KR102245670B1 - Novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound, or pharmaceutical composition comprising the same for preventing or treating inflammatory bowel disease and autoimmune diseases - Google Patents

Abstract

The present invention relates to a novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound, or a pharmaceutically acceptable salt thereof. Specifically, the present invention is a novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound having an effect of preventing or treating inflammatory bowel disease and autoimmune diseases, or pharmaceutically acceptable thereof It relates to possible salts.

Description

Novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound, or a pharmaceutical composition for the prevention or treatment of inflammatory bowel disease and autoimmune disease comprising the same {Novel 6-heteroarylamino-2,4 ,5-trimethylpyridin-3-ol compound, or pharmaceutical composition comprising the same for preventing or treating inflammatory bowel disease and autoimmune diseases}

The present invention relates to a novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound, or a pharmaceutically acceptable salt thereof. Specifically, the present invention is a novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound having an effect of preventing or treating inflammatory bowel disease and autoimmune diseases, or pharmaceutically acceptable thereof It relates to possible salts.

Inflammatory bowel disease (IBD) is clinically similar, but is classified into two diseases, ulcerative colitis and Crohn's disease, which are different in terms of histological findings, endoscopic and immunological aspects, and activation of inflammatory cells is an important etiology. It is known to be.

Persistent or inappropriate activation of the intestinal immune system plays an important role in the pathophysiology of chronic mucosal inflammation, in particular, by infiltration of neutrophils, macrophages, lymphocytes and mast cells, eventually leading to mucosal destruction and ulcers. Infiltrated and activated neutrophils are an important cause of reactive oxygen species and reactive nitrogen species, and these active species are cytotoxic substances that induce oxidative stress that degrades crosslinking proteins, lipids and nucleic acids, and causes epithelial dysfunction and damage. do.

In inflammatory diseases, various inflammatory cytokines are secreted from the mucous membrane of the intestine. TNF-α is high in the colon lumen and colon epithelial cells of patients with ulcerative colitis. According to a recent study, it is known that TNF-α plays an important role in the etiology of ulcerative colitis. Infliximab, an anti-TNF-α antibody, is known to be effective not only in the treatment of rheumatoid arthritis, but also in the treatment of previously untreated Crohn's disease. However, these treatments are expensive, and in some patients, side effects such as fluid reactions or infectious complications are caused.

IL-6 is one of the cytokines that induce inflammation, and is well known as one of the etiologies of inflammatory growth disease. After binding to the receptor, IL-6 activates JAK and subsequently induces phosphorylation of STAT3. Phosphorylated STAT3 acts as a transcription factor and expresses several types of target genes including TNF-α. As such, TNF-α and IL-6 form a close relationship with each other and are involved in inflammation amplification and tissue damage in inflammatory bowel disease.

In addition, it is known that IL-8 is significantly elevated in the mucous membrane of patients with inflammatory bowel disease and promotes capillary angiogenesis. The more severe the inflammation in the large intestine, the greater the expression of IL-8, and it was known that IL-8-specific antibodies reduce intestinal inflammation in an animal model using rodents. At this time, ^{changes in intracellular Ca 2+} act as an important factor in IL-8 induction.

Current inflammatory bowel disease treatments include 5-aminosalicylic acid (5-ASA) drugs that block the production of prostaglandins, such as sulfasalazine, or steroid immunosuppressants. I am using

Sulfasalazine is likely to cause side effects or adverse effects such as fullness, headache, rash, liver disease, leukopenia, agranulocytosis, and male infertility. In addition, it is unclear whether sulfasalazine has a sufficient inhibitory effect on recurrence in patients with incisions in the intestine or patients with remission.

Steroid-type immunosuppressants are adrenal cortical steroids, which are recognized for their short-term effects, but cannot improve long-term prognosis. In addition, in terms of side effects such as induced infectious diseases, secondary adrenal insufficiency, peptic ulcer, diabetes, mental disorders, steroidal nephropathy, etc., there is a limitation that should be used only in acute cases.

That is, since there is still no reliable oral therapy for inflammatory bowel disease, there is a demand for the development of an effective and low-cost oral therapeutic agent for such diseases.

On the other hand, T cells, one of the cell groups that play a central role in the immune system, which is a biological defense system against various pathogens, are generated in the thymus gland of the human body, and differentiate into T cells with unique characteristics through a series of differentiation processes. T cells that have completed differentiation are largely divided into CD8 T cells and CD4 T cells according to their functions. Among them, CD4 T cells are differentiated in detail according to environmental factors, and the most representative are Th1, Th2, Th17 and Treg cells.

Th1 cells are involved in cell-mediated immunity, Th2 cells are involved in humoral immunity, Th17 cells are involved in infection and inflammatory diseases, and Treg cells suppress immunity to maintain homeostasis. In the immune system, these cell populations maintain the balance of the immune system by checking each other so that they do not overactivate each other.

Most of the immune diseases can be seen to be due to the imbalance between these immune cells. For example, it is known that when the activity of Th1 cells and Th17 cells is abnormally increased, autoimmune diseases may occur, and when the activity of Th2 cells is abnormally increased, immune diseases caused by hypersensitivity reactions are known to occur. In addition, excessive immune suppression of Treg cells can lead to cancer.

Recently, according to the results of studies on autoimmune diseases caused by Th1 cells and Th17 cells, the mechanism of the function of immune-regulatory T cells (Treg) that can regulate the activity of these cells is known, and studies on the treatment of immune diseases using these cells have emerged. Has become. Treg cells have the property of controlling the inflammatory response by inhibiting the function of abnormally activated immune cells. Many experiments have been reported to treat immune diseases through the action of increasing the activity of Treg cells.

It has been found that Th1 cells and Th17 cells are involved in the forefront of the inflammatory response seen in immune diseases, 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 inhibition of Th1 and Th17 cell activity has been greatly highlighted.

Immunosuppressants that block signal transduction pathways in T cells are the most commonly used treatments for immune diseases currently being used.These immunosuppressants are toxicity, infection, lymphoma, diabetes, tremor, headache, diarrhea, high blood pressure, and nausea. Or there is a problem that side effects such as renal dysfunction occur.

In addition, in addition to the method of treating immune diseases through the method of inhibiting the activation of T cells, therapies that control the amount of cytokines secreted from immune cells, and therapies using antibodies targeting cytokines secreted from immune cells This is under development. However, such a method should take a lot of time to apply to patients through practical clinical trials. In addition, the method of using the antibody has a problem that too much cost is required in the process of producing the antibody.

Therefore, it is necessary to develop a new therapeutic agent for immune disease that has no side effects and is inexpensive and has excellent therapeutic effects.

Accordingly, the present inventors have a novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound and its pharmaceutically acceptable salts have excellent effects of preventing or treating inflammatory bowel disease and autoimmune diseases. By confirming the presence, the present invention was completed.

Specifically, the present invention provides a novel 6-heteroarylamino-2,4,5-trimethylpyridin-3-ol compound, or a pharmaceutically acceptable salt thereof. In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease or autoimmune disease comprising the compound as an active ingredient.

The present invention provides the following novel compound of Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

In the above formula,

Z is O or S,

R is hydrogen; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; Heteroarylalkyl; A fused ring group in which an aryl group is fused to a cycloalkyl group; A fused ring group in which a heteroaryl group is fused to a cycloalkyl group; Pyrrolidone substituted or unsubstituted with a benzyl group; Alkylbenzenesulfonate; Benzodioxol; Or 2,3-dihydro-1,4-benzodioxin,

The alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; And heteroarylalkyl is

Hydroxy; Boronate; halogen; Haloalkyl; Haloalkoxy; Haloaryl; Amino; Alkylamino; Dialkylamino; Acylamino; Arylcarbonylamino; Sulfonamino; Nitro; Cyano; Carbonyl; Acyl; Aminocarbonyl; Alkylaminocarbonyl; Dialkylaminocarbonyl; Carbohydrazide; Carboxyl; Alkoxycarbonyl; Arylsulfonyl; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Arylalkyl; Diarylalkyl; Arylalkoxy; Aryloxy unsubstituted or substituted with one or more groups selected from the group consisting of alkyl and halogen; Heterocyclic substituted or unsubstituted with alkyl; And unsubstituted or substituted with one or more groups selected from the group consisting of alkyl substituted or unsubstituted heteroaryl,

Where alkyl is C _1-30 alkyl,

Alkoxy is C _1-30 alkoxy,

Alkenyl is C _2-30 alkenyl,

Alkynyl is C _2-30 alkynyl,

Cycloalkyl is C _3-30 cycloalkyl,

Aryl is C _5-30 aryl,

Heterocyclic is a heterocyclic having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S,

Heteroaryl is a heteroaryl having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S.

The novel compound of Formula 1 or a pharmaceutically acceptable salt thereof of the present invention provides not only an excellent inflammatory bowel disease treatment effect, but also an autoimmune disease treatment effect.

