with the dead cell marker propidium iodide. From these results, it can be seen that the primary cytotoxic effect of primary CD4 + T cells by the compounds of Example 4 and Example 5 is excellent (see FIGS. 6 and 7 of Experimental Example 7).

As a result of analysis of proliferation factor IL-2 production in primary CD4 + T cells as inhibition of cell proliferation by concentration of the compound of Example according to the present invention was observed, it was found that the proliferation promoting cytokine IL-2 production was reduced. However, there was no decrease in the production of cytokine IL-2 promoting cell proliferation by the compound of Example 5. In addition, IL-2 mRNA expression was decreased in a concentration-dependent manner by the compound of Example 4, but IL-2 mRNA reduction by the compound of Example 5 was not observed (see FIGS. 8 and 9 of Experimental Example 8).

Furthermore, inhibition of cell proliferation by the concentration of the compound of the example according to the present invention was observed, so that intracellular cytokine production in activated primary CD4 + T cells was induced by intracellular cytokine staining As a result of evaluation, cell growth was inhibited by high concentration treatment and long time treatment of the compounds of Example 4 and Example 5, but generation of cytokines was confirmed by stimulation of phorbol 12-myristate 13-acetate / ionomycin (Ionomycin) . In addition, inhibition of IFN-y production was observed by both Example 4 and Example 5 compounds. Furthermore, inhibition of IL-2 production by the compound of Example 4 was confirmed, but it was reaffirmed that the compound of Example 5 did not affect IL-2 production (see FIG. 10 of Experimental Example 9).

As a result of experiments to evaluate the inhibitory effect of Th1 (T helper cells 1) cell differentiation according to the concentration of the compound according to the present invention, Th1 cells of CD4 + T cells (Fig. 11 of Experimental Example 10).

Further, as a result of conducting an experiment to evaluate the inhibitory activity of the compound of Example of the present invention according to the present invention, the compound of Example 4 according to the present invention was found to inhibit colitis in vivo using immune deficient RAG KO mice It was confirmed that the experiment also showed excellent colitis inhibitory effect (see FIGS. 12 and 13 of Experimental Example 11).

In addition, as a result of an experiment to evaluate the inflammatory cytokine IFN? Inhibitory activity in the colitis-induced animal model of the compound of Example according to the present invention, the compound of Example 4 according to the present invention showed that inflammatory cytokine It was confirmed that IFNg inhibitory activity was remarkably excellent (see FIG. 14 of Experimental Example 12).

In addition, as a result of conducting an experiment to perform colchicine-reduced histopathological analysis by the compound of the example according to the present invention, infiltration of inflammatory cells in the colon tissue and destruction of tissue were observed in the CD4 + T cell injected group, 4 compound injected group was decreased (see FIG. 15 of Experimental Example 13).

The compound of formula (I) according to the present invention may be administered orally or parenterally in a variety of formulations at the time of clinical administration. In the case of formulation, the compound of the present invention may be used as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients.

Solid form preparations for oral administration include tablets, patients, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like are included in addition to commonly used simple diluents such as water and liquid paraffin. .

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

The effective dose of the compound of the present invention on the human body may vary depending on the age, weight, sex, dosage form, health condition and disease severity of the patient, and is generally about 0.001-100 mg / kg / 0.0 > mg / kg / day. ≪ / RTI > It is generally from 0.07 to 7000 mg / day, preferably from 0.7 to 2500 / day, based on adult patients weighing 70, and may be administered once to several times per day It may be administered in divided doses.

Further, the present invention provides a health food composition for preventing or ameliorating an inflammatory disease containing the compound represented by the formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

In this case, the health food composition can be used for the production of interferon gamma (IFN gamma), which is an inflammatory cytokine, and 5-lipoxygenase (5-lipoxygenase) by the interleukin-2 (Interleukin-6, IL-6), and inhibits the differentiation into Th1 (T helper cells 1) cells in which the expression of Lipoxygenase, 5-LO is mainly expressed. The inflammatory diseases include Crohn's disease, inflammatory bowel disease, rheumatoid arthritis, asthma, atopy and the like.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following examples and experimental examples are illustrative of the present invention, and the present invention is not limited thereto.

< Example  1 (Z) -2 - ((4- ( Phenyldiazenyl ) Phenyl ) Amino) Benzo [d] Oxazole (7a) &lt; / RTI &gt;

Step 1: (Z) -l- (2- Hydroxy -5- Methoxyphenyl ) -3- (4- ( Phenyldiazenyl ) Phenyl ) Thio Production of urea

(Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazene was added in the same equivalent (1 eq) to 2-amino-4-methoxyphenol (100 mg, 25 mL was added to dissolve completely, and the mixture was stirred at room temperature for 24 hours. The precipitate formed after stirring was subjected to reduced pressure filtration or column chromatography to prepare (Z) -1- (2-hydroxy-5-methoxyphenyl) -3- (4- (phenyldiazenyl) phenyl) thiourea Respectively.

Step 2: (Z) -5- Methoxy -N- (4- ( Phenyldiazenyl ) Phenyl ) Benzo [d] Oxazole -2- Amine  Produce

(Z) -1- (2-hydroxy-5-methoxyphenyl) -3- (4- (phenyldiazenyl) phenyl) thiourea (100 mg, 1 eq) And 5 equivalents of potassium peroxide (KO ₂ ) was added 5 mL of acetonitrile. The mixture was reacted at room temperature for 12 hours with vigorous stirring, and poured into cooling water to dilute. After extraction with dichloromethane and drying over MgSO ₄ , the solvent was concentrated under reduced pressure to give (Z) -5-methoxy-N- (4- (phenyldiazenyl) phenyl) benzo [d] oxazol- .

Step 3: (Z) -2 - ((4- ( Phenyldiazenyl ) Phenyl ) Amino) Benzo [d] Oxazole (7a) &lt; / RTI &gt;

To 100 mg (1 eq) of the (Z) -5-methoxy-N- (4- (phenyldiazenyl) phenyl) benzo [d] oxazole-2- amine prepared in the above step 2, 5 mL of dichloromethane , And 0.2 mL of boron triborohydride (BBr ₃ ) was added thereto, followed by stirring at 0 ° C for 24 hours. After 24 hours, the reaction mixture was diluted with water, extracted with ethyl acetate, and then dried over MgSO ₄ , and the solvent was concentrated under reduced pressure. The residue was purified by column chromatography (ethyl acetate: hexane = 1: 3) to give the title compound as an orange powder (yield: 59%).

