Novel Compounds and Their Use in the Treatment of Autoimmune Diseases

Abstract

The present invention relates to novel compounds and their use in the treatment of autoimmune diseases. A pharmaceutical composition for the treatment or prevention of autoimmune diseases containing the novel compounds of the present invention is expected to restore the homeostasis of intestinal tissue not only by controlling inflammation but also by regulating the immune balance and repairing damaged tissues.

Description

Technical Field

[0001] The present invention relates to novel compounds and their use in the treatment of autoimmune diseases.

Background Art

[0002] Humans protect their bodies from pathogens through immune responses. The body's defense mechanisms against foreign microorganisms such as viruses and bacteria are divided into innate immunity and specific immunity, which are mediated by cytokines mainly secreted from immune-related cells.

[0003] The immune system plays a role in protecting the body from antigens, which are harmful external substances. Examples of such antigens include bacteria, viruses, toxins, cancer cells, and other types of blood and tissues. The immune system generates antibodies to destroy these harmful substances, but when an abnormality occurs in autoimmunity, the immune system cannot distinguish its own organs from harmful antigens and destroys normal tissues. The diseases induced by such reactions are autoimmune diseases.

[0004] The 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 that senses not only xenobiotic ligands such as environmental pollutants (e.g., dioxin) but also physiological ligands generated from cells, microorganisms, and food.

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

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a novel compound, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0007] An object of the present invention is to provide a novel compound, its stereoisomer or a pharmaceutically acceptable salt thereof that is useful for the prevention and treatment of autoimmune diseases.

[0008] An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases containing a novel compound, its stereoisomer or a pharmaceutically acceptable salt thereof.

Means for Solving the Problems

[0009] 1. A compound represented by the following Chemical Formula 1, its stereoisomer or a pharmaceutically acceptable salt thereof.

Chemical Formula

[0010] 2. The compound according to item 1 above, wherein the substituted 5-7 membered heterocyclic ring is a 5-7 membered heterocyclic ring substituted with a C1-C3 alkyl group, hydroxy group or dimethylamine, its stereoisomer or its pharmaceutically acceptable salt.

[0011] 3. The compound according to item 1 above, wherein the substituted or unsubstituted 5-7 membered heterocyclic ring is any one selected from the group consisting of the following heterocyclic rings, its stereoisomer or its pharmaceutically acceptable salt. JPEG2025520373000003.jpg8792

[0012] 4. The compound according to item 1 above, which is a compound selected from the group consisting of the following compounds, its stereoisomer or its pharmaceutically acceptable salt. JPEG2025520373000004.jpg207140 JPEG2025520373000005.jpg207140 JPEG2025520373000006.jpg197140 JPEG2025520373000007.jpg73138

[0013] 5. A pharmaceutical composition comprising the compound according to any one of items 1 to 4 above, its stereoisomer or its pharmaceutically acceptable salt.

[0014] 6. The pharmaceutical composition according to item 5 above, which is for the treatment or prevention of autoimmune diseases.

[0015] 7. The pharmaceutical composition according to item 5 above, which is for the treatment or prevention of any one autoimmune disease 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.

[0016] 8. The pharmaceutical composition according to item 5 above, which is for the treatment or prevention of cancer.

[0017] 9. The composition according to item 8 above, which is for the treatment or prevention of cancer, wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer, and lung cancer.

Advantages of the Invention

[0018] The novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt has the effect of regulating the immune balance and repairing damaged tissues in addition to controlling inflammation by inducing the activity of AHR, an immunomodulatory transcription factor.

[0019] The novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt has the effect of regulating an excessive immune reaction, specifically an autoimmune reaction, by suppressing the production of IL-6, an inflammatory factor.

[0020] The novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt has the effect of inducing the activity of regulatory T cells (Treg).

[0021] In addition, the novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt has the effect of preventing and treating autoimmune diseases by regulating the inflammatory factors.

Brief Description of the Drawings

[0022]

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Best Mode for Carrying Out the Invention

[0023] The present invention will be described in detail below.

[0024] All technical terms used in this specification have the same meaning as commonly understood by those of ordinary skill in the relevant art of the present invention, unless otherwise defined. Also, although preferred methods or samples are described in this specification, similar or equivalent ones are also included within the scope of the present invention.

[0025] In the chemical formula (structural formula) of the present invention, when no substituents are described despite being required at certain positions, it means that the hydrogen substituents are omitted. This applies equally to all chemical formulas (structural formulas) of the present invention.

[0026] The present invention relates to a compound represented by the following chemical formula 1, its stereoisomers, or its pharmaceutically acceptable salts.

[0027]

Chemical formula

[0028] In the above structural formula, when no substituents are described despite being required at certain positions, it means that the hydrogen substituents are omitted. This applies equally to all structural formulas of the present invention.

[0029] In Chemical Formula 1 above, A is hydrogen, halogen, hydroxy group, C1-C3 alkyl group, C2-C3 alkenyl group, C2-C3 alkynyl group, C1-C3 alkoxy group, dimethylamine, -NO2, -CN, -COOR2 or -S(=O)2R2; B is hydrogen, C1-C3 alkyl group, phenyl group, acetyl group, -CH2C(=O)OR2, -C(=O)OR2 or -S(=O)2R2; R1 is a substituted or unsubstituted 5-7 membered heterocyclic ring or -NH2; and R2 is a C1-C3 alkyl group.

[0030] R1 is substituted at any of the benzene ring carbon atoms of benzothiazole.

[0031] The 5- to 7-membered heterocyclic ring may be a compound in which 1 to 2 carbon atoms in the ring are substituted with nitrogen or oxygen atoms.

[0032] The substituted 5- to 7-membered heterocyclic ring may be a compound which is a 5- to 7-membered heterocyclic ring substituted with a C1-C3 alkyl group, a hydroxy group or dimethylamine.

[0033] The substituted or unsubstituted 5- to 7-membered heterocyclic ring may be any one selected from the group consisting of the following heterocyclic rings. JPEG2025520373000009.jpg8287

[0034] The compound represented by Chemical Formula 1 may be any one selected from the group consisting of the compounds shown in Table 1 below.

[0035]

Table 1

[0036] The compound represented by Chemical Formula 1 may be any one selected from the group consisting of the following compounds. 2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 6); 2-(5-chloro-1H-indol-3-yl)-N-(6-(4-methylpiperazine-1-yl)benzo[d]thiazol-2-yl)acetamide (Compound 9); 2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 21); 2-(5-hydroxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 32); and 2-(5-methoxy-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 39).

[0037] The present invention also relates to a pharmaceutical composition comprising the above compound, its stereoisomer or its pharmaceutically acceptable salt.

[0038] The pharmaceutical composition may be a pharmaceutical composition for the treatment or prevention of autoimmune diseases. Specifically, it 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), Behçet's disease, or Sjögren's syndrome. More specifically, it may be inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, asthma, atopy, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, or type 1 diabetes mellitus, but is not limited thereto.

[0039] In the present invention, the "autoimmune disease" refers to a disease that damages cells and tissues by humoral immunity, cellular immunity, or both, in which the immune system has an inappropriate reaction to autoantigens, and the autoimmune reaction appears systemically or specifically in specific organs, etc., and can induce chronic inflammation.

[0040] The "inflammatory bowel disease" refers to a disease in which abnormal chronic inflammation in the intestinal tract repeats remission and recurrence, and may be one or more diseases from the group consisting of Crohn's disease, ulcerative colitis, and intestinal Behçet's disease, but is not limited thereto.

[0041] The above-mentioned "multiple sclerosis" refers to a broad range of signs and symptoms caused by damage and / or depletion of the fatty myelin sheath that wraps around the axonal processes of the brain and spinal cord, and refers to an inflammatory disease that induces demyelination and scar formation. The types of multiple sclerosis include, but are not limited to, relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), progressive relapsing multiple sclerosis (PRMS), etc.

