WO2023017937A1

WO2023017937A1 - Novel inhibitor for multiple protein kinases

Info

Publication number: WO2023017937A1
Application number: PCT/KR2022/003249
Authority: WO
Inventors: 김신; 이진호; 홍빅터석봉; 백혜숙; 정승익; 추현성
Original assignee: 계명대학교 산학협력단
Priority date: 2021-08-10
Filing date: 2022-03-08
Publication date: 2023-02-16
Also published as: KR102566028B1; KR20230023206A

Abstract

The present invention relates to a novel inhibitor for multiple protein kinases, and provides: a novel indolin-2-one compound or a pharmaceutically acceptable salt thereof that exhibits excellent anti-inflammatory activity; and a composition for treating inflammatory diseases, containing same as an active ingredient. The novel compound strongly inhibits the activity of various protein kinases related to inflammation, and thus can be used as an inhibitor for multiple protein kinases, and has an excellent anti-inflammatory effect as compared to other anti-inflammatory drugs, and thus can be used in the development of therapeutic agents for inflammatory diseases.

Description

Novel multiple protein kinase inhibitors

The present invention relates to novel multiple protein kinase inhibitors, and to novel indolin-2-one derivatives exhibiting excellent anti-inflammatory activity.

Inflammation is an important and fundamental immune system response of the body that protects the host from harmful stimuli and maintains tissue homeostasis. An effective inflammatory response allows you to survive infection or injury, but excessive or persistent inflammation can lead to various pathological conditions such as asthma, cancer, chronic pain, gout, mental disorders, nervous breakdown, psoriasis, rheumatoid arthritis and vasculitis. . Moreover, it is equally important that the inflammatory response to a noxious stimulus ends when the stimulus is removed. Therefore, a balance between an effective inflammatory response and an anti-inflammatory response is one of the most important conditions for maintaining a healthy state.

Toll-like receptors (TLRs) constitute an important receptor family in the innate immune system that recognizes various pathogens and damaged tissues. TLRs are divided into several types according to specificities such as structure, subcellular localization and recognition molecular properties. Lipopolysaccharide (LPS), an endotoxin of bacteria constituting the outer wall of Gram-negative bacteria, is recognized by TLR4. When LPS binds to TLR4, inflammation-related gene expression is regulated through the transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells). Inflammation is regulated by soluble immune signaling molecules called cytokines, including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). The same inflammatory cytokines are induced by LPS. These cytokines are involved in various inflammatory diseases, and their inhibition is of great help in the treatment of inflammatory diseases.

The inflammasome, a complex of inflammatory molecules, is an innate immune response to pathogenic infections and plays an important role in the formation of proinflammatory cytokines. Among them, the functions of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome have been extensively studied, and the apoptosis-associated speck-like proteins (including the regulatory protein NLRP3, CARD) have been indispensable. -like protein containing a CARD (ASC) adapter molecule, procaspase-1 and NIMA-related kinase 7. After transcription and translation of NLRP3 and IL-1β by NF-κB, active caspase-1 can process pro-IL-1β into its mature secreted form. Therefore, it is very important to effectively modulate NLRP3 inflammasome-related signaling in inflammatory diseases.

Protein kinase (protein kinase) is responsible for phosphorylation of proteins, which plays an important role in intracellular signal transduction in inflammation. Protein kinases are very important drug targets in drug development for the treatment of inflammatory diseases. For example, tofacitinib, a potent Janus-associated kinase (JAK) 1 and JAK 3 antagonist developed by Pfizer, reduces the signs and symptoms of rheumatoid arthritis (RA) and is improved bodily functions. Moreover, the p38 kinase inhibitor semapimod improved clinical outcomes in patients with Crohn's disease. BAY 61-3606, a spleen tyrosine kinase inhibitor, has been approved by the Food and Drug Administration (FDA) as an indication for allergic asthma patients. In addition, various Bruton's tyrosine kinase (BTK) inhibitors are in clinical trials. As such, the need for development of promising protein kinase inhibitors is increasing.

An object of the present invention is to provide novel compounds having excellent protein kinase inhibitory activity.

Another object of the present invention is to provide a composition for treating inflammatory diseases comprising the compound.

In order to achieve the above object, the present invention provides a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

In Formula 1, R ¹ is selected from hydrogen, hydroxy, halogen, (C1-C4) alkyl, (C3-C10) cycloalkyl, or adamantanyl, and R ² is imidazolyl , pyrazolyl, pyrrolyl or pyrrolidinyl.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases containing the compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Since the novel compound according to the present invention strongly inhibits the activity of various protein kinases related to inflammation, it can be used as a multi-protein kinase inhibitor.

In addition, since the compound regulates cytokine and NLRP3 inflammasome activity and has a superior anti-inflammatory effect compared to other anti-inflammatory agents, it can be used for the development of therapeutic agents for inflammatory diseases.

1 is a schematic diagram showing a synthesis process of a novel KMU-series compound synthesized according to an embodiment of the present invention.

Figure 2 confirms the inhibitory effect of KMU-series compounds on LPS-induced up-regulated pro-inflammatory cytokines in THP-1 cells according to an experimental example of the present invention (*p < 0.05, **p < 0.01, #p < 0.001 compared to LPS alone)

Figure 3 confirms the inhibitory effect of KMU-11342 on RANKL-induced osteoclastogenesis in RAW 264.7 cells (**p < 0.01, #p < 0.001 compared to RANKL alone).

Figure 4 is a novel compound synthesized according to an embodiment of the present invention, KMU-11170 (hereinafter referred to as "KMU-1170" in the drawings and experimental results) and LPS-induced iNOS and COX-induced iNOS and COX- in the human monocyte cell line THP-1. 2, (A) is a schematic diagram showing the synthesis process of KMU-1170, (B) confirms the cytotoxic effect of KMU-1170 in THP-1 cells through XTT analysis, (C) to (I) are graphs confirming changes in mRNA or protein expression of iNOS, COX-1, and COX-2 using western blotting and Image-J software (†p < 0.05, *p < 0.01, and #p < 0.001 compared to LPS).

Figure 5 confirms the inhibitory effect of KMU-1170 on LPS-induced up-regulated pro-inflammatory cytokines in THP-1 cells according to an experimental example of the present invention. The protein expression levels of pro-IL-1β, TNF-α, IL-6, and β-actin were measured by blotting, (B) using Image-J software, pro-IL-1β, TNF-α, And analyzing the relative optical density of the IL-6 band, (C) extracting total RNA and confirming the mRNA expression levels of IL-1β, TNF-α, and IL-6 by RT-PCR, (D ) is the analysis of the relative optical density of IL-1β, TNF-α, and IL-6 bands using Image-J software, (E) to (G) are total RNA extracted and IL-1β by real-time PCR , TNF-α, and IL-6 mRNA expression levels were confirmed (†p < 0.05, *p < 0.01, and #p < 0.001 compared to LPS).

Figure 6 confirms the effect of KMU-1170 on LPS-induced TLR4 signaling-related proteins in THP-1 cells according to an experimental example of the present invention. , MyD88, and β-actin protein expression levels were measured, (B) analyzed the relative optical density of TLR4 and MyD88 bands using Image-J software, (C) was p-TAK1 by western blot , TAK1, and β-actin protein expression levels were measured, (D) analyzed the relative optical density of the p-TAK1 band using Image-J software, (E) was p-ERK by Western blot , ERK, p-JNK, JNK, p-p38, p38, and measuring the protein expression levels of β-actin, (F) using Image-J software to measure p-ERK, p-JNK, and p- The relative optical density of the p38 band was analyzed (#p < 0.001 compared to LPS).

Figure 7 confirms the effect of KMU-1170 on LPS-induced phosphorylation of IKKα / β and NF-κB and nuclear translocation of NF-κB in THP-1 cells according to an experimental example of the present invention, (A) The protein expression levels of p-IKKα/β, IKKα/β, p-NF-κB p65, NF-κB p65, and β-actin were measured by Western blotting, (B) was obtained using Image-J software. Analysis of relative optical densities of -IKKα/β and p-NF-κB p65 bands, (C) shows cells were treated with NF-κB p65 (green) and 4',6-diamidino-2-phenylindole (4 ',6-diamidino-2-phenylindole) (blue) and confirmed by fluorescence microscopy, arrows indicate 400-fold magnification (*p < 0.01 and #p < 0.001 compared to LPS).

Figure 8 relates to the inhibitory effect of KMU-1170 on LPS-induced NLRP3 inflammasome activation and anti-inflammatory potential of KMU-1170 in THP-1 cells according to an experimental example of the present invention, (A) is for cells Lysate and medium supernatant were separated, and the protein expression levels of pro-IL-1β and IL-1β in the supernatant and NLRP3, ASC, pro-caspase-1, pro- Measuring the protein expression levels of IL-1β and β-actin, (B) is the relative ratio of NLRP3, ASC, pro-caspase-1, and pro-IL-1β bands in the lysate using Image-J software The optical density was analyzed, (C) the protein expression levels of pro-IL-1β and β-actin were measured in the lysate by Western blot, (D) was pro-IL-1β using Image-J software. The relative optical density of the 1β band was analyzed (†p < 0.05, *p < 0.01, and #p < 0.001 compared to LPS).

