Novel benzene derivatives and their immunosuppressive applications

Abstract

To provide compounds that have excellent therapeutic, preventive, or ameliorative effects on immune diseases selected from autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases. [Solution] The present invention relates to novel benzene derivatives and their immunosuppressive applications. Specifically, the present invention relates to compositions comprising a compound of chemical formula 1, its stereoisomers, solvates, hydrates, crystalline forms, or pharmaceutically acceptable salts. The compositions have preventive or therapeutic applications for immune diseases such as autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to novel benzene derivatives and their immunosuppressive-related uses. Specifically, the present invention relates to a composition comprising a compound of Chemical Formula 1, its stereoisomers, solvates, hydrates, crystal forms or pharmaceutically acceptable salts. The composition has preventive or therapeutic uses for immune diseases such as autoimmune diseases, transplant rejection reactions, allergic diseases and inflammatory diseases.

Background Art

[0002] Autoimmunity refers to an inappropriate reaction against autoantigens of the immune system that causes damage to cells or tissues by humoral immunity, cellular immunity or both. Autoimmune diseases are associated with molecules, cells and tissues targeted by autoimmune reactions and may appear systemically depending on the distribution of the target antigen or specifically in certain organs. For example, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), etc. belong to systemic autoimmune diseases, and autoimmune anemia, insulin-dependent diabetes mellitus (IDDM), Graves' disease, etc. belong to organ-specific autoimmune diseases.

[0003] Transplant rejection occurs when the transplanted tissue is rejected by the immune system of the transplant recipient, destroying the transplanted tissue. This can be classified into hyperacute rejection, acute rejection, chronic rejection, etc.

[0004] Allergic diseases are conditions in which antibodies react hypersensitively to specific substances acting as allergens when exposed to or in contact with them, resulting in inflammation and allergic symptoms in the affected area. These include bronchial asthma, allergic rhinitis, atopic dermatitis, urticaria (angioedema, rash, etc.), anaphylactic reactions, allergic conjunctivitis, seasonal allergies, drug allergies, and food allergies.

[0005] Inflammatory diseases occur when abnormal inflammation develops, and can be caused by a variety of factors, including physical causes such as burns and trauma, biological causes such as pathogens, hypersensitivity, and stress, and chemical causes such as toxins and particulate matter.

[0006] Autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases, as described above, share the common characteristic of being caused by an excessive immune response. These immune diseases are prevalent, and research and development into their treatment or prevention has been ongoing for a long time. For example, antihistamines and steroids have been developed and are still commonly used today. However, as problems such as toxicity and resistance arise, there remains a significant need in the relevant technological field for substances that can exhibit the desired therapeutic or preventive effects against immune diseases without side effects or adverse effects. [Overview of the project] [Problems that the invention aims to solve]

[0007] The inventors recognized the problems of the prior art and conducted numerous trials and errors to find a compound that has excellent therapeutic, preventive, or ameliorative effects on immune diseases selected from autoimmune diseases, transplant rejection reactions, allergic diseases, and inflammatory diseases. As a result, they developed the compound of Chemical Formula 1 and completed the present invention. [Means for solving the problem]

