KR20150124880A - Novel compounds having treatment of immune diseases and use therof - Google Patents

Abstract

The present invention relates to a novel compound capable of effectively treating and preventing immune disease and to a use thereof. The novel compound of the present invention suppresses the production of inflammatory cytokines and increases activities of regulatory T cells having immune regulatory ability. In addition, the compound of the present invention controls over an excessive immune response by inhibiting the production of autoantibodies and differentiation of osteoclasts, thereby being able to be used for treating immune diseases induced by regulatory anomalies of various immune response such as autoimmune disease, inflammatory diseases, and transplant rejection.

Description

[0001] The present invention relates to novel compounds having an effect of treating immune diseases,

The present invention relates to novel compounds having excellent therapeutic and preventive effects on immune diseases and their use.

Immune disorders are diseases in which constituents of the mammalian immune system cause, mediate, or contribute to the pathology of mammals, particularly inflammatory disorders being one of the most important health problems in the world. Inflammation is generally a localized protective response of the body tissue to host infiltration by foreign substances or by harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial formulations; An immune response such as an allergic and autoimmune response, or a condition associated with oxidative stress.

Inflammation is characterized by pain, numbness, swelling, heat and ultimate loss of function in the affected area. These symptoms are the result of a series of complex interactions that take place between cells of the immune system. The reaction of cells results in a network of interaction groups of inflammatory mediators as a result: proteins (eg, cytokines, enzymes (eg proteases, peroxidases), major basic proteins, (E.g., VCAM), lipid mediators (e.g., eicosanoids, prostaglandins, leukotrienes, platelet activating factors (PAF)), reactive oxygen species such as hydroperoxides, superoxide anions O2-, ), Etc. However, many of these mediators of inflammation are also regulators of normal cellular activity, so that the deficiency of the inflammatory response leads to damage (i.e., infection) without control of the host, and thus partly due to chronic inflammation Inflammatory diseases mediated by the overproduction of several of the above-mentioned mediators.

Also, an autoimmune disease, one of the immune diseases, is characterized by the immune system attacking its own organ and causing a spontaneous reaction. These responses are due to the recognition of auto-antigen by T lymphocytes, which leads to immune responses in the body fluid (autoantigen production) and in the cellular phase (increased lymphocyte and macrophage cytotoxic activity). Examples of autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or aggravation of immune responses by antigens, such as allergies to asthma, drugs or food. These diseases are all limited and chronic diseases, and in some cases, they are fatal, and thus there is no effective treatment method for treating the diseases. Therefore, drugs, medicines or medicines that can alleviate or alleviate the disease during the course of the disease will be said to be an important solution for the health of the patient.

We have focused our efforts on finding suitable medicines and methods by exploring methods of treating autoimmune diseases. Today, the treatment of autoimmune diseases is mainly based on the use of immunosuppressive drugs such as glucocorticoids, calcineurin inhibitors and antiproliferatives-antimetabolites. However, such pharmacological therapies act on a variety of targets, and as a whole may impair immune function. Otherwise, long-term use of this pharmacotherapy can lead to a number of cytotoxic effects, which can lead to exposure of the patient to infection and cancer by inhibiting the immune system in a nonspecific manner. The use of calcineurin and glucocorticoids is limited in the case of some clinical symptoms (eg, renal insufficiency, diabetes, etc.), as they present another problem due to their nephrotoxicity and diabetes-inducing properties.

Therefore, there is a need to develop a therapeutic agent for a new immune disease which is capable of treating immune diseases such as autoimmune diseases and inflammatory diseases, and which has excellent therapeutic effect even without side effects.

Accordingly, the present inventors have found that a novel compound synthesized by the present invention can effectively treat an immune disease while searching for a material capable of effectively preventing or treating immune diseases while having fewer side effects on human body, and completed the present invention.

Korean Patent Publication No. 10-2013-0069737

It is therefore an object of the present invention to provide novel compounds.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating an immunological disease comprising the novel compound as an active ingredient.

Another object of the present invention is to provide an immunomodulator comprising the novel compound as an active ingredient.

Another object of the present invention is to provide a method for treating undifferentiated T cells to differentiate into Th17 cells and reduce the activity of Th17 cells by treating the undifferentiated T cells with the novel compounds of the present invention in vitro.

Another object of the present invention is to provide a method for treating undifferentiated T cells to differentiate into Treg cells and increase the activity of Treg cells by treating the undifferentiated T cells with the novel compounds of the present invention in vitro.

The present invention provides a novel compound, N-ethyl-4-fluorophenyl biguanide.

The present invention also provides a pharmaceutical composition for the prevention or treatment of immune diseases comprising the novel compound of the present invention as an active ingredient.

In one embodiment of the invention, the compound is capable of reducing or inhibiting the production of inflammatory cytokines, inhibiting autoantibody production, and inhibiting osteoclast differentiation.

In one embodiment of the present invention, the inflammatory cytokine may be IL-17, IL-6, TNF-a, IFN-r, MMP-9 or STAT-3.

