HK1237798B

HK1237798B - Stable peptide-conjugated ascorbic acid derivative, method for preparing same, and cosmetic composition containing same

Info

Publication number: HK1237798B
Application number: HK17111782.0A
Authority: HK
Inventors: 朴永濬; 金贞润; 郑镇姣; 金炯美; 赵银珠; 任周爀; 宋贤南; 朴善卿; 文源刚; 张宸在; 洪承绪
Original assignee: 赛特瑞恩股份有限公司
Priority date: 2014-11-27
Filing date: 2015-10-22
Publication date: 2021-07-16

Abstract

The present invention provides a stable peptide-conjugated ascorbic acid derivative, a method for preparing the same, and a cosmetic composition containing the same as an active ingredient. The peptide-conjugated ascorbic acid derivative of the present invention has a dual complex function of exhibiting a whitening effect through melanogenesis inhibition and a wrinkle reduction effect through collagenesis activation at the same time, and can be used for a cosmetic composition.

Description

Stable peptide-conjugated ascorbic acid derivatives, methods for preparing the same, and cosmetic compositions containing the same

Technical Field

The present invention relates to a stable peptide-conjugated ascorbic acid derivative exhibiting excellent wrinkle-reducing effect and excellent whitening effect, a method for preparing the same, and a cosmetic composition containing the same as an active ingredient.

Background Art

Typically, ascorbic acid acts as a reducing agent, collagen synthesis promoter or antioxidant, stimulates iron absorption in the small intestine, and is involved in carnitine biosynthesis and immunity. It is known that when the body lacks ascorbic acid, scurvy, abnormalities in connective tissue caused by the abnormality in collagen synthesis, bone pain, fractures, diarrhea, etc. may occur, and when there is excessive ascorbic acid in the body, gastrointestinal disorders may occur, including nausea, abdominal pain, diarrhea, etc. In particular, the excellent antioxidant effect of ascorbic acid is known to suppress the production of melanin in the skin, thereby preventing abnormal pigmentation such as freckles, and therefore ascorbic acid is attracting people's attention as a material for skin external use.

Although ascorbic acid exhibits various effects as described above, it has problems in that it is almost destroyed when heated in air, is unstable to alkali, and is easily oxidized in aqueous solution, thereby losing such effects. Therefore, in order to overcome this instability of ascorbic acid, various ascorbic acid derivatives have been developed.

The ascorbic acid derivatives developed before are as follows. The first, there are derivatives such as phosphorylated ascorbic acid or its metal salt. Compared with other derivatives, such derivatives are easily converted into the L-ascorbic acid form available in the human body, but the disadvantage is that they are difficult to be absorbed into the skin because they have a negative charge. The second, the derivatives of known multiple fatty acid conjugates, ascorbyl palmitate, ascorbyl laurate, ascorbyl stearate, etc. Among these derivatives, ascorbic acid-6-palmitate (see U.S. Patent number 5,409,693) is the most widely used. These derivative compounds are absorbed into the skin, but the disadvantage is that they are difficult to be converted into the L-ascorbic acid form. The third, the peptide-conjugated ascorbic acid derivatives of known collagen production, wherein succinyl is connected to the hydroxyl group at carbon 5 or carbon 6 of ascorbic acid by an ester bond and the peptide producing collagen is connected by an amide bond (see Korean Patent No. KR 10-0459679). Compared with ascorbic acid, these derivatives show excellent efficacy but have the disadvantage of poor stability.

As mentioned above, almost all ascorbic acid derivatives developed to date have not been improved in efficacy or stability compared to pure ascorbic acid, and peptide-conjugated ascorbic acid derivatives are almost all designed to simply enhance the whitening effect of ascorbic acid. Therefore, the widespread use of these derivatives for cosmetic applications is limited.

Summary of the Invention

Technical issues

Therefore, the inventors of the present invention have made extensive efforts to develop ascorbic acid derivatives that have both wrinkle-reducing and whitening effects while overcoming the problems of conventional ascorbic acid derivatives. As a result, the inventors of the present invention have found that when a peptide compound having 2 to 5 units is conjugated with an ascorbic acid aminopropanol phosphate diester compound, a stable ascorbic acid derivative having both wrinkle-reducing and whitening effects can be obtained, thereby completing the present invention.

Therefore, an object of the present invention is to provide a peptide-conjugated ascorbic acid derivative represented by the following formula I or a cosmetically acceptable salt thereof.

Another object of the present invention is to provide a method for preparing a peptide-conjugated ascorbic acid derivative represented by the following formula I or a cosmetically acceptable salt thereof.

Yet another object of the present invention is to provide a cosmetic composition comprising a peptide-conjugated ascorbic acid derivative represented by the following Formula I or a cosmetically acceptable salt thereof as an active ingredient, and use thereof for reducing skin wrinkles and whitening.

Technical Solution

The present invention relates to a peptide-conjugated ascorbic acid derivative represented by the following formula I or a cosmetically acceptable salt thereof:

Formula I

in

refers to a peptide in which identical or different amino acid residues selected from natural or unnatural amino acid residues are linked via amide bonds;

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

_R2 is absent when X is hydrogen, or is palmityl, lauryl, or stearyl when _X is carbonyl; and

n is an integer in the range of 2 to 5.

Preferably, represents a peptide in which the same or different amino acid residues selected from glycine, alanine, serine, threonine, cysteine, valine, leucine, isoleucine, proline, hydroxyproline, lysine, phenylalanine, methionine, tyrosine, tryptophan, asparagine, glutamine, histidine, aspartic acid, glutamic acid and arginine are bound by amide bonds; _R represents the side chain of the amino acid residue; and n is an integer ranging from 3 to 5.