[0008] The compound according to the present invention, which achieves the objective of the present invention, is represented by the following chemical formula 1. [ka] In the aforementioned chemical formula 1, One or two of the atoms X1, X2, X3, X4, and X5 are nitrogen atoms, and the remaining atoms are carbon atoms. R1, R2, R3, R4, and R5 are independently selected from hydrogen, halogen, C1-C14 alkyl group, C2-C14 alkenyl group, C2-C14 alkynyl group, C5-C14 aryl group, C5-C14 arylalkyl group, C3-C14 cycloalkyl group, C3-C14 cycloalkylalkyl group, C3-14 heteroaryl group, C3-14 heteroarylalkyl group, C3-14 heterocycloalkyl group, C3-14 heterocycloalkylalkyl group, C1-C14 alkoxy group, C5-C14 aryloxy group, C5-C14 heteroaryloxy group, -NO2, -NR6, -SO2R6, -SO2N(R6)2, -CF3, -OH, cyanide group, thiol group, triazine, triazole, tetrazole, thiazole, diazole, imidazole, alkylamine, cycloalkylamine, heterocycloamine, and biotin. Y is selected from -OH, thiol group, phenoxy, cyanide group, thiazole, diazole, imidazole, triazine, triazole, tetrazole, -CON(R6)2, -NR6COR6, -SO2N(R6)2, -NH2, -NO2, -COOR6, -R7COOR6, -R7CON(R6)2, -CONHSR6, -CONHR7SR6, -CONHSSR6, -CONHR7SSR6, -CONHR7SO2R6, -CONHOR6, -CONHR7OR6, -CON(R7)2CON(R6)2, and -CON(R7)2NR7COR6. Here, R6 and R7 are independently of each other: hydrogen, halogen, thiol group, phenoxy, cyanide group, thiazole, diazole, imidazole, triazine, triazole, tetrazole, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C12 aryl group, C6-C12 arylalkyl group, C3-C12 cycloalkyl group, C3-C12 cycloalkylalkyl group, C3-10 heteroaryl group, C3-10 heteroarylalkyl group, C3-10 heterocycloalkyl group, C3-10 heterocycloalkylalkyl group, C1-C6 alkoxy group, -CF3, A Hydrogen, C1-C14 alkyl group, C2-C14 alkenyl group, C2-C14 alkynyl group, C6-C12 aryl group, C6-C12 arylalkyl group, C3-C12 cycloalkyl group, C3-C12 cycloalkylalkyl group, C3-10 heteroaryl group, C3-10 heteroarylalkyl group, C3-10 heterocycloalkyl group, C3-10 heterocycloalkylalkyl group, alkylamine, cycloalkylamine, heterocycloamine, and biotin, which are substituted or unsubstituted with one or more of mine, C1-6 alkylamine, and -CN.

[0009] The aforementioned chemical formula 1 can be used interchangeably with the following chemical formula and has the same meaning. Tsu.

[0010] As used in this invention, the term "independently of each other" means that two or more substituents are defined individually and may be different from each other or the same. For example, X1, X2, X3, X4, X5, and X6 may be carbon or nitrogen and may be the same from each other or different from each other.

[0011] In this invention, terms such as alkyl, alkenyl, and alkynyl are intended to include all linear (or linear) or side-chain (or branched) components.

[0012] In this invention, the term "halogen" means fluoro, chlorine, bromine, or iodine.

[0013] In this invention, the term "alkoxy" means O-alkyl.

[0014] In this invention, the term "hetero" refers to a heteroatom selected from oxygen, nitrogen, and sulfur.

[0015] In this invention, the term "heterocycloalkyl" refers to a cycloalkyl group in which a heteroatom is contained within the ring.

[0016] The present invention provides compositions for the treatment or prevention of immune diseases selected from autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases, comprising a compound represented by chemical formula 1, its isomers, solvides, or pharmaceutically acceptable salts.

[0017] As used in this invention, the term "treatment" means all actions by which the symptoms of an immune disease are improved or completely cured by the administration of the composition according to the present invention.

[0018] As used in this invention, the term "prevention" means all actions by which an immune disease is suppressed or delayed by the administration of the composition according to the present invention.

[0019] The aforementioned immune disorders should be understood to include all symptoms, diseases, and conditions that develop as an immune disorder progresses or as an increase in the severity of an immune disorder.

[0020] For example, autoimmune diseases include systemic lupus erythematosus (systemic lupus erythematosus) Erythematosus (SLE), rheumatoid arthritis arthritis; RA), multiple sclerosis; MS), systemic sclerosis, autoimmune anemia, insulin-dependent diabetes mellitus; IDDM), type 1 diabetes, Grave's disease, se), Graves hyperthyroidism, Kikuchi disease, hemophagocytic lymphohistiocytosis, lymphohistiocytosis), adult-onset Still's disease), Behcet disease, IgG4-related diseases, psoriasis, and the like can be included, but are not limited thereto.

[0021] Allergic diseases may include, but are not limited to, bronchial asthma, allergic rhinitis, atopic dermatitis, urticaria (such as angioedema, rash, etc.), anaphylactic reaction, allergic conjunctivitis, seasonal allergy, drug allergy, food allergy, and the like.

[0022] Inflammatory diseases may include, but are not limited to, inflammatory diseases caused by various chemical causes such as physical causes such as burns and trauma, pathogens, biological causes such as allergies and stress, toxins, particulate matter, and the like.

[0023] The compound includes its stereoisomers, solvates, hydrates, crystal forms or pharmaceutically acceptable salts.

