KR102241064B1 - Novel indole derivatives and composition for preventing or treating inflammatory diseases comprising the same - Google Patents

Abstract

The present invention relates to a novel indole derivative and a composition for preventing or treating inflammatory diseases comprising the same, and more specifically, a series of substituted indole derivative compounds combining the core structure of an anti-inflammatory algae metabolite was designed and synthesized, and the As the inhibitory activity and anti-inflammatory activity against COX-1 and COX-2 were confirmed in the compound, the compound can be used as an effective pharmaceutical composition or health functional food composition for the prevention, improvement or treatment of inflammatory diseases.

Description

A novel indole derivative and a composition for preventing or treating inflammatory diseases containing the same {NOVEL INDOLE DERIVATIVES AND COMPOSITION FOR PREVENTING OR TREATING INFLAMMATORY DISEASES COMPRISING THE SAME}

The present invention relates to a novel indole derivative and a composition for preventing or treating inflammatory diseases comprising the same.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most frequently chosen treatments for inflammatory symptoms, but they often have undesirable side effects on the kidney, liver and cardiovascular system. . Therefore, the discovery of new NSAIDs with a better safety profile remains an important challenge.

In general, two different strategies are being used for the development of new NSAIDs. The first is the development of inhibitors of the production of _{prostaglandin (PG) E 2} and arachidonic acid metabolites, which are potent mediators of inflammation. Cyclooxygenases (COX-1 and -2) are essential for the biosynthesis of these mediators. Another strategy is the development of inhibitors of inducible nitric oxide synthase (iNOS). iNOS is a cytotoxic inflammatory mediator and contributes to acute and chronic inflammation through the production of nitric oxide.

Conventional NSAIDs exhibit pharmacological action through inhibition of in vivo cyclooxygenase (hereinafter, referred to as COX) activity and reduction of biosynthesis of PG in local tissues. Currently, the COX inhibitory effect of NSAIDs is believed to be the basis of their pharmacodynamic effects. Therefore, COX protein is recognized as an indispensable target for drug development.

COX contains two isoforms that play different physiological roles. Constitutively expressed in various tissues, COX-1 is described as a housekeeping enzyme that regulates common cellular processes such as gastric cell protection, plasma homeostasis, platelet aggregation, and renal function. In contrast, COX-2 is maintained at very low levels in most tissues and is rapidly upregulated only in the inflammatory phase, which increases the production of prostanoids that occur at the site of disease and inflammation.

Conventional non-selective COX inhibitors such as aspirin, phenazone and indomethacin are effective in the treatment of inflammatory diseases, but the cytoprotective action of the constitutive COX-1 isoform in the gastrointestinal tract is reduced. Is associated with major defects that cause ulcer formation, liver and kidney toxicity. Therefore, COX-2 selective inhibitors such as celecoxib, rofecoxib and valdecoxib have been developed as anti-inflammatory agents with reduced gastrointestinal side effects. However, these COX-2 selective inhibitors have also been found to be associated with other side effects, such as cardiovascular disorders due to imbalance in the COX pathway, and are often withdrawn from the market.

Recently, Wallace et al. showed that inhibition of COX-1 could prevent the detrimental effects caused by COX-1 inhibition, such as gastric hypermobility and subsequent disorders caused by PG deficiency. It has been shown to induce upregulation. In addition, inhibition of COX-1 contributes significantly to the resolution of inflammation. In the ulcer healing process, not only the COX-2 specific inhibitor but also the COX-1 specific inhibitor slowed the healing. These findings suggest that, although COX-2 plays a pivotal role in the inflammatory process, balanced inhibitors of COX-1/COX-2 are in some respects given the lethal side effects of both non-selective or selective inhibitors. It shows that it appears more advantageous, and accordingly, it is necessary to develop a new COX inhibitor.

Korean Patent Publication No. 10-2004-0047862 (published on June 5, 2004)

In order to solve the above problems, the present invention provides a novel indole derivative as a COX inhibitor.

In addition, the present invention provides a composition for preventing or treating inflammatory diseases comprising the novel indole derivative as an active ingredient.

A compound represented by the following Formula 1 according to the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof,

[Formula 1]

In Formula 1, n is 0 or 1, R ¹ may be selected from halogen, and R ² is linear or branched (C1 to C12) alkyl, (C3 to C8) cycloalkyl, phenyl (C1 to C4) )Alkyl, (C1~C2)alkylthiazol-5-yl, furyl, hydrogen or may be selected from the following formula (2),

[Formula 2]

At this time, R ³ and R ⁴ may be the same or different, respectively, and may be selected from hydrogen, halogen, cyano, (C1 ~ C4) alkoxy, hydroxy, or carboxy.

The pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention may contain a compound represented by Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The health functional food composition for preventing or improving inflammatory diseases according to the present invention may contain a compound represented by Chemical Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

The compound according to the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, is a novel indole derivative as a COX inhibitor, reducing oxidative stress, and at least one selected from the group consisting of IKK, IκBα and NF-κB. It inhibits the activity by reducing phosphorylation, and can inhibit the expression of one or more pro-inflammatory factors selected from the group consisting of _{iNOS, NO, PGE 2, TNF-α and IL-6.}

Since the compound, or its stereoisomer, or a pharmaceutically acceptable salt thereof, has excellent anti-inflammatory activity as described above and has few side effects, it can be used as a pharmaceutical composition or a health functional food composition for the prevention, improvement or treatment of inflammatory diseases. And, through this, it is possible to safely and effectively treat and improve the disease.

1 schematically shows the design process of a COX inhibitor according to the present invention combining a partial structure of Herdmanin and a conventional NSAID.
Figure 2 is a graph showing the results of in vitro COX-1 and COX-2 enzyme inhibitory activity analysis of compounds 5a-5u and 6a-6b according to an experimental example of the present invention.
3 is a graph showing the cytotoxicity results of compound 5m according to an experimental example of the present invention.
Figure 4 shows the changes in the protein expression of pro-inflammatory factors iNOS and COX-2 according to an experimental example of the present invention, the top first image is LPS-induced iNOS and COX according to the concentration of compound 5m in RAW264.7 cells The expression level of -2 is shown, (A) is a graph showing the relative protein expression level of iNOS, and (B) is a graph showing the relative protein expression level of COX-2.
5 shows the change in the generation of pro-inflammatory factors according to an experimental example of the present invention, (A) is nitric oxide (NO), (B) is interleukin-6 (IL-6), (C) is a graph showing the degree of production of tumor necrosis factor α (TNF-α), and (D) is prostaglandin E ₂ (prostaglandin E ₂ , PGE _{2 ).}
6 shows the change in the production of reactive oxygen species (ROS) according to an experimental example of the present invention, (A) is ROS in LPS-induced cells, indicated by green fluorescence by DCFH-DA, a fluorescent probe, (B) is a graph obtained by quantifying fluorescence intensity with a fluorescent microplate reader.
Figure 7 shows the change in phosphorylation of NF-κB according to an experimental example of the present invention, (A) is a confocal microscope image of NF-κB p65, (B) is phosphorylation of NF-κB, (C) is IKK phosphorylation and (D) are graphs showing the phosphorylation of IκBα.
8 shows the anti-inflammatory mechanism of compound 5m as a COX inhibitor identified through an experimental example of the present invention.
9 is a docking (docking) and crystal structure of the ligand / COX binding according to an experimental example of the present invention.

Hereinafter, the present invention will be described in detail.

In order to develop an effective anti-inflammatory agent having an improved safety profile by herdmanines, an anti-inflammatory algae metabolite, the inventors of the present invention are COX-2, iNOS in Herdmanin C and D isolated from Herdmania momus. A series of substituted indole derivatives that combine their core structures were designed and synthesized by confirming the effect of inhibiting the production of, IL-6, NO, etc., and their inhibitory activity against COX-1 and COX-2 and anti-inflammatory By confirming the activity, the present invention was completed.

In the present specification, "prevention" means any action of inhibiting the occurrence of or delaying the onset of an inflammatory disease or at least one symptom of the disease by administration of the pharmaceutical composition or health functional food composition according to the present invention. . In addition, it includes treatment of a subject with remission to the disease to prevent or prevent recurrence.

In the present specification, "treatment" refers to any act of improving or beneficially altering the symptoms of an inflammatory disease, or at least one symptom of the disease, such as alleviating, reducing, or eliminating at least one symptom of the disease by administration of the pharmaceutical composition according to the present invention. Means.

In the present specification, the term "improvement" refers to an inflammatory disease, or at least one symptom of the disease by ingestion of the health functional food composition according to the present invention, alleviating, reducing, or eliminating the symptoms, etc. It means all actions.

In the present specification, the term "pharmaceutical composition" means a composition administered for a specific purpose, and for the purposes of the present invention, it is meant to be administered to prevent or treat an inflammatory disease, or at least one symptom of the disease.

In the present specification, the term "health functional food" includes foods manufactured and processed using raw materials or ingredients having functions useful for the human body according to the Health Functional Food Act No. 6727, and in addition to supplying nutrition, the object of the present invention It refers to foods with high medical effects and medicines processed to efficiently exhibit biomodulatory functions such as prevention of inflammatory diseases, body defense, immunity, and recovery.

The present invention provides a compound represented by the following formula (1), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

[Formula 1]

In Formula 1, n is 0 or 1, R ¹ may be selected from halogen, and R ² is linear or branched (C1-C12)alkyl, (C3-C8)cycloalkyl, phenyl (C1-C4) )Alkyl, (C1~C2)alkylthiazol-5-yl, furyl, hydrogen, or may be selected from Formula 2 below,

[Formula 2]

At this time, R ³ and R ⁴ may be the same or different, respectively, and may be selected from hydrogen, halogen, cyano, (C1 ~ C4) alkoxy, hydroxy, or carboxy.

Preferably, in Formula 1, R ² is linear or branched (C3-C8)alkyl, (C3-C6)cycloalkyl, phenyl (C1-C2)alkyl, methylthiazol-5-yl, furyl or the above It may be selected from Formula 2, and in Formula 2, R ³ and R ⁴ may be the same or different, respectively, and in hydrogen, halogen, cyano, (C1 ~ C2) alkoxy, hydroxy or carboxy Can be chosen.

