Novel compounds and their use for treatment of autoimmune diseases

Abstract

The present invention relates to novel compounds and their use for the treatment of autoimmune diseases. The pharmaceutical composition for treating or preventing autoimmune diseases, comprising the novel compound of the present invention, not only can control inflammation, but also can repair immune balance and damaged tissues, thereby being hopeful to repair intestinal tissue homeostasis.

Description

Technical Field

[0001] The present invention relates to novel compounds and their use in treating autoimmune diseases. Background Art

[0002] The human body can be protected from pathogens by immune responses. Biological defense mechanisms against foreign microorganisms such as viruses and bacteria are generally divided into innate immunity and adaptive immunity, which are mediated by cytokines secreted mainly by immune-related cells.

[0003] The immune system is used to protect the body from antigens (i.e., harmful foreign substances). Types of these antigens include bacteria, viruses, toxins, cancer cells, and blood and tissues from other humans or animals. The immune system produces antibodies and cytokines to destroy these harmful substances. If there is an autoimmune disorder, the immune system cannot distinguish between its body organs and harmful antigens, and may destroy normal tissues. Diseases induced by such responses as described above are called autoimmune diseases.

[0004] Aryl Hydrocarbon Receptor (AHR) is a ligand-dependent transcription factor belonging to the PER-ARNT-SIM (PAS) superfamily and is mainly expressed in immune cells, epithelial cells, endothelial cells, and stromal cells of barrier tissues. AHR is an environmental sensor and detects not only xenobiotic ligands such as environmental pollutants (e.g., dioxins), but also physiological ligands produced by cells, microorganisms, and foods.

[0005] The inactivated form of AHR forms a complex (AHR chaperone complex) with the Hsp90:XAP2:p23:Src chaperone in the cytoplasm and maintains a structure with high affinity for the ligand. When AHR is activated after ligand binding, the complex moves to the nucleus, and AHR separates from the chaperone complex and binds to the AHR responsive DNA element (xenobiotic response element, XRE) located in the upstream regulatory region of the target gene to regulate the expression of the target gene. Non-toxic immunomodulatory ligands that can activate AHR in vivo can be developed as new therapeutic agents for autoimmune diseases. Summary of the invention

[0006] Problem that the invention aims to solve

[0007] The object of the present invention is to provide novel compounds, stereoisomers thereof or pharmaceutically acceptable salts thereof.

[0008] Furthermore, another object of the present invention is to provide a novel compound useful for preventing and treating autoimmune diseases, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0009] Furthermore, another object of the present invention is to provide a pharmaceutical composition for preventing or treating an autoimmune disease, which comprises the novel compound, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0010] Solutions for solving problems

[0011] 1. A compound represented by the following formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0012] [Formula 1]

[0013]

[0014] (wherein A is hydrogen, halogen, hydroxyl, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy, dimethylamine, -NO2, -CN, -COOR2 or -S(=O)2R2,

[0015] B is hydrogen, C1-C3 alkyl, phenyl, acetyl, -CH2C(=O)OR2, -C(=O)OR2 or -S(=O)2R2, R1 is a substituted or unsubstituted 5-7 membered heterocycle or -NH2, and

[0016] R2 is C1-C3 alkyl).

[0017] 2. The compound according to 1 above, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the substituted 5-7 membered heterocyclic ring is a 5-7 membered heterocyclic ring substituted by a C1-C3 alkyl group, a hydroxyl group or a dimethylamine group.

[0018] 3. The compound according to 1 above, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the substituted or unsubstituted 5-7 membered heterocycle is any one selected from the group consisting of the following heterocycles:

[0019]

[0020] 4. The compound according to 1 above, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein

[0021] The compound represented by Formula 1 is any one selected from the group consisting of the following compounds.

[0022]

[0023]

[0024]

[0025]

[0026] 5. A pharmaceutical composition comprising the compound according to any one of 1 to 4 above, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0027] 6. The pharmaceutical composition according to 5 above, wherein the composition is used to treat or prevent autoimmune diseases.

[0028] 7. The pharmaceutical composition according to 5 above, wherein the autoimmune disease is any one selected from the group consisting of inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, systemic lupus erythematosus and type 1 diabetes.

[0029] 8. The pharmaceutical composition according to 5 above, wherein the composition is used for treating or preventing cancer.

[0030] 9. The pharmaceutical composition according to 8 above, wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer and lung cancer.

[0031] 10. A method for treating an autoimmune disease, comprising administering the compound according to any one of 1 to 4 above to a subject in need thereof.

[0032] 11. A method for treating cancer, comprising administering the compound according to any one of 1 to 4 above to a subject in need thereof.

[0033] Effects of the Invention

[0034] The novel compound, stereoisomer or pharmaceutically acceptable salt thereof according to the present invention can induce the activity of AHR as an immunomodulatory transcription factor, thereby achieving the effects of not only controlling inflammation but also repairing immune balance and damaged tissues.

[0035] The novel compound, stereoisomer or pharmaceutically acceptable salt thereof according to the present invention can inhibit the production of IL-6 as an inflammatory factor, thereby achieving the effect of regulating excessive immune response, especially autoimmune response.

[0036] The novel compound according to the present invention, its stereoisomer or a pharmaceutically acceptable salt thereof can exhibit an effect of inducing the activity of regulatory T cells (Treg).

[0037] Furthermore, the novel compound, stereoisomer thereof or pharmaceutically acceptable salt thereof according to the present invention can exhibit the effect of preventing and treating autoimmune diseases by regulating the above-mentioned inflammatory factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 and Figure 2 Measurement of the expression level of the AHR target gene CYP1A1 is shown to confirm that the compounds of the present invention are AHR ligands under cell culture conditions.

[0039] Figure 3 The measurement of the inhibitory effect of the compound of the present invention on the production of inflammatory factor IL-6 is shown.

[0040] Figure 4 The measurement of the effect of the compounds of the present invention on promoting the generation of Foxp3+ regulatory T cells is shown.

[0041] Figure 5 and Figure 6 The compounds of the present invention are shown to have therapeutic effects on inflammatory bowel disease in an animal model of inflammatory bowel disease induced by dextran sodium sulfate (DSS). Specifically, Figure 5 showed that the lower the severity index (disease activity index) compared with the control (vehicle), the more treatment was completed, and Figure 6 It was shown that the smaller the weight of the large intestine compared to its length, the higher the treatment effect of inflammatory bowel disease.

[0042] Figure 7a , Figure 7b , Figure 8 and Fig. 9 The compounds of the present invention were shown to inhibit the expression of inflammatory factors (IL-1β, IL-6, IL-17a, TNF-α, S100a8 and S100a9) in an animal model of DSS-induced inflammatory bowel disease ( Figure 7a and Figure 7b ), increase the expression of immune regulatory factors (IL-10 and Foxp3) ( Figure 8 ) and increased expression of intestinal epithelial cell protective factors (Reg3b and Muc2) ( Fig. 9 ) effect.

[0043] Fig.10The mucosal healing effect of the compounds of the present invention in an animal model of DSS-induced inflammatory bowel disease using FITC-dextran is shown. Herein, this means that the lower the degree of detection, the higher the mucosal healing effect.

[0044] Fig.11 and Fig.12 The present invention shows the effect of the compound of the present invention in preventing colorectal cancer in the AOM / DSS colorectal cancer animal model, and specifically, shows the changes in body weight of mice not administered with a drug that induces colorectal cancer (AOM / DSS) (normal), mice administered with AOM / DSS (DSS+vehicle), and mice administered with compound 6 (DSS+compound 6) when AOM / DSS was administered ( Fig.11 ) and changes in the number and size of tumors ( Fig.12 ).

[0045] Fig.13 and Fig.14 The effect of the compound of the present invention on treating colorectal cancer in the AOM / DSS colorectal cancer animal model is shown, and specifically, the changes in body weight of mice not administered with AOM / DSS (normal), mice administered with AOM / DSS (DSS+vehicle), and mice administered with compound 6 after induction of colorectal cancer (DSS+compound 6) are shown ( Fig.13 ) and changes in the number and size of tumors ( Fig.14 ).

[0046] Figures 15 to 17 The compounds of the present invention are shown to be effective in treating psoriasis in animal models, and in particular, are shown to be effective in reducing epithelial thickness ( Fig.15 ), inhibiting the expression of skin lesion inflammatory factors (IL-17a and S100a8) Fig.16 ) and the effect of promoting the expression of inflammatory inhibitors (Foxp3 and IL-10) Fig.17 ).

[0047] Fig.18 and Fig.19 The present invention shows the effect of the compound of the present invention in treating graft-versus-host disease (GVHD) in the B6->BDF1 animal model, and specifically, shows the survival rate and clinical score of mice after transplantation ( Fig.18 ) and pathological score ( Fig.19 ).

[0048] Fig. 20The effect of the compounds of the present invention in treating multiple sclerosis in the autoimmune encephalomyelitis (EAE) model is shown. Here, this means that the lower the clinical score, the higher the therapeutic effect.

[0049] Fig.21 The effect of the compounds of the present invention in treating neutrophilic asthma in a neutrophilic asthma model is shown. Here, this means that the lower the number of neutrophils in the bronchoalveolar lavage (BAL), the higher the therapeutic effect.

[0050] Fig. 22 Shown are changes in blood concentrations over time of Compound 6 of the present invention and Comparative Example 1 after oral administration (PO) or intravenous administration (IV).

