Novel compounds and their therapeutic applications for 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] This invention relates to novel compounds and their therapeutic applications for autoimmune diseases. [Background technology]

[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 mainly mediated by cytokines secreted by immune-related cells.

[0003] The immune system plays a role in protecting the body from harmful external substances called antigens. These antigens include bacteria, viruses, toxins, cancer cells, and various types of blood and tissue. The immune system produces antibodies to destroy these harmful substances, but if autoimmunity malfunctions, the immune system cannot distinguish between its own organs and harmful antigens, leading to the destruction of healthy tissue. Diseases induced by this reaction are called autoimmune diseases.

[0004] Aryl hydrocarbon receptors (AHRs) are ligand-dependent transcription factors belonging to the PER-ARNT-SIM (PAS) superfamily and are primarily expressed in immune cells, epithelial cells, endothelial cells, and stromal cells of barrier tissues. AHRs are environmental sensors that sense not only xenobiotic ligands such as environmental pollutants (e.g., dioxins) but also physiological ligands produced from cells, microorganisms, and food.

[0005] In the inactive form, AHR forms a complex with the Hsp90:XAP2:p23:Src chaperone (AHR chaperone complex) in the cytoplasm, maintaining a structure with high affinity for ligands. When AHR is activated after ligand binding, the complex moves to the nucleus, and AHR detaches from the chaperone complex and binds to AHR-responsive DNA elements (xenobiotic response elements, XREs) located in the upstream regulatory regions of target genes, thereby regulating 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. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention aims to provide novel compounds, their stereoisomers, or pharmaceutically acceptable salts thereof.

[0007] The present invention aims to provide novel compounds useful for the prevention and treatment of autoimmune diseases, their stereoisomers, or pharmaceutically acceptable salts thereof.

[0008] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases, comprising a novel compound, its stereoisomer, or a pharmaceutically acceptable salt thereof. [Means for solving the problem]

[0009] 1. A compound represented by the following chemical formula 1, its stereoisomer, or a pharmaceutically acceptable salt thereof. [ka] (wherein, 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.)

[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, a stereoisomer thereof or a pharmaceutically acceptable salt thereof.

[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, a stereoisomer thereof or a pharmaceutically acceptable salt thereof. JPEG0007857690000002.jpg8792

[0012] [[ID=​​​​​​​​​​​​​​​​​​​7. In the above item 5, a pharmaceutical composition for treating or preventing 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. In the above item 5, a pharmaceutical composition for treating or preventing cancer.

[0017] 9. In the above item 8, 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 carcinoma, gastric cancer, breast cancer, metastatic cancer, prostate cancer, gallbladder cancer, pancreatic cancer, blood cancer, skin cancer, and lung cancer, and the composition is for treating or preventing cancer.

Advantages of the Invention

[0018] The novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt can, by inducing the activity of AHR, an immunomodulatory transcription factor, not only control inflammation, but also regulate the immune balance and repair damaged tissues.

[0019] The novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt can regulate 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 can induce the activity of regulatory T cells (Treg).

[0021] Also, the novel compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt can prevent and treat autoimmune diseases by regulating the above inflammatory factors.

Brief Description of the Drawings

[0022] [Figure 1]Figure 1 shows the measurement of the expression level of CYP1A1, an AHR target gene, to confirm that the compound of the present invention is an AHR ligand under cell culture conditions. [Figure 2] Figure 2 shows the measurement of the expression level of CYP1A1, an AHR target gene, to confirm that the compound of the present invention is an AHR ligand under cell culture conditions. [Figure 3] Figure 3 shows the measured inhibitory effect of the compound of the present invention on the production of the inflammatory factor IL-6. [Figure 4] Figure 4 shows the measured effect of the compound of the present invention on promoting the generation of FoxP3+ regulatory T cells. [Figure 5] Figure 5 shows the therapeutic effect of the compound of the present invention on inflammatory bowel disease in a DSS (dextran sodium sulfate)-induced inflammatory bowel disease animal model. A lower disease activity index compared to the control group (vehicle) indicates a greater therapeutic effect. [Figure 6] Figure 6 shows the therapeutic effect of the compound of the present invention on inflammatory bowel disease in a DSS (dextran sodium sulfate)-induced inflammatory bowel disease animal model. The lower the weight relative to the length of the colon, the greater the therapeutic effect on inflammatory bowel disease. [Figure 7a] Figure 7a shows the effect of the compound of the present invention on the suppression of the expression of inflammatory factors (IL-1β, IL-6, IL-17a, TNF-α, S100a8, S100a9) in an animal model of DSS-induced inflammatory bowel disease. [Figure 7b] Figure 7b shows the effect of the compound of the present invention on the suppression of the expression of inflammatory factors (IL-1β, IL-6, IL-17a, TNF-α, S100a8, S100a9) in an animal model of DSS-induced inflammatory bowel disease. [Figure 8] Figure 8 shows the effect of the compound of the present invention on increasing the expression of immunomodulatory factors (IL-10, Foxp3) in an animal model of DSS-induced inflammatory bowel disease. [Figure 9]Figure 9 shows the effect of the compound of the present invention on increasing the expression of intestinal epithelial cell protective factors (Reg3b, Muc2) in an animal model of DSS-induced inflammatory bowel disease. [Figure 10] Figure 10 shows the mucosal therapeutic effect of the compound of the present invention in an animal model of DSS-induced inflammatory bowel disease using FITC-dextran. A lower detection level indicates a greater mucosal therapeutic effect. [Figure 11] Figure 11 shows the preventive effect of the compound of the present invention on colorectal cancer in an AOM / DSS colorectal cancer animal model, illustrating the changes in body weight of mice that were not administered the colorectal cancer-inducing drug (AOM / DSS) (Normal), mice that received AOM / DSS (DSS+Vehicle), and mice that received compound 6 at the same time as AOM / DSS administration (DSS+compound 6). [Figure 12] Figure 12 shows the preventive effect of the compound of the present invention on colorectal cancer in an AOM / DSS colorectal cancer animal model, and shows the number and size of tumors in mice that were not administered the colorectal cancer-inducing drug (AOM / DSS) (Normal), mice that were administered AOM / DSS (DSS+Vehicle), and mice that were administered compound 6 at the same time as AOM / DSS administration (DSS+compound 6). [Figure 13] Figure 13 shows the therapeutic effect of the compound of the present invention on colorectal cancer in an AOM / DSS colorectal cancer animal model, illustrating the changes in body weight of mice that were not administered AOM / DSS (Normal), mice administered AOM / DSS (DSS+Vehicle), and mice that were administered AOM / DSS to induce colorectal cancer and then administered compound 6 (DSS+compound 6). [Figure 14] Figure 14 shows the therapeutic effect of the compound of the present invention on colorectal cancer in an AOM / DSS colorectal cancer animal model, showing the number and size of tumors in mice that were not administered AOM / DSS (Normal), mice administered AOM / DSS (DSS+Vehicle), and mice that were administered AOM / DSS to induce colorectal cancer and then administered compound 6 (DSS+compound 6). [Figure 15] Figure 15 shows the therapeutic effect of the compound of the present invention on psoriasis in an animal model, demonstrating an effect of reducing epithelial thickness. [Figure 16] Figure 16 shows the therapeutic effect of the compound of the present invention on psoriasis in an animal model, demonstrating suppression of the expression of skin lesion inflammation pro-inflammatory factors (IL-17a, S100a8). [Figure 17] Figure 17 shows the therapeutic effect of the compound of the present invention on psoriasis in an animal model, demonstrating its effect in promoting the expression of anti-inflammatory factors (Foxp3, IL-10). [Figure 18] Figure 18 shows the therapeutic effect of the compound of the present invention on graft-versus-host disease in a B6->BDF1 animal model, and displays the mouse survival rate and clinical score after transplantation. [Figure 19] Figure 19 shows the therapeutic effect of the compounds of the present invention on graft-versus-host disease in a B6->BDF1 animal model, and displays the pathological score. [Figure 20] Figure 20 shows the therapeutic effect of the compound of the present invention on multiple sclerosis using an autoimmune encephalomyelitis (EAE) model, where a lower clinical score indicates a greater therapeutic effect. [Figure 21] Figure 21 shows the therapeutic effect of neutrophilic asthma in a neutrophilic asthma model, indicating that a lower number of neutrophils in bronchoalveolar lavage (BAL) indicates a greater therapeutic effect. [Figure 22] Figure 22 shows the changes in blood concentration over time after oral administration (PO) or intravenous administration (IV) of Compound 6, which is the compound of the present invention, and Comparative Example 1. [Figure 23] Figure 23 shows the inhibitory effect of Compound 6, the compound of the present invention, on the activity of Th17 cells that produce IL-17A after treatment with Comparative Example 1. [Figure 24] Figure 24 shows the effect of Compound 6, the compound of the present invention, on promoting the generation of regulatory T cells expressing Foxp3 after treatment with Comparative Example 1. [Figure 25] Figure 25 shows the reduction in IL-6 and S100a9 mRNA expression levels after oral administration of compound 6 and comparative example 1, respectively, to mice with inflammatory bowel disease. [Modes for carrying out the invention]

