KR101221764B1 - Novel whitening compound - Google Patents

Abstract

[화학식 2]

(상기 식에서, X는 공유결합을 나타내거나, 또는 아미노산을 나타내고,
상기 아미노산의 아미노기는 coumaric acid 의 카르복시기와 결합하고, 상기 아미노산의 카르복시기는 Leu의 아미노기와 결합하고,
상기 식 1에서 아미노산은 Gly, Leu, Ile, Pro, Hyp 또는 Val이고,
상기 식 2에서 아미노산은 Gly, Leu, Ile, Pro 또는 Val이다.)The present invention relates to a compound that can be utilized as a new whitening material that can maintain its function longer than existing whitening materials and a whitening cosmetic composition comprising the compound. In order to achieve the above object, the present invention provides a compound for whitening represented by the following formula (1) or (2).
[Formula 1]

(2)

Wherein X represents a covalent bond or an amino acid,
The amino group of the amino acid binds to the carboxy group of coumaric acid, the carboxy group of the amino acid binds to the amino group of Leu,
Amino acid in Formula 1 is Gly, Leu, Ile, Pro, Hyp or Val,
Amino acid in Formula 2 is Gly, Leu, Ile, Pro or Val.)

Description

New whitening compound {NOVEL WHITENING COMPOUND}

The present invention relates to a compound that can be utilized as a new whitening material that can maintain its function longer than conventional whitening materials.

With the recent increase in national income, the aging of the population and the entry of women have increased significantly. In addition, as the culture of LOHAS and well-being rises, the desire to maintain fine skin is increasing day by day. Among these, consumer demand for clear and clean skin is increasing, and accordingly, technology development and discovery regarding whitening-related materials are attracting attention.

In particular, recently, a new mechanism has been studied and approached from one of the existing whitening mechanisms of inhibition of tyrosinase. This is to overcome this with a new approach as the limitations of previously commercialized whitening materials are gradually revealed. This new approach reduces the effects of ultraviolet light, cytokines and hormones that promote melanin formation, pre-melanin production that regulates tyrosinase transcription, melanin production that inhibits tyrosinase activity, and produced melanin In the form of melanosomes, materials for controlling various mechanisms, such as controlling the delivery stage of melanin delivered to keratinocytes, have been developed. Research into the use of peptides that form the basis of the substances involved in each step of these materials is being activated. (US 5731408, US 6054556, US 7084111, US 7582610, WO 2009010356A1)

Peptides are bio-friendly and have very high activity, and thus, many researches have been conducted in the cosmetic and pharmaceutical industries as new generation materials. However, since the stability is very low, it is easily decomposed from the skin and loses activity.

The present invention is to solve the above problems, the purpose of introducing a small organic material to the N-terminal of the peptide to stably increase the whitening activity. In addition, these small organic substances were used to further enhance the efficacy by using coumaric acid having whitening activity through conventional tyrosinase inhibition.

In order to achieve the above object, the present invention provides a compound for whitening represented by the following formula (1) or (2).

[Formula 1]

[Formula 2]

Wherein X represents a covalent bond or an amino acid,

The amino group of the amino acid binds to the carboxy group of coumaric acid, the carboxy group of the amino acid binds to the amino group of Leu,

Amino acid in Formula 1 is Gly, Leu, Ile, Pro, Hyp or Val,

Amino acid in Formula 2 is Gly, Leu, Ile, Pro or Val.)

The whitening material using the novel whitening compound according to the present invention can stably increase the whitening activity as compared to the existing whitening material, and has an effect of maintaining the whitening function longer.

