KR102307045B1 - Composition for preventing or treating atopic dermatitis comprising caffeic acid ester derivatives and synthesis method thereof - Google Patents

Abstract

The present invention relates to a composition for preventing or treating atopic dermatitis containing a caffeic acid ester derivative as an active ingredient and a method for synthesizing the same, wherein the synthesis method is a caffeic acid ester derivative using the Verley-Doebner transformation reaction of knephenylkel condensation. p -hydroxyphenol caffeate can be synthesized more simply and efficiently, and the p -hydroxyphenol caffeate synthesized by the method of the present invention significantly inhibits IL-13 production of epithelial T cells affecting atopic dermatitis. On the other hand, as it was confirmed to increase the IL-2 production ability of lymph node T cells, the p -hydroxyphenol caffeate can be provided as an effective therapeutic agent for atopic dermatitis.

Description

A composition for preventing or treating atopic dermatitis comprising a caffeic acid ester derivative as an active ingredient, and a method for synthesizing the same

The present invention relates to a method for efficiently synthesizing a caffeic acid ester derivative and a composition for preventing or treating atopic dermatitis containing the caffeic acid ester derivative synthesized by the above method as an active ingredient.

Atopy is a type of hypersensitivity that has recently been highlighted with a sharp increase in its prevalence. From an immunological point of view, the It is defined as a group of allergic diseases with a strong genetic tendency with major symptoms.

Compared with healthy skin, patients with atopic dermatitis show barrier abnormalities and complex immune modulation disorders. It has been reported that, at the same time, the expression of cytokine and chemokine genes associated with allergic inflammation is increased.

In the malignant stage of atopic dermatitis, the activation of T helper (Th) 2 cells is the most important feature, whereas in the chronic stage, immunoglobulin E production and Th1, Th17 and Th22 cell responses are more important.

Th2-type cytokines, including interleukin (IL)-4 and IL-13, are known to play multiple roles in diseases such as atopic dermatitis and vitiligo. IL-13 is a typical Th2-type cytokine, and the receptor of IL-13 was reported to be similar to IL-4, and it was confirmed that IL-13 and IL-4 were activated through JAK2-STAT6 signaling.

Abnormally elevated serum levels of IL-13 were confirmed in patients with atopic dermatitis or vitiligo, and an increase in the number of cells with increased IL-4, IL-5, and IL-13 mRNA expression was confirmed in patients with atopic dermatitis with malignant skin lesions. became

Accordingly, it has been reported that IL-4 plays an important role in the initiation of allergic reactions and in the triggering and development of Th2 cells, whereas IL-13 plays an important role in the production of immunoglobulin E and the pathogenesis of chronic atopic dermatitis. It was confirmed that the level of IL-13 in the epithelial T cells was higher than that of IL-4.

Although 0.5%-1% of the total population and 5-10% of children complain of severe symptoms of atopic dermatitis, it is estimated that it is related to genetic factors and immune system deficiency, but the exact cause has yet to be revealed. It is expected to be somewhat alleviated through improvement of the environment and diet, but there is no fundamental treatment method.

Although atopic dermatitis shows individual differences, most of it is accompanied by extreme itching, dry skin, rash, ooze, scabs, and scaly skin (scaly skin), resulting in emotional anxiety, stress, tension, frustration, and anger. It is frequently accompanied by other allergic diseases such as urticaria, metal allergy, asthma or allergic rhinitis, and is emerging as a serious social problem that threatens public health today.

On the other hand, representative drugs known to be effective for atopic dermatitis up to now include steroids, antihistamines, antibiotics, non-steroids, and other sedatives and nerve stabilizers.

While steroids have excellent anti-inflammatory and immunosuppressive effects, when used for a long period of time, the skin loses elasticity and becomes thin, revealing thread veins. appears, and symptoms worsen explosively when the drug is stopped.

Antihistamines are only a temporary measure to relieve itching symptoms by preventing the release of histamine from mast cells.

In addition, antibiotics are intended to prevent secondary bacterial infection caused by scratching, rather than as a fundamental treatment drug for atopic dermatitis. It can lead to aplastic anemia due to bone marrow function.

Other non-steroidal agents are only moisturizing agents for relieving symptoms of atopic dermatitis, and sedatives and nerve stabilizers are only adjuvants for symptom relief rather than for the purpose of fundamental treatment.