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

[0024] All technical terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, unless otherwise defined. While preferred methods or samples are described herein, similar or equivalent methods are also included within the scope of the present invention.

[0025] In the chemical formulas (structural formulas) of the present invention, if no substituents are listed where substituents are required, it means that hydrogen substituents have been 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 stereoisomer, or a pharmaceutically acceptable salt thereof.

[0027] [ka]

[0028] In the aforementioned structural formula, if no substituents are listed despite the presence of a substituent, it means that a hydrogen substituent has been omitted. This applies equally to all structural formulas of the present invention.

[0029] In the above chemical formula 1, A is hydrogen, halogen, hydroxyl 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 heterocycle or -NH2; and R2 is a C1-C3 alkyl group.

[0030] R1 is a substitution of any carbon atom in the benzene ring of benzothiazole.

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

[0032] The substituted 5-7 membered heterocycle may be a compound that is a 5-7 membered heterocycle substituted with a C1-C3 alkyl group, a hydroxyl group, or a dimethylamine.

[0033] The substituted or unsubstituted 5-7 membered heterogly ring may be any one selected from the following group of heterogly rings. JPEG0007857690000008.jpg8287

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

[0035] [Table 1] JPEG0007857690000010.jpg187126 JPEG0007857690000011.jpg178127 JPEG0007857690000012.jpg67127

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

[0037] Furthermore, the present invention relates to a pharmaceutical composition comprising the aforementioned compound, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0038] The aforementioned pharmaceutical composition may be a pharmaceutical composition for the treatment or prevention of autoimmune diseases, and may specifically be for inflammatory bowel disease (IBD), multiple sclerosis (MS), graft-versus-host disease (GVHD), asthma, atopic dermatitis, 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 for inflammatory bowel disease, multiple sclerosis, graft-versus-host disease, asthma, atopic dermatitis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, or type 1 diabetes, but is not limited to these.

[0039] In this invention, "autoimmune disease" refers to a disease in which cells or tissues are damaged by humoral immunity, cellular immunity, or both, in which the immune system has an inappropriate reaction to autoantigens, and the autoimmune reaction manifests systemically or specifically in certain organs, and can induce chronic inflammation.

[0040] The aforementioned "inflammatory bowel disease" refers to a disease characterized by repeated periods of improvement and relapse of abnormal chronic inflammation in the intestinal tract, and may include, but is not limited to, one or more diseases from the group consisting of Crohn's disease, ulcerative colitis, and intestinal Behcet's disease.

[0041] The term "multiple sclerosis" refers to a broad set of signs and symptoms resulting from damage and / or depletion of the fatty myelin sheath surrounding the axons of the brain and spinal cord, and is an inflammatory disease that induces demyelination and scar formation. 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), and progressive relapsing multiple sclerosis (PRMS).

[0042] The aforementioned "graft-versus-host disease" is a disease in which lymphocytes transfused during hematopoietic stem cell transplantation attack a host with weakened immune function, causing symptoms such as fever, rash, and liver dysfunction. It can invade the skin, lungs, intestines, liver, etc., but is not limited to these.

[0043] The aforementioned "asthma" is a disease in which symptoms such as cough and difficulty breathing repeatedly occur due to inflammation of the bronchi when exposed to specific triggering substances. It can be caused by infection, smoking, allergic antigens, etc., but is not limited to these.

[0044] The term "atopy" above refers to atopic dermatitis. It is a chronic, recurrent inflammatory skin disease and a representative allergic disease characterized by symptoms such as itching and dry skin.

[0045] The term "psoriasis" refers to an inflammatory disease that occurs in the skin or joints due to an abnormality in the immune system, causing problems such as discoloration, keratin buildup, erythematous plaques, and pain. Psoriasis can include one or more conditions selected from psoriatic arthritis, guttate psoriasis, pustular psoriasis, erythrodermic psoriasis, scalp psoriasis, onychopsoriasis, and enthesitis.

[0046] The aforementioned "rheumatoid arthritis" refers to a systemic autoimmune disease characterized by chronic inflammation of the joints.

[0047] The aforementioned "generalized erythematous lupus," also known as "lupus," is a chronic inflammatory autoimmune disease that affects various organs of the body, including connective tissue, skin, joints, blood, and kidneys. While the exact cause is unknown, previous research suggests a genetic component may be involved. The American College of Rheumatology (ACR) has published 11 symptoms, signs, and laboratory findings to facilitate the diagnosis of lupus and help differentiate it from other diseases; a diagnosis of lupus can be made if four or more of these symptoms are present.