^{mp 249-250 ℃, 1 H-NMR} (DMSO-d 6, 400MHz) δ 10.978 (s, 1H), 9.270 (s, 1H), 7.953 (s, 4H), 7.854 ~ 7.876 (m, 2H), 7.507 (M, 3H), 7.293 (d, J = 8.8 Hz, 1H), 6.868 (d, J = 2.4 Hz, 1H), 6.571 (dd, J = 2.0 Hz, J = _{-FABMS Calcd for C 19 H 15 N} 4 O 2 (M + + H): 331.1192, Found: 331.1190.

< Example  2 > 2- (Phenylamino) benzo [d] Oxazole -5- All  (7b)

Was carried out in the same manner as in Example 1, except that isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane to obtain the title compound Was obtained in the form of brown powder (yield: 36%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.459 (s, 1H), 9.210 (s, 1H), 7.719 (d, J = 7.6Hz, 2H), 7.353 (t, J = 8.0Hz, 2H) , 7.232 (d, J = 8.4 Hz, 1H), 7.012 (t, J = 7.2 Hz, 1H), 6.798 (d, J = 2.4 Hz, 1H), 6.513 (dd, J = 2.4 Hz, J = , 1H), HR-FABMS Calcd for C 13 H 11 N 2 O 2 (M + + H): 227.0815, Found: 227.0818.

< Example  3 > 2 - ((4- Ethyl phenyl ) Amino) Benzo [d] Oxazole (7c) &lt; / RTI &gt;

Was prepared in the same manner as in Example 1, except that 1-ethyl-4-isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) To give the title compound as a pale brown powder (yield: 64%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.350 (s, 1H), 9.205 (s, 1H), 7.612 (d, J = 8.4Hz, 2H), 7.199 (dd, J = 8.4Hz, J = J = 2.4 Hz, 1H), 6.495 (dd, J = 2.4 Hz, J = 8.4 Hz, 1H), 2.565 (dd, J = 7.6 Hz, J = ), 1.168 (t, J = 7.6Hz, 3H), HR-FABMS Calcd for C 15 H 15 N 2 O 2 (M + + H): 255.1129, Found: 255.1128.

< Example  4 > 2 - ((3,4- Dichlorophenyl ) Amino) Benzo [d] Oxazole (7d) &lt; / RTI &gt;

Except that 1,2-dichloro-4-isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane, To give the title compound in the form of a white powder (yield: 31%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.855 (s, 1H), 9.263 (s, 1H), 8.095 (d, J = 2.4Hz, 1H), 7.589 ~ 7.650 (m, 2H), 7.269 ( d, J = 8.8Hz, 1H) , 6.846 (d, J = 2.4Hz, 1H), 6.553 (dd, J = 2.4Hz, J = 8.4Hz, 1H), HR-FABMS Calcd for C 13 H 9 C l2 N ₂ O ₂ (M &lt; ⁺ & gt ^; + H): 295.0036, Found: 295.0036.

< Example  5 > 2 - ((4- Hydroxyphenyl ) Amino) Benzo [d] Oxazole (7e) &lt; / RTI &gt;

Was carried out in the same manner as in Example 1, except that 4-isothiocyanatophenol was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane The title compound was prepared in the form of a pale gray powder (yield: 37%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.100 (s, 1H), 9.157 (s, 2H), 7.459 ~ 7.498 (m, 2H), 7.173 (d, J = 8.8Hz, 1H), 6.729 ~ 6.770 (m, 3H), 6.458 (dd, J = 2.0Hz, J = 8.6Hz, 1H), HR-FABMS Calcd for C 13 H 11 N 2 O 3 (M + + H): 243.0765, Found: 243.0764.

< Example  6> 2 - ((2-ethyl Phenyl ) Amino) benzo [d] Oxazole -5- All  (7f)

Except that 1-ethyl-2-isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane, To give the title compound as a pale brown powder (yield: 35%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 9.518 (s, 1H), 9.119 (s, 1H), 7.708 (d, J = 7.6Hz, 1H), 7.112 ~ 7.266 (m, 4H), 6.686 ( 2H), 1.128 (t, J = 7.6 Hz, 3H), HR-NMR (CDCl3) _{FABMS Calcd for C 15 H 15 N} 2 O 2 (M + + H): 255.1125, Found: 255.1128.

< Example  7 > 2 - ((4-Iodo Phenyl ) Amino) benzo [d] Oxazole -5- All  (7 g)

Except that 1-iodo-4-isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane. The title compound was prepared in the form of a pale brown powder (yield: 39%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.623 (s, 1H), 9.234 (s, 1H), 7.662 ~ 7.691 (m, 2H), 7.550 ~ 7.587 (m, 2H), 7.242 (d, J = 8.8 Hz, 1 H), 6.805 (d, J = 2.4 Hz, 1H), 6.526 (dd, J = 2.4 Hz, J = 8.4 Hz, 1H), HR-FABMS Calcd for C ₁₃ H ₁₀ IN ₂ O ₂ M &lt; ⁺ & gt ^; + H): 352.9781, Found: 352.9781.

< Example  8 > 2 - ((4- Isopropylphenyl ) Amino) Benzo [d] Oxazole (7h) &lt; / RTI &gt;

Except that 1-isopropyl-4-isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane, The title compound was prepared in the form of a pale brown powder (yield: 16%).

¹ H-NMR (DMSO-d ₆ , 400 MHz)? 10.415 (s, 1H), 7.553-7.588 (m, 4H), 7.186 (d, J = 8.4 Hz, 2H), 6.936 d, J = 6.8Hz, 6H) , HR-FABMS Calcd for C 16 H 17 N 2 O 2 (M + + H): 269.1285, Found: 269.1283.