In particular, the novel compound of Formula 1 or a pharmaceutically acceptable salt thereof of the present invention exhibits a far superior effect compared to 5-ASA, an active metabolite of sulfasalazine, which is a drug currently used for the treatment of inflammatory bowel disease in clinical practice. In addition, since it exhibits specific activity only on inflammatory cells and is only involved in inflammatory differentiation of T cells, it has the advantage of having less side effects due to less influence on other cells.

Fig. 1A shows the results of an experiment for inhibiting cytokine-induced Th1 cell differentiation by administration of a compound.
Figure 1B shows the results of the cytokine-induced Th17 cell differentiation inhibition test results by the administration of the compound.
Fig. 1C shows the results of an experiment for inhibiting differentiation of immunosuppressive cells (FoxP3+, Treg cells) by administration of a compound.
2A to 2C show the results of toxicity experiments on lymphocyte cells extracted from the spleen after administration of Compound 2-036, tofacitinib and triamcinolone by concentration.
Figure 3A shows the results of the behavior pattern experiment of mice in the control group (EAE) and compound 2-036 administration group after antigen injection.
3B and 3C show the results of confirming the number of immune cells in the spleen (FIG. 3B) and the central nerve (brain and spinal cord) (FIG. 3C) in the control group (EAE) and the compound 2-036 administration group after antigen injection.
3D is a cross-sectional view (top panel) stained with H&E after extracting the spinal cord from the control group (EAE) and the compound 2-036 administration group on the 21st day after antigen injection, cut to a thickness of 5-10 μm, and staining white matter with Luxol fast blue. It is a cross-sectional view (bottom panel).
3E is a quantification of the number of cells in the white matter on the cross section stained with H&E in FIG. 3D.
3F is a quantification of the degree of myelination on the section stained with Luxol fast blue in FIG. 3D.
3G is a flow cytometric analysis result showing the fraction of Th1 and Th17 cells among cells extracted from the brain and spinal cord of the control group (EAE) and compound 2-036 administration group after antigen injection.
3H is a quantification of the degree of Th1 and Th17 cells distributed in the brain and spinal cord of the control group (EAE) and compound 2-036 administration group after antigen injection.
Figure 4A shows the weight of the negative control group without DSS treatment, the DSS treatment group, and DSS and 2-036 treatment together, and then the body weight was measured every day.
Figure 4B is a photograph of measuring the colon length of a mouse 10 days after treatment with a negative control group without DSS treatment and DSS treatment, or treatment with DSS and 2-036 together, and quantification thereof.
Figure 4C is a quantification of the cell number as a percentage of CD4 T cells and CD8 T cells in the lymph node closest to the intestine.
4D is a quantification of the degree of Th1 and Th17 cells in the lymph node closest to the intestine.

The present invention relates to the following novel compound of Formula 1, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In the above formula,

Z is O or S,

R is hydrogen; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; Heteroarylalkyl; A fused ring group in which an aryl group is fused to a cycloalkyl group; A fused ring group in which a heteroaryl group is fused to a cycloalkyl group; Pyrrolidone substituted or unsubstituted with a benzyl group; Alkylbenzenesulfonate; Benzodioxol; Or 2,3-dihydro-1,4-benzodioxin,

The alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; And heteroarylalkyl is

Hydroxy; Boronate; halogen; Haloalkyl; Haloalkoxy; Haloaryl; Amino; Alkylamino; Dialkylamino; Acylamino; Arylcarbonylamino; Sulfonamide; Nitro; Cyano; Carbonyl; Acyl; Aminocarbonyl; Alkylaminocarbonyl; Dialkylaminocarbonyl; Hydrazide; Carboxyl; Alkoxycarbonyl; Arylsulfonyl; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Arylalkyl; Diarylalkyl; Arylalkoxy; Acetamide; Benzamide; Aryloxy unsubstituted or substituted with one or more groups selected from the group consisting of alkyl and halogen; Heterocyclic substituted or unsubstituted with alkyl; And unsubstituted or substituted with one or more groups selected from the group consisting of alkyl substituted or unsubstituted heteroaryl,

Where alkyl is C _1-30 alkyl,

Alkoxy is C _1-30 alkoxy,

Alkenyl is C _2-30 alkenyl,

Alkynyl is C _2-30 alkynyl,

Cycloalkyl is C _3-30 cycloalkyl,

Aryl is C _5-30 aryl,

Heterocyclic is a heterocyclic having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S,

Heteroaryl is a heteroaryl having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S.

In addition, the present invention relates to the following novel compound of Formula 1, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In the above formula,

Z is O or S,

R is hydrogen; Alkyl; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; Heteroarylalkyl; A fused ring group in which an aryl group is fused to a cycloalkyl group; A fused ring group in which a heteroaryl group is fused to a cycloalkyl group; Pyrrolidone substituted or unsubstituted with a benzyl group; Alkylbenzenesulfonate; Benzodioxol; Or 2,3-dihydro-1,4-benzodioxin,

The alkyl; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; And heteroarylalkyl is

Hydroxy; Boronate; halogen; Haloalkyl; Haloalkoxy; Haloaryl; Amino; Dialkylamino; Acylamino; Arylcarbonylamino; Sulfonamide; Nitro; Cyano; Hydrazide; Carboxyl; Alkoxycarbonyl; Arylsulfonyl; Alkyl; Alkoxy; Aryl; Diarylalkyl; Arylalkoxy; Acetamide; Benzamide; Aryloxy unsubstituted or substituted with one or more groups selected from the group consisting of alkyl and halogen; Heterocyclic substituted or unsubstituted with alkyl; And unsubstituted or substituted with one or more groups selected from the group consisting of alkyl substituted or unsubstituted heteroaryl,

Where alkyl is C _1-18 alkyl,

Alkoxy is C _1-18 alkoxy,

Alkenyl is C _2-18 alkenyl,

Alkynyl is C _2-18 alkynyl,

Cycloalkyl is C _3-18 cycloalkyl,

Aryl is C _5-18 aryl,

Heterocyclic is a heterocyclic having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S,

Heteroaryl is a heteroaryl having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S.

Preferably, the compound of the present invention may be a compound represented by Table 1 below, or a pharmaceutically acceptable salt thereof.

More preferably, Formulas 2-036, 2-037, 2-039, 2-041, 2-045, 2-046, 2-047, 2-048, 2-049, 2-053 disclosed in Table 1 , 2-056, 2-061, 3-002, 3-010, 3-036, 3-041, 3-046, 3-047, 3-048, 3-049, 3-053, 3-056, 3 It may be a compound represented by -061, 3-065, 3-073, 3-086, 3-100 or 3-123, or a pharmaceutically acceptable salt thereof.

Most preferably, it is a compound represented by Formula 2-036, or a pharmaceutically acceptable salt thereof.

In the present invention, "alkyl" refers to a linear or branched hydrocarbon group that is not substituted or substituted with one or more functional groups. Unless otherwise specified, the “alkyl” group preferably contains 1 to 30 carbon atoms. Preferably, the “alkyl” group may be methyl, ethyl, propyl, isopropyl, butyl, heptadecyl, but is not limited thereto.

"Alkoxy" refers to -O-alkyl as a functional group to which an alkyl group as defined above is bonded through an oxygen bridge, and is substituted or unsubstituted with one or more functional groups. It preferably contains 1 to 30 carbon atoms.

"Alkenyl" refers to a linear, branched or cyclic hydrocarbon group having one or more unsaturated carbon-carbon bonds, which is not substituted or substituted with one or more functional groups. Unless otherwise specified, it preferably contains 2 to 30 carbon atoms.

“Alkynyl” means a linear or branched hydrocarbon group having one or more carbon-carbon triple bonds, which is not substituted or substituted with one or more functional groups. Unless otherwise specified, it preferably contains 2 to 30 carbon atoms.

“Cycloalkyl” refers to a cyclic or polycyclic hydrocarbon group substituted or unsubstituted with one or more functional groups. Unless otherwise specified, it preferably contains 3 to 30 carbon atoms. Preferably, the “cycloalkyl” group may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or adamantane, but is not limited thereto.

“Aryl” means a monocyclic or polycyclic aromatic group having 6 to 30 ring carbons, which is not substituted or substituted with one or more functional groups. Preferably, the “aryl” group may be phenyl or naphthyl, but is not limited thereto.

“Heterocyclic” refers to a monocyclic or polycyclic non-aromatic group in which one or more ring carbons are substituted with a heteroatom selected from B, N, O or S, respectively, which is not substituted or substituted with one or more functional groups. Unless otherwise specified, it preferably contains 3 to 30 ring atoms. Preferably, the "heterocyclic" group is piperidinyl, piperazinyl, azetidine, pyrrolidine, 1,3,2-dioxaborolane, morpholine, It may be tetrahydrofuranyl or tetrahydropyranyl, but is not limited thereto.

“Heteroaryl” refers to a monocyclic or polycyclic aromatic group in which at least one ring carbon is replaced with a heteroatom selected from B, N, O or S, which is not substituted or substituted with one or more functional groups. Unless otherwise specified, it preferably contains 3 to 30 ring atoms. Preferably, the “heteroaryl” group is pyrrolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiazolyl, thiadiazolyl, furanyl, It may be thiophenyl, pyridinyl, pyrazinyl, pyrimidinyl, indole, indazolyl, quinolinyl, isoquinolinyl, benzofuran, benzothiazolyl, benzothiophenyl, pyrazolopyridinyl, but is not limited thereto. .