[0048] The aforementioned "Type 1 diabetes" is an immune-mediated disease in which insulin-secreting β-cells are destroyed by an autoimmune reaction, and its causes include numerous genetic and environmental factors. This can be accompanied by progressive inflammatory infiltration of the islets of Langerhans due to immune cells specifically targeting insulin-secreting β-cells.

[0049] The pharmaceutical compositions of the present invention can be prepared using pharmaceutically appropriate and physiologically acceptable excipients in addition to the active ingredient, which is the compound of the present invention, or can be administered to mammals. Examples of such excipients include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or flavoring agents.

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

[0051] The “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit-risk ratio applicable to medical treatment, and the effective dose level can be determined according to factors including the type and severity of the disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, and the elimination ratio, the duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents. They may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. It is important to administer an amount that provides the greatest effect with the minimum amount without side effects, taking all of the above factors into consideration, 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, sex, condition, weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and the drugs used in combination. Typically, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight can be administered daily or every other day, or divided into 1 to 3 doses per day. However, the dosage can be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., so the above dosage does not limit the scope of the present invention in any sense.

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

[0054] Examples of the carrier, excipients, and diluents 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. The materials may further contain fillers, anti-flocculants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0055] Furthermore, the compositions of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an individual requiring the pharmaceutical composition of the present invention, including humans. The formulation may be a powder, granules, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterile injection solution, or sterile powder.

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

[0057] Acid addition salts can be formed from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrite, or phosphorous acid; non-toxic organic acids such as aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, and alkanediates; aromatic acids; and aliphatic and aromatic sulfonic acids. These 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, svelate, sebacate, fumarate, mariate, buty-1,4-dioate, hexane-1,6- It may contain geoate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandalate.

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

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

[0060] The present invention also relates to a method for inducing the activity of AHR, comprising the step of administering the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0061] Specifically, the compounds of the present invention target the aryl hydrocarbon receptor (AHR), an immunomodulatory transcription factor, and act as agonists that induce AHR activity, thereby controlling inflammation, regulating immune balance, and repairing damaged tissue, and can be used for the treatment of autoimmune diseases, but are not limited to these applications. Conventional ligands have problems that make them unsuitable for development as pharmaceutical compositions due to toxicity, low affinity and structural stability, and high target nonspecificity. In contrast, when AHR activity is induced with compounds having the "drug-like properties" of the present invention, they can be effectively used for the treatment and prevention of autoimmune diseases.

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

[0063] Specifically, the compounds of the present invention can be used to treat autoimmune diseases by suppressing the production of IL-6, an inflammatory factor known to induce autoimmune diseases. In fact, numerous therapeutic agents and related papers for autoimmune diseases that aim to suppress IL-6 are known. The compounds of the present invention have been confirmed to suppress the production of IL-6 from the experimental data below, and are expected to have the effect of reducing autoimmune reactions, so they can be used for the treatment and prevention of autoimmune diseases.

[0064] Furthermore, the present invention relates to a composition for the prevention or treatment of cancer comprising the aforementioned compound, its stereoisomer, or a pharmaceutically acceptable salt thereof.

[0065] In this invention, "cancer" broadly refers to the uncontrolled abnormal growth of host cells itself that invades the surrounding tissue of the initial abnormal cell growth site in the host and potential tissue distal to that site, and may include carcinoma, which is cancer of epithelial tissue (e.g., skin, squamous cells); sarcoma, which is cancer of connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemia, which is cancer of hematopoietic tissue (e.g., bone marrow); lymphoma and myeloma, which are cancers of immune cells; and cancers of the central nervous system, including cancers from brain and spinal tissue.

[0066] The aforementioned cancers can be selected from, but are not limited to, the group consisting of colorectal cancer, melanoma, liver cancer, gliocytoma, 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, hematological cancer, skin cancer, and lung cancer.

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

[0068] In the aforementioned treatment method, the compound, its stereoisomer, or a pharmaceutically acceptable salt thereof can be administered to a patient diagnosed with cancer at any stage of anticancer treatment, and is not limited to any particular stage.

[0069] Furthermore, the aforementioned compounds, their stereoisomers, or pharmaceutically acceptable salts may be administered in the form of the aforementioned pharmaceutical compositions, but are 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] The present invention will be described in detail below with reference to manufacturing examples and embodiments. [Examples]

[0072] Manufacturing example Manufacturing Example 1: Synthesis of 2-(5-chloro-1H-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 6) (1) Synthesis of perfluorophenyl 2-(5-chloro-1H-indole-3-yl)acetate JPEG0007857690000013.jpg39146 At room temperature, a solution of 2-(5-chloro-1H-indol-3-yl)acetic acid (56.00 g, 267.14 mmol), 2,3,4,5,6-pentafluorophenol (54.9 g, 293.85 mmol), and EDC-HCl (61.45 g, 320.57 mmol) in dichloromethane (890 mL) was stirred, and N,N-diisopropylethylamine (52.82 mL, 293.85 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours, and after confirming the completion of the reaction, distilled water (890 mL) was added. The separated 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 perfluorophenyl 2-(5-chloro-1H-indole-3-yl)acetate (50.00 g, yield 50%).

[0073] (2) Synthesis of 2-(5-chloro-1H-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 6) JPEG0007857690000014.jpg47142 A solution of 6-morpholinobenzo[d]thiazole-2-amine (26.00 g, 110.49 mmol) and perfluorophenyl 2-(5-chloro-1H-indole-3-yl) acetate (47.74 g, 127.07 mmol) in THF (1.1 L) was stirred at room temperature. The reaction mixture was heated under reflux for 24 hours, and after confirming the completion of the reaction, it was cooled to room temperature. The reaction mixture was concentrated under reduced pressure and purified by MPLC to obtain compound 6 (28.5 g, yield 60%).

[0074] 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)

[0075] Manufacturing Example 2: Synthesis of 2-(5-chloro-1H-indole-3-yl)-N-(6-(4-methylpiperazine-1-yl)benzo[d]thiazole-2-yl)acetamide (Compound 9) JPEG0007857690000015.jpg52158 At room temperature, 2-(5-chloro-1H-indole-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) were added to a 10 ml solution of DMF and stirred for 2 days. After the reaction was complete, the reaction mixture was placed in 50 mL of ice water, 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%).

[0076] 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)

[0077] Manufacturing Example 3: Synthesis of 2-(5-chloro-1-methyl-1H-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 21) (1) Synthesis of perfluorophenyl 2-(5-chloro-1-methyl-1H-indole-3-yl)acetate JPEG0007857690000016.jpg42152 At room temperature, a solution of 2-(5-chloro-1-methyl-1H-indole-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, and after confirming the completion of the reaction, distilled water (2 mL) was added. The separated 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 perfluorophenyl 2-(5-chloro-1-methyl-1H-indole-3-yl)acetate (150 mg, 78%).

