KR101740116B1 - Novel Composition comprising 5,6-dihydroergosterol glycoside derivatives - Google Patents

Abstract

The present invention provides novel 5,6-dihydroergosterol glycoside derivatives.
The novel 5,6-dihydroergosterol glycoside derivative according to the present invention is useful for the prevention and treatment of dermatitis by inhibiting the production of chemokines which are induced by skin inflammatory stimuli and by inhibiting the activity of transcription factors controlling the expression of various inflammatory mediators It can be useful as a cosmetic material for preventing and treating dermatitis and improving atopic appearance.

Description

Dihydroergosterol glycoside derivatives < RTI ID = 0.0 > (Novel Composition < / RTI > comprising 5,6-dihydroergosterol glycoside derivatives)

The present invention relates to a 5,6-dihydroergosterol glycoside derivative and a composition for preventing and treating dermatitis comprising the same.

Atopy refers to the Greek word 'a-topos', which means 'unusual', 'strange', or 'abnormal reaction'. Literally, a variety of causes are complicated, muddled, loosened and recurred.

Atopic diseases include allergic asthma, allergic conjunctivitis, allergic rhinitis, and atopic dermatitis. These diseases can occur alone or in combination of several diseases.

The cause of atopic disease is not known exactly yet, but it is universally believed that genetic and immunological factors are involved, and other environmental and psychological factors are exacerbated by experts.

Atopic dermatitis is one of the most common skin diseases in infants and children and starts at 45% in 6 months, 60% in 12 months and at least 85% in 5 years. It is usually known to be a sick child at a young age, but 50% of the patients disappear within two weeks, but 25% continue until adolescence, and the remaining 25% continue without disappearance.

The main symptom of atopic dermatitis is itch. When it gets scratched, it develops into eczematous lesion, and as the lesion progresses, a series of vicious cycle in which more severe pruritus is induced is repeated.

Currently, steroids, antihistamines, and immunomodulators are used in the treatment of skin immune diseases. Among them, steroid drugs are known to have the best anti-inflammatory and immunosuppressive effects. However, long-term use of steroids has been reported to cause side effects such as hair on the right skin, shrinking the skin, reducing skin pigmentation, and thinning the skin.

Dexamethasone, known as a steroid, is the most widely used treatment for atopic dermatitis. However, it is effective for short-term treatment. However, long-term treatment for more than one year does not have stability and efficacy, Problems such as skin atrophy, scarring, skin discoloration and other side effects are reported.

Therefore, it is necessary to study new drugs with excellent efficacy that can effectively prevent or treat atopic dermatitis, which is rapidly increasing while minimizing side effects of chemicals.

KR 10-2013-0058857 KR 10-1151718

The present invention is directed to provide novel 5,6-dihydroergosterol glycoside derivatives.

The present invention provides a pharmaceutical composition for preventing and treating dermatitis comprising a 5,6-dihydroergosterol glycoside derivative.

The present invention provides a cosmetic composition for improving or alleviating atopic skin comprising a 5,6-dihydroergosterol glycoside derivative.

The present invention is directed to a method for preventing or treating dermatitis, comprising administering a therapeutically effective amount of a 5,6-dihydroergosterol glycoside derivative.

The present invention provides the use of a 5,6-dihydroergosterol glycoside derivative for the preparation of a medicament for preventing or treating dermatitis, particularly atopic dermatitis.

As a result of efforts to find a substance capable of treating or ameliorating dermatitis, especially atopic dermatitis, the present inventors have found that 5,6-dihydroergosterol glycoside derivatives, especially novel 5,6-dihydroergosterol glycoside The present inventors completed the present invention by confirming that the seed derivative, 5,6-dihydroergergol-galactoside, has the effect of significantly improving and treating symptoms of dermatitis and atopic dermatitis.

The present invention provides a pharmaceutical composition for preventing or treating dermatitis comprising a 5,6-dihydroergosterol glycoside derivative. The 5,6-dihydroergosterol glycoside derivative according to the present invention may be a compound represented by the following formula (1).

[Chemical Formula 1]

Wherein R ₁ to R ₄ are each independently hydrogen or hydroxy and R ₅ is hydrogen or CH ₂ OH.

More preferably, R ₁ to R ₅ may have a structure as shown in Table 1 below.

[Table 1]

For example, in Table 1 above, when R ₁ is OH, R ₂ is OH, R ₃ is OH, R ₄ is H, and R ₅ is CH ₂ OH, the glycoside is glucose.

In Table 1, when R ₁ is OH, R ₂ is OH, R ₃ is H, R ₄ is OH, and R ₅ is CH ₂ OH, the glycoside is galactose.

In Table 1, when R ₁ is OH, R ₂ is OH, R ₃ is OH, R ₄ is H, and R ₅ is H, the glycoside is xylose.

In Table 1, when R ₁ is NH ₂ , R ₂ is OH, R ₃ is OH, R ₄ is H, and R ₅ is CH ₂ OH, the glycoside is glucosamine.