Figure 9 confirms the inhibitory effect of KMU-1170 on the LPS-induced inflammatory response in primary human osteoarthritis (OA) fibroblast-like synovial cells (FLS) according to an experimental example of the present invention, (A) to (C) is to extract total RNA and measure IL-1 β, TNF-α, and IL-6 mRNA expression levels by real-time PCR, (D) to (F) isolate whole cell lysates and use Western blot, respectively pro - protein expression levels of IL-1β, TNF-α, IL-6, and β-actin (D); protein expression levels of iNOS, COX-2, and β-actin (E); The protein expression levels (F) of p-IKKα/β, p-NF-κB p65, and β-actin; were measured (*p < 0.01 and #p < 0.001 compared to LPS).

Figure 10 is a schematic diagram of the anti-inflammatory effect mechanism of KMU-1170 confirmed according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

Protein kinase is an enzyme that phosphorylates a protein molecule by attaching a phosphoryl group to a serine, threonine or tyrosine residue, and binding to ATP is essential for kinase activity. Recently, reports on the involvement of protein kinases in various human diseases including cancer and inflammatory diseases have led to the development of various protein kinase inhibitors.

Accordingly, the present inventors synthesized KMU-series compounds, which are indolin-2-one derivatives, as novel multi-target protein kinase inhibitors, and synthesized human monocyte cell line THP-1 and primary human osteoarthritis fibroblast-like synovial membrane. By using the cell to confirm its anti-inflammatory mechanism, the present invention was completed.

The present invention provides a compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof.

In Formula 1, R ¹ may be selected from hydrogen, hydroxy, halogen, (C1-C4) alkyl, (C3-C10) cycloalkyl, or adamantanyl, and R ² is imidazolyl ( imidazolyl), pyrazolyl, pyrrolyl, and pyrrolidinyl.

Preferably, R ¹ may be selected from (C3-C8) cycloalkyl or adamantanyl, and R ² may be selected from imidazolyl or pyrrolyl. , but is not limited thereto.

More preferably, the compound or salt thereof is (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(cyclopropylamino)pyrazin-2-yl)indoline- 2-one [(Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(cyclopropylamino)pyrazin-2-yl)indolin-2-one] (4a; KMU-11170) , (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one [ (Z)-3- ((1H-imidazol-5-yl)methylene)-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one] ( 4b; KMU-11342), (Z)-3-((1H -imidazol-5-yl)methylene)-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one [ (Z)-3-((1H-Imidazol-5-yl) methylene)-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one ] (4c; KMU-11361), (Z)-3-((1H-pyrrol-2-yl)methylene)- 5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one [ (Z)-3-((1H- pyrrol -2- yl ) methylene )-5-(6-(cyclohexylamino) pyrazin-2-yl) indolin-2-one ] (4c'; KMU-11426), (Z) -3-((1H-imidazol-5-yl) methylene) -5-(6-(adamantane) -1-ylamino) pyrazin-2-yl) indolin-2-one [ (Z)-3-((1H-Imidazol-5-yl)methylene)-5-(6-(adamantan-1-ylamino) pyrazin-2-yl)indolin-2-one ] (4d; KMU-11421), and (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(cycloheptylamino) )pyrazin-2-yl)indolin-2-one [ (Z)-3-((1H- Imidazol -5- yl ) methylene )-5-(6-(cycloheptylamino)pyrazin-2-yl)indolin -2-one ] (4e; KMU-11427), but is not limited thereto.

In the present invention, the compound may be used in the form of a pharmaceutically acceptable salt within a range having the same efficacy.

As used herein, "pharmaceutically acceptable" means a salt that is not toxic to cells or humans exposed to the composition and has a safety and efficacy profile suitable for administration to humans.

The salt may be used in the form of either a pharmaceutically acceptable basic salt or acid salt. The basic salt can be used in the form of any one of an organic basic salt and an inorganic basic salt, and includes sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethylaminium salt, and pyrol. It may be selected from the group consisting of dinium salts.

As the acid salt, an acid addition salt formed by a free acid is useful. Inorganic acids and organic acids can be used as free acids, and hydrochloric acid, hydrobromic acid, sulfuric acid, sulfurous acid, phosphoric acid, double phosphoric acid, and nitric acid can be used as inorganic acids, and citric acid, acetic acid, maleic acid, malic acid, and fumaric acid can be used as organic acids. , glucoic acid, methanesulfonic acid, benzenesulfonic acid, camphorsulfonic acid, oxalic acid, malonic acid, glutaric acid, acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, Glutamic acid, citric acid, aspartic acid, stearic acid, etc. may be used, but are not limited thereto, and salts formed using various inorganic acids and organic acids commonly used in the art may all be included.

In addition, the compound may include all salts, hydrates, solvates, derivatives, and the like that can be prepared by conventional methods, as well as the salts. The addition salt can be prepared by a conventional method, and is dissolved in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile, and an excess organic base is added or an aqueous solution of an inorganic base is added, followed by precipitation or crystallization. can be manufactured by Alternatively, the solvent or excess base may be evaporated from the mixture and then dried to obtain an addition salt, or the precipitated salt may be suction filtered.

In the present invention, the compound or salt thereof may inhibit various inflammation-related protein kinases.

Preferably, the compound or salt thereof is MAPK (mitogen-activated protein kinase), TAK1 (TGF-beta activated kinase 1), Lck (lymphocyte-specific protein tyrosine kinase), TYK2 (non-receptor tyrosine-protein kinase 2) , JAK3 (janus kinase 3), and Txk (tyrosine-protein kinase) may inhibit one or more protein kinases selected from the group consisting of, but is not limited thereto.

According to one experimental example of the present invention, the compound is related to various cellular processes including apoptosis, differentiation, immunity, inflammation and survival in THP-1 cells, particularly related to LPS-TLR4-TAK1 signaling of pro-inflammatory cytokines. It was confirmed that TAK1 inhibits LPS-induced phosphorylation of ERK and JNK.

The compound or salt thereof may inhibit the activity of NLRP3 inflammasome and inhibit LPS-induced up-regulation of various cytokines, and thus may have excellent anti-inflammatory activity.

According to one experimental example of the present invention, the compound inhibits iNOS belonging to the family of catalytic enzymes necessary for the production of nitric oxide (NO), an important mediator of the inflammatory response, and various diseases such as inflammatory diseases, neoplastic diseases and Alzheimer's disease. LPS-induced IKKα/β and NF-κB p65 associated with NF-κB factor, a key mediator of the immune system that selectively inhibits COX-2 associated with human diseases and regulates key factors such as cytokines and inflammasomes in the inflammatory response It was confirmed that phosphorylation of , nuclear translocation of NF-κB p65, and upregulation of IL-1β, TNF-α, and IL-6 were inhibited. On the other hand, the compound did not inhibit the LPS-mediated upregulation of TLR4 and MyD88 in THP-1 cells, and these results suggest that the compound has an anti-inflammatory effect downstream of TLR4/MyD88 in the LPS-mediated inflammatory response of THP-1 cells. shows that it has

In addition, as a result of comparing the anti-inflammatory effect of the compound with other targeted anti-inflammatory drugs, COX-2 inhibitors (celecoxib), BTK inhibitors (ibrutinib), JAK inhibitors (ruxolitinib), and methotrexate were significantly superior to other drugs. It was confirmed that it exhibited an excellent anti-inflammatory effect.

Accordingly, the compound or salt thereof may be used as a composition for preventing or treating inflammatory diseases.

The inflammatory disease may be any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, osteoporosis, Paget's disease of bone, and ankylosing spondylitis, but is not limited thereto.

According to an experimental example of the present invention, it can be confirmed that the compound inhibits the LPS-induced inflammatory response in osteoarthritis FLS, which plays an important role in the progression of osteoarthritis characterized by continuous synovial inflammation and progressive cartilage degradation. Osteoarthritis can be prevented, treated or ameliorated.

The pharmaceutical composition according to the present invention can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition may be formulated with an appropriate pharmaceutically acceptable carrier according to the formulation and, if necessary, may further contain excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, and the like. there is. The appropriate carrier and the like do not inhibit the activity and characteristics of the compound according to the present invention or a pharmaceutically acceptable salt thereof, and may be selected differently depending on the dosage form and dosage form.

Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutical composition can be applied in any dosage form, and specifically, according to conventional methods, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. It may be formulated into a form and used, preferably formulated into a unit dosage form suitable for oral administration.

More specifically, the solid dosage form of the oral dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and includes at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, and mannitol. , cellulose, gelatin, etc. may be mixed, and lubricants such as magnesium stearate and talc may be included in addition to simple excipients. In addition, in the case of a capsule formulation, a liquid carrier such as fatty oil may be further included in addition to the above-mentioned materials.