The "alkyl" is a linear or branched saturated hydrocarbon, for example methyl, ethyl, isopropyl, isobutyl, tert-butyl, 2-methylbutyl, pentyl, hexyl, heptyl, octyl , Nonyl, and the like.

More specifically, the compound is (E)-1-(4-chlorobenzyl)-N'-(2-methylpropylidene)-1H-indole-3-carbohydrazide, (E)-1-( 4-chlorobenzyl)-N'-(2,2-dimethylpropylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(2-methyl Butylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(cyclopropylmethylene)-1H-indole-3-carbohydrazide, ( E)-1-(4-chlorobenzyl)-N'-(cyclohexylmethylene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-hexyl Den-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-nonylidene-1H-indole-3-carbohydrazide, (E)-1- (4-chlorobenzyl)-N'-(3-phenylpropylidene)-1H-indole-3-carbohydrazide, (E)-N'-benzylidene-1-(4-chlorobenzyl)-1H- Indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-hydroxybenzylidene)-1H-indole-3-carbohydrazide, (E)-1 -(4-chlorobenzyl)-N'-(4-chlorobenzylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-me Toxybenzylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydra Zide, (E)-N'-(4-bromobenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide, (E)-4-((2-(1 -(4-chlorobenzyl)-1H-indole-3-carbonyl)hydrazono)methyl)benzoic acid, (E)-1-(4-chlorobenzyl)-N'-(4-fluorobenzylidene)- 1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(3,5-dichlorobenzylidene)-1H-indole-3-carbohydrazide, (E )-1-(4-chlorobenzyl)-N'-(3,5-dibromobenzylidene)-1H-indole-3-carbohydrazide, (E)-N'-(3-chloro-5 -Methoxybenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-((4-methylthiazole -5-yl)methylene)-1H-indole-3-carbohydraji De, (E)-1-(4-chlorobenzyl)-N'-(furan-2-ylmethylene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzoyl) -N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide and (E)-1-(4-bromobenzoyl)-N'-(4-cyanobenzylidene)- 1H-indole-3-carbohydrazide may be selected from the group consisting of, more preferably (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole It may be -3-carbohydrazide, but is not limited thereto.

The compounds according to the invention may contain stereoisomers thereof. A "stereoisomer" refers to a molecular formula and a connection method of constituent atoms that are the same, but different spatial arrangements between atoms, and may be a diasteromer or an enantiomer. The enantiomer refers to an isomer that does not overlap with the mirror image, such as the relationship between the left hand and the right hand, and is also called an optical isomer. Enantiomers are classified into R (Rectus: clockwise) and S (sinister: counterclockwise) when four or more substituents are different on the chiral central carbon. Diastereomers are stereoisomers that are not in an enantiomeric relationship, and may include geometric isomers such as cis-trans isomers and E-Z isomers.

The compound according to the present invention may be used in the form of a pharmaceutically or food acceptable salt, and the salt may be a salt that does not cause serious irritation to the administered organism and does not impair the biological activity and physical properties of the compound.

The salt may be used in the form of either a pharmaceutically or food pharmaceutically acceptable basic salt or acid salt. The basic salt can be used in the form of either an organic base salt or an inorganic base salt, and sodium salt, potassium salt, calcium salt, lithium salt, magnesium salt, cesium salt, aminium salt, ammonium salt, triethyl salt It may be selected from the group consisting of an aminium salt and a pyridinium salt, but is not limited thereto.

Acid salts are useful acid addition salts formed by free acids. Inorganic acids and organic acids can be used as the free acid, hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, double phosphoric acid, nitric acid, etc. can be used as the inorganic acid, and citric acid, acetic acid, maleic acid, malic acid, fumaric acid, etc. can be used as the inorganic acid. , Glucose, 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, and the like can be used.

In addition, the compound according to the present invention may include all salts, hydrates, and solvates that can be prepared by conventional methods, as well as pharmaceutically or food acceptable salts. The addition salt can be prepared by a conventional method, and the compound is dissolved in a water-miscible organic solvent, such as acetone, methanol, ethanol, or acetonitrile, and an excessive amount of organic base is added or an aqueous base solution of an inorganic base is added, followed by precipitation. It can be prepared by making it or crystallizing it. Alternatively, the mixture may be prepared by evaporating a solvent or an excess base and drying to obtain an addition salt, or by suction filtration of the precipitated salt.

The compound according to the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, has a cyclooxygenase (COX) inhibitory activity, and thus can be used as a COX inhibitor, and COX-1 and COX-2 are used. I can restrain it in a balance. According to an experimental example of the present invention, it was confirmed that the compound has an inhibitory activity of COX-1 and/or COX-2, of which compound 5m has excellent inhibitory activity in both COX-1 and COX-2. appear. More details will be described later by the following experimental examples.

The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases containing a compound represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1, n is 0 or 1, R ¹ may be selected from halogen, and R ² is linear or branched (C1-C12)alkyl, (C3-C8)cycloalkyl, phenyl (C1-C4) )Alkyl, (C1~C2)alkylthiazol-5-yl, furyl, hydrogen, or may be selected from Formula 2 below,

[Formula 2]

At this time, R ³ and R ⁴ may be the same or different, respectively, and may be selected from hydrogen, halogen, cyano, (C1 ~ C4) alkoxy, hydroxy, or carboxy.

Preferably, in Formula 1, R ² is linear or branched (C3-C8)alkyl, (C3-C6)cycloalkyl, phenyl (C1-C2)alkyl, methylthiazol-5-yl, furyl or the above It may be selected from Formula 2, and in Formula 2, R ³ and R ⁴ may be the same or different, respectively, and in hydrogen, halogen, cyano, (C1 ~ C2) alkoxy, hydroxy or carboxy Can be chosen.

More specifically, the compound is (E)-1-(4-chlorobenzyl)-N'-(2-methylpropylidene)-1H-indole-3-carbohydrazide, (E)-1-( 4-chlorobenzyl)-N'-(2,2-dimethylpropylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(2-methyl Butylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(cyclopropylmethylene)-1H-indole-3-carbohydrazide, ( E)-1-(4-chlorobenzyl)-N'-(cyclohexylmethylene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-hexyl Den-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-nonylidene-1H-indole-3-carbohydrazide, (E)-1- (4-chlorobenzyl)-N'-(3-phenylpropylidene)-1H-indole-3-carbohydrazide, (E)-N'-benzylidene-1-(4-chlorobenzyl)-1H- Indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-hydroxybenzylidene)-1H-indole-3-carbohydrazide, (E)-1 -(4-chlorobenzyl)-N'-(4-chlorobenzylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-me Toxybenzylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydra Zide, (E)-N'-(4-bromobenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide, (E)-4-((2-(1 -(4-chlorobenzyl)-1H-indole-3-carbonyl)hydrazono)methyl)benzoic acid, (E)-1-(4-chlorobenzyl)-N'-(4-fluorobenzylidene)- 1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(3,5-dichlorobenzylidene)-1H-indole-3-carbohydrazide, (E )-1-(4-chlorobenzyl)-N'-(3,5-dibromobenzylidene)-1H-indole-3-carbohydrazide, (E)-N'-(3-chloro-5 -Methoxybenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-((4-methylthiazole -5-yl)methylene)-1H-indole-3-carbohydraji De, (E)-1-(4-chlorobenzyl)-N'-(furan-2-ylmethylene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzoyl) -N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide and (E)-1-(4-bromobenzoyl)-N'-(4-cyanobenzylidene)- 1H-indole-3-carbohydrazide may be selected from the group consisting of, more preferably (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole It may be -3-carbohydrazide, but is not limited thereto.

In the pharmaceutical composition according to the present invention, the composition can inhibit cyclooxygenase (COX), reduce the production of reactive oxygen species (ROS), and can reduce IKK (IκB-kinase), IκBα (An inhibitor of NF-κB) and NF-κB (nuclear factor-kappa B) can reduce one or more phosphorylation selected from the group consisting of. In addition, at least one selected from the group consisting of iNOS (inducible nitric oxide synthase), NO (nitric oxide), PGE ₂ (prostaglandin E ₂ ), TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) It can inhibit the expression of pro-inflammatory factors. As described above, the composition can suppress oxidative stress and have an excellent anti-inflammatory effect. It can be used as a pharmaceutical composition for the prevention or treatment of inflammatory diseases.

According to an experimental example of the present invention, compounds 5a to 5u and 6a to 6b synthesized according to the synthesis example of the present invention were confirmed to have inhibitory activity of COX-1 and/or COX-2, of which compound 5m Was shown to have excellent inhibitory activity in both COX-1 and COX-2. In addition, when compound 5m was treated in LPS-induced RAW264.7 cells, ROS production was inhibited, phosphorylation of IKK, IκBα and NF-κB was reduced, iNOS, NO, PGE ₂ , TNF-α, IL- It was confirmed that the expression of pro-inflammatory factors such as 6 is suppressed. More details will be described later by the following experimental examples.

In the pharmaceutical composition according to the present invention, the inflammatory disease may be an arthritis-related disease selected from the group consisting of degenerative arthritis (osteoarthritis), rheumatoid arthritis, ankylosing spondylitis, septic arthritis, and lupus arthritis.

In the present specification, the term "inflammatory diseases" is a generic term for diseases whose main lesion is the pathological condition of an abscess formed due to the invasion of bacteria, etc., arthritis, dermatitis, allergy, conjunctivitis, rhinitis, otitis media, gastritis, It may be one selected from the group consisting of colitis and acute and chronic inflammatory diseases, preferably arthritis, but is not limited thereto.

In the above diseases, "arthritis" is a representative cartilage-related disease, a disease name that collectively refers to inflammatory changes in the joint caused by some cause such as bacteria or trauma. This refers to the loss of cartilage. The arthritis may include degenerative arthritis (osteoarthritis), rheumatoid arthritis, ankylosing spondylitis, septic arthritis, lupus arthritis, and the like.

