KR100891970B1 - Hydroquinone derivatives grafted with tat peptide, its preparation method and cosmetic composition for skin whitening comprising the same - Google Patents

Abstract

The present invention relates to a hydroquinone derivative having a cell penetrating Tat peptide having a self-invasive signal, a preparation method thereof, and a cosmetic composition for skin whitening comprising the same. Since the hydroquinone derivative has high stability against pH, oxygen, and light and excellent skin permeability, the hydroquinone derivative provides a skin whitening agent having an excellent whitening effect and a sustaining effect thereof.

Skin Whitening, Hydroquinone, Tat Peptide, Cell Penetration, Self Infiltration Signal

Description

HYDROQUINONE DERIVATIVES GRAFTED WITH TAT PEPTIDE, ITS PREPARATION METHOD AND COSMETIC COMPOSITION FOR SKIN WHITENING COMPRISING THE SAME}

The present invention relates to a hydroquinone derivative to which a peptide is bound, and more particularly, to a hydroquinone derivative having high stability against pH, oxygen, and light and excellent skin permeability, and a cosmetic composition for skin whitening comprising the same. .

Human skin color depends on several factors, especially season, race, and sex, and is mainly determined by the amount of melanin, carotene, and hemoglobin, of which melanin is the most decisive factor. Melanin (melanin) is synthesized in the pigment cells (melanocyte) present in the basal layer of the skin and metastasized to the surrounding keratinocytes of human skin color. When abnormally low melanin causes skin lesions such as vitiligo, and when produced in excess, it is known to form spots and blemishes. It is well known that melanin is made from tyrosin, an amino acid, by tyrosinase, an enzyme. Tyrosinase is more activated by ultraviolet rays, and it is well known that skin becomes dark when exposed to a lot of sunlight. Therefore, cosmetics with the concept of skin whitening effect are forming a large market in Korea and Japan, and most of them are products that contain substances that inhibit the activity of tyrosinase.

Meanwhile, raw materials conventionally formulated in whitening cosmetics as active inhibitors of tyrosinase include ascorbic acid (vitamin C) and its derivatives, lettuce extract, green tea extract, aloe extract, and golden extract, as well as kojic acid, arbutin, and oil-soluble. Licorice extract and the like. However, various plant extracts are unstable over time, and when combined with a product, the effect is not sustained, and tyrosinase activity inhibitory power is insignificant. In addition, kojic acid is commonly used as a tyrosinase inhibitor, but may cause an allergic reaction (Contact allergy to kojic acid in skin care products, Nakagawa M. et.al., Contact Dermatitis, Jan 95, vol 43 (1). ), PP9-3) is unstable in the product, there is a problem that the production of the composition containing the material is difficult.

Hydroquinone is a water-soluble substance and is the most commonly used whitening agent because of its excellent function of inhibiting tyrosinase and inhibiting melanogenesis. However, hydroquinone is easily destabilized by oxygen, pH, and light to turn brown, and has a problem that it is difficult to use as a whitening agent due to severe side effects due to toxicity.

In order to solve the above problems, there are studies to reduce skin side effects by using steroid compounds (Kligman, AM and wills., Arch. Dermatocol., 75, vol 111, pp40-48) which are anti-irritants, but rather steroids There is a problem that side effects caused by the compound occur. On the other hand, research is being conducted to solve the stability by developing hydroquinone derivatives, for example, hydroquinone fatty acid esters such as arbutin, hydroquinone monocaprylate, hydroquinone monolaurate, hydroquinone monopalmitate, arbutin monoester, etc. Is used as a cosmetic raw material having a whitening effect (US Patent No. 4,764,505, US Patent No. 4,526,779, and Korean Patent Publication No. 1999-013691).

However, even though these hydroquinone derivatives are chemically stabilized, there is a problem that the skin is hardly absorbed percutaneously. Therefore, compared to hydroquinone, the whitening effect is insignificant, and the side effects due to the toxicity of the hydroquinone derivatives cannot be completely avoided. There is this. Therefore, even if the weakness and stability of the existing hydroquinone are overcome somewhat, this alone is insufficient, the absorption from the skin is increased to maximize the whitening function in vivo, it is urgent to develop a new skin whitening agent with improved stability.

Accordingly, there is a need for research and development of a new skin whitening agent having no skin irritation, an increase in absorption from the skin, and improved stability to maximize the whitening function in vivo.