Among the oral formulations, liquid formulations include suspensions, solutions for internal use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, an injection, a lyophilized formulation, a suppository, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, Tween 61, cacao butter, laurin fat, glycerogeratin, and the like may be used. It is not limited thereto, and all suitable agents well known in the art may be used.

In addition, an antioxidant may be further added to the pharmaceutical composition to enhance therapeutic efficacy. The antioxidants include thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), pyridoxine (vitamin B6) and cobalamin (cobalamin, Vitamin B group compounds such as vitamin B12), vitamin C, vitamin D, vitamin E, etc. may be used, but are not limited thereto, and suitable preparations widely known in the art may be used.

The pharmaceutical composition according to the present invention can be administered in a pharmaceutically effective amount.

In the present specification, "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects.

The effective dosage level of the pharmaceutical composition depends on the purpose of use, the patient's age, sex, weight and health condition, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route and excretion rate, treatment Duration, combination, or factors including drugs used concurrently and other factors well known in the medical arts may be determined differently. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg, may be administered once or several times a day. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to any animal that may develop an inflammatory disease, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs.

The pharmaceutical composition may be administered by an appropriate administration route according to the formulation form, and may be administered through various oral or parenteral routes as long as it can reach the target tissue. The method of administration is not particularly limited, and may be administered by conventional methods such as oral, rectal or intravenous, intramuscular, skin application, intraventricular inhalation, intrauterine dural or intracerebroventricular injection. there is.

The pharmaceutical composition may be used alone for the prevention or treatment of inflammatory diseases, or may be used in combination with surgery or other drug treatment.

In addition, the present invention provides a health functional food composition for preventing or improving inflammatory diseases containing a compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Corresponding features may be substituted in the foregoing.

In the health functional food composition according to the present invention, the health functional food may be prepared in powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving inflammatory diseases. There is no limit to the form that the health functional food can take, and it can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods.

In the health functional food composition, the health functional food may include all foods in a conventional sense. For example, beverages and various drinks, fruits and their processed foods (canned fruit, jam, etc.), fish, meat and their processed foods (ham, bacon, etc.), breads and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), etc., and may include all functional foods in a conventional sense. In addition, food used as feed for animals may also be included.

The health functional food composition may be prepared by further including food additives (food additives) commonly used in the art and appropriate other auxiliary components. The suitability as a food additive can be determined according to the standards and standards for the item in accordance with the general rules of the Food Additive Code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations; and the like.

The other auxiliary ingredients include, for example, flavoring agents, natural carbohydrates, sweeteners, vitamins, electrolytes, colorants, pectic acid, alginic acid, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents, etc. may additionally contain. In particular, as the natural carbohydrate, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol may be used. As the sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame may be used.

An effective dose of the compound or salt thereof contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as preventing or improving inflammatory diseases.

The health functional food composition uses food as a raw material and has the advantage of not having side effects that can occur when taking general medicines for a long time, and has excellent portability, so it can be taken as an adjuvant for preventing or improving inflammatory diseases.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<< 실시예Example 1> 1> KMUKMU -series (-series ( KMUKMU -11421, 11426, 11427, 11361, 11342, 11170) 화합물의 합성-11421, 11426, 11427, 11361, 11342, 11170) Synthesis of compounds

1. Synthesis Reagents and device

All reactions were performed using commercially available reagents and solvents without further purification under appropriate conditions specified by the manufacturer. A CEM Discover BenchMate was used for microwave-assisted reactions. Completion of the reaction was monitored on E. Merck silica gel F254 TLC plates. Purification of the synthesized compound was performed by flash column chromatography using Merck Silica Gel 60 (230-400 mesh). The synthesized compound was characterized by ¹ H on Bruker AVANCE 400 ( ¹ H: 400 MHz) and JEOL ECA 500 ( ¹ H: 500 MHz) spectrometer, and the chemical shift δ value was in ppm units with TMS as the reference standard. was measured as Mass spectra were acquired using Waters ACQUI-TY UPLC, Micromass Quattro microTM API.

2. Synthesis method

As shown in Figure 1, the KMU-series compounds were synthesized from 5-bromoindoline-2,3-dione under the following conditions:

Compound 1 was obtained by reduction and borylation [(a) i) NH ₂ NH ₂ , NaOH, EtOH; ii) bis(pincholato)diboron, PdCl ₂ (dppf), KOAc, 1,4-dioxane: EtOH (1: 1.5), microwave], compound 1 and pyrazine derivative compound 2 [(b) K ₂ CO ₃ , amines or alcohols, DMF] to obtain compound 3 [(c) PdCl ₂ (PPh ₃ ) ₂ , 2M K ₂ CO ₃ , 1,4-dioxane : EtOH (1: 1.5), microwave]. Finally, KMU-series compounds were synthesized through a Knoevenagel condensation reaction with 1H-imidazole-4-carbaldehyde [(d) piperidine, 1H-imidazole- 4-carbaldehyde, EtOH, microwave].

2-1. 5-(4,4,5,5-2-1. 5-(4,4,5,5- 테트라메틸tetramethyl -1,3,2--1,3,2- 디옥사보롤란Dioxaborolane -2-일)-2 days) 인돌린indoline -2-온 [-2-on [ 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)indolin-2-one5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)indolin-2-one ] 합성 : 화합물 (1)] Synthesis: Compound (1)

A microwave vessel was prepared with 5-bromoindoline-2,3-dione (0.50 g, 2.2 mmol), hydrazine hydrate (0.14 g, 4.4 mmol), and ethanol (EtOH, 2 mL) was filled. 100 W microwave was irradiated to the mixture at 100° C. for 10 minutes. After adding sodium hydroxide (0.18 g, 4.4 mmol), the mixture was irradiated with 100 W microwave at 80°C for 10 minutes. The mixture was acidified with 2 M hydrochloric acid (HCl) and cold water was added to form a precipitate, which was collected by filtration, washed with 2 M HCl and washed again with water. Drying under vacuum gave 0.42 g of 5-bromoindolin-2-one (89% yield), which was used in the next step without further purification.

After filling 5-bromoindoline-2-one (0.40 g, 1.9 mmol) and 1,4-dioxane (2.0 mL) in a microwave container, add the mixture to the mixture. bis (pinacolato) diboron [bis (pinacolato) diboron; 0.72 g, 3.8 mmol] and potassium acetate (KOAc; 0.56 g, 5.7 mmol) were added and purged with N ₂ gas for 5 minutes. [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II), PdCl ₂ (dppf) in N ₂ gas in the reaction mixture ; After removal of the solvent in vacuo, the residue was purified by silica gel chromatography using 1:1 hexane (HEX)/ethyl acetate (EA) to give 0.410 g (82%) of the title compound (1).

2-2-1. 6-2-2-1. 6- 클로로Chloro -N--N- 사이클로프로필피라진Cyclopropylpyrazine -2--2- 아민amine [ [ 6-6- ChloroChloro -N-cyclopropylpyrazin-2-amine-N-cyclopropylpyrazin-2-amine ] 합성 : 화합물 (2a)] Synthesis: compound (2a)

Cyclopropylamine (5.1 mmol, 0.35 mL) and potassium carbonate (K ₂ CO ₃ ; 6.7 mmol, 0.93 g) were mixed with 5.0 mL of N,N-dimethylformamide (DMF). was added and the mixture was stirred at room temperature for 30 minutes. After addition of 2,6-dichloropyrazine (3.4 mmol, 0.50 g), the reaction mixture was stirred at room temperature overnight, then the solvent was removed in vacuo. The residue was treated with dichloromethane (DCM) and filtered using celite, then the filtrate was collected and the solvent removed in vacuo. The final residue was purified by silica gel chromatography using 5:1 DCM/EA to give 0.21 g (36%) of the title compound (2a).

2-2-2. 6-2-2-2. 6- 클로로Chloro -N--N- 사이클로펜틸피라진Cyclopentylpyrazine -2--2- 아민amine [ [ 6-6- ChloroChloro -N-cyclopentylpyrazin-2-amine-N-cyclopentylpyrazin-2-amine ] 합성 : 화합물 (2b)] Synthesis: compound (2b)

Cyclopentylamine (5.0 mmol, 0.50 mL) and K ₂ CO ₃ (6.7 mmol, 0.93 g) were added to 5.0 mL of DMF and the mixture was stirred at room temperature for 30 minutes. After adding 2,6-dichloropyrazine (3.4 mmol, 0.50 g), the reaction mixture was stirred at room temperature overnight, then the solvent was removed in vacuo. The residue was treated with DCM and filtered using celite, then the filtrate was collected and the solvent removed in vacuo. The final residue was purified by silica gel chromatography using 5:1 DCM/EA to give 0.43 g (65%) of the title compound (2b).