"Degenerative arthritis" is arthritis caused by gradual damage to the cartilage that protects the joint, and it is the most frequent among joint diseases, and is also called osteoarthritis.

"Rheumatoid arthritis" is a chronic inflammatory disease that occurs due to inflammation of a tissue called the synovial membrane surrounding the joint, and is a type of autoimmune disease, and symmetric polyarthritis is the most common.

"Ankylosing spondylitis" is a type of chronic arthritis characterized by a major lesion of the spine in which the spine becomes inflamed and the vertebral joints gradually harden.

"Septic arthritis" is a disease caused by invading joints by bacteria that have spread to the blood circulation, and various types of pathogens can cause this disease, among which Staphylococcus aureus is the most common causative agent.

"Lupus arthritis" is an arthritis observed in patients with lupus, a chronic autoimmune disease that causes inflammation of the whole body due to an abnormality in the immune system, and movement disorders may occur due to tissue changes around the joint.

The pharmaceutical composition according to the present invention can be prepared according to a conventional method in the pharmaceutical field.

The pharmaceutical composition according to the present invention may be blended with an appropriate pharmaceutically acceptable carrier according to the above formulation, and further includes excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc., if necessary. It can be manufactured by. The term "pharmaceutically acceptable" means that it is not toxic to cells or humans exposed to the pharmaceutical composition, and the appropriate carrier or the like is a compound according to the present invention, or a stereoisomer thereof, or a pharmaceutically acceptable It does not inhibit the activity and properties of the salt, and may be selected differently depending on the dosage form and formulation.

The pharmaceutical composition according to the present invention may be applied in any dosage form, and more particularly, may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

Among the oral dosage forms, the solid dosage form is in the form of tablets, pills, powders, granules, capsules, etc., and at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. It can be prepared by mixing, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of the capsul formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included.

Among the oral dosage forms, liquid dosage forms correspond to suspensions, liquid solutions, emulsions, syrups, and the like.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The present invention is not limited thereto, and any suitable agent known in the art may be used.

In addition, the pharmaceutical composition according to the present invention may further add _{calcium or vitamin D 3 to improve therapeutic efficacy.}

In the pharmaceutical composition according to the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

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

The pharmaceutical composition according to the present invention may be administered to any animal that may cause 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 according to the present invention may be administered by an appropriate route of administration according to the form of the formulation, and may be administered through various routes, either oral or parenteral, 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, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrabronchial inhalation, intrauterine dura mater or intracere-broventricular injection. .

The pharmaceutical composition according to the present invention may be used alone to prevent or treat 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 represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1, n is 0 or 1, R ¹ may be selected from halogen, and R ² is linear or branched (C1-C12)alkyl, (C3-C8)cycloalkyl, phenyl (C1-C4) )Alkyl, (C1~C2)alkylthiazol-5-yl, furyl, hydrogen, or may be selected from Formula 2 below,

[Formula 2]

At this time, R ³ and R ⁴ may be the same or different, respectively, and may be selected from hydrogen, halogen, cyano, (C1 ~ C4) alkoxy, hydroxy, or carboxy.

Preferably, in Formula 1, R ² is linear or branched (C3-C8)alkyl, (C3-C6)cycloalkyl, phenyl (C1-C2)alkyl, methylthiazol-5-yl, furyl or the above It may be selected from Formula 2, and in Formula 2, R ³ and R ⁴ may be the same or different, respectively, and in hydrogen, halogen, cyano, (C1 ~ C2) alkoxy, hydroxy or carboxy Can be chosen.

More specifically, the compound is (E)-1-(4-chlorobenzyl)-N'-(2-methylpropylidene)-1H-indole-3-carbohydrazide, (E)-1-( 4-chlorobenzyl)-N'-(2,2-dimethylpropylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(2-methyl Butylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(cyclopropylmethylene)-1H-indole-3-carbohydrazide, ( E)-1-(4-chlorobenzyl)-N'-(cyclohexylmethylene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-hexyl Den-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-nonylidene-1H-indole-3-carbohydrazide, (E)-1- (4-chlorobenzyl)-N'-(3-phenylpropylidene)-1H-indole-3-carbohydrazide, (E)-N'-benzylidene-1-(4-chlorobenzyl)-1H- Indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-hydroxybenzylidene)-1H-indole-3-carbohydrazide, (E)-1 -(4-chlorobenzyl)-N'-(4-chlorobenzylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-me Toxybenzylidene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydra Zide, (E)-N'-(4-bromobenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide, (E)-4-((2-(1 -(4-chlorobenzyl)-1H-indole-3-carbonyl)hydrazono)methyl)benzoic acid, (E)-1-(4-chlorobenzyl)-N'-(4-fluorobenzylidene)- 1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-(3,5-dichlorobenzylidene)-1H-indole-3-carbohydrazide, (E )-1-(4-chlorobenzyl)-N'-(3,5-dibromobenzylidene)-1H-indole-3-carbohydrazide, (E)-N'-(3-chloro-5 -Methoxybenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzyl)-N'-((4-methylthiazole -5-yl)methylene)-1H-indole-3-carbohydraji De, (E)-1-(4-chlorobenzyl)-N'-(furan-2-ylmethylene)-1H-indole-3-carbohydrazide, (E)-1-(4-chlorobenzoyl) -N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide and (E)-1-(4-bromobenzoyl)-N'-(4-cyanobenzylidene)- 1H-indole-3-carbohydrazide may be selected from the group consisting of, more preferably (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole It may be -3-carbohydrazide, but is not limited thereto.

In the health functional food composition according to the present invention, the composition can inhibit cyclooxygenase (COX), reduce the production of reactive oxygen species (ROS), and the group consisting of IKK, IκBα and NF-κB Phosphorylation of one or more selected from may be reduced. In addition, it is possible to inhibit the expression of one or more pro-inflammatory factors selected from the group consisting of iNOS, NO, PGE _{2, TNF-α and IL-6.} As described above, the composition can suppress oxidative stress and have an excellent anti-inflammatory effect. It can be used as a health functional food composition for preventing or improving inflammatory diseases.

In the health functional food composition according to the present invention, the inflammatory disease may be an arthritis disease selected from the group consisting of degenerative arthritis (osteoarthritis), rheumatoid arthritis, ankylosing spondylitis, septic arthritis, and lupus arthritis.

In the health functional food composition according to the present invention, the health functional food may be prepared as a powder, granule, tablet, capsule, syrup or beverage, and there is no limitation on the form that the health functional food can take. It can include all foods of meaning. For example, beverages and various drinks, fruits and processed foods thereof (canned fruit, jam, etc.), fish, meat and processed foods thereof (ham, bacon, etc.), bread and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ) And the like, and may include all functional foods in the usual sense. It may also include food used as feed for animals.

The health functional food composition according to the present invention may be prepared by further comprising a food pharmaceutically acceptable food additive (food additive) and other appropriate auxiliary ingredients commonly used in the art. Unless otherwise specified, the suitability as a food additive can be determined according to the standards and standards for the relevant item in accordance with the general rules and general test methods for food additives approved by the Food and Drug Administration.

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

The effective dose of the compound, or a stereoisomer thereof, or a pharmaceutically acceptable 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 prevention or improvement of inflammatory diseases.

The composition has the advantage of not having side effects that may occur when taking a general drug for a long time by using food as a raw material, has excellent portability, and 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, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art.

Example 1 Design of COX inhibitors based on natural anti-inflammatory compounds and clinically used NSAIDs.

1 schematically shows the design process of a COX inhibitor according to the present invention combining a partial structure of Herdmanin and a conventional NSAID.

Referring to FIG. 1, the structures of Herdmanin C and D are characterized by guanidyl and indole moieties, respectively. Indomethacin is an indole derivative, and darbufelone and meloxicam have a partial structure chemically similar to the guanidyl group. Therefore, the target molecule was designed as an indole derivative having an acylhydrazone residue as an isostere of a guanidyl residue.

The acylhydrazone residue is characterized by an open chain R ¹ -acyl-NHN=CH-R ² group, and this hydrogen bond donor structure is the _{guanidyl residue of Herdmanin C (HNC(NH 2} ) ₂ ) Is similar to Indole and its derivatives can be used as a major template for drug design including anti-inflammatory agents, and the acylhydrazone residue can be used as a drug molecular structure block (pharmacophore block) for the synthesis of various novel bioactive compounds. , Hydrazone derivatives may have anti-inflammatory activity including COX inhibitory activity.

Halobenzyl or halobenzoyl residues also mimic the partial structure of indomethacin and promote hydrophobic interactions with major amino acid residues in the ligand binding domain of COX. Included in the target scaffold. Since halobenzyl or halobenzoyl residues are important for binding to the COX active site and stabilizing hydrophobic interactions with major amino acids of COX, synthetic derivatives are indole, acylhydrazone, and halobenzyl/halobenzoyl residues as shown in FIG. It was designed by combining.

In addition, based on the original design of the present invention, the hydrogen bond donor acylhydrazone residue was placed at the C-3 position of the indole, and the physicochemical properties of the alkyl and aryl groups were changed (compounds 4a-4u). , In compound 5a-5u, a chlorobenzyl residue was introduced at the N-1 position of indole, and a halobenzoyl residue was introduced in compound 6a-6b.

<Synthesis Example 1> Synthesis of a target compound as a COX inhibitor

1. Preparation for synthesis

All reagents used were commercially available, and some organic solvents were re-distilled under positive pressure if necessary. The reaction was observed by thin-layer chromatography (hereinafter, TLC) on a glass plate coated with silica gel using a fluorescent indicator (GF254, Merck, Germany). ¹ H and ¹³ C NMR spectral data were recorded with Varian Unity 500 MHz and 400 MHz NMR spectroscopy respectively. High-resolution fast-atom bombardment mass spectrometry (HRFABMS) data were obtained using an Agilent 1200 UHPLC accurate-mass Q-TOF MS spectrometer. All chemical reagents were purchased from Sigma-Aldrich and Alfa Aesar.