[0024] As used in the present invention, the term "pharmaceutically acceptable salt" refers to a salt that can be produced by conventional methods in the art, and is not limited to salts with inorganic acids such as hydrochloric acid, hydrogen brominated, sulfuric acid, sodium bisulfate, phosphoric acid, or carbonic acid, or with organic acids such as formic acid, acetic acid, oxalic acid, benzoic acid, citric acid, tartaric acid, gluconic acid, gentisic acid, fumaric acid, lactobionic acid, salicylic acid, or acetylsalicylic acid (aspirin), forming pharmaceutically acceptable salts of these acids, or reacting with alkali metal ions such as sodium or potassium to form metal salts of these acids, or reacting with ammonium ions to form other forms of pharmaceutically acceptable salts.

[0025] The pharmaceutical composition according to the present invention may contain, alone, the compound represented by Chemical Formula 1, its stereoisomer, solvide, hydrate, crystalline form, or pharmaceutically acceptable salt, or may further contain one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0026] The aforementioned pharmaceutically acceptable carriers may further include, for example, oral or parenteral carriers. Oral carriers may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Parenteral carriers may include water, suitable oils, saline solutions, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. For other pharmaceutically acceptable carriers, please refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0027] The pharmaceutical composition of the present invention can be administered to mammals, including humans, by any means. For example, it can be administered orally or parenterally. Parenteral administration methods are not limited to these, but may include intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal administration. For example, the pharmaceutical composition of the present invention can be prepared in an injectable dosage form and administered by lightly pricking the skin with a fine 30-gauge needle, or by directly applying it to the skin.

[0028] The pharmaceutical composition of the present invention can be formulated into oral or parenteral formulations via the administration route described above.

[0029] In the case of orally administered formulations, the composition of the present invention can be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., using methods known in the art. For example, an oral formulation can be obtained by compounding the active ingredient with a solid excipient, then grinding the mixture, adding a suitable adjuvant, and finally processing it with a granular mixture to obtain tablets or sugar-coated tablets. Examples of suitable excipients include lac. The composition may contain sugars such as tose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants. Furthermore, the composition of the present invention may further contain anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0030] For parenteral formulations, dosage forms can be developed in the form of injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalants in the manner known to the art. These dosage forms are described in the generally known prescription literature for all pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).

[0031] The total effective dose of the pharmaceutical composition of the present invention can be administered to a patient in a single dose, or in a fractionated treatment protocol using multiple doses over a long period. The content of the active ingredient in the pharmaceutical composition of the present invention can vary depending on the symptoms of the disease. Preferably, the preferred total dose of the composition of the present invention may be about 0.01 μg to 1,000 mg per kg of the patient's body weight per day, most preferably 0.1 μg to 100 mg. However, the dose of the pharmaceutical composition of the present invention should be determined by a person with ordinary knowledge in the art, taking into account various factors such as the patient's age, weight, health condition, sex, disease severity, diet, and excretion rate, as well as the route of administration and number of treatments. The pharmaceutical composition of the present invention is not particularly limited in its dosage form, route of administration, or method of administration, as long as it exhibits the effects of the present invention.

[0032] Furthermore, the pharmaceutical composition of the present invention can be administered as a standalone therapeutic agent or in combination with other therapeutic agents. When administered in combination with other therapeutic agents, the composition of the present invention and the other therapeutic agents can be administered simultaneously, individually, or sequentially. The other therapeutic agents may be substances already known to have therapeutic or ameliorative effects on immune diseases. The other therapeutic agents include all surgical procedures, surgeries, etc., other than drug therapy.

[0033] When the pharmaceutical composition of the present invention is administered in combination with other therapeutic agents, the composition of the present invention and the other therapeutic agents may be packaged separately in different containers, or they may be packaged together in the same container.

[0034] In another aspect of the present invention, a method for treating or preventing an immune disease is provided, comprising the step of administering a composition of the present invention to an individual.

[0035] In the method for treating or preventing an immune disease according to the present invention, unless otherwise specified, each term has the same meaning as described above in reference to the composition for treating or preventing the immune disease.

[0036] The term “individual” includes any human or non-human animal. The term “non-human animal” may be vertebrates, e.g., non-human primates, sheep, dogs, and rodents, e.g., mice, rats, and guinea pigs. The individual may preferably be a human. The term “individual” is this In the specification, the terms "subject" and "patient" are interchangeable.