2-2-3. 6-2-2-3. 6- 클로로Chloro -N--N- 사이클로헥실피라진Cyclohexylpyrazine -2--2- 아민amine [ [ 6-6- ChloroChloro -N-cyclohexylpyrazin-2-amine-N-cyclohexylpyrazin-2-amine ] 합성 : 화합물 (2c)] Synthesis: compound (2c)

Cyclohexylamine (5.0 mmol, 0.58 mL) and K ₂ CO ₃ (6.7 mmol, 0.93 g) were added to 5.0 mL of DMF, and the mixture was stirred at room temperature for 30 minutes. After adding 2,6-dichloropyrazine (3.4 mmol, 0.50 g), the reaction mixture was stirred at room temperature overnight, then the solvent was removed in vacuo. The residue was treated with DCM and filtered using celite, then the filtrate was collected and the solvent removed in vacuo. The final residue was purified by silica gel chromatography using 40:1 DCM/EA to give 0.25 g (35%) of the title compound (2c).

2-2-4. N-(2-2-4. N-( 아다만탄Adamantane -1-일)-6--1-day)-6- 클로로피라진Chloropyrazine -2--2- 아민amine [ [ N-(N-( AdamantanAdamantan -1--One- ylyl )-6-chloropyrazin-2-amine)-6-chloropyrazin-2-amine ] 합성 : 화합물 (2d)] synthesis: compound (2d)

1-adamantanamine (1-adamantanamine; 0.99 mmol, 0.15 g) and potassium tert-butyrate (potassium tert-butoxide, KOtbu; 4.0 mmol, 0.45 g) was added at room temperature and refluxed for 10 minutes under argon (Ar) gas. (2-biphenyl)tert-butylphosphine [(2-biphenyl)di-tert-butylphosphine (JohnPhos); 0.027 mmol, 0.0079 g] and tris(dibenzylideneacetone)dipalladium(0) (Pd ₂ (dba) ₃ ); 0.026 mmol, 0.024 g], the reaction mixture was refluxed in argon gas for 4 hours and then the solvent was removed in vacuo. The residue was treated with EA and filtered using celite, then the filtrate was collected and the solvent was removed under reduced pressure. The final residue was purified by silica gel chromatography DCM to give 0.033 g (13%) of the title compound (2d).

2-2-5. 6-2-2-5. 6- 클로로Chloro -N--N- 사이클로헵틸피라진Cycloheptylpyrazine -2--2- 아민amine [ [ 6-6- ChloroChloro -N-cycloheptylpyrazin-2-amine-N-cycloheptylpyrazin-2-amine ] 합성 : 화합물 (2e)] synthesis: compound (2e)

Cycloheptylamine (4.0 mmol, 0.31 mL) and K ₂ CO ₃ (6.7 mmol, 0.93 g) were added to 5.0 mL of DMF and the mixture was stirred at room temperature for 30 minutes. After adding 2,6-dichloropyrazine (3.4 mmol, 0.50 g), the reaction mixture was stirred at room temperature overnight, then the solvent was removed in vacuo. The residue was treated with DCM and filtered using celite, then the filtrate was collected and the solvent removed in vacuo. The final residue was purified by silica gel chromatography DCM to give 0.33 g (44%) of the title compound (2e).

2-3-1. 5-(6-(2-3-1. 5-(6-( 사이클로프로필아미노cyclopropylamino )) 피라진pyrazine -2-일)-2 days) 인돌린indoline -2-온 [-2-on [ 5-(6-(Cyclopropylamino)pyrazin-2-yl)indolin-2-one5-(6-(Cyclopropylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (3a)] Synthesis: compound (3a)

A microwave vessel was charged with compound 1 (0.45 g, 1.8 mmol), compound 2a (0.20 g, 1.2 mmol), 1,4-dioxane (2.0 mL), ethanol (0.40 mL), and 2 M aqueous potassium carbonate (1.8 mL, 2.54 mL). mmol) was filled. bis(triphenylphosphine) palladium(II) dichloride, PdCl ₂ (PPh ₃ ) ₂ in N ₂ atmosphere; 0.041 g, 0.059 mmol] was added, and the mixture was irradiated with 100 W microwave at 110° C. for 10 minutes. The solvent was removed in vacuo and the residue was treated with DCM, filtered using celite and the filtrate was collected, and the solvent was removed in vacuo. The residue was purified by silica gel chromatography using 5:1 DCM/EA to give 0.19 g (41%) of the title compound (3a).

2-3-2. 5-(6-(2-3-2. 5-(6-( 사이클로펜틸아미노Cyclopentylamino )) 피라진pyrazine -2-일)-2 days) 인돌린indoline -2-온 [-2-on [ 5-(6-(Cyclopentylamino)pyrazin-2-yl)indolin-2-one5-(6-(Cyclopentylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (3b)] Synthesis: compound (3b)

A microwave vessel was charged with compound 1 (0.23 g, 0.91 mmol), compound 2b (0.15 g, 0.76 mmol), 1,4-dioxane (2.0 mL), ethanol (0.40 mL), and 2 M aqueous potassium carbonate (0.91 mL, 1.5 mL). mmol) was filled. Bis(triphenylphosphine)palladium( _II ) dichloride [PdCl ₂ (PPh ₃ ) ₂ ; 0.027 g, 0.038 mmol] was added, and the mixture was irradiated with 100 W microwave at 110° C. for 10 minutes. The solvent was removed in vacuo and the residue was treated with DCM, filtered using celite and the filtrate was collected, and the solvent was removed in vacuo. The residue was purified by silica gel chromatography using 95:5 DCM/MeOH to give 0.11 g (49%) of the title compound (3b).

2-3-3. 5-(6-(2-3-3. 5-(6-( 사이클로헥실아미노cyclohexylamino )) 피라진pyrazine -2-일)-2 days) 인돌린indoline -2-온 [-2-on [ 5-(6-(Cyclohexylamino)pyrazin-2-yl)indolin-2-one5-(6-(Cyclohexylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (3c)] synthesis: compound (3c)

A microwave vessel was charged with compound 1 (0.12 g, 0.47 mmol), compound 2c (0.12 g, 0.56 mmol), 1,4-dioxane (2.0 mL), ethanol (0.40 mL), and 2 M aqueous sodium carbonate (0.70 mL). , 1.4 mmol). Bis(triphenylphosphine)palladium( _II ) dichloride [PdCl ₂ (PPh ₃ ) ₂ ; 0.017 g, 0.024 mmol] was added, and the mixture was irradiated with 100 W microwave at 110° C. for 10 minutes. The solvent was removed in vacuo and the residue was treated with DCM, filtered using celite and the filtrate was collected, and the solvent was removed in vacuo. The residue was purified by silica gel chromatography using 95:5 DCM/MeOH to give 0.070 g (48%) of the title compound (3c).

2-3-4. 5-(6-(2-3-4. 5-(6-( 아다만탄Adamantane -1--One- 일아미노ylamino )) 피라진pyrazine -2-일)-2 days) 인돌린indoline -2-온 [-2-on [ 5-(6-(Adamantan-1-ylamino)pyrazin-2-yl)indolin-2-one5-(6-(Adamantan-1-ylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (3d)] synthesis: compound (3d)

A microwave vessel was charged with compound 1 (0.062 g, 0.24 mmol), compound 2d (0.052 g, 0.20 mmol), 1,4-dioxane (2.0 mL), ethanol (0.40 mL), and 2 M aqueous sodium carbonate (0.30 mL, 0.6 mmol). ) was filled with Bis(triphenylphosphine)palladium( _II ) dichloride [PdCl ₂ (PPh ₃ ) ₂ ; 0.0070 g, 0.010 mmol] was added, and the mixture was irradiated with 100 W microwave at 110° C. for 10 minutes. The solvent was removed in vacuo and the residue was purified by dry column vacuum chromatography (DCVC) on silica gel using 90:10 DCM/MeOH. After removal of the solvent in vacuo, the residue was purified by silica gel chromatography using 95:5 DCM/MeOH to give 0.034 g (47%) of the title compound (3d).

2-3-5. 5-(6-(2-3-5. 5-(6-( 사이클로헵틸아미노cycloheptylamino )) 피라진pyrazine -2-일)-2 days) 인돌린indoline -2-온 [-2-on [ 5-(6-(Cycloheptylamino)pyrazin-2-yl)indolin-2-one5-(6-(Cycloheptylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (3e)] synthesis: compound (3e)

A microwave vessel was charged with compound 1 (0.12 g, 0.46 mmol), compound 2e (0.13 g, 0.56 mmol), 1,4-dioxane (2.0 mL), ethanol (0.40 mL), and 2 M aqueous sodium carbonate (0.69 mL, 1.4 mmol). ) was filled with Bis(triphenylphosphine)palladium( _II ) dichloride [PdCl ₂ (PPh ₃ ) ₂ ; 0.016 g, 0.023 mmol] was added, and the mixture was irradiated with 100 W microwave at 110° C. for 10 minutes. The solvent was removed in vacuo and the residue was treated with 97:3 DCM/MeOH, filtered using celite and the filtrate was collected, and the solvent was removed in vacuo. The residue was purified by silica gel chromatography using 97:3 DCM/MeOH and 95:5 DCM/MeOH to give 0.12 g (79%) of the title compound (3e).