[0078] (2) Synthesis of 2-(5-chloro-1-methyl-1H-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 21) JPEG0007857690000017.jpg511566-Morphorinobenzo[d]thiazole-2-amine (40 mg, 169.99 μmol) and perfluorophenyl 2-(5-chloro-1-methyl-1H-indole-3-yl) acetate (99 mg, 254.99 μmol) in THF (1.7 mL) were stirred under reflux for 12 hours. After the reaction mixture was cooled to room temperature, impurities were removed by filtering under reduced pressure, and the filtrate was concentrated under reduced pressure. The concentrated reaction mixture was recrystallized with diethyl ether to obtain compound 21 (40 mg, yield 54%).

[0079] 1H 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)

[0080] Manufacturing Example 4: Synthesis of 2-(5-hydroxy-1H-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 32) A solution of compound 39 (2-(5-methoxy-1H-indole-3-yl)-N-(6-morpholinobenzo[d]thiazole-2-yl)acetamide) (25 mg, 59.17 μmol) obtained in the following preparation example 5 was stirred at room temperature for 6 hours in borontribromide (7.9 μml, 71.01 μmol). Distilled water was added to the reaction mixture to terminate the reaction, and after neutralization with sat.NaHCO3, the layers were separated by DCM. The organic layer was washed, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was recrystallized over diethyl ether to obtain compound 32 (11 mg, yield 45%).

[0081] 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)

[0082] Manufacturing Example 5: Synthesis of 2-(5-methyl-1H-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 39) (1) Synthesis of perfluorophenyl 2-(5-methoxy-1H-indole-3-yl)acetate JPEG0007857690000019.jpg42161 At 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, and 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-indole-3-yl)-N-(6-morpholinebenzo[d]thiazole-2-yl)acetamide (compound 39) A solution of 6-morpholinobenzo[d]thiazole-2-amine (100 mg, 424.98 μmol) and perfluorophenyl 2-(5-methoxy-1H-indole-3-yl) acetate (189 mg, 509.98 μmol) in THF (4.3 mL) was stirred under reflux for 12 hours. After the reaction mixture was cooled to room temperature, impurities were removed by filtering under reduced pressure, and the filtrate was concentrated under reduced pressure. The concentrated reaction mixture was recrystallized with diethyl ether to obtain compound 39 (120 mg, yield 67%).

[0084] 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)

[0085] Comparative Example 1: Synthesis of N-(benzo[d]thiazole-2-yl)-2-(5-chloro-1H-indole-3-yl)acetamide (Comparative Example 1) JPEG0007857690000021.jpg30137 At room temperature, a solution of 2-(5-chloro-1H-indole-3-yl)acetic acid (7.0 g, 33.39 mmol) in DMF (100 mL) was stirred and benzo[d]thiazole-2-amine (4.51 mg, 30.05 mmol), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3-oxidehexafluorophosphate (HATU, 15.24 g, 40.07 mmol), and trimethylamine (9.4 mL, 66.78 mmol) were added sequentially. 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, the organic layer was washed with distilled water, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the concentrate was purified by column chromatography to obtain the compound of Comparative Example 1 (5.0 g, yield 45%).

[0086] 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)

[0087] Examples Example 1: Confirmation of the effect of inducing CYP1A1 expression To confirm the target specificity of the compound of the present invention manufactured as described above, we checked whether the expression levels of the AHR target gene, CYP1A1 mRNA, and CYP1A1 protein increased.

[0088] (1) Effect of inducing CYP1A1 mRNA expression HepG2 cells were harvested from DMEM-fetal bovine serum (FBS) 10% medium and their viability was confirmed to be 97% or higher by trypan blue staining. Afterward, the cells were centrifuged at 1200 rpm for 5 minutes at room temperature, and then 3 × 10⁶ cells were placed in DMEM-fetal bovine serum 10% medium. 5 The cells were resuspended at a concentration of cells / ml and prepared. Next, 3 ml of cells were dispensed into 60 mm dishes, and 50 μl of 1 μM concentrations of compounds 6, 21, 32, and 39, diluted in DMEM medium, were added to each dish. The cells were then cultured in a cell incubator (5% CO2 incubator) for 24 hours. The control group was treated with 50 μl of 0.05% dimethyl sulfoxide (DMSO) / DMEM medium.

[0089] Cultured cells were harvested, and mRNA samples were prepared. mRNA was extracted from the harvested cells using phenol-chloroform precipitation with Trizol reagent (Invitrogen, Cat No. 15596018). cDNA was synthesized from the isolated RNA by reverse transcription, and CYP1A1 expression was confirmed by real-time polymerase chain reaction (real-time PCR) using iQ SYBR-Green Supermix (Bio-rad) in a CFX96 (Bio-rad) detection system. Relative enzyme expression levels were compared using the ΔΔct method (delta-delta ct method) with GAPDH as the control enzyme. A 1x factor was established using the control group.

[0090] The real-time polymerase chain reaction was performed at an annealing temperature of 58°C for 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] This confirmed that the expression levels of CYP1A1 mRNA were higher in the groups treated with compounds 6, 21, 32, and 39 than in the control group (vehicle). Therefore, it was confirmed that compounds 6, 21, 32, and 39 significantly induce the expression of CYP1A1, an AHR target gene (Figure 1).

[0092] (2) Effect of inducing CYP1A1 protein expression HepG2 cells were harvested from DMEM-fetal bovine serum (FBS) 10% medium and their viability was confirmed to be 97% or higher by trypan blue staining. Afterward, the cells were centrifuged at 1200 rpm for 5 minutes at room temperature, and then transferred to DMEM-fetal bovine serum 10% medium in a 1.5 × 10⁶ solution. 5 The solution was resuspended at a concentration of 1 / ml and prepared.

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

[0094] After 24 hours, the plate medium was discarded, washed with dPBS, and then treated with 100 μl of 2 μM 7-ethoxyresorufin. Cells were cultured in a cell incubator (5% CO2 incubator) for 30 minutes. After 30 minutes, each cell was treated with 75 μl of 150 μg / ml fluorescamine (150 μg / ml), and resorufin excitation was measured at 535 nm and emission at 590 nm. A standard curve was used, measured with resorufin solution at 0–50 pmol.

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

[0096] After 15 minutes, the protein was quantified using the Bradford method, and the value was calculated using the formula (sample resorufin amount / sample protein amount). A 1x factor was then set using the control group.

[0097] This confirmed that the expression levels of CYP1A1 protein were significantly higher in the groups treated with compounds 6, 21, 32, and 39 compared to the control group (vehicle). Therefore, it was confirmed that compounds 6, 21, 32, and 39 significantly induce the protein of CYP1A1, an AHR target gene (Figure 2).