In order to solve the problems of the prior art, the present invention is non-irritating, easily and safely penetrates the outer skin and the endothelial, there is no problem of inducing skin diseases, cells having excellent tyrosinase inhibitory ability, and melanin synthesis inhibitory function, cells It is an object to provide a hydroquinone derivative to which a permeable Tat peptide is bound.

Another object of the present invention is to provide a method for preparing a hydroquinone derivative to which the cell penetrating Tat peptide is bound.

It is another object of the present invention to provide a cosmetic composition for skin whitening, comprising a hydroquinone derivative having the cell penetrating property, stability and non-irritating effect, and excellent in whitening effect and durability, the cell penetrating Tat peptide is combined as an active ingredient. It is.

The present invention relates to a hydroquinone derivative in which a peptide is bound, more specifically, a hydroquinone derivative in which a cell penetrating Tat peptide derived from a Tat peptide, which is a kind of human immunodeficiency virus protein having self penetration, and It relates to a cosmetic composition for skin whitening comprising.

We provide novel hydroquinone derivatives incorporating cell penetrating Tat peptides into hydroquinone. The hydroquinone derivative of the present invention has excellent stability, good skin absorption, sustained antioxidant, and an inhibitor of tyrosinase activity due to the characteristics of the Tat protein, compared to the existing hydroquinone derivative. Therefore, the cell penetrating Tat peptide of the present invention can be used as an excellent skin lightening agent.

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

The present invention provides a hydroquinone derivative to which the cell penetrating Tat peptide represented by the following Chemical Formula 1 is bound.

_Wherein [] _Tat is a cell penetrating Tat peptide, wherein R ₁ is at least one selected from the group consisting of glutamine, lysine, arginine, and glycine side chain, and n is an integer ranging from 4 to 12 .

Recently, according to the fact that various human diseases are caused by abnormal activity of cellular proteins, the development of substances capable of treating fatal human diseases by regulating the activity of these proteins has been of global interest. However, although peptides and proteins have excellent selectivity and utility for physiological action compared to other compounds, practical use as effective drug delivery means has been difficult due to the disadvantage that they cannot be delivered directly into cells.

In order to solve the above problems, it is possible to directly or efficiently deliver or absorb the substances necessary for the treatment of human diseases by using a protein penetration technology that efficiently penetrates various biofunctional proteins having important physiological activities into cells. It became. The protein penetration technology uses Tat peptide, a type of Human Immunodeficiency Virus type-1 (HIV) protein, having a self-penetration signal, spontaneously penetrates and migrates through the cell membrane. Turned out. This function is due to the nature of the protein transduction domain, which is the intermediate region of the Tat peptide sequence, and its exact mechanism is unknown (Frankel, AD and Pabo, CO (1988) Cell 55, 1189-1193). Green, M. and Loewenstein, PM (1988) Cell 55, 1179-1188. Ma, M. and Nath, A. (1997) J. Virol. 71, 2495-2499. Vives, E., Brodin, P. and Lebleu, B. (1997) J. Biol. Chem. 272, 16010-16017.).

The present inventors have investigated the penetration characteristics of the Tat peptides as described above. As a result, the desired skin active ingredient such as hydroquinone is attached to the Tat peptide having the self-invasive signal, thereby completing the direct and efficient penetration of these into the intracellular skin cells. By this, the present invention was completed.

As used herein, "cell penetrating Tat peptide" refers to a Tat peptide or peptide derived therefrom that has cell penetrating ability with itself or with a substance bound thereto. Specifically, the main characteristic of the Tat peptide of HIV-1 is that a sequence of a specific section of the protein vector has a signal that opens a cell membrane. The sequence is represented by the following Chemical Formula 2 as RKKRRQRRR.                     

Among the 20 amino acids, the constituent amino acids of the cell penetrating Tat peptide are lysine (hereinafter referred to as Lys or K), arginine (hereinafter referred to as Arg or R), and glutamine (hereinafter referred to as Gln or Q). Since it has an amine group and a carboxyl group, it can be combined with hydroquinone by an esterification reaction with a carboxyl group through reaction with an alcohol. Cell penetrating 9-Tat peptide (n = 9), for example 9-Tat peptide, Lys-Lys-Lys-Lys, represented by Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg or RKKRRQRRR 9-Tat peptides represented by -Lys-Lys-Lys-Lys-Lys or KKKKKKKKK, 9-Tat peptides represented by Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg or RRRRRRRRR, etc. It can be combined with the paddy by esterification. The cell penetrating Tat peptide is represented by the following formula (3),                     

The R ₁ includes one or more amino acid substituents selected from the group consisting of glutamine (Gln), lysine (lysine: Lys), arginine (arginine: Arg), and glycine (glycine: Gly), wherein the Tat peptide Is a peptide consisting of 4 to 12 amino acids, more preferably a peptide consisting of one or more amino acids selected from the group consisting of lysine and arginine. In Formula 3, n is an integer of 4 to 12, more preferably 8 to 10, most preferably n is 9.