Moreover, these existing treatments for atopic dermatitis have different effects depending on the individual's constitution, and in particular, they are premised on the addition and use of ingredients and artificial chemical compositions during the manufacturing process. There is a problem that it is difficult to trust its safety because there is a very high possibility of it.

Republic of Korea Patent No. 10-0837733 (published on Jun. 13, 2008)

An object of the present invention is to provide a method for more conveniently and efficiently synthesizing a caffeic acid ester derivative and a composition for preventing or treating atopic dermatitis containing the caffeic acid ester derivative synthesized by the above method as an active ingredient.

Caffeic acid, characterized in that the compound of Formula 1 is synthesized by reacting the phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] under a piperazine catalyst and a pyridine solvent A method for synthesizing an ester derivative is provided.

[Formula 1]

[Formula 1-1]

In Formula 1,

R ¹ is selected from the group consisting of hydrogen, —OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen.

The present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis comprising the caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention provides a cosmetic composition for preventing or improving atopic dermatitis comprising the caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving atopic dermatitis containing the caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The invention cafe acid ester derivative of p using the Verley-Doebner modification reaction of immense phenyl Kell condensation - were simpler and more synthetic efficiently the hydroxyphenyl cafe benzoate, p-synthesized by the synthesis-hydroxyphenyl cafe benzoate , the p as found to the other hand which significantly reduce the IL-13 production of epidermal T cells that affect the atopic dermatitis, increasing the IL-2-producing ability of lymph node T cells are conventional immune-hydroxyphenyl cafe benzoate It can be provided as an effective therapeutic agent for atopic dermatitis without inducing systemic inhibition of T cells, which has been a major drawback of inhibitors.

1 is p - hydroxy is the structure of the hydroxy phenol Cafe Eight (p -hydroxyphenyl caffeate).
Figure 2 is a p on viability of lymph node T cells - as a result of confirming the effect of the hydroxyphenyl cafe of solvents (1), p - hydroxyphenyl cafe benzoate (1) the presence or activated on non-existent condition lymph node T cells This is the result of confirming the viability by performing the propidium iodide exclusion method after culturing (* p <0.05 vs. control group).
3 is a result of confirming the IL-2 production ability of p -hydroxyphenylcaffeate (1) in lymph node T cells. Lymph node T activated in the presence or absence of p-hydroxyphenylcaffeate (1). This is the result of performing IL-2 ELISA analysis with the culture supernatant obtained by culturing cells (* p<0.05 vs. control group).
4 is a p on viability of epidermal T cells - as a result of confirming the effect of the hydroxyphenyl cafe of solvents (1), p - hydroxyphenyl cafe benzoate (1) the presence or activated on non-existent condition epidermal T cells This is the result of confirming the viability by performing the propidium iodide exclusion method after culturing (* p <0.05 vs. control group).
5 is a result of confirming the IL-13-producing ability of p -hydroxyphenylcaffeate (1) in epithelial T cells. Epithelial T activated in the presence or absence of p-hydroxyphenylcaffeate (1). This is the result of performing IL-13 ELISA analysis with the culture supernatant obtained by culturing cells (* p<0.05 vs. control group).

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention p - hydroxyphenyl malonate intermediate using Verley-Doebner modification reaction of immense phenyl Kell condensation of p - hydroxy been simpler to synthesize efficiently the hydroxyphenyl cafe benzoate, synthesized p to the synthetic methods -Hydroxyphenylcaffeate significantly reduced IL-13 production of epithelial T cells affecting atopic dermatitis, while confirming the effect of increasing IL-2 production ability of lymph node T cells, the p -hydr The present invention was completed by confirming that oxyphenylcaffeate can be used as an excellent modulator of IL-13 expression without inducing systemic suppression of T cells, which was a major drawback of conventional immunosuppressive agents.

Caffeic acid, characterized in that the compound of Formula 1 is synthesized by reacting the phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] under a piperazine catalyst and a pyridine solvent A method for synthesizing an ester derivative can be provided.

[Formula 1]

[Formula 1-1]

In Formula 1,

R ¹ may be selected from the group consisting of hydrogen, —OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen.