[0037] In the method for treating or preventing an immune disease according to the present invention, the composition of the present invention may be administered to an individual simultaneously, sequentially, or individually with other therapeutic agents. "Simultaneous" administration means that the composition of the present invention and other therapeutic agents are administered at once through the same infusion method. "Sequential" administration means that the composition of the present invention and other therapeutic agents are administered using separate infusion methods, but relatively continuously, accepting the minimum possible time to be consumed between administrations. "Individual" administration means that the composition of the present invention and other therapeutic agents are administered at regular time intervals. The method of administering the composition of the present invention and other therapeutic agents can be appropriately selected by a person with ordinary skill in the art, taking into consideration the patient's therapeutic efficacy and side effects. [Effects of the Invention]

[0038] Compositions comprising the compound of chemical formula 1 according to the present invention, its stereoisomers, solvates, hydrates, crystalline forms, or pharmaceutically acceptable salts can effectively prevent or treat immune diseases selected from autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases. [Brief explanation of the drawing]

[0039] [Figure 1] This FACS graph illustrates the relationship between CFSE fluorescence staining and cell proliferation. [Figure 2] This graph shows the proliferation of cells after administering each compound of the present invention, as read by FACS. [Figure 3] The graphs show the secretion levels of IL-2 24 hours after drug treatment, and IFN-γ, TNF-α, and IL-17A 72 hours after treatment. [Figure 4] The graphs show the secretion levels of IL-2 24 hours after drug treatment, and IFN-γ, TNF-α, and IL-17A 72 hours after treatment. [Figure 5] The graphs show the secretion levels of IL-2 24 hours after drug treatment, and IFN-γ, TNF-α, and IL-17A 72 hours after treatment. [Figure 6] The graphs show the secretion levels of IL-2 24 hours after drug treatment, and IFN-γ, TNF-α, and IL-17A 72 hours after treatment. [Figure 7] This document describes a method and results for observing the efficacy of the compound of the present invention in a mouse animal model of psoriasis. [Figure 8] This document describes a method and results for observing the efficacy of the compound of the present invention in a mouse animal model of psoriasis. [Figure 9] This document describes a method and results for observing the efficacy of the compound of the present invention in a mouse animal model of psoriasis. [Figure 10a] The results of evaluating the efficacy of the compounds of the present invention from a mouse animal model of psoriasis relate to the composition ratio of CD11b granulocytes, B cells, and T cells. [Figure 10b] The results of evaluating the efficacy of the compounds of the present invention from a mouse animal model of psoriasis relate to the composition ratio of CD11b granulocytes, B cells, and T cells. [Figure 10c] The results of evaluating the efficacy of the compounds of the present invention from a mouse animal model of psoriasis relate to the composition ratio of CD11b granulocytes, B cells, and T cells. [Modes for carrying out the invention]

[0040] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples. Furthermore, a person with ordinary skill in the art will understand the present invention. Various modifications and alterations can be made to the present invention as long as they do not impair the intent of the invention. Terms not specifically defined herein should be understood to have the meanings commonly used in the art to which the present invention pertains.

[0041] Manufacturing example Compounds belonging to chemical formula 1 were typically prepared by reaction formulas 1 or 2 below. Compounds with different substituents were also prepared through similar steps, but not all of these are explicitly shown herein. A person with ordinary skill in the art can easily prepare compounds belonging to chemical formula 1 by referring to the representative examples below.

[0042] [ka]

[0043] In the above reaction formula 1, compound C is obtained through a coupling reaction between compound A and compound B, using 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and hydroxybenzotriazole (HOBt).

[0044] [ka]

[0045] In the above reaction equation 2, compound C is obtained by reacting compound A and compound B.

[0046] Representative examples of compounds produced in this manner are shown below, along with their specific chemical structures. However, not all compounds shown in the table below are included in Chemical Formula 1 of the present invention.