[0051] Fig.23 The results show the inhibitory effect of the activity of Th17 cells producing IL-17A after treatment with Compound 6 of the present invention and Comparative Example 1.

[0052] Fig.24 The results show that the treatment with Compound 6 of the present invention and Comparative Example 1 promotes the generation of regulatory T cells expressing Foxp3.

[0053] Fig.25 The results show that the compound 6 of the present invention and the comparative example 1 respectively have the effect of reducing the expression levels of IL-6 and S100a9 mRNA after oral administration to mice with inflammatory bowel disease. DETAILED DESCRIPTION

[0054] Hereinafter, the present invention will be described in detail.

[0055] Unless otherwise defined, all technical terms used in the present invention are used with the same meaning as those generally understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples will be described in this specification, similar or equivalent ones are also included in the scope of the present invention.

[0056] In the formulae (structural formulae) of the present invention, if a position requires a substituent but no substituent is specified at that position, this means that the hydrogen substituent is omitted, and this applies to all formulae (structural formulae) of the present invention.

[0057] The present invention relates to a compound represented by the following formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0058] [Formula 1]

[0059]

[0060] In the above formula 1, A is hydrogen, halogen, hydroxyl, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy, dimethylamine, -NO2, -CN, -COOR2 or -S(=O)2R2, B is hydrogen, C1-C3 alkyl, phenyl, acetyl, -CH2C(=O)OR2, -C(=O)OR2 or -S(=O)2R2, R1 is a substituted or unsubstituted 5-7 membered heterocycle or -NH2, and R2 is a C1-C3 alkyl.

[0061] R1 is substituted on any of the carbon atoms in the benzene ring of benzothiazole.

[0062] The 5- to 7-membered heterocyclic ring may be a compound in which 1 to 2 carbon atoms in the ring are substituted by a nitrogen atom or an oxygen atom.

[0063] The substituted 5- to 7-membered heterocyclic ring may be a compound having a 5- to 7-membered heterocyclic ring substituted by a C1-C3 alkyl group, a hydroxyl group or a dimethylamine.

[0064] The substituted or unsubstituted 5-7 membered heterocyclic ring may be any one selected from the group consisting of the following heterocyclic rings:

[0065]

[0066] The compound represented by the above formula 1 may be any one selected from the group consisting of the compounds listed in the following Table 1:

[0067] [Table 1]

[0068]

[0069]

[0070]

[0071]

[0072] The compound represented by Formula 1 may be any one selected from the group consisting of the following compounds:

[0073] 2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide, compound 6;

[0074] 2-(5-chloro-1H-indol-3-yl)-N-(6-(4-methylpiperazin-1-yl)benzo[d]thiazol-2-yl)acetamide, compound 9;

[0075] 2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide, compound 21;

[0076] 2-(5-Hydroxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide, compound 32; and

[0077] 2-(5-Methoxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide, compound 39.

[0078] Furthermore, the present invention relates to a pharmaceutical composition comprising the above-mentioned compound, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0079] The pharmaceutical composition may be a pharmaceutical composition for treating or preventing an autoimmune disease. Specifically, the disease may be inflammatory bowel disease (IBD), multiple sclerosis (MS), graft-versus-host disease (GVHD), asthma, atopy, psoriasis, rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1D), Behcet's disease or Sjogren's syndrome. More specifically, the disease may be inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, but is not limited thereto.

[0080] In the present invention, "autoimmune disease" may cause damage to cells or tissues through humoral immunity, cellular immunity, or both. That is, autoimmune disease is a disease in which the immune system produces an inappropriate response to self-antigens, thereby inducing autoimmune responses systemically or specifically in specific organs, which may cause chronic inflammation.

[0081] “Inflammatory bowel disease” refers to a disease in which abnormal chronic inflammation in the intestine repeatedly improves and relapses, and may correspond to one selected from the group consisting of Crohn's disease, ulcerative colitis, and intestinal Behcet's disease, but is not limited thereto.

[0082] "Multiple sclerosis" refers broadly to an inflammatory disease that induces demyelination and scarring as signs and symptoms, caused by damage and / or consumption of the fatty myelin sheaths around the axons of the brain and spinal cord. Types of multiple sclerosis may include relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), and progressive recurrent multiple sclerosis (PRMS), but they are not limited thereto.

[0083] "Graft-versus-host disease" is a disease in which lymphocytes infused during hematopoietic stem cell transplantation attack a host with reduced immune function to cause symptoms such as fever, rash, and abnormal liver function, and may affect the skin, lungs, intestines, liver, etc., but is not limited thereto.

[0084] "Asthma" refers to a disease in which symptoms such as coughing and breathing difficulties recur due to inflammation of the bronchial tubes when exposed to a specific pathogen, and may be caused by infection, smoking, allergens, etc., but is not limited thereto.

[0085] “Atopy” refers to atopic dermatitis, and is a representative allergic disease that presents symptoms such as itching and dry skin as a chronic recurring inflammatory skin disease.

[0086] "Psoriasis" refers to an inflammatory disease that occurs in the skin or joints due to abnormalities in the immune system, and may cause problems such as the appearance of ugly, increased keratin or erythematous plaques and pain. Psoriasis may include any one or more diseases selected from psoriatic arthritis, guttate psoriasis, pustular psoriasis, red skin psoriasis, scalp psoriasis, nail psoriasis, and enthesitis.

[0087] "Rheumatoid arthritis" refers to a systemic autoimmune disease characterized by chronic inflammation of the joints.

[0088] "Systemic lupus erythematosus" is also called "lupus," and refers to a systemic disease that is a chronic inflammatory autoimmune disease that attacks various organs of the body, such as connective tissue, skin, joints, blood, and kidneys. The exact cause is unknown, but according to previous studies, genetic factors are known to be associated with the development of the disease. To help diagnose lupus, the American College of Rheumatology (ACR) publishes 11 symptoms, signs, and test results to help distinguish the disease from other diseases. According to published research, if more than 4 of the 11 symptoms are present, lupus can be diagnosed.

[0089] "Type 1 diabetes" is an immune-mediated disease in which the insulin-secreting beta cells are destroyed by an autoimmune response. The causes of the disease can include a number of genetic and environmental factors that specifically target the insulin-secreting beta cells. The disease may be accompanied by progressive inflammatory infiltration of the pancreatic islets by immune cells.

[0090] In the present invention, in addition to the active ingredient of the compound of the present invention, a pharmaceutically suitable and physiologically acceptable additive can also be used to prepare a pharmaceutical composition. The composition can be administered to a mammal. As the above-mentioned additive, for example, an excipient, a disintegrant, a sweetener, a binder, a coating agent, a bulking agent, a lubricant, a glidant or a flavoring agent can be used.

[0091] Furthermore, the pharmaceutical composition of the present invention may be preferably formulated as a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier in addition to the above-mentioned pharmaceutically effective amount of the active ingredient for administration.

[0092] "Pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio suitable for drug therapy, and can be based on the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, route of administration and excretion rate, treatment duration, factors including drugs used simultaneously, and other factors known to the medical field to determine the effective dose level. Pharmaceutical compositions according to the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents. In addition, compositions can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single doses or multiple doses. Taking all of the above factors into account, it is important to use the minimum amount that can achieve maximum effect without side effects, and such an amount can be easily determined by those skilled in the art.

[0093] Particularly, the effective amount of the pharmaceutical composition according to the present invention can depend on the patient's age, sex, patient's condition and / or body weight, absorption of active ingredient in vivo, inactivation rate and excretion rate, disease type and the medicine to be used in combination and change.Usually, 0.001 to 150mg, preferably 0.01 to 100mg per 1kg body weight can be administered every day or every other day, or can be divided into 1 to 3 times a day.However, dosage can increase or decrease depending on route of administration, severity of obesity, sex, body weight, age, etc., therefore, will not limit the scope of the present invention in any way.

[0094] In addition, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not generally cause allergic reactions or the like, such as gastrointestinal disorders and dizziness, when administered to humans.

[0095] The example of carrier, excipient and diluent can comprise lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, filler, anti-aggregating agent, lubricant, wetting agent, flavoring agent, emulsifier and preservative can be comprised in addition.

[0096] In addition, the compositions of the present invention can be formulated using any method known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a subject, including a human, who needs to be treated with the pharmaceutical composition of the present invention. The preparation can be a powder, granules, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection or sterile powder.

[0097] The expression "pharmaceutically acceptable salt" refers to salts prepared using specific compounds according to the present invention and relatively non-toxic acids or bases. Pharmaceutically acceptable salts may include, for example, acid addition salts or metal salts.

[0098] Acid addition salts can be formed by inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic monocarboxylates and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, and nontoxic organic acids such as aromatic acids, aliphatic sulfonic acids and aromatic sulfonic acids. These pharmaceutically nontoxic salts can include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, fluorides, acetates, propionates, decanoates, octanoates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates , maleate, butyne-1,4-dioate, nucleoside-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

[0099] The metal salt may be a sodium salt, a potassium salt or a calcium salt. The metal salt may be prepared using a base, for example, an alkali metal salt or an alkaline earth metal salt may be obtained by dissolving the compound in an excess of an alkali metal hydroxide solution or an alkaline earth metal hydroxide solution, filtering the undissolved compound salt and evaporating or drying the filtrate.