2. Synthesis method

Scheme 1 below shows the process of synthesizing target compounds according to the present invention.

<Reaction Scheme 1>

* Reagents and conditions: (i) diluted H ₂ SO ₄ , MeOH, reflux, 24 h; (ii) NH ₂ -NH ₂ .H ₂ O, EtOH, reflux, 2 h; (iii) RCHO/EtOH, propionic acid, reflux, 2.5 h; (iv) 4-Cl-PhCH ₂ Cl, NaH, DMF, RT, 24 h; (v) 4-Cl-PhCOCl/4-Br-PhCOCl, NaH, DMF, RT, 24 h.

2-1. Synthesis of compound 2

Indole-3-carboxylic acid (1.0 mmol-equiv.) solution and an appropriate amount of anhydrous methyl alcohol (25 mL) in the presence of a few drops of sulfuric acid (97%) for 24 hours During reflux (reflux). The progress of the reaction was monitored by TLC (Silica gel 60F ₂₅₄ , Merck, Germany). The precipitated 1H-indole-3-carboxylate methyl ester was filtered and recrystallized from an ethanol-water (3:2) mixture to give compound 2 (77%).

Compound 2: Methyl 1H-indole-3-carboxylate , white powder, 77% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.94 (s, 1H), 8.09 (s, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.26-7.14 (m, 2H) ), 3.81 (s, 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.8, 136.4, 132.4, 125.7, 122.4, 121.3, 120.4, 112.4, 106.3, 50.6. HRFABMS m/z 176.0673 [M+H] ⁺ (calcd for C ₁₀ H ₉ NO ₂ , 176.0706).

2-2. Synthesis of indole N-arylhydrazone derivatives 4a-4u

Hydazine hydrate (12.5g, 0.25M) and Compound 2 1H-indole-3-carboxylate methyl ester (1H-Indole-3-carboxylate methyl ester, 1.0 mmol-equiv.) in an appropriate amount of ethanol (30 mL) It was refluxed for 2 hours. The progress of the reaction was monitored by TLC. After the reaction mixture was cooled to room temperature, the mixture was filtered without purification to obtain a white solid crude product, compound 3 (96%).

Next, indole-hydrazide (1.0 mmol) of Compound 3 dissolved in ethanol (30 mL) was variously substituted with appropriate amounts of aldehyde (1.5 mmol-equiv.) and a few drops of propionic acid. Was added dropwise, stirred for 2.5 hours, and refluxed. After cooling, the precipitate was filtered and washed several times with methanol to obtain a crystalline indole N-arylhydrazone derivative 4a-4u. The resulting compounds 4a-4u were used as starting compounds for the synthesis of indolyl-N-substituted benzyl/benzoyl derivatives.

2-3. Synthesis of indolyl-N-substituted benzyl/benzoyl derivatives 5a-5u/6a-6b

DMF solution in which compound 4a-4u (1 mmol) was dissolved was added to a solution of dimethylfomamide (hereinafter, DMF, 10 mL) in which sodium hydride (NaH, 1.5 mmol) was dissolved, and the reaction mixture was added at room temperature. Stir for 1 hour. Benzyl chloride/benzoyl chloride (1.5 mmol) was added, and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was poured into ice water, filtered, and washed several times with methanol to obtain a target compound, an indolyl-N-substituted benzyl/benzoyl derivative 5a-5u/6a-6b.

The final product was characterized through ¹ H-NMR, ¹³ C-NMR and mass spectrometric analysis. The geometry of the carbon-nitrogen double bond in 5a-5u/6a-6b was determined as (E) through comparison ^{of the 1 H-NMR data reported for the N-arylhydrazone derivative.} The chemical shift of HC=N hydrogen in 5a-5h (δ _H 7.49-7.55), 5i-5u and 6a-6b (δ _H 8.22-8.37) is (E) chemical shift of the N-arylhydrazone derivative of the geometry. Is almost the same as The sterically stable E-isomer predominated over the sterically hindered Z-isomer.

Compound 5a: (E)-1-(4-chlorobenzyl)-N'-(2-methylpropylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl)- N'-(2-methylpropylidene)-1H-indole-3-carbohydrazide ], white powder, 35% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.92 (s, 1H), 8.20 (s, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.41 (m, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.18 (m, 2H), 7.17 (m, 2H), 5.49 (s, 2H), 2.53 (m, 1H), 1.06 (d, J = 6.5 Hz, 6H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.1, 155.2, 136.0, 132.3, 129.3, 128.7 (×6), 122.4, 121.0 (×2), 110.6 (×2), 48.7, 30.9, 19.7 (×2). HRFABMS m/z 354.1358 [M+H] ⁺ (calcd for C ₂₀ H ₂₀ ClN ₃ O, 354.1368).

Compound 5b : (E)-1-(4-chlorobenzyl)-N'-(2,2-dimethylpropylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl )-N'-(2,2-dimethylpropylidene)-1H-indole-3-carbohydrazide ], white powder, 55% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.87 (s, 1H), 8.22 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 7.7 Hz, 2H), 7.19 (m, 2H), 7.17 (m, 2H), 5.48 (s, 2H), 1.07 (s, 9H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 165.6, 156.1, 135.9, 132.3, 129.3, 128.7 (×6), 122.4, 121.0 (×2), 110.6 (×2), 48.7, 34.4, 19.7 (×3). HRFABMS m/z 368.1525 [M+H] ⁺ (calcd for C ₂₁ H ₂₂ ClN ₃ O, 368.1524).

Compound 5c: (E)-1-(4-chlorobenzyl)-N'-(2-methylbutylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl) -N'-(2-methylbutylidene)-1H-indole-3-carbohydrazide ], white powder, 42% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.95 (s, 1H), 8.20 (s, 1H), 7.54 (d, J = 7.9 Hz, 1H), 7.42 (m, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.20 (m, 2H), 7.17 (m, 2H), 5.50 (s, 2H), 2.31 (m, 1H), 1.50 (m, 1H), 1.40 (m, 1H), 1.05 (d, J = 6.2 Hz , 3H), 0.90 (t, J = 7.4 Hz, 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ=ppm) δ 164.3, 153.9, 136.3, 132.3, 129.3, 128.7 (×6), 122.4, 121.1 (×2), 110.6 (×2), 48.7, 37.6, 27.0, 17.3, 11.4. HRFABMS m/z 368.1525 [M+H] ⁺ (calcd for C ₂₁ H ₂₂ ClN ₃ O, 368.1524).

Compound 5d : (E)-1-(4-chlorobenzyl)-N'-(cyclopropylmethylene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl)-N' -(cyclopropylmethylene)-1H-indole-3-carbohydrazide ], white powder, 60% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.91 (s, 1H), 8.15 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39 (m, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.16 (m, 2H), 7.14 (m, 2H), 5.48 (s, 2H), 1.66 (m, 1H), 0.86 (dd, J = 8.1, 2.2 Hz, 2H), 0.63 (dd, J = 6.6, 4.1 Hz, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 165.3, 154.1, 136.2, 132.2, 129.1 (×2), 128.6 (×4), 122.3, 121.5, 120.9 (×2), 110.5 (×2), 48.7, 13.4, 5.8 (×2) . HRFABMS m/z 352.1205 [M+H] ⁺ (calcd for C ₂₀ H ₁₈ ClN ₃ O, 352.1211).

Compound 5e : (E)-1-(4-chlorobenzyl)-N'-(cyclohexylmethylene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl)-N' -(cyclohexylmethylene)-1H-indole-3-carbohydrazide ], white powder, 45% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.93 (s, 1H), 8.13 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 7.18 (m, 2H), 7.16 (m, 2H), 5.49 (s, 2H), 2.23 (m, 1H), 1.73 (m, 4H), 164 (m, 1H), 1.29 (m , 1H), 1.24 (m, 4H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.1, 152.8, 136.2, 132.3, 129.2, 128.6 (×4), 122.3 (×2), 121.5, 121.0 (×2), 110.5 (×2), 48.7, 29.8 (×2), 25.6 , 25.1 (×3). HRFABMS m/z 394.1668 [M+H] ⁺ (calcd for C ₂₃ H ₂₄ ClN ₃ O, 394.1681).

Compound 5f: (E)-1-(4-chlorobenzyl )-N'-hexylidene-1H-indole-3-carbohydrazide[ (E)-1-(4-Chlorobenzyl)-N'-hexylidene -1H-indole-3-carbohydrazide ], white powder, 50% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.96 (s, 1H), 8.19 (s, 1H), 7.52 (d, J = 7.9 Hz, 1H), 7.40 (m, 2H), 7.25 (m, 2H), 7.18 (m, 2H) ), 7.15 (m, 2H), 5.50 (s, 2H), 2.24 (m, 2H), 1.49 (m, 2H), 1.29 (m, 4H), 0.87 (t, J = 5.6 Hz, 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 166.3, 153.8, 136.2 (×2), 132.2, 129.0 (×2), 128.6 (×2), 122.3 (×2), 121.5, 120.9 (×2), 110.5 (×2), 48.7 , 31.7, 30.7, 25.7, 21.8, 13.7. HRFABMS m/z 382.1695 [M+H] ⁺ (calcd for C ₂₂ H ₂₄ ClN ₃ O, 382.1681).

Compound 5g: (E)-1-(4-chlorobenzyl )-N'-nonylidene-1H-indole-3-carbohydrazide[ (E)-1-(4-Chlorobenzyl)-N'-nonylidene -1H-indole-3-carbohydrazide ], white powder, 55% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 10.95 (s, 1H), 8.17 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.16 (m, 2H), 7.15 (m, 2H), 5.48 (s, 2H), 2.23 (dd, J = 12.9, 7.3 Hz, 2H), 1.47 (m, 2H), 1.24 (m , 10H), 0.83 (t, J = 6.4 Hz, 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 165.5, 154.5, 136.2 (×2), 132.2, 128.9 (×2), 128.5 (×2), 122.3×2, 121.5, 120.9×2, 110.5 (×2), 48.7, 31.7, 31.1 , 28.7, 28.5, 26.0, 23.2, 21.9, 13.8. HRFABMS m/z 424.2143 [M+H] ⁺ (calcd for C ₂₅ H ₃₀ ClN ₃ O, 424.2150).