[0047] TIFF2026086653000005.tif177168TIFF2026086653000006.tif178168TIFF20260866530 00007.tif179168TIFF2026086653000008.tif176168TIFF2026086653000009.tif164168

[0048] The molecular weight, properties, and 1H NMR values ​​of the compound were confirmed upon preparation, as shown in Table 1 below. [Table 1] TIFF2026086653000011.tif215168TIFF2026086653000012.tif200168TIFF2026086653000013.tif227168TIFF2026086653000014.t if232168TIFF2026086653000015.tif206168TIFF2026086653000016.tif221168TIFF2026086653000017.tif211168TIFF20260866530 00018.tif224168TIFF2026086653000019.tif200168TIFF2026086653000020.tif206168TIFF2026086653000021.tif224168TIFF202 6086653000022.tif217168TIFF2026086653000023.tif230168TIFF2026086653000024.tif221168TIFF2026086653000025.tif222168

[0049] Experimental Example 1: Verification of the effect of suppressing T cell proliferation To investigate whether the compounds obtained in the above production example have an effect of suppressing T cell proliferation, T cell proliferation was measured ex vivo using the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE), which covalently binds to intracellular molecules. After removing and pulverizing the spleen from 7-week-old C57BL / 6 mice, strain After isolating single cells using er (40 μM pore size, SPL), red blood cells were removed with ACK (Ammonium-Chloride-Potassium) lysis buffer to isolate only white blood cells. CD90.2 microbeads (130-121-278, Miltenyi Biotech.) were added and the mixture was reacted at 4°C for 20 minutes. Finally, spleen T cells were isolated using a MACS Magnetic Stand and LS column. The T cells isolated from the spleen were resuspended in 1 mL of Free media (RPMI-1640 + 200 U / mL penicillin + 200 μg / mL streptomycin), and then 0.3 μL of CFSE (10 mM) was added and the mixture was reacted at 37°C for 5 minutes. Subsequently, 10 mL of Free media was added to stop the reaction, and the cell precipitate was obtained by centrifugation. To this, RPMI 1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin was added to suspend the cells, and 2 × 10⁶ cells were placed per well in a 96-well plate. 5 Cells were aliquoted from the cell culture and treated with CD3 and CD28 antibodies (0.5 μg / mL each) to activate T cells. The compound, diluted in DMSO, was treated in three wells each at concentrations of 10 μM, 30 μM, and 50 μM. After incubation for 3 days in a 37°C, 5% CO2 incubator, FACS was performed. The degree of cell proliferation was read using a Canto instrument.

[0050] In the FACS graph in Figure 1, the vertical axis represents the quantity (number) of cells, and the horizontal axis represents the fluorescence level of the cells. Cells stained with CFSE decrease in the amount of CFSE as they undergo repeated cell division. This decrease in CFSE fluorescence is reflected by a shift to the left on the horizontal axis of the graph, which indicates cell proliferation.

[0051] Figure 2 shows graphs of cell proliferation read by FACS after administration of compound 9, compound 69, and compound 84, respectively. Each graph shows the drug concentration increasing from bottom to top, corresponding to drug concentrations of 10 μM, 30 μM, and 50 μM, respectively. Compounds 69 and 84 show inhibition of cell proliferation at concentrations of 10-30 μM, while compound 9 shows no inhibition of cell proliferation even at a concentration of 50 μM.

[0052] To express the growth inhibition rate collectively for the aforementioned experimental results, the growth inhibition rate was calculated by determining the relative growth amount to the amount grown after compound administration, based on the growth amount measured after administration of CD3 / CD28+DMSO, and then converting this to an inhibition amount and expressing it as a percentage. Specifically, the growth inhibition rate was calculated as follows.

[0053] Growth inhibition rate (%) = {1 - growth rate in drug-administered cells ÷ CD3 / CD28 + growth rate after DMSO administration} × 100

[0054] [Table 2] TIFF2026086653000027.tif248168TIFF2026086653000028.tif80168

[0055] On the other hand, as can be seen in Table 3 below, compounds that do not belong to Chemical Formula 1 according to the present invention were found to have a very low growth inhibition rate.

[0056] [Table 3]

[0057] Experiment Example 2: Cytotoxicity Experiment The following experiments were conducted to evaluate the cytotoxicity of the compounds according to the present invention.

[0058] Jurkat E6-1 T lymphocyte cell line, purchased from the Korea Cell Line Bank, was cultured in RPMI-1640 medium containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. The culture was then harvested and centrifuged at 1,300 rpm for 5 minutes. After removing the supernatant, the cells were transferred to a medium under the same conditions, resulting in approximately 2.2 × 10⁶ cells. 5 The compounds were resuspended at a concentration of cells / mL and dispensed into 96-well plates (Costar 96-well cell culture plates, Corning) at a rate of 90 μL per well. After treating each plate with 10 μL of each compound at different concentrations, the plates were incubated in a 5% CO2 incubator at 37°C for 24 hours. The cells were cultured for 2 hours. Subsequently, 10 μL of CCK-8 was added to each well, and the cells were cultured for another 2 hours at 37°C in a 5% CO2 incubator. The absorbance was then measured at 450 nm, and the relative cell viability compared to untreated cells is shown in Table 4.