The present invention is to prepare and purify the cell penetrating Tat peptide, by binding the Tat peptide to the hydroquinone or a derivative thereof in the presence of an organic solvent, a catalyst and a condensation agent by an ester reaction, a hydroquinone derivative to which the cell penetrating Tat peptide is bound It provides a method of manufacturing. In addition, it is preferable to perform an ester reaction with hydroquinone or a derivative thereof after activating a halide at the C-terminus of the cell penetrating Tat peptide.

In one embodiment of the present invention, a cesium salt of hydroquinone is used as a starting material to provide a method of preparing a compound of formula 1 by reacting a Tat peptide-halide of formula (4), such as chlorine, bromine, or iodine.                     

상기 화학식 4의 식에서, X는 염소, 브롬, 요오드 등의 할로이드, 토실레이트, 트리플레이트, 또는 트레실레이트로 이루어지는 군으로부터 선택될 수 있다.

In the formula (4), X may be selected from the group consisting of halides, tosylate, triflate, or tresylate such as chlorine, bromine, iodine and the like.

delete

In one example of the present invention, before reacting a cell penetrating Tat peptide with a hydroquinone or a derivative thereof in the presence of an organic solvent, a catalyst and a condensing agent, the hydroquinone derivative is prepared after changing the terminal functional group of the hydroquinone or its derivative with an alcohol. Provide a way to.

More specifically, the preparation of a hydroquinone derivative in which the cell penetrating Tat peptide of the present invention is bound may be divided into a step of preparing a Tat peptide and a step of combining the Tat peptide with a hydroquinone or a derivative thereof.

Preparation of Cell Penetrating Tat Peptides

The first is a biological method using a recombinant expression vector, which comprises: a) six histidine tags (N-terminal) having an N-terminal signal region gene from HIV-1 Tat gene of pSVC21. Cloning the Tat gene of HIV-1 to a pET vector, which is a protein expression vector, using a PCR technique to produce a vector that can be expressed in the form of a fusion protein, and b) the pET-Tat It is a method for producing a Tat peptide comprising the step of expressing in large quantities in the form of recombinant His-Tat peptide in E. coli using an expression vector, followed by pure separation and purification.

The second method comprises a) inserting a target protein, such as catalase or superoxide dismutase (SOD), into a pET-Tat expression vector and mass-expressing it as a fusion protein of His-Tat-target protein. It is a method for producing a Tat peptide comprising the step of pure separation and purification.

The third method is to prepare the Tat peptide, which synthesizes pure His-Tat peptide using an organic synthesizer for peptide synthesis, by using Merrifield's solid phase peptide synthesis method (J. Am. Chem. Soc. 85, 2149). -2154 (1963)) ABI's peptide synthesizer (Model 431A) was synthesized.

Solid phase synthesis begins by coupling an amino acid having an α-amino group protected to a suitable resin starting from the amino acid at the carboxyl end. At this time, the amino acid in which the α-amino group is protected is attached to hydroxy methyl resin or chloromethylated resin as an ester bond. Fmoc (9-fluorenyl methoxycarbonyl) or Boc (t-butyloxycarbonyl) was used as the group protecting the α-amino group, and the amino acid protected by Fmoc was purchased through ABI, Pharmacia or Calbiochem. Amino acids whose amino acid residues are reactive, for example, Arg residues are protected by suitable groups such as trityl (Trt), 4-methoxy-2,3, trimethylbenzene sulfonyl (Mrt), tert-butyl (t-Bu) Fmoc-amino acid was used. Peptide synthesis sequentially couples activated and protected amino acids with α-amino groups to the amino terminus of the peptide chain attached to the solid support resin, and after synthesis, the peptide is cleaved from the resin, and the protecting group is tripled. Remove with a reagent such as uroacetic acid (TPA). Peptides are separated from the TFA solution by filtration, centrifugation or diethyl ether and separated and purified by high performance liquid chromatography (HPLC) or other methods.