[0042] The above-mentioned "graft-versus-host disease" refers to a disease in which lymphocytes transfused during hematopoietic stem cell transplantation attack a host with reduced immune function, causing symptoms such as fever, rash, and abnormal liver function, and can invade the skin, lungs, intestines, liver, etc., but are not limited thereto.

[0043] The above-mentioned "asthma" refers to a disease in which symptoms such as cough and dyspnea repeatedly occur due to bronchial inflammation when exposed to specific causative substances, and can be caused by, but are not limited to, infection, smoking, allergic antigens, etc.

[0044] The above-mentioned "atopy" means atopic dermatitis. It is a chronic relapsing inflammatory skin disease and a typical allergic disease with symptoms such as itching and dry skin.

[0045] The above-mentioned "psoriasis" refers to an inflammatory disease that occurs in the skin or joints due to abnormalities in the immune system, causing problems such as deterioration of appearance, accumulation of keratin, erythematous plaques, and pain. Psoriasis can include any one or more diseases selected from psoriatic arthritis, guttate psoriasis, pustular psoriasis, erythrodermic psoriasis, scalp psoriasis, nail psoriasis, and enthesitis.

[0046] The above-mentioned "rheumatoid arthritis" refers to a systemic autoimmune disease characterized by chronic inflammation of the joint area.

[0047] The "systemic lupus erythematosus" is a chronic inflammatory autoimmune disease, also known as "lupus", which refers to a systemic disease that invades various organs of the body such as connective tissue, skin, joints, blood, and kidneys. Although the exact cause is unknown, previous studies have shown that genetic factors are involved. The American College of Rheumatology (ACR) has published 11 symptoms, signs, and test findings that are useful for differentiating lupus from other diseases to make the diagnosis of lupus easier. If there are more than 4 symptoms among the 11 items, lupus can be diagnosed.

[0048] The "type 1 diabetes" is an immune-mediated disease in which insulin-secreting β-cells are destroyed by an autoimmune reaction, and the causes include a number of genetic and environmental factors. This can be accompanied by progressive inflammatory infiltration of the islets of Langerhans by immune cells specifically targeted at insulin-secreting β-cells.

[0049] The pharmaceutical composition of the present invention can be prepared using pharmaceutically appropriate and physiologically acceptable adjuvants in addition to the active ingredient which is the compound of the present invention, or can be administered to mammals. As the adjuvants, excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents can be used.

[0050] In addition, the pharmaceutical composition of the present invention can further contain one or more pharmaceutically acceptable carriers in addition to the pharmaceutically effective amount of the active ingredient for administration, and can be preferably formulated into a pharmaceutical composition.

[0051] The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined according to factors including the type of disease, its severity, the activity of the drug, the sensitivity to the drug, the administration time, the administration route, and the excretion ratio, the treatment period, elements including drugs used simultaneously, and other elements well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents. It may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above elements, and this can be easily determined by those skilled in the art.

[0052] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, weight, absorption degree, inactivation rate and excretion rate of the active ingredient in the body, the type of disease, and the drugs used in combination. Usually, it can be administered at 0.001 - 150 mg per kg of body weight, preferably 0.01 - 100 mg, daily or every other day, or divided into 1 - 3 doses per day. However, since it can be increased or decreased depending on the administration route, severity of obesity, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any sense.

[0053] Also, the term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and usually does not cause gastrointestinal disorders, allergic reactions such as dizziness or similar reactions when administered to humans.

[0054] Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Further, it may further contain a filler, an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.

[0055] In addition, the composition of the present invention can be formulated by a method known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after being administered to an individual in need of the pharmaceutical composition of the present invention, including humans. The dosage form may be a powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

[0056] In the present invention, "pharmaceutically acceptable salt" means a salt prepared using an acid or base that is relatively non-toxic compared to a specific compound according to the present invention, and the pharmaceutically acceptable salt may be, for example, an acid addition salt or a metal salt.

[0057] Acid addition salts can be formed from non-toxic organic acids such as inorganic acids like hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

[0058] The metal salt may be a sodium, potassium or calcium salt. The metal salt can be produced using a base. For example, an alkali metal or alkaline earth metal salt can be obtained by dissolving the compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering off the insoluble compound salt, and evaporating and drying the filtrate.

[0059] The present invention relates to a method for treating an autoimmune disease, which includes the step of administering the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0060] In addition, the present invention relates to a method for inducing the activity of AHR, which includes the step of administering the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0061] Specifically, the compound of the present invention targets the aryl hydrocarbon receptor (AHR), an immune regulatory transcription factor, and acts as an agonist that induces the activity of AHR, thereby controlling inflammation, regulating immune balance, and repairing damaged tissues, and can be used for the treatment of autoimmune diseases, but is not limited thereto. Conventional ligands have problems that are not suitable for development as pharmaceutical compositions due to toxicity, low affinity and structural stability, and high target non-specificity. In contrast, inducing the activity of AHR with the compound having "Drug-like properties" of the present invention can be effectively used for the treatment and prevention of autoimmune diseases.

[0062] The present invention relates to a method for suppressing the production of IL-6, which includes the step of administering the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0063] Specifically, since it is known that IL-6, an inflammatory factor, induces autoimmune diseases, the compound of the present invention can be used for the treatment of autoimmune diseases by a mechanism that suppresses its production. In fact, there are many known therapeutic drugs and related papers for treating autoimmune diseases with the suppression of IL-6 as the treatment purpose. It has also been confirmed from the following experimental data that the compound of the present invention can also suppress the production of IL-6, and it is expected to have the effect of reducing the autoimmune reaction, so it can be used for the treatment and prevention of autoimmune diseases.

[0064] Furthermore, the present invention relates to a composition for preventing or treating cancer, which comprises the compound, its stereoisomer or its pharmaceutically acceptable salt.

[0065] In the present invention, "cancer" broadly refers to the uncontrolled abnormal growth of host cells that invade the surrounding tissues of the initial abnormal cell growth site in the host and the potential tissues distal to that site, and includes carcinomas that are cancers of epithelial tissues (e.g., skin, squamous cells); sarcomas that are cancers of connective tissues (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias that are cancers of hematopoietic tissues (e.g., bone marrow tissue); lymphomas and myelomas that are cancers of immune cells; and cancers of the central nervous system including cancers from brain and spinal tissues.

[0066] Specifically, the cancer can be selected from the group consisting of, but not limited to, colorectal cancer, melanoma, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer, and lung cancer.

[0067] The present invention relates to a method for treating cancer, which comprises the step of administering the compound, its stereoisomer, or its pharmaceutically acceptable salt to a subject in need thereof.

[0068] In the above treatment method, the compound, its stereoisomer or its pharmaceutically acceptable salt can be administered to a patient who has received a cancer diagnosis at any stage of anti-cancer treatment, and is not limited to a specific stage.

[0069] Also, the compound, its stereoisomer or its pharmaceutically acceptable salt can be administered in the form of the aforementioned pharmaceutical composition, but is not limited thereto.

[0070] The compound represented by Chemical Formula 1 of the present invention can be produced by methods known in various literatures.

[0071] Hereinafter, the present invention will be described in detail with reference to the production examples and examples of the present invention.

Examples

[0072] Production Example Production Example 1: Synthesis of 2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzothiazol-2-yl)acetamide (Compound 6) (1) Synthesis of perfluorophenyl 2-(5-chloro-1H-indol-3-yl)acetate JPEG2025520373000014.jpg39146 A solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (56.00 g, 267.14 mmol), pentafluorophenol (54.9 g, 293.85 mmol), and EDC-HCl (61.45 g, 320.57 mmol) in dichloromethane (890 mL) was stirred at room temperature, and N,N-diisopropylethylamine (52.82 mL, 293.85 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours. After confirming the completion of the reaction, distilled water (890 mL) was added. The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by column chromatography to obtain perfluorophenyl 2-(5-chloro-1H-indol-3-yl)acetate (50.00 g, yield 50%).