[0059] The values ​​entered in Table 4 represent the relative cell survival rates for each group treated with different concentrations of each drug, with the survival rate of untreated cells set at 100%.

[0060] [Table 4] TIFF2026086653000031.tif248168TIFF2026086653000032.tif126168

[0061] Experimental Example 3: Verification of the effect of reducing cytokine expression To confirm the effect of the compound of the present invention on changes in the amount of cytokines secreted by T cells, the following experiment was performed.

[0062] After removing and pulverizing the spleen from 7-week-old C57BL / 6 mice, single cells were isolated using a strainer (40 μM pore size, SPL). Red blood cells were then removed using ACK (Ammonium-Chloride-Potassium) lysis buffer to isolate only white blood cells. These were then reacted with 10 v / v% CD90.2 microbeads (130-121-278, Miltenyi Biotech.) at 4°C for 20 minutes. Finally, T cells from the spleen were isolated based on the magnetic-based tablet. The T cells isolated through the above method totaled 2 × 10⁶ cells. 5 After culturing the T cells in a 96-well plate at a cell / well concentration, each well was treated with 1 μg / mL of anti-CD3 / CD28 antibody to activate the T cells. Subsequently, each well was treated with Vehicle (DMSO) and 30 μM of compounds (compounds 14, 19, 26, 31, 35, 40, 41, 56, 69, and 75), and the cells were incubated at 37°C in a 5% CO2 incubator. After 24 and 72 hours, the T cell culture medium was taken and centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant.

[0063] To measure the concentrations of cytokines present in the T cell culture medium, the Mouse Th1 / Th2 / Th17 Cytokine CBA Kit (Cytometric Bead Array kit) 560485 (BD Science) was used to measure the secretion levels of IL-2 24 hours after each drug treatment, and IFN-γ, TNF-α, and IL-17A 72 hours after each drug treatment via flow cytometry.

[0064] As can be seen from Figures 3-6 showing the experimental results, T cells stimulated with anti-CD3 / CD28 antibody without treatment with the compounds of the present invention secreted large amounts of IL-2, IFN-γ, TNF-α, and IL-17A. In contrast, when treated with the compounds (compounds 14, 19, 26, 31, 35, 40, 41, 56, 69, and 75), the secretion of these cytokines was significantly reduced. Based on this, it has been proven that the compounds of the present invention have the effect of reducing cytokine secretion in the process of suppressing T cell proliferation.

[0065] Experimental Example 4: Verification of the effectiveness of treatment in a psoriasis model To verify the therapeutic effect of the compounds according to the present invention on psoriasis, an animal model of psoriasis was constructed as described below, and the alleviation of skin lesions and the changes in immune cells were evaluated by injecting the compounds corresponding to compound 69 and compound 83.

[0066] To construct a psoriasis mouse animal model, BALB / c mice (7 weeks old, 20g, female) were purchased from Hana Biotech Co., Ltd. and allowed to acclimate to the living environment for one week. Subsequently, the hair on the backs of the mice was first removed using a depilator, and then completely removed using a depilatory cream (Nikurin Cream (80% thioglycolac estide, ILDONG Pharmaceuticals)). The mice were then kept for 24 hours to check for wounds on their backs.

[0067] Subsequently, in order to verify the preventive and therapeutic effects of the compounds according to the present invention on psoriasis, five experimental groups were set up as shown in Figure 7 and Table 5. Mice in the second to fifth groups were treated with Aldara® cream, 80 mg (imiquimod, 4 mg) once daily for two weeks (14 days, a total of 14 applications) to induce psoriasis. Simultaneously, to compare the preventive and therapeutic effects of the compounds according to the present invention (compounds 69 and 83), in the groups treated with the drugs, 40 mg / kg of the compound per mouse was completely dissolved in DMSO equivalent to 10% of the total dose, and finally, the ratio of DMSO:cremofor EL:PEG 400:distilled water was 1:1:4:4 (v / Add this to the Cremofort EL-PEG 400-distilled water mixture to make it v / v / v. The solution was diluted. Subsequently, 100 μL of the solution was administered intraperitoneally to each mouse twice a day at 12-hour intervals. In the vehicle administration group, 100 μL of the drug-free solution (DMSO: Cremofor EL: PEG 400: Distilled water = 1:1:4:4 (v / v / v / v)) was administered intraperitoneally twice a day at 12-hour intervals.