[0100] The present invention may relate to a method for treating an autoimmune disease, comprising administering the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0101] Furthermore, the present invention may relate to a method for inducing the activity of AHR, which comprises administering the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0102] Specifically, the compounds of the present invention can target the aryl hydrocarbon receptor (AHR) as the immunomodulatory transcription factor of the present invention, and can be used as an agent for inducing AHR activity, thereby controlling inflammation, regulating immune balance and repairing damaged tissues. Therefore, the compounds can be used for the treatment of autoimmune diseases, but are not limited to this. Existing ligands are toxic, have low affinity and structural stability and high target non-specificity, which brings about the problem that these compounds are not suitable for development into pharmaceutical compositions. On the other hand, when AHR activity is induced by a compound of the present invention having "drug-like properties", it can be effectively used to treat and prevent autoimmune diseases.

[0103] The present invention may relate to a method for inhibiting the production of IL-6, comprising administering the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[0104] Specifically, since the known inflammatory factor IL-6 causes autoimmune diseases, the compounds of the present invention can be used to treat autoimmune diseases by inhibiting the mechanism of IL-6 production. In fact, there are many known therapeutic agents for autoimmune diseases that inhibit the inhibition of IL-6, and related papers. According to the following experimental data, the compounds of the present invention are also confirmed to inhibit the production of IL-6 and are therefore expected to have the effect of reducing autoimmune responses, thus, the compositions of the present invention can be used to treat and prevent autoimmune diseases.

[0105] Furthermore, the present invention relates to a composition for preventing or treating cancer, which comprises the compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[0106] In the present invention, "cancer" refers to the uncontrolled abnormal growth of the host's own cells, which invades the surrounding tissues of the initial abnormal cell growth site in the host and potential tissues away from these sites. In addition, it can include carcinoma as a cancer of epithelial tissue (e.g., skin, squamous cells); sarcoma as a cancer of connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemia as a cancer of hematopoietic tissue (e.g., bone marrow tissue); lymphoma and myeloma as cancer of immune cells; cancer of the central nervous system, including cancer in brain and spinal cord tissue.

[0107] Specifically, the cancer can be selected from the group consisting of colorectal cancer, melanoma, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer and lung cancer, but is not limited thereto.

[0108] The present invention relates to a method for treating cancer, which comprises administering the compound, its stereoisomer or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0109] The method of treatment may include administering the compound, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a patient diagnosed with cancer at any stage of chemotherapy and is not limited to a specific stage.

[0110] Furthermore, the compound, its stereoisomer or a pharmaceutically acceptable salt thereof may be administered in the form of the above-mentioned pharmaceutical composition, but is not limited thereto.

[0111] The compound represented by Formula 1 according to the present invention may be prepared by any method known in various documents.

[0112] Hereinafter, the present invention will be described in detail through preparation examples and examples of the present invention.

[0113] Preparation Example

[0114] Preparation Example 1: Synthesis of 2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 6)

[0115] (1) Synthesis of perfluorophenyl 2-(5-chloro-1H-indol-3-yl)acetate

[0116]

[0117] While stirring a solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (56.00 g, 267.14 mmol), 2,3,4,5,6-pentafluorophenol (54.09 g, 293.85 mmol) and EDC-HCl (61.45 g, 320.57 mmol) in dichloromethane (890 mL) at room temperature, N,N-diisopropylethylamine (52.82 mL, 293.85 mmol) was added. After the reaction mixture was stirred at room temperature for 24 hours and the reaction was confirmed to be complete, distilled water (890 mL) was added. The layers were separated, and the organic layer was dried over anhydrous Na2SO4 and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain 2-(5-chloro-1H-indol-3-yl) perfluorophenyl acetate (50.00 g, yield 50%).

[0118] (2) Synthesis of 2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 6)

[0119]

[0120] A solution of 6-morpholinobenzo[d]thiazole-2-amine (26.00 g, 110.49 mmol) and perfluorophenyl 2-(5-chloro-1H-indol-3-yl)acetate (47.74 g, 127.07 mmol) in THF (1.1 L) was stirred. The reaction mixture was refluxed for 24 hours and confirmed to be complete, and then cooled to room temperature. The reaction mixture was concentrated under reduced pressure and purified by MPLC to obtain compound 6 (28.5 g, 60% yield).

[0121] 1 H NMR (DMSO-d6, 400MHz): δ12.37(s,1H),11.12(s,1H),7.59(t,2H,J=16.0Hz),7.43(d,2H,J=16.0Hz ),7.34(s,1H),7.12(d,1H,J=8.0Hz),7.02(d,1H,J=12.0Hz),3.88(m,2H),3.74(m,4H),3.11(m,4H)

[0122] Preparation Example 2: Synthesis of 2-(5-chloro-1H-indol-3-yl)-N-(6-(4-methylpiperazin-1-yl)benzo[d]thiazol-2-yl)acetamide (Compound 9)

[0123]

[0124] A solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (210 mg, 1.00 mmol), 6-(4-methylpiperazine-1-yl)benzo[d]thiazole-2-amine (249 mg, 1.00 mmol), HBTU (760 mg, 2.00 mmol) and DIPEA (0.7 mL, 4.01 mmol) was added to DMF (10 ml) and stirred for 2 days. After the reaction was complete, the reaction mixture was put into ice water (50 mL), stirred for 30 minutes, filtered and washed with water. The resulting solid mixture was purified by column chromatography to obtain compound 9 (100 g, 22%).

[0125] 1 H NMR (DMSO-d6, 400MHz): δ12.37 (s, 1H), 11.20 (s, 1H), 7.67 (d, 1H, J = 4.0Hz), 7.56 (d, 1H, J = 8.0Hz), 7.44(d,1H,J=4.0Hz),7.37(m,2H),7.06(m,2H),3.87(s,2H),3.14(m,4H),2.50(m,4H),2.24(s,3H)

[0126] Preparation Example 3: Synthesis of 2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 21)

[0127] (1) Synthesis of perfluorophenyl 2-(5-chloro-1-methyl-1H-indol-3-yl)acetate

[0128]

[0129] While stirring a solution of 2-(5-chloro-1-methyl-1H-indol-3-yl)acetic acid (110 mg, 491.83 μmol), 2,3,4,5,6-pentafluorophenol (100 mg, 541.01 μmol), EDC-HCl (113 mg, 590.19 μmol) in dichloromethane (1.6 mL), N,N-diisopropylethylamine (0.1 mL, 541.01 μmol) was added. After the reaction mixture was stirred at room temperature for 24 hours and the reaction was confirmed to be complete, distilled water (2 mL) was added. The layers were separated, and the organic layer was dried over anhydrous Na2SO4 and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain 2-(5-chloro-1-methyl-1H-indol-3-yl) perfluorophenyl acetate (150 mg, 78%).

[0130] (2) Synthesis of 2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 21)

[0131]

[0132] A solution of 6-morpholinobenzo [d] thiazole-2-amine (40 mg, 169.99 μmol) and 2- (5-chloro-1-methyl-1H-indol-3-yl) perfluorophenyl acetate (99 mg, 254.99 μmol) in THF (1.7 mL) was stirred at reflux for 12 hours. The reaction mixture was cooled to room temperature, then filtered under reduced pressure to remove impurities, and the filtrate was concentrated under reduced pressure. The concentrated reaction mixture was recrystallized with ether to obtain compound 21 (40 mg, yield 54%).

[0133] 1 H NMR (DMSO-d6, 500MHz): δ12.38 (s, 1H), 7.69 (s, 1H), 7.58 (d, 1H, J = 10.0Hz), 7.44 (d, 2H, J=5.0Hz),7.36(s,1H),7.11(m,2H),3.86(s,2H),3.77(s,3H),3.73(m,4H),3.11(m,4H)

[0134] Preparation Example 4: Synthesis of 2-(5-hydroxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 32)

[0135]

[0136] Compound 39 (2-(5-methoxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide) (25 mg, 59.17 μmol) obtained in the following Preparation Example 5 in boron tribromide (7.9 μml, 71.01 μmol) was stirred at room temperature for 6 hours. Distilled water was added to the reaction mixture to terminate the reaction. After neutralization with NaHCO3, the layers were separated with DCM. The organic layer was washed, dried over anhydrous Na2SO4 and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was recrystallized from ether to obtain compound 32 (11 mg, yield 45%).

[0137] 1 H NMR(DMSO-d6,500MHz): δ12.30(s,1H),10.65(s,1H),8.64(s,1H),7.57(d,1H,J=10.0Hz),7.45(s,1H ),7.19(s,1H),7.11(m,2H),6.90(s,1H),6.59(d,1H,J=5.0Hz),3.78(s,2H),3.74(m,4H),3.11(m,4H)

[0138] Preparation Example 5: Synthesis of 2-(5-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 39)

[0139] (1) Synthesis of perfluorophenyl 2-(5-methoxy-1H-indol-3-yl)acetate

[0140]

[0141] While stirring a solution of 2-(5-methoxy-1H-indol-3-yl)acetic acid (200 mg, 974.61 μmol), 2,3,4,5,6-pentafluorophenol (198 mg, 1.07 mmol) and EDC-HCl (225 mg, 1.17 mmol) in dichloromethane (3.3 mL), (N,N-diisopropylethylamine (0.2 mL, 1.07 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours and after confirming the completion of the reaction, distilled water (4 mL) was added. The layers were separated, and the organic layer was dried over anhydrous Na2SO4 and filtered. After concentrating the filtrate under reduced pressure, the concentrate was purified by column chromatography to obtain the title compound (288 mg, 80%).

