WO2009051460A2

WO2009051460A2 - Peptide coupled ascorbic acid derivatives

Info

Publication number: WO2009051460A2
Application number: PCT/KR2008/006204
Authority: WO
Inventors: Yoon-Sik Lee; Han-Young Kim; Jin-Mi Noh
Original assignee: Seoul National University Industry Foundation
Priority date: 2007-10-20
Filing date: 2008-10-20
Publication date: 2009-04-23
Also published as: KR20090040187A; KR101413939B1; WO2009051460A3

Abstract

The present invention relates to a peptide linked ascorbic acid derivative and preparation method thereof, which has a whitening effect.

Description

[DESCRIPTION] [Invention Title]

PEPTIDE COUPLED ASCORBIC ACID DERIVATIVES [Technical Field]

<1> The present invention relates to a peptide coupled ascorbic acid derivative of the following formula (I) with whitening effect. Particularly, the present invention is directed to peptide coupled ascorbic acid derivative of formula (I) and a method for preparing thereof:

<2> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a side chain of amino acid residue! R is NH2 or OH; and n is an integer of 2 to 5.

<3>

<4> Formula (I) [Background Art]

<5> There are 20 kinds of amino acids as being a minimum unit of protein, of which 9 kinds of amino acids are not synthesized in human body, so they are classified as essential amino acids which should be taken through foods. Here, amino acid would be classified into hydrophilic amino acid and hydrophobic amino acid. The amino acid having hydrocarbon chain or ring is known hydrophobic, and there are alanine, glycine, isoluecine, luecine, methionine, phenylalanine, tryptophan, proline, and valine.

<6> The peptide is a polymer and resides in a form that a few number of amino acids is coupled. When it is coupled with a lot of amino acids, it is generally called protein. The peptide coupling is formed in a form of -CO-NH- formed as water is released from between -COOH and NH₂. Namely, the peptide is formed in a form of a compound in which at least two amino acids are peptide coupled, which amino acids are major components in the construction of protein. Generally, 2 to 50 amino acids are coupled, with the amount of molecular being less than 100,000. The peptide of which number of amino acids is 2, 3, 4 is called dipeptide, tripeptide, tetrapeptide depending on their coupled numbers. Here, the peptide coupled with less than about 10 peptides is called oligopeptide. What is coupled with multiple peptides is called polypeptide.

<7> Ascorbic acid is so-called vitamin C and is hydrophilic and has an excellent whitening effect.

<8> In recent years, society becomes aged, and leisure people increase, so many people is easily exposed to ultraviolet ray. Furthermore, social stress is on the rise, and the radiation of ultraviolet ray increases depending on the destruction of ozone layer due to an environmental pollution, so skin pigmentation phenomenon occurs. So, a lot of people are interested in a skin whitening. As a social and economical demand with respect to a skin whitening increases, a biologically friendly and high whitening activation substance is urgently needed in the industry.

<9> The above research is widely conducted in Korea. About 40 chemicals are introduced for curing pigmentation. In a cosmetic field, various kinds of cosmetics are available, which have excellent skin whitening effects.

<10> The skin color of people is determined depending on the amount of melanin, carotene and hemoglobin of which melanin functions as a major element. The melanin is synthesized in melanocyte which resides in a substrate layer of a skin as a coloring cell. The melanin is transferred to a neighboring horny substance (keratinoside)), so a specific skin color is shown. The melanin residing in an epidermis which is an outer skin layer functions for blocking ultraviolet ray and holds a free radical created in a skin biological body while protecting a skin organ below dermis. Namely, the melanin basically protects proteins and genes in a skin.

<1i> However, the melanin, which is created by means of internal or external stress-based stimulus, is a stable substance which is not discharged to the outside through a skin horning process even when stress disappears. Over created melanin causes liver spots or freckle which are big problems to women.

<12> The synthesizing process of melanin in a human body will be described. It is known that melanin is created through a copolyperization and oxidation process using an enzyme such as tyrosinase as a catalyst with tyrosine or DOPA being adapted as a substrate. In addition, the creation of melanin increases when a lot of free radicals increases in skin, or inflammation reaction occurs, or a certain stimulus such as ultraviolet ray is applied.

<13> The presently available whitening agent contains a component for inhibiting a creation of melanin and a component for removing a melanin pigment which resides in a horn layer.

<14> As a whitening agent which is able to inhibit a formation of a melanin pigment, there are Albutine, Kojic acid, ascorbic acid, etc. As a substance for removing a horny substance with a pigment, there are AHA (alpha hydroxy acid), BHA (butylated hydroxyanisole) , retinoid.