Binding of Cell-penetrating Tat Peptides to Hydroquinone or Its Derivatives

The hydroquinone derivative to which the Tat peptide is bound is reacted with a ceat salt of the hydroquinone represented by Formula 4 and a Tat peptide derivative (Tat-X) to which a halide group is bound in the presence of an organic solvent, a catalyst, and a condensation agent. Get it.

The ester reaction proceeds under anhydrous conditions. Hydroquinone is insoluble in water and therefore anhydrous organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), hexamethyl phosphoramide (HMPA), N-methylpyrrolidone, pyrrolidone, dimethylacet Amide (DMAC) and 1,3-dimethyl-3,4,5,6-tetrahydro-2 (1H) -pyrimidinone (DMPU) may be used alone or in combination. Although the esterification reaction proceeds even in the case of a non-catalyst, a catalyst using activated Lewis acid or a halide such as thionyl chloride is used to activate the carboxylic acid. After the reaction with alcohol or methanesulfonic acid (methanesulfonic acid) or the like using a catalyst to prepare a compound of the present invention. In addition, since esterification reaction is a reversible reaction, it is preferable to use 10 equivalent or more of hydroquinone and / or its derivative (s) normally in order to send reaction forward. The condensing agent is preferably N, N-carbonyldiimidazole (CDI) or N, N-dicyclohexylcarbodiimide (DCC), and N, N-dimethyl as a catalyst for promoting the condensation reaction. Preference is given to using aminopyridine (DMAP) or 1-hydroxybenzotriazole [1-HOBT].

Referring to Scheme 1, hydroquinone cesium salt (A) prepared by treating hydroquinone with water as a solvent and cesium bicarbonate and crystallizing by addition of isopropyl alcohol (Tat peptide-halide (eg, chlorine, Bromine, iodine or the like) to prepare a hydroquinone (B) to which a Tat-peptide is bound.

Condensing agents, catalysts, and solvents that can be used in the present invention can be used as is commonly known in the art within the scope that does not adversely affect the reaction.

In addition, the hydroquinone derivative to which the Tat peptide represented by Chemical Formula 1 prepared by the above method is bound may be purified using a conventional separation and purification method, for example, recrystallization or column chromatography.

Hydroquinone derivatives bound to the Tat peptide represented by Formula 1 prepared by the preparation method of the present invention act as inhibitors of chemical stability and tyrosinase activity, and can be used as novel whitening agents with excellent stability. Detailed activity assessments for this are described in the Examples.

The present invention provides a cosmetic composition for skin whitening containing the compound represented by the formula (1). The composition according to the present invention contains 0.001 to 5% (w / w), preferably 0.01 to 3% (w / w) of the compound represented by the formula (1). It is difficult to expect a substantial whitening effect at concentrations less than 0.001%, and if it exceeds 5% concentration, the low solubility may adversely affect the stability of the formulation and the product.

Ethanol, Glycerine, Butylene Glycol, Propylene Glycol, Glyceres-26, Methylgluses-20, Isocetyl Myristate, Isocetyl Octanoate, Octyldodecyl as a solvent when preparing the composition for skin whitening of the present invention One or more selected from myristate, octyldodecanol, isostearyl isostearate, cetyloctanoate and neopentyl glycol dicaprate are used. When the composition of the present invention is prepared using such a solvent, the solubility of the compound in the solvent varies slightly depending on the kind of the compound and the mixing ratio of the solvent. The type and amount of usage can be selected and applied appropriately.

The composition according to the present invention may be prepared in the form of cosmetics such as skin external ointment, softening longevity, nourishing cosmetics, nutrition cream, massage cream, essence, pack. Here, the skin external ointment is prepared to contain 50.0 to 97.0% by weight of petrolatum, and 0.1 to 5.0% by weight of polyoxyethylene oleyl-ether phosphate in addition to the active ingredient of the compound represented by the formula (1), the softening longevity is propylene glycol, glycerin 1.0 to 10.0 weight% of polyhydric alcohols, such as these, and 0.05 to 2.0 weight% of surfactants, such as polyethylene oleyl ether and polyoxyethylene hardened castor oil, are manufactured. Nutrients and nourishing creams are 5.0 to 20.0 wt% of oils such as squalane, petrolatum, octyldodecanol, and wax components 3.0 to 15.0 wt% of cetanol, stearyl alcohol, beeswax, etc. %, And the essence contains 5.0 to 30.0% by weight of polyhydric alcohols such as glycerin and propylene glycol. Massage cream is prepared by containing 30.0 to 70.0% by weight of oils such as liquid paraffin, petrolatum and isononylisononanoate in addition to the active ingredient of the compound represented by the formula (1), the pack contains 5.0 to 20.0% by weight of polyvinyl alcohol It is prepared as a wash-off pack containing 5.0 to 30.0% by weight of pigments such as kaolin, talc, zinc oxide and titanium dioxide in a peel-off pack or a general emulsion cosmetic.