In one embodiment of the invention, the antibody may be IgG, IgGl or IgG2a.

In one embodiment of the present invention, the compound may promote or increase the activity of regulatory T cells and decrease or inhibit the activity of Th17 cells, the pathological cells.

In one embodiment of the present invention, the compound may be contained in the composition at a concentration of 0.1 mM to 10 mM.

In one embodiment of the invention, the immune disease is autoimmune disease; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs.

In one embodiment of the invention, the immunological disorder is selected from the group consisting of rheumatoid arthritis, Behcet's disease, multiple myelitis or skin myositis, autoimmune hemocytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, , Autoimmune meningitis, Sjogren's syndrome, Lupus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis epididymitis, glomerulonephritis, Related syndromes and ulcerative colitis associated with acute myelogenous leukemia, including but not limited to acute myelogenous leukemia, Bile syndrome, Hashimoto's disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigoid, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthropathies, ≪ / RTI >

In one embodiment of the present invention, the graft rejection disease may be graft versus host disease.

The present invention also provides an immunomodulator comprising the novel compound of the present invention as an active ingredient.

The present invention also provides a method for treating undifferentiated T cells to differentiate into Th17 cells and reduce the activity of Th17 cells by treating the undifferentiated T cells with the novel compounds of the present invention in vitro.

The present invention also provides a method for treating undifferentiated T cells to differentiate into Treg cells and to increase the activity of Treg cells by treating the undifferentiated T cells with the novel compounds of the present invention in vitro.

The present invention relates to novel compounds capable of effectively preventing and treating immune diseases and their use. The novel compounds of the present invention inhibit the production of inflammatory cytokines, increase the activity of regulatory T cells having immunomodulatory functions, inhibit autoantibody production and control excessive immune responses, There is an effect that can be used for the treatment of immune diseases such as autoimmune diseases, inflammatory diseases and graft rejection diseases caused by abnormalities in regulation of various immune responses.

FIG. 1 shows cytotoxicity, autoantibody production, inflammatory cytokine production, and inflammatory gene expression level of SD-282 compounds treated with spleen cells in normal mice.
FIG. 2 is a graph showing the results of analysis of Th17 inhibition, Treg activity promotion, osteoclast differentiation inhibition and hyperactive Th17 inhibition by SD-282 compound treatment in normal mouse spleen cells.
FIG. 3 shows cytotoxicity, autoantibody production, inflammatory cytokine production, and inflammatory gene expression level of SD-282 compounds treated with splenocytes in mice in which rheumatoid arthritis was induced .
FIG. 4 shows the results of analysis of the inhibition of Th17, the promotion of Treg activity, and the inhibition of osteoclast differentiation by SD-282 compound treatment for each concentration in spleen cells of rheumatoid arthritis-induced mice.
FIG. 5 shows cytotoxicity, autoantibody production, inflammatory cytokine production, and inflammatory gene expression levels of SD-282 compounds treated with splenocytes in the lymphocyte-spleen mice.
FIG. 6 shows the results of analysis of the degree of Th17 inhibition and promoting Treg activity by SD-282 compound treatment for each concentration in spleen cells of the mice in which the disease was developed.
FIG. 7 shows the results of analysis of cytotoxicity and the degree of inflammatory cytokine production by SD-282 compound treatment for each concentration of lymphocytes isolated from human peripheral blood.

The present invention is characterized in that the following novel compounds can be used as new therapeutic agents for effectively preventing or treating immune diseases.

Therefore, the present invention can provide a novel compound represented by the following formula.

More preferably, the compound of the above formula is N-Ethyl-4-fluorophenyl biguanide.

The present inventors have conducted an experiment to confirm whether the synthesized novel compound can treat an immune disease. According to one embodiment of the present invention, the compound reduces or suppresses the production of inflammatory cytokines, produces autoantibodies IL-6, TNF-a, IFN-r, MMP-9 or IL-17, but not limited to, inflammatory cytokines. STAT-3. ≪ / RTI >

In the present invention, it was also found that the compound is capable of regulating the production of IgG, IgG1 or IgG2a, which is an autoantibody, to suppress excessive immune response.

Furthermore, the compounds of the present invention can activate or increase the activity of regulatory T cells and reduce or inhibit the activity of Th17 cells, which are pathogenic cells.

Therefore, the inventors of the present invention have confirmed the possibility that the above-synthesized compound of the present invention as a novel therapeutic agent for effectively treating an immunological disease.

On the other hand, there are T cells as one of the cell groups which plays a central role in the immune system as a bio-defense system for various pathogens. T cells are produced in the thymus of the human body, and undergo a series of differentiation processes, resulting in differentiation into T cells with inherent characteristics. The differentiated T cells are largely classified into type 1 (Th1) Auxiliary cells (Th2). Among these, Th1 cells are mainly involved in cell mediated immunity, Th2 cells are involved in humoral immunity, and in the immune system, these two cell populations maintain the balance of the immune system through mutual checks to prevent mutual activation with each other.