The fused ring group in which the aryl group is fused to a cycloalkyl group may be preferably tetrahydronaphthyl, but is not limited thereto.

The fused ring group in which the heteroaryl group is fused to a cycloalkyl group may be preferably tetrahydrocyclopentapyrazolyl, tetrahydroindazolyl, or tetrahydrobenzothiophenyl, but is not limited thereto.

"Haloalkyl" means a branched and straight chain saturated hydrocarbon group in which at least one carbon is substituted with halogen. Preferably, “haloalkyl” may be trifluoromethyl, but is not limited thereto.

The arylalkoxy may be preferably benzyloxy, and the aryloxy may preferably be phenoxy, but is not limited thereto.

In addition, the present invention relates to a pharmaceutical composition having an effect of preventing or treating inflammatory bowel disease, including Formula 1 or a pharmaceutically acceptable salt thereof.

In addition, the present invention relates to a pharmaceutical composition having an effect of preventing or treating an autoimmune disease comprising Formula 1 or a pharmaceutically acceptable salt thereof.

In addition, the present invention relates to a method for preventing or treating inflammatory bowel disease, comprising administering a compound of Formula 1 or a pharmaceutically acceptable salt thereof to a subject.

In addition, the present invention relates to a method for preventing or treating autoimmune diseases, comprising administering a compound of Formula 1 or a pharmaceutically acceptable salt thereof to a subject.

Autoimmune diseases include Behcet's disease, polymyositis, skin myositis, autoimmune hemocytopenia, autoimmune myocarditis, atopic dermatitis, asthma, primary cirrhosis, dermatitis, fibromyalgia, Goodfighter syndrome, autoimmune meningitis, Sjogren's syndrome, systemic Lupus erythematosus, Addison's disease, alopecia areata, ankylosing myelitis, autoimmune hepatitis, autoimmune mumps, insulin dependent diabetes, dystrophy bullous epidermis, epididymitis, glomerulonephritis, Graves' disease, Celiac disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, multiple sclerosis, myasthenia gravis, amyotrophic lateral sclerosis, vulgaris vulgaris, psoriasis, rheumatoid fever, rheumatoid arthritis, sarcoidosis, skin sclerosis, spondyloarthritis, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, antiphospholipid syndrome, solid form It may be late organ transplantation and chronic rejection, or graft versus host disease.

The inflammatory bowel disease may be selected from the group consisting of ulcerative colitis, Crohn's disease, enteric Behcet's disease, hemorrhagic rectal ulcer and pouchitis.

The pharmaceutically acceptable salt is an organic acid selected from the group consisting of oxalic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid and benzoic acid, or an acid part formed by an inorganic acid selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid and hydrobromic acid. It may be in the form of salt, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention may be prepared in an oral or parenteral formulation. Pharmaceutical preparations for oral or parenteral administration may be, for example, tablets, dispersible tablets, coated tablets, effervescent tablets, capsules, suspension powders, suspensions or suppositories, or unit dosage forms such as ampoules. These are prepared by known methods, for example by processes such as mixing, granulation, coating or freeze drying.

Pharmaceutical preparations for oral administration are prepared by combining the active ingredient with a solid carrier, granulating the obtained mixture if necessary, and processing the mixture or granules, if necessary, after the addition of suitable additives to obtain tablets or coated tablet cores. Can be.

Such suitable carriers may contain sugar (eg, lactose, sucrose, mannitol or sorbitol), cellulose, calcium phosphate (eg, tricalcium phosphate or calcium hydrogen phosphate), etc. as fillers; As a binder, starch (eg, corn, wheat, rice or potato starch), gelatin, tragacanth, methylcellulose, polyvinylpyrrolidone, and the like may be included; As a disintegrant, starch, polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be included.

The additive may include salicylic acid, talc, stearic acid, magnesium stearate, calcium stearate, polyethylene glycol, and the like as a lubricant.

In addition, the pharmaceutical preparations that can be administered orally may be hard gelatin capsules, and soft sealed capsules of gelatin and a plasticizer (eg, glycerol or sorbitol).

For parenteral administration, an aqueous solution of the active ingredient in a water-soluble form, for example a water-soluble salt form, is mainly suitable.

The dosage of the active ingredient can be determined according to various factors such as the activity of the active ingredient and the duration of action, the severity of the disease to be treated or its symptoms, the method of administration, the species, sex, age, weight and the individual condition of the warm-blooded animal. have.

The compound represented by Formula 1 of the present invention was prepared by the same method as in Schemes 1 to 3.

[Scheme 1]

In VIII and IX above,

R is hydrogen; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; Heteroarylalkyl; A fused ring group in which an aryl group is fused to a cycloalkyl group; A fused ring group in which a heteroaryl group is fused to a cycloalkyl group; Pyrrolidone substituted or unsubstituted with a benzyl group; Alkylbenzenesulfonate; Benzodioxol; Or 2,3-dihydro-1,4-benzodioxin,

The alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; And heteroarylalkyl is

Hydroxy; Boronate; halogen; Haloalkyl; Haloalkoxy; Haloaryl; Amino; Alkylamino; Dialkylamino; Acylamino; Arylcarbonylamino; Sulfonamide; Nitro; Cyano; Carbonyl; Acyl; Aminocarbonyl; Alkylaminocarbonyl; Dialkylaminocarbonyl; Hydrazide; Carboxyl; Alkoxycarbonyl; Arylsulfonyl; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Arylalkyl; Diarylalkyl; Arylalkoxy; Acetamide; Benzamide; Aryloxy unsubstituted or substituted with one or more groups selected from the group consisting of alkyl and halogen; Heterocyclic substituted or unsubstituted with alkyl; And unsubstituted or substituted with one or more groups selected from the group consisting of alkyl substituted or unsubstituted heteroaryl,

Where alkyl is C _1-30 alkyl,

Alkoxy is C _1-30 alkoxy,

Alkenyl is C _2-30 alkenyl,

Alkynyl is C _2-30 alkynyl,

Cycloalkyl is C _3-30 cycloalkyl,

Aryl is C _5-30 aryl,

Heterocyclic is a heterocyclic having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S,

Heteroaryl is a heteroaryl having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S.

[Scheme 2]

From the compound of Formula IX obtained in Scheme 1, a compound of Formula 2 was synthesized according to Scheme 2 below.

The compounds of the present invention prepared by the above method are shown in Table 2 below.

[Scheme 3]

From the compound of Formula IX obtained in Scheme 1, a compound of Formula 3 was synthesized according to Scheme 3.

The compounds of the present invention prepared by the above method are shown in Table 3 below.

[Example 1]

Synthesis of 4,5-bis(chloromethyl)-2-methylpyridin-3-ol hydrochloride (Formula II)

[4,5-bis(chloromethyl)-2-methylpyridin-3-ol hydrochloride]

_{SOCl 2} (30 mL) and DMF (0.2 mL) were added to pyridoxine hydrochloride (Chemical Formula I) (5 g), followed by reflux stirring at 80° C. for 3 hours. After the reaction mixture was cooled to room temperature, Et ₂ O was added, followed by stirring for 1 hour under ice cooling. The precipitated solid was filtered under reduced pressure, and the filtered solid _{was washed with Et 2} O and dried to obtain the title compound II (5.5 g) as a white solid.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 8.42 (s, 1H), 4.99 (s, 2H), 4.96 (s, 2H), 2.63 (s, 3H).

[Example 2]

Synthesis of 2,4,5-trimethylpyridin-3-ol (Chemical Formula III)

[2,4,5-trimethylpyridin-3-ol]

Zinc powder (8.08 g) was added in small portions to a suspension of compound II (10 g) in acetic acid (50 mL), followed by reflux stirring at 130° C. for 2 hours. The reaction mixture was cooled to room temperature, filtered under reduced pressure, and the pH of the filtrate was adjusted to 6 using 10 M NaOH solution. The filtrate was saturated with salt and extracted with EtOAc.

The EtOAc solution was washed with saturated salt water, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl ₃ :MeOH=20:1) to obtain the title compound III (5.2 g) as a white solid.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 8.49 (s, 1H), 7.72 (s, 1H), 2,31 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H).

[Example 3]

Synthesis of 6-bromo-2,4,5-trimethylpyridin-3-ol (Formula IV)

[6-bromo-2,4,5-trimethylpyridin-3-ol]

1,3-dibromo-5,5-dimethylhydantoin (DBDMH, 2.5 g) was added to a suspension of compound III (2.5 g) in THF (30 mL), followed by stirring at room temperature for 3 hours. After the reaction mixture was concentrated, the residue was diluted with EtOAc and water, and the aqueous layer was extracted with EtOAc. The EtOAc solution was washed with saturated salt water, dried over MgSO ₄ , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (EtOAc:Hex=1:4) to obtain the title compound IV (3.22 g) as a pale yellow solid.