< Example  9> 2 - ((4- ( Methyl thio ) Phenyl ) Amino) Benzo [d] Oxazole (7i) &lt; / RTI &gt;

Except that (4-isothiocyanatophenyl) (methyl) sulfine was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane. 1, the title compound was prepared in the form of a pale brown powder (yield: 28%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.486 (s, 1H), 9.226 (s, 1H), 7.666 ~ 7.695 (m, 2H), 7.287 ~ 7.316 (m, 2H), 7.224 (d, J J = 2.4 Hz, 1H), 6.508 (dd, J = 2.0 Hz, J = 8.6 Hz, 1H), 2.502 (dd, J = 2.0 Hz, J = 3H), HR-FABMS Calcd for C 14 H 13 N 2 O 2 S (M + + H): 273.0693, Found: 273.0692.

< Example  10 > 2 - ((3- Bromophenyl ) Amino) Benzo [d] Oxazole (7j) &lt; / RTI &gt;

Except that 1-bromo-3-isothiocyanatobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane. The title compound was prepared in the form of a pale brown powder (yield: 11%).

¹ H-NMR (DMSO-d ₆ , 400 MHz)? 10.767 (s, 1H), 9.237 (s, 1H), 8.047 (t, J = 1.8 Hz, 1H), 7.626 , 7.159 ~ 7.319 (m, 3H ), 6.821 (d, J = 2.4Hz, 1H), 6.525 (dd, J = 2.4Hz, J = 8.6Hz, 1H), HR-FABMS Calcd for C 13 H 10 BrN 2 ^{_{O 2 (M + + H)}} : 304.9922, Found: 304.992.

< Example  11 > 2 - ((4- Nitrophenyl ) Amino) Benzo [d] Oxazole (7k) &lt; / RTI &gt;

Except that 1-isothiocyanato-4-nitrobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane. The title compound was prepared in the form of a yellow powder (yield: 32%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 11.344 (s, 1H), 9.329 (s, 1H), 8.257 ~ 8.288 (m, 2H), 7.924 ~ 7.955 (m, 2H), 7.315 (d, J = 8.4Hz, 1H), 6.878 ( d, J = 2.4Hz, 1H), 6.598 (dd, J = 2.4Hz, J = 8.4Hz, 1H), HR-FABMS Calcd for C 13 H 10 N 3 O 4 ( M &lt; ⁺ & gt ^; + H): 272.0666, Found: 272.0667.

< Example  12 > 2- (p-tolyl Amino ) Benzo [d] Oxazole -5- All  (7l)

Except that 1-isothiocyanato-4-methylbenzene was used in place of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane To give the title compound as a pale brown powder (yield: 27%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.344 (s, 1H), 9.163 (s, 1H), 7.596 (d, J = 8.4Hz, 2H), 7.211 (d, J = 8.8Hz, 1H) , 7.155 (d, J = 7.6 Hz, 2H), 6.778 (d, J = 2.4 Hz, 1H), 6.493 (dd, J = 2.4 Hz, J = 8.6 Hz, 1H) _{-FABMS Calcd for C 14 H 13 N} 2 O 2 (M + + H): 241.0972, Found: 241.0975.

< Example  13 > 2 - ((4- Butylphenyl ) Amino) Benzo [d] Oxazole 5-ol (7 m)

Except that 1-butyl-4-isothiocyanatobenzene was used in place of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane To give the title compound in the form of a white powder (yield: 33%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 10.344 (s, 1H), 9.183 (s, 1H), 7.597 (dd, J = 2.8Hz, J = 6.6Hz, 2H), 7.211 (d, J = (Dd, J = 2.0 Hz, J = 8.8 Hz, 1H), 2.534 (d, J = 8.8 Hz, 1H), 7.162 , J = 7.8Hz, 2H), 1.496 ~ 1.571 (m, 2H), 1.231 ~ 1.349 (m, 2H), 0.896 (t, J = 7.4Hz, 3H), HR-FABMS Calcd for C 17 H 19 N 2 ^{_{O 2 (M + + H)}} : 283.1441, Found: 283.1442.

< Example  14 > 2 - ((2- Methoxy -4- Nitrophenyl ) Amino) Benzo [d] Oxazole (7n) &lt; / RTI &gt;

Except that 1-isothiocyanato-2-methoxy-4-nitrobenzene was used instead of (Z) -1- (4-isothiocyanatophenyl) The title compound was prepared in the form of a yellow powder (yield: 33%) by the same procedure as in Example 1 above.

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 8.623 (d, J = 9.2Hz, 1H), 8.015 (dd, J = 2.4Hz, J = 8.8Hz, 1H), 7.825 (d, J = 2.4Hz (D, J = 2.4 Hz, 1H), 7.313 (d, J = 8.8 Hz, 1H), 6.883 ), HR-FABMS Calcd for C 14 H 12 N 3 O 5 (M + + H): 302.0775, Found: 302.0771.

< Example  15 > 2 - ((4-amino Phenyl ) Amino) benzo [d] Oxazole -5- All  (7o)

Except that 4-isothiocyanatobenzenamine was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane, the procedure of Example 1 was repeated The title compound was prepared in the form of a black powder (yield: 34%).

^{1 H-NMR (DMSO-d} 6, 400MHz) δ 9.893 (s, 1H), 7.668 ~ 7.727 (m, 2H), 7.324 (dd, J = 2.0Hz, J = 6.8Hz, 1H), 7.143 (d, J = 8.8 Hz, 1H), 6.703 (d, J = 2.0 Hz, 1H), 6.564 (dd, J = 2.0 Hz, J = 6.8 Hz, 1H), 6.43 , 1H), 4.821 (s, 1H), 4.20 (d, J = 3.2Hz, 2H), HR-FABMS Calcd for C 13 H 12 N 3 O 2 (M + + H): 242.0924, Found: 242.0924.

< Example  16 > 2 - ((3-hydroxy Phenyl ) Amino) benzo [d] Oxazole -5- All  (7p)

Was carried out in the same manner as in Example 1, except that 3-isothiocyanatophenol was used instead of (Z) -1- (4-isothiocyanatophenyl) -2-phenyldiazane The title compound was prepared in the form of a light powder (yield: 20%).