More preferably, represents a peptide in which the same or different amino acid residues selected from valine, lysine, glycine, arginine, aspartic acid, threonine and serine are bound by amide bonds; _R1 represents a side chain of the amino acid residue; and n is an integer ranging from 3 to 5.

Even more preferably, it represents lysine-valine-lysine or arginine-glycine-aspartic acid.

As used herein, the term "natural amino acid" refers to an α-amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, and glutamic acid.

As used herein, the term "unnatural amino acid" refers to an amino acid that is not encoded by a nucleic acid codon, and examples of unnatural amino acids include, but are not limited to, the D-isomers of the natural α-amino acids described above; Aib (aminobutyric acid), bAib (3-aminoisobutyric acid), Nva (norvaline), β-Ala, Aad (2-aminoadipic acid), bAad (3-aminoadipic acid), Abu (2-aminobutyric acid), Gaba (γ-aminobutyric acid), Acp (6-aminohexanoic acid), Dbu (2,4-diaminobutyric acid), α-aminopimelic acid, TMSA (trimethylaminobutyric acid), silyl-Ala), alle (alloisoleucine), Nle (norleucine), tert-Leu, Cit (citrulline), Orn, Dpm (2,2'-diaminopimelanediol), Dpr (2,3-diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), etc.; cyclic amino acids; Nα-alkylated amino acids, for example, MeGly (Nα methylglycine), EtGly (Nα ethylglycine) and EtAsn (Nα ethylasparagine); and amino acids having two side chain substituents on the α-carbon.

The peptide-conjugated ascorbic acid derivative according to the present invention is selected from the group consisting of:

(6S,9S,12S)-6,12-bis(4-aminobutyl)-9-isopropyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazanonacosyl(5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)hydrogen phosphate (I-1); and

(3S)-3-(2-((S)-2-amino-5-((diaminomethylene)amino)pentanamido)acetamido)-4-((3-((((5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)propyl)amino)-4-oxobutanoic acid (I-2).

In the present invention, "cosmetically acceptable salts" include, but are not limited to, all non-toxic inorganic and organic acid salts, for example, hydrochloride, sulfate, nitrate, phosphate, acetate, trifluoroacetate, benzenesulfonate, citrate, and the like.

The present invention also relates to a method for preparing a peptide-conjugated ascorbic acid derivative represented by Formula I. The preparation method of the present invention comprises the steps of subjecting an ascorbic acid aminopropanol phosphodiester compound of the following Formula II to a condensation reaction with a peptide compound of the following Formula IV having 2 to 5 units, and then subjecting the product to a deprotection reaction if a protecting group is present in the product:

Formula II

Formula IV

in

_R1 represents the side chain of the amino acid residue;

R ₁ ′ is the same as R ₁ or is R ₁ in which the amino group or the carboxyl group is protected;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

As the amino protecting group of the compound of formula IV, there can be used, but not limited to, tert-butyloxycarbonyl (t-Boc), benzyloxycarbonyl (carbobenzyloxy, Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), etc. In addition, the deprotection of the amino protecting group of the compound of formula IV can be carried out using hydrochloric acid or trifluoroacetic acid, but is not limited thereto.

As the carboxyl protecting group of the compound of formula IV, there can be used but not limited to methyl, ethyl, tert-butyl, 2,2,2-trichloroethyl, etc. In addition, the deprotection of the carboxyl protecting group of the compound of formula IV can be carried out using lithium hydroxide or trifluoroacetic acid, but is not limited thereto.

The condensation reaction can be carried out in the presence of a condensing agent. The example of the condensing agent that can be used in the condensation reaction includes, but is not limited to, dicyclohexylcarbodiimide (DCC), diphenylphosphoryl azide (DPPA), diethyl cyanophosphonate (DEPC), benzotriazole-1-base-oxygen base-tris (dimethylamino) phosphorus hexafluorophosphate (BOP reagent) etc. In the condensation reaction, if necessary, an organic base such as triethylamine or diisopropylethylamine (DIPEA) can be used together with the condensing agent, and if necessary, an activator such as 4-(dimethylamino) pyridine (DMAP) or N-hydroxysuccinimide (HOSu) can be used.

Examples of solvents that can be used in the condensation reaction include halogenated aliphatic hydrocarbons such as chloroform and dichloromethane, ethyl acetate, tetrahydrofuran (THF), dimethylformamide (DMF), acetonitrile, 1,4-dioxane, methanol, etc. The condensation reaction can be carried out at a temperature of -10°C to 50°C, preferably 0°C to 25°C.

The compound of formula II can be synthesized according to a known method (see Bull. Korean Chem. Soc., 2003, Vol. 24, No. 8, pp 1169-1171), or is a commercially available product.

In the compound of formula IV, the peptide (wherein R ₂ is absent) or the fatty acid-peptide (wherein R ₂ is palmityl, lauryl or stearyl) may be a peptide synthesized using a solid phase synthesis method or a fatty acid-peptide synthesized by polymerizing a synthetic peptide with a fatty acid, or may be a commercially available product.

Hereinafter, a method for preparing a peptide-conjugated ascorbic acid derivative of Formula I according to the present invention will be described in detail with reference to the following Reaction Scheme 1. The method shown in the following Reaction Scheme 1 merely represents a possible preparation method, and the order of unit operations, reagents, reaction conditions, etc. shown in the following Reaction Scheme 1 can be appropriately changed if necessary.