[0073] (2) Synthesis of 2-(5-chloro-1H-indol-3-yl)-N-(6-morpholinobenzothiazol-2-yl)acetamide (Compound 6) JPEG2025520373000015.jpg47142 A solution of 6-morpholinobenzothiazol-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 at room temperature. The reaction mixture was heated to reflux for 24 hours. After confirming the completion of the reaction, it was cooled to room temperature. The reaction mixture was concentrated under reduced pressure and then purified by MPLC to obtain Compound 6 (28.5 g, yield 60%).

[0074] 1 H NMR (DMSO-d6, 400 MHz): δ 12.37 (s, 1H), 11.12 (s, 1H), 7.59 (t, 2H, J = 16.0 Hz), 7.43 (d, 2H, J = 16.0 Hz), 7.34 (s, 1H), 7.12 (d, 1H, J = 8.0 Hz), 7.02 (d, 1H, J = 12.0 Hz), 3.88 (m, 2H), 3.74 (m, 4H), 3.11 (m, 4H)

[0075] Production 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) JPEG2025520373000016.jpg52158 At room temperature, 2-(5-chloro-1H-indol-3-yl)acetic acid (210 mg, 1.00 mmol), 6-(4-methylpiperazin-1-yl)benzo[d]thiazol-2-amine (249 mg, 1.00 mmol), HBTU (760 mg, 2.00 mmol), and DIPEA (0.7 mL, 4.01 mmol) were added to a DMF (10 ml) solution and stirred for 2 days. After completion of the reaction, the reaction mixture was poured into ice water (50 mL), stirred for 30 minutes, filtered, and washed with water. The obtained solid mixture was purified by column chromatography to obtain Compound 9 (100 g, 22%).

[0076] 1 H NMR (DMSO-d6, 400 MHz): δ 12.37 (s, 1H), 11.20 (s, 1H), 7.67 (d, 1H, J = 4.0 Hz), 7.56 (d, 1H, J = 8.0 Hz), 7.44 (d, 1H, J = 4.0 Hz), 7.37 (m, 2H), 7.06 (m, 2H), 3.87 (s, 2H), 3.14 (m, 4H), 2.50 (m, 4H), 2.24 (s, 3H)

[0077] Production Example 3: Synthesis of 2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 21) (1) Synthesis of Perfluorophenyl 2-(5-chloro-1-methyl-1H-indol-3-yl)acetate JPEG2025520373000017.jpg42152 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), and EDC-HCl (113 mg, 590.19 μmol) in dichloromethane (1.6 mL) was stirred, and N,N-diisopropylethylamine (0.1 mL, 541.01 μmol) was added. The reaction mixture was stirred at room temperature for 24 hours. After confirming the completion of the reaction, distilled water (2 mL) was added. The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by column chromatography to obtain perfluorophenyl 2-(5-chloro-1-methyl-1H-indol-3-yl)acetate (150 mg, 78%).

[0078] (2) Synthesis of 2-(5-chloro-1-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 21) JPEG2025520373000018.jpg51156 A solution of 6-morpholinobenzo[d]thiazol-2-amine (40 mg, 169.99 μmol) and perfluorophenyl 2-(5-chloro-1-methyl-1H-indol-3-yl)acetate (99 mg, 254.99 μmol) in THF (1.7 mL) was refluxed and stirred for 12 hours. After cooling the reaction mixture to room temperature, it was filtered under reduced pressure to remove impurities, and the filtrate was concentrated under reduced pressure. The concentrated reaction mixture was recrystallized from diethyl ether to obtain Compound 21 (40 mg, yield 54%).

[0079] 11H NMR (DMSO-d6, 500 MHz): δ 12.38 (s, 1H), 7.69 (s, 1H), 7.58 (d, 1H, J = 10.0 Hz), 7.44 (d, 2H, J = 5.0 Hz), 7.36 (s, 1H), 7.11 (m, 2H), 3.86 (s, 2H), 3.77 (s, 3H), 3.73 (m, 4H), 3.11 (m, 4H)

[0080] Production Example 4: Synthesis of 2-(5-Hydroxy-1H-indol-3-yl)-N-(6-morpholinobenzothiazol-2-yl)acetamide (Compound 32) JPEG2025520373000019.jpg56158 A solution of boron tribromide (7.9 μl, 71.01 μmol) in 2-(5-methoxy-1H-indol-3-yl)-N-(6-morpholinobenzothiazol-2-yl)acetamide (Compound 39) (25 mg, 59.17 μmol), which was obtained in the following Production Example 5, was stirred at room temperature for 6 hours. Distilled water was added to the reaction mixture to terminate the reaction. After neutralization with sat. NaHCO3, the layers were separated with DCM. The organic layer was washed, dried over anhydrous Na2SO4, and filtered. After concentrating the filtrate under reduced pressure, the concentrate was recrystallized from diethyl ether to obtain Compound 32 (11 mg, yield 45%).

[0081] 1 1H NMR (DMSO-d6, 500 MHz): δ 12.30 (s, 1H), 10.65 (s, 1H), 8.64 (s, 1H), 7.57 (d, 1H, J = 10.0 Hz), 7.45 (s, 1H), 7.19 (s, 1H), 7.11 (m, 2H), 6.90 (s, 1H), 6.59 (d, 1H, J = 5.0 Hz), 3.78 (s, 2H), 3.74 (m, 4H), 3.11 (m, 4H)

[0082] Production Example 5: Synthesis of 2-(5-Methyl-1H-indol-3-yl)-N-(6-morpholinobenzothiazol-2-yl)acetamide (Compound 39) (1) Synthesis of Perfluorophenyl 2-(5-methoxy-1H-indol-3-yl)acetate JPEG2025520373000020.jpg42161At room temperature, 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) was stirred, and N,N-diisopropylethylamine (0.2 mL, 1.07 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours. After confirming the completion of the reaction, distilled water (4 mL) was added. The separated organic layer was dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by column chromatography to obtain the title compound (288 mg, yield 80%).

[0083] (2) Synthesis of 2-(5-methyl-1H-indol-3-yl)-N-(6-morpholinobenzo[d]thiazol-2-yl)acetamide (Compound 39) JPEG2025520373000021.jpg331466A solution of 6-morpholinobenzo[d]thiazol-2-amine (100 mg, 424.98 μmol) and perfluorophenyl 2-(5-methoxy-1H-indol-3-yl)acetate (189 mg, 509.98 μmol) in THF (4.3 mL) was refluxed and stirred for 12 hours. After the reaction mixture was cooled to room temperature, it was filtered under reduced pressure to remove impurities, and the filtrate was concentrated under reduced pressure. The concentrated reaction mixture was recrystallized from diethyl ether to obtain Compound 39 (120 mg, yield 67%).

[0084] 1 H NMR (DMSO-d6, 500 MHz): δ 12.34 (s, 1H), 10.82 (s, 1H), 7.57 (d, 1H, J = 10.0 Hz), 7.45 (s, 1H), 7.24 (d, 2H, J = 10.0 Hz), 7.11 (m, 2H), 6.72 (dd, 1H, J = 10.0 Hz), 3.84 (s, 2H), 3.74 (m, 7H), 3.10 (m, 4H)

[0085] Comparative Example 1: Synthesis of N-(benzothiazol-2-yl)-2-(5-chloro-1H-indol-3-yl)acetamide (Comparative Example 1) JPEG2025520373000022.jpg30137 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, and benzothiazol-2-amine (4.51 mg, 30.05 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazol[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU, 15.24 g, 40.07 mmol), and trimethylamine (9.4 mL, 66.78 mmol) were sequentially added. 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%).

[0086] 1 1H NMR (DMSO-d6, 400 MHz): δ 12.57 (s, 1H), 11.21 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 4.0 Hz, 1H), 7.37 (m, 3H), 7.27 (t, J = 16.0 Hz, 1H), 7.07 (dd, J = 8.0 4.0 Hz, 1H), 3.91 (s, 2H)

[0087] Examples Example 1: Confirmation of the CYP1A1 expression induction effect To confirm the target specificity of the compound of the present invention produced as described above, it was confirmed whether the expression levels of CYP1A1 mRNA and CYP1A1 protein, which are AHR target genes, increased.