[0142] (2) Synthesis of 2-(5-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 39)

[0143]

[0144] A solution of 6-morpholinobenzo [d] thiazole-2-amine (100 mg, 424.98 μmol) and 2- (5-methoxy-1H-indol-3-yl) perfluorophenyl acetate (189 mg, 509.98 μmol) in THF (4.3 mL) was stirred at reflux for 12 hours. The reaction mixture was cooled to room temperature, then filtered under reduced pressure to remove impurities, and the filtrate was concentrated under reduced pressure. The concentrated reaction mixture was recrystallized with ether to obtain compound 39 (120 mg, yield 67%).

[0145] 1 H NMR (DMSO-d6, 500MHz): δ12.34 (s, 1H), 10.82 (s, 1H), 7.57 (d, 1H, J = 10.0Hz), 7.45 (s, 1H), 7.24 (d,2H,J=10.0Hz),7.11(m,2H),6.72(dd,1H,J=10.0Hz),3.84(s,2H),3.74(m,7H),3.10(m,4H)

[0146] Comparative Example 1: Synthesis of N-(Benzo[d]thiazol-2-yl)-2-(5-chloro-1H-indol-3-yl)acetamide (Comparative Example 1)

[0147]

[0148] While a solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (7.0 g, 33.39 mmol) in DMF (100 mL) was stirred at room temperature, benzo[d]thiazole-2-amine (4.51 mg, 30.05 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU, 15.24 g, 40.07 mmol) and trimethylamine (9.4 mL, 66.78 mmol) were added in sequence. The reaction mixture was stirred at room temperature for 3 days. Distilled water was added to the mixture to terminate the reaction. The layers were separated with ethyl acetate, and the organic layer was washed with distilled water, dried over anhydrous Na2SO4 and filtered. After the filtrate was concentrated under reduced pressure, the concentrate was purified by column chromatography to obtain the compound of Comparative Example 1 (5.0 g, yield 45%).

[0149] 1 H NMR (DMSO-d6, 400MHz): δ12.57(s,1H),11.21(s,1H),7.94(d,J=8.0Hz,1H),7.73(d,J= 8.0Hz,1H),7.68(d,J=4.0Hz,1H),7.37(m,3H),7.27(t,J=16.0Hz,1H),7.07(dd,J=8.0 4.0Hz,1H),3.91(s,2H)

[0150] Example

[0151] Example 1: Confirmation of the CYP1A1 expression induction effect

[0152] In order to confirm the targeting specificity of the compound of the present invention prepared as described above, it was confirmed whether the expression levels of CYP1A1 mRNA and CYP1A1 protein, which are AHR target genes, were increased.

[0153] (1) Induction effect of CYP1A1 mRNA expression

[0154] After HepG2 cells cultured in DMEM-fetal bovine serum (FBS) 10% medium were recovered and the survival rate was confirmed to be 97% or more by trypan blue staining, the recovered product was centrifuged at 1200 rpm for 5 minutes at room temperature, and the cells were centrifuged at 3×10 5Cells were prepared by resuspending cells at 10% DMEM-fetal bovine serum (FBS) at 10 cells / ml. Thereafter, the cells were dispensed into 60 mm culture dishes in 3 ml portions, and each dish was treated with 50 μl of 1 μM compounds 6, 21, 32, and 39 diluted in DMEM culture medium, and then cultured in a cell culture incubator (5% CO2 incubator) for 24 hours. As a control, 50 μl of 0.05% dimethyl sulfoxide (DMSO) / DMEM culture medium was used for treatment.

[0155] Recover cultured cells to prepare mRNA samples. Specifically, mRNA was extracted from the recovered cells by phenol-chloroform precipitation using TRIZOL reagent (Invitrogen, catalog number 15596018). From the isolated RNA, cDNA was synthesized by reverse transcription, and the expression of CYP1A1 was confirmed by real-time polymerase chain reaction (PCR) using iQ SYBR-Green Supermix (Bio-rad) in the CFX96 (Bio-rad) detection system. GAPDH was used as a control enzyme to compare the relative values ​​of enzyme expression levels by the ΔΔct method. Herein, a control was used to set one (1) times.

[0156] Real-time polymerase chain reaction was performed under the conditions of 45 cycles at an annealing temperature of 58° C., using the following primer sequences: human CYP1A1 forward, 5′-CAC CCT CAT CAG TAA TGG TCA GA-3′ (SEQ ID NO: 1) and reverse, 5′-AAC GTG CTT ATC AGG ACC TC-3′ (SEQ ID NO: 2); human GAPDH forward, 5′-TGA TGA CAT CAAGAA GGT GG-3′ (SEQ ID NO: 3) and reverse, 5′-TTA CTC CTT GGA GGC CAT GT-3′ (SEQ ID NO: 4).

[0157] From the above results, it can be confirmed that the groups treated with compounds 6, 21, 32 and 39 showed higher CYP1A1 mRNA expression levels than the control (vehicle). Therefore, it was confirmed that compounds 6, 21, 32 and 39 significantly induced the expression of the AHR target gene CYP1A1 ( Figure 1 ).

[0158] (2) Induction effect of CYP1A1 protein expression

[0159] After HepG2 cells cultured in DMEM-fetal bovine serum (FBS) 10% medium were recovered and the survival rate was confirmed to be 97% or more by trypan blue staining, the recovered product was centrifuged at 1200 rpm for 5 minutes at room temperature, and the cells were centrifuged at 1.5×10 5 Prepare cells by resuspending at 100 cells / ml in DMEM-10% fetal bovine serum medium.

[0160] Thereafter, the cells were dispensed into 96-well plates in 200 μl portions, and each plate was treated with 3 μl of 1 μM compound 6, 21, 32, and 39 diluted in DMEM medium, and then cultured in a cell culture incubator (5% CO2 incubator) for 24 hours. As a control, 3 μl of 0.05% dimethyl sulfoxide (DMSO) / DMEM medium was used for treatment.

[0161] After 24 hours, the medium of the plate was discarded, washed with dPBS, treated with 100 μl of 2 μM 7-ethoxyresorufin, and cultured in a cell culture incubator (5% CO2 incubator) for 30 minutes. After 30 minutes, 75 μl of fluorescamine (150 μg / ml) was treated, and then the resorufin excitation was measured at 535 nm and the emission was measured at 590 nm. The standard curve was measured and used by 0 to 50 pmol of resorufin solution.

[0162] After the measurement, all the solution was removed, and then treated with 25 μl of 0.5 M sodium hydroxide (NaOH), and the cells were scraped and reacted on a shaker at room temperature for 15 minutes.

[0163] After 15 minutes, the protein was quantified using the Bradford method, and the value was obtained using the calculation formula (sample resorufin amount / sample protein amount), and then set to one (1) times using the control.

[0164] From the above results, it can be confirmed that the CYP1A1 protein expression level in the groups treated with compounds 6, 21, 32 and 39 was significantly higher than that of the control (vehicle), and therefore, it was confirmed that the protein of the AHR target gene CYP1A1 was significantly induced by compounds 6, 21, 32 and 39 ( Figure 2 ).

[0165] Example 2: Inhibitory effect on the production of inflammatory factor IL-6

[0166] In order to evaluate the inhibitory effect of the compound of the present invention prepared as described above on IL-6 production, an IL-6 production test of epithelial cells stimulated with IL-1β was performed.

[0167] After A549 cells cultured in DMEM-fetal bovine serum (FBS) 10% medium were recovered and the survival rate was confirmed to be 97% or more by trypan blue staining, the recovered product was centrifuged at 1200 rpm for 5 minutes at room temperature, and the cells were centrifuged at 1×10 6 Cells were prepared by resuspending cells / 3ml in DMEM-10% fetal bovine serum medium. Thereafter, the cells were dispensed into 60mm plates in 3ml, and each dish was treated with 50μl of compounds 6, 9, 21, 32 and 39 diluted in DMEM medium at a concentration of 5μM, and then cultured in a cell culture incubator (5% CO2 incubator) for 48 hours. Then, the cells were treated with 50μl of human recombinant IL-1β diluted in DMEM medium at a concentration of 12.5ng / ml, and then cultured in a cell culture incubator (5% CO2 incubator) for 24 hours. As a control, 50μl of 0.05% dimethyl sulfoxide (DMSO) / DMEM medium was used for treatment.

[0168] Recover the cultured cells to prepare mRNA samples. Specifically, mRNA was extracted from the recovered cells by phenol-chloroform precipitation using TRIZOL reagent (Invitrogen, catalog number 15596018). From the isolated RNA, cDNA was synthesized by reverse transcription, and the expression of CYP1A1 was confirmed by real-time polymerase chain reaction (PCR) using iQ SYBR-Green Supermix (Bio-rad) in the CFX96 (Bio-rad) detection system. GAPDH was used as a control enzyme to compare the relative values ​​of enzyme expression levels by the ΔΔct method. Herein, one (1) times of the control setting was used.

[0169] Real-time polymerase chain reaction was performed under the conditions of 45 cycles at an annealing temperature of 58° C., using the following primer sequences: human IL-6 forward, 5′-GAT GGC TGA AAA AGA TGG ATG C-3′ (SEQ ID NO: 5) and reverse, 5′-TGG TTG GGT CAG GGG TGG TT-3′ (SEQ ID NO: 6); human GAPDH forward, 5′-TGA TGA CAT CAA GAAGGT GG-3′ (SEQ ID NO: 3) and reverse, 5′-TTA CTC CTT GGA GGC CAT GT-3′ (SEQ ID NO: 4).