Therefore, most of the immune diseases are caused by the imbalance between these two immune cells. For example, when the activity of Th1 cells abnormally increases, an autoimmune disease may occur and the activity of Th2 cells abnormally increases It is known that immune diseases are caused by hypersensitivity reactions.

On the other hand, recent studies on the differentiation of Th1 cells have revealed the existence of a new group of immunoregulatory T cells (Tregs) capable of regulating the activity of Th1 cells, , Treg cells have the property of suppressing the function of abnormally activated immune cells and controlling the inflammatory reaction, and there have been reported many experiments for treating immune diseases through the action of increasing the activity of Treg cells.

In addition, Th17 cells are known to be formed through a process similar to the differentiation of Treg cells in the differentiation process of undifferentiated T cells. That is, the differentiation of Treg cells and Th17 cells is commonly performed in the presence of TGF-β, whereas IL-6 is not required for Treg cells, while IL-6 is present along with TGF-β for Th17 cells Differentiate in situations. In addition, differentiated Th17 cells are characterized by secretion of IL-17.

Th17 cells, unlike Treg cells, are involved in the forefront of the inflammatory response seen in immune diseases, thereby maximizing the signal of the inflammatory response and accelerating the progression of the disease. Therefore, in the case of autoimmune diseases that are not controlled by Treg cells among autoimmune diseases, the development of therapeutic agents for autoimmune diseases targeting the inhibition of Th17 cell activation has been greatly emphasized.

However, immunosuppressants that block signal transduction pathways in T cells are the most commonly used therapeutic agents for immune diseases. These immunosuppressants are toxic, infectious, lymphoid, diabetes, tremor, headache, diarrhea, hypertension, Nausea, and renal dysfunction.

In addition to the method of treating immune diseases through the method of inhibiting the activation of T cells, a method of regulating the amount of cytokine secreted from the immune cells and a method of using the antibody targeting the cytokine secreted from the immune cells have been developed . However, this method has a problem in that it takes much time to actually apply to patients through clinical experiments, and that the method using antibody is too expensive in the process of antibody production.

 In this regard, the novel compounds provided by the present invention are capable of simultaneously inhibiting the production of inflammatory cytokines and inhibiting Th17 cells and activating Treg cells, so that they can treat immune diseases more effectively than conventional therapeutic agents .

In addition, the results of one embodiment of the present invention show that the compound of the present invention has an activity of suppressing STAT3 gene expression. In recent years, active forms of STAT1, STAT3 and STAT5 have been found in various cancer species, STAT3 is an important cancer target because it is activated not only in blood cancer such as leukemia but also in various solid tumors such as breast cancer, head and neck cancer, melanoma, ovarian cancer, lung cancer, pancreatic cancer and prostate cancer (Hua Yu and Richard Jove, Nature Review Cancer ., 2004, 8, 945).

In addition, the activity of STAT3 has been known to inhibit apoptosis, induce angiogenesis, and induce immune evasion (Wang T. et al., Nature Medicine ., 2004, 10, 48). Therefore, inhibition of STAT3 activity has an effect of controlling the tumor by a complex anticancer mechanism. STAT3 protein is involved not only in tumor but also various cellular functions, so that its inhibitor can be developed as an immunosuppressant.

In addition, for reference, the immune system controls a specific immune response to an autoantigen in a normal state, and also suppresses an immune response to an external antigen. For example, in response to a fetus of a pregnant woman and a microorganism in a chronic infection state Immune response against the immune response. These phenomena are known to be induced by clonal deletion, clonal anergy, and active control by immunoregulatory T cells (Treg) as a mechanism by which antigen-specific immune tolerance can be induced. Some of the patients who have been successfully obtained immunostimulants for the transplantation antigen or those who have experimentally induced immune tolerance have been confirmed to be involved in the transplantation immunity tolerance in all three mechanisms. In particular, recently, immunocontrol T lymphocytes Has been attracting attention as an important cell involved in controlling virtually all immune responses in living bodies such as autoimmune, tumor immunity, and infectious immune response as well as transplantation immune response.

Immunoregulatory T lymphocytes (Tregs), which have recently been identified, can be divided into natural Treg and adaptive Treg cells. CD4 + CD25 + T cells, which are natural Tregs, , And is present in a frequency of 5-10% of the peripheral CD4 + T lymphocytes of normal individuals. Although the immunosuppressive mechanism of this cell has not yet been elucidated yet, it has recently been shown that the expression control gene of Foxp3 plays an important role in the differentiation and activity of this cell. In addition, peripheral T cells can be differentiated into cells exhibiting an immunosuppressive effect upon being stimulated by a self or external antigen under a specific environment, which is called an adaptive or inducible Treg and secretes IL-10 Tr1 that secretes TGF-beta, Th3 and CD8 Ts that secrete TGF-beta, and the like.

In addition, T cells are differentiated into Th17 cells through differentiation of Treg cells. Th17 cells are commonly found in the presence of TGF-β in the presence of Treg cells, whereas Treg cells do not require IL-6, In the case of Th17 cells, it is differentiated in the presence of TGF-beta and IL-6, and secretes IL-17.