[0088] (1) CYP1A1 mRNA expression induction effect HepG2 cells cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) were harvested. Trypan blue staining was used to confirm that the viability was 97% or higher. Subsequently, the cells were centrifuged at 1200 rpm for 5 minutes at room temperature, and then the cells were resuspended in DMEM medium supplemented with 10% fetal bovine serum at a density of 3×10 5 cells / ml. Next, 3 ml of the cell suspension was dispensed into each 60-mm dish, and 50 μl of compounds 6, 21, 32, and 39 at a concentration of 1 μM diluted in DMEM medium was added to each dish. The cells were then cultured in a cell incubator (5% CO2 incubator) for 24 hours. In the control group, 50 μl of 0.05% dimethylsulfoxide (DMSO) / DMEM medium was added.

[0089] The cultured cells were harvested to prepare mRNA samples. mRNA was extracted from the harvested cells by phenol-chloroform precipitation using Trizol reagent (Invitrogen, Cat No. 15596018). cDNA was synthesized from the isolated RNA by reverse transcription, and the expression of CYP1A1 was confirmed by real-time polymerase chain reaction (real-time PCR) using iQ SYBR-Green Supermix (Bio-rad) on a CFX96 (Bio-rad) detection system. The relative values of enzyme expression levels were compared by the ΔΔct method (delta-delta ct method) using GAPDH as a control enzyme. A fold change of 1 was set using the control group.

[0090] The annealing temperature of real-time polymerase chain reaction was set at 58°C and performed under the conditions of 45 cycles, 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 CAA GAA GGT GG-3’ (SEQ ID NO: 3) and reverse, 5’-TTA CTC CTT GGA GGC CAT GT-3’ (SEQ ID NO: 4).

[0091] As a result, in the compound 6, 21, 32, 39 treatment groups, it was confirmed that the expression level of CYP1A1 mRNA was higher than that of the control group (vehicle). From this, it was confirmed that compounds 6, 21, 32, 39 significantly induced the expression of CYP1A1, an AHR target gene (Figure 1).

[0092] (2) Inductive effect on the expression of CYP1A1 protein HepG2 cells cultured in DMEM - 10% fetal bovine serum (FBS) medium were collected, and it was confirmed by trypan blue staining that the survival rate was 97% or more. Then, after centrifuging at a speed of 1200 rpm for 5 minutes at room temperature, the cells were resuspended in DMEM - 10% fetal bovine serum medium at 1.5×10 5 cells / ml for preparation.

[0093] Next, the cells were dispensed into 96-well plates at 200 μl each. To each dish, 3 μl of compounds 6, 21, 32, 39 at a concentration of 1 μM diluted in DMEM medium were added, and then cultured in a cell incubator (5% CO2 incubator) for 24 hours. In the control group, 3 μl of 0.05% dimethylsulfoxide (DMSO) / DMEM medium was added.

[0094] After 24 hours, the medium on the plate was discarded, and after washing with dPBS, 100 μl of 2 μM ethoxyresorufin was treated, and the cells were cultured in a cell incubator (5% CO2 incubator) for 30 minutes. After 30 minutes, 75 μl of 150 μg / ml fluorescamine (150 μg / ml) was treated, and resorufin excitation was measured at 535 nm and emission at 590 nm. The standard curve was measured and used with a resorufin solution of 0 to 50 pmol.

[0095] After measurement, all the solutions were removed, 25 μl of 0.5 M sodium hydroxide (NaOH) was treated, and then the cells were scraped off and reacted on a shaker at room temperature for 15 minutes.

[0096] After 15 minutes, the protein was quantified using the Bradford method, the value was obtained using the calculation formula (sample resorufin amount / sample protein amount), and then a 1-fold value was set using the control group.

[0097] As a result, it was confirmed that in the compound 6, 21, 32, 39 treatment groups, the expression level of CYP1A1 protein was significantly higher compared to the vehicle control group. From this, it was confirmed that compounds 6, 21, 32, and 39 significantly induced the protein of CYP1A1, which is an AHR target gene (Figure 2).

[0098] Example 2: Inhibitory effect on the production of inflammatory factor IL-6 To evaluate the IL-6 production inhibitory effect of the compound of the present invention produced as described above, an IL-6 production experiment of epithelial cells by stimulation with IL-1β was conducted.

[0099] A549 cells cultured in DMEM-10% fetal bovine serum (FBS) medium were harvested, and it was confirmed by trypan blue staining that the viability was 97% or more. Then, after centrifugation at 1200 rpm for 5 minutes at room temperature, the cells were resuspended in DMEM-10% fetal bovine serum medium at 1×10 6 cells / 3 ml. Then, 3 ml of the cells were dispensed into 60 mm plates, and 50 μl of compounds 6, 9, 21, 32, and 39 at a concentration of 5 μM diluted in DMEM medium were added to each dish, followed by culturing in a cell incubator (5% CO2 incubator) for 48 hours. Then, 50 μl of human recombinant IL-1β at a concentration of 12.5 ng / ml diluted in DMEM medium was added, and the cells were cultured in a cell incubator (5% CO2 incubator) for 24 hours. In the control group, 50 μl of 0.05% dimethylsulfoxide (DMSO) / DMEM medium was added.

[0100] The cultured cells were harvested to prepare mRNA samples. mRNA was extracted from the harvested cells by phenol-chloroform precipitation using Trizol reagent (Invitrogen, Cat No. 15596018). cDNA was synthesized from the isolated RNA by reverse transcription, and the expression of CYP1A1 was confirmed by real-time polymerase chain reaction (real-time PCR) using iQ SYBR-Green Supermix (Bio-rad) with a CFX96 (Bio-rad) detection system. The relative values of enzyme expression levels were compared by the ΔΔct method using GAPDH as a control enzyme. A value of 1-fold was set using the control group.

[0101] The annealing temperature of real-time polymerase chain reaction was set at 58°C and performed under the conditions of 45 cycles, 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 GAA GGT GG-3' (SEQ ID NO: 3) and reverse, 5'-TTA CTC CTT GGA GGC CAT GT-3' (SEQ ID NO: 4).

[0102] As a result, the production of IL-6 in A549 by IL-1β stimulation was significantly decreased by the treatment with Compounds 6, 9, 21, 32, and 39. From this, it was confirmed that Compounds 6, 9, 21, 32, and 39 effectively suppressed the production of IL-6 (Figure 3).

[0103] Example 3: Confirmation of the effect of promoting the generation of FoxP3-expressing regulatory T cells (Tregs) The effect of promoting the generation of "FoxP3-expressing regulatory T cells", which play an important role in maintaining immune tolerance, was evaluated (Figure 4).

[0104] The spleens of C57BL / 6 mice (8 - 12 weeks old, female) were excised, added with RPMI medium and ground, and then passed through a 40 μm cell strainer (BD Falcon) to obtain a single cell suspension. After centrifugation of the single cell suspension (1200 rpm, 5 minutes), the supernatant was discarded, 1 ml of ACK lysis buffer was added and stirred for 1 minute, and then washed with RPMI medium. After centrifugation of the single cell suspension, T cells were separated using a mouse CD4 naive T cell enrichment kit (Invitrogen). The separated T cells were placed in RPMI-fetal bovine serum (FBS) 10% + 2-ME (mercaptoethanol) medium at 1×10 6Resuspended in / ml for preparation. For T cell activation, 5 μg / ml of anti-CD3 (eBioscience TM ) was dispensed at 100 μl per well into a 96-well plate, reacted in a cell incubator (37 °C, 5% CO2 incubator) for 4 hours, and then washed with phosphate-buffered saline for preparation. 200 μl of the resuspended T cells were dispensed per well into the prepared plate, and 2 μg / ml of anti-CD28 (eBioscience TM ), 2 ng / ml of TGF-β1 (R&D systems), and 100 U / ml of IL-2 were added to each well. 5 μl each of compound 6 and compound 9 at a concentration of 2.5 μM diluted in RPMI-10% fetal bovine serum + 2ME medium were added, and the cells were cultured in a cell incubator (37 °C, 5% CO2 incubator) for 3 days. In the control group, 5 μl of 0.05% dimethylsulfoxide (DMSO) / RPMI medium was added. After 3 days, to confirm the production effect of regulatory T cells, the cultured cells were collected and the expression of Foxp3 protein was measured.