[0068] [Table 5]

[0069] Fourteen days after the experiment, the degree of erythema, thickness, and scaling of the skin on the back of mice belonging to each group was observed, and histopathological analysis of the relevant areas was performed by H&E staining. As can be seen in Figure 8, the group in which psoriasis was induced alone (Psoriasis, Non-injection) and the group in which psoriasis was induced and the injection solvent was injected into the peritoneal cavity simultaneously (Psoriasis, Vehicle) showed severe erythema, thickness, and scaling of the skin on the back. However, in the groups administered compound 69 or compound 83, skin lesions were mild or hardly observed.

[0070] As can be seen in Figure 9, in the group where psoriasis was induced alone (psoriasis, non-injection) and in the group where psoriasis was induced and the injection solvent was injected into the peritoneal cavity simultaneously (psoriasis, vehicle), not only were hyperkeratosis, parakeratosis, and irregular acanthosis severely present, but inflammatory cell infiltration was also severe. In contrast, in the groups administered compound 69 or compound 83, these phenomena were mild or hardly observed.

[0071] To confirm the effects of each compound on immune cells in vivo, the spleens of mice administered each compound were removed, cells were extracted, and then suspended in MACS buffer (2 mM EDTA, 0.5% FBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, Miltenyi Biotech). For flow cytometry, 5 × 10⁶ mice's spleen cells were placed in FACS tubes (5 mL round tubes, Falcon). 5 After dispensing the antibodies into individual tubes, to prevent nonspecific binding, each tube was treated with 1 μg / mL anti-mouse CD16 / CD32 (InVivoMAb) for 30 minutes, then centrifuged at 13,000 rpm for 5 minutes and resuspended in 100 μL of MACS buffer. Subsequently, the experiment proceeded as follows, depending on the type of immune cells to be identified.

[0072] For B cells, each FACS tube was stained with 1 μg / mL of FITC-conjugated anti-B220 antibody (eBioscience®) at 4°C for 30 minutes, and then washed with 2 mL of MACS buffer. Subsequently, each tube was centrifuged at 15,000 rpm for 5 minutes, the supernatant was discarded, and the settled cells were fixed using 200 μL of 4% paraformaldehyde aqueous solution.

[0073] For T cells, each FACS tube was stained at 4°C for 30 minutes with 1 μg / mL of anti-CD3 (Biolegend) conjugated with PerCP-Cy5.5, anti-CD8 (eBioscience®) conjugated with FITC, and anti-CD4 antibody (eBioscience®) conjugated with Cy7. Subsequently, each tube was washed with 2 mL of MACS buffer and centrifuged at 15,000 rpm for 5 minutes. The supernatant was discarded, and the settled cells were fixed using 200 μL of 4% paraformaldehyde aqueous solution.

[0074] For macrophages, each FACS tube was stained with 1 μg / mL of FITC-conjugated anti-CD11b antibody (eBioscience®) at 4°C for 30 minutes, and then washed with 2 mL of MACS buffer. Subsequently, each tube was centrifuged at 15,000 rpm for 5 minutes, the supernatant was discarded, and the settled cells were fixed using 200 μL of 4% paraformaldehyde aqueous solution.

[0075] Through the above process, when immunocells were stained and flow cytometry was performed, as can be seen in Figures 10a to 10c, in the group in which only psoriasis was induced by topical application of imiquimod (psoriasis, non-injection) and in the group in which imiquimod was applied and Vehicle was administered intraperitoneally (psoriasis, vehicle), CD11b-positive granulocytes rapidly increased in the spleen, and the relative proportion of B cells and T cells decreased. However, in the group in which compound 69 or compound 83 was administered intraperitoneally, the proportion of CD11b granulocytes decreased, and along with this, the proportion of B cells and T cells increased. Finally, it was confirmed that the proportion of CD11b-positive granulocytes, B cells, and T cells in the spleen became similar to that observed in normal mice when the active compound was administered, confirming that the drug exhibits a therapeutic effect.