The compound represented by Formula 1 of the present invention may include asymmetric carbon. In such a case, the present invention may include not only the compound represented by Formula 1, but also optical isomers thereof.

As a preferred aspect of the present invention, the 5,6-dihydroergosterol glycoside derivative of Formula 1 may be a compound represented by Formula 2 below.

(2)

R ₁ to R ₅ in the above formula (2) are as shown in Table 2 below.

[Table 2]

For example, in Table 2 above, when R ₁ is OH, R ₂ is OH, R ₃ is OH, R ₄ is H, and R ₅ is CH ₂ OH, the glycoside is glucose.

In Table 2, when R ₁ is OH, R ₂ is OH, R ₃ is H, R ₄ is OH, and R ₅ is CH ₂ OH, the glycoside is galactose.

In Table 2, when R ₁ is OH, R ₂ is OH, R ₃ is OH, R ₄ is H, and R ₅ is H, the glycoside is xylose.

In Table 2, when R ₁ is NH ₂ , R ₂ is OH, R ₃ is OH, R ₄ is H, and R ₅ is CH ₂ OH, the glycoside is glucosamine.

In another preferred embodiment of the present invention, the 5,6-dihydroergosterol glycoside derivative represented by Formula 1 is selected from the group consisting of 5,6-dihydroergosterol glucose, 5,6-di Hydroergosterol galactose or 5,6-dihydroergosterol xylose.

(3)

[Chemical Formula 4]

[Chemical Formula 5]

The present invention includes all pharmacological or physiologically acceptable salt forms of the compounds of Formulas 1 to 5 above. For example, inorganic ion salts made of calcium, potassium, sodium and magnesium, inorganic acid salts made of hydrochloric acid, nitric acid, phosphoric acid, bromic acid, iodic acid, perchloric acid, tartaric acid and sulfuric acid, acetic acid, trifluoroacetic acid, , Malonic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, Organic acid salts prepared from organic acids such as acetic acid, acetic acid, acetic acid, acetic acid, acetic acid, vanillyric acid, hydroiodic acid, mandelic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, succinic acid, tartaric acid, Sulfonic acid salts prepared with camphorsulfonic acid, or naphthalenesulfonic acid, amino acid salts prepared with glycine, arginine, lysine, etc., and amino acid salts prepared with trimethylamine, triethylamine, ammonia, pyridine, Picoline, and the like. However, the types of salts as defined in the present invention are not limited by these listed salts. For example, the pharmaceutically acceptable salt may be hydrochloric acid as inorganic acid, or methanesulfonic acid as organic acid.

Known compounds among the above 5,6-dihydroergosterol glycoside derivatives can be chemically synthesized according to methods known in the art.

According to a preferred embodiment of the present invention, the 5,6-dihydroergosterol glycoside derivative is obtained by synthesizing 5,6-dihydroergosterol by performing a selective reduction reaction of ergosterol with DIBAL and synthesizing 5,6-dihydro The ergosterol is synthesized by performing a glycosylation reaction with glycosyl halide, glycosyl sulfide or glycosyl trichloroacetimidate.

The 5,6-dihydroergosterol glycoside derivatives of the above formulas (1) to (5) according to the present invention inhibit the production of chemokines which increase in response to skin inflammatory stimuli and inhibit the activity of transcription factors controlling the expression of various inflammatory mediators It has a remarkable effect on treating dermatitis. The dermatitis of the present invention includes atopic dermatitis, contact dermatitis, and seborrheic dermatitis, and is preferably atopic dermatitis.

In the present invention, the pharmaceutical composition may be a formulation selected from the group consisting of ointments, gels, creams, patches and sprays. In addition, the above pharmaceutical composition may be an oral preparation or a main ingredient. The oral preparation may be a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, suspension, solution, emulsion or syrup, and the injectable preparation may be in the form of a sterile injectable preparation.

The pharmaceutical composition of the present invention can be formulated by adding a pharmaceutically acceptable carrier. For formulation, refer to Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutically acceptable carrier means those conventionally used in the manufacture of a pharmaceutical composition for a person skilled in the art to which the present invention belongs. For example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl Pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Pharmaceutically acceptable carriers also include diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. For example, tablets, pills, powders, granules, capsules and the like may be prepared by dissolving at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin Etc., and may include lubricants such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solutions, emulsions, syrups, and the like, and may contain diluents such as water and liquid paraffin, wetting agents, sweetening agents, fragrances, preservatives and the like. Examples of the non-aqueous solution include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions and freeze-dried preparations, and non-aqueous solvents and suspensions include vegetable oils such as propylene glycol, polyethylene glycol and olive oil, And the like. However, the present invention is not limited to the above-listed pharmaceutically acceptable carriers and the like, and these are merely illustrative.