[0170] As a result, it was confirmed that the IL-6 production of A549 stimulated by IL-1β was significantly reduced by treatment with compounds 6, 9, 21, 32 and 39. Therefore, compounds 6, 9, 21, 32 and 39 effectively inhibited IL-6 production ( Figure 3).

[0171] Example 3: Confirmation of the effect of promoting the production of regulatory T cells (Treg) expressing Foxp3

[0172] The effect of promoting the production of "FoxP3-expressing regulatory T cells" which play an important role in maintaining immune tolerance was evaluated. Figure 4 ).

[0173] The spleen of C57BL / 6 mice (8 to 12 weeks old, female) was removed, RPMI medium was added and crushed, and then passed through a 40μm cell strainer (BD Falcon) to obtain a single cell suspension. The single cell suspension was centrifuged (1200rpm, 5 minutes), and after discarding the supernatant, 1ml of ACK lysis buffer was added, followed by stirring for 1 minute and then washing with RPMI medium. After centrifuging the single cell suspension, T cells were isolated using a mouse CD4 naive T cell enrichment kit (Invitrogen). The isolated T cells were separated at 1×10 6 The isolated T cells were prepared by resuspending the cells in RPMI-fetal bovine serum (FBS) 10% + 2-ME (mercaptoethanol) medium. For T cell activation, 5 μg / ml anti-CD3 (eBioscience TM ) was dispensed into 96-well plates in 100 μL, reacted in a cell culture incubator (37°C, 5% CO2 incubator) for 4 hours, and washed with phosphate-buffered saline to prepare the plate. T cells were dispensed into the prepared plate in 200 μL and incubated with 2 μg / ml anti-CD28 (eBioscience TM ), 2ng / ml TGFβ-1 (R&D systems) and 100U / ml IL-2 were used to treat each well. 5μL of compounds 6 and 9 diluted in RPMI-fetal bovine serum 10% + 2-ME medium at a concentration of 2.5μM were used for treatment, and then cultured in a cell culture incubator (37°C, 5% CO2 incubator) for 3 days. As a control, 5μl of 0.05% dimethyl sulfoxide (DMSO) / RPMI medium was used for treatment. After 3 days, in order to confirm the effect of regulatory T cell production, the cultured cells were recovered to measure the expression of Foxp3 protein.

[0174] To confirm the expression of Foxp3 protein, the recovered cells were placed in a 5 ml FACS tube (BD Falcon) and washed with 1 ml phosphate buffered saline. The cells were resuspended in 0.1 ml FACS buffer (0.1% NaN3, 1% FBS) and stained with 0.5 μg of CD16 / CD32 antibody (eBioscience TM) to prevent nonspecific binding of the antibody, and then react at 4°C for 15 minutes. Thereafter, 0.25 μg of CD4 monoclonal antibody (GK1.5) and PE-Cyanine7 (eBioscience TM ) and stained at 4°C for 30 minutes, followed by washing with 1 ml of FACS buffer. Then, 1 ml of fixation / permeabilization solution (eBioscience TM ) was added to the FACS tube containing each sample, followed by reaction at 4°C for 1 hour. Then, the product was washed with permeabilization buffer (eBioscience TM ) were washed twice. Thereafter, 0.5 μg of Foxp3 monoclonal antibody (eBioscience TM ) and then stained for 30 minutes at 4° C. The cells were washed twice with permeabilization buffer, suspended in 0.3 ml of FACS buffer, and measured by flow cytometry.

[0175] As a result, it was confirmed that Compound 6 and Compound 9 significantly increased the production of regulatory T cells expressing Foxp3 compared to the vehicle. Therefore, Compound 6 and Compound 9 effectively promoted the production of FoxP3+ regulatory T cells ( Figure 4 ).

[0176] Example 4: Confirmation of therapeutic effect on inflammatory bowel disease

[0177] In order to study the therapeutic effect of the compound according to the present invention prepared as described above on inflammatory bowel disease, inflammatory bowel disease was induced in C57BL / 6 mice, and compound 6 was administered to the mice to evaluate the efficacy as follows ( Figures 5 to 10 ).

[0178] (1) Confirmation of the therapeutic effect of compound 6 on inflammatory bowel disease

[0179] On day 0 of the experiment, a 2.0% DSS solution prepared by dissolving DSS (dextran sulfate sodium, MP biomedicals, catalog number 160110) in 2.0% sterile distilled water was given to C57BL / 6 mice (11 weeks old, female, 20±2 g) for drinking for 7 days. The 2.0% DSS solution was replaced at intervals of 2 days. Sterile distilled water was provided for drinking from day 8 of the experiment. To confirm the onset of inflammatory bowel disease, body weight and severity index were measured at intervals of 2 days from day 0 of the experiment.

[0180] 10 mg / kg of compound 6 per mouse was completely dissolved in a 7.5% (v / v) ethanol-Cremophor EL mixture corresponding to the administered dose, then diluted in phosphate buffered saline to prepare a final ethanol: Cremophor EL: phosphate buffered saline (0.375:0.375:9.25, v / v / v), and then 200 μl was orally administered daily from day 1 to day 14 of the experiment, for a total of 14 times. The inflammatory bowel disease severity index was visually observed and recorded at intervals of 2 days according to a severity index system classified into 0 to 10 levels.

[0181] Inflammatory bowel disease symptoms were evaluated by summing the scores of the three items according to the following items (Table 2).

[0182] [Table 2]

[0183]

[0184] As a result of the analysis, it was confirmed that the body weight of the solvent control group (vehicle) began to decrease from the 6th day of the experiment, decreased by more than 10% on the 10th day of the experiment, and 100% induced enteritis, accompanied by an increase in the severity index of more than 5. The mice in the solvent control group showed a severity index of 7.50±0.50 on the 10th day of the experiment when the severity index reached the maximum. On the other hand, on the 10th day of the experiment, the experimental group administered with 10 mg / kg of the present compound 6 showed a statistically significant therapeutic effect compared with the solvent control group. In addition, comparison of the large intestine weight: length ratio (weight: length ratio, mg / cm) on the 15th day of the experiment showed that, in terms of morphology, intestinal inflammation could be significantly suppressed (compared with the solvent control group (vehicle): ***, p<0.001, see Figure 5 and Figure 6 Specifically, when administered at an amount of 10 mg / kg, compound 6 of the present invention exhibited an excellent anti-inflammatory effect ( Figure 7a and Figure 7b ).

[0185] On the 15th day of the experiment, the large intestine of the mice was removed to prepare mRNA samples. To extract mRNA, the large intestine tissue was ground with a homogenizer to obtain a homogenous suspension. TM(DNA-free) Total RNA Extraction Kit (Intronbiotechnology, catalog number 17221), mRNA was extracted from the homogenized suspension by phenol-chloroform precipitation. cDNA was synthesized from the isolated RNA by reverse transcription, and then the expression of inflammatory cytokines was confirmed by real-time polymerase chain reaction (PCR) using iQ SYBR-Green Supermix (Bio-rad) in the CFX96 (Bio-rad) detection system. GAPDH was used as a control enzyme to compare the relative values ​​of enzyme expression levels by the ΔΔct method. Herein, normal mouse colon was used as a control to set one (1) times.

[0186] Real-time polymerase chain reaction was performed under the conditions of 45 cycles at an annealing temperature of 58°C, and the following primer sequences were used.

[0187] mouse IL-1β forward, 5′-CTC GTG CTG TCG GAC CCA TAT-3′ (SEQ ID NO: 7) and reverse, 5′-TTG AAG ACA AAC CGC TTT TCC A-3′ (SEQ ID NO: 8);

[0188] mouse IL-6 forward, 5′-CAT GTT CTC TGC GAA ATC GTG G-3′ (SEQ ID NO: 9) and reverse, 5′-AAC GCA CTA GGT TTG CCG AGT A-3′ (SEQ ID NO: 10);

[0189] mouse IL-17A forward, 5′-TTT AAC TCC CTT GGC GCA AAA-3′ (SEQ ID NO: 11) and reverse, 5′-CTT TCC CTC CGC ATT GAC AC-3′ (SEQ ID NO: 12);

[0190] mouse TNF-α forward, 5′-CCA CAC CGT CAG CCG ATT TG-3′ (SEQ ID NO: 13) and reverse, 5′-CAC CCA TTC CCT TCA CAG AGC-3′ (SEQ ID NO: 14);

[0191] mouse S100a8 forward, 5′-AAA TCA CCA TGC CCT CTA CAA G-3′ (SEQ ID NO: 15) and reverse, 5′-CCC ACT TTT ATC ACC ATC GCA A-3′ (SEQ ID NO: 16);

[0192] mouse S100a9 forward, 5′-ATA CTC TAG GAA GGA AGG ACA CC-3′ (SEQ ID NO: 17) and reverse, 5′-TCC ATG ATG TCA TTT ATG AGG GC-3′ (SEQ ID NO: 18);

[0193] mouse IL-10 forward, 5′-CAA GGC AGT GGA GCA GGT GAA-3′ (SEQ ID NO: 19) and reverse, 5′-CGG AGA GAG GTACAAACG AGG TT-3′ (SEQ ID NO: 20);

[0194] mouse Foxp3 forward, 5′-CCC ATC CCC AGG AGT CTT G-3′ (SEQ ID NO: 21) and reverse, 5′-ACC ATG ACT AGG GGC ACT GTA-3′ (SEQ ID NO: 22);

[0195] mouse Reg3b forward, 5′-ACT CCC TGA AGA ATA TAC CCT CC-3′ (SEQ ID NO: 23) and reverse, 5′-CGC TAT TGA GCA CAG ATA CGA G-3′ (SEQ ID NO: 24);

[0196] mouse Muc2 forward, 5′-ATG CCC ACC TCC TCA AAG AC-3′ (SEQ ID NO: 25) and reverse, 5′-GTA GTT TCC GTT GGA ACA GTG AA-3′ (SEQ ID NO: 26);

[0197] Mouse GAPDH forward, 5′-TTC ACC ACC ATG GAG AAG GC-3′ (SEQ ID NO: 27) and reverse, 5′-GGC ATG GAC TGT GGT CAT GA-3′ (SEQ ID NO: 28).