2-4-1. (Z)-3-((1H-이미다졸-5-일)메틸렌)-5-(6-(2-4-1. (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-( 사이클로프로필아미노cyclopropylamino )) 피라진pyrazine -2-일)인돌린-2-온 [-2-day) indoline-2-one [ (Z)-3-((1H-(Z)-3-((1H- imidazolimidazol -5--5- ylyl )) methylenemethylene )-5-(6-(cyclopropylamino)pyrazin-2-yl)indolin-2-one)-5-(6-(cyclopropylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (4a; ] Synthesis: compound (4a; KMUKMU -11170)-11170)

In a microwave container, compound 3a (0.15 g, 0.056 mmol), piperidine (6.0 μL, 0.06 mmol), 1H-imidazole-4-carbaldehyde (1H-imidazole-4-carbaldehyde; 0.081 g, 0.85 mmol) , and ethanol (2.0 mL), and the mixture was irradiated with 100 W microwave at 80° C. for 10 minutes. The solvent was removed in vacuo and the residue was purified by silica gel chromatography using 95:5 DCM/MeOH to give 0.042 g (22%) of the title compound (4a).

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ 11.27-11.14 (1H), 8.41 (s, 1H), 8.38 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.69 (s, 1H), 7.29 (s, 1H), 7.01 (d, J = 8.0 Hz, 1H), 2.71 (s, 1H) , 0.79 (m, 2H), 0.57–0.45 (m, 2H); ESI MS: m/z = 345[M+H] ⁺ , 173[(M+H)/2] ⁺ .

2-4-2. (Z)-3-((1H-이미다졸-5-일)메틸렌)-5-(6-(2-4-2. (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-( 사이클로펜틸아미노Cyclopentylamino )) 피라진pyrazine -2-일)인돌린-2-온 [-2-day) indoline-2-one [ (Z)-3-((1H-(Z)-3-((1H- imidazolimidazol -5--5- ylyl )) methylenemethylene )-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one)-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (4b; ] Synthesis: compound (4b; KMUKMU -11342)-11342)

A microwave vessel was charged with compound 3b (0.080 g, 0.27 mmol), piperidine (6.0 μL, 0.05 mmol), 1H-imidazole-4-carboaldehyde (0.031 g, 0.33 mmol), and ethanol (2.0 mL); The mixture was irradiated with 100 W microwave at 80°C for 10 minutes. The solvent was removed in vacuo and the residue was purified by silica gel chromatography using 80:10 DCM/MeOH to give 0.042 g (22%) of the title compound (4b).

¹ H-NMR (500 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.37 (s, 1H), 8.30 (s, 1H), 8.03 (s, 1H), 8.00 (s, 1H), 7.97 ( d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.09 (d, J = 6.3 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 4.26 (q, J = 6.1 Hz, 1H), 2.18-1.91 (m, 2H), 1.82-1.67 (m, 2H), 1.67-1.57 (m, 2H), 1.57-1.32 (m, 2H); ESI MS: m/z = 373[M+H] ⁺ , 187[(M+H)/2] ⁺ .

2-4-3-1. (Z)-3-((1H-이미다졸-5-일)메틸렌)-5-(6-(2-4-3-1. (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-( 사이클로헥실아미노cyclohexylamino )) 피라진pyrazine -2-일)인돌린-2-온 [-2-day) indoline-2-one [ (Z)-3-((1H-(Z)-3-((1H- ImidazolImidazol -5--5- ylyl )) methylenemethylene )-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one)-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (4c; ] Synthesis: compound (4c; KMUKMU -11361)-11361)

A microwave vessel was charged with compound 3c (0.056 g, 0.18 mmol), piperidine (1.8 μL, 0.018 mmol), 1H-imidazole-4-carboaldehyde (0.021 g, 0.22 mmol), and ethanol (2.0 mL); The mixture was irradiated with 100 W microwave at 80°C for 10 minutes. The solvent was removed under reduced pressure, and the resulting solid was filtered by adding distilled water. The residue was purified by silica gel chromatography using 100:5:0.5 chloroform (CHCl ₃ )/MeOH/ammonia water (NH ₄ OH) to give 0.053 g (75%) of the title compound (4c).

¹ H-NMR (500 MHz, DMSO-d6): δ 8.31 (s, 1H), 8.24 (s, 1H), 7.98 (s, 1H), 7.93 (s, 1H), 7.89 (d, J = 8.6 Hz , 1H), 7.78 (s, 1H), 6.96 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 7.4 Hz, 1H), 3.88–3.74 (m, 1H), 2.00–1.89 (m, 2H), 1.76-1.65 (m, 2H), 1.61-1.52 (m, 1H), 1.41-1.30 (m, 2H), 1.28-1.13 (m, 4H); ESI MS: m/z = 387[M+H] ⁺ , 194[(M+H)/2] ⁺ .

2-4-3-2-4-3- 2.(2.( Z)-3-((1H-피롤-2-일)메틸렌)-5-(6-(Z)-3-((1H-pyrrol-2-yl)methylene)-5-(6-( 사이클로헥실아미노cyclohexylamino )) 피라진pyrazine -2-일)인돌린-2-온 [-2-day) indoline-2-one [ (Z)-3-((1H-(Z)-3-((1H- pyrrolpyrrol -2--2- ylyl )) methylenemethylene )-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one)-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (4c'; ] Synthesis: compound (4c'; KMUKMU -11426) -11426)

A microwave vessel was charged with compound 3c (0.079 g, 0.26 mmol), piperidine (2.5 μL, 0.025 mmol), 1H-imidazole-4-carboaldehyde (0.029 g, 0.31 mmol), and ethanol (2.0 mL); The mixture was irradiated with 100 W microwave at 80°C for 10 minutes. The solvent was removed under reduced pressure, and the resulting solid was filtered by adding distilled water. The residue was purified by silica gel chromatography using 100:3:0.3 CHCl ₃ /MeOH/NH ₄ OH to give 0.068 g (68%) of the title compound (4c′).

¹ H-NMR (500 MHz, DMSO-d6): δ 11.03 (s, 1H), 8.28 (s, 1H), 8.24 (s, 1H), 7.85 (m, 2H), 7.77 (s, 1H), 7.35 (br, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.91 (d, J = 7.4 Hz, 1H), 6.82 (t, J = 1.7 Hz, 1H), 6.39–6.32 (m, 1H) , 3.85–3.75 (m, 1H), 1.99–1.90 (m, 2H), 1.71 (dt, J = 13.5, 3.5 Hz, 2H), 1.58 (dt, J = 12.5, 4.0 Hz, 1H), 1.36 (q , J = 11.1 Hz, 2H), 1.28–1.14 (m, 3H); ESI MS: m/z = 386[M+H] ⁺ .

2-4-4. (Z)-3-((1H-이미다졸-5-일)메틸렌)-5-(6-(2-4-4. (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-( 아다만탄Adamantane -1--One- 일아미노ylamino )) 피라진pyrazine -2-일)인돌린-2-온 [-2-day) indoline-2-one [ (Z)-3-((1H-(Z)-3-((1H- ImidazolImidazol -5--5- ylyl )) methylenemethylene )-5-(6-()-5-(6-( adamantanadamantan -1-ylamino)pyrazin-2-yl)indolin-2-one-1-ylamino) pyrazin-2-yl) indolin-2-one ] 합성 : 화합물 (4d; ] synthesis: compound (4d; KMUKMU -11421)-11421)

A microwave vessel was charged with compound 3d (0.034 g, 0.094 mmol), piperidine (0.9 μL, 0.0091 mmol), 1H-imidazole-4-carboaldehyde (0.011 g, 0.11 mmol), and ethanol (1.0 mL), The mixture was irradiated with 100 W microwave at 80°C for 10 minutes. The solvent was removed under reduced pressure, and the resulting solid was filtered by adding distilled water. The residue was purified by silica gel chromatography using 90:10 DCM/MeOH to give 0.030 g (72%) of the title compound (4d).

¹ H-NMR (500 MHz, DMSO-d6): δ 11.20 (br, 1H), 8.38 (s, 1H), 8.27 (s, 1H), 8.05 (s, 1H), 7.97 (d, J = 8.0 Hz , 1H), 7.91 (s, 1H), 7.80 (s, 1H), 7.61 (s, 1H), 7.03 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 2.27–2.14 (m, 6H), 2.14-2.04 (m, 3H), 1.83-1.64 (m, 6H); ESI MS: m/z = 439[M+H] ⁺ , 220[(M+H)/2] ⁺ .