¹ H-NMR (CDCl ₃ ) δ 5.56 (br s, 1H), 2.42 (s, 3H), 2.31 (s, 3H), 2.25 (s, 3H).

[Example 4]

2-bromo-5-((t-butyldiphenylsilyl)oxy)-3,4,6-trimethylpyridine (Formula V)

[2-bromo-5-((tert-butyldiphenylsilyl)oxy)-3,4,6-trimethylpyridine]

To a solution of compound IV (5.11 g) in DMF (34 mL), imidazole (3.54 g) and TBDPSCl (9.1 mL) were added, followed by stirring at room temperature for 16 hours. Water was added to the reaction mixture, followed by extraction with _{CH 2} Cl _2. The extract was washed sequentially with water and saturated salt water, _{dried over MgSO 4} , filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (1% EtOAc in hexanes) to obtain the title compound V (9.73 g) as a white solid.

¹ H-NMR (CDCl ₃ ) δ 7.67-7.62 (m, 4H), 7.44-7.31 (m, 6H), 2.21 (s, 3H), 2.16 (s, 3H), 1.98 (s, 3H), 1.10 ( s, 9H).

[Example 5]

N-(5-((t-butyldiphenylsilyl)oxy)-3,4,6-trimethylpyridin-2-yl)-1.1-diphenylmethanimine (Formula VI)

[N-(5-((tert-butyldiphenylsilyl)oxy)-3,4,6-trimethylpyridin-2-yl)-1,1-diphenylmethanimine]

_{Pd 2} (dba) ₃ (375 mg), BINAP (504 mg), NaO ^t Bu (860 mg), and benzophenoneimine (1.37 mL) were sequentially added to a solution of Compound V (3.7 g) in toluene (32 mL). It was stirred under reflux under an argon stream for 3 hours. After cooling to room temperature, the reaction mixture was diluted with EtOAc and washed with water and saturated salt water. The EtOAc solution _{was dried over MgSO 4} and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (3% to 5% EtOAc in hexanes) to obtain the title compound VI (3.72 g) as a yellow solid.

¹ H-NMR (CDCl ₃ ) δ 7.85-7.75 (m, 2H), 7.66-7.61 (m, 4H), 7.48-7.28 (m, 11H), 7.25-7.10 (m, 3H), 2.09 (s, 3H) ), 1.84 (d, J = 9.1 Hz, 6H), 1.10 (s, 9H).

[Example 6]

5-((t-butyldiphenylsilyl)oxy)-3,4,6-trimethylpyridin-2-amine (Formula VII)

[5-((tert-butyldiphenylsilyl)oxy)-3,4,6-trimethylpyridin-2-amine]

Compound VI (6.78 g) was dissolved in a mixed solvent of MeOH (60 mL) and THF (7 mL), and the solution was ice-cooled. AcCl (1.92 mL) was slowly added dropwise thereto, the temperature was raised to room temperature, and the mixture was stirred for 28 hours. After the reaction mixture was concentrated under reduced pressure, the residue was diluted with EtOAc, and 2M NaOH was added to adjust the pH of the solution to 7. The solution was washed with saturated salt water, dried over MgSO ₄ , filtered, and then concentrated under reduced pressure. The residue was purified by column chromatography ( _{2.5% MeOH in CH 2} Cl ₂ ) to give the title compound VII (2.52 g) as a brown solid.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 7.65 (d, J = 1.4 Hz, 4H), 7.48-7.36 (m, 6H), 5.02 (s, 2H), 1.92 (s, 3H), 1.86 ( d, J = 2.4 Hz, 6H), 1.04 (s, 9H).

[Example 7]

General synthesis method of 2,4,5-trimethyl-6-((5-R-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol (Formula 2)

[2,4,5-trimethyl-6-((5-R-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol]

A solution of compound VII (1.0 mmol) and compound VIII (1.1 mmol) having various substituents R in CH ₂ Cl ₂ (5 mL) was stirred at room temperature. After the reaction mixture was concentrated under reduced pressure, the residue was purified by column chromatography to obtain compound IX.

Compound IX (1.0 mmol) was dissolved in N-methylpyrrolidine (10 mL), Et ₃ N (2.4 mmol) and p-TsCl (1.2 mmol) were sequentially added, followed by stirring at room temperature. The reaction mixture was diluted with EtOAc, washed with water and saturated salt water, _{dried over MgSO 4} , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound X.

After dissolving compound X (1 mmol) in THF (5 mL), 1M n-Bu ₄ NF THF solution (1.2 mmol) was added, followed by stirring at room temperature. The reaction mixture was diluted with EtOAc, washed with water and saturated salt water, _{dried over MgSO 4} , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound 2.

[Example 8]

General synthesis method of 2,4,5-trimethyl-6-((5-R-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Formula 3)

[2,4,5-trimethyl-6-((5-R-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

A solution of compound VII (1.0 mmol) and compound VIII (1.1 mmol) having various substituents R in CH ₂ Cl ₂ (5 mL) was stirred at room temperature. After the reaction mixture was concentrated under reduced pressure, the residue was purified by column chromatography to obtain compound IX.

Compound IX (1.0 mmol) was dissolved in DMSO (5 mL), EDCI (1.2 mmol) was added, and the mixture was stirred at 50 to 60 °C. The reaction mixture was cooled to room temperature and then diluted with EtOAc. The EtOAc solution was washed sequentially with water and saturated salt water, _{dried over MgSO 4} , filtered and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound XI.

Compound XI (1 mmol) was dissolved in THF (5 mL), 1M n-Bu ₄ NF THF solution (1.2 mmol) was added, and the mixture was stirred at room temperature. The reaction mixture was diluted with EtOAc, washed with water and saturated salt water, _{dried over MgSO 4} , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain compound 3.

[Example 9]

2,4,5-trimethyl-6-((5-phenyl-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol (Formula 2-036)

[2,4,5-trimethyl-6-((5-phenyl-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.55 (s, 1H), 8.29 (s, 1H), 7.96-7.87 (m, 2H), 7.56-7.43 (m, 3H), 2.43 (s, 3H ), 2.23 (s, 3H), 2.17 (s, 3H).

[Example 10]

2,4,5-trimethyl-6-((5-(p-tolyl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol (Formula 2-037)

[2,4,5-trimethyl-6-((5-(p-tolyl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.50 (s, 1H), 8.28 (s, 1H), 7.79 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H) , 2.42 (s, 3H), 2.36 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H).

[Example 11]

6-((5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol (Formula 2-039)

[6-((5-(4-bromophenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.61 (s, 1H), 8.31 (s, 1H), 7.86 (d, J = 8.5 Hz, 2H), 7.73-7.68 (m, 2H), 2.43 ( s, 3H), 2.23 (s, 3H), 2.17 (s, 3H).

[Example 12]

6-((5-(4-(dimethylamino)phenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol (Formula 2-041 )

[6-((5-(4-(dimethylamino)phenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.29 (s, 1H), 8.23 (s, 1H), 7.70 (d, J = 8.8 Hz, 2H), 6.79 (d, J = 9.0 Hz, 2H) , 2.98 (s, 6H), 2.41 (s, 3H), 2.21 (s, 3H), 2.16 (s, 3H).

[Example 13]

6-((5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol (Formula 2-045)

[6-((5-(4-methoxyphenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.44 (s, 1H), 8.26 (s, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.09-7.03 (m, 2H), 3.82 ( s, 3H), 2.42 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H).

[Example 14]

2,4,5-trimethyl-6-((5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol (Formula 2-046)

[2,4,5-trimethyl-6-((5-(4-(trifluoromethyl)phenyl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.73 (s, 1H), 8.33 (s, 1H), 8.12 (d, J = 8.1 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H) , 2.44 (s, 3H), 2.23 (s, 3H), 2.19-2.14 (m, 3H).

[Example 15]

2,4,5-trimethyl-6-((5-(4-(trifluoromethoxy)phenyl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol (Formula 2 -047)

[2,4,5-trimethyl-6-((5-(4-(trifluoromethoxy)phenyl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.63 (s, 1H), 8.31 (s, 1H), 8.04 (d, J = 8.8 Hz, 2H), 7.53-7.48 (m, 2H), 2.43 ( s, 3H), 2.23 (s, 3H), 2.17 (s, 3H).

[Example 16]

6-((5-(4-(benzyloxy)phenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol (Formula 2-048 )

[6-((5-(4-(benzyloxy)phenyl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.43 (s, 1H), 8.26 (s, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.51-7.46 (m, 2H), 7.44- 7.38 (m, 2H), 7.35 (dt, J = 9.6, 4.3 Hz, 1H), 7.17-7.12 (m, 2H), 5.18 (s, 2H), 2.42 (s, 3H), 2.22 (s, 3H) , 2.16 (s, 3H).

[Example 17]

6-((5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridine- 3-ol (Chemical Formula 2-049)

[6-((5-(benzo[d][1,3]dioxo-5-yl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol ]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.47 (s, 1H), 8.27 (s, 1H), 7.46 (d, J = 1.6 Hz, 1H), 7.36 (dd, J = 8.1, 1.7 Hz, 1H), 7.03 (d, J = 8.1 Hz, 1H), 6.11 (s, 2H), 2.42 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H).