[0198] By administering compound 6, the expression levels of inflammatory cytokines IL-1β, IL-6, IL-17A, TNF-α, S100a8, and S100a9 in colorectal lesions were significantly decreased compared with the solvent control group (vehicle) (compared with the solvent control group (vehicle): **, p<0.01, see Figure 7a and Figure 7bIn addition, the expression levels of immunomodulatory factors IL-10 and Foxp3 in colorectal lesions were significantly increased by the administration of compound 6 compared with the solvent control group (vehicle) (compared with the solvent control group (vehicle): *, p<0.05; **, p<0.01, see Figure 8 In addition, the expression levels of intestinal epithelial cell protective factors Reg3b and Muc2 in colorectal lesions were significantly increased by the administration of compound 6 compared with the vehicle control group (vehicle) (compared with the solvent control group (vehicle): *, p < 0.05; ***, p < 0.001, see Fig. 9 ). From these results, it can be seen that compound 6 of the present invention can significantly reduce the expression of inflammatory factors in the intestine, while significantly increasing the expression of intestinal immune regulatory factors and intestinal epithelial cell protective factors.

[0199] To investigate the mucosal healing effect of the compounds according to the present invention, inflammatory bowel disease was induced in C57BL / 6 mice, and the degree of restoration of intestinal epithelial barrier integrity was evaluated by administering Compound 6 as follows.

[0200] On day 0 of the experiment, a 2.0% DSS solution prepared by dissolving 2.0% DSS in sterile distilled water was given to C57BL / 6 mice (11 weeks old, female, 20±2 g) for drinking for 7 days. The 2.0% DSS solution was replaced at intervals of 2 days. Sterile distilled water was provided for drinking from day 8 of the experiment. Body weight and severity index were measured at intervals of 2 days from day 0 of the experiment to confirm the onset of inflammatory bowel disease.

[0201] In the compound 6 administration group according to the present invention, 10 mg / kg of the compound per mouse was completely dissolved in a 7.5% (v / v) ethanol-Cremophor EL mixture corresponding to the administered dose, and then diluted in phosphate buffered saline to prepare a final ethanol:Cremophor EL (0.375:0.375:9.25, v / v / v), and then 200 μl of the prepared solution was orally administered daily from day 1 to day 14 of the experiment, for a total of 14 times.

[0202] The day before FITC-dextran was used, mice were deprived of water overnight. On the 15th day of the experiment, 600mg / kg of FITC-dextran (fluorescein isothiocyanate-dextran, Sigma Aldrich, catalog number FD40) was diluted in phosphate buffered saline and orally administered once to mice with 200 μl. 4 hours after oral administration, fluorescence was measured in serum extracted from the heart (fluorimeter, excitation 485-490nm, emission 528-530nm).

[0203] Serum FITC-dextran was significantly decreased by the administration of compound 6 compared with the solvent control group (vehicle) (compared with the solvent control group (vehicle): ***, p<0.001, see Fig.10 ). From the above results, it can be seen that the compound 6 of the present invention exhibits a significant mucosal healing effect. From the above results, it is confirmed that the compound 6 of the present invention has an oral therapeutic effect in a mouse model of inflammatory bowel disease.

[0204] Example 5: Confirmation of the preventive and therapeutic effects on colorectal cancer

[0205] In order to investigate the prevention of ( Fig.11 and Fig.12 ) and treatment( Fig.13 and Fig.14 )The effect of compound 6 on colitis-associated colorectal cancer (CA-CRC) was evaluated by administering compound 6 to a colorectal cancer model (AOM / DSS mice).

[0206] AOM (Sigma Aldrich, catalog number A5486) was diluted with physiological saline to a concentration of 10 mg / kg, and then intraperitoneally administered to C57BL / 6 mice (8 weeks old, female, 18±2 g) at 200 μl for a total of 3 times at 7-day intervals (experimental days 0, 7, and 14). On the 7th day of the experiment, a 1.5% DSS solution prepared by dissolving 1.5% DSS in sterile distilled water was given to the mice for drinking for 7 days. In addition, the 1.5% DSS solution was replaced at intervals of 2 days. Sterile distilled water was provided for drinking from the 8th day of the experiment.

[0207] In the compound 6 administration group according to the present invention, 10 mg / kg of compound 6 per mouse was completely dissolved in a 7.5% (v / v) ethanol-Cremophor EL mixture corresponding to the administered dose, and then diluted in phosphate buffered saline to prepare a final ethanol: Cremophor EL: phosphate buffered saline (0.375: 0.375: 9.25, v / v / v), and then 200 μl of the prepared solution was orally administered. In order to confirm the preventive effect, compound 6 was administered 14 times from the 7th day to the 20th day of the experiment.

[0208] As a result, the control group (AOM / DSS+vehicle) showed a decrease in body weight after 70 days, but the group administered with compound 6 (AOM / DSS+compound 6) showed an increase in body weight similar to that of normal mice (normal) ( Fig.11In addition, it was confirmed that the number of tumors in the compound 6 administration group was significantly smaller than that in the control group, and the size was also significantly smaller than that in the control group. That is, it was confirmed that compound 6 has the effect of preventing colorectal cancer caused by inflammation ( Fig.11 and Fig.12 ).

[0209] The effect of compound 6 according to the present invention on the treatment of colorectal cancer was confirmed. From the 50th day to the 63rd day of the experiment, DSS was administered to the mice on the 7th and 14th day after AOM was administered to induce colorectal cancer, and compound 6 was administered 14 times from the 50th day to the 63rd day. As a result, the control group (AOM / DSS+vehicle) showed a decrease in body weight after 70 days, but the group administered with compound 6 (AOM / DSS+compound 6) showed an increase in body weight similar to that of normal mice (normal) ( Fig.13 In addition, it was confirmed that the number of tumors in the compound 6-administered group was significantly smaller than that in the control group, and the size was also significantly smaller than that in the control group ( Fig.14 ). That is, it was confirmed that compound 6 is effective in treating colorectal cancer ( Fig.13 and Fig.14 ). Therefore, it was confirmed that compound 6 can be used as an effective preventive and therapeutic agent for colorectal cancer.

[0210] Example 6: Confirmation of the therapeutic effect of psoriasis

[0211] In order to study the therapeutic effect of the compound according to the present invention prepared as described above on psoriasis, the following experiment was carried out ( Figures 15 to 17 ).

[0212] The hair on the back of female BALB / C mice (8-10 weeks old) was shaved using a razor and shaving cream, and 62.5 mg of 5% imiquimod cream (imiquimod cream) was applied to the shaved skin every day from the second day for 6 days. Vaseline cream was applied to control mice (vehicle). In the compound 6 administration group according to the present invention, the compound of each mouse 10 mg / kg was completely added to the ethanol-polyoxyethylene castor oil EL (Cremophor EL) mixture (1: 1, v / v) corresponding to 15% (v / v) of the applied dose, and then diluted in phosphate buffered saline to prepare the final ethanol: polyoxyethylene castor oil EL (Cremophor EL): phosphate buffered saline (7.5: 7.5: 85, v / v / v), and then 200 μl of the prepared solution was orally administered every day from the 0th day to the 5th day of the experiment, for a total of 6 times. On the 6th day of the experiment, the back skin tissue of the mouse was obtained for histology and mRNA experiments.

[0213] As a result, it was confirmed that in the oral administration group of compound 6, the epithelial thickness was significantly reduced ( Fig.15), and the mRNA expression levels of IL-17a and S100a8, which promote inflammation in skin lesions, were significantly decreased ( Fig.16 ), as well as a significant increase in Foxp3 and IL-10, which induces suppression of inflammation in skin lesions ( Fig.17 ). Therefore, it was confirmed that compound 6 can be used as an effective therapeutic agent for psoriasis.

[0214] Example 7: Confirmation of the therapeutic effect of graft-versus-host disease

[0215] In order to study the therapeutic effect of the compound according to the present invention prepared as described above on graft-versus-host disease, compound 6 was administered to a graft-versus-host disease model to evaluate its efficacy ( Fig.18 and Fig.19 ).