Compound 5h: (E)-1-(4-chlorobenzyl)-N'-(3-phenylpropylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl)- N'-(3-phenylpropylidene)-1H-indole-3-carbohydrazide ], white powder, 65% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.04 (s, 1H), 8.17 (s, 1H), 7.59 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 7.26 (m, 6H), 7.17 (m, 2H), 7.14 (m, 2H), 5.47 (s, 2H), 2.82 (t, J = 7.7 Hz, 2H), 2.57 (dt, J = 12.7, 6.3 Hz , 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ=ppm) δ 163.8, 153.3, 141.1, 136.2 (×2), 135.9, 132.3, 129.1, 128.7 (×2), 128.4 (×2), 128.3 (×2), 125.9, 122.4 (×2), 121.5 , 121.1 (×2), 110.6 (×2), 48.8, 33.6, 32.2. HRFABMS m/z 416.1517 [M+H] ⁺ (calcd for C ₂₅ H ₂₂ ClN ₃ O, 416.1524).

Compound 5i : (E)-N'-benzylidene-1-(4-chlorobenzyl)-1H-indole-3 -carbohydrazide[ (E)-N'-benzylidene-1-(4-chlorobenzyl)- 1H-indole-3-carbohydrazide ], white powder, 45% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.48 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.68 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.44 (m, 5H) ), 7.34 (s, 2H), 7.22 (m, 2H), 7.21 (m, 2H), 5.56 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.4, 154.3, 136.2 (×2), 135.9, 134.6 (×2), 132.3, 129.5, 129.3, 128.8 (×2), 128.7 (×2), 126.7, 122.5 (×2), 121.5 , 121.2 (×2), 110.6 (×2), 48.8. HRFABMS m/z 388.1198 [M+H] ⁺ (calcd for C ₂₃ H ₁₈ ClN ₃ O, 388.1211).

Compound 5j : (E)-1-(4-chlorobenzyl)-N'-(4-hydroxybenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl) -N'-(4-hydroxybenzylidene)-1H-indole-3-carbohydrazide ], white powder, 80% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.70 (s, 1H), 11.26 (s, 1H), 8.23 (s, 1H), 8.20 (s, 1H), 7.66 (d, J = 8.6 Hz, 1H), 7.50 (m, 5H) ), 7.18 (m, 2H), 7.16 (m, 2H), 7.10 (d, J = 8.7 Hz, 2H), 5.17 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 168.9, 159.2, 156.4, 135.8, 132.4, 129.4 (×3), 128.4 (×3), 128.2 (×2), 127.7, 122.1 (×2), 121.1, 120.6 (×2), 115.2 (×2), 111.8, 68.5. HRFABMS m/z 404.1165 [M+H] ⁺ (calcd for C ₂₃ H ₁₈ ClN ₃ O ₂ , 404.1160).

Compound 5k: (E)-1-(4-chlorobenzyl)-N'-(4-chlorobenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl)- N'-(4-chlorobenzylidene)-1H-indole-3-carbohydrazide ], white powder, 85% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.46 (s, 1H), 8.30 (s, 1H), 8.22 (s, 1H), 7.67 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 7.30 (d, J = 7.3 Hz, 2H), 7.21 (m, 2H), 7.18 (m, 2H), 5.53 (s, 2H) ). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 166.1, 154.3, 136.1, 135.8, 133.8, 133.5, 132.3, 129.2 (×2), 128.8 (×2), 128.6 (×2), 128.2 (×2), 122.5 (×2), 121.4 , 121.2 (×2), 110.6 (×2), 48.7. HRFABMS m/z 422.0828 [M+H] ⁺ (calcd for C ₂₃ H ₁₇ Cl ₂ N ₃ O, 422.0821).

Compound 5l : (E)-1-(4-chlorobenzyl)-N'-(4-methoxybenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl) -N'-(4-methoxybenzylidene)-1H-indole-3-carbohydrazide ], white powder, 85% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.31 (s, 1H), 8.24 (s, 2H), 7.60 (s, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.45 (m, 2H), 7.33 (m, 2H) ), 7.22 (m, 2H), 7.20 (m, 2H), 7.01 (d, J = 8.3 Hz, 2H), 5.54 (s, 2H), 3.81 (s, 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 163.9, 160.4, 154.3, 136.2 (×2), 135.2, 132.4, 129.3, 128.6 (×2), 128.2 (×2), 127.2, 122.4 (×2), 121.5, 121.1 (×2) , 114.3 (×2), 110.5 (×2), 55.2, 48.7. HRFABMS m/z 418.1322 [M+H] ⁺ (calcd for C ₂₄ H ₂₀ ClN ₃ O ₂ , 418.1317).

Compound 5m : (E)-1-(4-chlorobenzyl)-N'-(4-cyanobenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl) -N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide ], white powder, 58% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.49 (s, 1H), 8.32 (s, 1H), 8.23 (s, 1H), 7.64 (m, 3H), 7.57 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 7.2 Hz, 2H), 7.22 (m, 2H), 7.21 (m, 2H), 5.55 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 168.2, 157.4, 139.8, 136.8, 136.6, 133.3 (×2), 133.1, 130.0, 129.4 (×4), 129.2, 127.9 (×2), 123.3, 122.1, 122.0 (×2), 119.3 , 111.9, 111.4, 49.4. HRFABMS m/z 413.1168 [M+H] ⁺ (calcd for C ₂₄ H ₁₇ ClN ₄ O, 413.1164).

Compound 5n : (E)-N'-(4-bromobenzylidene)-1-(4-chlorobenzyl)-1H-indole-3 -carbohydrazide[ (E)-N'-(4-bromobenzylidene )-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide ], yellow powder, 65% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.66 (s, 1H), 8.35 (s, 1H), 8.23 (s, 1H), 7.90 (m, 1H), 7.86 (s, 2H), 7.58 (d, J = 8.2 Hz, 1H ), 7.45 (d, J = 7.2 Hz, 2H), 7.32 ( J = 7.7 Hz, 2H), 7.23 (m, 2H), 7.21 (m, 2H), 5.56 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.8, 155.7, 136.1 (×2), 135.8, 133.9, 132.3 (×2), 131.7, 129.2 (×2), 128.6 (×2), 128.5 (×2), 122.5 (×2) , 121.5, 121.2 (×2), 110.6 (×2), 48.7. HRFABMS m/z 466.0314 [M+H] ⁺ (calcd for C ₂₃ H ₁₇ BrClN ₃ O, 466.0316).

Compound 5o : (E)-4-((2-(1-(4-chlorobenzyl)-1H-indole-3-carbonyl)hydrazono)methyl)benzoic acid [ (E)-4-((2- (1-(4-Chlorobenzyl)-1H-indole-3-carbonyl)hydrazono)methyl)benzoic acid ], white powder, 78% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.61 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.82 (s, 1H), 7.58 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.49 (m, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.32 (d, J = 7.8 Hz, 2H), 7.24 (m, 2H), 7.22 (m, 2H) ), 5.37 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 166.3, 165.1, 155.2, 139.2, 136.1, 135.0, 132.3, 129.8 (×2), 129.7 (×2), 129.2, 128.6 (×2), 128.4 (×2), 126.8, 122.5 (× 2), 121.4, 121.2 (×2), 110.6, 48.7. HRFABMS m/z 432.0966 [M+H] ⁺ (calcd for C ₂₄ H ₁₈ ClN ₃ O ₃ , 432.0964).

Compound 5p : (E)-1-(4-chlorobenzyl)-N'-(4-fluorobenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl) -N'-(4-fluorobenzylidene)-1H-indole-3-carbohydrazide ], white powder, 85% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.43 (s, 1H), 8.31 (s, 1H), 8.23 (s, 1H), 7.72 (s, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 2H), 7.30 (m, 2H), 7.27 (d, J = 8.7 Hz, 2H), 7.21 (m, 2H), 7.17 (m, 2H), 5.54 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ=ppm) δ 165.6, 161.5, 156.9, 136.1, 132.3, 131.2, 129.2 (×2), 128.8 (×2), 128.7 (×2), 128.6 (×2), 122.4, 121.4, 121.1 (×2) , 115.8, 115.6, 110.6 (×2), 48.7. HRFABMS m/z 406.1112 [M+H] ⁺ (calcd for C ₂₃ H ₁₇ ClFN ₃ O ₃ , 406.1117).

Compound 5q : (E)-1-(4-chlorobenzyl)-N'-(3,5-dichlorobenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl )-N'-(3,5-dichlorobenzylidene)-1H-indole-3-carbohydrazide ], white powder, 86% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.71 (s, 1H), 8.37 (s, 1H), 8.21 (s, 1H), 7.74 (s, 2H), 7.58 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 7.7 Hz, 2H), 7.22 (m, 2H), 7.20 (m, 2H), 5.55 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.5, 155.2, 136.2 (×2), 134.5 (×2), 129.0 (×2), 128.9 (×2), 128.6 (×2), 128.4, 124.9 (×2), 122.5 (× 2), 121.8, 121.2 (×2), 110.6 (×2), 48.7. HRFABMS m/z 456.0426 [M+H] ⁺ (calcd for C ₂₃ H ₁₆ Cl ₃ N ₃ O, 456.0286).

Compound 5r : (E)-1-(4-chlorobenzyl)-N'-(3,5-dibromobenzylidene)-1H-indole-3-carbohydrazide[ (E)-1-(4 -Chlorobenzyl)-N'-(3,5-dibromobenzylidene)-1H-indole-3-carbohydrazide ], yellow powder, 75% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.71 (s, 1H), 8.36 (s, 1H), 8.21 (s, 1H), 7.88 (s, 2H), 7.57 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 7.4 Hz, 2H), 7.22 (m, 2H), 7.20 (m, 2H), 5.54 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 162.8, 157.8, 140.8, 138.8, 136.2, 133.6, 132.3, 129.0 (×3), 128.6 (×3), 128.1 (×2), 122.8 (×2), 122.5, 121.2 (×2) , 110.6 (×2), 48.7. HRFABMS m/z 543.9271 [M+H] ⁺ (calcd for C ₂₃ H ₁₆ Br ₂ ClN ₃ O, 543.9276).