The dosage of the pharmaceutical composition varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but may be suitably selected depending on the case. For example, the 5,6-dihydroergosterol glycoside derivative may be administered at a dose of 0.01 mg / kg to 10 mg / kg, preferably 2.5 mg / kg to 5 mg / kg per day, The administration or application may be administered once a day or divided into several times. In addition, the pharmaceutical composition may contain 0.0001 to 80% by weight of the 5,6-dihydroergosterol glycoside derivative based on the total weight of the composition. The pharmaceutical composition may be administered to mammals, such as humans, by a variety of routes, for example, by oral, intravenous, intramuscular, or subcutaneous injection.

In addition, the pharmaceutical composition of the present invention may contain, in addition to the 5,6-dihydroergosterol glycoside derivative, at least one active ingredient showing improvement, alleviation, treatment or prevention of dermatitis.

In addition, the pharmaceutical composition of the present invention can be used alone, for improving, alleviating, treating or preventing dermatitis, or in combination with methods using surgery, hormone therapy, drug therapy and biological response modifier.

The present invention provides a cosmetic composition for improving or alleviating atopic skin comprising a 5,6-dihydroergosterol glycoside derivative.

In the present invention, the cosmetic composition of the present invention can be used in cosmetics such as soft longevity, astringent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, A body oil, a body essence, a makeup base, a foundation, a hair dye, a shampoo, a rinse, and a body cleansing agent.

The cosmetic composition of the present invention can be prepared in various forms using the above-mentioned 5,6-dihydroergosterol derivatives according to a conventional method for producing a cosmetic, and can be used as a stabilizer, a dissolving agent, a vitamin , Pigments, and flavoring agents.

When the cosmetic composition is prepared in the form of a perfume product containing the 5,6-dihydroergosterol derivative, a shampoo, a hair lotion, a hair cream, a hair gel or the like, it is diluted with a normal cleansing liquid, Can be used.

The cosmetic composition of the present invention may contain 0.001% to 20% by weight of the 5,6-dihydroergosterol derivative based on the total weight of the composition.

The present invention is directed to a method for preventing or treating dermatitis, comprising administering a therapeutically effective amount of a 5,6-dihydroergosterol glycoside derivative.

As used herein, the term "therapeutically effective amount" refers to the amount of 5,6-dihydroergosterol glycoside derivative effective for the prevention or treatment of dermatitis.

The method for preventing or treating dermatitis of the present invention, particularly, a method of preventing or treating atopic dermatitis, comprises administering (by applying to the skin) the 5,6-dihydroergosterol glycoside derivative described above, before the manifestation of the disease itself As well as inhibiting or avoiding the sign of it. In the management of disease, the prophylactic or therapeutic dose of a particular active ingredient will vary depending on the nature and severity of the disease or condition, and the route by which the active ingredient is to be administered. Preferably at a dose of 0.01 mg / kg to 10 mg / kg per day, more preferably 2.5 mg / kg to 5 mg / kg per day, or may be applied to skin or the like. The administration or application may be performed once or several times a day . However, the frequency of dosages and dosages thereof will vary with the age, body weight, and response of the individual patient, and suitable dosing regimens can be readily selected by those of ordinary skill in the art which will of course consider these factors.

In addition, the method for preventing or treating dermatitis of the present invention may further comprise administration of a therapeutically effective amount of an additional active agent that is useful for treating a disease together with the 5,6-dihydroergosterol glycoside derivative, The active agent may exhibit synergistic or adjunctive effects with the 5,6-dihydroergosterol glycoside derivative.

The present invention provides the use of a 5,6-dihydroergosterol glycoside derivative for the preparation of a medicament for preventing or treating dermatitis, particularly atopic dermatitis. The 5,6-dihydroergosterol glycoside derivative for the manufacture of a medicament may be admixed with an acceptable adjuvant, diluent, carrier or the like, and may be prepared in combination with other active agents to have synergistic action of the active ingredients have.

The matters mentioned in the use, composition and treatment method of the present invention are applied equally unless they are mutually contradictory.

The present invention provides 5,6-dihydroergosterol galactoside as a novel 5,6-dihydroergosterol glycoside derivative. The 5,6-dihydroergosterol galactose according to the present invention has a structure represented by the following formula (4).

[Chemical Formula 4]

The 5,6-dihydroergosterol galactose is synthesized by selective reduction of ergosterol with DIBAL (Diisobutylaluminium hydride) to synthesize 5,6-dihydroergosterol and the synthesized 5,6-dihydroergosterol is reacted with glyco Glycosylation reaction is performed by glycosylation with glycosyl halide, glycosyl sulfide or glycosyl trichloroacetimidate.

According to one embodiment, the reaction of 5,6-dihydroergosterol with 2,3,4,6-tetra-O-benzoyl- beta -D-galactopyranosyl trichloroacetimidate under methylene chloride gives Cu (OTf) ₂ is further treated with triethylamine and neutralized to give 2,3,4,6-tetrabenzoylgalactopyranosyl dihydroergosterol. The resulting 2,3,4,6-tetrabenzoylgalactopyranosyl dihydroergosterol was dissolved in methylene chloride and methanol, and then NaOMe was added. After the reaction, the mixture was neutralized by adding Dowex Mac-3 and then purified by column chromatography to obtain the product .