¹ H-NMR (DMSO-d ₆ , 400 MHz)? 10.332 (s, IH), 9.420 (s, IH), 9.215 J = 2.4 Hz, 1H), 6.506 (dd, J = 2.4 Hz, J = 8.6 Hz, 1H), 6.395-6.424 (m, m, 1H), HR-FABMS Calcd for C 13 H 11 N 2 O 3 (M + + H): 243.0764, Found: 243.0767.

The chemical structures of the compounds prepared in Examples 1-16 are summarized in Table 1 below.

Example Chemical structure Example Chemical structure One

9

2

10

3

11

4

12

5

13

6

14

7

15

8

16

< Experimental Example  1 > 5- Lipoxygenase (5- Lipoxygenase ) Evaluation of inhibitory activity

In order to evaluate the inhibitory effect of 5-Lipoxygenase activity of the compounds of the examples according to the present invention, the following experiment was conducted.

1-1. Confirmed bone marrow  Derived bone marrow-derived mast cells, BMMCs ) Preparation

Herein, LTC4 represented by the following formula (6) is a kind of cysteinyl leukotrienes, and leukotriene is an eicosanoid inflammatory mediator acting as a signal molecule.

[Chemical Formula 6]

First, to prepare bone marrow-derived mast cells (BMMC), bone marrow was collected from male Balb / c mice, and the cells were treated with interleukin-3 (IL-3, Sigma I4144, 2 ng / (2 mM L-glutamine, 25 mM HEPES buffer, 2 mg / mL sodium bicarbonate, 100 units / mL penicillin G, 100 lg / mL streptomycin sulfate and 0.25 0.0 &gt; lg / ml &lt; / RTI &gt; amphotericin B) for 4 weeks.

Three weeks after the culture, it was confirmed that more than 98% of BMMC was present by staining using toluidine blue.

2-2. TLC4  Evaluation of release suppression effect

The BMMCs were suspended in a nutrient enrichment medium at a concentration of 1 x 106 cells / mL, treated with the compounds of Examples 3, 4, 9 and 15 dissolved in DMSO (final DMSO concentration of less than 0.5%), % CO2 incubator.

Stimulation was then performed with stem cell factor (SCF, Sigma S9915, 100 ng / mL) for 20 min and assayed using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) The concentration of LTC4 was measured in the supernatants according to the manufacturer's instructions. All experiments were performed in triplicate and the inhibitory effect of LTC4 release was assessed by calculating the% reduction in LTC4 release. The results are shown in Table 2 below.

Example 5-LO
IC ₅₀ ([mu] g / mL) One - 2 - 3 16.11 4 4.97 5 - 6 - 7 - 8 - 9 6.43 10 - 11 - 12 - 13 - 14 - 15 5.59 16 -

As shown in the above Table 2, the compounds of Examples 3, 4, 9 and 15 according to the present invention showed that the 5-LO IC ₅₀ (㎍ / mL) value showing the inhibitory effect of LTC4 release was less than 20 μg / mL. In particular, the compounds of Examples 4, 9 and 15 appeared to have a 5-LO IC ₅₀ (μg / mL) value of less than 7 μg / mL and thus had a significantly superior 5-LO inhibitory effect.

< Experimental Example  2> interleukin-6 ( Interleukin -6, IL-6)

In order to evaluate the inhibitory effect of the compound of the example according to the present invention on the production of interleukin-6 (IL-6), it was confirmed through the degree of inhibition of the STAT3 reporter gene.

Human liver cancer cell line HepG2 was cultured in MEM supplemented with 10% (v / v) fetal bovine serum, streptomycin (100 U / ml) and penicillin (100 U / Korea) medium at 37 ° C and 5% CO ₂ . Cells were allowed to grow to 80% of 6 well cell culture dishes.

Subsequently, 50 쨉 l of the serum-free medium was exchanged and 0.1 쨉 g pSTAT3-TA-Luc6 construct was transfected. The transformed cells were serum starvated with 1% BSA (Bovine Serum Albumin) / DMEM (Dulbecco's Modified Eagle Medium), treated with the compounds of Examples 3, 5, 6, 13 and 14 for 1 hour, Interleukin-6 (IL-6) was added and incubated for 3 hours.

After washing with PBS, 50 μl of lysis buffer was added and shaken vigorously for 1 minute. 30-100 μl of luciferase substrate (luminescent enzyme substrate, Promega luciferase assay system) Min. The absorbance at OD 540 nm (optical density) of the standard diluent expressed by various concentrations of interleukin-6 (IL-6) was confirmed. The results are shown in Table 3 below.

Example IL-6 (STAT 3)
IC ₅₀ ([mu] g / mL) One - 2 - 3 9.12 4 - 5 4.15 6 3.47 7 - 8 - 9 - 10 - 11 - 12 - 13 3.69 14 7.27 15 - 16 -

As shown in Table 3, the compounds of Examples 3, 5, 6, 13, and 14 according to the present invention had IL-6 IC ₅₀ (㎍ / mL) Respectively. In particular, the compounds of Examples 5, 6 and 13 showed an IL-6 IC ₅₀ (㎍ / mL) value of less than 5 μg / mL and thus showed a remarkably superior IL-6 inhibitory effect.

< Experimental Example  3> in splenic T cells IFN γ (Interferon gamma) production inhibition

In order to evaluate the inhibitory effect of IFNγ (interferon gamma) production in spleen-existing T cells of the compound of Example according to the present invention, the following experiment was conducted. Here, the ability to inhibit IFN? Production is due to the anti-inflammatory effect.

3-1. Isolation and Culture of Splenic T Cells

Prepare the spleen of male C57BL6 mouse single cell suspension (single cell suspension) the process for manufacturing and, RBC (Red Blood Cell) dissolution (lysis) buffer (155 mM NH4Cl, 10 mM KHCO 3, 0.5 M EDTA (pH 8.0)) After erythrocytes were removed, the same volume of 10% FBS RPMI (Roswell Park Memorial Institute) medium was added to neutralize. To completely remove the RBC lysis buffer, cells washed twice with 10% FBS RPMI medium were counted, and 10% FBS RPMI medium was added to make 5x10 ⁶ cells / mL. The culture plate coated with anti-CD3 (1 μg / mL) at 4 ° C. on the previous day was washed once with PBS and 10% FBS RPMI medium, and the cells counted from the above were inoculated seeding) to induce activation. After 2 hours of cell stimulation, the compounds of Example 4 and Example 5 were treated at various concentrations, respectively.