Compound 5s : (E)-N'-(3-chloro-5-methoxybenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-carbohydrazide[ (E)-N'- (3-chloro-5-methoxybenzylidene)-1-(4-chlorobenzyl)-1H-indole-3-Carbohydrazide ], white powder, 75% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.45 (s, 1H), 8.30 (s, 1H), 8.23 (s, 1H), 7.94 (d, J = 10.2 Hz, 1H), 7.65 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.32 (s, 2H), 7.21 (m, 2H), 7.19 (m, 2H), 5.53 (s, 2H), 3.90 (s , 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 166.0, 156.2, 136.2 (×2), 132.3, 130.6, 129.1 (×2), 128.7, 128.6 (×4), 127.6, 122.4 (×2), 121.4, 121.1 (×2), 112.7 , 111.1, 110.6, 56.4, 48.7. HRFABMS m/z 496.0259 [M+H] ⁺ (calcd for C ₂₄ H ₁₉ BrClN ₃ O ₂ , 496.0276).

Compound 5t : (E)-1-(4-chlorobenzyl)-N'-((4-methylthiazol-5-yl)methylene)-1H-indole-3-carbohydrazide[ (E)-1 -(4-Chlorobenzyl)-N'-((4-methylthiazol-5-yl)methylene)-1H-indole-3-carbohydrazide ], yellow powder, 88% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.49 (s, 1H), 9.00 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H), 8.20 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H ), 7.40 (d, J = 8.2 Hz, 2H), 7.29 (s, 2H), 7.19 (m, 2H), 5.49 (s, 2H), 2.45 (s, 3H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 164.5, 154.1, 153.0, 136.2, 132.4, 129.3, 128.8 (×4), 128.1 (×2), 122.6 (×2), 121.4, 121.3 (×2), 110.7 (×2), 48.8 , 15.3. HRFABMS m/z 409.0873 [M+H] ⁺ (calcd for C ₂₁ H ₁₇ ClN ₄ OS, 409.0884).

Compound 5u : (E)-1-(4-chlorobenzyl)-N'-(furan-2-ylmethylene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzyl) -N'-(furan-2-ylmethylene)-1H-indole-3-carbohydrazide ], yellow powder, 67% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.50 (s, 1H), 8.36 (s, 1H), 8.25 (s, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 7.1 Hz, 2H), 7.21 (m, 2H), 7.20 (m, 2H), 6.86 (d, J = 3.0 Hz, 1H), 6.63 (dd, J = 3.1, 1.6 Hz, 1H ), 5.53 (s, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 166.8, 149.8, 144.5, 136.1 (×2), 135.8, 132.3, 129.2 (×2), 128.6 (×4), 122.5 (×2), 121.5, 121.2 (×2), 112.0, 110.6 , 48.8. HRFABMS m/z 378.0840 [M+H] ⁺ (calcd for C ₂₁ H ₁₆ ClN ₃ O ₂ , 378.0858).

Compound 6a : (E)-1-(4-chlorobenzoyl)-N'-(4-cyanobenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Chlorobenzoyl) -N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide ], white powder, 88% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.84 (s, 1H), 8.32 (s, 1H), 8.26 (s, 1H), 7.89 (s, 1H), 7.87 (m, 2H), 7.85 (m, 2H), 7.70 (m , 2H), 7.66 (s, 1H), 7.41 (m, 2H), 7.39 (m, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ=ppm) δ 167.7, 163.5, 159.6, 132.5 (×3), 131.3, 128.9 (×6), 127.4, 125.4 (×2), 124.7 (×2), 122.0, 118.5, 115.6 (×2), 111.7 (×2). HRFABMS m/z 427.0961 [M+H] ⁺ (calcd for C ₂₄ H ₁₅ ClN ₄ O ₂ , 427.0956).

Compound 6b: (E)-1-(4-bromobenzoyl)-N'-(4-cyanobenzylidene)-1H-indole-3 -carbohydrazide[ (E)-1-(4-Bromobenzoyl )-N'-(4-cyanobenzylidene)-1H-indole-3-carbohydrazide ], white powder, 85% yield;

¹ H-NMR (500 MHz, DMSO- d ₆ , δ = ppm) δ 11.88 (s, 1H), 8.36 (s, 1H), 8.29 (s, 1H), 7.90 (s, 1H), 7.88 (m, 2H), 7.87 (m, 2H), 7.83 (m , 2H), 7.72 (s, 1H), 7.46 (m, 2H), 7.43 (m, 2H). ¹³ C-NMR (125 MHz, DMSO- d ₆ , δ = ppm) δ 168.4, 160.2, 157.4, 145.3, 142.9, 139.3, 133.3 (×2), 132.6 (×5), 132.1, 128.0, 126.1, 125.4 (×2), 122.4, 119.2, 116.3 (×2) , 112.4 (×2). HRFABMS m/z 471.0441 [M+H] ⁺ (calcd for C ₂₄ H ₁₅ BrN ₄ O ₂ , 471.0451).

<Experimental Example 1> Evaluation of the physiological activity of the target compound

1. Experimental method

1-1. Cell culture

RAW264.7 murine macrophages were purchased from Korean Cell Line Bank (KCLB®, Seoul, Korea), rat liver cells Ac2F and mouse fibroblasts L929 were ATCC (American Type Culture Collection, Rockville, MD, USA). Cells were cultured in a humidified incubator at 37° C., 5% CO ₂ , and 100 mg/mL streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated cattle. It was maintained in high-glucose Dulbecco's Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan) to which fetal bovine serum (FBS) was added.

1-2. Cytotoxicity assay

A suspension of the tested cell line (1.0×10 ⁴ cells/well) was seeded into a 96-well culture plate and incubated for 12 hours, followed by treatment with 5m of a compound of various dilution concentrations for 24 hours. Control cells were treated with culture medium alone. The test compound was evaluated in three dilutions (10, 20 and 50 μM), and the highest concentration was 50 μM. A water soluble tetrazolium (WST) reagent (EZ-CyTox, Daeil Lab Service Co., Ltd., Seoul, Korea) was added to each well (10 μL) and cultured at 37°C for 1 hour to ensure cell viability ( Cell viability) was evaluated. The absorbance at 450 nm was measured using a microplate absorbance reader (iMark Microplate Absorbance Reader, Bio-Rad Laboratories, Hercules, CA, USA). Cells in the exponential phase were used in all experiments.

1-3. Cyclooxygenase (COX) inhibitory ability analysis

Analysis was performed according to the manufacturer's protocol using a COX fluorescence inhibitor screening assay kit (Cayman Chemical, MI, USA). COX-1 (sheep) and COX-2 (human recombination) were _{used to catalyze arachidonic acid to PGG 2} , which is 10-acetyl-3,7-dihydroxyphenoxazine (10-acetyl-3,7-dihydroxyphenoxazine). , ADHP) to produce a solid fluorescent compound resorufin. Resorupine fluorescence can be analyzed using an excitation wavelength of 510 nm and an emission wavelength of 580 nm. Briefly, 150 μL assay buffer, 10 μL heme, 10 μL enzyme (COX-1 or COX-2), and 10 μL vehicle were mixed in one well to serve as 100% initial active wells; 160 μL assay buffer, 10 μL heme and 10 μL vehicle were mixed in one well and served as background wells; 150 μL assay buffer, 10 μL heme, and 10 μL enzyme (COX-1 or COX-2) and 10 μL compound were mixed in one well and served as inhibitor wells.

After incubating the plate for 5 minutes at room temperature, 10 μL ADHP was added to each well. To start the reaction, 10 μL of arachidonic acid solution was quickly added to each well, incubated for 2 minutes at room temperature, and then excitation wavelength of 510 nm with a multimode microplate reader (TriStar LB 941 Multimode Microplate Reader, Bad Wildbad, Germany). And an emission wavelength of 580 nm was used to measure the fluorescence spectrum.

1-4. Proinflammatory factor activity assay

RAW264.7 macrophages (cal. 1×10 ⁴ cells/well) were inoculated into a 96-well culture plate and cultured for 12 hours. Cells were pretreated with drugs of various concentrations (2.5, 5, 10 μM) for 1 hour, and then co-cultured with 25 ng/mL LPS (Lipopolysaccharide) for 24 hours.

The NO concentration in the medium was determined using a Grease assay. Grease reagent (80 μL) was added to the supernatant of the medium, followed by incubation at 37°C for 15 minutes in the dark. Absorbance at 520 nm was measured using an iMark Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA). NO concentration was calculated using 0-100 μM sodium nitrite standards.

TNF-α and IL-6 expression in the culture medium was quantified using a sandwich-type ELISA kit (Biolegend, San Diego, CA, USA), and absorbance at 450 nm was measured. According to _{the protocol of the PGE 2} expression ELISA kit (Cayman Chemical, MI, USA) _{, the production of prostaglandin E 2} (PGE ₂ ) in the medium was determined using this, and absorbance was measured at 415 nm.

1-5. Reactive oxygen species (hereinafter, ROS) analysis

RAW264.7 macrophages were cultured in a confocal dish and the test compound was treated for 1 hour. The LPS solution was added to the dish at a final concentration of 1 μg/mL, and incubation was continued for 24 hours. The medium was removed and washed with PBS; The diluted DCFH-DA was added to the FBS-free medium and incubated at 37° C. for 30 minutes so that the final concentration became 100 μM. The medium was removed and the cells were washed once in PBS. Fluorescence was confirmed using a confocal microscope (FluoView FV10i; Olympus, Australia) having an excitation wavelength of 485 nm and an emission wavelength of 520 nm.