The novel 5,6-dihydroergosterol glycoside derivatives of the present invention inhibit the production of chemokines that increase in response to skin inflammatory stimuli in the prevention and treatment of dermatitis and inhibit the activity of transcription factors that control the expression of various inflammatory mediators Thereby being useful as an agent for preventing or treating dermatitis or improving atopic appearance.

FIG. 1 is a graph showing the cytotoxicity of 5,6-dihydroergosterol glycoside derivative in HaCaT cells, a keratinocyte cell line. FIG.
FIG. 2 is a graph showing the results of confirming that the 5,6-dihydroergosterol glycoside derivative in HaCaT cells, a keratinocyte cell line, inhibits the expression of CCL17 and CCL22 according to TNF-α and IFN-γ.
FIG. 3 shows the results of confirming the effect of inhibiting the activation of NF-.kappa.B and STAT1, the major transcription factors of CCL17 and CCL22 expression, by the 5,6-dihydroergosterol glycoside derivative.
FIG. 4 is a graph showing the results of examining appearance skin lesions and immune cell changes upon treatment with 5,6-dihydroergosterol glycoside derivative in a mouse model of atopic dermatitis.
FIG. 5 is a diagram showing the results of confirming epidermal thickness and changes in mast cell number according to 5,6-dihydroergosterol glycoside derivative treatment in a mouse model of atopic dermatitis.
FIG. 6 is a graph showing the results of confirming changes in IgE and histamine concentrations upon treatment with a 5,6-dihydroergosterol glycoside derivative in a mouse model of atopic dermatitis.

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

Example 1 Preparation of 5,6-dihydroergosterol

Ergosterol (0.75 g), toluene (10 ml) and diisobutyl aluminum hydride (1 M in toluene, 8 ml) were placed in a sealed tube and stirred at 140 ° C for 20 hours. After adding a small amount of methanol, the solution was quickly added to a 3M aqueous HCl solution and extracted with methylene chloride. The extracted organic layer was washed with NaCl aqueous solution three times, dried with MgSO ₄ and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography to obtain a product with a yield of 96%.

The hydrogen NMR peak is as shown below.

¹ H NMR (300 MHz, CDCl ₃ ):? 0.55-2.02 (m, 42H),? 3.60 (brs,

Example 2: Preparation of 5,6-dihydroergosterol glucose

(1) Preparation of 2,3,4,6-tetrabenzoyl glucopyranosyl dihydroergosterol

5,6-Dihydroergosterol (0.5 g) and 2,3,4,6-tetra-O-benzoyl-aD-glucopyranosyl trichloroacetimidate (1.21 g) were dissolved in methylene chloride (27 ml) Followed by stirring at -20 占 폚 for 30 minutes with 4 Å molecular sieves. Cu (OTf) ₂ (49 mg) was added thereto, followed by stirring at -20 占 폚 for one day. Triethylamine was added and the mixture was sufficiently neutralized for 30 minutes, then filtered through celite, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography to give the product in 68% yield.

The hydrogen NMR peak is as shown below.

_{^{1 H NMR (300MHz, CDCl 3}} ): δ 0.51-1.86 (42H, dihydroergosterol peaks), δ3.59 (brs, 1H), δ4.14-4.20 (m, 1H), δ4.52 (dd, 1H, J = 5.9,12.1 Hz),? 4.61 (dd, 1H, J = 3.3, 11.9 Hz),? 4.95 (d, 1H, J = 7.9 Hz),? t, 1H, J = 6.3 Hz ), δ5.47-5.53 (m, 1H), δ5.63 (t, 1H, J = 9.7 Hz), δ5.90 (t, 1H, J = 9.7 Hz), δ7 29-7.57 (m, 12H),? 7.82-8.03 (m, 8H)

(2) Preparation of 5,6-dihydroergosterol glucose

2,3,4,6-tetrabenzoylglucose Pyranoyl dihydroergosterol (0.7 g) was dissolved in methylene chloride (7.0 ml) and methanol (7.0 ml), and then 0.5M NaOMe (8.6 ml) was added thereto. Lt; / RTI > Dowex Mac-3 was added to neutralize, and the filtrate was concentrated under reduced pressure. Purification by column chromatography gave the product in 89% yield.

Melting point, hydrogen NMR and carbon NMR peak are as shown below.