In addition, Th17 cells have cytotoxic properties that maximize signal of inflammatory response and accelerate disease progression. Therefore, inhibition of differentiation or activity into Th17 cells is one of the methods for treating immune diseases.

In addition, Treg cells express Foxp3, Foxp3 is predominantly in immunoregulatory T cells derived from thymus, and is a transcriptional factor present in cells with CD4 + CD25 + , Which functions as a potent autoimmune disorder among CD4 + CD25- T cells that do not express Foxp3, which has low reactivity to antigen upon recognition of T cells expressing Foxp3 and differentiation from thymus T cells, and suppressor T cells (suppressor T cells) that inhibit IL-2 production and cell division. In addition, Foxp3 stimulates not only IL-2 but also IL-4, which is influenced by NFAT, which is a transcription factor, in CD25- T cells through regulatory T cells expressing Foxp3 and cell-cell contact with them, Lt; RTI ID = 0.0 > IFN-gamma. ≪ / RTI > Therefore, in the case of T cells expressing Foxp3, which has the above-mentioned action, it is applied to the treatment of immune diseases through the action of suppressing or modulating the immune response, and it is also applied to the treatment of CD4 T cells expressing Foxp3 To increase the number of autoantigen-specific T cells (self-antigen specific T cell clone) by treatment with high concentration of IL-2 cytokine and combination treatment with anti-CD3 and anti-CD28 antibody, There have been attempts to.

Therefore, in view of the state of the art development related to the conventional immunological disease treatment, the novel compound provided by the present invention can solve the problems that have not been solved in the past and exhibit more effective pharmacological effects, and thus can be very useful as a therapeutic agent for immune diseases.

Therefore, the novel compound contained in the pharmaceutical composition for preventing or treating the immunological disease provided by the present invention can be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

The compounds according to the present invention can be isolated from nature or prepared by chemical synthesis methods known in the art. The present inventors synthesized these compounds by the method described in the following examples.

In the present invention, the term "immune disease" means diseases in which constituents of the mammalian immune system cause, mediate, or contribute to a pathological condition of a mammal. In addition, the stimulation or discontinuation of the immune response may include all diseases that have a compensatory effect on the progress of the disease, and the present invention may include diseases caused by an irritable immune response. Examples of such immune disorders include, but are not limited to, autoimmune diseases; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs, and the like.

In addition, one of the most important properties of all normal individuals is that they do not react negatively to the antigenic material that constitutes the self, while non-self antigens are recognized and reacted to remove I have the ability. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance.

However, when problems arise in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs, thereby attacking the tissue of the self. The disease caused by this process is called "autoimmune disease" .

The term "inflammatory disease" refers to a disease caused by excessive stimulation of the human immune system by inflammatory factors such as an inflammatory inducer or radiation, resulting in the release of TNF-α, IL-1 refers to a disease caused by an inflammatory agent (inflammatory cytokine) such as interleukin-1, IL-6, prostagladin, luecotriene or nitric oxide (NO).

Successful organ transplants also need to overcome the recipient's immune rejection response to cells and organs to be transplanted. The main mediator of transplantation immune rejection is T cell, which is expressed in the graft. Major histocompatibility complex (MHC) is recognized by T cell receptor (T cell receptor) A reaction occurs. The main histocompatibility molecule is determined by the type of glycoprotein. The immune response caused by the inconsistency of the histocompatibility antigens is an obstacle to successful transplantation. Therefore, it is very important to investigate the accuracy of the histocompatibility antigen test.

There are several types of histocompatibility antigens in humans, including Class I antigens that include HLA-A, -B, and -C, and Class II antigens that include HLA-DR, -DP, and -DQ. The biological function of these antigens is to transmit antigens to T lymphocytes. Class I antigens are expressed in most nucleated cells, and the antigens transferred through them are recognized by CD8 + cytotoxic T lymphocytes. Class II antigens are expressed in dendritic cells known as antigen presenting cells, B lymphocytes, activated T lymphocytes, and large predominant cells, and function to transfer antigen to CD4 + T lymphocytes. The T lymphocytes recognize the antigen by binding to the T lymphocyte receptor, and the transplantation recognizes the tissue-compatible antigen from other people as a high frequency. About 1% to 10% of the total T lymphocytes of the donor or patient recognize a tissue-compatible antigen derived from the patient or donor and multiply in response to this, resulting in a series of immune responses called "alloresponse" do. In addition, the donor T lymphocyte responds to the tissue-specific antigen (GVDH) of the patient, and the response of the patient's T lymphocytes to donor tissue-positive antigen is called graft rejection rejection ".

Immunosuppressive agents have been used to reduce abnormal responses due to immune reactions during transplantation. The common goal of these immunosuppressive agents is to inhibit T cell-mediated immune responses to grafts. Recently, Methods have been attempted to treat graft rejection by inhibiting the immune response.