On the other hand, the skin whitening cosmetic composition containing the compound represented by the formula (1) of the present invention is a common ingredient, such as oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents formulated in general skin cosmetics , Colorants, preservatives, fragrances and the like can be applied and blended as necessary.

As a result of performing transdermal absorption experiment, skin irritation experiment, tyrosinase inhibitory activity, and radical scavenging experiment conducted to confirm the physiological activity of the compound of the present invention, the compound of the present invention is better than conventional arbutin and hydroquinone. It has excellent transdermal absorption and has proved to be excellent in experiments such as tyrosinase activity inhibitory activity and free radical scavenging activity. Therefore, the compound of the present invention can be added and used in all forms such as creams, lotions, gels, etc. of cosmetics due to its activity, non-irritability, and excellent skin absorption and long lasting effect.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited to the following examples.

EXAMPLE

Example 1 Preparation of 9-Tat Peptide (R = kkkkkkkkk) by Genetic Recombination Technique

(1) Preparation of 9-Tat fusion protein (KKKKKKKKK: oligolysine) expression vector

First, pLys, a fusion peptide expression vector capable of delivering a target peptide having a penetrating function into cells, was prepared. To facilitate the analysis of the ability to deliver nine lysine peptides into cells, a DNA fragment corresponding to the base sequence of GFP was selected by selecting Green Fluorescence Protein (abbreviated as "GFP") as the target peptide. Was inserted into the pLys vector to prepare a pLys-GFP fusion peptide, a vector capable of expressing the oligo-Lys-GFP fusion peptide. In order to overexpress GFP, the control peptide to the oligo Lys-GFP fusion peptide, only 9 lysines were included and the remaining sites developed identical pGFP expression vectors.

Oligonucleotide having a nucleotide sequence capable of expressing 9 lysines, forward primer: 5'-TAAGAAGAAAAAGAAAAAGAAAAAGAAGC-3 '; Reverse primer: pET-15b vector digested with NdeI and XhoI restriction enzymes using a double-stranded oligonucleotide made by annealing two 5'-TCGAGC TTCTTTTTCTTTTTCTTTTTCTTCT-3's for 5 minutes at 100 ° C. PLys, a vector capable of expressing 9 lysines, was prepared by insertion into Invitrogen, Carlsbad, CA). Therefore, the newly prepared vector is designed to express 6 histidines, 9 lysine residues and target peptides in the form of fusion peptides from the amino terminus.

Next, the target peptide was selected as a green fluorescent protein (GFP), and two kinds of oligonucleotides were synthesized based on the cDNA sequence of GFP. The forward initiator has a Xho I restriction enzyme site of 5'-CTCGAGGTGAGCAAGGGCG AGGAGCTG-3 ', and the sequence of the reverse initiator has a BamH I restriction site of 5'-GGATCCTTACTTGT ACAGCTCGTCCATGCCGAG-3'.

DNA fragments having GFP sequences were prepared by polymerase chain reaction (PCR) from pEGFP-C2 (clontech) using the synthesized oligonucleotides as follows. PCR was performed in a thermal cycler (Perkin-Elmer, model 9600), the reaction mixture was placed in a 50 μl silicon tube (siliconized reaction tube) and heated at 94 ° C for 3 minutes. PCR reactions were carried out for 30 extensions at 94 ° C for 30 seconds, denaturation at 51 ° C for 45 seconds, annealing at 70 ° C for 1 minute and 30 seconds, and 10 minutes at 72 ° C, and 5 minutes at 20 ° C. The final extension reaction was induced.

After PCR, the reaction was separated by agarose gel electrophoresis and inserted into the pGEM-T vector (Promega, USA). Appropriate cells were then transformed with this vector, and plasmids from the transformed bacteria were isolated by alkaline lysis method (Sambrook et al., 1989). PGEM-T vector containing GFP cDNA was digested with Xho I and BamH I and inserted into pET-15b and pLys expression vectors. The vectors obtained here were named pGFP and pLys-GFP, respectively.