[0216] The spleen of C57BL / 6 mice (8 to 12 weeks old, female, 18±3 g) was removed, RPMI medium was added and crushed, and then passed through a 40μm cell strainer (BD Falcon) to obtain a single-cell suspension. The single-cell suspension was centrifuged (1200rpm, 5 minutes), and after discarding the supernatant, 1 ml of ACK (ammonium chloride / potassium bicarbonate) lysis buffer (0.15M NH4Cl, 1mM KHCO3, 0.1mM Na2EDTA) was added, followed by stirring for 1 minute and then washing with RPMI medium. After centrifugation, the cell suspension was reacted on mouse CD90.2 microbeads (Miltenyi Biotec, catalog number 130-121-278) at 4°C for 20 minutes. After the reaction was completed, the cell suspension was centrifuged and filtered with 10 ml of The cells were washed with running buffer (Miltenyi Biotec, catalog number 130-091-221) and then washed with 3 ml of Then, CD90.2 was obtained from the cell suspension using Auto MACS pro (Miltenyi Biotec). + T cells (positive selection). + The bone marrow cells transplanted with T cells were aseptically obtained from the femur and tibia of normal (wild type) C57BL / 6 mice (8-12 weeks old, female, 18 ± 3g). The ends of the femur and tibia were cut off, and the bone marrow was extracted by perfusing RPMI culture medium to the bone tissue with a syringe (femur 21G, tibia 26G). The extracted bone marrow was passed through a 40 μm cell strainer to obtain a single cell suspension.

[0217] The bone marrow single cell suspension was centrifuged, and after discarding the supernatant, 500 μl of ACK lysis buffer was added, followed by stirring for 30 seconds and washing the solution with RPMI medium. After centrifugation, the suspension was reacted on mouse CD90.2 microbeads at 4°C for 20 minutes. After the reaction was completed, the cell suspension was centrifuged and washed with 10 ml of Wash with running buffer and then 3 ml of Then, the CD90.2 - T cell-depleted bone marrow cells (TCD-BM) (negative selection). + T cells and normal TCD-BM were washed with phosphate-buffered saline. T cells were plated at 4 × 10 6 T cells were prepared by resuspending TCD-BM at 5 × 10 6 Prepare TCD-BM by resuspending the cells in phosphate-buffered saline.

[0218] Normal BDF1 mice (9 to 11 weeks old, female, 19±3 g) were irradiated with 950 cGy of radiation at 3-hour intervals using a radiation irradiator. + The graft prepared by mixing T cells and TCD-BM in a ratio of 1:1 was injected through the tail vein of BDF1 mice at a flow rate of 100 μl. In the compound 6 administration group according to the present invention, 10 mg / kg of the compound per mouse was completely dissolved in an ethanol-Cremophor EL mixture corresponding to 7.5% (v / v) of the administered dose, and then diluted in phosphate buffered saline to prepare the final ethanol: Cremophor EL: phosphate buffered saline (0.375: 0.375: 9.25, v / v / v), and then 200 μl of the prepared solution was orally administered every day from the 3rd day to the 14th day of the experiment, for a total of 12 times.

[0219] The GVHD severity index was evaluated by visual observation at intervals of 3 to 4 days in a severity index system divided into a total of 10 points, with 0 to 2 points for each item of weight loss, hair condition, posture, activity, and skin changes.

[0220] As a result of the analysis, the severity index of graft-versus-host disease (8±1) occurring in the solvent control group (vehicle) was significantly reduced (2.2±1.2) by administration of compound 6 (***, p<0.001 compared to the solvent control group, see Fig.18). Histopathological analysis of the colon tissues of mice in each experimental group confirmed that the administration of compound 6 significantly reduced the inflammatory pathological index of the colon tissues (see Fig.19 ). These results show that compound 6 can be used as a preventive and therapeutic drug for graft-versus-host disease.

[0221] Example 8: Confirmation of the effect of multiple sclerosis treatment

[0222] In order to study the therapeutic effect of the compound according to the present invention prepared as described above on multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) was induced in C57BL / 6 mice as follows, and compound 6 was administered to the mice to evaluate its efficacy ( Fig. 20 ).

[0223] On day 0 of the experiment, myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide (MOG 35-55 , Peptron) (200 μg), heat-killed Mycobacterium tuberculosis, Difco, catalog number 231141) (500 μg) and adjuvant (incomplete Freund's adjuvant, Sigma Aldrich, catalog number F5506) were mixed for 7 minutes to form an emulsion. 35-55 The emulsion was subcutaneously injected into the flanks of both sides of C57BL / 6 mice (7-8 weeks old, female), and then 100 μl of pertussis toxin (Sigma Aldrich, catalog number P2980) (200 ng) was administered into the tail vein. On the second day of the experiment, an equal amount of pertussis toxin was administered intravenously. The leakage of the emulsion from the injection site of the mouse was checked by visual observation to confirm the onset of multiple sclerosis by visual observation starting from the seventh day of the experiment.

[0224] In the compound 6 administration group according to the present invention, 10 mg / kg of compound 6 per mouse was completely dissolved in a 7.5% (v / v) ethanol-Cremophor EL mixture corresponding to the administered dose, and then diluted in phosphate buffered saline to prepare a final ethanol: Cremophor EL: phosphate buffered saline (0.375: 0.375: 9.25, v / v / v), and then 200 μl of the prepared solution was orally administered daily from the 15th day to the 20th day of the experiment, for a total of 6 times. The multiple sclerosis index was visually observed and recorded in a severity index system divided into 0-5 levels.

[0225] The symptoms of autoimmune encephalomyelitis were evaluated by an index based on the following items.

[0226] 0: No symptoms

[0227] 1: Tail weakness

[0228] 2: Tail weakness and hind limb weakness

[0229] 3: Paralysis of hind limbs

[0230] 4: Paralysis of the hind limbs and weakness of the forelimbs

[0231] 5: Dead or dying

[0232] As a result of the analysis, on day 21 of the experiment (acute reaction period), the disease severity index was 2.9±1.92 in the solvent control group (vehicle) and was 0.92±0.58 in the compound 6-administered group, so that the compound 6-administered group showed a lower severity index than the solvent control group (vehicle) (*, p<0.05, Fig. 20 ), and thus confirmed the therapeutic effect of compound 6 on multiple sclerosis.

[0233] Example 9: Confirmation of the therapeutic effect of neutrophilic asthma

[0234] In order to study the therapeutic effect of the compound according to the present invention prepared as described above on neutrophilic asthma, compound 6 was administered to a neutrophilic asthma mouse model to evaluate its efficacy ( Fig.21 ).

[0235] Sensitization was induced in C57BL / 6 mice (7 weeks old, female) by intranasal administration of 10 μg of LPS (lipopolysaccharide, Sigma Aldrich, catalog number L2630) and 75 μg of OVA (ovalbumin, Sigma Aldrich, catalog number A5503) diluted in phosphate buffered saline (20 μl in total) on days 0, 1, 2, and 7 of the experiment. From day 14 of the experiment, challenges were performed by intranasal administration of 50 μg of OVA diluted in phosphate buffered saline at 2-day intervals per week, for a total of 8 times until day 36. Intranasal administration was performed after anesthesia using isoflurane inhalation anesthetic. In the compound 6 administration group according to the present invention, 10 mg / kg of compound 6 per mouse was completely dissolved in a 7.5% (v / v) ethanol-Cremophor EL mixture corresponding to the administered dose, and then diluted in phosphate-buffered saline to prepare a final ethanol: Cremophor EL: phosphate-buffered saline (0.375: 0.375: 9.25, v / v / v), and then 200 μl of the prepared solution was orally administered daily from the 27th day to the 36th day of the experiment, for a total of 10 times.

[0236] As a result, the number of neutrophils in the bronchoalveolar lavage fluid (BAL) was significantly decreased in the compound 6-administered group compared with the solvent control group (vehicle). Fig.21 ), and thus confirmed the therapeutic effect of compound 6 on neutrophilic asthma.

[0237] Example 10: Comparison of the effects between the compounds of the present invention and Comparative Example 1

[0238] In order to confirm the superiority of the compounds according to the embodiments of the present invention compared with the compounds of Comparative Example 1 (referred to as "Comparative Example 1"), the following experiments were performed: (1) measurement of pharmacokinetic parameters; (2) measurement of solubility; (3) confirmation of the inhibitory effect on the inflammatory activity of Th17 cells; (4) confirmation of the promoting effect on the production of regulatory T cells expressing Foxp3; (5) confirmation of whether the activity of drug metabolism-related enzymes is inhibited; and (6) confirmation of the therapeutic effect on inflammatory bowel disease.

[0239] (1) Measurement of pharmacokinetic parameters

[0240] The pharmacokinetic parameters of compound 6 and comparative example 1 were measured (Table 3 and Fig. 22 ).

[0241] For rats, the oral administration group was fasted for 16 hours, and the intravenous administration group was intravenously administered without fasting, at the dose concentration listed in Table 3, with 10 mL / kg of the following solution, which was prepared by completely dissolving Compound 6 in a 7.5% (v / v) ethanol-Cremophor EL mixture corresponding to the administered dose, and then diluting in phosphate buffered saline to become a final ethanol: Cremophor EL: phosphate buffered saline (0.375: 0.375: 9.25, v / v / v). The oral administration group was fed food about 4 hours after administration. Blood was collected from the intravenous administration group 0.083, 0.5, 1, 2, 4, 8, 24 hours after administration, and blood was collected from the oral administration group 0.5, 1, 2, 4, 8, 12, 24 hours after administration, and plasma was recovered using centrifugation. In the recovered plasma, the drug concentration in plasma was measured using the UHPLC-MS / MS method.

[0242] As shown in Table 3, it was confirmed that the bioavailability (F%) and AUC were significantly higher when Compound 6 was orally administered at a concentration of 10 mg / kg than when Comparative Example 1 was orally administered at a concentration of 10 mg / kg. last and C maxIn addition, it was confirmed that the bioavailability (F%), AUC last and C max The increases were 67.2 times, 66.8 times, and 30.2 times, respectively. From the above results, it was confirmed that Compound 6 exhibited medicinal effects even in a smaller amount than in Comparative Example 1.