More preferably, the caffeic acid ester derivative may be a compound represented by the following formula (2).

[Formula 2]

In addition, the phenolic compound of Formula 1-1 may be 3-(4-hydroxyphenoxy)-3-oxopropanoic acid [3-(4-Hydroxyphenoxy)-3-oxopropanoic acid], but is not limited thereto.

The synthesis method may be to synthesize the caffeic acid ester derivative of Compound 1 through Verley-Doebner transformation reaction of knephenylkel condensation between the phenol compound of Formula 1-1 and 3,4-dihydroxybenzaldehyde.

In more detail, the synthesis method comprises the steps of: refluxing Meldrum's acid and 4-benzyloxyphenol for 10 to 12 hours in a toluene solvent to obtain a malonate monoester compound;

reacting the malonate monoester compound under hydrogen gas, a palladium catalyst, and a methanol solvent to obtain a phenol compound of Formula 1-1; and

It may include reacting the phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] with a piperazine catalyst in a pyridine solvent for 40 to 50 hours.

More specifically, the malonate monoester compound may be 3-(4-(benzyloxy)phenoxy)-3-oxopropanoic acid [3-(4-(Benzyloxy)phenoxy)-3-oxopropanoic acid] , but not limited thereto.

In addition, the phenolic compound of Formula 1-1 may be 3-(4-hydroxyphenoxy)-3-oxopropanoic acid [3-(4-Hydroxyphenoxy)-3-oxopropanoic acid], but is not limited thereto.

The present invention may provide a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising a caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

R ¹ may be selected from the group consisting of hydrogen, —OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen.

More preferably, the caffeic acid ester derivative may be a compound represented by the following formula (2).

[Formula 2]

The caffeic acid ester derivative represented by Formula 1 may inhibit the production of IL-13 in skin epithelial T cells and increase the production of IL-2 in lymph node T cells.

In one embodiment of the present invention, the pharmaceutical composition for the prevention or treatment of atopic dermatitis containing the caffeic acid ester derivative or a pharmaceutically acceptable salt thereof as an active ingredient is prepared by injection, granule, powder, tablet, or pill according to a conventional method. , capsules, suppositories, gels, suspensions, emulsions, drops or liquids may be used in any one formulation selected from the group consisting of.

In another embodiment of the present invention, the pharmaceutical composition is a suitable carrier, excipient, disintegrant, sweetener, coating agent, swelling agent, lubricant, lubricant, flavoring agent, antioxidant, buffer, bacteriostatic agent commonly used in the preparation of pharmaceutical compositions. , may further include one or more additives selected from the group consisting of diluents, dispersants, surfactants, binders and lubricants.

Specifically, carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil can be used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules. agent and the like, and such a solid preparation may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like in the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base material for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

According to an embodiment of the present invention, the pharmaceutical composition is administered intravenously, intraarterially, intraperitoneally, intramuscularly, intraarterially, intraperitoneally, intrasternally, transdermally, intranasally, inhalation, topical, rectal, oral, intraocular or intradermal The route may be administered to a subject in a conventional manner.

A preferred dosage of the caffeic acid ester derivative or a pharmaceutically acceptable salt thereof may vary depending on the subject's condition and weight, the type and extent of disease, drug form, administration route and period, and may be appropriately selected by those skilled in the art. . According to an embodiment of the present invention, although not limited thereto, the daily dose may be 0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more specifically 0.1 to 100 mg/kg. Administration may be administered once a day or may be administered in several divided doses, thereby not limiting the scope of the present invention.

In the present invention, the 'subject' may be a mammal including a human, but is not limited to these examples.

In addition, the present invention can provide a cosmetic composition for preventing or improving atopic dermatitis containing a caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

R ¹ may be selected from the group consisting of hydrogen, —OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen.

The cosmetic composition may include, in addition to the active ingredient, a caffeic acid ester derivative or a pharmaceutically acceptable salt thereof, a stabilizer, a solubilizer, a conventional adjuvant such as vitamins, pigments and fragrances, and a carrier.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation and spray, but is not limited thereto. More specifically, it may be prepared in the form of a sun cream, a softening lotion, astringent lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a pack, a spray, or a powder.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. .

When the formulation is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane or a propellant such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butylglycol oil, glycerol fatty ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , aluminum metahydroxide, bentonite, agar or tracanth may be used.