3-2. IFN Confirmation of production of γ

After the compound was treated and cultured for 48 hours, the cell culture was recovered and the production of IFNγ was confirmed by ELISA method.

Specifically, purified anti-mIFN gamma (1 μg / ml, BD Pharmingen) antibody and 0.1 M Na ₂ HPO ₄ are mixed in a 96-well plate (Costar) and left overnight at 4 ° C. The plates were washed 3 times with 0.05% Tween 20 / PBS and blocked with 1% BSA / PBS for 2 hours at room temperature. After washing the plate three times with 0.05% Tween 20 / PBS once more, the cell culture solution of Experimental Example 3-1 was diluted to 1:10, and left overnight at 4 ° C. After washing the plate three times with 0.05% Tween 20 / PBS, add biotinylated anti-IFNγ (1 μg / ml, BD Pharmingen) to 1% BSA / PBS and leave at room temperature for 1 hour. The plate was washed three times with 0.05% Tween 20 / PBS, and then alkaline phosphatase-conjugated streptavidin (1 μg / ml, BD Pharmingen) was added to 1% BSA / do. After washing the plate three times with 0.05% Tween 20 / PBS, diluted 1: 500 phosphatase substrate was added to diethanol amine buffer (10% Diethanolamine, 0.5 mM MgCl ₂ ) Add absorbance read at 405 nm and 450 nm with an ELISA plate reader (Molecular devices). The purified recombinant mIFNγ was used as a standard cytokine, and a linear curve was quantified by serial dilution. Then, a standard curve graph was prepared and used for analysis. The results of IFNγ measurement are shown in FIG. 1 Respectively.

FIG. 1 is a graph showing the results of evaluating IFN gamma (interferon gamma) production inhibitory effects of the compounds of Examples 4 and 5 according to the present invention.

As shown in Fig. 1, IFN gamma (interferon gamma) cytokines produced in splenic T cells were decreased in a concentration-dependent manner by the treatment of the compounds of Example 4 and Example 5 according to the present invention.

< Experimental Example  4> primary (primary) CD4 + T cell growth inhibition

The following experiment was conducted to evaluate the inhibitory effect of the compound of Example according to the present invention on the primary CD4 + T cell growth.

4-1. CD4 + Positive T cells ( CD4 + T cell)

First, CD4 + T cells were isolated from the spleen and lymph nodes of male C57BL6 mice. A single cell suspension was prepared from the spleen and lymph node, and red blood cells were removed. The cells were washed with Mini Macs buffer (2 mM EDTA, 0.5% BSA in PBS) per 10 x ¹⁰ cells And 25 μl of CD 4 (L3T4) microbeads (Miltenyi Biotec) were mixed and shaded and left at 4 ° C for 20 minutes. The MACS separation LS column was installed in the magnetic field and the LS column was equilibrated with 3 mL of Mini MACS buffer. Anti-CD4 Ab (Antibody) bound cells were added to the LS column, washed twice with 3 mL of Mini MACS buffer, the LS column was separated from the magnetic field, and 5 mL of Mini MACS buffer was added to the LS column to remove the plunger ) Were used to isolate CD4 + T cells.

The separated cells were seeded on a coated plate coated with anti-CD3 (1 μg / mL) and anti-CD28 (1 μg / mL) at a concentration of 2 × 10 ⁶ cells / mL of recombinant human IL-2 was added to promote cell growth.

4-2. Example  4 and Example  5 Compound treatment CD4 + T cell growth inhibition

The isolated CD4-positive T cells were stimulated with anti-CD3 and anti-CD28 antibodies to induce activation. At the same time, the compounds of Example 4 and Example 5 were further treated by concentration, and after 48 hours, morphological changes, degree of proliferation, and IFN-y cytokine concentration were confirmed. The results are shown in Fig.

FIG. 2 is a graph showing the results of evaluating IFN gamma (interferon gamma) production inhibitory effects of the compounds of Examples 4 and 5 according to the present invention.

As shown in FIG. 2, the treatment of the concentration of the compounds of Examples 4 and 5 showed that CD4 + T cell growth and IFN-y cytokine production were significantly reduced.

< Experimental Example  5> primary (primary) CD4 + Evaluation of Toxicity to T Cells

To evaluate the toxicity of the compounds of the examples according to the present invention to primary CD4 + T cells, the degree of cytotoxicity was analyzed using a cytotoxicity kit (EZ-CyTox).

More specifically, after seeding the CD4 + T cells isolated in Experimental Example 4-1, the compounds of Example 4 and Example 5 were treated for 24 hours or 48 hours depending on the concentration. After 24 hours or 48 hours, the cell culture was dispensed into a 96-well plate. The EZ-CyTox solution was added to each well, and incubated for 30 minutes in a 5% CO ₂ incubator at 37 ° C. The plate was gently shaken for 1 minute and absorbance was measured at 450 nm using a plate reader (Molecular devices). Samples not treated with the example compounds were set as a control group to be 100%, and compared to other samples,% was expressed as a number of surviving cells. The results are shown in Fig.

FIG. 3 is a graph showing the results of evaluating the toxicity of the compounds of Examples 4 and 5 according to the present invention to primary CD4 + T cells. FIG.

As shown in FIG. 3, no cytotoxicity was observed in primary and secondary CD4 + T cells when 24 hours had elapsed. However, at 48 hours after active cell proliferation, A significant reduction in the number of cells around 20% was observed.

< Experimental Example  6> primary culture CD4 + T cell growth inhibition

In order to evaluate the growth inhibitory effect on the primary cultured CD4 + T cells of the compounds of the examples according to the present invention, the following experiment was conducted.