Reaction Scheme I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

R ₂ is absent when X is hydrogen, or is palmityl, lauryl or stearyl when _X is carbonyl;

_R3 is hydrogen, methyl, ethyl, tert-butyl or 2,2,2-trichloroethyl; and

n is an integer in the range of 2 to 5.

The preparation method according to the present invention comprises the following steps:

(i) protecting the compound of formula III with an amino protecting group such as t-butyloxycarbonyl (t-Boc) and a carboxyl protecting group such as t-butyl to obtain a compound of formula IV in which the amino and carboxyl groups are protected, or deprotecting the C-terminal carboxyl group of the compound of formula III with a base such as lithium hydroxide to obtain a compound of formula IV

(ii) subjecting the compound of formula IV to a condensation reaction with the compound of formula II (3-aminopropyl (5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl) hydrogen phosphate) in the presence of a condensing agent such as dicyclohexylcarbodiimide (DCC), and deprotecting the amino and carboxyl groups of the amino acid side chain with hydrochloric acid or trifluoroacetic acid to obtain a compound of formula I.

If the compound of formula III does not require the protection or deprotection step (i) of the preparation process, the compound of formula I can be synthesized by performing step (ii) without performing step (i).

The peptide-conjugated ascorbic acid derivative of the present invention prepared according to the above-mentioned method is stable and also exhibits both an excellent wrinkle-reducing effect and an excellent whitening effect (see Experimental Examples 1, 2, and 3 below).

The present invention also relates to a cosmetic composition comprising: a peptide-conjugated ascorbic acid derivative of Formula I or a cosmetically acceptable salt thereof; and a cosmetically acceptable base. The cosmetic composition of the present invention particularly has both skin wrinkle reduction and skin whitening effects.

The cosmetic composition of the present invention comprises, as an active ingredient, 0.0001 to 2 wt%, preferably 0.01 to 0.20 wt%, of the compound of formula I, based on the total weight of the cosmetic composition.

There is no particular limitation on the preparation of the cosmetic composition according to the present invention, and depending on the preparation to be prepared, the cosmetic composition of the present invention can include conventional cosmetic composition components as known in the art. The cosmetic composition of the present invention can be prepared as preparations such as lotions, emulsions, nutritional creams, facial masks (pack), beauty serums, essential oils, etc., and depending on the preparation to be prepared, it can also include one or more components selected from the following: oil, water, surfactants, wetting agents, lower alcohols, thickeners, chelating agents, pigments, preservatives, spices, etc. In addition, in view of the fact that the main cause of melanin formation is UV rays, the cosmetic composition of the present invention can include UV screening agents, light scattering agents, etc. However, the preparation and components of the cosmetic composition are not limited to the foregoing.

Beneficial effects

The peptide-conjugated ascorbic acid derivative according to the present invention is a novel synthetic compound and exhibits both the effect of whitening the skin by inhibiting melanin formation and the effect of reducing skin wrinkles by activating collagen production, and thus can be advantageously used in cosmetic compositions.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in more detail with reference to Preparation Examples and Examples. It will be apparent to those skilled in the art that these Preparation Examples and Examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Preparation Example 1: Preparation of a compound of formula IV wherein the amino (-NH ₂ )- and carboxyl (-COOH)-groups are protected

Preparation Example 1-1: (10S,13S,16S)-16-(4-((tert-Butoxycarbonyl)amino)butyl)-13-isopropyl-2,2-dimethyl-4,11,14-trioxo-10-palmitamido-3-oxa-5,12,15-triazaheptadecan-17-oate (IV-1)

Palmitoyl tripeptide-5 (Pal-KVK) (30.6 g, 50 mmol) was dissolved in 100 mL of methanol and 50 mL of triethylamine. The reaction solution was cooled to 0° C., and di-tert-butyl dicarbonate (29 mL, 125 mmol) was added thereto, followed by stirring at room temperature for 18 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent, and saturated sodium bicarbonate solution (150 mL) was added to the residue, which was then washed twice with diethyl ether (150 mL). The aqueous layer was adjusted to pH 2 by adding 1 M sodium bisulfate aqueous solution (200 mL) and extracted three times with ethyl acetate (300 mL), and the organic layer was washed successively with distilled water (300 mL) and saturated sodium chloride aqueous solution (300 mL), then dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain the title compound (34 g, 84%).

ES-MS m/z：812[M+H] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ0.89(m, 3H), 0.97(m, 6H), 1.28-1.43(m, 48H), 1.59-1.99(m, 8H), 2.06(m, 1 H), 2.24 (brt, J=7.2Hz, 2H), 3.02 (m, 4H), 4.20 (d, J=7.2Hz, 1H), 4.37 (m, 2H).

Preparation Example 1-2: (11S,17S)-17-(2-(tert-butoxy)-2-oxoethyl)-6,11-bis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12,15-trioxo-3-oxa-5,7,13,16-tetraazaoctadec-6-ene-18-oate (IV-2)

N-carbobenzoxyglycine (Z-Gly-OH) (11.48 g, 54.8 mmol) and 4-tert-butyl-1-methyl L-aspartate hydrochloride (13.14 g, 54.8 mmol) were dissolved in tetrahydrofuran (275 ml) and then cooled to 0° C. 1-hydroxybenzotriazole hydrate (HOBt) (8.90 g, 65.8 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (11.31 g, 65.8 mmol) were added to the reaction solution, and triethylamine (23.1 mL, 164.6 mmol) was added dropwise. The reaction solution was stirred at 0° C. for 30 minutes and then at room temperature for 16 hours. After the reaction was complete, water (200 mL) was added to the reaction mixture solution, which was then washed three times with ethyl acetate (100 mL). Next, the organic layers were combined, washed with a saturated aqueous sodium chloride solution (200 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to give (S)-4-tert-butyl 1-methyl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)succinate (16.0 g, 74%).