In addition, the present invention can provide a health functional food composition for preventing or improving atopic dermatitis containing a caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In Formula 1,

R ¹ may be selected from the group consisting of hydrogen, —OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen.

The health functional food is used together with other foods or food additives other than the caffeic acid ester derivative represented by Formula 1 or a pharmaceutically acceptable salt thereof, and may be appropriately used according to a conventional method. It can be suitably determined according to the intended use, for example, prophylactic, health or therapeutic treatment.

The effective dose of the compound contained in the health functional food may be used according to the effective dose of the therapeutic agent, but in the case of long-term intake for health and hygiene or health control, it may be less than the above range, It is certain that the active ingredient can be used in an amount beyond the above range because there is no problem in terms of safety.

The type of health functional food is not particularly limited, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. 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 explain the present invention to those of ordinary skill in the art.

<Example 1> Compound synthesis and synthesis process

1. Synthesis process

Previous studies have reported that it is difficult to prepare ester and amide derivatives corresponding to hydroxycinnamic acid derivatives containing caffeic acid without protection of the phenol group of the derivative during esterification or amidation (Han, 2016; Xia and Hu, 2005). ).

Therefore, in order to minimize this long and complicated protection or deprotection operation, the Verley-Doebner modification of knephenylkel condensation (Xia and Hu, 2005), which is a form of decarboxyl group condensation reaction of malonic acid intermediate (2) as shown in Scheme 1 below The synthesis of compound (1) was planned through , and it was expected that the malonic acid intermediate (2) could be obtained in high yield through the transesterification of commercially available Meldrum's acid 3 with the malonic acid intermediate (2).

[Scheme 1]

First, 3-(p-hydroxyphenoxy)-2-oxopropanoic acid [3-(p-hydroxyphenoxy)-2-oxopropanoic acid] as a Verley-Doebner modification of knephenylkel condensation for an intermediate as shown in Scheme 2 The synthesis of (2) was started.

It is very difficult to achieve mono-esterification between malonic acid and hydroquinone to obtain compound (2) in high yield without phenol addition and protection of the carboxylic acid moiety (Xia and Hu, 2005). Therefore, an attempt was made to synthesize compound (2) using commercially available compound (3) and 4-benzyloxyphenol as protected synthetic equivalents of malonic acid and hydroquinone, respectively.

The transesterification and subsequent recrystallization between compound (3) and 4-benzyloxyphenol in refluxed toluene was expected to lead to a high yield (77%) of pure monoester (4).

Hydrolysis of the benzyl ester of compound (4) provided compound (2) without hydrolysis of the phenolic ester, whereas hydrolysis of the phenolic ester resulted in compound (2) via protection of the MOM-protected or TBS-protected precursor. ) was observed in the generation trial.

Since propanoic acid (2) exhibits strong polarity, it was used in the substitution reaction without further purification.

[Scheme 2]

Finally, as in Scheme 3 below, compound 1 through Verley-Doebner transformation of knephenylkel condensation between 3,4-dihydroxybenzaldehyde (5) and oxopropanoic acid (2) using piperazine as a catalyst was obtained in a suitable yield.

[Scheme 3]

2. Compound Synthesis

All starting materials and reagents were purchased commercially and used without further purification. All solutions used for general product separation and chromatography were reagent grade and distilled.

The reaction flask was dried at 100° C. before use, and air and moisture sensitive reactions were performed under argon. Flash column chromatography was performed with each solvent using silica gel 60 (230-400 mesh, Merck). In addition, thin layer chromatography was performed using a 0.25 mm silica gel plate.

Mass spectra were obtained using VG Trio-2 GC-MS equipment, and high resolution mass spectra were obtained using JEOL JMS-AX 505WA unit.

¹ H and ¹³ C spectra were recorded with deuteriodimethylsulfoxide (DMSO-d ₆ ) or deuteriomethanol (CD ₃ OD) at JEOL JNM-LA 300 MHz, Bruker advance III 400 MHz or Bruker advance III 800 MHz.

Chemical shifts are expressed downfield per million (ppm, δ) from tetramethylsilane and are based on deuterated solvents.