<15> The chemicals mainly used as a whitening agent are substances well known s a melanin creation inhibition agent and are polyhydroxyphenol compound such as phenol, catechol, Resorcinol and hydroquinone. Here, the polyhydroxy compound is a compound for inhibiting an activation of tyrosinase with the helps of antioxidation effect while providing a positive effect in view of an inhibition of enzyme activation, whereas it is known to cause a serious skin trouble, which might lead to leukoplakia by destroying a melanin creation cell, so there is a big problem in its stability for an application to a skin.

<16> In addition, the ascorbic acid derivatives or plant extracts such as Mori Radicis Cortex, aloe, licorice root, etc. which are a skin whitening agent generally used in cosmetics have some problems because they do not have substantial whitening effects. Since the above substances almost have reduction functions since they are coupled with a plurality of peptides, the whitening effects might decrease when they are stored at a room temperature for a long time, and the colors might change. So, in the industry, a more effective whitening agent development having no problems is urgently needed.

<17> In the development of such whitening agents, it is very important to develop a biologically friendly agent having stability with respect to long time storage along with an excellent whitening activation. Namely, a new agent should not have a skin trouble with its biologically friendly function while effectively inhibiting an activation of biological tyrosinase, and a development of a whitening agent which can be stored for a long time is very important. In order to synthesize a whitening agent which is able to satisfy the above criteria, it is needed to properly combine a whitening activation and a biologically friendly function by introducing a biologically friendly substance to a substance which is previously known as having a whitening activation.

<18> Though many researches and developments are conducted so far with respect to the substances having a whitening activation and biologically friendly function, a whitening agent having a stability in a long time storage function is not developed in the industry.

<19> The whitening agents, which can be used as an activation inhibition agent of tyrosinase, are substances having reduction functions having a structure similar with phenol in a chain of Tyrosine, and there are Kojic acid, Albutine, ascorbic acid, etc. <20> In case of Kojic acid, it is known as having a melanin creation inhibition function and can be used as a material of a whitening cosmetic. However, since it is not stable with respect to light, heat and pH, it cannot be well applied to a combination of cosmetic materials.

<2i> In order to overcome the above disadvantages, the Kojic acid derivatives such as Kojic acid-vitamin C complex (chem.. Pharm. Bull., 44(9), 1647-1655(1996), Kojic acid-amino acid complex (Biosci. Biotech.Biochem. , 59(9), 1745-1746(1995), Kojic acid-Kojic acid complex (WO 02/053562), Kojic acid-cinnamic acid complex (US5,486,624), Kojic acid-oxothiazolidine complex (US5,968,487) , Kojic acid mono fatty acid ester (Japan patent laid-open Sho60-9722), Kojic acid di-fatty acid ester (Japan patent laid-open 61- 60801), Kojic acid glycoside (Japan patent laid-open Hei6-56872), etc. are developed.

<22> Since Albutine has a relatively lower stimulus as a skin whitening component, it is generally used for a skin de-pigmentation. Albutine is formed as β-D- glucose is coupled to hydroquinone. When it is applied to skin, β-D- glucose is slowly decomposed by means of a decomposition enzyme residing in a human body for thereby obtaining an excellent whitening effect. The above prior art for using a whitening function of Albutine is disclosed in Korean patent laid-open number 2001-0109730. In the above prior art, a technology for preparing a whitening cosmetic using cowberry is disclosed.

<23> Since Albutine has an excellent whitening effect, but has a disadvantage that decoloration problem occurs in the course of solidifying as cosmetic. So, its application is limited. A number of studies are performed so as to overcome the above problems. As disclosed in Korean patent laid-open number 1999-0065147, there is a trial for stabilizing Albutine by adding leutin along with Albutine, and according to Korean patent registration number 10-0198508, there is a trial for obtaining a stabilization in a Albutine -contained whitening cosmetic by capsulating Albutine by using PLA as wall material .

<24> However, in case of a whitening agent which contains only Albutine, its stability is poor, and when adding another stabilization component such as leutin so as to obtain a certain stability, the added stabilization agent interferes with a whitening effect. In addition, a capsulation is non- economical, and a desired whitening effect is not obtained.

<25> As the most representative one among cosmetic materials, vitamin has been used. Vitamin is one of the necessary substances having metabolism acceleration, antioxidation effect, cell wall protection, immunity acceleration, infection resistance enhancement, etc. In case of almost primates, it is impossible to biologically synthesize vitamin, so it should be taken through foods. In lack of vitamin, various deflection problems occur. In case of skin caring and therapy, vitamin functions as an important component for pigmentation prevention, collagen synthesize acceleration, ultraviolet ray block, skin dry and horning prevention, wrinkle prevention, skin moisturizing effect, etc.