(2) Expression and Identification of Fusion Peptides

Escherichia coli cells induced overexpression of the fusion peptide with IPTG were sonicated at 4 ° C, and then centrifuged to separate peptides in the supernatant by 12% SDS-polyacrylamide gel electrophoresis.

E. coli BL21 (DE3) transformed with pGFP and pLys-GFP were selected, then colonies were inoculated into 50 mL LB medium and IPTG (0.5 mM) was added to the medium to induce overexpression of the fusion peptide and then cell extract. Was prepared. Overexpression of recombinant GFP and oligo Lys-GFP fusion peptides was induced. Overexpressed GFP and oligo Lys-GFP fusion peptides present in the cell extracts were identified by SDS-polyacrylamide gel electrophoresis and Western blot analysis.

(3) Purification of 9-Tat Fusion Peptide

E. coli BL21 transformed with pLys-GFP vector was placed in LB medium containing ampicillin and incubated with stirring at 250 rpm at 37 ° C. When the bacterial concentration in the culture medium showed O.D 600 = 0.6 to 1.0, IPTG was added to the medium to bring the final concentration to 0.5 mM, followed by further incubation for 3 hours. The cultured cells were collected by centrifugation, followed by sonication with 5 mL of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Recombinant oligo Lys-GFP fusion peptides were purified under two conditions, respectively, in denatured condition and native condition. 10 g of bacterial pellet was placed in 40 mL buffer A (6 M urea, 20 mM HEPES, pH 8.0, 100 mM NaCl) and sonicated to disrupt the cells, followed by two centrifugations (16,500 rpm × 30 min, 40,000 rpm x 30 min, 4 ° C.) was performed continuously to remove insoluble cell debris.

2.5 mL Ni ²⁺ -nitrilotriacetic acid sepharose column equilibrated with buffer B (6M urea, 20 mM HEPES, pH 8.0, 100 mM NaCl, 20 mM imidazole); Quagen, USA) was immediately loaded with the supernatant obtained by centrifugation, and 10 times the binding buffer and 6 times the washing buffer (60 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) After washing with Elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) and eluted Lys-GFP fusion peptide. Subsequently, the fractions containing the oligo Lys-GFP fusion peptide were collected and PD-10 column chromatography containing sephadex G-25M was performed to remove salts contained in the fractions. It was freeze-dried again to obtain a Tat peptide (KKKKKKKKK) consisting of nine lysines in powder form, from which moisture was completely removed.

Example 2. Preparation of 9-Tat Peptide (R = kkkkkkkkk) by Chemical Synthesis

(1) Synthesis of 9-Tat Peptide (KKKKKKKKK: Oligolysine)

The 9-Tat peptide (oligolysine) used in the present invention was synthesized by a solid phase method using a 431A autosynthesizer of Applied Biosystems. 0.25 mmol of parahydroxy methyl phenyloxymethyl polystyrene (HMP) resin was added to a standard reaction vessel (38 mL), and Fmoc-amino acid at the carboxy terminus of the peptide to synthesize Fmoc-amino acid was added. . Cartridges containing 1 mmol of Fmoc-amino acids are arranged in the guideway in the order of C-terminal amino acids to N-terminal amino acids. The metal lid of the cartridge was removed and empty cartridges containing no amino acids were placed at the beginning and end. Peptide synthesis was performed before editing according to the standard scale Fmoc coupling protocol developed by ABI and following the autosynthesis menu (see ABI User's Manual. Jan, 1992). When using standard scale Fmoc, deprotection was performed for 21 minutes with 20% piperidine diluted with NMP (N-Meting pyrrolidine), washed for 9 minutes with NMP and coupling for 71 minutes. Was carried out. HOBT (1-hydroxy-benzotriazole) was used for the coupling and the system was washed with NMP for 7 minutes.