[Example 18]

6-((5-([1,1'-biphenyl]-4-yl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3- All (Chemical Formula 2-053)

[6-((5-([1,1'-biphenyl]4-yl)-1,3,4-thiadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.58 (s, 1H), 8.30 (s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.84-7.79 (m, 2H), 7.77- 7.72 (m, 2H), 7.51 (dd, J = 10.4, 4.8 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 2.45 (s, 3H), 2.24 (s, 3H), 2.17 (s , 3H).

[Example 19]

2,4,5-trimethyl-6-((5-(naphthalen-1-yl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol (Formula 2-056)

[2,4,5-trimethyl-6-((5-(naphthalene-1-yl)-1,3,4-thiadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.62 (s, 1H), 8.76-8.69 (m, 1H), 8.30 (s, 1H), 8.11-8.03 (m, 2H), 7.85 (dd, J = 7.2, 1.1 Hz, 1H), 7.70-7.58 (m, 3H), 2.38 (s, 3H), 2.26 (s, 3H), 2.18 (s, 3H).

[Example 20]

4-(5-((5-hydroxy-3,4,6-trimethylpyridin-2-yl)amino)-1,3,4-thiadiazol-2-yl)phenyl 4-methylbenzenesulfonate ( Chemical Formula 2-061)

[4-(5-((5-hydroxy-3,4,6-trimethylpyridin-2-yl)amino)-1,3,4-thiadiazol-2-yl)phenyl 4-methylbenzenesulfonate]

It was prepared according to the method of Example 7.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.60 (s, 1H), 8.30 (s, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.81-7.75 (m, 2H), 7.53- 7.46 (m, 2H), 7.18-7.12 (m, 2H), 2.43 (s, 3H), 2.41 (s, 3H), 2.22 (s, 3H), 2.16 (s, 3H).

[Example 21]

2,4,5-trimethyl-6-((5-methyl-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Formula 3-002)

[2,4,5-trimethyl-6-((5-methyl-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.38 (s, 1H), 8.44 (s, 1H), 2.33 (s, 3H), 2.28 (s, 3H), 2.10 (d, J = 34.2 Hz, 6H).

[Example 22]

2,4,5-trimethyl-6-((5-(trifluoromethyl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Formula 3-010)

[2,4,5-trimethyl-6-((5-(trifluoromethyl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.44 (s, 1H), 8.67 (s, 1H), 2.33 (s, 3H), 2.17 (d, J = 21.1 Hz, 6H).

[Example 23]

2,4,5-trimethyl-6-((5-phenyl-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Chemical Formula 3-036)

[2,4,5-trimethyl-6-((5-phenyl-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.79 (s, 1H), 8.53 (s, 1H), 7.91 (d, J = 57.9 Hz, 2H), 7.58-7.51 (m, 3H), 2.45- 2.06 (m, 9H).

[Example 24]

6-((5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol (Formula 3-041 )

[6-((5-(4-(dimethylamino)phenyl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.53 (s, 1H), 8.43 (s, 1H), 7.61 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H) , 2.98 (s, 6H), 2.18 (dd, J = 47.2, 24.9 Hz, 9H).

[Example 25]

2,4,5-trimethyl-6-((5-(4-trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Formula 3 -046)

[2,4,5-trimethyl-6-((5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.99 (s, 1H), 8.60 (s, 1H), 8.05 (s, 2H), 7.91 (d, J = 8.3 Hz, 2H), 2.34 (s, 3H), 2.18 (d, J = 16.9 Hz, 6H).

[Example 26]

2,4,5-trimethyl-6-((5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Formula 3 -047)

[2,4,5-trimethyl-6-((5-(4-(trifluoromethoxy)phenyl)-1,3,4-oxadazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.88 (s, 1H), 8.54 (s, 1H), 7.96 (s, 2H), 7.55 (dd, J = 8.9, 0.9 Hz, 2H), 2.42- 2.06 (m, 9H).

[Example 27]

6-((5-(4-(benzyloxy)phenyl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol (Formula 3-048 )

[6-((5-(4-(benzyloxy)phenyl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.68 (s, 1H), 8.49 (s, 1H), 7.78 (s, 2H), 7.51-7.32 (m, 5H), 7.20-7.15 (m, 2H) ), 5.19 (s, 2H), 2.40-2.06 (m, 9H).

[Example 28]

6-((5-(benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridine- 3-ol (Chemical Formula 3-049)

[6-((5-(benzo[d][1,3]dioxo-5-yl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol ]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.71 (s, 1H), 8.47 (s, 1H), 7.46-7.26 (m, 2H), 7.07 (d, J = 8.1 Hz, 1H), 6.13 ( s, 2H), 2.35-2.03 (m, 9H).

[Example 29]

6-((5-([1,1'-biphenyl]-4-yl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3- All (Chemical Formula 3-053)

[6-((5-([1,1'-biphenyl]4-yl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.83 (s, 1H), 8.54 (s, 1H), 8.04-7.81 (m, 4H), 7.78-7.70 (m, 2H), 7.55-7.46 (m) , 2H), 7.44-7.38 (m, 1H), 2.31 (s, 3H), 2.26-2.01 (m, 6H).

[Example 30]

2,4,5-trimethyl-6-((5-(naphthalen-1-yl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Chemical Formula 3-056)

[2,4,5-trimethyl-6-((5-(naphthalene-1-yl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.92 (s, 1H), 9.05 (d, J = 8.1 Hz, 1H), 8.50 (s, 1H), 8.16-7.94 (m, 4H), 7.75- 7.61 (m, 4H), 2.34 (s, 3H), 2.16 (d, J = 9.3 Hz, 6H).

[Example 31]

4-(5-((5-hydroxy-3,4,6-trimethylpyridin-2-yl)amino)-1,3,4-oxadiazol-2-yl)phenyl 4-methylbenzenesulfonate ( Chemical Formula 3-061)

[4-(5-((5-hydroxy-3,4,6-trimethylpyridin-2-yl)amino)-1,3,4-oxadiazol-2-yl)phenyl 4-methylbenzenesulfonate]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.84 (s, 1H), 8.50 (s, 1H), 7.92-7.72 (m, 4H), 7.49 (d, J = 8.1 Hz, 2H), 7.24- 7.18 (m, 2H), 2.43 (s, 3H), 2.34-2.02 (m, 9H).

[Example 32]

2,4,5-trimethyl-6-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylamino)pyridin-3-ol (Chemical Formula 3-065)

[2,4,5-trimethyl-6-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.16-9.95 (m, 1H), 8.77-8.73 (m, 2H), 8.62-8.48 (m, 1H), 7.87-7.70 (m, 2H), 2.32 -1.96 (m, 9H).

[Example 33]

2,4,5-trimethyl-6-((5-(pyrimidin-4-yl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol (Chemical Formula 3-073)

[2,4,5-trimethyl-6-((5-(pyrimidin-4-yl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 10.21 (s, 1H), 9.30 (s, 1H), 8.98 (d, J = 5.3 Hz, 1H), 8.61 (s, 1H), 8.03 (s, 1H), 2.37-2.02 (m, 9H).

[Example 34]

2,4,5-trimethyl-6-(5-(5-methyl-1H-pyrazol-3-yl)-1,3,4-oxadiazol-2-ylamino)pyridin-3-ol (chemical formula 3-086)

[2,4,5-trimethyl-6-((5-(5-methyl-1H-pyrazol-3-yl)-1,3,4-oxadiazol-2-yl)amino)pyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 13.07 (s, 1H), 9.65 (s, 1H), 8.48 (s, 1H), 6.46 (s, 1H), 2.28 (s, 3H), 2.27 ( s, 3H), 2.15 (s, 3H), 2.08 (s, 3H).

[Example 35]

6-(5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-ylamino)-2,4,5-trimethylpyridin-3-ol (Chemical Formula 3-100)

[6-((5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin-3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 11.79 (s, 1H), 9.53 (s, 1H), 8.41 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 2.7 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.20 (dd, J = 15.9, 7.5 Hz, 2H), 2.29 (s, 3H), 2.17 (s, 3H), 2.12 ( s, 3H).

[Example 36]

6-(5-(2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-1,3,4-oxadiazol-2-ylamino)-2,4,5- Trimethylpyridin-3-ol (Chemical Formula 3-123)

[6-((5-(2,3-dihydrobenzo[b][1,4]dioxin-2-yl)-1,3,4-oxadiazol-2-yl)amino)-2,4,5-trimethylpyridin -3-ol]

It was prepared according to the method of Example 8.

¹ H-NMR ((CD ₃ ) ₂ SO) δ 9.78 (s, 1H), 8.49 (s, 1H), 6.92-6.86 (m, 4H), 5.62-5.60 (m, 1H), 4.56 (dd, J = 11.8, 2.3 Hz, 1H), 4.42 (dd, J = 11.8, 5.8 Hz, 1H), 2.25 (s, 3H), 2.13 (s, 3H), 2.03 (s, 3H).