[0105] To confirm the expression of Foxp3 protein, the collected cells were placed in a 5-ml tube for FACS (BD Falcon) and washed with 1 ml of phosphate-buffered saline. The cells were resuspended in 0.1 ml of FACS buffer solution (0.1% NaN3, 1% FBS), and 0.5 μg of CD16 / CD32 antibody (eBioscience TM ) was added to prevent non-specific binding of the antibody, and the reaction was carried out at 4 °C for 15 minutes. Then, 0.25 μg of CD4 monoclonal antibody (GK1.5), PE-Cyanine7 (eBioscience TM ) was added, and the cells were stained at 4 °C for 30 minutes and then washed with 1 ml of FACS buffer solution. 1 ml of Fixation / Permeabilization solution (eBioscience TM ) was added to the FACS tube containing the sample, and the reaction was carried out at 4 °C for 1 hour. After that, permeabilization buffer (eBioscience TM) It was washed twice. Then, 0.5 μg of Foxp3 monoclonal antibody (FJK-16s), PE (eBioscience TM ) was added, and after staining at 4°C for 30 minutes, it was washed twice with the buffer for permeabilization treatment, suspended in 0.3 ml of FACS buffer solution, and then measured by flow cytometry.

[0106] As a result, the generation of Foxp3-expressing regulatory T cells was significantly increased by Compound 6 and Compound 9 compared with the control group (Vehicle). From this, it was confirmed that Compound 6 and Compound 9 effectively promoted the generation of FoxP3+ regulatory T cells (Figure 4).

[0107] Example 4: Therapeutic effect on inflammatory bowel disease To examine the therapeutic effect of the compound of the present invention produced as described above on inflammatory bowel disease, inflammatory bowel disease was induced in C57BL / 6 mice as follows, and Compound 6 was administered to evaluate its effect (Figures 5 to 10).

[0108] (1) Therapeutic effect of Compound 6 on inflammatory bowel disease On the 0th day of the experiment, a 2.0% DSS solution prepared by dissolving DSS (Dextran sulfate sodium, MP biomedicals, Cat No. 160110) in sterile distilled water was given to C57BL / 6 mice (11 weeks old, female, 20 ± 2 g) to drink for 7 days. The 2.0% DSS solution was replaced at 2-day intervals. From the 8th day of the experiment, sterile distilled water was given to drink. The body weight and disease activity index were measured at 2-day intervals from the 0th day of the experiment to confirm the onset of inflammatory bowel disease.

[0109] After dissolving 10 mg / kg of Compound 6 per mouse completely in a mixture of ethanol-Cremophor EL corresponding to 7.5% (v / v) of the administration dose, it was diluted with phosphate-buffered saline so that the final ratio of ethanol:Cremophor EL:phosphate-buffered saline was (0.375:0.375:9.25, v / v / v), and 200 μl each was orally administered daily for a total of 14 times from the first day to the 14th day of the experiment. The severity index of inflammatory bowel disease is a severity index system classified into 0 to 10 levels, and was visually observed and recorded at two-day intervals.

[0110] Regarding the symptoms of inflammatory bowel disease, the scores of the following three items (Table 2) were totaled for index evaluation.

[0111]

Table 2

[0112] As a result of the analysis, it was confirmed that the vehicle control group started to lose weight from the 6th day of the experiment, and by the 10th day of the experiment, the body weight decreased by more than 10%, and 100% of the enteritis with a severity index increase of 5 or more was induced. The mice in the vehicle control group showed a severity index of 7.50 ± 0.50 on the 10th day of the experiment, which was the maximum value. The 10 mg / kg administration group of Compound 6 of the present invention showed a statistically significant therapeutic effect compared to the vehicle control group on the 10th day of the experiment (Figure 5), and by comparing the weight:length ratio (mg / cm) of the large intestine on the 15th day of the experiment, it was confirmed that intestinal inflammation was also significantly suppressed morphologically (*** compared to the vehicle control group, p < 0.001, see Figures 5 and 6). Compound 6 of the present invention showed an excellent anti-inflammatory effect when administered at 10 mg / kg (Figures 7a and 7b).

[0113] On the 15th day of the experiment, the large intestine of the mouse was excised, and an mRNA sample was prepared. To extract mRNA, the large intestine tissue was pulverized with a homogenizer to obtain a homogeneous suspension. The mRNA in the homogeneous suspension was extracted by the phenol-chloroform precipitation method using the easy-spinTM (DNA free) total RNA extraction kit (Intron biotechnology, Cat No. 17221). cDNA was synthesized from the separated RNA by reverse transcription, and the expression of inflammatory cytokines was confirmed by real-time polymerase chain reaction (real-time PCR) using iQ SYBR-Green Supermix (Bio-rad) with the CFX96 (Bio-rad) detection system. The relative value of the enzyme expression level was compared by the ΔΔct method using GAPDH as a control enzyme. A fold change of 1 was set using the large intestine of normal mice as a control group.

[0114] The annealing temperature of the real-time polymerase chain reaction was set at 58 °C and performed under the conditions of 45 cycles using the following primer sequences.

[0115] 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); 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); 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); 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); 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); 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); Mouse IL-10 forward, 5’-CAA GGC AGT GGA GCA GGT GAA-3’ (SEQ ID NO: 19) and reverse, 5’-CGG AGA GAG GTA CAA ACG AGG TT-3’ (SEQ ID NO: 20); 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); 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); 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); 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).

[0116] The expression levels of inflammatory cytokines IL-1β, IL-6, IL-17A, TNF-α, S100a8, and S100a9 in colonic lesions were significantly decreased by the administration of compound 6 compared with the vehicle control group (**, p < 0.01, see Figures 7a and 7b). The expression levels of immunomodulatory factors IL-10 and Foxp3 in colonic lesions were significantly increased by the administration of compound 6 compared with the vehicle control group (*, p < 0.05; **, p < 0.01, see Figure 8). The expression levels of intestinal epithelial cell protective factors Reg3b and Muc2 in colonic lesions were significantly increased by the administration of compound 6 compared with the vehicle control group (*, p < 0.05; ***, p < 0.001, see Figure 9). From these results, it was found that compound 6 of the present invention significantly decreased the expression of intestinal inflammatory factors and significantly increased the expression of intestinal immunomodulatory factors and intestinal epithelial cell protective factors.

[0117] In addition, to examine the mucosal healing effect of the compounds according to the present invention, inflammatory bowel disease was induced in C57BL / 6 mice as follows, and compound 6 was administered to evaluate the degree of recovery of the intestinal epithelial barrier integrity.

[0118] On day 0 of the experiment, a 2.0% DSS solution prepared by dissolving DSS in sterile distilled water was administered to C57BL / 6 mice (11 weeks old, female, 20 ± 2 g) for 7 days. The 2.0% DSS solution was changed at 2-day intervals. From day 8 of the experiment, sterile distilled water was administered. Body weight and disease activity index were measured at 2-day intervals from day 0 of the experiment to confirm the onset of inflammatory bowel disease.

[0119] In the group administered with Compound 6 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 administration dose, and then diluted with phosphate-buffered saline so that the final ethanol:Cremophor EL:phosphate-buffered saline was (0.375:0.375:9.25, v / v / v). A total of 14 times, 200 μl each, were orally administered daily from the first day to the 14th day of the experiment.

[0120] One day before the administration of FITC-dextran, the mice were deprived of water overnight. On the 15th day of the experiment, 600 mg / kg of FITC-dextran (Fluorescein isothiocyanate-dextran, Sigma aldrich, Cat No. FD40) was diluted with phosphate-buffered saline and orally administered once at 200 μl. Four hours after the oral administration, the fluorescence in the serum extracted from the heart was measured (fluorometer, excitation 485 - 490 nm, emission 528 - 530 nm).