In addition, the immunological diseases that can be prevented and treated in the present invention include, but are not limited to, rheumatoid arthritis, Behcet's disease, multiple myositis or skin muscularitis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, asthma, Aplastic anemia, autoimmune hepatitis, autoimmune mumps, Crohn ' s disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis , Pyrethroiditis, glomerulonephritis, Graves' disease, Guilin Barre syndrome, Hashimoto disease, hemolytic anemia, multiple sclerosis, myasthenia gravis, pemphigus pneumoniae, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, Malignant anemia, mitochondrial related syndrome, and ulcerative colitis.

Therefore, the composition according to the present invention can be used as a pharmaceutical composition capable of preventing or treating immune diseases.

Refers to reversing, alleviating, inhibiting, or preventing the disease or disorder to which the term applies, or one or more symptoms of the disease or disorder, unless stated otherwise , And the term treatment as used herein refers to the act of treating when "treating" is defined as above. Thus, "treatment" or "treatment regimen" of an immune disorder in a mammal may include one or more of the following:

(1) inhibiting the growth of the immune system, i.e., inhibiting its development,

(2) preventing the spread of immune diseases, i.e., preventing metastasis,

(3) Reducing immune diseases.

(4) preventing the recurrence of immune disorders, and

(5) palliating symptoms of immune disorders

The composition for the prevention or treatment of an immune disorder according to the present invention may comprise a pharmaceutically effective amount of the novel compound of the present invention or a salt thereof alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents . A pharmaceutically effective amount as used herein refers to an amount sufficient to prevent, ameliorate, and treat symptoms of an immune disease.

The pharmaceutically effective amount of the novel compound or a salt thereof according to the present invention may be appropriately changed depending on the degree of symptoms of the immune disease, the age, body weight, health condition, sex, administration route and treatment period of the patient.

Also, the pharmaceutically acceptable hereinabove refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, the compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders.

In addition, the composition for preventing or treating immunological diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may be appropriately determined depending on the route of administration, The composition of the present invention can be appropriately selected according to various factors such as body weight and the severity of the patient and the composition for preventing or treating immune diseases according to the present invention can be used in combination with known compounds having the effect of preventing, Lt; / RTI >

The present invention also provides the use of a composition comprising said compound as an active ingredient for the manufacture of a medicament for the prevention or treatment of immune diseases. The composition of the present invention comprising the compound of the present invention as an active ingredient can be used for the preparation of a medicament for the prevention or treatment of immune diseases.

The present invention also provides methods of preventing or treating immune disorders comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition of the invention.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like.

Further, the present invention can provide an immunomodulator comprising the compound of the present invention as an active ingredient.

The present invention also provides a method for reducing the activity of Th17 cells in a cell by treating the compound of the present invention with a compound of the present invention in vitro, A method of increasing the activity of Treg cells can also be provided.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  1>

Synthesis of novel compounds having the therapeutic effect of the immunological diseases according to the present invention

The present inventors synthesized a new novel compound having the following structural formula as a candidate substance of a new therapeutic agent for the treatment of immune diseases by the following method. First, aniline compound (1.0 mmol) was dissolved in acetonitrile solvent. Dicyanodiamide (1.0 mmol) and concentrated sulfuric acid (1.0 mmol) were added to the sealed reactor and sealed. The mixture was stirred at 175 ° C for 1 hour. After cooling to room temperature, a white solid was formed, the solvent was removed, and the solid was washed with hexane and isopropyl alcohol to synthesize the following compound (SD-282) in the form of a white solid.

The synthesized compounds were identified by ¹ H-NMR analysis and the results are as follows.

¹ H-NMR analysis result of the SD-282 compound ^{1 H-NMR (600MHz, DMSO} ) δ (ppm): 7.36-7.34 (m, 2H), 7.31-7.28 (m, 2H) 7.105 (s, 2H) 6.89 (s, 4H) 3.69 (t, 2H, J = 7.2Hz), 1.07 (t, 3H, J = 7.2Hz). MS (m / z): [M + H] ⁺ 224. Other compounds, SD-281, SD-283 and SD-284, were also synthesized by methods similar to those described above.

<Experimental Method>

&Lt; 1 >

The present inventors conducted the following experiments based on the following experimental groups to confirm whether the novel compounds of the present invention newly synthesized herein can treat and prevent immune diseases.

Experiments performed in the experiments of the present invention Experimental group Subject cell Normal mouse group The spleen of normal mouse DBA1J mice, BM cells Rheumatoid arthritis mouse group The spleen of DBA1J mice induced with rheumatoid arthritis by CIA, BM cells Lupus mouse group Sanroque with a lupus Mouse spleen, BM cells Human P.B Lymphocytes isolated from human peripheral blood (P.B)

More specifically, in the normal mouse group, spleen cells obtained from normal DBA / 1J mice were cultured for 72 h with anti-CD3 0.5 ug / ml stimulation before treating the compound of the present invention, Respectively.