[Test Example 1]

Intestinal epithelial cell adhesion inhibition activity test of monocytes induced by TNF-α or IL-6

<Test method>

HT-29 human colon cancer-derived epithelial cells and U937 human-derived mononuclear cells were cultured in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin (PS) under conditions of _{37°C and 5% CO 2.} When growing more than 80% in the culture flask, it was passaged at a ratio of 1:3 and used in this experiment. HT-29 cells were cultured in a 24 well plate at ^{a concentration of 2×10 5} cells/cm ² , and the test drug was pretreated for 1 hour in a medium containing only 1% FBS and 1% PS. After that, 10 μg/mL BCECF-AM was treated and reacted at 37°C for 30 minutes to test U937 cells loaded with BCECF and TNF-α (10 ng/mL) or IL-6 (10 ng/mL) in advance. The drug-treated HT-29 cells were reacted at 37° C. for 3 hours. When the reaction was over, the medium was removed and washed twice with PBS to remove non-adherent U937 cells. In the next step, 0.1% Triton X-100 (0.1 M Tris) was treated for cell lysis and reacted at room temperature for 30 minutes, and then fluorescence was measured and quantified using a Fluostar optima microplate reader (BMG Labtechnologies, Germany) (Carvalho et al., 1996; Thapa et al., 2008).

<TNF-α-induced mononuclear cell adhesion inhibition activity of intestinal epithelial cells>

The results of examining the inhibitory activity of the test drug (1 μM) on the adhesion of intestinal epithelial cells (HT-29) and mononuclear cells (U937) induced by TNF-α are shown in Table 4 below.

compound Compound concentration (μM) Inhibitory activity (%) 5-ASA 1000 9.9±2.1 ^* 5-ASA One 3.8±1.0 2-036 One 78.7±2.5 ^* 2-037 One 16.6±3.1 ^* 2-039 One 13.7±2.3 ^* 2-041 One 62.5±6.5 ^* 2-045 One 13.2±1.9 ^* 2-046 One 52.3±1.1 ^* 2-047 One 30.9±6.9 ^* 2-048 One 19.5±3.3 2-049 One 19.4±6.6 2-053 One 38.4±2.6 ^* 2-056 One 86.8±3.5 ^* 2-061 One 54.6±3.9 ^* 3-002 One 26.2±4.6 ^* 3-010 One 35.8±5.0 ^* 3-036 One 1.4±3.0 3-041 One 15.3±4.2 3-046 One 55.7±4.9 ^* 3-047 One 60.3±6.5 ^* 3-048 One 13.6±2.5 3-049 One 45.0±5.1 ^* 3-053 One 22.5±4.8 3-056 One 81.4±3.0 ^* 3-061 One 32.3±3.9 ^* 3-065 One 61.9±3.9 ^* 3-073 One 87.2±4.1 ^* 3-086 One 24.2±2.8 3-100 One 28.3±4.2 3-123 One 27.4±6.1 ^*

* P <0.05 compared to vehicle-treated control group.

As shown in Table 4, in the case of 5-ASA (positive control), an active metabolite of sulfasalazine, a drug currently used in the treatment of inflammatory bowel disease, it exhibits an inhibition rate of 9.9% at a concentration of 1000 μM, and the test concentration of the compounds is 1 μM. The effect was very insignificant even at a concentration of 1000 times higher, whereas the 3-073 compound and the 2-056 compound showed an inhibition rate of about 87%, and the 3-056 compound showed an inhibition rate of about 81%, and the 2-036 compound Showed an inhibition rate of about 79%, showing very good activity. Most of the compounds, including compounds 2-041, 2-046, 2-061, and 3-046, were found to have superior inhibitory activity compared to the control substance, 5-ASA.

<IL-6-induced mononuclear cell adhesion inhibition activity of intestinal epithelial cells>

In the same manner as described above, the results of investigating the inhibitory activity of the test drug (1 μM) on the adhesion of intestinal epithelial cells (HT-29) and mononuclear cells (U937) induced by IL-6 are shown in Table 5 below. .

compound Compound concentration (μM) Inhibitory activity (%) 5-ASA 1000 1.7±7.8 2-036 One 68.6±5.4 ^* 2-037 One 13.1±7.1 ^* 2-039 One 9.3±12.3 ^* 2-041 One 51.7±4.8 ^* 2-045 One 8.7±6.3 ^* 2-046 One 50.4±5.4 ^* 2-047 One 21.4±5.8 2-048 One 9.6±5.5 2-049 One 11.4±4.4 2-053 One 30.5±4.9 ^* 2-056 One 77.7±3.2 ^* 2-061 One 46.3±5.6 ^* 3-002 One 16.4±6.2 3-010 One 30.8±7.0 ^* 3-036 One 9.2±3.5 3-041 One 8.1±3.3 3-046 One 44.7±5.7 ^* 3-047 One 49.8±7.0 ^* 3-048 One 6.0±4.6 3-049 One 31.6±4.2 ^* 3-053 One 14.3±4.1 3-056 One 72.5±3.3 ^* 3-061 One 25.0±2.9 ^* 3-065 One 57.0±2.9 ^* 3-073 One 77.4±3.9 ^* 3-086 One 15.2±5.3 3-100 One 20.3±2.0 3-123 One 19.3±3.0

* P <0.05 compared to vehicle-treated control group.

As shown in Table 5, for IL-6-induced monocyte-intestinal epithelial cell adhesion, 5-ASA (positive control) showed an inhibitory rate of 1.7% at a concentration of 1000 μM, which is 1000 times higher than the test concentration of 1 μM. While the effect was hardly seen in the concentration, the 2-056 compound showed an inhibition rate of about 78%, the 3-073 compound showed an inhibition rate of about 77%, and the 3-056 compound showed an inhibition rate of about 73%. Showed excellent activity. Compound 2-036 showed an inhibition rate of about 69%, 3-065 showed an inhibition rate of about 57%, and 3-047 showed an inhibition rate of about 50%, including compounds 2-041, 2-046, and 3-046. It was confirmed that most of the compounds have superior inhibitory activity compared to the control substance, 5-ASA.

[Test Example 2]

In vivo efficacy test of compound oral administration in an animal model of inflammatory bowel disease induced by TNBS

<Test method>

As for the animals, 7-8 weeks old Sprague Dawley Rats were purchased from Orient Bio (Korea), stabilized with normal solid feed for 3 days, and used for experiments. During the experiment, feed and water were supplied freely, and the temperature of the breeding room was maintained at 25±1℃, and the relative humidity was maintained at 50±10%. Lighting management was controlled in a 12-hour light-dark cycle by an automatic lighting controller. Experimental groups consist of 6 animals per group, and 5 groups (control group, TNBS alone group, TNBS+sulfasalazine 300mg/kg administration group, TNBS+test drug 1mg/kg administration group) by the egg mass method (randomized block design) so that the average weight is 180±10g. Divided into the experiment.

(1) TNBS rectal administration causes enteritis

After 24 hours of fasting rats were anesthetized with diethyl ether, 5% TNBS (2,4,6-trinitrobenzene sulfonic acid) diluted with 50v/v% ethanol in the lumen of the colon using a 1 mL syringe connected with a polyethylene catheter. After slowly injecting 0.8 mL, the rat was left standing for 60 seconds in an upside-down state to prevent leakage of 5% TNBS into the anus. As a control group, only vehicle [50v/v% ethanol] was injected in the same manner as other groups (Thapa et al., 2008).

(2) drug administration

In order to investigate the effect of the drug, the drug was administered at a constant time every day for 5 days from the day after TNBS treatment.

(3) weight observation

Weight changes of each rat were observed during the TNBS administration and drug administration from the fasting stage using a digital mass meter.

(4) sheet weight measurement

The large intestine of the rat was excised, and a tissue between 5-6 cm from the anus was cut into a length of 1 cm, and the weight of the tissue was measured.

<Effect of oral administration of the compound on TNBS-induced intestinal inflammation>

In the in vitro adhesion inhibition test, the in vivo enteritis inhibitory activity was measured for the 2-036 compound having excellent activity and is shown in Table 6 below.

compound Dosage Bowel weight recovery rate (%) Weight recovery rate (%) Sulfasalazine 300 mg/kg 64 49 2-036 1 mg/kg 79 59

A. Changes in weight

In a colitis model that induced inflammation in the intestine using 5% TNBS in rats weighing 180-190 g, a change in body weight was observed daily for 5 days based on the weight before TNBS treatment, and the vehicle-treated control group continued. As a result, the body weight increased, and the TNBS-treated group continued to lose weight and slightly recovered from the 5th day, but the body weight decreased significantly compared to the normal group. The group treated with sulfasalazine 300mg/kg, the positive control group, recovered gradually and decreased compared to the vehicle-treated control group, but the body weight increased significantly compared to the group administered with TNBS alone, resulting in a 49% weight recovery rate. Compound 2-036 showed a body weight recovery rate of 59% even when only 1 mg/kg, which is 1/300 of the dose of sulfasalazine, was administered.