ES-MS m/z: 395[M+H] ⁺ , 417[M+Na] ⁺

^1H NMR (400MHz, DMSO-d6) δ1.41 (s, 9H), 2.68-2.83 (m, 2H), 3.61 (s, 3H), 3.62 (s, 3H), 3.62-3 .64(m, 2H), 4.64-4.69(m, 1H), 5.03(s, 2H), 7.31-7.39(m, 5H), 7.46(t, 1H), 8.35(d, 1H).

The obtained compound (16.0 g, 40.57 mmol) was dissolved in methanol (200 mL), and 10% palladium on carbon (2.02 g) was added thereto, followed by stirring at room temperature for 3 hours in a hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through celite, washed several times with methanol, and then concentrated under reduced pressure to remove the solvent, thereby obtaining (S)-4-tert-butyl 1-methyl 2-(2-aminoacetamido) succinate (8.04 g, 91%) as a white solid.

ES-MS m/z: 261[M+H] ⁺ , 283[M+Na] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ) δ 1.41 (s, 9H), 2.64-2.78 (m, 2H), 3.47 (s, 3H), 3.62 (s, 3H), 3.65-3.77 (m, 2H), 4.10-4.12 (m, 1H), 8.10 (s, 1H).

(S)-4-tert-butyl 1-methyl 2-(2-aminoacetamido) succinate (8.04 g, 30.89 mmol) and N _α Boc-N _δ Cbz-L-ornithine (13.5 g, 38.8 mmol) were dissolved in tetrahydrofuran (185 mL) and cooled to 0° C. 1-hydroxybenzotriazole hydrate (HOBt) (5.98 g, 44.2 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (7.6 g, 44.2 mmol) were added to the reaction solution, and triethylamine (11.39 mL, 84.1 mmol) was added dropwise. The reaction mixture was stirred at 0° C. for 30 minutes and then at room temperature for 16 hours. After completion of the reaction, water (150 mL) was added to the reaction mixture solution, which was then washed twice with ethyl acetate (100 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to give (S)-4-tert-butyl 1-methyl 2-(2-((S)-5-(((benzyloxy)carbonyl)amino)-2-((tert-butoxycarbonyl)amino)pentanamido)acetamido)succinate (9.76 g, 47%) as a white solid.

ES-MS m/z: 609[M+H] ⁺ , 631[M+Na] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ1.44(s, 18H), 1.58-1.66(m, 4H), 1.82-1.92(m, 1H), 2.81-2.87(m, 1H), 2.96-3.01(m, 1H), 3.15-3.39(m, 2H), 3.67( s, 3H), 3.72 (s, 3H), 3.94 (m, 2H), 4.25 (m, 1H), 4.83 (m, 1H), 5.08 (s, 2H), 5.13 (m, 2H), 7.00 (m, 2H), 7.32-7.35 (m, 5H).

The obtained compound (9.76 g, 17.2 mmol) was dissolved in methanol (85 mL), and 10% palladium on carbon (0.86 g) was added thereto, followed by stirring at room temperature for 3 hours in a hydrogen atmosphere. After completion of the reaction, the reaction mixture was filtered through celite, washed several times with methanol, and then concentrated under reduced pressure to remove the solvent, thereby obtaining (S)-4-tert-butyl 1-methyl 2-(2-((S)-5-amino 2-((tert-butoxycarbonyl)amino)pentanoylamido)acetamido)succinate (7.45 g, 99%) as a white solid.

ES-MS m/z: 475[M+H] ⁺ , 497[M+Na] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ1.44(s, 18H), 1.52-1.61(m, 2H), 1.72-1.77(m, 1H), 1.76-1.87(m, 1H), 2.20(brd, 4H), 2.75(m, 1H), 2.79-2.85(m, 1H), 2 .98-3.03 (m, 1H), 3.69 (s, 3H), 3.75 (s, 3H), 3.94 (m, 2H), 4.20 (m, 1H), 4.85 (m, 1H), 5.84 (m, 1H), 7.16 (m, 1H), 7.54 (m, 1H).

(S)-4-tert-Butyl 1-methyl 2-(2-((S)-5-amino 2-((tert-butoxycarbonyl)amino)pentanamido)acetamido)succinate (7.45 g, 17.2 mmol) was dissolved in dichloromethane (85 mL), and 1,3-di-Boc-2-(trifluoromethanesulfonyl)guanidine (7.08 g, 18.1 mmol) and triethylamine (2.5 mL, 18.1 mmol) were added thereto, followed by stirring at room temperature for 16 hours. After completion of the reaction, water (85 mL) was added to the reaction mixture solution, which was then washed twice with dichloromethane (50 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The residue was purified by column chromatography to give (S)-4-tert-butyl 1-methyl 2-((S)-6,11-bis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12-dioxo-3-oxa-5,7,13-triazapentadecan-6-enamide)succinate (11.43 g, 98%) as a white solid.

ES-MS m/z: 718[M+H] ⁺ , 740[M+Na] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ1.44(s, 18H), 1.48(s, 18H), 1.64-1.72(m, 4H), 1.87(m, 1H), 2.81-2.86(m, 1H), 2.96-3.01(m, 1H), 3.30(m, 1H), 3.48(m, 1H), 3. 68 (s, 3H), 3.74 (s, 3H), 3.96 (m, 2H), 4.24 (m, 1H), 4.85 (m, 1H), 5.54 (m, 1H), 7.04 (m, 1H), 7.10 (m, 1H), 8.41 (m, 1H), 11.43 (m, 1H).