Quantification of cellular oxidative stress was performed with a slight modification of the previously reported method. RAW264.7 macrophages (cal. 1×10 ⁴ cells/well) were inoculated into a black 96-well cell culture plate and cultured for 12 hours. The cells were pretreated with the test compound for 1 hour and then co-cultured with 1 μg/mL LPS for 24 hours. The medium was removed and washed with PBS; 100 μL of diluted DCFH-DA was added to the FBS-free medium to a final concentration of 100 μM, followed by incubation at 37°C for 30 minutes. The medium was removed, and 100 μL PBS was added to each well, and fluorescence was detected at 485 nm excitation wavelength and 520 nm emission wavelength with TriStar LB 941 Multimode Microplate Reader (Bad Wildbad, Germany).

1-6. Immunofluorescence staining analysis of NF-κB p65

RAW264.7 cells were cultured in a confocal dish to treat the test compound for 24 hours. Then, the cells were fixed in 10% formalin solution for 15 minutes, washed 3 times with PBS, treated with 0.5% (v/v) Triton X-100/PBS for 15 minutes, washed 3 times with PBS, and then washed 3 times with PBS at room temperature. Blocked with 10% FBS/PBS for 30 minutes at.

Cells were treated with rabbit anti-NFκB-p65 antibody (Cell Signaling Technology, USA) and incubated overnight at 4°C, washed three times with PBS, and then a second antibody as a molecular probe at room temperature for 30 minutes. (anti-rabbit Alexa 488, Cell Signaling Technology, USA) was treated and cultured, washed again with PBS three times, and incubated with DAPI (5 μg/mL) for 20 minutes at room temperature.

The location of NFκB-p65 was observed using a confocal microscope (FluoView FV10i, Olympus, Australia) with an excitation wavelength of 499 nm and an emission wavelength of 520 nm.

1-7. Western blotting

RAW264.7 cells were harvested and suspended in a lysis buffer containing a cocktail of protease and phosphatase inhibitors. Protein concentration was determined using BCA protein analysis (Thermo Scientific, Rockford, IL, USA). The same amount of protein was analyzed by 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride (PVDF) membrane by electrophoresis. Non-specific binding to the membrane was obtained by incubating the membrane in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 5% skim milk for 1 hour at room temperature. After blocking, a specific primary antibody (Cell Signaling Technology, Danvers, MA, USA) was incubated overnight at 4°C. Anti-rabbit IgG-HRP was used as a secondary antibody. The signal was developed using the ChemiDoc™ Touch Imaging System (Bio-Rad Laboratories, Hercules, CA, USA).

1-8. Molecular modeling

Docking calculations were performed using AutoDock Vina 1.1.2 software. Vina's default settings and scoring function were applied. To prepare the ligand, the 2D structure of the candidate was converted into 3D structure data with minimal energy using Chem3D Ultra 8.0 software. Protein coordinates were downloaded from Protein Data Bank (accession code 1EQH/4COX). Chain A was prepared for docking within the molecular modeling software package, Chimera 1.5.3, by calculation of the protonation state of the protein as well as the removal of additional chains and all ligands and water molecules. The addition of polar hydrogen and the setting of the grid box parameters were performed using MGLTools 1.5.4, and the exhaustiveness parameter was set to 8. PyMol v1.5 was used for the analysis and visual investigation of ligand-protein interactions in docking poses.

2. Experiment result

2-1. Confirmation of COX-1 and COX-2 inhibitory activity of synthetic compounds

The COX-1 and COX-2 inhibitory ability of the compounds (5a-5u and 6a-6b) synthesized according to the above Synthesis Example was tested using an enzyme immunoassay (EIA).

Figure 2 is a graph showing the results of in vitro COX-1 and COX-2 enzyme inhibitory activity analysis of compounds 5a-5u and 6a-6b according to an experimental example of the present invention. SC-560 is a COX-1 selective inhibitor, DUP-697 and celecoxib (Cel.) are COX-2 selective inhibitors, and Con is a control (DMSO). Results are expressed as mean±SD (n=3) values of 3 independent experiments. (Compared to the control group, * p <0.05, ** p <0.01, * **p <0.001)

Referring to FIG. 2, compound 5m exhibited high inhibitory effects in both COX-1 and COX-2, and accordingly, various activity evaluations were conducted around compound 5m.

Table 1 shows the in vitro COX-1 and COX-2 inhibition (IC ₅₀ , μM) and selectivity index for compound 5m and standard reagents. Results are the average of three values obtained using the COX Fluorescence Inhibitor Screening Assay Kit (Cayman Chemical, MI, USA). The COX-2 selectivity index was used as the value of COX-1 IC ₅₀ /COX-2 IC ₅₀ , SC-560 was used as a COX-1 selective inhibitor, and celecoxib and DUP-697 were used as COX-2 selective inhibitors.

compound COX-1 COX-2 COX-2-selectivity 5m 39.20 7.59 5.16 SC-560 0.01 ＞3.30 <0.003 Ibuprofen 4.12 8.91 0.46 Aspirin 6.12 12.36 0.49 Mesalazine 7.73 7.53 1.02 Diclofenac 18.79 1.24 15.18 Celecoxib ＞100.00 1.31 ＞76.39 Dup-697 ＞3.00 0.02 ＞139.53

Referring to Table 1, 5m of the tested compounds showed significant COX-1 and COX-2 inhibitory activity with _{IC 50 values of 39.20 and 7.59 μM, respectively.} The COX-1 inhibitory activity of 5m was found to be less active than the tested NSAIDs, but for the COX-2 inhibitory activity, 5m showed significant efficacy and was found to be similar to the value of the tested NSAIDs.

The COX inhibitory efficacy of 5 m and the COX-2 selectivity index (IC ₅₀ (COX-1)/IC ₅₀ (COX-2)) are COX-1 selective (SC-560), COX-2 selective (celecoxib and Dup-697). ), and commercial drugs. COX-2 selectivity of 5 m was higher than that of ibuprofen, aspirin and mesalazine, but was found to be much lower than that of celecoxib and Dup-697. The 5 m COX-2 selectivity was found to be similar to diclofenac. In particular, diclofenac is widely used for inflammatory diseases, but has been associated with some GI side effects in clinical trials and epidemiological studies. In addition, diclofenac does not have the cardiovascular side effects characteristic of COX-2 selective inhibitors such as celecoxib. These results suggest that a balanced COX-2 selectivity of 5 m may be beneficial for the development of NSAIDs with reduced gastrointestinal and cardiovascular side effects. Thereafter, the 5m in vitro anti-inflammatory activity was evaluated at the cell level using RAW264.7 murine macrophages.

2-2. In vitro cytotoxicity confirmation of compound 5m

Prior to the cell-based anti-inflammatory assay, cytotoxicity was evaluated in murine macrophages (RAW264.7), rat liver cells (Ac2F) and mouse fibroblasts (L929) to determine the appropriate concentration of compound 5m for the anti-inflammatory assay.

3 is a graph showing the cytotoxicity results of compound 5m according to an experimental example of the present invention.

Referring to Figure 3, 5m showed little toxicity to all of the cell lines at a concentration of up to 50 μM for 24 hours. Therefore, anti-inflammatory analysis was performed by treating rat macrophages (RAW264.7) with 5 m at a concentration of 20 μM or less.

2-3. Confirmation of LPS-induced inhibition of pro-inflammatory cytokine expression of compound 5m

To evaluate the in vitro anti-inflammatory effect of compound 5m, protein expression of pro-inflammatory factors iNOS and COX-2 was tested using Western blotting. Cells treated with different concentrations of 5 m were cultured for 24 hours in the presence or absence of LPS (1 μg/mL). Dexamethasone (DEX, 10 μM) was used as a positive control, and the results were expressed as the mean±SD (n=3) of three independent experiments ( ^### p <0.001, LPS-stimulated compared to the control group). Compared to group ** p <0.01, ***p <0.001)

4 shows changes in protein expression of pro-inflammatory factors iNOS and COX-2 according to an experimental example of the present invention. The first image at the top shows the level of expression of LPS-induced iNOS and COX-2 according to the concentration of compound 5m in RAW264.7 cells, (A) is a graph showing the relative protein expression level of iNOS, and (B) is COX- 2 is a graph showing the relative protein expression level.

Referring to Figure 4, LPS stimulation significantly increased the protein expression of iNOS and COX-2, but this increase was found to be significantly concentration-dependently down-regulated in cells pretreated to 5m. In particular, 20 μM of 5 m strongly attenuated the protein expression of iNOS and COX-2 than 10 μM of dexamethasone.

In inflammatory processes, proinflammatory stimulation of nitric oxide (nitric oxide; hereinafter, NO), prostaglandin E ₂ (prostaglandin E _2; hereinafter, PGE _2), tumor necrosis factor-α (tumor necrosis factor α; hereinafter, TNF-α) and It induces the production of various cytokines and inflammatory mediators such as interleukin-6 (hereinafter, IL-6). Since compound 5m inhibits iNOS expression, it is expected that NO production in macrophages will be reduced, and the amount of NO in the RAW264.7 cell supernatant was measured using a grease reagent. In addition, the production of TNF-α and IL-6 was determined by enzyme-linked immunosorbent assay (ELISA).

5 shows the change in the generation of pro-inflammatory factors according to an experimental example of the present invention, (A) is nitric oxide (NO), (B) is interleukin-6 (IL-6), (C) is a graph showing the degree of production of tumor necrosis factor α (TNF-α), and (D) is prostaglandin E ₂ (prostaglandin E ₂ , PGE _{2 ).} Results are represented as representative values of three independent experiments ( ^### p <0.001 compared to the untreated control group, ** p <0.01, ***p <0.001 compared to the LPS-treated cells).

Referring to FIG. 5, in (A), NO generation was found to decrease in a concentration-dependent manner of compound 5m. _{The PGE 2} production of (D) also decreased in a concentration-dependent manner according to the 5m treatment. Inflammatory mediator is that high production of PGE ₂ is caused by the inflammatory stimulus, the inhibition of the COX-2 enzyme metabolism of PGE ₂ reduces the formation of PGE _2.