2-4-5. (Z)-3-((1H-이미다졸-5-일)메틸렌)-5-(6-(2-4-5. (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-( 사이클로헵틸아미노cycloheptylamino )) 피라진pyrazine -2-일)인돌린-2-온 [-2-day) indoline-2-one [ (Z)-3-((1H-(Z)-3-((1H- ImidazolImidazol -5--5- ylyl )) methylenemethylene )-5-(6-(cycloheptylamino)pyrazin-2-yl)indolin-2-one)-5-(6-(cycloheptylamino)pyrazin-2-yl)indolin-2-one ] 합성 : 화합물 (4e; ] Synthesis: compound (4e; KMUKMU -11427)-11427)

A microwave vessel was charged with compound 3e (0.12 g, 0.7 mmol), piperidine (4.1 μL, 0.037 mmol), 1H-imidazole-4-carboaldehyde (0.042 g, 0.44 mmol), and ethanol (2.0 mL); The mixture was irradiated with 100 W microwave at 80°C for 10 minutes. The solvent was removed under reduced pressure, and the resulting solid was filtered by adding distilled water. The residue was purified by silica gel chromatography using 100:3:0.3 CHCl ₃ /MeOH/NH ₄ OH to give 0.062 g (42%) of the title compound (4e).

¹ H-NMR (500 MHz, DMSO-d6): δ 11.20 (s, 1H), 8.38 (s, 1H), 8.28 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.66 (s, 1H), 7.09–6.98 (m, 2H), 4.14–3.94 (m, 1H), 2.06–1.89 (m, 2H) ), 1.76-1.65 (m, 2H), 1.65-1.47 (m, 8H); ESI MS: m/z = 401[M+H] ⁺ , 201[(M+H)/2] ⁺ .

<실험방법><Experiment method>

1. 실험 및 분석방법1. Experiment and analysis method

1-1. reagent

LPS ( Escherichia coli serotype) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Celecoxib, ibrutinib, methotrexate, and ruxolitinib were purchased from Selleck Chemicals (Houston, TX, USA). Receptor activator of NF-κB ligands (RANKL) was purchased from PeproTech (Rocky Hill, NH, USA).

iNOS, TLR4, MyD88, p-TAK1 (Ser412), p-ERK, ERK, p-JNK, p-IKKα/β, IKKα/β, p-NF-κB p65, NF-κB, ASC, caspase-1, and primary antibodies against NLRP3 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-IL-1β antibody was purchased from Novus Biologicals (Centennial, CO, USA). Antibodies against IL-6, COX-1, COX-2, TAK1, p-p38, and p38 were respectively Santa Cruz Bio-technology (Santa Cruz, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA). ) was purchased. Anti-TNF-α antibody was purchased from Abcam (Cambridge, MA, USA). Anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-mouse IgG-horseradish peroxidase (HRP) and anti-mouse IgG-HRP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

1-2. Cell lines and cell culture

Human monocytic cell line THP-1 cells (Korean Cell Line Bank, Seoul, Korea) were cultured in RPMI1640 medium (Welgene Inc., Gyeongsan, Korea), 10% fetal bovine serum (FBS; Welgene Inc.). ., Gyeongsan, Korea), and 1% antibiotic-antimycotic solution (Welgene Inc., Gyeongsan, Korea). Cells were cultured at 37.5° C., 5% CO ₂ in completely humid air.

alpha-MEM was developed by Welgene Inc. (Gyeongsan, Korea).

1-3. Osteoclast differentiation ( osteoclast differentiation

RAW 264.7 cells were grown in osteoclast differentiation medium (alpha-MEM+50 ng/ml RANKL) to generate osteoclast-like cells. After differentiation, multinucleated cells (MNCs) were visualized using a TRAP staining kit (TaKaRa, Japan) according to the manufacturer's protocol.

1-4. Primary human osteoarthritis fibroblast-like of synovial cells Isolation and culture

Isolation of human osteoarthritis (OA) fibroblast-like synoviocytes (FLS) was performed by enzymatic digestion of synovial tissues of osteoarthritis patients who underwent synovectomy at Keimyung University Dongsan Medical Center.

Enrolled osteoarthritis patients met American College of Rheumatology (ACR) criteria and agreed to provide informed consent. The experimental study was approved by the Institutional Review Board (IRB) of Keimyung University Dongsan Medical Center on November 20, 2020 [IRB No. 2020-11-031].

Osteoarthritis tissue was pulverized into 2-3 mm pieces, and then the minced tissue was treated with 0.5 mg/mL type II collagenase in Dulbecco's modified Eagle's medium (DMEM; Welgene Inc., Gyeongsan, Korea) at 37.5°C under 5% CO ₂ . (collagenases; Thermo Fisher Scientific, Waltham, MA, USA). The isolated cells were centrifuged (3,000 rpm, 5 min), suspended in DMEM, 10% FBS, and 1% antibiotic-antimycotic solution, and stirred in a 75 cm ² flask. After 1 day, adherent cells were cultured in DMEM, 10% FBS, and 1% antibiotic-antimycotic solution at 37.5° C. under 5% CO ₂ , and the medium was changed every 3 days. When 90-95% of the bottom of the flask is filled with fibroblasts, the medium is diluted 1:3 with fresh culture.

1-5. Kinase-profiling assay

*To evaluate the specificity of the novel multi-protein kinase inhibitor KMU-1170, a kinase-profiling service was performed by Eurofins Cerep S.A. Kinase-profiling assays were performed with ATP concentrations of 1 μM of compound and K values for each individual kinase and kinase substrate according to Eurofin's protocol.

1-6. Cell viability assay

Cell viability assay was performed using XTT assay (Welgene Inc., Gyeongsan, Korea). Briefly, 2×10 ⁵ THP-1 cells were plated in 96-well plates for 24 hours, and then cells were treated with various concentrations of KMU-1170 and cultured for 24 hours at 37° C. under 5% CO ₂ . . Thereafter, a 0.5 mg/mL XTT solution was added to the culture medium, and cultured at 37° C. for 3 hours under 5% CO ₂ . Optical density (OD) was measured with a microplate reader (BMG Labtech, Ortenberg, Germany) at a wavelength of 450 nM.

1-7. Western blotting analyze

For Western blotting analysis, THP-1 cells (2×10 ⁶ cells/well in 60-mm dishes) and FLS (2×10 ⁶ cells/well in 100-mm dishes) were plated. After culturing for 24 hours, the cells were dissolved in RIPA buffer (20 mM HEPES and 0.5% Triton X-100, pH 7.6), and the supernatant fraction was collected. Protein concentration was measured using the BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were electrophoresed on SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Science, Pittsburgh, PA, USA). Then, the membrane was incubated for 1 hour with blocking buffer (0.05% Tween 20 with 5% non-fat dry milk in Tris-buffered saline (TBS)) and then incubated for 24 hours with an appropriately diluted primary antibody against a specific target protein. cultured for a while. Next, the membrane was washed in TBS with Tween 20 and incubated with the appropriate secondary antibody for 1 hour at room temperature.

Specific proteins were detected with the ECL western blotting kit (EMD Millipore, Darmstadt, Germany) according to the manufacturer's protocol, and signal intensities were measured using the Chemi Image System Fusion FX (Vilber Lourmat, France). Protein bands were quantified using Image-J software. Quantitative analysis of band density using Image-J was expressed as the mean±standard deviation (SD) of three independent experiments.

1-8. RNA isolation and reverse transcription Reverse Transcription-Polymerase Chain Reaction (RT- PCR ) and real-time quantification PCR ( qPCR )

Total cellular RNA was isolated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and each RNA was quantified using a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). Each cDNA was obtained from 1 μg of isolated RNA using Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA) according to the manufacturer's instructions.

Table 1 below shows the primer sequences used in this experimental example.

PrimersPrimers	방향direction	Sequences ( 5’→ 3’)Sequences ( 5’→ 3’)	서열번호sequence number
Cathepsin K Cathepsin K	Forward Forward	CAG CAG AAC GGA GGC ATT GACAG CAG AAC GGA GGC ATT GA	1One
Cathepsin K Cathepsin K	ReverseReverse	CCT TTG CCG TGG CGT TAT ACCCT TTG CCG TGG CGT TAT AC	22
iNOS iNOS	ForwardForward	CTG TCT GGT TCC TAC GTC ACCCTG TCT GGT TCC TAC GTC ACC	33
iNOS iNOS	ReverseReverse	CCC ACG TTA CAT GGG AGG ATACCC ACG TTA CAT GGG AGG ATA	44
COX-1 COX-1	ForwardForward	ACC TTG AAG GAG TCA GGC ATG AGACC TTG AAG GAG TCA GGC ATG AG	55
COX-1 COX-1	ReverseReverse	TGT TCG GTG TCC AGT TCC AAT ATGT TCG GTG TCC AGT TCC AAT A	66
COX-2 COX-2	ForwardForward	ATC ACA GGC TTC CAT TGA CCATC ACA GGC TTC CAT TGA CC	77
COX-2 COX-2	ReverseReverse	TAT CAT CTA GTC CGG AGG GGTAT CAT CTA GTC CGG AGG GG	88
IL-1β IL-1β	ForwardForward	CCT TGG GCC TCA AGG AAA ACCT TGG GCC TCA AGG AAA A	99
IL-1β IL-1β	ReverseReverse	CTC CAG CTG TAG AGT GGG CTT ACTC CAG CTG TAG AGT GGG CTT A	1010
TNF-α TNF-α	ForwardForward	GGA GAA GGG TGA CCG ACT CAGGA GAA GGG TGA CCG ACT CA	1111
TNF-α TNF-α	ReverseReverse	CTG CCC AGA CTC GGC AACTG CCC AGA CTC GGC AA	1212
IL-6 IL-6	ForwardForward	ATG GCA CAG TAT CTG GAG GAGATG GCA CAG TAT CTG GAG GAG	1313
IL-6 IL-6	ReverseReverse	TAA GCT GGA CTC ACT CTC GGATAA GCT GGA CTC ACT CTC GGA	1414
β-actin β-actin	ForwardForward	AAT CTG GCA CCA CAC CTT CTAAAT CTG GCA CCA CAC CTT CTA	1515
β-actin β-actin	ReverseReverse	ATA GCA CAG CCT GGA TAG CAAATA GCA CAG CCT GGA TAG CAA	1616