(S)-4-tert-Butyl 1-methyl 2-((S)-6,11-bis((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,12-dioxo-3-oxa-5,7,13-triazapentadeca-6-enamido)succinate (11.43 g, 16.9 mmol) was dissolved in tetrahydrofuran (127 mL) and cooled to 0° C., and 1 M lithium hydroxide solution (45 mL) was added thereto, followed by stirring at room temperature for 16 hours. After the reaction was completed, the reaction solution was cooled to 0° C., adjusted to pH 2 by 1 M HCl aqueous solution, and extracted twice with ethyl acetate (80 mL). The organic layers were combined, washed with saturated sodium chloride aqueous solution (100 mL), dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the title compound (8.96 g, 82%) as a white solid.

ES-MS m/z: 703[M+H] ⁺ , 725[M+Na] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ1.28(t, 1H), 1.42(s, 18H), 1.48(s, 9H), 1.49(s, 9H), 1.62-1.65(m, 3H), 1.85(m, 1H), 2.81-2.85(m, 1H), 2.99-3.04(m, 1H), 3.40 (m, 1H), 3.85 (m, 1H), 4.13 (m, 1H), 4.76 (m, 1H), 5.24 (m, 1H), 5.85 (m, 1H), 5.82 (m, 1H), 7.71 (m, 2H), 8.67 (m, 1H).

Example: Preparation of compounds of formula I

Example 1: (6S,9S,12S)-6,12-bis(4-aminobutyl)-9-isopropyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazanonacosyl(5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-hydrofuran-3-yl)hydrogen phosphate hydrochloride (I-1)

Compound IV-1 (31.2 g, 38.4 mmol) obtained in Preparation Example 1-1, in which the amino group is protected, was dissolved in dimethylformamide (250 mL), and N, N'-dicyclohexylcarbodiimide (DCC) (9.5 g, 46.1 mmol) and N-hydroxysuccinimide (5.3 g, 46.1 mmol) were added thereto, followed by reaction at room temperature for 3 hours. After the reaction, N, N'-dicyclohexylurea produced as a by-product was removed by filtration under reduced pressure. 3-aminopropyl (5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl) hydrogen phosphate of Formula II (Vitagen, 13.2 g, 42.2 mmol) and diisopropylethylamine (13.5 mL, 77 mmol) were added to the remaining filtrate and stirred at room temperature for 20 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure to remove the solvent. The residue was recrystallized by adding MeOH, dichloromethane and isopropyl ether in sequence and filtered to obtain the compound of formula I-1 in which the amino group was protected (47 g, 112%).

ES-MS m/z：1107[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD) δ0.96 (m, 3H), 1.01 (m, 6H), 1.35-1.55 (m, 48H), 1.65-1.88 (m, 10H), 2.14 (m, 1H), 2.30 (brt, J=7.2Hz, 2H), 3.09 (m, 4H), 3.28 (m, 2H), 3.73-3.82 (m, 3H), 4.00 (m, 1H), 4.06 (m, 2H), 4.22 (d, J=6.8Hz, 1H), 4.37 (m, 2H), 4.86-5.01 (m, 1H).

The obtained compound (47g, 37.9mmol) of the protected formula I-1 of amino group is dissolved in 1,4-dioxane (160mL), and the 4M hydrochloric acid (110mL, 440mmol) in 1,4-dioxane solution is added thereto, then at room temperature stirred for 15 hours.After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove the solvent. Acetonitrile (600mL) is added in the residue, and the solid formed is filtered to obtain title compound (33.0g, 89%).

ES-MS m/z：907[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD) δ0.90 (m, 3H), 0.92 (d, J=6.4Hz, 6H), 1.28-1.39 (m, 26H), 1.44-1.88 (m, 14H), 2.12 (m, 1H), 2.25 (brt, J=7.6Hz, 2H), 2.96 (m, 4 H), 3.24-3.35 (m, 2H), 3.68 (m, 3H), 3.95-3.99 (td, J=1.6, 6.8Hz, 1H), 4.08-4.15 (m, 3H), 4.29 (m, 1H), 4.37 (m, 1H), 4.93 (m, 1H).

Example 2: (3S)-3-(2-((S)-2-amino-5-((diaminomethylene)amino)pentanamido)acetamido)-4-((3-((((5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)propyl)amino)-4-oxobutanoate hydrochloride (I-2)

Compound IV-2 (2.46 g, 3.5 mmol) obtained in Preparation Example 1-2, wherein the amino and carboxyl groups are protected, was dissolved in dimethylformamide (20 mL), and N, N'-dicyclohexylcarbodiimide (DCC) (0.72 g, 3.5 mmol) and N-hydroxysuccinimide (0.4 g, 3.5 mmol) were added thereto, followed by reaction at room temperature for 1 hour. After the reaction, N, N'-dicyclohexylurea produced as a by-product was removed by filtration under reduced pressure. 3-aminopropyl (5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl) hydrogen phosphate (Vitagen, 1.32 g, 4.2 mmol) and diisopropylethylamine (1.22 mL, 7.0 mmol) of Formula II were added to the filtrate obtained and stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure to remove the solvent. The residue was recrystallized by adding methanol, dichloromethane and isopropyl ether in sequence and filtered to give the compound of formula I-2 in which the amino and carboxyl groups were protected (11S, 17S)-tert-butyl 6,11-di((tert-butoxycarbonyl)amino)-17-((3-((((5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)propyl)carbamoyl)-2,2-dimethyl-4,12,15-trioxo-3-oxa-5,7,13,16-tetraazanonadeca-6-ene-19-oate) (3.28 g, 94%).