The suspension was prepared so that the CD4 + T cells isolated in Experimental Example 4-1 were 2 x 10 ⁶ cells / mL, and CFSE (Carboxyfluorescein succinimidyl ester) was added thereto to give a concentration of 5 μM. The cells were shaken vigorously, shaded, reacted at room temperature for 5 minutes, washed with 10% FBS RPMI medium, and then washed once more with RPMI medium. Live cells were counted again and seeded into anti-CD3-coated plates at 2 x 10 ⁶ cells / mL to stimulate. At the same time as the inoculation, the compounds of Example 4 and Example 5 were treated by concentration and cultured for 48 hours or 72 hours, and the cells were recovered and staining buffer (2% FBS dissolved in PBS) was added. Subsequently, the degree of division of cells labeled with CFSE (Carboxyfluorescein succinimidyl ester) was measured by FACs Calibur (BD Bioscience) and cell density was analyzed using CellQuest software. The results are shown in Fig. 4 and Fig.

FIG. 4 is an image showing evaluation of growth inhibitory effect of primary cultured CD4 + T cells of Example 4 compound according to the present invention. FIG.

FIG. 5 is an image showing evaluation of growth inhibitory effect of primary cultured CD4 + T cells of Example 5 compound according to the present invention. FIG.

As shown in Figs. 4 and 5, the compounds of Example 4 and Example 5 inhibited the growth of primary cultured CD4 + T cells in a concentration-dependent manner.

< Experimental Example  7> CD4 + T cell death ( apoptotic  cell death induction effect evaluation

In order to explain the cause of the numerical reduction of activated CD4 + T cells by the compound of the example according to the present invention, the influence on cell death was analyzed.

The activated CD4 + T cells, obtained for Example 4 and Example handle 5 compound, and the cells were collected 72 hours after 1x10 ⁶ cells / mL are to 1x binding buffer (binding buffer) (10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2). Then, 1 × 10 ⁵ cells were mixed with FITC (fluorescein isothiocyanate) -buffered annexin V antibody and propidium iodide, mixed and shaded, and allowed to react at room temperature for 15 minutes. Staining buffer, Cells were washed with 2% FBS in PBS, resuspended and analyzed with FACs Calibur (BD Bioscience). The killed cells were quantitatively analyzed using CellQuest software, and the results are shown in FIG. 6 and FIG.

FIG. 6 is an image obtained by treating the activated CD4 + T cells with the compounds of Example 4 and Example 5 for 72 hours at different concentrations, and then staining with annexin V to confirm cell death.

FIG. 7 is an image obtained by treating the activated CD4 + T cells with the compounds of Example 4 and Example 5 for 72 hours for each concentration and then staining with propidium iodide to confirm cell death.

As shown in FIGS. 6 and 7, the expression of annexin V, which is an apoptotic cell death marker, was confirmed by the compounds of Examples 4 and 5, and at the same time, Propidium iodide confirmed the increase in stained cells. From these results, it can be seen that the cytotoxic effect of primary cultured CD4 + T cells by the compounds of Examples 4 and 5 is excellent.

< Experimental Example  8> CD4 + IL-2 (IL-2) production in T cells

IL-2 production was assayed in CD4 + T cells as inhibition of cell proliferation by concentration of the compound of the example according to the present invention was observed.

8-1. Measurement of IL-2 concentration

First, the separated CD4 + T cells were seeded into anti-CD3 (1 μg / mL) / CD28 (1 μg / mL) coated plates for activation, and reconstituted human IL-2 and cells were expanded by adding RPMI medium supplemented with 10 U / mL recombinant human IL-2. Cells were seeded to a plate coated with anti-CD3 (0.5 μg / mL) at 1 × 10 ⁶ cells / mL, and after 48 hours, the cell culture medium and cells were obtained and subjected to ELISA Linked ImmunoSorbent Assay). Antibodies were purified using purified anti-mIL-2 (1 μg / mL, BD Pharmingen) and purified linear recombinant mIL2 was used as a standard cytokine to quantify the primary linearity by serial dilution, A standard curve graph was created and used for analysis. The results are shown in Fig.

FIG. 8 is a graph showing the results of analysis of proliferation factor IL-2 production in primary CD4 + T cells according to the concentrations of the compounds of Examples 4 and 5 according to the present invention.

As shown in Fig. 8, the production of the cell proliferation promoting cytokine IL-2 by the compound of Example 4 was decreased. However, there was no decrease in the production of cytokine IL-2 promoting cell proliferation by the compound of Example 5.

8-2. IL-2 mRNA  Measure level

Add 500 μl of TRIzol solution (Invitrogen) to CD4 + T cells to dissolve the cells, add 100 μl of chloroform, and shake vigorously for 15 seconds. Thereafter, the transparent water layer is separated by centrifugation at 12,000 rpm at 4 DEG C for 15 minutes, and the RNA is precipitated by mixing with 100 mu l of isopropanol. After centrifugation at 12,000 rpm for 15 minutes at 4 ° C, the remaining solution except for the precipitated RNA is completely removed. Then, 1 ml of 75% ethanol is added thereto, and the mixture is centrifuged at 12,000 rpm for 5 minutes at 4 ° C. The total RNA precipitated (total RNA) is dissolved in 0.1% DEPC (diethyl pyrocarbonate) water and the RNA concentration is quantified.

MuLV reverse transcriptase (Promega), and 5 μg / ml of RNA were mixed with 0.5 μg of oligo-dT and allowed to stand at 72 ° C for 5 minutes. 10 mM dATP, 10 mM dGTP, 10 mM dCTP, and 25 units of ribonuclease inhibitor (Promega). Thereafter, cDNA was synthesized by reacting at 42 DEG C for 1 hour.

The synthesized cDNA was mixed with 0.25 μl and 2X cyber green (SYBR green, Toyobo), and analyzed by real-time PCR (ABI) using the following primers. The results are shown in FIG.

beta-actin-FWD 5'-AGA GGG AAA TCG TGC GTG AC-3 '

β-actin-REV 5'-CAA TAG TGA TGA CCT GGC CGT-3 '

IL-2-FWD 5'-CCT GAG CAG GAT GGA GAA TTA CA-3 '

IL-2-REV 5'-TCC AGA ACA TGC CGC AGA G-3 '

FIG. 9 is a graph showing the results of analysis of proliferation factor IL-2 mRNA expression in CD4 + T cells according to the concentrations of the compounds of Examples 4 and 5 according to the present invention.