For PCR, DNA polymerase was used with primers targeting IL-1β, TNF-α, and IL-6. Amplified PCR products were electrophoretically separated on a 2% agarose gel and detected under ultraviolet light. For qPCR using specific primers and SYBR GREEN Premix (Toyobo, Osaka, Japan), it was performed with a LightCycler® 480 real-time PCR system (Roche Diagnostics, Mannheim, Germany). The experiment was performed three times, and the threshold cycle number (Ct) of each target mRNA was normalized to β-actin. The delta-delta Ct (ΔΔ Ct) values of each gene were expressed as the mean±standard deviation of three independent experiments.

1-9. immunofluorescence staining

THP-1 cells (1×10 ³ ) were cultured on 8-chamber glass slides for 24 hours, treated with 1 μmol/L of KMU-1170 for 1 hour, and then stimulated with LPS for 6 hours. Cells were washed with phosphate-buffered saline (PBS), fixed with 4% formaldehyde, permeabilized with PBS containing 0.2% Triton X-100, and treated with bovine serum albumin for 1 hour. Incubation was performed to block non-specific binding. Then, the cells were incubated with the primary antibody for 24 hours at 4°C and then FITC-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) and 4',6-diamidino-2 After treatment with -phenylindole (4',6-diamidino-2-phenylindole; Thermo Fisher Scientific, Wal-tham, MA, USA; for nuclear staining), the stained cells were examined under a fluorescence microscope.

1-10. statistical analysis

Quantitative results were expressed as mean and standard deviation. Data were analyzed by one-was ANOVA and post-hoc comparisons (Student-Newman-Keuls) using the Statisti-cal Package for Social Science 26.0 software (SPSS Inc.; Chicago, IL, USA). A p value of 0.05 or less was considered significant.

<< 실험예Experimental example 1> 1> 실시예Example 1에 따른 according to 1 KMUKMU -시리즈 화합물의 항염 활성 분석-Analysis of anti-inflammatory activity of series compounds

To investigate the potential of KMU-series compounds as anti-inflammatory agents, the anti-inflammatory effects of KMU-series compounds on LPS-mediated inflammation in THP-1 cells were analyzed.

THP-1 cells were differentiated into macrophages using 100 nM of phorbol-12-myristate-13-acetate (PMA) for 24 hours. The cells were treated with KMU-11421, 11426, 11427, 11361, 11342, 11170 (1 μM), and Ruxolitinib (50 μM) for 1 hour, then LPS (1 μg/mL) was added for 3 hours . Total RNA was extracted and mRNA expression levels of IL-1β, TNF-α, and IL-6 were determined using real-time PCR.

As a result, as shown in Figures 2A and 2B, when cells were pretreated with KMU-series compounds other than KMU-11426 and ruxolitinib (JAK inhibitor), LPS-induced upregulation of IL-1β and TNF-α was attenuated. It became. On the other hand, as shown in Figure 2C, all KMU-series compounds and ruxolitinib inhibited the LPS-induced upregulation of IL-6.

<< 실험예Experimental example 2> 2> KMUKMU -11342의 of -11342 RANKLRANKL -유도 파골세포 분화 억제 확인-Confirmation of inhibition of induced osteoclast differentiation

To evaluate the effect of KMU-11342 on RANKL-induced osteoclast differentiation, after treatment with KMU-11342 (0.01, 0.25, 0.5 μM) for 1 hour, RAW 264.7 cells were treated with RANKL (50 ng/mL) for 5 days. The treated cells were differentiated into osteoclasts, and the cells were stained using a TRAP staining kit. TRAP-positive multinucleated cells (MNC) were stained purple red, and the number of TRAP-stained cells indicating the degree of osteoclast differentiation increased in a time-dependent manner for 5 days.

As a result, as shown in Figure 3A, the higher the concentration of KMU-11342, the fewer TRAP-positive MNCs were observed, indicating that KMU-11342 inhibited RANKL-induced osteoclast differentiation of RAW 264.7 cells in a dose-dependent manner. indicates

Then, total RNA was extracted and the effect of KMU-11342 on the expression of cathepsin K, a biomarker specific to osteoclasts involved in the final regulatory step of osteoclast differentiation, was analyzed using real-time PCR. As shown in 3B, KMU-11342 inhibited RANKL-induced upregulation of cathepsin K mRNA expression levels.

<< 실험예Experimental example 3> 3> KMUKMU -11170 (이하, 실험결과 및 도면에서는 "-11170 (Hereinafter, in the experimental results and drawings, " KMUKMU -1170"으로 표기) 화합물의 여러 가지 활성 분석-1170") Analysis of various activities of the compound

3-1. 다양한 단백질 키나아제의 활성에 대한 3-1. activity of various protein kinases. KMUKMU -1170의 영향 확인Check the impact of -1170

Table 2 below shows the kinase activities of 15 protein kinases according to 1 μM KMU-1170 treatment.

Referring to Table 2 below, protein kinase analysis shows that KMU-1170 inhibits various kinases related to the inflammatory response, in particular, mitogen-activated protein kinase 1 (MAPK1), Lck, TYK2 , JAK3, and Txk.

3-2. 3-2. THPTHP -1 세포에서 in -1 cell KMUKMU -1170의 -1170's LPSLPS -유도 상향 조절된 -induced upregulated iNOSiNOS 및 COX-2의 억제 효과 확인 and confirmation of the inhibitory effect of COX-2

To investigate the toxicity of KMU-1170 in THP-1 cells, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide [2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, XTT] analysis was performed.

THP-1 cells were differentiated into macrophages using 100 nM of PMA for 24 hours, and then the cells were treated with various concentrations of KMU-1170 (0.01, 0.1, 0.5, 1, 5, and 10 μM), Fig. 4B As shown, KMU-1170 showed no toxicity up to 1 μM concentration.

After that, KMU-1170 (0.1, 0.5, and 1μM) was pretreated for 1 hour, followed by LPS (1μg/mL) for 6 hours to confirm the upregulated changes in iNOS and COX-2, 1 μM of KMU-1170 inhibited LPS-induced up-regulation of inducible nitric oxide synthase (iNOS) mRNA and protein (Fig. 4C, D, E), and as a pretreatment, LPS-induced cyclook Upregulation of cyclooxygenase-2 (COX-2) mRNA and protein was suppressed, but no significant changes were observed in COX-1 mRNA and protein (FIG. 4F, G, H, I).

3-3. 3-3. THPTHP -1 세포에서 in -1 cell KMUKMU -1170의 -1170's LPSLPS -유도 -Judo 전염증성pro-inflammatory 사이토카인 생성 억제 효과 확인 Confirmation of cytokine production inhibitory effect

To evaluate the anti-inflammatory mechanism of action of KMU-1170, the regulation of the levels of proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 was investigated. THP-1 cells were differentiated into macrophages using PMA (100 nM) for 24 hours, followed by pretreatment with KMU-1170 (0.1, 0.5, and 1 μM) for 1 hour followed by LPS (1 μg/mL) for 6 hours. As a result of confirming by processing, as shown in FIG. 5, cells pretreated with 1 μM KMU-1170 showed LPS-induced up-regulated IL-1β (interleukin-1β), TNF-α (tumor necrosis factor-α), and It has been shown to inhibit IL-6.

3-4. 3-4. THPTHP -1 세포에서 in -1 cell KMUKMU -1170의 -1170's LPSLPS -유도 -Judo TAK1TAK1 및 and MAPKs의MAPKs 인산화 억제 효과 확인 Confirmation of phosphorylation inhibitory effect

The effects of KMU-1170 on TLR4 and MyD88 expression were investigated. THP-1 cells were differentiated into macrophages using PMA (100 nM) for 24 hours, followed by pretreatment with KMU-1170 (0.1, 0.5, and 1 μM) for 1 hour followed by LPS (1 μg/mL) for 6 hours. 6A and 6B, as shown in FIGS. 6A and 6B, THP-1 cells pretreated with 1 μM KMU-1170 exhibited LPS-induced upregulation of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). did not appear to weaken.