(2) Isolation and Purification of the 9-Tat Peptide (KKKKKKKKK: Oligolysine)

After the synthesis was completed, the separation of the 9-Tat peptide was cleaved from the solid support using trifluoroacetic acid (TFA). Peptides were isolated by referring to the ABI manual (Introduction to Cleavage Techniques, P6-19 (1990)). After synthesis, the resin with the peptide was placed in a round flask and cooled. The lid was closed and reacted at room temperature for 1 to 2 hours. After the reaction, the resin and the reaction solution were filtered through low vacuum through a sintered glass funnel to separate the resin and the peptide solution. The flask and glass funnel were washed with 5-10 mL DCM (dichloromethane) to mix the solution with the peptide solution. At least 50 mL of cold diethylether was added to obtain a precipitate of peptides. The precipitate was filtered with a funnel under low vacuum to dry the precipitate collected on the funnel, and then dissolved in 30% acetic acid and freeze-dried. The peptide thus obtained was purified by HPLC (High Performance Liquid Chromatography). The column was a C ₁₈ analytical column (Pharmacia), buffer A was equilibrated with 10% acetonitrile + 0.05% TFA, buffer B was peptide using 80% acetonitrile + 0.05% TFA Eluted. As a result, a highly purified peptide was obtained and the synthetic yield was about 30 ± 5%.

Example 3 Synthesis of Hydroquinone Cesium Salt (Compound A)

Hydroquinone (3.12 g, 28.4 mmol) was dissolved in 20 mL of water, and then solid cesium bicarbonate (4.63 g, 14.2 mmol) was slowly added while maintaining the solution at 0 ° C to 5 ° C. After complete addition, 40 mL of isopropyl alcohol is added slowly and the solid is obtained when carbon dioxide gas is completely stopped. The resulting solid was filtered, washed and dried to yield 7.36 g (95%) of a white solid (Compound A).

Example 4 Preparation of Tat Peptide-Hyroquinone (Compound 1)

The cell penetrating Tat peptide (3.1 g, 5.45 mmol) prepared in Example 1 or 2 was dissolved in 10 mL of DMF. Anhydrous SOCl ₂ was added and then stirred for 2 hours. Hydroquinone cesium salt (Compound A) (760 mg, 3.15 mmol) was added and the reaction was then reacted at 60 ° C. for 24 hours. After removal of the solvent under reduced pressure, crystallization with 20 mL of diethyl ether, followed by drying and purification using reverse phase column chromatography gave 1.23 g (66%) of Tat peptide-hydroquinone (Compound 1).

Example 5. Tyrosinase Inhibitory Activity

The tyrosinase enzyme was extracted from mushrooms, and a sigma company was used. First, L-tyrosine, a substrate, was dissolved in a phosphate buffer solution (0.05 M concentration, pH 6.8) to a concentration of 1.5 mM, and then 0.01 mL of this solution was added to a 0.3 mL spectrophotometer cuvette and cofactor. Phosphorus waveguide was added to 0.016 mL at a concentration of 0.06 mM. To this, the compound represented by Chemical Formula 1 of the present invention was added, and the phosphate buffer solution was added to 0.1 mL. The reaction was advanced by adding 0.1 mL of an enzyme solution in which tyrosinase was dissolved in phosphate buffer at 60 U / mL. And arbutin and hydroquinone were used for comparative experiments. In this case, 0.1 mL of a buffer solution was added instead of tyrosinase as a control (blank).

The reaction was let proceed at 37 ℃ 10 minutes, a spectrophotometer (spectrophotometer, Beckman DU-7500) by measuring the absorbance at 475 nm using a seek inhibition rate on tyrosinase, IC ₅₀ values for the 50% activity inhibition Determined by the concentration of inhibitors to reach. The formula for calculating the inhibition rate is as shown below, and as a result, the compound represented by Formula 1 showed relatively superior tyrosinase inhibitory ability than arbutin.

A: absorbance at 475 nm without addition of inhibitor

B: absorbance at 475 nm with inhibitor added

Example 6 Stability Test

In order to evaluate the stability over time of the compound represented by the formula (1), arbutin, and hydroquinone, each prepared in 2% concentration using purified water and stored in an opaque container at room temperature (25 ℃), high temperature (40 ℃) Quantitative analysis by high performance liquid chromatography (HPLC). The pH of the aqueous solution was calibrated to 7.0 using 0.1 N sodium hydroxide, and as a result, the compound represented by Chemical Formula 1 was found to have excellent temperature change and stability over time compared to arbutin and hydroquinone.

Example 7 Percutaneous Absorption Experiment

Intradermal cell penetration of the compound represented by Formula 1, which is commonly referred to as transdermal absorption, is referred to herein as transdermal absorption. Percutaneous absorption experiments were compared with arbutin and hydroquinone, which are used as known cosmetic ingredients for whitening, and purified water as carrier media (Ref .: LehmanPA, Slattery JT, Franz TJ.Percutaneous absorption of retinoids: Influence of vehicle, light exposure, and dose, J. Invest Dermatol., 91; 56-61, 1988).