As a result, it was confirmed that the level of IL-2 mRNA was decreased in dependence on the compound concentration of Example 4 as shown in Fig. 9, but not by the compound treatment of Example 5.

< Experimental Example  9> Evaluation of cytokine production inhibitory effect

As the inhibition of cell proliferation by the concentration of the compound of the example according to the present invention was observed, intracellular cytokine production in activated CD4 + T cells was evaluated by intracellular cytokine staining method.

First, CD4 + T cells obtained by isolation were activated by seeding on anti-CD3 (1 μg / mL) / anti-CD28 (1 μg / mL) coated plate and incubated with recombinant human IL- 2 (recombinant human IL-2) supplemented with 10 U / mL of RPMI medium was added to expand the cells. The cells were seeded onto anti-CD3 (0.5 μg / mL) coated plates at 1 × 10 ⁶ cells / mL, and then cultured for 24 hours to obtain intracellular cytokine analysis.

CD4 + T cells activated for 4 days were treated for 4 hours with 10 ng / mL of phorbol 12-myristate 13-acetate and 1 μg / mL of ionomycin and then incubated for 2 hours before harvesting (Monensin) 4 [mu] M to obtain cells. 1x10 ⁵ cells were fixed with Cytofix / cytoperm solution (BD Biosciences) at 4 ° C for 20 minutes, washed with 1 ml of 1X Perm / wash solution and then incubated with PE-conjugated IFN- γ antibody and an APC-conjugated IL-2 antibody were added to the perm / wash solution. The cells were washed with perm / wash solution (500 μl), and then stained with FACs Calibur (BD Bioscience) by adding staining buffer (2% FBS dissolved in PBS). Cell population was analyzed using CellQuest software. The results are shown in Fig.

10 is an image showing the results of analysis of the effects of the compounds of Examples 4 and 5 according to the present invention on the intracellular cytokine production in CD4 + T cells by concentration.

As shown in FIG. 10, the cell growth was inhibited by the high-concentration treatment and the long-time treatment of the compounds of Examples 4 and 5, but the stimulation of cytokine (PMA) by phorbol 12-myristate 13-acetate / ionomycin Generation was confirmed. In addition, inhibition of IFN-y production was observed by both Example 4 and Example 5 compounds. Furthermore, inhibition of IL-2 production by the compound of Example 4 was confirmed, but it was reaffirmed that the compound of Example 5 did not affect IL-2 production.

< Experimental Example  10> CD4 + T cells Th1 (T helper cells 1) cells

The following experiments were conducted to evaluate the inhibitory effect of Th1 (T helper cells 1) cell differentiation according to the concentration of the compound of the example according to the present invention.

After primary CD4 + T cells were separated, the compounds of Example 4 and Example 5 according to the present invention were treated by concentration while inducing differentiation into Th1 cells producing IFN-y. After induction of differentiation in vitro for 5 days, anti-CD3 was re-stimulated for 24 hours, and cytokine IFN-γ production was confirmed by ELISA according to the method of Experimental Example 3-1, and anti -mIFN? (1 占 퐂 / mL, BD Pharmingen) antibody was used. The results are shown in Fig.

11 is a graph showing the results of evaluating the inhibitory effect of Th1 (T helper cells 1) cell differentiation according to the concentration of the compounds of Examples 4 and 5 according to the present invention.

As shown in Fig. 11, the treatment of Example 4 and Example 5 showed that the differentiation of CD4 + T cells into Th1 cells was inhibited in a concentration-dependent manner.

< Experimental Example  11> Assessment of inhibitory activity against irritable colitis

The following experiments were conducted to evaluate the inhibitory activity of the compounds of the examples according to the present invention on irritable colitis.

Immune deficient RAG KO mice were injected with CD4 + T cells and induced T cell mediated enteritis for 6 weeks, followed by intraperitoneal injection of DMSO (vehicle) or Example 4 at 10 mg / kg for 2 weeks Symptoms of irritable colitis were observed. The results are shown in Fig. 12 and Fig.

12 is a graph showing the results of evaluating the inhibitory activity against the stool consistency of the compound of Example 4 according to the present invention.

13 is a graph showing the results of evaluating how the compound of Example 4 according to the present invention affects the colon length.

As shown in FIG. 12, the symptoms of enteritis were significantly increased in the DMSO-treated group, while the symptoms of enteritis were decreased in the compound-treated group of Example 4.

In addition, as shown in Fig. 13, colon shortening clearly observed in Dextran sulfate sodium-mediated enteritis in the compound group treated with Example 4 was not confirmed.

Therefore, it can be seen that the compound of Example 4 according to the present invention exhibits an excellent colitis inhibitory effect even in an in vivo experiment using immune deficient RAG KO mice that induced colitis.

< Experimental Example  12> Inflammatory cytokines in colitis induced animal models IFN γ inhibitory activity evaluation

The following experiment was conducted to evaluate the inhibitory activity of the compound of Example according to the present invention on inflammatory cytokine IFN? Inhibition in a colitis-induced animal model.

Immune deficient RAG KO mice were injected with CD4 + T cells and induced inflammatory cytokine IFNγ production after inducing T cell mediated enteritis for 6 weeks. Thereafter, intraperitoneal injection of DMSO (vehicle) or the compound of Example 4 at 10 mg / kg for 2 weeks was confirmed to decrease IFN gamma production.

Cells were isolated from the mesenteric lymph nodes and stimulated with anti-CD3 antibody. The production of cytokines ex vivo was confirmed by intracellular cytokine staining and ELISA. The results are shown in Fig.

14 is a graph showing the results of evaluating the inflammatory cytokine IFN? Inhibitory activity of the compound of Example 4 according to the present invention in a colitis-induced animal model.

As shown in FIG. 14, IFN gamma production was increased in the group injected with CD4 + T cells in RAG KO mice, and IFN gamma production was significantly inhibited in the group injected with the compound of Example 4.

Therefore, it can be seen that the compound of Example 4 according to the present invention is remarkably superior in the IFNg inhibitory activity of the inflammatory cytokine in a colitis-induced animal model.