Melting point: 257-270 ℃

^? H (300 MHz, pyridine-d ⁵ ):? 0.59 (s, 3H),? 0.74 (s, 3H),? 0.89 (d, 6H, J = 6.8 Hz) J = 6.8 Hz),? 1.08 (d, 3H, J = 6.6 Hz),? 1.15-1.28 (m, 4H),? 1.35-1.62 (m, 8H),? 1.66-1.73 , 4H),? 1.73-1.83 (m, 2H),? 1.86-1.95 (m, 2H),? 1.99-2.06 (m, 3H),? 3.95-4.08 (m, 3H),? (D, 1H, J = 11.5Hz),? 4.86 (brs, -OH peak),? 5.04 (d, , 1H, J = 7.7Hz),? 5.19 (brs, 1H),? 5.26-5.29 (m, 1H)

¹³ C NMR (300 MHz, Pyridine-d ⁵ ):? 12.27, 13.06, 17.84, 19.83,20.14,21.37,21.73,23.27,28.53,30.02,33.33,34.53,34.72,37.31,39.60,40.15,40.85,43.06, 43.46, 49.56, 55.28, 56.05, 62.90, 71.75, 75.38, 77.04, 78.54, 78.67, 79.80, 102.24, 117.86, 132.06, 136.18, 139.54

Example 3: Preparation of 5,6-dihydroergosterol galactose

(1) Preparation of 2,3,4,6-tetrabenzoylgalactopyranosyl dihydroergosterol

Dihydroergosterol (1.0 g) and 2,3,4,6-tetra-O-benzoyl- beta -D-galactopyranosyl trichloroacetimidate (2.23 g) were dissolved in methylene chloride (36 ml ), 4 Å molecular sieves were added thereto, and the mixture was stirred at -20 ° C for 30 minutes. Cu (OTf) ₂ (90 mg) was added thereto, and the mixture was stirred at -20 占 폚 for 4 hours. Triethylamine was added and the mixture was sufficiently neutralized for 30 minutes, then filtered through celite, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography to give the product in 74% yield.

^{1 H NMR (300 MHz, CDCl3} ) δ 0.52-2.33 (42H, dihydroergosterol peaks), 3.67 (brs, 1H), 4.31-4.36 (m, 1H), 4.41-4.47 (m, 1H), 4.66-4.72 (m 1H), 4.93 (d, 1H, J = 7.7 Hz), 5.10-5.21 (m, 2H), 5.60 (dd, 1H, J = 3.2,10.4 Hz), 5.75-5.81 2H, J = 7.5 Hz), 7.96 (d, 2H, J = 7.5 Hz), 7.21-7.26 (m, 2H), 7.35-7.63 , 8.03 (d, 2H, J = 8.1 Hz), 8.11 (d, 2H, J = 7.3 Hz)

(2) Preparation of 5,6-dihydroergosterol galactose

2,3,4,6-tetrabenzoylgalactopyranosyl dihydroergosterol (0.2 g) was dissolved in methylene chloride (2.0 ml) and methanol (2.0 ml), followed by the addition of 0.5 M NaOMe (2.5 ml) Lt; / RTI > Dowex Mac-3 was added to neutralize, and the filtrate was concentrated under reduced pressure. Purification by column chromatography gave the product in 87% yield.

Melting point, hydrogen NMR and carbon NMR peak are as shown below.

Melting point: 267-269 ℃

^{1 H NMR (300 MHz, Pyridine} -d5) δ0.59 (s, 3H), 0.73 (s, 3H), 0.83 (dd, 1H, J = 2.9, 7.0 Hz), 0.89 (dd, 6H, J = 1.9 , 6.7 Hz), 0.99 (d, 4H, J = 6.8 Hz), 1.08 (d, 3H, J = 6.4 Hz), 1.18-1.30 (m, 4H), 1.34-1.62 (m, 4H), 1.77-1.86 (m, IH), 1.88-1.94 (m, IH), 1.98-2.12 (m, 3H), 4.01 1H, J = 7.5 Hz), 4.24 (dd, 1H, J = 3.0,9.4 Hz), 4.47-4.52 (m, 5.18 (brs, 1 H), 5.27 (t, 1 H, J = 5.8 Hz).

^{13 C NMR (75MHz, Pyridine-} d5) δ12.27, 13.05, 17.83, 19.82, 20.14, 21.36, 21.73, 23.27, 28.52, 30.02, 33.33, 34.52, 34.75, 37.33, 39.61, 40.17, 40.84, 43.06, 43.47, 49.59, 55.28, 56.07, 62.62, 70.39, 72.73, 75.46, 76.93, 76.99, 102.86, 117.86, 132.06, 136.19, 139.55.

Example 4 Preparation of 5,6-dihydroergosterol xylose

(1) Preparation of 2,3,4-tribenzoyl-pyropyranosyl dihydroergosterol

To a solution of 5,6-dihydroergosterol (0.3 g) and 2,3,4-tri-O-benzoyl- beta -D-xylopyranosyl trichloroacetimidate (0.5 g) in methylene chloride After dissolving, 4 Å molecular sieves were added and the mixture was stirred at -20 ° C for 30 minutes. Cu (OTf) ₂ (90 mg) was added thereto, and the mixture was stirred at -20 占 폚 for 10 hours and 30 minutes. Triethylamine was added and the mixture was sufficiently neutralized for 30 minutes, then filtered through celite, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography to give the product in 65% yield.