¹ H-NMR data were recorded as follows; chemical shift, multiplicity; s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet, and/or multiple resonance, number of protons, and coupling constant in hertz (Hz).

1. 3-(4-(Benzyloxy)phenoxy)-3-oxopropanoic acid [3-(4-(Benzyloxy)phenoxy)-3-oxopropanoic acid; (4)] synthesis

To a solution of Meldrum's acid (1.00 g, 6.94 mmol) dissolved in toluene (20 mL) at room temperature, 4-benzyloxyphenol (1.39 g, 6.94 mmol) was added.

The reaction mixture was refluxed for 12 hours and concentrated in vacuo.

Malonate monoester (4) (malonate monoester 4; 1.54 g, 77%) was obtained as a white solid crystallized from diethyl ester/n-hexane.

¹ H NMR (DMSO- d ₆ , 400 MHz) δ 7.46-7.44 (m, 2H), 7.41-7.31 (m, 2H), 7.35-7.31 (m, 1H), 7.11-7.03 (m, 4H), 5.11 (s, 2H), 4.04 (s, 1H); LRMS (FAB) m/z 287 (M+H ⁺ ).

2. 3-(4-hydroxyphenoxy)-3-oxopropanoic acid [3-(4-Hydroxyphenoxy)-3-oxopropanoic acid; (2)]

To a solution of benzyloxypropanoic acid (4); 1.54 g, 5.36 mmol) dissolved in methanol (80 mL) at room temperature, palladium on an activated carbon carrier was added in a catalytic amount of 5%.

The reaction mixture was stirred at room temperature and _{under H 2} gas until no more starting material was detected. Thereafter, the reaction mixture was filtered through a pad of celite, filtered and concentrated in vacuo to obtain crude phenol (2) (1.03 g, 99%), which was a highly polar intermediate and was subjected to the following reaction without further purification. was used for

¹ H NMR (DMSO- d ₆ , 300 MHz) δ 6.90 (d, 2H, J = 8.8 Hz), 6.76 (d, 2H, J = 8.8 Hz), 6.55 (s, 1H), 3.56 (s, 2H) ; LRMS (FAB) m/z 197 (M+H ⁺ ).

3. p -Hydroxyphenol caffeate [ p -Hydroxyphenylcaffeate; (One)]

After dissolving crude phenol (2) (199 mg, 1.01 mmol) in pyridine (7 mL), 3,4-dihydroxybenzaldehyde (5) (3,4-dihydroxybenzaldehyde; 56 mg, 0.41 mmol) and Piperazine (7 mg, 0.08 mmol) was added to the reaction mixture.

The reaction mixture was stirred at room temperature for 48 h, concentrated in vacuo and diluted with ethyl acetate and water.

The organic layer was washed with 1N HCl solution and brine, dried over MgSO ₄ and concentrated in vacuo. The residue was purified by column chromatography on silica gel (MeOH/CH ₂ Cl ₂ = 1:40 to 1:10) to obtain a yellow solid p -hydroxyphenolcaffeate (1) (52 mg, 47%).

¹ H-NMR (800 MHz, CD ₃ OD) δ 7.68 (d, 1H, J = 15.8 Hz), 7.09 (d, 1H, J = 2.0 Hz), 7.00 (dd, 1H, J = 2.1, 8.2 Hz) , 6.93 (d, 2H, J = 8.9 Hz), 6.80 (d, 1H, J = 8.7 Hz) 6.78 (d, 2H, J = 9.0 Hz), 6.42 (d, 1H, J = 15.8 Hz); ¹³ C-NMR (200 MHz, CD ₃ OD) δ 169.0, 157.1, 150.8, 149.2, 147.7, 145.7, 128.4, 124.2, 124.2, 124.1, 117.4, 117.4, 117.3, 116.1, 115.2; LRMS (FAB) m/z 273 (M+H ⁺ ).

<Example 2> p - Confirmation of immunomodulatory effect of hydroxyphenol caffeate (1)

1. Confirmation of cell viability and IL-2 production in mouse lymph node T cells

To confirm the effect of p -hydroxyphenolcaffeate (1), a cell viability experiment was performed to confirm the viability of mouse lymph node T cells, and IL-2 assay was performed to determine the p- The effect of hydroxyphenol caffeate (1) was confirmed.