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

[0099] A549 cells cultured in DMEM - 10% fetal bovine serum (FBS) medium were harvested, and it was confirmed by trypan blue staining that the survival rate was 97% or higher. Then, after centrifuging 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 value of enzyme expression was compared by the ΔΔct method using GAPDH as a control enzyme. A value of 1 - fold was set using the control group.

[0101] The real-time polymerase chain reaction was performed at an annealing temperature of 58°C for 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, IL-6 production by A549 induced by IL-1β stimulation was significantly reduced by treatment with compounds 6, 9, 21, 32, and 39. This confirmed that compounds 6, 9, 21, 32, and 39 effectively suppress IL-6 production (Figure 3).

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

[0104] The spleen of C57BL / 6 mice (8-12 weeks old, female) was excised, RPMI medium was added and the cells were ground. A single-cell suspension was then obtained by passing the spleen through a 40 μm cell strainer (BD Falcon). The single-cell suspension was centrifuged (1200 rpm, 5 minutes), the supernatant was discarded, 1 ml of ACK lysis buffer was added and mixed for 1 minute, and then washed with RPMI medium. After centrifugation, T cells were isolated from the single-cell suspension using a mouse CD4 naive T cell enrichment kit (Invitrogen). The isolated T cells were then mixed in RPMI-fetal bovine serum (FBS) 10% + 2-ME (mercaptoethanol) medium at a rate of 1 × 10⁶ 6Prepared by resuspending in / ml. For T cell activation, 5 μg / ml of anti-CD3 (eBioscience TM ) was dispensed into 96-well plates in 100 μl increments and incubated in a cell culture incubator (37°C, 5% CO2 incubator) for 4 hours, then washed with phosphate-buffered saline to prepare the plates. 200 μl of resuspended T cells were dispensed into each prepared plate, and 2 μg / ml of anti-CD28 (eBioscience) was added to each well. TM Cells were treated with 2 ng / ml of TGF-β1 (R&D systems) and 100 U / ml of IL-2. 5 μl each of 2.5 μM concentrations of compound 6 and compound 9, diluted in RPMI-fetal bovine serum 10% + 2 ME medium, were treated and cultured in a cell incubator (37°C, 5% CO2 incubator) for 3 days. The control group was treated with 5 μl of 0.05% dimethyl sulfoxide (DMSO) / RPMI medium. After 3 days, cultured cells were harvested and Foxp3 protein expression was measured to confirm the effect of regulatory T cell generation.

[0105] To confirm Foxp3 protein expression, the harvested cells were placed in 5 ml FACS tubes (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) was added to prevent nonspecific binding of the antibody. TM The mixture was treated with ) and reacted at 4°C for 15 minutes. Then, 0.25 μg of CD4 monoclonal antibody (GK1.5) and PE-Cyanine7 (eBioscience) were added. TM After treating with ) and staining at 4°C for 30 minutes, the cells were washed with 1 ml of FACS buffer solution. Fixation / Permeabilization solution (eBioscience) was then applied to the FACS tube containing the sample. TM Add 1 ml of ) at a time, react at 4°C for 1 hour, then add permeabilization buffer (eBioscience TMThe sample was washed twice with ) . Then, 0.5 μg of Foxp3 monoclonal antibody (FJK-16s) and PE (eBioscience) were added. TM The samples were treated with ( ), stained at 4°C for 30 minutes, washed twice with permeabilization buffer, suspended in 0.3 ml of FACS buffer solution, and then measured by flow cytometry.

[0106] As a result, the generation of Foxp3-regulated T cells was significantly increased by compounds 6 and 9 compared to the control group (Vehicle). This confirms that compounds 6 and 9 effectively promote the generation of FoxP3+ regulated T cells (Figure 4).

[0107] Example 4: Therapeutic effect on inflammatory bowel disease To investigate the therapeutic effect of the compound of the present invention, manufactured as described above, on inflammatory bowel disease, inflammatory bowel disease was induced in C57BL / 6 mice as follows, and the effect was evaluated after administering compound 6 (Figures 5-10).

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

[0109] Compound 6 at a dose of 10 mg / kg per mouse was completely dissolved in an ethanol-cremofol EL mixture equivalent to 7.5% (v / v) of the administered dose. This was then diluted in phosphate-buffered saline to a final concentration of ethanol:cremofol EL:phosphate-buffered saline (0.375:0.375:9.25, v / v / v), and 200 μl of this solution was administered orally daily for a total of 14 doses from day 1 to day 14 of the experiment. The severity index for inflammatory bowel disease was classified into 0-10 levels and recorded visually at 2-day intervals.

[0110] For the symptoms of inflammatory bowel disease, the scores of the following three items (Table 2) were summed up to form an index.

[0111] [Table 2]

[0112] Analysis revealed that the solvent control group (Vehicle) began to lose weight from day 6 of the experiment, and by day 10, their weight had decreased by more than 10%, and 100% of them developed enteritis with a severity index of 5 or more. The solvent control mice showed a severity index of 7.50 ± 0.50 on day 10 of the experiment, which is the maximum severity index. The group administered 10 mg / kg of compound 6 of the present invention showed a statistically significant therapeutic effect compared to the solvent control group on day 10 of the experiment (Figure 5), and on day 15, a comparison of the weight:length ratio (mg / cm) of the large intestine confirmed a significant suppression of enteritis morphologically (compared to the solvent control group (Vehicle) ***, p<0.001, see Figures 5 and 6). Compound 6 of the present invention showed excellent anti-inflammatory effects when administered at 10 mg / kg (Figures 7a and 7b).

[0113] On day 15 of the experiment, the large intestine of a mouse was excised and mRNA samples were prepared. To extract mRNA, the colon tissue was pulverized using a homogenizer to obtain a homogeneous suspension. mRNA from the homogeneous suspension was extracted using the phenol-chloroform precipitation method with the easy-spin™ (DNA free) total RNA extraction kit (Intron biotechnology, Cat No. 17221). cDNA was synthesized from the isolated 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) in a CFX96 (Bio-rad) detection system. The relative values ​​of enzyme expression levels were compared using the ΔΔct method with GAPDH as the control enzyme. A 1x factor was set using the large intestine of a normal mouse as the control group.

[0114] The real-time polymerase chain reaction was performed with an annealing temperature of 58°C for 45 cycles, using the following primer sequences.