ES-MS m/z：999[M+H] ⁺

The compound of formula I-2 (3.28g, 3.29mmol) wherein amino and carboxyl are protected is dissolved in 1,4-dioxane (35mL), and 4M hydrochloric acid (8.3mL, 33mmol) in 1,4-dioxane solution is added thereto, then stirred at room temperature for 15 hours. After completion of the reaction, the reaction solution is concentrated under reduced pressure to remove the solvent, and the residue is recrystallized by adding acetonitrile, dichloromethane and isopropyl ether in sequence, and filtered to obtain title compound (2.25g, 91%).

ES-MS m/z：642[M+H] ⁺

¹ H NMR (400 MHz, CDCl ₃ )δ1.28(t, 1H), 1.62-1.65(m, 3H), 1.85(m, 1H), 1.91(m, 2H), 2.81-2.8 5(m, 1H), 2.99-3.04(m, 1H), 3.17(m, 2H), 3.20(m, 2H), 3.40(m, 1H), 3. 45(m, 2H), 3.63(s, 1H), 3.85(m, 1H), 3.91(m, 1H), 4.13(m, 1H), 4.76(m , 1H), 5.24(m, 1H), 5.85(m, 1H), 5.82(m, 1H), 7.71(m, 2H), 8.67(m, 1H).

Experimental Example 1: Analysis of melanin inhibition

B16F1 melanoma cell line (ATCC CRL-6323) is added to DMEM culture medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100μg/ml streptomycin and cultured under the conditions of 37°C and 5% CO _2. After cultivation, the culture medium is replaced with DMEM culture medium (Invitrogen-Gibco-BRL, cells/well) and treated with an appropriate amount of sample solution so that the final concentration of each test compound reaches 0μg/ml, 10μg/ml, 20μg/ml or 40μg/ml. After 72 hours, the culture medium is removed, and the cells are washed, harvested with PBS (phosphate buffered saline), and dried at 60°C. Next, a melanin extraction solution (1N NaOH+10% DMSO) is added to the cells, which are then dissolved at 80°C. Next, the amount of melanin is quantified using synthetic melanin as a standard, and the amount of protein is quantified. The amount of melanin per unit protein was calculated and expressed as % control value relative to the control.

The results are shown in Formula 1 below.

Formula 1

Experimental Example 2: Analysis of the effect on collagen activation

Human newborn skin fibroblasts (Cascade Co.) are seeded into 24-well microplates (5 × 10 ⁴ cells/well) with DMEM culture medium supplemented with 10% fetal bovine serum and cultured for 24 hours at 37°C and 5% CO _2. Culture medium is replaced with serum-free DMEM culture medium, and cells are cultured for 24 hours and treated with each sample solution so that the final concentration of each test compound reaches 1 μg/ml or 5 μg/ml. After 48 hours of culture, cell culture medium is collected, and the amount of procollagen in the culture medium is measured using a collagen measurement kit (Takara Shuzo Co., Ltd., Japan). Specifically, the collected cell culture medium is added to a 96-well plate with a primary collagen antibody uniformly coated thereon, and antigen-antibody reaction is performed at 37°C for 3 hours. Cell culture medium is removed from each well, and each well is then washed four times with PBS. Chromogenic substance is added to each well and incubated at room temperature for 15 minutes, and then 1N sulfuric acid solution is added to each well to stop the reaction. The absorbance at 450 nm was measured using a spectrophotometer.A standard curve was prepared using the standard solution, and the absorbance obtained as described above was substituted into the standard curve to determine the production of procollagen in the cell culture medium to which each test compound was added.

The results are shown in Table 2 below.

Table 2

Experimental Example 3: Test for stability against water and heat

The stability of the active ingredients used in the cosmetic composition after water and heat is an important factor not only during the preparation process of the composition but also in the long-term storage of the composition. Therefore, the stability of compounds I-1 and I-2 prepared in Examples 1 and 2, respectively, against water and heat was measured. Specifically, each of compounds I-1 and I-2 was stored in a sealed state under the harsh conditions of a temperature of 40°C and a relative humidity of 75%, and the percentage of the active ingredient in each sample after 3, 7, 14 and 28 days was analyzed by high performance liquid chromatography (HPLC) relative to the initial value of the active value of the active ingredient on day 0.

The results are shown in Table 3 below.

Table 3

As can be seen in Table 3 above, each of Compounds I-1 and I-2 was tested under severe conditions for 28 days, and as a result, it can be seen that these compounds exhibited excellent stability comparable to that of commercially available LAAP (Vitagen).

Experimental Example 4: Inhibition of DNA methyltransferase (DNMT) activity

In order to investigate the mechanism of the effect of compound 1-1 on promoting procollagen synthesis, the inhibitory effect of compound for DNMT activity was investigated using nuclear extracts and DNA methyltransferase activity/inhibitor screening assay kit isolated from Hela cells. Hela nuclear extracts comprising DNMT, compound 1-1 and Adomet were added to the band coated with DNA rich in cytosine (cystosine). As a positive control, 5-aza-2'-deoxycytidine (5-azadC) was used. The capture antibody and detection antibody bound to methylated DNA were sequentially added to the band, followed by addition of developer solution to induce chromogenic reaction, followed by addition of stop solution to stop the reaction. The absorbance at 450nm was measured, and based on the absorbance measured, the percentage inhibition of DNMT activity was calculated using the following formula 1. Calculation results are shown in Table 4 below.