<26> Vitamin is formed of Retinol (vitamin A), Ascorbic acid (vitamin C, Tocophirol (vitamin E) and their derivatives.

<27>

<28> (Ascorbic acid)

<29>

<30> Vitamin C has a function such as a skin wrinkle improvement through UV block effect, antioxidation effect and collagen formation acceleration, pigmentation improvement effect such as liver spot/freckle/dark spot, etc, and immune system enhancement effect. The above vitamin C effects are as follows.

<3i> Vitamin C has a blocking function with respect to UVA (Darr, D. et al., 1996, Acta Derm. Venereol . (Strckh). 76: 264-268; Black, H. S. et al . , 1975, J. Invest. Dermatol. 65: 412-414). In addition, vitamin functions for protecting a skin damage which might occur due to UVB. When vitamin C is applied to a pig and a human skin where UVB is applied before the UVB is radiated, red burn problem and sun burn-like phenomenon are prevented (Darr, D. et al., 1992, Brit. J. Dermatil. 127: 247-253; Murry, J. et al., 1991, J. Invest. Dermatol. 96: 587).

<32> In a skin, blood and other tissues, vitamin C operates as a strong biological ant ioxidation agent which neutralize ROS (reactive oxygen species) caused by means of UV. Vitamin C receives two electrons and is easily oxidized in a form of dehydro-L-ascorbic acid. Vitamin C is an important component in a non-enzyme ant ioxidation protection system of skin. When vitamin C resides very intensively, ROS such as singlet oxygen, superoxide anion and hydroxy radical neutralizes oxides a biological component such as protein, hexane and cell membrane substrate before oxidizing or transforming their properties (Buettner, G. R. et al. 1996.Cadenas, E., Packer, L., eds. Handbook of antioxidants, pp. 91-115). As vitamin C is supplied to a horny tissue in percutaneous manner, it has a skin luster effect, skin color improvements, wrinkle decrease and elastic force enhancement (US patent number 4983382). In addition, it is known that collagen synthesization is accelerated with the help of vitamin C. Hydroxyproline, which occupies about 10% of collagen polypeptide, is biologically synthesized by means of proline hydroxylase. At this time, vitamin C functions as cofactor (Tomia, Y. el al., 1980, J. Invest. Dermatol. 75(5): 379-382). Namely, vitamin C accelerates an activation of proline hydroxylase and then accelerates a synthesization of Hydroxyproline, so that a collagen biological synthesization with a triple chain structure is accelerated, so that it is possible to improve a skin function such as a skin wrinkle formation inhibition effect.

<33> The whitening effect by means of vitamin C can be obtained since tyrosinase activation, which functions a key role in a formation of melanin, and melanin formation are inhibited (Tomia, Y. el al., 1980, J. Invest. Dermatol. 75(5): 379-382).

<34> The function of strengthening an immune system by means of vitamin C is proved as an emission of histamine, which is an allergy reaction substance, is inhibited from a cell membrane, and an allergy reaction is inhibited from a sensitive skin, and it is protected from an UV-guided immune inhibition (Nakamura, T. et al . , 1997, J. Invest. Dermatol. 109: 20-24).

<35> In addition, vitamin C helps a phagocytosis operation of white blood corpuscle and inhibites an injection by accelerating a white blood corpuscle move in the course of infection, and enhances a biological synthesization of interferon which is a virus growth inhibition substance, and even increases a biological resistance ability with respect to various infectious diseases. In addition, vitamin C functions in a metabolism of folic acid in body and amino acid.

<36> The vitamin C, which functions the above roles, is a water soluble substance having the formula of C6H806. Vitamin C has hydrophi licity because of hydration group in the positions of second, third, fifth and sixth carbons. In neutral pH such as water, hydration group, in particular second and third carbons have negative electric charges, so vitamin C well melts in water. However its solubility is limited in an organic environment like a skin.

<37> It is known that vitamin C does not well melts in an organic solvent officially used in an external use agent, for example, glycerine, propylene glycol and various fats. So, there is a certain limit for carrying vitamin C to a skin tissue. When vitamin C is not ionized, it can easily pass through a skin wall and is well absorbed. In order to maintain a non-ionized state, its pH should be less than 4.2.