First, Orient Bio Inc. Mouse lymph node T cells were prepared from whole body lymph nodes of 6-week-old C57BL/6 female mice purchased from (Seongnam-si, Korea).

The lymph node cell suspension harvested using Histopaque 1083 (Sigma Aldrich, St. Louis, USA) was subjected to gradient centrifugation at room temperature at 400×g for 30 minutes to concentrate T cells.

Lymph node T cells were transfected with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 100 mM non-essential amino acids, 25 mM pH 7.0 HEPES solution, 1 mM sodium pyrate and 50 μM 2-β-mercaptoethanol in RPMI 1640 complete medium and adjusted to a cell density of ^{1×10 6 /mL.}

T cells were stimulated with 4 μg/mL of concanavalin A (Con A, Sigma Aldrich), a potent mitogen for T cells, and the stimulated T cells were placed in a 96-well round-bottom microtiter plate at 1×10 ⁵ Cells/well were dispensed, and p-hydroxyphenol caffeate (1) was mixed in various concentrations in each well, and then T cells were cultured at 37° C., humidified, 5% CO _{2 in an} incubator.

The control group was stimulated only with Con A, and all reagents used for T cell culture were purchased from Gibco (Waltham, USA).

After 5 days of culture, the culture supernatant was collected and the viability of activated T cells was confirmed by performing the propidium iodide (Sigma Aldrich) exclusion method.

Briefly, cells were suspended in ice-cold PBS containing 1 μg/mL of propidium iodide and subjected to flow cytometry using a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, USA) for a weakly red fluorescence signal in the whole cell population. Viable cells representing

The results were analyzed with Cell Quest software (BD Biosciences), and the relative cell viability was confirmed by the following calculation formula.

% cell viability = 100 × (live cell count in test group) / (live cell count in control group)

At the same time, using the Mouse IL-2 ELISA Ready-Set-Go kit (eBioscience, Waltham, USA) according to the manufacturer's instructions, ELISA (enzyme-linked immunosorbent assay) was performed to collect IL-2 cytokines in the culture supernatant. The concentration was confirmed.

The relative IL-2 production rate was confirmed by the following formula.

% IL-2 production = 100 × (IL-2 concentration in test group) / (IL-2 concentration in control group)

As a result, as shown in FIG. 3, p -hydroxyphenol caffeate (1) in a concentration range of 3 to 13 μM did not reduce the viability of activated lymph node T cells. However, p -hydroxyphenol caffeate (1) at a concentration of 25 μM slightly decreased cell viability, but the effect was not statistically significant.

Therefore, in a subsequent experiment to confirm the immunomodulatory activation of p -hydroxyphenolcaffeate (1) using activated lymph node T cells, the experiment was performed using p -hydroxyphenolcaffeate (1) up to a concentration of 25 μM. carried out.

As a result, p ranging from 3 to 25 μM concentration range as shown in FIG. 4-hydroxy-phenol cafe benzoate (1) did not reduce the IL-2 produced, the interesting 25 μM concentration of p-hydroxy-phenol cafe benzoate (1 ) markedly enhanced IL-2 production in mouse lymph node T cells.

From the above results, it was confirmed that p -hydroxyphenol caffeate (1) may not induce systemic inhibition of T cells, which was a major disadvantage of conventional immunosuppressants.

2. Confirmation of Cell Viability and IL-13 Production in Mouse Epithelial T Cells

Next, cell viability experiments were performed to confirm the viability of mouse epithelial T cells, and IL-13 analysis was performed to confirm the effect of p-hydroxyphenolcaffeate (1) on skin-specific T cell responses. did.

First, mouse epithelial T cells were purified from the skin epithelial tissue of 6-week-old C57BL/6 female mice. Briefly, whole skin biopsy specimens of C57BL/6 mice (Orient Bio Inc.) were treated with RPMI medium containing 2.4 U/mL dispase II (Roche Applied Science, Indianapolis, USA) overnight at 4°C. incubated.

The epidermal sheet was separated from the skin using forceps, treated with 0.3% trypsin-GNK solution (Sigma Aldrich) at 37° C. for 10 minutes, and then vigorously stirred.

T cells were concentrated by gradient centrifugation of the collected epithelial cell suspension using Histopaque 1083 (Sigma Aldrich, St. Louis, USA) at room temperature at 400 x g for 30 min.