[0115] Mouse IL-1β in the forward direction: 5'-CTC GTG CTG TCG GAC CCA TAT-3' (SEQ ID NO: 7), and in the reverse direction: 5'-TTG AAG ACA AAC CGC TTT TCC A-3' (SEQ ID NO: 8); Mouse IL-6 forward direction: 5'-CAT GTT CTC TGC GAA ATC GTG G-3' (sequence number 9) and reverse direction: 5'-AAC GCA CTA GGT TTG CCG AGT A-3' (sequence number 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 in the forward direction: 5'-CCA CAC CGT CAG CCG ATT TG-3' (SEQ ID NO: 13) and in the reverse direction: 5'-CAC CCA TTC CCT TCA CAG AGC-3' (SEQ ID NO: 14); Mouse S100a8, forward direction: 5'-AAA TCA CCA TGC CCT CTA CAA G-3' (SEQ ID NO: 15), and reverse direction: 5'-CCC ACT TTT ATC ACC ATC GCA A-3' (SEQ ID NO: 16); Mouse S100a9: Forward direction, 5'-ATA CTC TAG GAA GGA AGG ACA CC-3' (SEQ ID NO: 17); Reverse direction, 5'-TCC ATG ATG TCA TTT ATG AGG GC-3' (SEQ ID NO: 18); Mouse IL-10 in the forward direction: 5'-CAA GGC AGT GGA GCA GGT GAA-3' (SEQ ID NO: 19) and in the reverse direction: 5'-CGG AGA GAG GTA CAA ACG AGG TT-3' (SEQ ID NO: 20); Mouse Foxp3 forward direction: 5'-CCC ATC CCC AGG AGT CTT G-3' (sequence number 21) and reverse direction: 5'-ACC ATG ACT AGG GGC ACT GTA-3' (sequence number 22); Mouse Reg3b in the forward direction: 5'-ACT CCC TGA AGA ATA TAC CCT CC-3' (SEQ ID NO: 23), and in the reverse direction: 5'-CGC TAT TGA GCA CAG ATA CGA G-3' (SEQ ID NO: 24); Mouse Muc2 forward direction: 5'-ATG CCC ACC TCC TCA AAG AC-3' (sequence number 25) and reverse direction: 5'-GTA GTT TCC GTT GGA ACA GTG AA-3' (sequence number 26); Mouse GAPDH in the forward direction: 5'-TTC ACC ACC ATG GAG AAG GC-3' (SEQ ID NO: 27) and in the reverse direction: 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 colorectal lesions were significantly reduced by administration of compound 6 compared to the solvent control group (Vehicle) (compared to the solvent control group (Vehicle)**, p<0.01, see Figures 7a and 7b). The expression levels of immunomodulatory factors IL-10 and Foxp3 in colorectal lesions were significantly increased by administration of compound 6 compared to the solvent control group (Vehicle) (compared to the solvent control group (Vehicle)*, p<0.05;**, p<0.01, see Figure 8). The expression levels of intestinal epithelial cell protective factors Reg3b and Muc2 in colorectal lesions were significantly increased by administration of compound 6 compared to the solvent control group (Vehicle) (compared to the solvent control group (Vehicle)*, p<0.05;***, p<0.001, see Figure 9). These results show that compound 6 of the present invention significantly reduces the expression of intestinal inflammatory factors and significantly increases the expression of intestinal immunomodulatory factors and intestinal epithelial cell protective factors.

[0117] Furthermore, in order to investigate the mucosal healing effect of the compound according to the present invention, inflammatory bowel disease was induced in C57BL / 6 mice as follows, and the degree of recovery of the intestinal epithelial barrier integrity was evaluated after administering compound 6.

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

[0119] In the group receiving compound 6 according to the present invention, 10 mg / kg of the compound per mouse was completely dissolved in an ethanol-cremofol EL mixture equivalent to 7.5% (v / v) of the dose. This mixture was then diluted in phosphate-buffered saline to a final ethanol:cremofol EL:phosphate-buffered saline (0.375:0.375:9.25, v / v / v), and 200 μl of this solution was administered orally daily for a total of 14 times from day 1 to day 14 of the experiment.

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

[0121] Serum FITC-dextran levels were significantly reduced by administration of compound 6 compared to the solvent control group (Vehicle) (*** compared to the solvent control group (Vehicle), p<0.001, see Figure 10). This result indicates that compound 6 of the present invention exhibits a significant mucosal healing effect. This confirms that compound 6 of the present invention has an oral therapeutic effect in a mouse model of inflammatory bowel disease.

[0122] Example 5: Preventive and therapeutic effects on colorectal cancer To investigate the preventive (Figures 11 and 12) and therapeutic effects (Figures 13 and 14) of the compound of the present invention manufactured as described above on colitis-associated colorectal cancer (CA-CRC), compound 6 was administered to a colorectal cancer model (AOM / DSS mouse) and its effects were evaluated as follows.

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

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

[0125] As a result, the control group (AOM / DSS + Vehicle) showed a decrease in body weight from day 70, while the compound 6 treatment group (AOM / DSS + compound 6) showed an increase in body weight, similar to that of normal mice (Figure 11). Furthermore, the compound 6 treatment group showed a significantly lower number of tumors and significantly smaller tumor size. In other words, compound 6 was confirmed to have an effect in preventing the development of colorectal cancer due to inflammation (Figures 11, 12).

[0126] The therapeutic effect of compound 6 according to the present invention on colorectal cancer was confirmed. From day 50 to day 63 of the experiment, mice were administered AOM, and then DSS was administered on days 7 and 14 to induce colorectal cancer. Compound 6 was administered 14 times from day 50 to day 63. As a result, the control group (AOM / DSS + Vehicle) showed a decrease in body weight from day 70, while the compound 6 administration group (AOM / DSS + compound 6) showed an increase in body weight, similar to that of normal mice (Figure 13). Furthermore, it was confirmed that the number of tumors and their size were significantly smaller in the compound 6 administration group (Figure 14). In other words, it was confirmed that compound 6 is effective in treating colorectal cancer (Figures 13, 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 To investigate the therapeutic effect of the compound of the present invention, manufactured as described above, on psoriasis, the following experiments were conducted (Figures 15-17).

[0128] Female BALB / C mice (8-10 weeks old) had their back hair removed using a depilatory device and depilatory cream. From the following day, 62.5 mg of 5% imiquimod cream was applied daily for 6 days to the depilated skin. Control mice (Vehicle) were treated with Vaseline cream. In the group administered compound 6 according to the present invention, 10 mg / kg of the compound per mouse was completely dissolved in an ethanol-cremofol EL mixture (1:1, v / v) equivalent to 15% (v / v) of the dose. This mixture was then diluted in phosphate-buffered saline to a final ethanol:cremofol EL:phosphate-buffered saline (7.5:7.5:85, v / v / v), and 200 μl was administered orally daily for a total of 6 times from day 0 to day 5 of the experiment. On day 6 of the experiment, skin tissue from the backs of the mice was taken and subjected to histological and mRNA experiments.