[0243] [Table 3]

[0244]

[0245] (2) Solubility measurement

[0246] The solubility of Compound 6 and Comparative Example 1 was measured.

[0247] After dissolving Compound 6 in ethanol, the solubility was measured and analyzed using a SIRIUS T3 machine.

[0248] As listed in Table 4, it was confirmed that the logP value of Compound 6 was greater than that of Comparative Example 1, and the pH-metric solubility molar concentration was about 3.92 times.

[0249] [Table 4]

[0250]

[0251] (3) Confirmation of the inhibitory effect on Th17 cell inflammatory activity

[0252] Comparison of the inhibitory effects of Compound 6 and Comparative Example 1 on the inflammatory activity of Th17 cells, which are key pathogenic cells of autoimmune diseases ( Fig.23 ).

[0253] The spleen of C57BL / 6 mice (8 to 12 weeks old, female) was removed, crushed by adding IMDM medium, and then passed through a 40μm cell strainer (BD Falcon) to obtain a single cell suspension. The single cell suspension was centrifuged (1200rpm, 5 minutes), and after discarding the supernatant, 1ml of ACK lysis buffer was added, followed by stirring for 1 minute and then washing with RPMI medium. After centrifuging the single cell suspension, T cells were isolated using a mouse CD4 naive T cell enrichment kit (Invitrogen). The isolated T cells were separated at 1×10 6The isolated T cells were prepared by resuspending the cells in RPMI-fetal bovine serum (FBS) 10% + 2-ME (mercaptoethanol) medium. For T cell activation, 5 μg / ml anti-CD3 (eBioscience TM ) was dispensed into 96-well plates in 100 μl, reacted in a cell culture incubator (37°C, 5% CO2 incubator) for 4 hours, and washed with phosphate-buffered saline to prepare the plate. T cells were dispensed into the prepared plate in 200 μl and incubated with 2 μg / ml anti-CD28 (eBioscience TM ), 3ng / ml TGFβ-1 (R&D systems), 10ng / ml IL-2 (R&D systems), 5μg / ml anti-mouse IFN-γ (Bio X cell) and 5μg / ml anti-mouse IL-4 (Bio X cell) were treated in each well. 5μl of each compound diluted in RPMI-fetal bovine serum 10% + 2-ME medium at a concentration of 1.0μM, 2.5μM and 5μM was treated, and then cultured in a cell culture incubator (37°C, 5% CO2 incubator) for 3 days. As a control, 5μl of 0.05% dimethyl sulfoxide (DMSO) / RPMI medium was used for treatment. After 3 days, in order to confirm the inhibitory effect of the compound on the inflammatory activity of Th17 cells, the culture medium was recovered to confirm IL-17A using flex set (BD biosciences).

[0254] In order to confirm the IL-17A cytokine, 25 μl of recovery culture medium and 25 μl of assay dilution buffer were placed in a FACS tube (BD falcon), and the sample was diluted 1 / 2. 1 μl of capture beads (capture bead) was placed in 49 μl of capture bead diluent to prepare 50 μl of capture bead solution for each sample. After the capture bead solution was mixed by vortex, it was added to the FACS tube containing each sample with 50 μl, then vortexed again, and placed at room temperature for 1 hour. After 1 hour, 1 μl of PE detection reagent was added to 49 μl of PE detection reagent diluent to prepare 50 μl of PE detection solution for each sample. After the PE detection solution was vortexed, it was added to the FACS tube containing capture bead solution and sample with 50 μl. After vortexing the FACS tube, they were placed at room temperature for 1 hour. After 1 hour, 1 ml of CBA washing buffer was added to each tube, and centrifuged at 400 g for 5 minutes, and then the supernatant was removed. After gentle vortexing, 150 μl of fixation buffer was added and gently vortexed before analysis using flow cytometry.

[0255] As a result, it was confirmed that Compound 6 significantly reduced the production of IL-17A by Th17 cells compared with Comparative Example 1 ( Fig.23).

[0256] (4) Confirmation of the effect of promoting the generation of regulatory T cells (Treg) expressing Foxp3

[0257] Comparison of the effects of Compound 6 and Comparative Example 1 on the generation of "FoxP3-expressing regulatory T cells" which play an important role in maintaining immune tolerance ( Fig.24 ). Figure 4 The same experimental method was used to isolate and culture CD4 + T cells were then treated with 5 μl of each compound diluted in RPMI-10% fetal bovine serum + 2ME medium at a concentration of 2.5 μM and 5 μM. After culturing in a cell culture incubator (37°C 5% CO2 incubator) for 3 days, the analysis was performed. As a control, 5 μl of 0.05% dimethyl sulfoxide (DMSO) / RPMI medium was treated. After 3 days, in order to confirm the effect of generating regulatory T cells, the cultured cells were recovered and incubated with the same Figure 4 Foxp3 protein expression was measured in the same manner as described above.

[0258] As a result, it was confirmed that Compound 6 significantly increased the production of regulatory T cells expressing Foxp3 compared with Comparative Example 1 ( Fig.24 ).

[0259] (5) Confirmation of whether the activity of drug metabolism-related enzymes is inhibited

[0260] The activity of CYP isozymes, which are enzymes involved in drug metabolism, is measured.

[0261] Live human hepatocytes (Corning, catalog number 454551) were collected using plate culture medium and seeded on 24-well plates to a cell density of 0.4 × 10 6 Cells / well were added and cultured for 2 to 4 hours. After replacing the plate medium with hepatocyte culture medium, the hepatocytes were cultured for 24 hours. After 24 hours of culture, the hepatocytes were treated with 1, 5, and 25M of compound 6. The culture medium containing the compound was replaced every 24 hours for 2 days. The cultured cells were recovered to prepare mRNA samples. mRNA was extracted from the recovered cells by phenol-chloroform precipitation using TRIZOL reagent (Invitrogen, catalog number 15596018). cDNA was synthesized from the isolated RNA by reverse transcription and then analyzed using Quant Studio TMThe 7Flex real-time PCR system (Applied Biosystems, CA) was used to measure drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2B6, and CYP3A4 by real-time PCR analysis. GAPDH was used as a control enzyme to compare the relative values ​​of enzyme expression levels by the ΔΔct method. Herein, a control setting of one (1) times was used. The results are expressed as % of the control group compared to the control sample.

[0262] Table 5 shows the changes in CYP isozyme activity after treatment with 10 μM of each compound (Comparative Example 1, Compound 6, Ketoconazole) (% of control activity)

[0263] [Table 5]

[0264]

[0265] As a result, it was confirmed that Comparative Example 1 inhibited the enzyme activity of CYP2C19 by 24.4% at 10 μM, while Compound 6 had almost no effect on the enzyme activity of CYP2C19. That is, it was confirmed that Compound 6 can be administered in combination with a drug metabolized by CYP2C19.

[0266] (6) Confirmation of therapeutic effects on inflammatory bowel disease

[0267] The therapeutic effects of Compound 6 and Comparative Example 1 on inflammatory bowel disease were compared. Inflammatory bowel disease was induced in C57BL / 6 mice in the same manner as in Example 4, and after oral administration of 10 mg / kg of Compound 6 and Comparative Example 1 to the mice, the expression levels of IL-6 and S100a9 mRNA were confirmed in the large intestine tissue.

[0268] As a result, it was confirmed that Compound 6 significantly reduced the expression levels of IL-6 and S100a9 mRNA in colon tissue compared with Comparative Example 1 ( Fig.25 ). That is, it was confirmed that Compound 6 showed a higher therapeutic effect on inflammatory bowel disease than Comparative Example 1.

Claims

1. A compound represented by the following formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof: [Formula 1] (Among them, A is hydrogen, halogen, hydroxy, C1-C3 alkyl, C2-C3 alkenyl, C2-C3 alkynyl, C1-C3 alkoxy, dimethylamine, -NO2, -CN, -COOR2 or -S(=O)2R2, B is hydrogen, C1-C3 alkyl, phenyl, acetyl, -CH2C(=O)OR2, -C(=O)OR2 or -S(=O)2R2, R1 is a substituted or unsubstituted 5-7 membered heterocycle or -NH2, and R2 is C1-C3 alkyl).

2. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the substituted 5-7 membered heterocyclic ring is a 5-7 membered heterocyclic ring substituted by a C1-C3 alkyl group, a hydroxyl group or a dimethylamine group.

3. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the substituted or unsubstituted 5-7 membered heterocycle is any one selected from the group consisting of the following heterocycles:

4. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound represented by Formula 1 is any one selected from the group consisting of the following compounds:

5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4, its stereoisomer or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 5 , wherein the composition is used to treat or prevent an autoimmune disease.

7. The pharmaceutical composition according to claim 5, wherein the autoimmune disease is any one selected from the group consisting of inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, systemic lupus erythematosus and type 1 diabetes.

8. The pharmaceutical composition according to claim 5, wherein the composition is used for treating or preventing cancer.

9. The pharmaceutical composition of claim 8, wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer and lung cancer.

10. A method for treating an autoimmune disease, comprising administering a compound according to any one of claims 1 to 4 to a subject in need thereof.

11. A method for treating cancer, comprising administering a compound according to any one of claims 1 to 4 to a subject in need thereof.

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