^{1 H NMR (300 MHz, CDCl3} ) δ 0.62-2.15 (42H, dihydroergosterol peaks), 3.65-3.71 (m, 2H), 4.44 (d, 1H, J = 11.7 Hz), 4.96 (brs, 1H), 5.26- 5.35 (m, 2H), 5.75 (t, 1H, J = 7.1 Hz), 7.33-7.40 (m, 6H), 7.49-7.53 (m, 3H), 7.98-8.05 (m, 6H).

(2) Preparation of 5,6-dihydroergosterol xylose

2,3,4-Tribenzoylgylopyranosyl dihydroergosterol (0.7 g) was dissolved in methylene chloride (12 ml) and methanol (6 ml), followed by the addition of 0.5 M NaOMe (6.6 ml) at room temperature for 3 hours Lt; / RTI > Dowex Mac-3 was added to neutralize, and the filtrate was concentrated under reduced pressure. Purification by column chromatography gave the product in 86% yield.

Melting point, hydrogen NMR and carbon NMR peak are as shown below.

Melting point: 220-224 ℃

^{1 H NMR (300 MHz, Pyridine} -d5) δ 0.59 (s, 3H), 0.75 (s, 3H), 0.89 (dd, 6H, J = 2.2, 6.8 Hz), 0.99 (d, 3H, J = 6.8 Hz ), 1.08 (d, 3H, J = 6.4 Hz), 1.19-1.53 (m, 11H), 1.62-1.79 (m, 8H), 1.88-1.94 1H, J = 8.1 Hz), 4.24-4.37 (m, 2H), 4.42 (m, 2H) 1H, J = 7.5 Hz), 4.19 (m, 1H), 4.95 (d, 1H, J = 7.5 Hz).

^{13 C NMR (75 MHz, Pyridine} -d5) δ 12.28, 13.06, 17.83, 19.83, 20.14, 21.37, 21.75, 23.27, 28.54, 30.03, 30.17, 33.33, 34.56, 34.81, 37.41, 39.59, 40.31, 40.86, 43.05, 43.47, 49.56, 55.28, 56.06, 67.22, 71.26, 75.16, 77.36, 78.61, 103.20, 117.85, 132.06, 136.19, 139.59.

Example 5: Cytotoxicity of 5,6-dihydroergosterol glycoside derivative

The cytotoxicity of 5,6-dihydroergosterol glucose (DHE-Glc) and 5,6-dihydroergergol galactose (DHE-Gal) in HaCaT cells, a human skin keratinocyte line, was confirmed.

2.0 x 10 ⁴ cells were transferred to 96-well microplates and cultured in DMEM containing 10% (v / v) FBS for 12 hours. The cultured cells were treated with 5,6-dihydroergosterol glucose or 5,6-dihydroergol sterol galactose (10, 20, 50, 100 占 퐂 / ml) and further cultured for 24 hours. The cultured cells were incubated with 1 mg / ml MTT-containing medium for 1 hour, and the absorbance at 570 nm was measured by MTT assay.

The results are shown in Fig. As shown in Fig. 1, when the concentration of 5,6-dihydroergosterol glucose (DHE-Glc) or 5,6-dihydroergol sterol galactose (DHE-Gal) was 0-100 g / ml, And it did not affect growth significantly.

Example 6 Confirmation of Inhibitory Effect of 5,6-Dihydroergosterol Glycoside Derivatives on Expression of CCL-17 and CCL-22

TNF-alpha and / or IFN-alpha, which are known as inflammation inducing molecules upon treatment with 5,6-dihydroergosterol glucose (DHE-Glc) and 5,6-dihydroergerol glycolate (DHE- γ-induced expression of CCL-17 and CCL-22.

1X10 ⁶ HaCaT cells were transferred to 6-well microplates, cultured in DMEM containing 10% (v / v) FBS for 12 hours, washed with PBS, and then 2 ml of free medium was added with 5,6-dihydroergosterol Glucose (DHE-Glc) or 5,6-dihydroergol sterol galactose (DHE-Gal) were treated at 2.5, 5, 10 and 20 ㎍ / ml for 1 hour, respectively. Thereafter, the inflammation-inducing molecules TNF-a, IFN-y, or TNF-a and IFN-y were treated and further cultured for 18 hours.

RNA was isolated according to the manufacturer's manual using a Trizol reagent kit (Invitrogen, Gaithersburg, Md.) To confirm the expression changes of CCL-17 and CCL-22 at the RNA level. The separated RNA was subjected to reverse transcription using a template to obtain cDNA. cDNA was added to 2 ug of reverse transcription buffer (5 mM MgCl 2, 1 mM dNTP, 2.5 U / ul reverse transcriptase, and 2.5 uM oligo (dT) 15asa primer (omniscript RT kit, Qiagen), 0.5 units of RNase inhibitor, 2.5 uM oligo (oligo (dT)) and 2.5 units of reverse transcriptase were added and reacted at 37 ° C to obtain cDNA.