< Experimental Example  13> Reduced colitis Histopathological analysis

Reduction of Colitis by Example Compounds According to the Present Invention The following experiments were conducted to perform histopathological analysis.

Immune deficient RAG KO mice were injected with CD4 + T cells and induced T cell mediated enteritis for 6 weeks, followed by intraperitoneal injection of DMSO (vehicle) or Example 4 at 10 mg / kg for 2 weeks Symptoms of irritable colitis were observed. The results are shown in Fig.

Fig. 15 is an image showing the result of performing colitis-reduced histopathological analysis by the compound of Example according to the present invention.

As shown in Fig. 15, infiltration and tissue destruction of inflammatory cells in the colon tissue were observed in the CD4 + T cell injected group, but the inflammatory findings were decreased in the compound injected group of Example 4 as histopathologically.

< Formulation example  1> Preparation of pharmaceutical preparations

1-1. Manufacture of Powder

2 g of the compound of formula 1 according to the invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

1-2. Manufacture of tablets

100 mg of the compound of formula 1 according to the present invention

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

1-3. Preparation of capsules

100 mg of the compound of formula 1 according to the present invention

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

1-4. Manufacture of granules

The compound of formula 1 according to the present invention, 150 mg

Soybean extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 μl of 30% ethanol was added and the mixture was dried at 60 ° C to form granules, which were filled in a capsule.

Claims (10)

Claims 1. A compound represented by the following formula (1), an optical isomer thereof, or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

(In the formula 1,
R ¹ , R ² and R ³ are independently selected from the group consisting of -H, -OH, halogen, C _{1 -10} linear or branched alkyl, C _1-10 linear or branched alkoxy, -SR ⁴ , -NO ₂ , -NH ₂ Or a -N = N-Ph, wherein R ⁴ is a straight or branched alkyl of C _{1 -10).}
The method according to claim 1,
R ¹ is -H, -OH, halogen, C _{1 -5} straight or branched chain alkyl or C _{1 -5} straight or branched alkoxy;
R ² is selected from the group consisting of -H, -OH, halogen, C _{1 -5} straight or branched chain alkyl, C _{1 -5} straight or branched alkoxy, -SR ⁴ , -NO ₂ , -NH ₂ Or a -N = N-Ph, wherein R ⁴ is a straight or branched alkyl of C _{1 -5;} And
R ³ is -H, C _{1 -5} straight or branched alkyl or C _{1 -5} straight or branched alkoxy, an optical isomer thereof, or a pharmaceutically acceptable salt thereof.
The method according to claim 1,
R ¹ is -H, -OH, -Cl or -Br;
R ² is -H, -OH, -CH ₃ , -CH ₂ CH ₃ , -CH (CH ₃ ) ₂ , - (CH ₂ ) ₃ CH ₃ , -Cl, -I, -SCH ₃ , -NO ₂ , -NH ₂ or -N = N-Ph; And
R ³ is -H, -CH ₂ CH ₃ or -OCH ₃ , an optical isomer thereof, or a pharmaceutically acceptable salt thereof.
The method according to claim 1,
The compound represented by the formula (1) is any one selected from the group consisting of the following compounds, an optical isomer thereof, or a pharmaceutically acceptable salt thereof:
(1) (Z) -2 - ((4- (phenyldiazenyl) phenyl) amino) benzo [d] oxazol-5-ol;
(2) 2- (phenylamino) benzo [d] oxazol-5-ol;
(3) 2 - ((4-ethylphenyl) amino) benzo [d] oxazol-5-ol;
(4) 2 - ((3,4-dichlorophenyl) amino) benzo [d] oxazol-5-ol;
(5) 2 - ((4-hydroxyphenyl) amino) benzo [d] oxazol-5-ol;
(6) 2 - ((2-ethylphenyl) amino) benzo [d] oxazol-5-ol;
(7) 2 - ((4-iodophenyl) amino) benzo [d] oxazol-5-ol;
(8) 2 - ((4-isopropylphenyl) amino) benzo [d] oxazol-5-ol;
(9) 2 - ((4- (methylthio) phenyl) amino) benzo [d] oxazol-5-ol;
(10) 2 - ((3-bromophenyl) amino) benzo [d] oxazol-5-ol;
(11) 2 - ((4-nitrophenyl) amino) benzo [d] oxazol-5-ol;
(12) 2- (p-Tolylamino) benzo [d] oxazol-5-ol;
(13) 2 - ((4-butylphenyl) amino) benzo [d] oxazol-5-ol;
(14) 2 - ((2-methoxy-4-nitrophenyl) amino) benzo [d] oxazol-5-ol;
(15) 2 - ((4-aminophenyl) amino) benzo [d] oxazol-5-ol; And
(16) 2 - ((3-hydroxyphenyl) amino) benzo [d] oxazol-5-ol.
As shown in Scheme 1 below,
Reacting a compound represented by formula (2) with a compound represented by formula (3) to prepare a compound represented by formula (4) (step 1);
A step of cyclizing the compound of formula (4) prepared in step 1 to prepare a compound of formula (5) (step 2); And
(Step 3) of converting the methoxy group of the compound represented by the formula (5) prepared in the step 2 into a hydroxy group to prepare a compound represented by the formula (1) Way:
[Reaction Scheme 1]

(In the above Reaction Scheme 1,
R ¹ , R ² And R &lt; ³ &gt; are independently as defined in formula (1) of claim 1).
A pharmaceutical composition for the prophylaxis or treatment of inflammatory diseases containing the compound represented by the general formula (1) of claim 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
The method according to claim 6,
Wherein said pharmaceutical composition inhibits the proliferation of T cells and the expression of IFNy (interferon gamma), an inflammatory cytokine.
The method according to claim 6,
Wherein said inflammatory disease is a disease selected from the group consisting of Crohn's disease, inflammatory bowel disease, rheumatoid arthritis, asthma and atopy.
A health food composition for preventing or ameliorating an inflammatory disease containing the compound represented by the general formula (1) of claim 1, an optical isomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.
10. The method of claim 9,
Wherein said inflammatory disease is a disease selected from the group consisting of Crohn's disease, inflammatory bowel disease, rheumatoid arthritis, asthma and atopy.

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