<38> The skin accumulation level of vitamin C is as high as 20 times through 40 times when it is used as external use agent rather than oral administration. In case of a skin external use agent used for the purposes of UV block effect, antioxidation effect, collagen formation acceleration-based skin wrinkle improvement, pigmentation improvement effects with respect to liver spot/freckle/dark spot, an effective substance should reach a cell of a skin layer through a horny tissue of a skin, so it is needed to increase a Percutaneous absorption rate of an effective substance.

<39> Generally, a skin penetration rate of a certain substance depends on a lipophilicity of the same.

<40> In case of a substance similar with a skin lipophilicity, since a distribution coefficient toward a skin is high, percutaneous adsorption is better. In case of vitamin C, lipophilicity is high, and a distribution to a skin is low, so percutaneous adsorption is worse.

<4i> Ascorbic acid, namely, vitamin C is a competent substance for a competent coupling with Tyrosine with respect to tyrosinase which is a rate limiting enzyme in the course of melanin biological synthesis process, and is an inhibitor with respect to a generation of tyrosinase gene. A full activation with respect to a whitening related biological process is obtained by inhibiting a unnecessary melanin biological synthesis by reducing oxidized melanin (Postaire E et al . , Biochem MoI Int , 42, 1023pp-1033pp,1997) . However there is a problem for actually using vitamin C due to its stability problem. In particular, the color of vitamin changes and turns a dark brown, nearly black color, through yellow and brown colors depending on its destroy level, so the product quality is worsened.

<42> In order to overcome the above problems, a method for preparing various derivatives based on a coupling by a chemical reaction with various salts at a portion where ascorbic acid can easily change (Korean patent number 10- 0459679-0000 and Korean patent laid-open number 2001-0109730).

<43> However all ascorbic acid derivatives, which are prepared so far, are worse in their effects than pure ascorbic acids, and skins might be stimulated since harmful residues remain, which is generated in the course of a derivative synthesis process. In addition, ascorbic acid can be easily decomposed and is hydrophilic, a penetration with respect to a cell is low, so a lot of ascorbic acid should be used so as to obtain a whitening effect.

<44> A technology for enhancing a biological friendship and stability while maintaining a reduction of a whitening agent is urgently needed in the industry.

<45> In the present invention, a biologically friendly peptide is induced into ascorbic acid which is widely used as a whitening substance, so it is possible to maintain a stability in its long time storage while maintaining a whitening effect without a side effect in skin. [Disclosure] [Technical Problem]

<46> The primary object of the present invention is to provide a peptide coupled ascorbic acid derivative of formula (I), which has biocompatibility, whitening effect and long-term storage stability without side effects by the introduction of peptide into ascorbic acid:

<47> <48> Formula (I)

<49> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a side

2 chain of amino acid residue; R is NH₂ or OH; and n is an integer of 2 to 5.

<50> Another object of the present invention is to provide a method for preparing an ascorbic acid peptide derivative of the above formula (I), which comprises reacting an ascorbic acid of which a hydroxyl group at the 3- position is activated with a peptide formed from 2 to 5 amino acids.

<51 > Yet another object of the present invention is to provide a skin whitening agent which comprises a compound of the above formula (I). [Technical Solution]

<52> The above-mentioned object of the present invention can be achieved by providing a peptide coupled ascorbic acid derivative of formula (I):

<53> <54> Formula (I)

<55> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a side chain of amino acid residue; R is NH₂ or OH; and n is an integer of 2 to 5.

^<56> Ascorbic acid used in the preparation of the skin whitening cosmetic composition the persent invention is an ascorbic acid produced by the conventional method.

<57>

<58> Ascorbic acid

<59>

<60> Ascorbic acid(L-threo-2,3,4,5,6-pentahydroxy- 2-hexenoic acid-4- lactone) is an essential amino acid for human and is mainly contained in fresh vegetables. The ascorbic acid is a compound having a strong reducing power, and L-ascorbic acid is called vitamin C. The oxidation type and reduction type of the ascorbic acid can change in reverse. Its aqueous solution is acidic since one of enol-type hydroxyls is dissociated. It operates in an oxidation-reduction system in vivo as vitamin.

<6i> There are 20 amino acids (phenylalanine(F) , tyrosine(Y), tryptophan(Y) , serine(S), threonine(T), asphalt ic acid(D), asparagin(N) , Glutamic Acid(E), Glutamin(Q), lyzine(K), arginine(R), histidine(H), cysteine(C), methionine(M), proline(P), glycine(P), alanine(A), valnine(V), luecine(L), isoluecine(D) and amino acids has an L- and D-forms. Amino acids might be classified into hydrophile and hydrophobe. The amino acid having a hydrocarbon chain or ring is hydrophobic, examples of which are alanine, glycine, isoleucine, leucine, methione, phenyl alanine, tryptophan, proline and valine.