The inflammatory cytokines TNF-α and IL-6 are part of the host's response to the inflammatory situation and maintain a normal cellular state. In (B) and (C), when RAW264.7 murine macrophages were exposed to LPS, the amount of pro-inflammatory mediator was remarkably increased, but the increase was suppressed in a dose-dependent manner of 5 m. As a result of these results, it can be confirmed that compound 5m clearly attenuates the excessive immune response in LPS-stimulated RAW264.7 cells.

2-4. Confirmation of the inhibition of the generation of reactive oxygen species (ROS) by compound 5m

Reactive oxygen species (ROS) are chemically reactive species containing oxygen. In biological systems, ROS is formed as a natural by-product of the normal metabolism of oxygen and plays an important role in cellular signaling. ROS is also produced by COX-2 from arachidonic acid as a natural by-product of prostaglandin biosynthesis.

Meanwhile, it is known that the inflammatory state induces ROS production and affects transcription through the regulation of phosphorylation of transcription factors. Among the transcription factors, nuclear factor-kappa B (hereinafter, NF-κB) appears as an important step in the production of pro-inflammatory cytokines such as TNF-α, IL-6, iNOS and COX-2. Since it is involved in the regulation of pro-inflammatory genes, the oxidative stress of cells was detected and quantified.

6 shows the change in the production of reactive oxygen species (ROS) according to an experimental example of the present invention, (A) is ROS in LPS-induced cells, indicated by green fluorescence by DCFH-DA, a fluorescent probe, (B) is a graph of quantification of fluorescence intensity with a fluorescent microplate reader ( ^### p <0.001 compared to untreated control, ***p <0.001 compared to LPS-treated cells)

6, compound 5m significantly decreased ROS production in a dose-dependent manner compared to the LPS-treated group. In particular, 5 m at 20 μM concentration significantly reduced ROS production comparable to 20 μM diclofenac. Therefore, it was speculated that compound 5m inhibited the activity of NF-κB through reduction of ROS and subsequent blocking of NF-κB transcriptional activity of pro-inflammatory molecules.

2-5. Confirmation of LPS-induced NF-κB signal inhibition effect by compound 5m treatment

To further investigate the possible anti-inflammatory mechanism of compound 5m, the effect of 5m in the NF-κB pathway was tested. First, Western blotting was used to investigate the induction of NF-κB p65 phosphorylation according to 5m treatment in LPS-activated RAW264.7 cells. Cell extracts were collected for 30 minutes after LPS stimulation to detect the initial state of the NF-κB p65 signal.

Figure 7 shows the change in phosphorylation of NF-κB according to an experimental example of the present invention, (A) is a confocal microscope image of NF-κB p65, (B) is phosphorylation of NF-κB, (C) is IKK phosphorylation and (D) are graphs showing the phosphorylation of IκBα. β-actin was used as an internal control. Results are represented as representative values of three independent experiments ( ^## p <0.01 compared to untreated control, * p <0.05, ** p <0.01, *** compared to LPS-treated cells. p <0.001)

Referring to FIG. 7, in (A), NF-κB p65 was visualized by green fluorescence and the cell nucleus was confirmed by cyan fluorescence by DAPI staining. As a result, when 20 μΜ of compound 5m was treated, LPS-stimulated NF- It has been shown that the migration of κB into the nucleus is reduced.

In (B), the level of phosphorylated NF-κB p65 was significantly increased by LPS treatment, but it was found that the phosphorylation of NF-κB p65 was dose-dependently inhibited by treatment with compound 5m.

Phosphorylation of IκB (an inhibitor of NF-κB) and IKK (IκB-kinase) is essential for the nuclear translocation and activation process of NF-κB. Inflammatory stimuli such as toxins, pathogens or oxidative stress induce phosphorylation of IKK. After phosphorylation, IKK is an essential element in promoting IκBα phosphorylation, and phosphorylated IκBα is ubiquitinated and decomposed to release active NF-κB. Subsequently, the activated NF-κB migrates to the nucleus, binds to DNA, and promotes the expression of pro-inflammatory factors.

In Figure 7 (C) and (D), LPS stimulation significantly increased the level of phosphorylated IKK, but significantly decreased by treatment with compound 5m. In addition, the level of phosphorylated IκBα was also reduced compared to the LPS-treated group. These results indicate that compound 5m can exhibit anti-inflammatory activity through reduction of ROS level and inhibition of NF-κB activity in RAW264.7 cells.

8 shows the anti-inflammatory mechanism of compound 5m as a COX inhibitor identified through an experimental example of the present invention.

_{Referring to FIG. 8, compound 5m expresses pro-inflammatory factors including iNOS, NO, COX-2, PGE 2} , TNF-α and IL-6 in LPS-stimulated RAW264.7 macrophages through the above experimental example. It showed anti-inflammatory activity through inhibition, and ROS reduction was also confirmed. In addition, compound 5m reduced the phosphorylation of IKK, IκBα and NF-κB. A possible anti-inflammatory mechanism of compound 5m was proposed based on the results of these in vitro experiments.

That is, compound 5m can first inhibit COX-2 to reduce ROS production, and the production of reduced ROS reaches its peak due to NF-κB activation and inhibition of endonuclear translocation, iNOS, COX-2, It reduces the expression of pro-inflammatory mediators such as TNF-α and IL-6. These results can have a significant impact on the therapeutic potential of compound 5m, and compound 5m can act as a potential anti-inflammatory agent.

2-6. Molecular docking

The inhibitory effect of COX-1 and COX-2 and the in vitro anti-inflammatory activity of compound 5m allowed the ligand-protein interaction to be understood in detail by performing intermolecular docking. Docking simulation was performed using ovine COX-1 (PDB ID: Ovis aries 1EQH) and rat COX-2 (PDB ID: Mus musculus 4COX) to gain insight into the interaction between compound 5m and COX enzyme. Performed. The calculated docking poses of compound 5m were compared with the same poses as indomethacin crystals using COX-2.

9 is a docking (docking) and crystal structure of the ligand / COX binding according to an experimental example of the present invention. (A) is an enlarged view of the physiologically active pocket of COX-2 (4COX), and the hydrophobic or hydrophilic portion of COX-2 is shown in red or white, respectively, and additional large spaces are Val ⁵²³ , Arg ⁵¹³ , And His ⁹⁰ amino acids. (B) is an enlarged crystal structure of COX-2 combined with indomethacin (4COX), and (C) is a hydrogen bond interaction between ^{compound 5m and the surrounding amino acids of COX-1 (1EQH), Arg 120} and Tyr ^355. The action is expanded, and (D) shows an expanded hydrogen bond interaction between ^{compound 5m and the surrounding amino acids Arg 120} and Tyr ^{355 of COX-2 (4COX).} 4COX is rat COX-2 (PDB ID: Mus musculus 4COX), 1EQH is positive COX-1 (PDB ID: Ovis aries 1EQH).

In general, COX-1 and COX-2 share 60% homology in the amino acid sequence, but referring to FIG. 9, the morphology for the substrate-binding site and the catalytic region is slightly different. Comparison of COX-1 and COX-2 enzymes shows that COX-2 has a larger and more flexible substrate channel than COX-1, and in (A) COX-2 is a selective inhibitor consisting ^{of the amino acids Val 523} , Arg ⁵¹³ , and His ⁹⁰ It shows that it has a larger space in the area that can be combined with.

COX-2 selective inhibitors such as SC-558 are located deep in large bioactive cavities and specific sites can form hydrogen bonds to ^{Arg 513} and His ^90. In contrast, non-selective COX inhibitors such as indomethacin and ibuprofen also bind to long hydrophobic channels, but do not occupy the additional pockets present in COX-2.

In addition, there are two important polar amino acids inside the active site of the curved narrow cleft. ^{The amino acids Arg 120} and Tyr ^355, located deeper in the active site, play an important role in the stabilization of the carboxyl groups of classical non-selective NSAIDs in hydrophobic COX channels. The carboxyl group of the drug forms a hydrogen bond with the guanidium ^{group of Arg 120} and Tyr ^{355 at the bonding site.} In (C) and (D). Compound 5m shows the same hydrogen bonding pose as NSAIDs in COX-1 and COX-2, indicating that the carboxylate groups of NSAIDs can be replaced by aryl-hydrazone moieties. This interaction is almost essential for COX-1/COX-2 inhibitory activity, as exemplified by the binding interaction of indomethacin. On the other hand, compound 5m did not form a hydrogen bond with ^{Arg 513} and His ^90. This lack of binding can explain the 5m selectivity for COX-2.

As described above, a specific part of the present invention has been described in detail, and for those of ordinary skill in the art, it is obvious that this specific description is only a preferred embodiment, 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 (13)

A compound represented by the following Formula 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
[Formula 1]

delete delete A pharmaceutical composition for preventing or treating inflammatory diseases containing a compound represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

delete The method of claim 4,
The composition,
A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that it inhibits cyclooxygenase (Cyclooxygenases, COX). The method of claim 4,
The composition,
A pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that reducing the production of reactive oxygen species (ROS). The method of claim 4,
The composition,
IKK, IκBα and NF-κB pharmaceutical composition for preventing or treating inflammatory diseases, characterized in that reducing the phosphorylation of one or more selected from the group consisting of. The method of claim 4,
The composition,
iNOS, NO, PGE ₂ , TNF-α and a pharmaceutical composition for the treatment or prevention of inflammatory diseases, characterized in that inhibiting the expression of one or more pro-inflammatory factors selected from the group consisting of IL-6. The method of claim 4,
The inflammatory disease,
Degenerative arthritis (osteoarthritis), rheumatoid arthritis, ankylosing spondylitis, septic arthritis and lupus arthritis. A health functional food composition for preventing or improving inflammatory diseases containing a compound represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

delete The method of claim 11,
The inflammatory disease,
Degenerative arthritis (osteoarthritis), rheumatoid arthritis, ankylosing spondylitis, septic arthritis and lupus arthritis.

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