Formula 1

Inhibition % = [1-(OD _inhibitor -OD _blank )/(OD _control -OD _blank )] × 100

Table 4

As can be seen in Table 4 above, compound I-1 inhibited DNA methylation by more than 50% at concentrations of 5 μg/ml and above, and exhibited a concentration-dependent inhibitory effect on DNA methylation within the concentration range of 1-10 μg/ml. Therefore, it can be seen that compound I-1 has the effect of increasing procollagen levels by inhibiting the methylation of the collagen gene.

Formulation Example: Cosmetic Composition Containing a Peptide-Conjugated Ascorbic Acid Derivative

Based on the above experimental examples, cosmetic compositions containing peptide-conjugated ascorbic acid derivatives according to embodiments of the present invention were prepared according to the compositions shown in the following table. The following formulation examples are for illustrative purposes and are not intended to limit the scope of the present invention.

Preparation Example 1: Preparation of skin softener

According to the composition shown in Table 5 below, a skin softening agent containing the peptide-conjugated ascorbic acid derivative according to the Examples was prepared.

Table 5

Preparation method

(1) Each of the aqueous phase and the dissolved phase is uniformly mixed and dissolved.

(2) The dissolved phase is added to the aqueous phase and mixed to obtain an oil-in-water emulsion.

(3) Then, additional phase I dissolved in the phase containing the peptide-conjugated ascorbic acid derivative dissolved therein is added and mixed with the emulsion, and then additional phase II is added and mixed with the emulsion.

Preparation Example 2: Preparation of Lotion

According to the composition shown in Table 6 below, a lotion containing the peptide-conjugated ascorbic acid derivative according to the Examples was prepared.

Table 6

Preparation method

(1) Each of the water phase and the oil phase is heated, uniformly mixed, and dissolved.

(2) At 75°C, the oil phase was added and mixed with the water phase to achieve dissolution.

(3) Next, the mixture is cooled to 50° C., and then additional phase I dissolved in the phase containing the peptide-conjugated ascorbic acid derivative dissolved therein is added and mixed with the emulsion, and then additional phase II is added and mixed with the emulsion.

Preparation Example 3: Preparation of Cream

According to the composition shown in Table 7 below, a skin lotion containing the peptide-conjugated ascorbic acid derivative according to the Examples was prepared.

Table 7

Preparation method

Technical Field

Background Art

Summary of the Invention

Technical issues

Technical Solution

Formula I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

Formula II

Formula IV

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

Reaction Scheme I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

_R3 is hydrogen, methyl, ethyl, tert-butyl or 2,2,2-trichloroethyl; and

n is an integer in the range of 2 to 5.

Beneficial effects

DETAILED DESCRIPTION

ES-MS m/z：812[M+H] ⁺

ES-MS m/z: 395[M+H] ⁺ , 417[M+Na] ⁺

ES-MS m/z: 261[M+H] ⁺ , 283[M+Na] ⁺

ES-MS m/z: 609[M+H] ⁺ , 631[M+Na] ⁺

ES-MS m/z: 475[M+H] ⁺ , 497[M+Na] ⁺

ES-MS m/z: 718[M+H] ⁺ , 740[M+Na] ⁺

ES-MS m/z: 703[M+H] ⁺ , 725[M+Na] ⁺

Example: Preparation of compounds of formula I

ES-MS m/z：1107[M+H] ⁺

ES-MS m/z：907[M+H] ⁺

ES-MS m/z：999[M+H] ⁺

ES-MS m/z：642[M+H] ⁺

Experimental Example 1: Analysis of melanin inhibition

The results are shown in Formula 1 below.

Formula 1

Experimental Example 2: Analysis of the effect on collagen activation

The results are shown in Table 2 below.

Table 2

Experimental Example 3: Test for stability against water and heat

The results are shown in Table 3 below.

Table 3

Experimental Example 4: Inhibition of DNA methyltransferase (DNMT) activity

Formula 1

Inhibition % = [1-(OD _inhibitor -OD _blank )/(OD _control -OD _blank )] × 100

Table 4

Preparation Example 1: Preparation of skin softener

Table 5

Preparation method

Preparation Example 2: Preparation of Lotion

Table 6

Preparation method

Preparation Example 3: Preparation of Cream

Table 7

Preparation method

Technical Field

Background Art

Summary of the Invention

Technical issues

Technical Solution

Formula I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

Formula II

Formula IV

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

Reaction Scheme I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

_R3 is hydrogen, methyl, ethyl, tert-butyl or 2,2,2-trichloroethyl; and

n is an integer in the range of 2 to 5.

Beneficial effects

DETAILED DESCRIPTION

ES-MS m/z：812[M+H] ⁺

ES-MS m/z: 395[M+H] ⁺ , 417[M+Na] ⁺

ES-MS m/z: 261[M+H] ⁺ , 283[M+Na] ⁺

ES-MS m/z: 609[M+H] ⁺ , 631[M+Na] ⁺

ES-MS m/z: 475[M+H] ⁺ , 497[M+Na] ⁺

ES-MS m/z: 718[M+H] ⁺ , 740[M+Na] ⁺

ES-MS m/z: 703[M+H] ⁺ , 725[M+Na] ⁺

Example: Preparation of compounds of formula I

ES-MS m/z：1107[M+H] ⁺

ES-MS m/z：907[M+H] ⁺

ES-MS m/z：999[M+H] ⁺

ES-MS m/z：642[M+H] ⁺

Experimental Example 1: Analysis of melanin inhibition

The results are shown in Formula 1 below.

