KR20210053622A - 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 including a caffeic acid ester derivative as an active ingredient, and a method for preparing the same. According to the method of the present invention, p-hydroxyphneol caffeate as the caffeic acid ester derivative can be prepared more simply and efficiently by using Varley-Doebner modification of the Knoevenagel condensation. The p-hydroxyphneol caffeate prepared by the method according to the present invention significantly reduces the production of IL-13 of epithelial T cells affecting atopic dermatitis, while increasing IL-2 producibility of limp node T cells. Therefore, the p-hydroxyphneol caffeate can be provided as an effective agent for treating atopic dermatitis.

Description

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

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, which has become a major concern as the prevalence increases rapidly in recent years.From an immunological point of view, atopy is caused by the immune response of immunoglobulin E and allergin. It is defined as a group of allergic diseases with a strong genetic tendency with major symptoms.

Compared to healthy skin, patients with atopic dermatitis exhibit characteristics of barrier abnormalities and complex immune dysregulation. Patients with atopic dermatitis have symptoms such as modified lipid synthesis, decreased moisture, decreased epidermal differentiation, and penetration of T cells. It has been reported, and 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 activity of T helper (Th) 2 cells is the most important feature, whereas in the chronic stage, the production of immunoglobulin E and the response of Th1, Th17 and Th22 cells are more important.

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

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

Accordingly, IL-4 has been reported to play an important role in the initiation of allergic reactions and in the triggering and development of Th2 cells, while IL-13 has been reported to play an important role in the development of immunoglobulin E and chronic atopic dermatitis. It was found 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 serious symptoms of such atopic dermatitis, it is estimated that it is related to genetic factors and immune system deficiency. There is no fundamental treatment method, but it is expected to be somewhat alleviated through environmental and dietary improvements.

Although atopic dermatitis shows individual differences, most of them involve extreme itching, dry skin, rash, swelling, scab, and skin with scaly crusts (phosphorus scales), which result from emotional anxiety, stress, tension, frustration, and anger. In addition to the emotions, other allergic diseases such as hives, metal allergies, asthma or allergic rhinitis are frequently accompanied, and these days, it is emerging as a serious social problem that threatens the public health.

Meanwhile, representative drugs known to be effective against atopic dermatitis so far include steroids, antihistamines, antibiotics, nonsteroidal drugs, and other sedatives and stabilizers.

Among these, steroids are excellent in anti-inflammatory and immunosuppressive effects, but when used for a long time, the elasticity of the skin disappears and becomes thin, resulting in unpleasantness, such as revealing thread veins. Appears, and if the drug is stopped, the symptoms explosively worsen.

Antihistamines are only temporary measures to relieve itchy symptoms by preventing histamine from being released from mast cells, and when used for a long time, side effects such as insomnia, anxiety, and loss of appetite appear.

In addition, antibiotics are intended to prevent secondary bacterial infection caused by scratching, rather than a fundamental treatment drug for atopic dermatitis, and when used for a long time, resistance increases and gastrointestinal function decreases, as well as liver damage or pseudomembranous colitis, It can lead to aplastic anemia due to bone marrow function.

Other nonsteroidal drugs are only moisturizers for relieving symptoms of atopic dermatitis, and sedatives and tranquilizers are also only adjuvants for symptom relief rather than for the purpose of fundamental treatment.

Moreover, these existing atopic dermatitis treatments have different effects depending on the individual's constitution, and in particular, they are premised on the addition and use of artificial chemical compositions during the manufacturing process and ingredients. Apart from pharmacological ingredients, they act as serious allergens for atopic dermatitis patients There is a problem that it is difficult to trust its safety because it is very likely to be.

Korean Patent Registration No. 10-0837733 (announced on June 13, 2008)

The present invention is to provide a simpler and more efficient method for 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.

The present invention is caffeic acid, characterized in that the compound of Formula 1 is synthesized by reacting a phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] in a piperazine catalyst and a pyridine solvent. It provides a method for synthesizing an ester derivative.

[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 containing a 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 containing a 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 Chemical 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 atopic dermatitis treatment without inducing systemic inhibition of T cells, which was a major disadvantage of the inhibitor.