[0121] Serum FITC-dextran was significantly decreased by the administration of Compound 6 compared to the vehicle control group (***, p < 0.001 compared to the vehicle control group, see Figure 10). From this result, it was found that Compound 6 of the present invention exhibits a significant mucosal healing effect. Thus, it was confirmed that Compound 6 of the present invention has an oral administration therapeutic effect in an inflammatory bowel disease mouse model.

[0122] Example 5: Preventive and Therapeutic Effects on Colorectal Cancer To examine the preventive (Figures 11 and 12) and therapeutic effects (Figures 13 and 14) of the compound of the present invention produced as described above on colitis-associated colorectal cancer (CA-CRC) related to inflammatory bowel disease, Compound 6 was administered to a colorectal cancer model (AOM / DSS mice) as follows to evaluate its effect.

[0123] AOM (Sigma Aldrich, Cat No. A5486) was diluted with physiological saline to a concentration of 10 mg / kg, and 200 μl of the diluted solution was intraperitoneally administered to C57BL / 6 mice (8-week-old, female, 18 ± 2 g) at 7-day intervals (days 0, 7, and 14 of the experiment) for a total of 3 times. On the 7th day of the experiment, a 1.5% DSS solution prepared by dissolving DSS in sterile distilled water was given to the mice for drinking for 7 days. The 1.5% DSS solution was changed every 2 days. From the 8th day of the experiment, sterile distilled water was given for drinking.

[0124] In the administration group of Compound 6 according to the present invention, 10 mg / kg of Compound 6 per mouse was completely dissolved in an ethanol-Cremophor EL mixture corresponding to 7.5% (v / v) of the administration dose, and then diluted with phosphate buffered saline to a final ethanol:Cremophor EL:phosphate buffered saline ratio of (0.375:0.375:9.25, v / v / v), and 200 μl of the diluted solution was orally administered daily. To confirm the preventive effect, Compound 6 was administered 14 times in total from the 7th day to the 20th day of the experiment.

[0125] As a result, in the control group (AOM / DSS + Vehicle), weight loss was observed from the 70th day, while in the Compound 6 administration group (AOM / DSS + Compound 6), weight gain was shown, approximating that of normal mice (Normal) (Figure 11). Also, in the Compound 6 administration group, it was confirmed that the number of tumors was significantly less and their size was also significantly smaller. That is, it was confirmed that Compound 6 has an effect of preventing the development of colorectal cancer caused by inflammation (Figures 11 and 12).

[0126] The therapeutic effect of Compound 6 according to the present invention on colorectal cancer was confirmed. From the 50th day to the 63rd day of the experiment, AOM was administered to mice, and DSS was administered on the 7th and 14th days after AOM administration to induce colorectal cancer. Compound 6 was administered 14 times from the 50th day to the 63rd day. As a result, in the control group (AOM / DSS + Vehicle), weight loss was shown from the 70th day, while in the Compound 6-administered group (AOM / DSS + Compound 6), weight gain was shown, approximating that of normal mice (Normal) (Figure 13). Also, in the Compound 6-administered group, it was confirmed that the number of tumors was significantly smaller and their size was also significantly smaller (Figure 14). That is, it was confirmed that Compound 6 is effective in the treatment of colorectal cancer (Figures 13 and 14). Therefore, it was confirmed that Compound 6 can be used as an effective preventive and therapeutic agent for colorectal cancer.

[0127] Example 6: Therapeutic effect on psoriasis In order to examine the therapeutic effect on psoriasis of the compound of the present invention produced as described above, the following experiment was conducted (Figures 15 to 17).

[0128] The hair on the backs of female BALB / C mice (8 - 10 weeks old) was removed using a hair remover and hair removal cream, and starting the next day, 5% imiquimod cream was applied to the depilated skin at 62.5 mg per day for 6 days. Vaseline cream was applied to the control group mice (Vehicle). In the group administered with Compound 6 according to the present invention, after completely dissolving 10 mg / kg of the compound per mouse in an ethanol - Cremophor EL mixture (1:1, v / v) corresponding to 15% (v / v) of the administration dose, it was diluted with phosphate - buffered saline so that the final ethanol:Cremophor EL:phosphate - buffered saline was 7.5:7.5:85 (v / v / v), and 200 μl each was orally administered daily for a total of 6 times from the 0th day to the 5th day of the experiment. On the 6th day of the experiment, skin tissue on the backs of the mice was taken for histological and mRNA experiments.

[0129] As a result, in the oral administration group of Compound 6, the epithelial thickness was significantly decreased (Figure 15), the expression levels of IL-17a and S100a8 mRNAs that promote the inflammation of skin lesions were significantly decreased (Figure 16), and it was confirmed that Foxp3 and IL-10 that induce the suppression of skin lesion inflammation were significantly increased (Figure 17). From this, it was confirmed that Compound 6 can be used as an effective psoriasis treatment agent.

[0130] Example 7: Therapeutic effect on graft-versus-host disease To examine the therapeutic effect of the compound of the present invention produced as described above on graft-versus-host disease, Compound 6 was administered to a graft-versus-host disease model to evaluate its effect (Figures 18 and 19).

[0131] The spleen of C57BL / 6 mice (8 - 12 weeks old, female, 18 ± 3 g) was excised, added with RPMI medium and crushed, and then passed through a 40 μm cell strainer (BD Falcon) to obtain a single cell suspension. The single cell suspension was centrifuged (1200 rpm, 5 minutes), the supernatant was discarded, 1 ml of ACK (ammonium chloride / potassium bicarbonate lysis buffer) (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM Na2EDTA) was added and stirred for 1 minute, and then washed with RPMI medium. The cell suspension was reacted with mouse CD90.2 microbeads (Miltenyi Biotec, Cat No. 130 - 121 - 278) at 4°C for 20 minutes after centrifugation. The cell suspension after the reaction was centrifuged and washed with 10 ml of autoMACS® running buffer (Miltenyi Biotec, Cat No. 130 - 091 - 221), and then resuspended with 3 ml of autoMACS® running buffer. Using Auto MACS pro (Miltenyi Biotec), CD90.2 + T cells were obtained (positive selection). The obtained CD90.2 +To obtain bone marrow cells for transplantation together with T cells, the bilateral femurs and tibias of normal (wild-type) C57BL / 6 mice (8 - 12 weeks old, female, 18 ± 3 g) were aseptically obtained. The tips of the femurs and tibias were cut, and RPMI medium was perfused into the bone tissue with syringes (21G for femurs, 26G for tibias) to extract bone marrow. The extracted bone marrow was passed through a 40-μm cell strainer to obtain a single-cell suspension.

[0132] For the bone marrow single-cell suspension, after centrifugation, the supernatant was discarded, 500 μl of ACK lysis buffer was added, and it was stirred for 30 seconds and then washed with RPMI medium. After centrifugation, it was reacted with mouse CD90.2 microbeads at 4°C for 20 minutes. The cell suspension after the reaction was centrifuged and washed with 10 ml of autoMACS® running buffer, and then resuspended with 3 ml of autoMACS® running buffer. CD90.2 - T cell-depleted bone marrow cells (TCD-BMs) were obtained (negative selection). The obtained normal CD90.2 + T cells and normal TCD-BMs were washed with phosphate-buffered saline. The T cells were resuspended in phosphate-buffered saline at 4 × 10 6 / ml, and the TCD-BM was resuspended in phosphate-buffered saline at 5 × 10 6 / ml for preparation.

[0133] Normal BDF1 mice (9 weeks old, female, 19 ± 3 g) were irradiated with 950 cGy of radiation in two divided doses at 3-hour intervals using a radiation irradiator. Transplanted grafts prepared by mixing the prepared CD90.2+ T cells and TCD-BM at a ratio of 1:1 were injected into the tail veins of BDF1 mice at 100 μl each. In the administration group of Compound 6 according to the present invention, after dissolving 10 mg / kg of the compound per mouse completely in an ethanol-Cremophor EL mixture corresponding to 7.5% (v / v) of the administration dose, it was diluted with phosphate-buffered saline so that the final ethanol:Cremophor EL:phosphate-buffered saline was (0.375:0.375:9.25, v / v / v), and 200 μl each was orally administered daily for a total of 12 times from day 3 to day 14 of the experiment.