To identify potential molecular targets of KMU-1170 in inflammation, phosphorylation of transforming growth factor-β-activated kinase 1 (TAK1), which regulates the function of MAPKs and NF-κB, was investigated. As a result of the investigation, as shown in FIGS. 6C and 6D , pretreatment with KMU-1170 in THP-1 cells inhibited LPS-induced phosphorylation of TAK1.

In addition, THP-1 cells were differentiated into macrophages using PMA (100 nM) for 24 hours, treated with or without KMU-1170 for 24 hours, and induced with LPS (1 μg/mL). 6E and 6F, among MAPKs, only ERK (extracellular signal-regulated kinases) and JNK (c-Jun N-terminal kinases) were phosphorylated in response to LPS, and pretreatment with 1 μM KMU-1170 It was shown to inhibit LPS-induced phosphorylation of ERK and JNK in 1 cells.

3-5. THP-1 세포에서 KMU-1170의 LPS-유도 NF-κB의 활성 억제능 확인3-5. Confirmation of KMU-1170's ability to inhibit LPS-induced NF-κB activity in THP-1 cells

In THP-1 cells, LPS induced phosphorylation of NF-κB kinase α/β inhibitors (inhibitor NF-κB kinase α/β, IKKα/β) and NF-κB p65, but referring to FIGS. 7A and 7B, KMU It was shown that pretreatment with -1170 inhibits LPS-induced phosphorylation.

As a result of examining whether KMU-1170 affects nuclear translocation of NF-κB p65 using a fluorescence microscope, as shown in FIG. 7C , pretreatment with KMU-1170 inhibited NF-κB It has been shown to inhibit LPS-induced nuclear translocation of p65.

3-6. THP-1 세포에서 KMU-1170의 LPS-유도 NLRP3의 활성 약화 확인3-6. Confirmation of attenuation of LPS-induced NLRP3 activity of KMU-1170 in THP-1 cells

To confirm whether KMU-1170 affects NLRP3 inflammasome signaling, THP-1 cells were differentiated into macrophages using PMA (100 nM) for 24 hours, and then pretreated with 1 μM KMU-1170 for 1 hour. After that, LPS (1 μg/mL) and/or ATP (1 mM) were treated for 6 hours.

As a result, as shown in FIG. 8A , pretreatment of THP-1 cells with 1 μM KMU-1170 inhibited LPS-induced pro-IL-1β and IL-1β cytoplasmic release. Furthermore, pretreatment with 1 μM KMU-1170 upregulated NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), pro-caspase-1, and pro-IL-1β induced by LPS and co-treatment with LPS and ATP. control was weakened.

These quantitative results show that KMU-1170 can significantly inhibit NLRP3 inflammasome activity (FIG. 8B).

3-7. THP-1 세포에서 KMU-1170의 강력한 항염증 활성 확인3-7. Confirmation of strong anti-inflammatory activity of KMU-1170 in THP-1 cells

Considering the action of KMU-1170 as an anti-inflammatory agent, the potential superiority of anti-inflammatory efficacy of KMU-1170 compared to various other anti-inflammatory agents was analyzed. THP-1 cells were differentiated into macrophages using PMA (100 nM) for 24 hours, followed by KMU-1170 (1 μM), celecoxib (25 μM), ibrutinib (5 μM), Luxority After pre-treatment with nip (luxolitinib, 50 μM) and methotrexate (1 μM) for 1 hour, LPS (1 μg/mL) was treated for 6 hours.

As a result, as shown in FIGS. 8C and 8D, 1 μM KMU-1170 significantly suppressed the LPS-induced increased IL-1β protein in THP-1 cells, whereas celecoxib (COX-2 inhibitor), Other tested drugs such as ibrutinib (a BTK inhibitor), ruxolitinib (a JAK inhibitor) and methotrexate (an anti-inflammatory in RA) did not show this effect even at high concentrations. Only 1 μM methotrexate was shown to inhibit LPS-induced up-regulation of IL-1β, similar to treatment with KMU-1170.

3-8. 골관절염 섬유아세포 유사 3-8. Osteoarthritis fibroblast-like 활막세포에서in synovial cells KMUKMU -1170의 -1170's LPSLPS -유도 염증 억제 활성 확인-Confirmation of induced inflammation suppression activity

Finally, the anti-inflammatory activity of KMU-1170 against osteoarthritis was confirmed using human osteoarthritis (OA) fibroblast-like synovial cells (FLS). Human osteoarthritis fibroblast-like synovial cells were obtained from the synovial tissues of patients with knee osteoarthritis, pretreated with KMU-1170 (0.1, 0.5, and 1 μM) for 1 hour and then treated with LPS (1 μg/mL) for 6 hours. did

As a result, as shown in FIG. 9, KMU-1170 pretreated FLS showed LPS-induced up-regulated IL-1β, TNF-α, and IL-6 mRNA and protein levels (FIG. 9A to 9D), iNOS and COX -2 protein levels (FIG. 9E). In addition, KMU-1170 pretreatment inhibited LPS-induced phosphorylation of IKKα/β and NF-κB p65 in FLS (FIG. 9F).

That is, the present invention is a novel multi-protein kinase inhibitor KMU-1170 is anti-inflammatory effect by regulating the p-TAK1 / p-IKKα / β / p-NF-κB / pro-inflammatory cytokine signaling pathway and NLRP3 inflammasome It was confirmed that it had (FIG. 10). These results suggest that the KMU-series compounds are very advantageous compounds for the development of potent anti-inflammatory drugs.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

A compound represented by Formula 1 or a pharmaceutically acceptable salt thereof:

<Formula 1>

In Formula 1,

R 1 is selected from hydrogen, hydroxy, halogen, (C1-C4) alkyl, (C3-C10) cycloalkyl, or adamantanyl;

R 2 is selected from imidazolyl, pyrazolyl, pyrrolyl or pyrrolidinyl.
According to claim 1,

The compound or salt thereof,

wherein R 1 is selected from (C3-C8) cycloalkyl or adamantanyl;

The R 2 is characterized in that selected from imidazolyl (imidazolyl) or pyrrolyl (pyrrolyl), a compound or a pharmaceutically acceptable salt thereof.
According to claim 1,

The compound or salt thereof,

(Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(cyclopropylamino)pyrazin-2-yl)indolin-2-one [(Z)-3-( (1H-imidazol-5-yl)methylene)-5-(6-(cyclopropylamino)pyrazin-2-yl)indolin-2-one] (4a; KMU-11170), (Z)-3-((1H- imidazol-5-yl)methylene)-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one [(Z)-3-((1H-imidazol-5-yl)methylene )-5-(6-(cyclopentylamino)pyrazin-2-yl)indolin-2-one] (4b; KMU-11342), (Z)-3-((1H-imidazol-5-yl)methylene)- 5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one [(Z)-3-((1H-Imidazol-5-yl)methylene)-5-(6-(cyclohexylamino) pyrazin-2-yl)indolin-2-one] (4c; KMU-11361), (Z)-3-((1H-pyrrol-2-yl)methylene)-5-(6-(cyclohexylamino)pyrazine -2-yl)indolin-2-one [(Z)-3-((1H-pyrrol-2-yl)methylene)-5-(6-(cyclohexylamino)pyrazin-2-yl)indolin-2-one ] (4c'; KMU-11426), (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(adamantan-1-ylamino)pyrazin-2-yl )indolin-2-one [(Z)-3-((1H-Imidazol-5-yl)methylene)-5-(6-(adamantan-1-ylamino)pyrazin-2-yl)indolin-2-one ] (4d; KMU-11421), and (Z)-3-((1H-imidazol-5-yl)methylene)-5-(6-(cycloheptylamino)pyrazin-2-yl)indolin-2 -one [(Z)-3-((1H-Imidazol-5-yl)methylene)-5-(6-(cycloheptylamino)pyrazin-2-yl)indolin-2-one] (4e; KMU-11427) characterized in that selected from the group consisting of , A compound or a pharmaceutically acceptable salt thereof.
According to claim 1,

The compound or salt,

A compound or pharmaceutically acceptable salt thereof, characterized in that it inhibits at least one protein kinase selected from the group consisting of MAPK, TAK1, Lck, TYK2, JAK3, and Txk.
According to claim 1,

The compound or salt,

A compound or a pharmaceutically acceptable salt thereof characterized in that it inhibits the activity of NLRP3 inflammasome.
According to claim 1,

The compound or salt,

A compound characterized by having anti-inflammatory activity or a pharmaceutically acceptable salt thereof.
A pharmaceutical composition for preventing or treating inflammatory diseases containing the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 7,

The inflammatory disease,

A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it is any one selected from the group consisting of osteoarthritis, rheumatoid arthritis, osteoporosis, Paget's disease of bone, and ankylosing spondylitis.
A health functional food composition for preventing or improving inflammatory diseases containing the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
According to claim 9,

The inflammatory disease,

Osteoarthritis, rheumatoid arthritis, osteoporosis, bone Paget's disease, and ankylosing spondylitis, characterized in that any one selected from the group consisting of inflammatory disease prevention or improvement functional health food composition.