1 is p - hydroxy is the structure of the hydroxy phenol Cafe Eight (p -hydroxyphenyl caffeate).
2 is a result of confirming the effect of p -hydroxyphenylcaffeate (1) on the viability of lymph node T cells, and as a result of confirming the effect of p -hydroxyphenylcaffeate (1), lymph node T cells activated in the presence or absence of conditions 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, and 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 the cells (* p<0.05 vs. control group).
FIG. 4 is a result of confirming the effect of p -hydroxyphenylcaffeate (1) on the viability of epithelial T cells, and as a result, epithelial T cells activated in the presence or absence of p-hydroxyphenylcaffeate (1) 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 production ability of p -hydroxyphenylcaffeate (1) in epithelial T cells, and epithelial T activated in the presence or absence of p-hydroxyphenylcaffeate (1). These are the results of 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 - on the other hand hydroxyphenyl cafe benzoate is that significantly reduce the IL-13 production of epidermal T cells that affect the atopic dermatitis, as the confirm the effect of increasing the IL-2-producing ability of lymph node T cells, the p-hydroxy It was confirmed that oxyphenyl caffeate can be used as an excellent IL-13 expression regulator without inducing systemic suppression of T cells, which was a major disadvantage of conventional immunosuppressants, and the present invention was completed.

The present invention is caffeic acid, characterized in that the compound of Formula 1 is synthesized by reacting a phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] in 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 a caffeic acid ester derivative of Compound 1 through a 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 obtaining a malonate monoester compound by reflux reaction of Meldrum's acid and 4-benzyloxyphenol for 10 to 12 hours in a toluene solvent;

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

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

More specifically, the malonate monoester compound may be 3-(4-(benzyloxy)phenoxy)-3-oxopropanoic acid [3-(4-(Benzyloxy)phenoxy)-3-oxopropanoic acid] , Is 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 can provide a pharmaceutical composition for preventing or treating 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.

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 preventing or treating atopic dermatitis containing the caffeic acid ester derivative or a pharmaceutically acceptable salt thereof as an active ingredient is an injection, granule, powder, tablet, 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 solution, bacteriostatic agent commonly used in the manufacture of pharmaceutical compositions. , A diluent, a dispersant, a surfactant, a binder, and a lubricant may further include one or more additives selected from the group consisting of.

Specifically, carriers, excipients and diluents are 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, and solid preparations for oral administration include tablets, pills, powders, granules, capsules. And the like, and these solid preparations 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 preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, and the like. 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 material for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

According to an embodiment of the present invention, the pharmaceutical composition is intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal It can be administered to the subject in a conventional manner via the route.

The preferred dosage of the caffeic acid ester derivative or a pharmaceutically acceptable salt thereof may vary depending on the condition and weight of the subject, the type and extent of the disease, the form of the drug, the route and duration of administration, 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 dosage 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 divided into several doses, whereby the scope of the present invention is not limited.

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 a stabilizer, a solubilizing agent, vitamins, pigments and flavors, as well as a carrier, in addition to a caffeic acid ester derivative or a pharmaceutically acceptable salt thereof, which is an active ingredient.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation or spray, but is not limited thereto. In more detail, it may be prepared in the form of a sun cream, a flexible lotion, a converging lotion, a nutrition lotion, a nutrition 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, tracant, cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, or zinc oxide may be used as a carrier component. .

When the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. 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, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the above formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, and the like 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 Chemical Formula 1 or a pharmaceutically acceptable salt thereof, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient is It may be appropriately determined according to the purpose of use, for example prophylactic, health or therapeutic treatment.

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

There are no particular restrictions on the types of health functional foods, examples of which 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, 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> Compound synthesis and synthesis process

1. Synthesis process

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

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

[Scheme 1]

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

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

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

Hydrolysis of the benzyl ester of compound (4) gave compound (2) without hydrolysis of the phenolic acid ester, whereas hydrolysis of the phenolic acid ester gave compound (2) through protection of MOM-protected or TBS-protected precursors. ) Was observed in the generation attempt.

Propanoic acid (2) was used in the substitution reaction without further purification because it showed strong polarity.

[Scheme 2]

Finally, compound 1 through Verley-Doebner modification of knephenyl kel condensation between 3,4-dihydroxybenzaldehyde (5) and oxopropanoic acid (2) using piperazine as a catalyst in the same manner as in Scheme 3 below. Was obtained in an appropriate 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.

Before use, the reaction flask was dried at 100° C., 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 were expressed as downfield per million parts per million from tetramethylsilane (ppm, δ) and were based on deuterated solvent.

¹ H-NMR data were recorded as follows; Chemical shift, multiplicity 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 in which Meldrum's acid (1.00 g, 6.94 mmol) was dissolved in toluene (20 mL) at room temperature was added 4-benzyloxyphenol (1.39 g, 6.94 mmol).

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

As a white solid crystallized with diethyl ester/n-hexane, malonate monoester 4 (1.54 g, 77%) was obtained.