To quantify the obtained cDNA, PCT was performed using the primers shown in Table 3 below, and the amplified DNA was confirmed by 1% agarose gel.

To examine the effect of 5,6-dihydroergosterol glucose (DHE-GIc) and 5,6-dihydroergergerol galactose (DHE-Gal) at the protein level, 5,6-dihydroergosterol glucose (DHE-GIc) or 5,6-dihydroergosterol galactose (DHE-Gal) at concentrations of 2.5, 5, 10 and 20 μg / ml for 1 hour, IFN-y, or TNF-a and IFN-y, and then cultured for 18 hours. The amount of protein for CCL-17 and CCL-22 in the medium was confirmed by ELISA kit (R & D Systems, Minneapolis, MN, USA). 50 μl of 2 μg / ml capture antibody (anti-CCL17 antibody, anti-CCL22 antibody) was coated on a 96 well plate for 2 hours, and then blocked with PBS containing 1% BSA for 1 hour. Subsequently, 100 μl of the recovered sample was added to each well, followed by reaction for 2 hours. After washing three times with PBS-T, 50 ul of 100 ng / well of detector antibody (anti-CCL17 antibody, anti-CCL22 antibody) was added to each well and reacted for 2 hours. After washing three times with PBS-T, HRP-conjugated antibody was diluted 1: 200, and 50 μl of each antibody was added and reacted for 30 minutes. After sufficient washing with PBS-T, the substrate solution (TMB) was added and absorbance was measured at 450 nm.

The experimental results are shown in Fig. As shown in FIG. 2, CCL-17 and CCL-22 (FIG. 2) were assayed in a concentration-dependent manner by treatment with 5,6-dihydroergosterol glucose (DHE-Glc) or 5,6-dihydroergeraldegalactose The levels of RNA and protein in the cells were decreased.

Example 7 Confirmation of Inhibitory Effect of 5,6-Dihydroergosterol Glycoside Derivative on Inflammatory Response Regulator Expression

NF-κB and STAT1 (signal transducer and activator of transcription 1) are important regulators of increased expression of CCL-17 and CCL-22 by TNF-α and / or IFN-γ (JE Darnell Jr et al., Science, 264: 1415-1421, 1994; A. Dhar, et al., MoI. Cell Biochem., 234-235: 185-193, 2002) The expression of NF-κB and STAT1 was determined by treatment with dihydroergo sterol glucose (DHE-Glc) and 5,6-dihydroergergol galactose (DHE-Gal).

1 × 10 ⁶ HaCaT cells were transferred to 6-well microplates, cultured in DMEM containing 10% (v / v) FBS for 12 hours, washed with PBS, and 2 ml of free medium was added with 5,6-dihydroergone Sterol glucose (DHE-Glc) was treated at 2.5, 5, 10, and 20 ㎍ / ml for 1 hour. (10 ng / ml), IFN-γ (10 ng / ml), or TNF-α (10 ng / ml) and IFN-γ Min. ≪ / RTI > The cultured cells were treated with 40 μl of cell lysis buffer (150 ml NaCl, 1 mM EDTA, 1 mM PASF, 1 μg / ml leupeptin, 1 mM Na3VO4, 1 mM NaF, 1% NP-40, 0.25% sodium deoxycholate) And allowed to stand at 4 DEG C for 30 minutes. After centrifugation at 13000 rpm at 4 ° C for 10 minutes, 10% SDS-PAGE was performed with supernatant. Western blotting was performed using rabbit-anti-phosphor-p65 antibody, rabbit-anti-IκBa antibody, rabbit-anti-phosphor-STAT1 antibody, rabbit-anti-STAT1 antibody and rabbit-anti-actin antibody.

The results are shown in Fig. As shown in Fig. 3 (1 hr treatment sample), cells stimulated with TNF-α showed increased phosphorylation of p65 and degradation of IkB, and cells stimulated with IFN-γ showed phosphorylation of STAT1 , But it was confirmed that p65 phosphorylation and IkB degradation were decreased and phosphorylation of STAT1 was decreased in a concentration dependent manner by 5,6-dihydroergosterol glucose treatment.

Example 8 Confirmation of Therapeutic Effect of 5,6-Dihydroergosterol Glycoside Derivative in Atopic Animal Model

In order to confirm the effect of 5,6-dihydroergosterol glycoside derivative on atopy, mice were experimented to induce atopy and effect of 5,6-dihydroergosterol glycoside derivative.