[0129] As a result, in the group administered compound 6 orally, epithelial thickness was significantly reduced (Figure 15), the expression levels of IL-17a and S100a8 mRNA, which promote inflammation of skin lesions, were significantly reduced (Figure 16), and Foxp3 and IL-10, which induce suppression of inflammation of skin lesions, were significantly increased (Figure 17). From this, it was confirmed that compound 6 can be used as an effective treatment for psoriasis.

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

[0131] The spleen of a C57BL / 6 mouse (8-12 weeks old, female, 18±3g) was removed, ground with RPMI medium, and then passed through a 40μm cell strainer (BD Falcon) to obtain a single-cell suspension. After centrifugation (1200 rpm, 5 min) of the single-cell suspension, 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 mixed for 1 minute, then washed with RPMI medium. After centrifugation, the cell suspension was reacted with mouse CD90.2 microbeads (Miltenyi Biotec, Cat No. 130-121-278) at 4°C for 20 minutes. After the reaction was complete, the cell suspension 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 was extracted from the cell suspension. + T cells were obtained (positive selection). CD90.2 was obtained. +To obtain bone marrow cells for transplantation along with T cells, bilateral femurs and tibias of normal (wild-type) C57BL / 6 mice (8-12 weeks old, female, 18±3g) were aseptically obtained. The ends of the femurs and tibias were cut, and bone marrow was extracted by perfusing the bone tissue with RPMI medium using syringes (21G for femurs, 26G for tibias). The extracted bone marrow was passed through a 40 μm cell strainer to obtain a single-cell suspension.

[0132] After centrifugation, the single-cell bone marrow suspension was discarded, 500 μl of ACK lysis buffer was added and mixed for 30 seconds, then washed with RPMI medium. After centrifugation, the cells were reacted with mouse CD90.2 microbeads at 4°C for 20 minutes. After the reaction was complete, the cell suspension was centrifuged and washed with 10 ml of autoMACS® running buffer, and then resuspended with 3 ml of autoMACS® running buffer. CD90.2 was extracted from the cell suspension using autoMACS pro. - 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. 6 At / ml, TCD-BM is 5 × 10 6 The solution was prepared by resuspending it in phosphate-buffered saline at a concentration of / ml.

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

[0134] The severity index for graft-versus-host disease is a system that classifies each item—weight loss, hair condition, posture, activity level, and skin changes—as a score of 0 to 2 points, totaling 10 points. The severity was evaluated visually at intervals of 3 to 4 days.

[0135] Analysis revealed that the severity index (8±1) of graft-versus-host disease in the solvent control group (Vehicle) was significantly reduced by administration of compound 6 (2.2±1.2) (compared to the solvent control group***, p<0.001, see Figure 18). Histopathological analysis of the colon tissue of mice from each experimental group confirmed that the inflammatory pathology index of the colon tissue was significantly reduced by administration of compound 6 (see Figure 19). This indicates that compound 6 can be used as a preventive and therapeutic agent for graft-versus-host disease.

[0136] Example 8: Therapeutic effect on multiple sclerosis To investigate the therapeutic effect of the compound of the present invention, manufactured as described above, on multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) was induced in C57BL / 6 mice as follows, and the effect of compound 6 was evaluated after administration (Figure 20).

[0137] On day 0 of the experiment, myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide (MOG 35-55 An emulsion was prepared by mixing 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) for 7 minutes. MOG 35-55 The emulsion was subcutaneously injected into both flanks of C57BL / 6 mice (7-8 weeks old, female), followed by the administration of 100 μl of pertussis toxin (Sigma aldrich, Cat No. P2980) (200 ng) via the tail vein. The same amount of pertussis toxin was administered intravenously on the second day of the experiment. Leakage of the emulsion from the injection site was checked, and from the seventh day of the experiment, the mice were visually observed to check for the onset of multiple sclerosis.

[0138] In the group administered compound 6 according to the present invention, 10 mg / kg of compound 6 per mouse was completely dissolved in an ethanol-cremofol EL mixture equivalent to 7.5% (v / v) of the administration dose. This mixture was then diluted in phosphate-buffered saline to a final ethanol:cremofol EL:phosphate-buffered saline (0.375:0.375:9.25, v / v / v), and 200 μl was administered orally daily for a total of six times from day 15 to day 20 of the experiment. The multiple sclerosis index is a severity index system classified into 0 to 5 stages, and was recorded by visual observation.

[0139] The symptoms of autoimmune encephalomyelitis were evaluated using an index based on the following criteria. 0: No symptoms 1: The tail loses its strength. 2: As the tail loses its strength, the hind legs weaken. 3: The hind legs become paralyzed. 4. The hind legs become paralyzed and the forelegs weaken. 5: Near death or dead

[0140] Analysis revealed that the disease severity index on day 21 of the experiment, the acute reaction period, was 2.9 ± 1.92 in the solvent control group (Vehicle) and 0.92 ± 0.58 in the compound 6 administration group. The compound 6 administration group showed a lower severity index compared to the solvent control group (Vehicle) (*, p<0.05, Figure 20). This confirmed the therapeutic effect of compound 6 on multiple sclerosis.

[0141] Example 9: Therapeutic effect on neutrophilic asthma To investigate the therapeutic effect of the compound of the present invention, manufactured 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 intranasal administration of 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 onwards, 50 μg of OVA diluted in phosphate-buffered saline was administered intranasally for two days each week until day 36, for a total of eight times. Intranasal administration was performed after anesthesia using an isoflurane inhalation anesthesia machine. In the group receiving compound 6 according to the present invention, 10 mg / kg of compound 6 per mouse was completely dissolved in an ethanol-cremofol EL mixture equivalent to 7.5% (v / v) of the dose. This mixture was then diluted in phosphate-buffered saline to a final ethanol:cremofol EL:phosphate-buffered saline (0.375:0.375:9.25, v / v / v), and 200 μl of this mixture was administered orally daily for a total of 10 times from day 27 to day 36 of the experiment.

[0143] As a result, the number of neutrophils in bronchoalveolar lavage (BAL) was significantly reduced in the compound 6 administration group compared to the solvent control group (Vehicle) (Figure 21). This confirmed the therapeutic effect of compound 6 on neutrophilic asthma.

[0144] Example 10: Comparison of effects with Comparative Example 1 To confirm the superiority of the compound in the example of the present invention compared to the compound in Comparative Example 1, the following experiments were conducted: (1) measurement of pharmacokinetic parameters, (2) measurement of solubility, (3) suppression of inflammatory activity in Th17 cells, (4) confirmation of the effect of promoting the generation of FoxP3 expression-regulating T cells, (5) whether or not the activity of drug metabolism-related enzymes is suppressed, and (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-cremofol EL mixture equivalent to 7.5% (v / v) of the administration dose at the dose concentrations shown in Table 3. This was then diluted in phosphate buffered saline to a final concentration of ethanol:cremofol EL:phosphate buffered saline (0.375:0.375:9.25, v / v / v), and administered orally and intravenously at a dose of 10 mL / kg. In the oral administration group, food was supplied approximately 4 hours after administration. Blood was collected in the intravenous administration group at 0.083, 0.5, 1, 2, 4, 8, and 24 hours after administration, and in the oral administration group at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration. Plasma was then collected by centrifugation. The drug concentration in the collected plasma was measured using UHPLC-MS / MS.