[0134] The severity index of graft-versus-host disease was a severity index system in which weight loss, hair condition, posture, activity, and skin changes were classified as 0 to 2 points for each item, totaling 10 points, and was visually observed and evaluated at 3- to 4-day intervals.

[0135] As a result of the analysis, the severity index of graft-versus-host disease (8 ± 1) that occurred in the solvent control group (Vehicle) was significantly reduced by the administration of Compound 6 (2.2 ± 1.2) (***, p < 0.001 compared with the solvent control group, see Figure 18). By histopathologically analyzing the large intestine tissues of mice in each experimental group, it was confirmed that the inflammatory pathology index of the large intestine tissues was significantly reduced by the administration of Compound 6 (see Figure 19). This indicates that Compound 6 can be used as a prophylactic and therapeutic agent for graft-versus-host disease.

[0136] Example 8: Therapeutic effect on multiple sclerosis To examine the therapeutic effect of the compound of the present invention produced as described above on multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) was induced in C57BL / 6 mice as follows, and Compound 6 was administered to evaluate its effect (Figure 20).

[0137] On the 0th day of the experiment, myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide (MOG 35-55 , Peptron) (200 μg), heat-killed Mycobacterium tuberculosis (Difco, Cat No. 231141) (500 μg), and an adjuvant (incomplete Freund's adjuvant, Sigma Aldrich, Cat No. F5506) were mixed for 7 minutes to prepare an emulsion. MOG 35-55 The emulsion was subcutaneously injected into both flanks of C57BL / 6 mice (7-8 weeks old, female). Then, 100 μl of pertussis toxin (Sigma Aldrich, Cat No. P2980) (200 ng) was administered via the tail vein. On the 2nd day of the experiment, the same amount of pertussis toxin was intravenously administered. The injection sites of the mice were checked for leakage of the emulsion, and visual observation was carried out from the 7th day of the experiment to confirm the onset of multiple sclerosis.

[0138] In the group administered with Compound 6 according to the present invention, after dissolving 10 mg / kg of Compound 6 per mouse completely in an ethanol-Cremophor EL mixture corresponding to 7.5% (v / v) of the administration dose, it was diluted with phosphate-buffered saline so that the final ethanol:Cremophor EL:phosphate-buffered saline was (0.375:0.375:9.25, v / v / v), and 200 μl each was orally administered daily for a total of 6 times from the 15th day to the 20th day of the experiment. The multiple sclerosis index is a severity index system classified into 0 to 5 levels and was visually observed and recorded.

[0139] The symptoms of autoimmune encephalomyelitis were evaluated according to the following items. 0: No symptoms 1: Loss of tail strength 2: Loss of tail strength and weakness of the hind legs 3: Paralysis of the hind legs 4: Paralysis of the hind legs and weakness of the front legs 5: Dying or dead

[0140] As a result of the analysis, the disease severity index on the 21st day of the experiment, which is the acute reaction period, was 2.9 ± 1.92 in the vehicle control group and 0.92 ± 0.58 in the compound 6 administration group. The compound 6 administration group showed a lower severity index compared to the vehicle control group (*, p < 0.05, Figure 20). From this, the therapeutic effect of compound 6 on multiple sclerosis was confirmed.

[0141] Example 9: Therapeutic effect on neutrophilic asthma To examine the therapeutic effect of the compound of the present invention produced as described above on neutrophilic asthma, compound 6 was administered to a neutrophilic asthma mouse model and its effect was evaluated (Figure 21).

[0142] C57BL / 6 mice (7 weeks old, female) were sensitized by nasal administration with 10 μg of LPS (lipopolysaccharide, Sigma aldrich, Cat No. L2630) and 75 μg of OVA (Ovalbumin, Sigma aldrich, Cat No. A5503) diluted in phosphate-buffered saline (total 20 μl) on days 0, 1, 2, and 7 of the experiment. From day 14 of the experiment, 50 μg of OVA diluted in phosphate-buffered saline was administered by nasal administration 8 times in total, 2 days a week until day 36, for challenge. Nasal administration was performed after anesthesia using an 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 an ethanol-Cremophor EL mixture corresponding to 7.5% (v / v) of the administration dose, and then diluted with phosphate-buffered saline to a final ethanol:Cremophor EL:phosphate-buffered saline (0.375:0.375:9.25, v / v / v), and 200 μl each was orally administered daily for a total of 10 times from day 27 to day 36 of the experiment.

[0143] As a result, in the group administered with Compound 6, the number of neutrophils in bronchoalveolar lavage (BAL) fluid was significantly decreased compared to the vehicle control group (Figure 21). From this, the therapeutic effect of Compound 6 on neutrophilic asthma was confirmed.

[0144] Example 10: Comparison of effects with Comparative Example 1 In order to confirm the superiority of the compounds of the examples of the present invention compared to the compound of Comparative Example 1, experiments were conducted as follows: (1) Measurement of pharmacokinetic parameters, (2) Measurement of solubility, (3) Inhibitory effect on the inflammatory activity of Th17 cells, (4) Confirmation of the promoting effect on the generation of FoxP3-expressing regulatory T cells, (5) Whether the activity of drug-metabolizing enzymes can be inhibited, (6) Confirmation of the therapeutic effect on inflammatory bowel disease.

[0145] (1) Measurement of pharmacokinetic parameters The pharmacokinetic parameters of Compound 6 and Comparative Example 1 were measured (Table 3, Figure 22).

[0146] Rats were fasted for 16 hours in the oral administration group and not fasted in the intravenous administration group. Compound 6 was completely dissolved in an ethanol-Cremophor EL mixture corresponding to 7.5% (v / v) of the administration dose at the administration concentrations shown in Table 3, and then diluted with phosphate-buffered saline to a final ethanol:Cremophor EL:phosphate-buffered saline (0.375:0.375:9.25, v / v / v), and orally administered and intravenously administered at 10 mL / kg. In the oral administration group, food was supplied about 4 hours after administration. Blood was collected 0.083, 0.5, 1, 2, 4, 8, 24 hours after administration in the intravenous administration group and 0.5, 1, 2, 4, 8, 12, 24 hours after administration in the oral administration group, and then plasma was collected using centrifugation. In the collected plasma, the drug concentration in the plasma was measured using the UHPLC-MS / MS method.

[0147] As shown in Table 3, when compound 6 was orally administered at a concentration of 10 mg / kg, it was confirmed that the bioavailability (F%) increased by 1.9 times, AUClast increased by 11.9 times, and Cmax increased by 5.9 times compared to the case when Comparative Example 1 was orally administered at a concentration of 10 mg / kg. Also, when compound 6 was orally administered at a concentration of 50 mg / kg, it was confirmed that the bioavailability (F%) increased by 67.2 times, AUClast increased by 66.8 times, and Cmax increased by 30.2 times compared to the case when Comparative Example 1 was orally administered at a concentration of 100 mg / kg. From this, it was confirmed that compound 6 can exhibit medicinal effects even in small amounts compared to Comparative Example 1.

[0148]

Table 3

[0149] (2) Measurement of solubility The solubilities of compound 6 and Comparative Example 1 were measured. After dissolving compound 6 in ethanol, measurement and analysis were performed using a SIRIUS T3 machine. As shown in Table 4, it was confirmed that compound 6 has a larger logP value and the pH-metric solubility molarity is about 3.92 times that of Comparative Example 1.

[0150]

Table 4

[0151] (3) Inhibitory effect on the inflammatory activity of Th17 cells The inhibitory effect on the inflammatory activity of Th17 cells, which are important causative cells for developing autoimmune diseases, was compared (Figure 23).