[0076] Through the results described above, we verified that compound 69 or compound 83, which are compounds of the present invention, exhibit therapeutic efficacy against an animal model of psoriasis, a type of autoimmune disease, and in the process, we verified that they affect the action of immune cells.

Claims

1. Compounds of the following chemical formula 1, their stereoisomers, solvides, hydrates, crystalline forms, or pharmaceutically acceptable salts: 【Chemistry 1】 In the aforementioned chemical formula 1, X １ , X ２ , X ３ , X ４ and X ５ Of these, one or two atoms are nitrogen, and the remaining atoms are carbon. R １ 、 R ２ 、 R ３ 、 R ４ and R ５ are, independently of each other, hydrogen, halogen, a C1-C14 alkyl group, a C2-C14 alkenyl group, a C2-C14 alkynyl group, a C5-C14 aryl group, a C5-C14 arylalkyl group, a C3-C14 cycloalkyl group, a C3-C14 cycloalkylalkyl group, a C3-14 heteroaryl group, a C3-14 heteroarylalkyl group, a C3-14 heterocycloalkyl group, a C3-14 heterocycloalkylalkyl group, a C1-C14 alkoxy group, a C5-C14 aryloxy group, a C5-C14 heteroaryloxy group, -NO ２ 、 -NR ６ 、 -SO ２ R ６ 、 -SO ２ N(R ６ ) ２ 、 -CF ３ 、 -OH, a cyano group, a thiol group, triazine, triazole, tetrazole, thiazole, diazole, imidazole, alkylamine, cycloalkylamine, heterocycloamine and biotin, and are selected from among Y is -OH, thiol group, phenoxy, cyanide group, thiazole, diazole, imidazole, triazine, triazole, tetrazole, -CON(R) ６ ) ２ , -NR ６ COR ６ , -SO ２ N(R) ６ ) ２ , -NH ２ , -NO ２ , -COOR ６ , -R ７ COOR ６ , -R ７ CON(R ６ ) ２ , -CONHSR ６ , -CONHR ７ SR ６ ,-CONHSSR ６ , -CONHR ７ SSR ６ , -CONHR ７ SO ２ R ６ , -CONHOR ６ , -CONHR ７ OR ６ , -CON(R ７ ) ２ CON(R ６ ) ２ and -CON(R ７ ) ２ NR ７ COR ６ Selected from among, Here, R ６ and R ７ These are, independently of each other, hydrogen, halogen, thiol group, phenoxy, cyanide group, thiazole, diazole, imidazole, triazine, triazole, tetrazole, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C6-C12 aryl group, C6-C12 arylalkyl group, C3-C12 cycloalkyl group, C3-C12 cycloalkylalkyl group, C3-10 heteroaryl group, C3-10 heteroarylalkyl group, C3-10 heterocycloalkyl group, C3-10 heterocycloalkylalkyl group, C1-C6 alkoxy group, -CF ３ , substituted or unsubstituted by one or more of amines, C1-6 alkylamines and -CN, hydrogen, C1-C14 alkyl group, C2-C14 alkenyl group, C2-C14 alkynyl group, C6-C12 aryl group, C6-C12 arylalkyl group, C3-C12 cycloalkyl group, C3-C12 cycloalkylalkyl group, C3-10 heteroaryl group, C3-10 heteroarylalkyl group, C3-10 heterocycloalkyl group, C3-10 he Selected from telocycloalkylalkyl groups, alkylamines, cycloalkylamines, heterocycloamines, and biotin.

2. A composition comprising the compound of chemical formula 1 as described in claim 1, its stereoisomers, solvates, hydrates, crystalline forms, or pharmaceutically acceptable salts.

3. The composition according to claim 1, wherein the composition is a pharmaceutical composition for the prevention or treatment of an immune disease selected from autoimmune diseases, transplant rejection, allergic diseases, and inflammatory diseases.

4. The composition according to claim 2, wherein the composition is a food composition for preventing or improving an immune disease selected from autoimmune diseases, transplant rejection reactions, allergic diseases, and inflammatory diseases.

5. The composition according to claim 2, wherein the composition is a cosmetic composition for the prevention or improvement of an immune disease selected from autoimmune diseases, transplant rejection reactions, allergic diseases, and inflammatory diseases.

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