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)]

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

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 passed through a pad of Celite, filtered, and concentrated in vacuo to obtain unrefined phenol (2) (1.03 g, 99%), and the phenol (2) was a highly polar intermediate and was subjected to the next reaction without further purification. Used in

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 unpurified 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 hours, concentrated in vacuo, and then 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. Silica gel (MeOH/CH ₂ Cl ₂ = 1: 40 ~ 1: 10) The residue was purified by column chromatography to obtain a yellow solid p -hydroxyphenol caffeate (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 -Checking the 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 -hydroxyphenol caffeate (1), cell viability experiments were performed to confirm the viability of mouse lymph node T cells, and IL-2 analysis was performed to p- The influence of hydroxyphenol caffeate (1) was confirmed.

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

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

Lymph node T cells in 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 dermatate And 50 μM 2-β-mercaptoethanol and resuspended 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 of T cells, and 1×10 ^{5 of the stimulated T cells were placed in a 96-well round-bottom microtiter plate.} After dispensing into cells/well, p-hydroxyphenol caffeate (1) was mixed in each well at various concentrations, and then T cells were cultured in an incubator at _{37° C., humidified and 5% CO 2.}

The control group was stimulated with only 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 the activated T cells was confirmed by performing a propidium iodide (Sigma Aldrich) exclusion method.

Briefly, cells were suspended in ice-cold PBS containing 1 μg/mL of propidium iodine, and flow cytometry was performed using a BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, USA) to show a weakly red fluorescence signal in the entire cell population. To obtain viable cells showing.

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, IL-2 cytokines in the culture supernatant collected by performing ELISA (enzyme-linked immunosorbent assay) according to the manufacturer's instructions using the Mouse IL-2 ELISA Ready-Set-Go kit (eBioscience, Waltham, USA) The concentration was checked.

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

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

As a result, p -hydroxyphenol caffeate (1) in a concentration range of 3 to 13 μM as shown in FIG. 3 did not decrease 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 -hydroxyphenol caffeate (1) using activated lymph node T cells , p -hydroxyphenol caffeate (1) was used up to a concentration of 25 μM. Performed.

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 suppression 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 -hydroxyphenol caffeate (1) on skin-specific T cell responses. I did.

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

The epidermal sheet was separated from the skin using forceps, and a 0.3% trypsin-GNK solution (Sigma Aldrich) was treated at 37° C. for 10 minutes, followed by vigorous stirring.

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

To restore CD3 expression, the collected epithelial cells were resuspended in complete medium containing 10 U/mL mouse IL-2 and incubated overnight in _{a 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 -hydroxyphenol caffeate (1), and the control was 0 mM. The p -hydroxyphenol caffeate (1) was treated and stimulated in the same manner.

After 5 days of cultivation, the culture supernatant was collected and carried out by the propidium iodide exclusion method to confirm the viability of the activated T cells.

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

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

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

As a result, p -hydroxyphenol caffeate (1) in a concentration range of 3 to 13 μM as shown in FIG. 5 did not decrease 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, specific parts of the present invention have 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. something to do. Therefore, it will be said that the practical scope of the present invention is 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 a phenol compound of Formula 1-1 with 3,4-dihydroxybenzaldehyde [3,4-dihydroxybenzaldehyde] in 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 of claim 1, wherein the caffeic acid ester derivative is a compound represented by the following formula (2).
[Formula 2]

The cafe 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] Synthesis method of ic acid ester derivative. The method of claim 1, wherein the synthesis method comprises the steps of obtaining a malonate monoester compound by reflux reaction of Meldrum's acid and 4-benzyloxyphenol for 10 to 12 hours in a toluene solvent;
Reacting the malonate monoester compound in a 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] in a piperazine catalyst and a pyridine solvent for 40 to 50 hours Methods of 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] Method for synthesizing a caffeic acid ester derivative, characterized in that. The cafe 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] Synthesis method of ic acid ester derivative. A pharmaceutical composition for preventing or treating 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 is selected from the group consisting of hydrogen, -OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen. The pharmaceutical composition for preventing or treating atopic dermatitis according to claim 7, wherein the caffeic acid ester derivative is a compound represented by the following formula (2).
[Formula 2]

The method of claim 7, wherein the caffeic acid ester derivative represented by Formula 1 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 containing a caffeic acid ester derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

In Formula 1,
R ¹ is selected from the group consisting of hydrogen, -OH, (C1-C4)alkyl, (C1-C4)alkoxy and halogen. A health functional food composition for preventing or improving atopic dermatitis containing a caffeic acid ester derivative represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

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

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