<62> Peptide is a short polymer of amino acids. When a lot of amino acids are linked, it is called protein. A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amine group of the other molecule, thereby releasing a molecule of water. Namely, the peptide is a compound in which at least two amino acids, which are major components for forming protein, are linked through the peptide bond. Generally, 2 to 50 amino acids are linked to form a peptide, with the amount of molecular weight being less than 100,000. The peptide of which number of amino acids is 2, 3, 4 etc. is called dipeptide, tripeptide, tetrapeptide etc., respectively, depending on the number of peptide bonds.

<63> Since proteins which const itutess a biological membrane contains a lot of hydrophobic amino acids such as alanine, leucine, isoleucine, etc., the biological membrane is strongly hydrophobic. Ascorbic acid has a low biological membrane permeability due to its hydrophilicity and, however, an ascorbic acid peptide derivative having a hydrophobic amino acid residues has a high biological membrane permeability.

<64> In addition, an ascorbic acid peptide derivative containing an amino acid residues of phenyl alanine, tryptophan, tyrosine, lysine and aspartic acid has a high inhibition of tyrosinase activity, and much higher inhibition of tyrosinase activity when the tyrosine amino acid residue includes tyrosine residue.

<65> Another object of the present invention can be achieved by providing a method for preparing an ascorbic acid peptide derivative of the above formula (I), which comprises reacting an ascorbic acid of which a hydroxyl group at the 3-position is activated with a peptide formed from 2 to 5 amino acids.

<66> In order to activate only the 3-position hydroxyl, prepared substance and 1,1-carbonyldidimidazole (CDI), which is a precursor of carbamate, are reacted in anhydride THF. The peptide is synthesized using a solid phase synthesis. A novel ascorbic acid peptide derivative is prepared by coupling the peptide with the ascorbic acid of which only 3-position hydroxy1 is activated.

<67> Another object of the present invention can be achieved by providing a method for preparing an ascorbic acid peptide derivative of the above formula (I), which comprises reacting an ascorbic acid of which hydroxy1 groups at the 5- and 6- positions are protected and a hydroxyl group at the 3-position is activated with a peptide formed from 2 to 5 amino acids.

<68> The tyrosinase inhibition of the ascorbic acid which is conventionally known as a whitening agent can be enhanced by the introduction of biocompatible peptide, as well as biocompatibi lity and stability. In order to synthesize an ascorbic acid derivative, an ascorbic acid is reacted by adding acetone and acetyl chloride, and the resulting product is washed with acetone/hexane, thereby producing 5,6-0-isopropylidene ascorbic acid which is protected in an acetal form in its 5- and 6-positions. It can be confirmed by comparing H-NMR data of L-ascorbic acid and H-NMR data of 5,6-0- isopropylidene-L-ascorbic acid that acetals are bonded at the 5- and 6- positions.

<69> In addition, in order to activate only the 3-position hydroxyl, prepared substance and 1,1-carbonyldidimidazole (CDI), which is a precursor of carbamate, are reacted in anhydride THF.

2

<70> In the case that R of the formula (I) is NH₂, the peptide is synthesized on a rink amide resin via a solid phase synthesis, and then the peptide is reacted with the ascorbic acid of which only hydroxyl at the 3- position is activated, thereby producing a novel ascorbic acid peptide derivat ive.

<7i> Yet another object of the present invention can be achieved by providing a skin whitening agent which comprises a compound of the above formula (I). The compound of the above formula (I) has a skin whitening effect. <72> Still another object of the present invention can be achieved by providing a skin whitening cosmetic composition which comprises a compound of the above formula (I) and a cosmetic substrate which is physiologically acceptable.

<73> The skin whitening cosmetic composition of the present invention is not limited to its formulation, and a conventional components of skin cosmetics typically used in the art might be included depending on the formulation to be prepared.

<74> For example, the skin whitening cosmetic composition of the present invention may be formulated as one selected from the group consisting of cosmetic water, emulsion, cream, pack, cosmetic solution, etc. and may be added by an appropriate amount of oily powder, water, surfactant, moisturizing agent, low grade alcohol, thickener, chelating agent, coloring agent, preservative, flavoring agent, etc. depending on the formulation to be prepared. In consideration that a major factor, which affects a melanin formation, is UV, the whitening cosmetic composition according to the present invention might contain a UV blocking agent, a light scattering agent, etc. and, however, it is obvious that the formulations and additives are not limited thereto.