[0152] The spleen of C57BL / 6 mice (8 - 12 weeks old, female) was excised, added with IMDM medium and ground. After that, it was passed through a 40 μm cell strainer (BD Falcon) to obtain a single cell suspension. The single cell suspension was centrifuged (1200 rpm, 5 minutes), the supernatant was discarded, 1 ml of ACK lysis buffer was added, stirred for 1 minute, and then washed with RPMI medium. The single cell suspension was centrifuged, and T cells were separated using a mouse CD4 naive T cell enrichment kit (Invitrogen). The separated T cells were resuspended in IMDM - 10% fetal bovine serum (FBS) + 2 - ME (mercaptoethanol) medium at 1×10 6 / ml for preparation. For the activation of T cells, 5 μg / ml of anti - CD3 (eBioscience TM ) was dispensed at 100 μl per well in a 96 - well plate, reacted in a cell incubator (37 °C, 5% CO2 incubator) for 4 hours, and then washed with phosphate - buffered saline for preparation. 200 μl of the resuspended T cells were dispensed into the prepared plate, and 2 μg / ml of anti - CD28 (eBioscience TM ), 3 ng / ml of TGF - β1 (R&D systems), 100 ng / ml of IL - 6 (R&D systems), 5 μg / ml of anti - mouse IFN - γ (Bio X cell), and 5 μg / ml of anti - mouse IL - 4 (Bio X cell) were added to each well. 5 μl of compounds at concentrations of 1.0 μM, 2.5 μM, and 5 μM diluted in IMDM - 10% fetal bovine serum + 2ME medium were added to each well respectively, and cultured in a cell incubator (37 °C, 5% CO2 incubator) for 3 days. In the control group, 5 μl of 0.05% dimethylsulfoxide (DMSO) / RPMI medium was added. After 3 days, to confirm the inhibitory effect of the inflammatory activity of Th17 cells, the cultured medium was collected and IL - 17A was confirmed using a flex set (BD biosciences).

[0153] To confirm the IL-17A cytokine, 25 μl of the collected medium and 25 μl of assay diluent buffer were placed in a FACS tube (BD falcon) to dilute the sample by half. 1 μl of capture beads was placed in 49 μl of capture bead diluent to prepare 50 μl of capture bead solution per sample. After mixing the capture bead solution by vortex, 50 μl of the capture bead solution was added to each FACS tube containing the sample, vortexed again, and left at room temperature for 1 hour. After 1 hour, 1 μl of PE detection reagent was placed in 49 μl of PE detection diluent to prepare 50 μl of PE detection solution per sample. After vortexing the PE detection solution, 50 μl of the PE detection solution was added to each FACS tube containing the capture bead solution and the sample. After vortexing the FACS tube, it was left at room temperature for 1 hour. After 1 hour, 1 ml of CBA wash buffer was added per tube, centrifuged at 400 g for 5 minutes, and the supernatant was removed. After gently vortexing, 150 μl of Fix buffer was added and gently vortexed, and then analyzed using flow cytometry.

[0154] As a result, it was confirmed that the production of IL-17A in Th17 cells was significantly decreased by Compound 6 compared to Comparative Example 1 (Figure 23).

[0155] (4) Confirmation of the effect of promoting the generation of T cells that regulate FoxP3 expression (Treg) The effect of promoting the generation of "T cells that regulate FoxP3 expression", which plays an important role in maintaining immune tolerance, was compared (Figure 24). By the same experimental method as in Figure 4, CD4 +After separating T cells, they were cultured and treated with 5 μl each of compounds at concentrations of 2.5 μM and 5 μM diluted in RPMI - 10% fetal bovine serum + 2ME medium. After culturing in a cell incubator (37 °C, 5% CO2 incubator) for 3 days, they were analyzed. In the control group, 5 μl of 0.05% dimethylsulfoxide (DMSO) / RPMI medium was treated. After 3 days, in order to confirm the production effect of regulatory T cells, the cultured cells were collected and the expression of Foxp3 protein was measured in the same manner as in Figure 4.

[0156] As a result, it was confirmed that the production of Foxp3 - expressing regulatory T cells was significantly increased by Compound 6 compared to Comparative Example 1 (Figure 24).

[0157] (5) Feasibility of inhibiting the activity of drug - metabolizing enzymes The activity of CYP isoenzymes, which are drug - metabolizing enzymes, was measured.

[0158] Viable human hepatocyte cell line (Corning, Cat No. 454551) was collected using plating medium and plated at a cell density of 0.4×10 6After dispensing to form cell / wells, the cells were cultured for 2 to 4 hours. After replacing the plating medium with hepatocyte culture medium, the hepatocytes were cultured for 24 hours. After 24-hour culture, the hepatocytes were treated with Compound 6 at 1, 5, and 25 μM. The medium containing the compound was replaced every 24 hours for 2 days. The cultured cells were collected to prepare mRNA samples. mRNA was extracted from the collected cells by the phenol-chloroform precipitation method using Trizol reagent (Invitrogen, Cat No. 15596018). cDNA was synthesized from the isolated RNA by reverse transcription, and the drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2B6, and CYP3A4 were measured by real-time PCR analysis using a Quant StudioTM 7 Flex Real-Time PCR System (Applied Biosystems, CA). The relative values of enzyme expression levels were compared by the ΔΔct method using GAPDH as a control enzyme. A fold change of 1 was set using the control group. The results were shown as a percentage of the control group compared to the control group samples.

[0159] Table 5 shows the changes in the activities of CYP isoenzymes (% of Control Activity) after treatment with each compound (Comparative Example 1, Compound 6, ketoconazole) at 10 μM.

[0160]

Table 5

[0161] As a result, it was confirmed that Comparative Example 1 suppressed the enzyme activity of CYP2C19 to 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 co-administered with CYP2C19-metabolizing drugs.

[0162] (6) Therapeutic effect on inflammatory bowel disease 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. After orally administering Compound 6 and Comparative Example 1 to the mice at 10 mg / kg, the expression levels of IL-6 and S100a9 mRNA in the large intestine tissue were confirmed.

[0163] As a result, it was confirmed that the expression levels of IL-6 and S100a9 mRNA in the large intestine tissue were significantly decreased by Compound 6 compared to Comparative Example 1 (Figure 25). That is, it was confirmed that Compound 6 exhibits a higher therapeutic effect on inflammatory bowel disease than Comparative Example 1.

Claims

1. A compound represented by the following Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. 【Chemical 1】 (wherein, A is hydrogen, halogen, hydroxy group, C １ -C ３ alkyl group of, C ２ -C ３ alkenyl group of, C ２ -C ３ alkynyl group of, C １ -C ３ alkoxy group of, dimethylamine, -NO ２ , -CN, -COOR ２ or -S(=O) ２ R ２ and B is hydrogen, C １ -C ３ alkyl group of, phenyl group, acetyl group, -CH ２ C(=O)OR ２ -C(=O)OR ２ or -S(=O) ２ R ２ wherein R １ is a substituted or unsubstituted 5- to 7-membered heterocyclic ring or -NH ２ and is R ２ is C １ -C ３ is an alkyl group of.)

2. The 5- to 7-membered heterocyclic ring of the replacement is C １ -C ３ The compound according to claim 1, its stereoisomer or its pharmaceutically acceptable salt, which is a 5- to 7-membered heterocyclic ring substituted with an alkyl group, a hydroxy group or a dimethylamine of

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

4. The compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is 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, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5, which is for the treatment or prevention of an autoimmune disease.

7. The pharmaceutical composition according to claim 5, which is for the treatment or prevention of any one autoimmune disease 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, which is for the treatment or prevention of cancer.

9. The cancer is selected from the group consisting of colorectal cancer, melanoma, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell cancer, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer, and lung cancer. The composition according to claim 8, which is for the treatment or prevention of cancer.

10. A method for treating an autoimmune disease, comprising the step of administering to a patient a compound according to any one of claims 1 to 4.

11. A method for treating cancer, comprising the step of administering to a patient a compound according to any one of claims 1 to 4.

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