B. Morphological observation

After 5 days of drug administration, the large intestine was excised and examined with the naked eye. As a result, edema and congestion were observed in the large intestine of rats treated with TNBS compared to the control, and swelling and congestion of the appendix and adhesion of intestinal tissue were observed. . In the group treated with 300 mg/kg of sulfasalazine, the positive control group, adhesions between other organs and congestion of the large intestine were significantly suppressed, and 300 mg of sulfasalazine in the group administered compound 2-036 at a dose of 1 mg/kg. Symptoms improved more than the group treated with /kg.

C. Intestinal Weighing

As a result of extracting the large intestine of rats and measuring the tissue weight between 5-6 cm from the anus, the weight of the intestine with edema was significantly increased in the TNBS alone treatment group compared to the vehicle-treated control group, and the positive control sulfasalazine 300mg In the group treated with /kg, the intestinal tissue weight recovery rate was 64%. Compound 2-036 showed an excellent intestinal weight recovery rate of 79% even when only 1 mg/kg, which is 1/300 of sulfasalazine, was administered.

[Test Example 3]

Inflammatory T cell differentiation inhibition activity test (in vitro)

CD4 T cells were isolated from the spleen of B57BL/6 mice using a CD4 microbead through a magnetic device called MACS. In order to stimulate CD4 T cells, a CD3 antibody (1 mg/mL) was coated on a plate for growing cells, and then CD4 T cells were added and another stimulating agent, CD28 antibody (1 mg/mL), was added.

There are several types of inflammatory cells in T cells, and the most representative inflammatory cells are Th1, Th2, or Th17 cells. These inflammatory cells differ in differentiation methods. To make Th1 cells, 10% FBS and 1% penicillin/streptomycin (PS) were included in RPMI-1640 medium, and then the isolated CD4 T cells were cultured under conditions of _{37° C. and 5% CO 2.} When incubating, the cytostimulating agents CD3 antibody and CD28 antibody were added as before. In order to differentiate into Th1 cells, IL-12 (10 ng/mL) was added as a cytokine and grown for 4 days. At this time, compound 2-036 (1 μM) (treatment group) was included to confirm the inhibitory ability of the drug.

Tofacitinib (1 μM) and triamcinolone (1 μM), which are T cell inhibitors of rheumatoid arthritis disease, were used as controls. After 4 days, the cells were restimulated with PMA/ionomycin and golgi stop for 4 hours and washed twice with PBS. After washing, a hole was made on the cell surface using Fix/Perm buffer, and Th1 cells secreting IFN-γ were identified through flow cytometry using PE-anti-IFN-γ antibody.

For the differentiation of Th17 cells, which are other inflammatory cells, it is the same as the differentiation method of Th1 cells, but instead of IL-12 as a cytokine, IL-6 (10 ng/mL), TGF-β (1 ng/mL), anti- IFN-γ antibody (5 mg/mL) and anti-IL-4 antibody (5 mg/mL) were added. Regulatory T cells, which are anti-inflammatory cells, were carried out in the same manner as described above, but IL-2 (10 ng/mL) and TGF-β (10 ng/mL) were added as cytokines.

Table 7 shows the inhibitory effect of the compounds according to the present invention on the differentiation of inflammatory cells.

Compound (1μM) Th 1 cell differentiation
Inhibitory activity (%) Th 17 cell differentiation
Inhibitory activity (%) 2-036 55 55 2-037 22 24 2-039 27 16 2-041 37 23 2-045 10 30 2-046 26 14 2-047 55 -11 2-048 26 13 2-049 38 22 2-053 18 37 2-056 28 3 2-061 8 9 3-002 23 6 3-010 40 37 3-036 43 8 3-041 33 30 3-046 24 18 3-047 37 16 3-048 27 10 3-049 34 19 3-053 -3 6 3-056 8 -41 3-061 -16 15 3-065 14 20 3-073 One 20 3-086 22 11 3-100 23 23 3-123 25 19

As shown in Table 7, as a result of examining the degree of inhibition of in vitro differentiation of Th1 and Th17 cells by the compounds of the present invention, the 2-036 compound showed a differentiation inhibition rate of 50% or more in both Th1 and Th17 cells. In addition, Compound 2-036 was a compound that exhibited high inhibition rate and no cytotoxicity under T cell differentiation stimulation conditions. Th1 cell differentiation induction was measured through a flow cytometer 3 days after cytokine treatment and shown in FIG. 1. The degree of Th1 differentiation by cytokines was about 30%.

The degree of cytokine-induced Th1 cell differentiation by the administration of the compound was 13% or less for compound 2-036, and tofacitinib and triamcinolone used as positive controls were found to be about 8% (FIG. 1A).

In addition, about the differentiation of Th17 cells, compound 2-036 showed less than 10% differentiation, tofacitinib 5%, and triamcinolone 1% differentiation (FIG. 1B).

However, tofacitinib and triamcinolone severely inhibit the differentiation of immunosuppressive cells (FoxP3+, Treg cells), while compound 2-036 inhibited the differentiation of Th1 and Th17 cells and did not show any effect on the differentiation of Treg cells, which are immunosuppressive cells. (Figure 1C).

Therefore, it can be confirmed that compound 2-036 specifically reacts only to inflammatory cells.

[Test Example 4]

Cytotoxicity test (in vitro)

Cells were stimulated under inflammatory cell stimulation conditions for 3 days and then stained with PI and Annexin V to differentiate between dying or dead cells, and cytotoxicity was confirmed.

To accurately measure the cytotoxicity of Compound 2-036, toxicity tests were performed at various concentrations, and the results are shown in FIG. 2. As shown in the cell division graph of Fig. 2, compound 2-036 did not show toxicity at all concentrations (Fig. 2A).

On the other hand, positive control drugs, tofacitinib and triamcinolone, exhibited significantly severe cytotoxicity from low drug treatment concentrations (FIGS. 2B and 2C).

These control drugs exhibit cytotoxicity, affecting both inhibition of differentiation and toxicity, resulting in poor specificity of the drug. On the other hand, compound 2-036 is evaluated as a drug with very high specificity for T cell differentiation, showing only the ability to inhibit T cell differentiation, and showing no toxicity to T cells themselves. Therefore, since compound 2-036 is only involved in inflammatory differentiation of T cells, it has little effect on other cells, has little side effects, and shows the possibility of maximizing the effect.

[Test Example 5]

In vivo efficacy test of compound 2-036 in multiple sclerosis animal model (EAE)

B57BL/6 mice aged 8-12 weeks were used as animals, and feed and water were freely supplied during the breeding period, and the temperature of the sauksil was maintained at 25±1℃ and the relative humidity at 50±10%. Lighting management was controlled by a 12-hour contrast cycle by an automatic lighting controller. The experimental group was divided into 2 groups (control group, drug administration group) by the egg mass method (randomized block design) with 5 animals per group.

(1) Experimental autoimmune encephalomyelitis (EAE) induction

By subcutaneous injection, 6 mg/mL of the synthetic peptide myelin oligodendrocyte glycoprotein (MOG, nervous system antigen, MEVGWYRSPFSRVVHLYRNGK; peptide sequence) was used as a complete fluid adjuvant (CFA, 10 mg/ml heat-killed H37Ra, an immune adjuvant containing strain of Mycobacterium tuberculosis). It was mixed and injected. Two days later, 250 ng of pertussis toxin was injected by intraperitoneal injection.

In the control group, PBS was injected intraperitoneally every other day, and the drug-administered group was injected with compound 2-036 in an amount of 3 mg/kg into the abdominal cavity every other day. The severity of the disease was divided from 0 to 5.

0: no symptoms

1: the tail is drooped

2: Symptoms of partial paralysis of the extremities

3: bilateral lower limb paralysis

4: all four legs are paralyzed

5: death

It is usually observed up to 3 weeks after antigen injection, and after that, it recovers spontaneously in animal models.

(2) Confirmation of drug suppression effect through immune cell analysis

Two weeks after antigen injection, the spleen, lymph nodes (LN), brain and spinal cord (CNS) were excised from mice to calculate the number of immune cells, and immune cells obtained from each organ to check the distribution of Th1 and Th17 cells. Was stimulated with PMA/Ionomycin and golgi stop.

Then, after staining with anti-IFN-γ antibody that can identify Th1 cells and anti-IL-17 antibody that can identify Th17 cells, inflammatory cells are The specific gravity was checked. The results are shown in FIG. 3.

Referring to the graph of FIG. 3, after a week after antigen injection, the behavioral pattern of the mouse begins to show an abnormal appearance.In the control group, the movement of the mouse starts to show an abnormal appearance from one week, and the tail movement decreases significantly from the 10th day, and the tail movement decreases markedly from the 10th day. Paralysis occurred, and paralysis of the lower body began 2-3 days later.

However, in the group administered with compound 2-036 every other day, abnormal behavioral patterns appeared only after 10 days, and symptoms appeared 2-3 days later than the control group. In addition, the degree of abnormality was also weak, and only tail paralysis appeared, and no symptoms of paraplegia were observed (Fig. 3A).