<75> Moreover, the compound of the formula (I) may include medicinal composition for a parenteral (subcutaneous) administration, and further comprise a biologically and pharmatically available carrier. The above medicinal composition may include a medicinal composition for typical subcutaneous administration. [Advantageous Effects] <77> First, the ascorbic acid peptide derivative according to the present invention enhances a whitening effect through tyrosinase activity inhibition.

Also, the ascorbic acid peptide derivative has a melanin synthesis inhibition function in cells. <78> Secondly, an easier skin penetration can be achieved due to a biocompatible peptide of ascorbic acid peptide derivative. <79> Thirdly, the ascorbic acid peptide derivative of the present invention is more stable than the conventional ascorbic acid derivatives. [Description of Drawings] <80> Figure 1 is a reaction scheme illustrating an activation process for coupling the amine end group of peptide to an ascorbic acid. <81> Figure 2 is a reaction scheme illustrating a process that peptide is synthesized on a rinkamide-resin through a solid phase synthesis and is linked with an activated ascorbic acid according to the present invention. <82> Figure 3 is a graph which compares stabilities of a conventional ascorbic acid whitening agent and the whitening agent comprising the ascorbic acid derivative of the present invention.

[Best Mode] <83> Hereinafter, the technical elements and features of the present invention will be described in greater detail with reference to the following examples. However, the examples are given only for illustration of the present invention and not to be limiting the present invention.

<84>

<85> Example 1. Synthesis of activated ascorbic acid derivative

<86> In order to link an amine end group of peptide to the 3-hydroxyl of ascorbic acid which is a whitening substance, two hydroxyls at the 5- and 6- positions of the ascorbic acid were protected as an acetal form. Ascorbic acid(25g, 142mmol) was suspended in 200ml of acetone and was agitated for 30 minutes, and then acetyl chloride (2.81ml) was added and reacted for 12 hours, thereby producing white liquid. <87> The liquid was maintained at 4^°C for 24 hours, and white precipitates were obtained. The precipitates were filtered and washed using 20% acetone/hexane, thereby obtaining 5,6-0-isopropylidene ascorbic acid of which 5- and 6-positions were protected in acetal form.

<88> In order to activate only the 3-hydroxyl , the obtained substance (4g, 19mmol) and carbonyldidiinidazole (2.9g, lδmmol) which is a precursor of carbamate, were melted in anhydride THF(150ml) and were reacted for 72 hours, thereby obtaining a white solid.

<89>

<90> Example 2. Test for checking whether the hydroxyIs at the 5- and 6-positions of ascorbic acid were protected or not.

<91> The Jung's method (Jung's method, M. E. Jung; T. J. Shaw; J. Am.

Chem. Soc. 1980, 102, 6304) was used in order to protect the hydroxyls at the 5- and 6-positions of ascorbic acid in an acetal form using acetone. The ascorbic acid of which hydroxyls at the 5- and 6-positions were protected are commercially available. The H-NMR of the ascorbic acid protected in the above manner is open in the data of Aldrich, which is matched with a result of the synthesis of the present invention. The following H-NMR data show the data of the non-protected ascorbic acid and the H-NMR data of the ascorbic acid of which the hydroxyls at the fifth and sixth positions are protected using acetone. As shown below, there are two CH₃ peaks of the acetal portions at

1.33ppm, which means that the hydroxyls at the 5- and 6-positions of ascorbic acid were protected.

<92> ¹H-NMR of L-ascorbic acid (300MHz, d6-DMS0) δ 3.44-3.85(4H, m),

4.54(1H, d, J = 3Hz)) <93> ¹H-NMR of 5,6-0-Isopropylidene-L-ascorbic acid (300MHz, d6-DMS0 ) δ

1.33(6H, s), 3.90-4.45(4H, m), 4.6K1H, d, J = 3Hz).

<94>

<95> Example 3. Solid phase peptide synthesis using rink amide resin <%> Peptide was synthesized, via solid phase synthesis, by reacting 2 equivalents of Fmoc-amino acid for 2 hours under N-methylpyrrolidone (NMP) on the aminomethyl resin to which Rink amide linker was introduced. Then, amino acids were further introduced to the peptide using the Fmoc amino acid, BOP (benzotriazole-l-yloxy-tris(dimethylamino)-phosphoniumhexaf luorophosphate) and DIPEA(Di isopropylethylamine) coupling reagents. At this time, the coupling time was set to 2 hours, and the separation of Fmoc was conducted by treating twice the mixture using 20% piper idine/NMP for 3 minutes and 7 minutes, respectively.