Balb / c mice were induced with atopy. After epilating, 300 μl of 1% DNCB was applied to the dorsal area to boost the immune response. One week later, 300 μl of 0.1% DNCB was treated three times a week. After 3 weeks of atopy induction, IgE levels were determined in the serum and the mice were divided into 5 groups. In order to confirm the improvement effect, cream containing 50ug or 100ug of 5,6-dihydroergosterol glucose was applied on each of the backs while the induction was performed for 3 weeks each day, and only the same amount of cream was treated as a control. After treatment for the last 3 weeks, mice were sacrificed and the changes in the thickness of the atopy, the thickness of the atopy, and the pathological changes of the ear were examined.

8.1. Visually check the effect

The change in appearance was confirmed to confirm the efficacy of 5,6-dihydroergosterol glucose treatment, and the results are shown in FIG.

As shown in FIG. 4 (A), it was confirmed that atopic lesions were significantly increased in the control group in which the atopy was induced for a total of 6 weeks, the skin thickness was significantly increased, the keratin was dried, and the skin was formed due to cell necrosis. On the other hand, when dihydrogen ergosterol glucose was treated, all the visual symptoms were significantly alleviated compared with the control group, and it was confirmed that the condition was healthy. In addition, as shown in Fig. 4 (B), it was confirmed that DNCB significantly increased the cells in the spleen and lymph node, but greatly decreased in accordance with 5,6-dihydroergosterol glucose treatment.

8.2. Determination of skin layer thickness and number of mast cells

At the center of the lesion, the skin tissue was sampled at a size of 1 cm x 1 cm, fixed in 10% formaldehyde solution, and embedded in paraffin to prepare a 4 μm section. Changes in the thickness of skin epidermis were confirmed by H & E staining. Hematoxylin, which is treated in H & E staining, stains the nucleus to reveal a deep blue or purple color. Eosin stains the cytoplasm to produce red purple. For this purpose, the lesion was cut on the back of each group and fixed with paraffin, and the section was cut and stained.

The number of mast cells was measured in 0.5 cm x 1 cm of skin tissue in the middle of the mouse ring and fixed in IHC zinc fixative formalin-free solution. The skin tissue was embedded with paraffin and then serial sections were made at a thickness of 4 μm and each slice was attached to a slide coated with poly-L-lysine. After paraffin removal and hydration, the cells were stained with toluidine blue, and then magnified 200 times using an optical microscope. The number of mast cells was measured by selecting 3 fields of view.

The results are shown in Fig. As can be seen in Figure 5 (A), comparing the skin of each test group, the number of immune cells stained as a whole dark blue or violet in the group treated with 5,6-dihydroergosterol glucose as compared to the control group Respectively. In addition, as shown in Fig. 5 (B), it was confirmed that the number of mast cells decreased in comparison with the control group according to 5,6-dihydroergosterol glucose treatment.

8.3. serum IgE  And histidine change confirmation

Serum IgE and histidine concentrations were measured by ELISA (enzyme-linked immunosorbent assay) and blood samples taken on a plate coated with IgE and histamine antibodies were collected at 1/250, 1, / 10, reacted for 2 hours, and the secondary antibody with HRP was added. After reacting for 1 hour, the absorbance was measured at 450 nm and quantified using a regression curve. The results are shown in FIG. As shown in FIG. 6, the concentration of IgE and histidine was greatly increased in the control group treated with DNCB, but the concentration of IgE and histidine was decreased in a concentration-dependent manner by 5,6-dihydroergosterol glucose treatment.

All the measurements obtained above were calculated as mean ± standard deviation, and statistical significance was analyzed by ANOVA. The level of significance was less than 5%.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Novel Composition comprising 5,6-dihydroergosterol glycoside          derivatives <130> P14-017-REA-KHU <150> 10-2014-0041275 <151> 2014-04-07 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Foward primer of CCL-17 <400> 1 cttctctgca gcacatcc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of CCL-17 <400> 2 aagacctctc aaggctttg 19 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of CCL-22 <400> 3 aggacagagc atggctcgcc tacaga 26 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of CCL-22 <400> 4 taatggcagg gaggtagggc tcctga 26 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Foward primer of b-actin <400> 5 gcgggaaatc gtgcgtgaca tt 22 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of b-actin <400> 6 gatggagttg aaggtagttt cgtg 24

Claims (10)

delete Pharmaceutical composition for preventing or treating atopic dermatitis comprising 5,6-dihydroergosterol glucose as an active ingredient: delete delete delete 3. The pharmaceutical composition according to claim 2, wherein the composition is selected from the group consisting of ointments, gels, creams, patches and sprays. A cosmetic composition for improving or alleviating atopic skin comprising 5,6-dihydroergosterol glucose as an active ingredient: delete delete The cosmetic composition according to claim 7, wherein the cosmetic composition is at least one selected from the group consisting of softening agents, convergent lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, A cosmetic composition for improving or alleviating atopic skin, wherein the composition is any one selected from the group consisting of a cream, a body oil, a body essence, a makeup base, a foundation, a hair dye, a shampoo, a rinse and a body cleanser.

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