To restore CD3 expression, the collected epithelial cells were resuspended in complete medium containing 10 U/mL mouse IL-2 and incubated overnight at _{37° C., 5% CO 2 incubator.}

Cells were stimulated for 5 days with immobilized 10 mg/mL anti-mouse CD3 mAb (BD Biosciences) + 100 U/mL IL-2 and various concentrations of p -hydroxyphenolcaffeate (1), and the control group was 0 mM of p -hydroxyphenol caffeate (1) was treated and stimulated in the same way.

After 5 days of culture, the culture supernatant was collected and the viability of activated T cells was confirmed by performing the propidium iodide exclusion method.

At the same time, using the Mouse IL-13 ELISA Ready-Set-Go kit (eBioscience), ELISA was performed according to the manufacturer's instructions to confirm the IL-13 cytokine concentration in the collected culture supernatant.

The relative IL-13 production rate was confirmed by the following formula.

% IL-13 production = 100 × (IL-13 concentration in test group) / (IL-13 concentration in control group)

As a result, as shown in FIG. 5, p -hydroxyphenol caffeate (1) in a concentration range of 3 to 13 μM did not reduce the viability of activated epithelial T cells. However, p -hydroxyphenol caffeate (1) at a concentration of 25 μM slightly decreased cell viability, but the effect was not statistically significant.

However, as shown in FIG. 6, p -hydroxyphenol caffeate (1) at a concentration of 25 μM significantly reduced the production of IL-13, and even in p -hydroxyphenol caffeate (1) at a concentration of 12 μM, IL- 13 A decrease in production was confirmed, but the effect was not statistically significant.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

A caffeic acid ester derivative, characterized in that the compound of Formula 1 is synthesized by reacting the phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] under a piperazine catalyst and a pyridine solvent synthesis method.
[Formula 1]

[Formula 1-1]

In Formula 1,
R ¹ is selected from the group consisting of hydrogen, —OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen. The method for synthesizing a caffeic acid ester derivative according to claim 1, wherein the caffeic acid ester derivative is a compound represented by the following formula (2).
[Formula 2]

The method according to claim 1, wherein the phenolic compound of Formula 1-1 is 3-(4-hydroxyphenoxy)-3-oxopropanoic acid [3-(4-Hydroxyphenoxy)-3-oxopropanoic acid] Method for synthesizing caffeic acid ester derivatives. The method according to claim 1, wherein the synthesis method comprises the steps of: obtaining a malonate monoester compound by refluxing Meldrum's acid and 4-benzyloxyphenol in a toluene solvent for 10 to 12 hours;
reacting the malonate monoester compound under hydrogen gas, a palladium catalyst, and a methanol solvent to obtain a phenol compound of Formula 1-1; and
Caffeic acid ester comprising the step of reacting the phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] with a piperazine catalyst in a pyridine solvent for 40 to 50 hours Method for synthesizing derivatives. The method according to claim 4, wherein the malonate monoester compound is 3-(4-(benzyloxy)phenoxy)-3-oxopropanoic acid [3-(4-(Benzyloxy)phenoxy)-3-oxopropanoic acid] A method for synthesizing a caffeic acid ester derivative. The method according to claim 4, wherein the phenolic compound of Formula 1-1 is 3-(4-hydroxyphenoxy)-3-oxopropanoic acid [3-(4-Hydroxyphenoxy)-3-oxopropanoic acid] Method for synthesizing caffeic acid ester derivatives. delete A pharmaceutical composition for preventing or treating atopic dermatitis comprising a caffeic acid ester derivative represented by the following formula (2) as an active ingredient.
[Formula 2]

The method for preventing or treating atopic dermatitis according to claim 8, wherein the caffeic acid ester derivative represented by Formula 2 inhibits the production of IL-13 in skin epithelial T cells and increases the production of IL-2 in lymph node T cells. pharmaceutical composition. A cosmetic composition for preventing or improving atopic dermatitis comprising a caffeic acid ester derivative represented by Formula 2 as an active ingredient.
[Formula 2]

A health functional food composition for preventing or improving atopic dermatitis comprising a caffeic acid ester derivative represented by Formula 2 as an active ingredient.
[Formula 2]

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