<97>

<98> Example 4. Preparation of novel whitening agent of ascorbic acid peptide derivative.

<99> A novel ascorbic acid peptide derivative was prepared by coupling the activated ascorbic acid derivative synthesized in Example 1 on the Rink amide resin in which the peptide was synthesized in Example 3. 2 equivalents of the ascorbic acid derivative were added to the resin from which the above N- termianl protecting group was removed, under the NMP and was reacted for 72 hours. Then, desired substance was separated from the resin by treating with 20% TFA/MC, and ascorbic acid peptide product was obtained by precipitation using ether.

<10o>

<10i> Example 5. Check for coupling of carbonyldidimidazole at the 3-hydroxyl of ascorbic acid.

<102> The reaction that carbonyldidimidazole, a precursor of carbamate, was selectively introduced only to the hydroxyl at the 3-position of ascorbic

13 13 acid was confirmed by means of C-NMR. Analysing the below data of C-NMR, it was confirmed that the carbonyldidimidazole was introduced only at the 3- position of the final product since 3-carbons of 5,6-0-isopropyl idene-L- ascrobic acid and the final product appeared at 152.92 and 153.09, respectively. Numerals following C without any mark mean carbon of ascorbic acid, numerals with ' mean carbon of the acetal portion oat the 5- and 6- positions, and numerals with ' ' mean carbon of carbonyldidimidazole introduced at the 3-position.

<103> ¹³C-NMR of 5,6-0-Isopropylidene-L-ascorbic acid (75MHz, d6-DMS0 ) δ

61.97(C-2¹), 64.82(C-3¹), 68.39(C-6), 73.38(C-5), 74.20(C-I'), 74.60(C-4), 118.13(0-2), 152.92(C-3), 170.65(C-I)

<104> ¹³C-NMR of Activated-L-ascorbic acid (75MHz, d6-DMS0) δ 64.9KC-2¹),

67.05(C-3'), 67.43(C-6), 73.73(C-5), 74.38(C-I¹), 75.48(C-4), 77.45(C-2"), 108.93(0-3"), 120.22(0-2), 134.68(C-4" ), 153.09(C-3), 170.52(C-I), 175.04(C-I").

<105>

<106> Example 6. Confirmation of ascorbic acid peptide derivative

<107> It was confirmed that the ascorbic acid peptide derivative was a desired substance through the LC-Mass upon measuring the purity of the ascorbic acid peptide derivative using HPLC. As a result of the analysis using HPLC, it was confirmed that this substance is a single material, and as a result of the mass analysis using LC-Mass, it was confirmed that the above substance was ascorbic acid peptide derivative.

<108>

<!09>

<πo> (Result of HPLC analysis)

<1π>

555.

100

480.0

% 452.0

556.1

! i

481.1

453.0 I

Ϊ

!

700

<1 12>

<1 i3> (Resul t of LC-Mass analysi s)

<1 14>

<1i5> Example 7. Whitening activity test of ascorbic acid polypeptide derivative of the present invention.

<1i6> Tyrosinase and tyrosine in phosphate buffer solution (pH 6.8, 0.1M) was reacted with the product of Example 3 at 37 ^°C for 10 minutes, and the reaction was stopped by placing the reaction mixture into an ice bath. Absorbance was measured at 475nm, and the tyrosinase activivity inhibition was measured based on the following formula.

<1i7> % activation inhibition = (A-B)/A x 100

<1i8> In the above formula, A is absorbance when the product of Example 3 was not added, and B is absorbance when the product of Example 3 was added. Each tyrosinase activivity inhibition was compared depending on the kinds of introduced peptides. Results of the above comparison are shown in Table 1. In case of unmodified ascorbic acid, it shows 50% activity inhibition at the concentration of about 675umol and, therefore, the concentration of whitening agent in the reaction solution is 500μmol.

<1 19> Table 1.

<120>

< 121 > As shown in Table 1, it can be seen that the activity inhibition of enzyme was on the rise when the peptide containing tyrosine was introduced to conventional whitening agent, i.e. ascorbic acid. In addition, a few other amino acids have more enhanced or similar enzyme activity inhibition than ascorbic acid.