Specifically, the skin of the dorsal region of 8-week-old female rats (hairless mice) was cut to a size of 1.7 cm ² , and 250 μl of a 35 nM sample was applied thereto, followed by a transdermal absorption device (Franz cell) for 24 hours. After the receptor solution (PBS buffer solution containing 2% surfactant) and the material absorbed in the dorsal skin of the rat was extracted and quantitatively analyzed by high pressure liquid chromatography (HPLC) of the present invention represented by the formula (1) Compounds, arbutin and hydroquinone were quantitatively compared for transdermal uptake. As a result, the compound represented by the formula (1) showed a relatively superior percutaneous absorption increase rate compared to the conventional arbutin, and hydroquinone.

Example 8. Allergy Test (LLNA)

As a safety test for the compound represented by Chemical Formula 1 as a cosmetic raw material, ethanol was used as a transport medium (Kimber I (1990): Identification of contact allergens using the murine local lymph node assay, J. Appl.Toxicol. 10 (3); 173-180). 3-0-ethylhydroquinone and the compound represented by Formula 1 were used as 0.3% and 1% solutions, respectively. That is, 50 μl was applied to both ears of the mouse (Balb / c) for 3 days, and then auricular lymph nodes were isolated from the mice. Lymph nodes were pulverized into a single cell state and incubated for 24 hours with the addition of radioisotope ( ³ H-thymidine), and then the amplification degree (cpm) of the cells was measured. In all 5%, 10% and 20% solutions, allergens were lower than ethanol.

Example 9 Skin Irritancy Test of Hydroquinone with Tat Peptide

For the skin irritation test of the compound represented by Formula 1, a patch test using a guinea pig (Guinea Pig) was performed (Reference: ①Draize, JH (1959): Dermal toxicity.Assoc.Food and Drug officials, US.Appraisal of the safety of chemicals in Food, Drugs and Cosmetics., pp 46-59, Texas State Dept. of Health, Austin, Texas. (2) Federal Register (1973): Method of testing primary irritant substances 38 (187). : pp1500-1541). The sample was tested by preparing an oil-in-water emulsion having various concentrations of the compound represented by Formula 1, albumin, and hydroquinone.

First, remove the hair from the sample application area (back) and adjust the environment for 24 hours to minimize skin irritation, and then set the sample application area (1.5 cm × 1.5 cm) and apply the sample and gauze, and evaporate and lose the sample. In order to prevent the sealing with solid foil and fixed with an elastic bandage for 48 hours. The degree of stimulation was determined 2 hours and 24 hours (48 hours and 72 hours after the patch) after removal of the closed patch. As a result, it was found that the compound represented by Chemical Formula 1 is very safe for the skin compared to hydroquinone.

Example 10 Cytotoxicity Test of Hydroquinone with Tat Peptide

Method of MTT test by culturing V79-4 cells (Chinese hamster, continuous cell line of lung tissue fibroblasts) on compound represented by Formula 1 to verify primary safety as a raw material used in cosmetics (Reference: Mossman T. (1983) .Rapid colorimetric assay for cellular growth & survival: Application to proliferation & cytotoxicity assays.Journal of Immunological Methods 65, 55-63). Compared to quinone, it showed almost no toxicity and showed better stability than arbutin.

The present invention provides a hydroquinone derivative having a Tat peptide having a self-invasive signal, and a skin whitening agent comprising the same, and the whitening agent of the present invention has a pH compared to a conventional hydroquinone derivative whitening agent due to the presence of a cell penetrating Tat peptide. It is stable to oxygen, and light, has excellent absorption of skin, is non-irritating, stable in vivo, and has an excellent whitening effect and its durability.

Claims (6)

Hydroquinone derivative represented by the following formula (1): [Formula 1]

Where [] _Tat is a cell penetrating Tat peptide and is selected from the group consisting of RKKRRQRRR, KKKKKKKKK, and RRRRRRRRR. delete delete A method of preparing a hydroquinone derivative according to claim 1, wherein the hydroquinone derivative is prepared in a solvent in the presence of a catalyst and a condensation agent with a halide at the C-terminus of the cell penetrating Tat peptide, followed by an esterification reaction with the hydroquinone or its derivative. Way. The method of claim 4, wherein the cell penetrating Tat peptide is prepared by a biological method or a chemical synthesis method using a genetic recombination technique. A cosmetic composition for skin whitening comprising the compound according to claim 1 as an active ingredient.