Formula 1

Experimental Example 2: Analysis of the effect on collagen activation

The results are shown in Table 2 below.

Table 2

Experimental Example 3: Test for stability against water and heat

The results are shown in Table 3 below.

Table 3

Experimental Example 4: Inhibition of DNA methyltransferase (DNMT) activity

Formula 1

Inhibition % = [1-(OD _inhibitor -OD _blank )/(OD _control -OD _blank )] × 100

Table 4

Preparation Example 1: Preparation of skin softener

Table 5

Preparation method

Preparation Example 2: Preparation of Lotion

Table 6

Preparation method

Preparation Example 3: Preparation of Cream

Table 7

Preparation method

Technical Field

Background Art

Summary of the Invention

Technical issues

Technical Solution

Formula I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

Formula II

Formula IV

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

Reaction Scheme I

in

_R1 represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

_R3 is hydrogen, methyl, ethyl, tert-butyl or 2,2,2-trichloroethyl; and

n is an integer in the range of 2 to 5.

Beneficial effects

DETAILED DESCRIPTION

ES-MS m/z：812[M+H] ⁺

ES-MS m/z: 395[M+H] ⁺ , 417[M+Na] ⁺

ES-MS m/z: 261[M+H] ⁺ , 283[M+Na] ⁺

ES-MS m/z: 609[M+H] ⁺ , 631[M+Na] ⁺

ES-MS m/z: 475[M+H] ⁺ , 497[M+Na] ⁺

ES-MS m/z: 718[M+H] ⁺ , 740[M+Na] ⁺

ES-MS m/z: 703[M+H] ⁺ , 725[M+Na] ⁺

Example: Preparation of compounds of formula I

ES-MS m/z：1107[M+H] ⁺

ES-MS m/z：907[M+H] ⁺

ES-MS m/z：999[M+H] ⁺

ES-MS m/z：642[M+H] ⁺

Experimental Example 1: Analysis of melanin inhibition

The results are shown in Formula 1 below.

Formula 1

Experimental Example 2: Analysis of the effect on collagen activation

The results are shown in Table 2 below.

Table 2

Experimental Example 3: Test for stability against water and heat

The results are shown in Table 3 below.

Table 3

Experimental Example 4: Inhibition of DNA methyltransferase (DNMT) activity

Formula 1

Inhibition % = [1-(OD _inhibitor -OD _blank )/(OD _control -OD _blank )] × 100

Table 4

Preparation Example 1: Preparation of skin softener

Table 5

Preparation method

Preparation Example 2: Preparation of Lotion

Table 6

Preparation method

Preparation Example 3: Preparation of Cream

Table 7

Preparation method

Claims

1. A peptide-conjugated ascorbic acid derivative represented by Formula I, or a cosmetically acceptable salt thereof:

Formula I

in

This refers to a peptide in which the same or different amino acid residues selected from valine, lysine, glycine, arginine, and aspartic acid are linked by an amide bond.

R ₁ represents the side chain of the amino acid residue;

X is hydrogen or carbonyl (C=O);

n is an integer in the range of 2 to 5.

2. The peptide-conjugated ascorbic acid derivative or its cosmetically acceptable salt according to claim 1, wherein n is an integer in the range of 3 to 5.

3. The peptide-conjugated ascorbic acid derivative or its cosmetically acceptable salt according to claim 1 or 2, wherein the derivative is selected from lysine-valine-lysine and arginine-glycine-aspartic acid.

4. The peptide-conjugated ascorbic acid derivative or a cosmetically acceptable salt thereof according to claim 3, wherein the derivative is selected from the following compounds:

(6S,9S,12S)-6,12-bis(4-aminobutyl)-9-isopropyl-5,8,11,14-tetraoxo-4,7,10,13-tetraazanonadecanyl(5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)hydrophosphate (I-1); and

(3S)-3-(2-((S)-2-amino-5-((diaminomethylene)amino)pentamido)acetamido)-4-((3-((((5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-yl)oxy)(hydroxy)phosphoryl)oxy)propyl)amino)-4-oxobutyric acid (I-2).

5. A method for preparing a peptide-conjugated ascorbic acid derivative represented by Formula I, the method comprising the steps of: subjecting a compound of Formula II to a condensation reaction with a compound of Formula IV, and then, if a protecting group is present in the product of the condensation reaction, subjecting the product to a deprotection reaction:

Formula II

Formula IV

Formula I

in

This refers to a peptide in which the same or different amino acid residues selected from valine, lysine, glycine, arginine, and aspartic acid are linked by amide bonds.

R ₁ represents the side chain of the amino acid residue;

_R1 ' is the same as _R1 or _R1 in which the amino or carboxyl group is protected;

X is hydrogen or carbonyl (C=O);

_R2 is absent when X is hydrogen, or _R2 is palmityl, lauryl, or stearyl when X is carbonyl;

n is an integer in the range of 2 to 5.

6. A cosmetic composition comprising a peptide-conjugated ascorbic acid derivative or a cosmetically acceptable salt thereof according to any one of claims 1 to 4, and a cosmetically acceptable base.

7. The cosmetic composition according to claim 6, wherein the cosmetic composition is used for reducing skin wrinkles and whitening the skin.

8. The cosmetic composition according to claim 6 or 7, wherein the compound of formula I is contained in an amount of 0.0001 to 2% by weight based on the total weight of the cosmetic composition.