<122> <I23> Example 8. Stability test for the prepared whitening agent

<124> The whitening activity test was conducted through a mushroom tyrosinase inhibition analysis like in Example 6 while storing the prepared whitening agent and the conventional whitening agent, ascorbic acid, at a room temperature for one month. Referring to Figure 3, there is a significant difference in the stability between the prepared ascorbic acid peptide derivative and the ascorbic acid. As shown in the graph of Figure 3, in the case of the conventional whitening agent of ascorbic acid to which peptide was not introduced, the enzyme activity inhibition continued to decrease when it was stored at 50^°C in an aqueous solution state, but the enzyme activity inhibition of the peptide-introduced ascorbic acid peptide derivative was maintained even after one month. According to the present invention, it is possible to enhance an enzyme activity inhibition by introducing a biocompatible peptide to the conventional whitening agent, ascorbic acid, while achieving excellent stability.

<125>

<126> Example 9. Melanin synthesis inhibition effect test of ascorbic acid peptide derivative in eel 1 <127> In order to test the melanin synthesis inhibition effect in vivo of the ascorbic acid peptide derivative of the present invention, the melanin synthesis inhibition effect in cell of the ascorbic acid peptide derivative was tested using two kinds of cell systems. <128> Table 2.

* (Note) ASA: ascorbic acid, F: phenyl alanine, W: tryptophan, Y: Tyrosine

<131> <132> Ascorbic acid peptide derivatives with the C^~end of carboxyl and amide were synthesized and compared. In the case of ascorbic acid-phenylalanine- tryptophan-tyrosine (ASA-FWY-OF), an enhanced effect was obtained in the tyrosinase inhibition and the melanin synthesis inhibition effect test using the MeI-Ab cell, compared to ascorbic acid. In the case of ascorbic acid- phenylalanine-tryptophan-tyrosine-amide (ASA-FWY-NH₂), an enhanced effect was obtained in the tyrosinase inhibition and the melanin synthesis inhibition test using B16 cell. In particular, in the case of acid-phenylalanine- tryptophan-tyrosine-amide (ASA-FWY-NH₂), an enhanced whitening effect was obtained in the tyrosinase inhibition and the two kinds of cell systems even at a low concentration, compared to ascorbic acid.

Claims

[CLAIMS] [Claim 1] <134> A peptide coupled ascorbic acid derivative of formula (I):

<135> <136> Formula (I)

<137> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a side

2 chain of amino acid residue! R is NH2 or OH; and n is an integer of 2 to 5.

[Claim 2]

<138> The ascorbic acid derivative of Claim 1, wherein said R comprises at least one side chain of hydrophobic amino acid residue.

[Claim 3]

<139> The ascorbic acid derivative of Claim 1, wherein said R comprises at least one amino acid residue selected from the group consisting of phenyl alanine, tryptophan, tyrosine, lysine and aspartic acid.

[Claim 4] <14O> The ascorbic acid derivative of Claim 1, wherein said R comprises a side chain of tyrosine amino acid residue. [Claim 5]

< 141 > A method for preparing an ascorbic acid peptide derivative of formula (I), which comprises reacting an ascorbic acid of which a hydroxyl group at the 3-position is activated with a peptide formed from 2 to 5 amino acids:

<142> <143> Formula (I)

<144> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a

2 side chain of amino acid residue; R is NH₂ or OH; and n is an integer of 2 to

5. [Claim 6]

<145> A method for preparing an ascorbic acid peptide derivative of formula (I), which comprises reacting an ascorbic acid of which hydroxyl groups at the 5- and 6-positions are protected and a hydroxyl group at the 3-position is activated with a peptide formed from 2 to 5 amino acids:

<146> <147> Formula (I)

<148> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a

2 side chain of amino acid residue! R is NH₂ or OH; and n is an integer of 2 to

5.

[Claim 7]

<149> The method of Claim 5 or Claim 6, wherein said hydroxyl group at the 3- position is activated by introduction of carbonyldi imidazole.

[Claim 8]

<150> The method of Claim 5 or Claim 6, wherein said peptide is synthesized

2 on rink amide resin and R is NH2.

[Claim 9] <151> The method of Claim 5 or Claim 6, wherein said peptide is synthesized

2 on 2-tritylchloride resin and R is OH. [Claim 10]

<152> A skin whitening agent which comprises a compound of formula (I):

<153> <154> Formula (I)

<155> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a

2 side chain of amino acid residue! R is NH2 or OH; and n is an integer of 2 to

5. [Claim 11]

<156> A skin whitening cosmetic composition which comprises a compound of formula (I) and a cosmetic substrate which is physiologically acceptable:

<157> <158> Formula (I)

<159> wherein is a peptide to which same or different amino acid residue selected among amino acids is bonded through amide bond; R is a

5. [Claim 12]

<160> The composition of Claim 11, wherein said composition is formulated as one selected from the group consisting of cosmetic water, emulsion, cream, pack and cosmetic solution.