As a result of checking through a biopsy, the invasion of immune cells into the spinal cord is reduced by drugs (Figs. 3D, 3E), and myelin sheaths surrounding nerves are also greatly destroyed in the disease-inducing group, whereas myelin sheaths in the 2-036 drug-treated group Was well preserved. (Figure 3D, 3F)

Therefore, it was confirmed that the drug according to the present invention has a good disease inhibitory effect in vivo.

In the results of separating and analyzing immune cells from the spleen, lymphocytes, brain, and spinal cord, the total number of immune cells itself decreased in all of the drug-administered groups (Figs. 3B, 3C), and the results of analysis of Th1 and Th17 cells, which are inflammatory cells, were also found in spleen, lymphocyte, It was confirmed that it was significantly reduced in the brain and spinal cord (Figs. 3G, 3H).

Therefore, it was confirmed that compound 2-036 has excellent ability to inhibit inflammatory differentiation of T cells even in vivo and also has an excellent effect of inhibiting EAE disease.

[Test Example 6]

In vivo efficacy test of compound oral administration in an animal model of DSS-induced inflammatory bowel disease

<Test method>

B57BL/6 mice aged 8-12 weeks were used as animals, and feed and water were freely supplied during the breeding period, and the temperature of the breeding room was maintained at 25±1℃ and the relative humidity at 50±10%. Lighting management was controlled by a 12-hour contrast cycle by an automatic lighting controller. The experimental group was divided into 3 groups (control group, DSS alone administration group, and DSS + drug administration group) by the egg mass method (randomized block design).

(1) DSS oral administration causes enteritis

DSS was dissolved in water at a concentration of 2.5%, and the water was ingested for 6 days to induce enteritis.

(2) weight observation

Using a digital mass meter, the change in body weight of each mouse was observed during the DSS administration and drug administration from the fasting stage.

(3) measure the length of the sheet

The large intestine of the mouse was excised and the length of the tissue was measured.

<Effect of oral administration of the compound on DSS-induced enteritis>

In the in vitro inflammatory T cell differentiation inhibition test, the effect was confirmed by measuring the in vivo enteritis inhibitory activity for the 2-036 compound having excellent activity.

A. Changes in weight

In a colitis model that induced inflammation in the intestine using 2.5% DSS in a mouse weighing about 20 g, a change in body weight was observed at a certain time every day for 10 days based on the weight before DSS treatment. As a result, the weight increased, and the weight of the DSS-treated group began to decrease from day 3 and continued to decrease. Compared to the normal group, the weight decreased significantly. On the other hand, in the group administered with the drug DSS and 2-036, the weight was not increased compared to the control group, but it was confirmed that the weight loss was significantly suppressed (FIG. 4A).

B. Morphological observation

After 6 days of drug administration, after 4 days, the large intestine was excised and examined with the naked eye. As a result, it was confirmed that the large intestine of the DSS-treated mice was significantly reduced in length compared to the control group, and DSS+2-036 was treated with the drug. In the group, the length of the intestine was recovered (Fig. 4B).

C. Immune Cell Analysis

In addition, in the results of isolation and analysis of immune cells from lymph nodes, it was confirmed that the total number of immune cells itself was reduced in all of the drug-administered groups (Fig. 4C), and the results of analysis of inflammatory cells, Th1 and Th17 cells, were also significantly reduced (Fig. 4D).

Claims (9)

A compound of formula 1, or a pharmaceutically acceptable salt thereof:

[Formula 1]

In the above formula,
Z is O or S,
R is hydrogen; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; Heteroarylalkyl; A fused ring group in which an aryl group is fused to a cycloalkyl group; A fused ring group in which a heteroaryl group is fused to a cycloalkyl group; Pyrrolidone substituted or unsubstituted with a benzyl group; Alkylbenzenesulfonate; Benzodioxol; Or 2,3-dihydro-1,4-benzodioxin,
The alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; And heteroarylalkyl is
Hydroxy; Boronate; halogen; Haloalkyl; Haloalkoxy; Haloaryl; Amino; Alkylamino; Dialkylamino; Acylamino; Arylcarbonylamino; Sulfonamide; Nitro; Cyano; Carbonyl; Acyl; Aminocarbonyl; Alkylaminocarbonyl; Dialkylaminocarbonyl; Hydrazide; Carboxyl; Alkoxycarbonyl; Arylsulfonyl; Alkyl; Alkoxy; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Arylalkyl; Diarylalkyl; Arylalkoxy; Acetamide; Benzamide; Aryloxy unsubstituted or substituted with one or more groups selected from the group consisting of alkyl and halogen; Heterocyclic substituted or unsubstituted with alkyl; And unsubstituted or substituted with one or more groups selected from the group consisting of alkyl substituted or unsubstituted heteroaryl,
Where alkyl is C _1-30 alkyl,
Alkoxy is C _1-30 alkoxy,
Alkenyl is C _2-30 alkenyl,
Alkynyl is C _2-30 alkynyl,
Cycloalkyl is C _3-30 cycloalkyl,
Aryl is C _6-30 aryl,
Heterocyclic is a heterocyclic having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S,
Heteroaryl is a heteroaryl having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S.
A compound of formula 1, or a pharmaceutically acceptable salt thereof:

[Formula 1]

In the above formula,
Z is O or S,
R is hydrogen; Alkyl; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; Heteroarylalkyl; A fused ring group in which an aryl group is fused to a cycloalkyl group; A fused ring group in which a heteroaryl group is fused to a cycloalkyl group; Pyrrolidone substituted or unsubstituted with a benzyl group; Alkylbenzenesulfonate; Benzodioxol; Or 2,3-dihydro-1,4-benzodioxin,
The alkyl; Alkenyl; Alkynyl; Cycloalkyl; Aryl; Heterocyclic; Heteroaryl; Arylalkyl; Heterocyclic alkyl; And heteroarylalkyl is
Hydroxy; Boronate; halogen; Haloalkyl; Haloalkoxy; Haloaryl; Amino; Dialkylamino; Acylamino; Arylcarbonylamino; Sulfonamide; Nitro; Cyano; Hydrazide; Carboxyl; Alkoxycarbonyl; Arylsulfonyl; Alkyl; Alkoxy; Aryl; Diarylalkyl; Arylalkoxy; Acetamide; Benzamide; Aryloxy unsubstituted or substituted with one or more groups selected from the group consisting of alkyl and halogen; Heterocyclic substituted or unsubstituted with alkyl; And unsubstituted or substituted with one or more groups selected from the group consisting of alkyl substituted or unsubstituted heteroaryl,
Where alkyl is C _1-18 alkyl,
Alkoxy is C _1-18 alkoxy,
Alkenyl is C _2-18 alkenyl,
Alkynyl is C _2-18 alkynyl,
Cycloalkyl is C _3-18 cycloalkyl,
Aryl is C _6-18 aryl,
Heterocyclic is a heterocyclic having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S,
Heteroaryl is a heteroaryl having 3 to 30 ring atoms in which one or more ring carbons are each replaced with a heteroatom selected from B, N, O, S.
Formulas 2-036, 2-037, 2-039, 2-041, 2-045, 2-046, 2-047, 2-048, 2-049, 2-053, 2-056, 2-061, 3-002, 3-010, 3-036, 3-041, 3-046, 3-047, 3-048, 3-049, 3-053, 3-056, 3-061, 3-065, 3- A compound represented by 073, 3-086, 3-100 or 3-123, or a pharmaceutically acceptable salt thereof.
[Chemical Formula 2-036]

[Chemical Formula 2-037]

[Chemical Formula 2-039]

[Chemical Formula 2-041]

[Chemical Formula 2-045]

[Chemical Formula 2-046]

[Chemical Formula 2-047]

[Chemical Formula 2-048]

[Chemical Formula 2-049]

[Chemical Formula 2-053]

[Chemical Formula 2-056]

[Chemical Formula 2-061]

[Chemical Formula 3-002]

[Chemical Formula 3-010]

[Chemical Formula 3-036]

[Chemical Formula 3-041]

[Chemical Formula 3-046]

[Chemical Formula 3-047]

[Chemical Formula 3-048]

[Chemical Formula 3-049]

[Chemical Formula 3-053]

[Chemical Formula 3-056]

[Chemical Formula 3-061]

[Chemical Formula 3-065]

[Chemical Formula 3-073]

[Chemical Formula 3-086]

[Chemical Formula 3-100]

[Chemical Formula 3-123]

A compound represented by the following Formula 2-036, or a pharmaceutically acceptable salt thereof.
[Chemical Formula 2-036]

A compound represented by the following Formula 2-056, or a pharmaceutically acceptable salt thereof.
[Chemical Formula 2-056]

A compound represented by the following Formula 3-056, or a pharmaceutically acceptable salt thereof.
[Chemical Formula 3-056]

A compound represented by the following Formula 3-073, or a pharmaceutically acceptable salt thereof.
[Chemical Formula 3-073]

A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising the compound of claim 1. A pharmaceutical composition for preventing or treating autoimmune diseases comprising the compound of claim 1.

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