In addition, rheumatoid arthritis mice were prepared by using normal DBA / 1J mice. The normal mice were dissolved in 0.1N acetic acid solution so as to have type 2 collagen (ClI) of 4 mg / ml, and dialyzed buffer (50 mM Tris , 0.2 N NaCl) and mixed with the same amount of complete Freund's adjuvant (CFA, Chondrex) containing M. tuberculosis. Subcutaneously injected into the base of the tail of the mouse, 100 μl / 100 [micro] g) (primary injection). Two weeks later, the same CII was mixed with the same amount of incomplete Freud's adjuvant (IFA, Chondrex) and then 100 μl (ie 100 μl / 100 μg) was injected secondarily on one hind paw (foot pad) to induce rheumatoid arthritis , And splenocytes were obtained from the prepared arthritic mice and used for stimulation with anti-CD3 at a concentration of 0.5 ug / ml for 72 h to activate the cells.

Also, a model of a mouse with an onset of lupus was a sanroque mouse used in the art as a lupus model, and a human PB group was used by obtaining lymphocytes from human peripheral blood. Separation of the splenocytes from the respective mouse and human peripheral blood and separation of lymphocytes was performed using a general method well known in the art. All of the cells obtained were incubated for 72 h with anti-CD3 stimulation at 0.5 ug / ml, Activated and used.

<2> Cytotoxicity analysis (MTT analysis)

MTT assay was performed to determine whether the compound synthesized in the present invention caused toxicity to the cells. Each cell was subjected to MTT assay in a 96-well plate at a density of 2 × 10 ⁵ cells / well The compounds of the present invention were treated for each concentration and incubated for 72 hours. MTT solution (0.5% 3-4,5-dimethyl thiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide After incubation for 4 hours, absorbance was measured at 540 nm using an enzyme-linked immunospecific assay (ELISA), and cytotoxicity was observed.

<3> Autoantibody production immunoreactivity control assay

Enzyme Linked Immunosorbent Assay (ELISA) was performed to determine whether the novel compounds of the present invention affect the production of total IgG and total IgG1 and IgG2a antibodies in serum. Therefore, total IgG and antibody specific IgG1 and IgG2a were measured using sandwich ELISA for the cells treated with the compound of the present invention. After reacting each monoclonal anti-mouse IgG and CII in 96 well plate at room temperature for 1 hour, non-specific binding was blocked with blocking solution (1% BSA / PBST). Mouse contorl serum was serially diluted to 1/2 standard, and the cell culture supernatant was added and reacted at room temperature for 1 hour. After incubation for 1 hour at room temperature with anti-mouse IgG-HRP and anti-mouse IgG2a-HRP, the cells were washed four times and then developed with TMB system and absorbance was measured at 450 nm.

&Lt; 4 > Effect on Inflammatory Cytokine Production

The present inventors examined IL-17, IL-6, TNF-a, IFN, and IL-6 in order to confirm whether the novel compounds of the present invention could affect the production of inflammatory cytokines which are causative agents of inflammation and immune diseases. -r, MMP-9, and STAT-3 and the degree of expression of mRNA of these cytokines were analyzed. The degree of inflammatory cytokine production was determined by obtaining the supernatant from the cells cultured and treated with the compound of the present invention (Anti-IL-17, anti-IL-6, anti-TNF-a, and anti-IFN-γ) (1% BSA / PBST) to block nonspecific binding after overnight incubation with anti-MMP-9, anti-STAT-3. Each of the above biotinylated antibodies was reacted at room temperature for 2 hours and then washed 4 times. Then, the ExtraAvidin-Alkaline Phosphatase conjugate was diluted and reacted at room temperature for 2 hours. After that, the solution was developed with PNPP / DEA solution and absorbance was measured at a wavelength of 405 nm.

The expression level of inflammatory cytokines was analyzed by RT-PCR using primers specifically binding to IL-17, IL-6, TNF-a and IFN-r, Was performed to analyze the amount of mRNA.

<5> Th17 cell inhibition and Treg cell induction activity assay

Further, the inventors of the present invention conducted experiments using flow cytometry to determine whether the novel compounds of the present invention can inhibit the differentiation and activity of Th17 cells associated with inflammation induction and simultaneously promote the activity of Treg cells having immunoregulatory ability Respectively. In other words, the cells were cultured under conditions of differentiation of T 17 cells or T reg cells, and the number of cells of Foxp3 + T reg cells or il-17 + Th 17 cells was analyzed by flow cytometry.

&Lt; 6 > Inhibitory effect on osteoclast differentiation

To investigate the effect of the novel compounds of the present invention in osteoclast differentiation, mouse bone marrow cells were obtained and bone marrow cells were induced to differentiate in the presence of MCSF (macrophage colony-stimulating factor) and soluble RANKL (Sugatani et al. 2003, J. Cell. Biochem. 90, 59-67). Bone marrow cells were prepared from the femur and tibia of 6-week-old mice and cultured in M-CSF (30 ng / ml: R & D Systems, USA) in an 8-well chamber slide (3 × 10 ⁵ cells / well; Nalge Nunc International, Naperville, IL) (30 ng / ml) and RANKL (30 ng / ml; Strathmann, Minneapolis, Minn.). After 3 days, nonadherent cells including lymphocytes were removed, Hamburg, Germany) for an additional 4 days to obtain osteoclasts. At this time, the cell culture medium was changed once while M-CSF and RANKL were added. Cells were fixed and TRAP (tartrate-resistant acid phosphatase) was stained using a staining kit (sigma) according to the manufacturer's protocol. Microscopic examination of each chamber (40 magnification) showed that TRAP-positive polynuclear cells containing at least three nuclei were counted as osteoclasts (Sugatani et al., 2003, J. Cell. Biochem., 90, 59-67) .