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

[0148] [Table 3]

[0149] (2) Measurement of solubility The solubility of compound 6 and comparative example 1 was measured. Compound 6 was dissolved in ethanol, and then measured and analyzed using a SIRIUS T3 instrument. As shown in Table 4, compound 6 had a higher logP value and approximately 3.92 times greater pH-metric solubility molarity than comparative example 1.

[0150] [Table 4]

[0151] (3) Inhibitory effect on the inflammatory activity of Th17 cells We compared the inhibitory effects on inflammatory activity of Th17 cells, which are important causative cells in the development of autoimmune diseases (Figure 23).

[0152] The spleen of C57BL / 6 mice (8-12 weeks old, female) was excised, pulverized with IMDM medium, and 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 and mixed for 1 minute, and then washed with RPMI medium. After centrifugation, T cells were isolated from the single-cell suspension using a mouse CD4 naive T cell enrichment kit (Invitrogen). The isolated T cells were enriched in IMDM-fetal bovine serum (FBS) 10% + 2-ME (mercaptoethanol) medium at a rate of 1 × 10⁶ 6 Prepared by resuspending in / ml. For T cell activation, 5 μg / ml of anti-CD3 (eBioscience TM ) was dispensed in 100 μl portions into 96-well plates and incubated in a cell incubator (37°C, 5% CO2 incubator) for 4 hours, then washed with phosphate-buffered saline to prepare the plates. 200 μl portions of resuspended T cells were dispensed into each prepared plate, and 2 μg / ml of anti-CD28 (eBioscience) was added to each well. TM Cells were treated with 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). 5 μl each of compounds diluted in 10% IMDM-fetal bovine serum + 2 ME medium at concentrations of 1.0 μM, 2.5 μM, and 5 μM were treated and cultured in a cell incubator (37°C, 5% CO2 incubator) for 3 days. The control group was treated with 5 μl of 0.05% dimethyl sulfoxide (DMSO) / RPMI medium. After 3 days, to confirm the inhibitory effect on the inflammatory activity of Th17 cells, the cultured medium was collected and IL-17A was detected using a flex set (BD biosciences).

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

[0154] As a result, we confirmed that IL-17A production in Th17 cells was significantly reduced by compound 6 compared to comparative example 1 (Figure 23).

[0155] (4) Confirmation of the effect of promoting the generation of FoxP3-expressing regulatory T cells (Treg). The effect of promoting the generation of "FoxP3-regulated T cells," which play an important role in maintaining immune tolerance, was compared (Figure 24). CD4 +After isolating T cells, they were cultured and treated with 5 μl each of 2.5 μM and 5 μM compounds diluted in RPMI-fetal bovine serum 10% + 2 ME medium. The cells were cultured for 3 days in a cell incubator (37°C, 5% CO2 incubator) before analysis. The control group was treated with 5 μl of 0.05% dimethyl sulfoxide (DMSO) / RPMI medium. After 3 days, to confirm the effect of regulatory T cell generation, the cultured cells were harvested and Foxp3 protein expression was measured using the same method as in Figure 4.

[0156] As a result, we confirmed that the generation of Foxp3-regulating T cells was significantly increased by compound 6 compared to comparative example 1 (Figure 24).

[0157] (5) Whether or not the activity of drug metabolism-related enzymes can be suppressed. The activity of CYP isotopes, which are enzymes involved in drug metabolism, was measured.

[0158] Live human hepatocyte cell line (Corning, Cat No. 454551) was collected using plating medium, and a cell density of 0.4 × 10⁶ was obtained in 24 wells. 6After dispensing the cells into wells, they were cultured for 2 to 4 hours. The plating medium was replaced with hepatocyte culture medium, and the hepatocytes were cultured for 24 hours. After 24 hours of culture, the hepatocytes were treated with compound 6 at 1, 5, and 25 M concentrations. The culture medium containing the compound was replaced every 24 hours for 2 days. The cultured cells were harvested and mRNA samples were prepared. 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 drug-metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2B6, and CYP3A4 were measured by real-time PCR analysis using the Quant Studio™ 7 Flex Real-Time PCR System (Applied Biosystems, CA). The relative values ​​of enzyme expression levels were compared using the ΔΔct method with GAPDH as the control enzyme. A 1x factor was established using the control group. The results were shown as a percentage of the control group sample compared to the control group sample.

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

[0160] [Table 5]

[0161] As a result, while Comparative Example 1 suppressed the enzyme activity of CYP2C19 by 24.4% at 10 μM, compound 6 had almost no effect on the enzyme activity of CYP2C19. In other words, compound 6 was confirmed to be suitable for co-administration with CYP2C19 metabolites.

[0162] (6) Therapeutic effects 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 using the same method as in Example 4. Compound 6 and comparative example 1 were orally administered to the mice at a dose of 10 mg / kg, and the expression levels of IL-6 and S100a9 mRNA were checked in the colon tissue.

[0163] As a result, we confirmed that the expression levels of IL-6 and S100a9 mRNA in colon tissue were significantly reduced by compound 6 compared to comparative example 1 (Figure 25). In other words, we 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, its stereoisomer, or a pharmaceutically acceptable salt thereof. 【Chemistry 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, phenyl group, acetyl group, -CH ２ C (=O) OR ２ , -C (=O) OR ２ or -S (=O) ２ R ２ And, R １ is a substituted or unsubstituted six-membered heterocycle, where the six-membered heterocycle contains one or two heteroatoms selected from nitrogen and oxygen. R ２ is C １ -C ３ It is an alkyl group.

2. The aforementioned six-membered heterocycle of substitution is C １ -C ３ The compound according to claim 1, which is a heterocycle substituted with an alkyl group, a hydroxyl group, or a dimethylamine, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

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

4. A compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, selected from the group consisting of the following compounds.

5. A pharmaceutical composition comprising a 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, for the treatment or prevention of autoimmune diseases.

7. The pharmaceutical composition according to claim 5, 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, atopic dermatitis, psoriasis, rheumatoid arthritis, systemic lupus erythematous erythematous disease, and type 1 diabetes mellitus.

8. The pharmaceutical composition according to claim 5, for the treatment or prevention of cancer.

9. The composition according to claim 8, for the treatment or prevention of cancer, wherein the cancer is selected from the group consisting of colorectal cancer, melanoma, liver cancer, gliocytoma, 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, hematological cancer, skin cancer, and lung cancer.

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