In addition, the bone marrow cells were differentiated into osteoclasts, treated with the new compounds of the present invention at various concentrations in the presence of M-CSF and RANKL, and then cultured for 48 hours. Then, the cells were stained for TRAP and stained with TRAP- Respectively.

<Experimental Example 1>

The cytotoxicity analysis results of the novel compounds according to the present invention

MTT analysis was performed on the cells of each experimental group described in Table 2 to confirm whether the new compounds synthesized in Example 1 were cytotoxic. That is, the cells of each test group were divided into 2 × 10 ⁵ cells per well, and each of the four compounds was treated at each concentration and cell viability was analyzed.

As a result, it was found that the group treated with the novel compound of the present invention, SD-282, did not show cytotoxicity due to treatment at the cell concentration as compared with the control group (the group treated with nothing) or the group treated with metformin, Were not cytotoxic to normal cells and disease group cells.

< Experimental Example  2>

The effect of the novel compounds according to the present invention on the autoimmune response modulating immune response

In order to investigate whether the novel compounds synthesized in Example 1 had an effect on the immune response by autoantibody production, blood was obtained from each mouse group by orbital blood collection from the above-described four groups of mice, The amounts of IgG, IgG1, and IgG2a were measured.

The analysis showed that the amount of immunoglobulin (total IgG, IgG1, IgG2) in which the compounds of the present invention were autologous antibody producing conditions in the disease condition was decreased by the treatment concentration of the compound. Therefore, it was found that the novel compound of the present invention has an immunoregulatory function for suppressing an excessive immune response (see Table 3 below).

Total IgG IgG1 IgG2 A B C A B C A B C Control group 11 83.2 141.5 311.4 286 422.3 103.2 109.6 151.9 SD-281 2.3 58.1 124.5 83.8 135.2 258.2 35.6 33.5 84.3 SD-282 0.7 60.3 127.7 One 218.7 425.4 37.1 38.3 110.9 SD-283 2.9 49.6 113.5 41 135.3 296.5 97.6 99.4 76

Unit: ng / ml

A: normal mouse cells, B: rheumatoid arthritis cells, C: lupus cells

< Experimental Example  3>

Inflammatory cytokine production and inhibition of gene expression

The effect of the novel compounds of the present invention on the production of inflammatory cytokines described in Experimental Method < 4 > in order to confirm the inhibition of the production of inflammatory cytokines inducing inflammation and immune diseases and further suppressing these factors at the gene level Experiments were performed as described in the analysis.

As a result of the analysis, all of the novel compounds of the present invention were found to be capable of producing IL-17, IL-6, TNF-a, IFN-r, MMP-9 and STAT- Respectively. Thus, these results show that the novel compounds can prevent and treat immune diseases through inhibition of the production of inflammatory cytokines (see Table 4 below).

Effects of four compounds on the production of inflammatory cytokines IL-17 IL-6 TNF-a IFN-r A B C D A B C D A B C D A B C D Control group 3.2 3.7 0.9 0.3 2.2 1.9 1.8 0.9 0.1 1.3 9.4 8.3 14.5 0.2 SD-281 1.7 2.2 0.6 0.1 1.4 1.4 One 0.5 0.4 0.7 5.8 5.3 10 0.1 SD-282 1.7 2.1 0.6 0.1 1.3 1.3 One 0.4 0.4 0.7 3.3 2.9 7.6 0.1 SD-283 1.6 2.2 0.4 0.1 1.4 1.1 0.9 0.5 0.3 0.6 7.2 5.4 5.5 0.1 SD-284 0.1 0.08

Unit: ng / ml

A: normal mouse cell, B: rheumatoid arthritis cell, C: Lupus cell, D: lymphocyte of human peripheral blood

Effect of SD-284 Compounds on the Production of Inflammatory Cytokines in Normal Mouse Cells IL-17 IL-6 TNF-a IFN-r Control group 7.6 1.8 0.7 10 SD-284 6.5 0.7 0.5 4.9

Unit: ng / ml

Inhibition of Inflammatory Factor Activity of the Compounds of the Invention TNF-a MMP-9 STAT3 A B C A B C A Control group One One One One One One One SD-281 0.2 0.6 0.1 0.5 0.4 0.4 1.2 SD-282 0.07 0.06 0.8 0.1 0.08 1.3 0.6 SD-283 0.4 0.06 0.1 0.3 One 0.04 1.3

Unit: gene expression

A: normal mouse cells, B: rheumatoid arthritis cells, C: lupus cells

<Experimental Example 4>

The control activity of Th17 and Treg cells was analyzed

In order to investigate whether the Th17 cells secreting IL-17, which is an inflammatory cytokine, and the activity of differentiation and activity of Treg cells having immunoregulatory ability can be controlled simultaneously, And inhibition of Th17 cells and assay of Treg cell induction activity were performed.

As a result of the analysis, the novel compounds of the present invention were found to have an activity of reducing IL-17 expression in diseased cells and suppressing the differentiation into Th17 cells, which are disease cells, under the condition of Th17 differentiation, and at the same time, Foxp3 and Foxp3-expressing cells. In addition, it was found that Th17 cells effectively inhibited the activation of Th17 cells.

Th17 inhibition and Treg activity assay of compounds according to the present invention TCR condition Th17 Treg A B C A B C Control group 2 3.4 1.1 2.6 3.3 2.8 SD-281 1.2 1.5 0.8 2 4.6 2.8 SD-282 1.2 1.5 0.9 2.5 4.6 2.9 SD-283 1.1 2.3 0.5 3 5.1 3.3

A: normal mouse cell, B: rheumatoid arthritis cell, C: Lupus cell, D: lymphocyte of human peripheral blood

The effect of the compound according to the present invention on Th17 under Th17 differentiation conditions Th17 condition Th17 A Control group 5.7 SD-281 0.4 SD-282 0.5 SD-283 0.9 SD-284 2

Accordingly, the present inventors have found that the novel compounds of the present invention can regulate Th17 and Treg independently, but not simultaneously, and thus can induce immunomodulatory activity more effectively, thereby providing a more effective immunomodulator or immunogen It was found that the novel compounds synthesized in the present invention as a therapeutic agent for diseases can be utilized.

<Experimental Example 5>

Inhibition of osteoclast differentiation

In order to confirm that the novel compounds of the present invention can effectively treat immune diseases, the inventors of the present invention have conducted studies on the inhibition of osteoclast differentiation by compounds of the present invention in M-CSF and RANKL stimulated cells by TRAP staining for osteoclast differentiation factor Respectively.

As a result of the analysis, it was shown that the novel compound of the present invention decreased the number of cells expressing osteoclast differentiation marker TRAP. These results indicate that the novel compounds of the present invention can be effectively used for the prevention and treatment of diseases caused by differentiation of osteoclasts by effectively reducing the ability of osteoclasts to induce joint destruction.

Inhibitory effect of compounds according to the present invention on osteoclast differentiation TRAP + cells A B Control group 310 246 SD-281 196 57 SD-282 157 89 SD-283 123 85

A: normal mouse cell, B: rheumatoid arthritis cell

The results of the above-described experiment are shown in the results of FIGS. 1 to 7 in addition to the results of the above-described table.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (13)

A novel compound represented by the formula: or a salt thereof;

. A pharmaceutical composition for preventing or treating an immunological disease comprising the compound of claim 1 as an active ingredient. 3. The method of claim 2,
Wherein said compound reduces or inhibits the production of inflammatory cytokines, inhibits autoantibody production, and inhibits osteoclast differentiation. The method of claim 3,
Wherein said inflammatory cytokine is IL-17, IL-6, TNF-a, IFN-r, MMP-9 or STAT-3. The method of claim 3,
Wherein said antibody is IgG, IgG1 or IgG2a. 3. The method of claim 2,
Wherein said compound promotes or increases the activity of regulatory T cells and reduces or inhibits the activity of Th17 cells that are pathogenic cells. 3. The method of claim 2,
Wherein the compound is contained in the composition at a concentration of 0.1 mM to 10 mM. 3. The method of claim 2,
The immunological disease is autoimmune disease; Inflammatory disease; And transplantation rejection diseases of cells, tissues or organs. &Lt; Desc / Clms Page number 19 &gt; 9. The method of claim 8,
The immunological diseases are selected from the group consisting of rheumatoid arthritis, Behcet's disease, multiple myelitis or skin myositis, autoimmune hemocytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatomyositis, Goodfyther's syndrome, autoimmune meningitis, , Addison's disease, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis, epididymitis, glomerulonephritis, Graves' disease, Hiramoto disease, hemolytic anemia, Wherein the disease is selected from the group consisting of multiple sclerosis, acute myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, sarcoidosis, scleroderma, spondyloarthropathy, thyroiditis, vasculitis, leukoplakia, mucinous anemia, mitochondrial related syndrome and ulcerative colitis A pharmaceutical composition for preventing or treating. 9. The method of claim 8,
Wherein the graft rejection disease is a graft versus host disease. An immunomodulator comprising the compound of claim 1 as an active ingredient. Treating the undifferentiated T cells with the compound of claim 1 in vitro to differentiate the undifferentiated T cells into Th17 cells and reduce the activity of Th17 cells. The method of treating the undifferentiated T cells with the compound of claim 1 in vitro to differentiate undifferentiated T cells into T reg cells and increase the activity of T reg cells.

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