Figure 1 shows the HPLC analysis of the whitening compound coumaric acid-PLG-NH2 according to an embodiment of the present invention.
Figure 2 shows the MALDI-Tof analysis of the whitening compound coumaric acid-PLG-NH2 according to an embodiment of the present invention.
Figure 3 shows the HPLC analysis of the whitening compound coumaric acid-GLG-OH according to an embodiment of the present invention.
Figure 4 shows the MALDI-Tof analysis of the whitening compound coumaric acid-GLG-OH according to an embodiment of the present invention.
5 shows the results of HPLC analysis of coumaric acid-VLG-OH, a compound for whitening according to an embodiment of the present invention.
Figure 6 shows the MALDI-Tof analysis of the whitening compound coumaric acid-VLG-OH according to an embodiment of the present invention.
7 shows the results of HPLC analysis of coumaric acid-ILG-OH, a compound for whitening according to an embodiment of the present invention.
Figure 8 shows the MALDI-Tof analysis of the whitening compound coumaric acid-ILG-OH according to an embodiment of the present invention.
9 shows the results of HPLC analysis of coumaric acid-PLG-OH, a compound for whitening according to an embodiment of the present invention.
Figure 10 shows the MALDI-Tof analysis of the whitening compound coumaric acid-PLG-OH according to an embodiment of the present invention.
FIG. 11 shows the results of HPLC analysis of coumaric acid-LG-OH, a compound for whitening according to an embodiment of the present invention. FIG.
Figure 12 shows the MALDI-Tof analysis of the whitening compound coumaric acid-LG-OH according to an embodiment of the present invention.
Figure 13 shows the HPLC analysis of the whitening compound coumaric acid-GLG-NH2 according to an embodiment of the present invention.
Figure 14 shows the MALDI-Tof analysis of the whitening compound coumaric acid-GLG-NH2 according to an embodiment of the present invention.
FIG. 15 shows the results of HPLC analysis of coumaric acid-VLG-NH 2 as a whitening compound according to an embodiment of the present invention. FIG.
Figure 16 shows the MALDI-Tof analysis of the whitening compound coumaric acid-VLG-NH2 according to an embodiment of the present invention.
FIG. 17 shows the results of HPLC analysis of coumaric acid-LG-NH2 as a whitening compound according to an embodiment of the present invention. FIG.
Figure 18 shows the MALDI-Tof analysis of the whitening compound coumaric acid-LG-NH2 according to an embodiment of the present invention.
19 shows the results of HPLC analysis of coumaric acid-HyP-LG-NH2 as a whitening compound according to an embodiment of the present invention.
20 shows the results of MALDI-Tof analysis of coumaric acid-HyP-LG-NH2, a whitening compound according to an embodiment of the present invention.
FIG. 21 shows the results of HPLC analysis of coumaric acid-ILG-NH2 as a whitening compound according to an embodiment of the present invention. FIG.
22 shows the results of MALDI-Tof analysis of coumaric acid-ILG-NH 2, a compound for whitening according to an embodiment of the present invention.

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

The present invention provides a compound for whitening represented by the following formula (1) or (2).

[Formula 1]

[Formula 2]

Wherein X represents a covalent bond or represents an amino acid, wherein the amino group of the amino acid binds to the carboxy group of coumaric acid, the carboxy group of the amino acid binds to the amino group of Leu, and wherein in Formula 1 the amino acid is Gly, Leu , Ile, Pro, Hyp or Val, wherein the amino acid in Formula 2 is Gly, Leu, Ile, Pro or Val.

In a preferred embodiment of the present invention, X is Pro in the above formula, in which case the compound represented by Formula 1 is coumaric acid-Pro-Leu-Gly-NH2, and the compound represented by Formula 2 is coumaric acid -Pro-Leu-Gly-OH.

In addition, one preferred embodiment in the present invention, wherein X is a covalent bond in the above formula, in this case, the compound represented by the formula (1) is the same as the formula (3), the compound represented by the formula (2) is the same as the formula (4) .

(3)

[Formula 4]

Among the novel whitening compounds represented by Chemical Formula 1, coumaric acid-Pro-Leu-Gly-NH2 has a high biocompatibility, including a peptide having a whitening mechanism called melanostatin, and the coumaric acid at the N terminus of the peptide. It is designed to further improve the efficacy of the peptide material by introducing. In another aspect, the present invention can provide a whitening compound more easily commercially by replacing the amino acid Pro of the terminal of the melanostatin with an amino acid having another side chain group.

The whitening compound represented by Chemical Formula 1 or Chemical Formula 2 of the present invention is prepared through the combination of a peptide and coumaric acid.

The present invention was intended to solve the problem by introducing a small organic compound to the N terminal of the peptide to overcome the disadvantages of the peptide-based material. The peptide is based on melanostatin, the shortest peptide known to be efficacious for easy commercialization, and the small organic compound is made of coumaric acid, known as a conventional tyrosinase inhibitor, thereby improving the whitening effect. .

The present invention includes the application of replacing the terminal Pro with an amino acid having a side chain group of another alkyl group in the tripeptide sequence of melanostatin.

The reason why the peptide is attracting attention as a new material is because it is not only a high biocompatibility but also a high activity because it is the basis for constituting the human body. However, peptides are difficult to maintain their efficacy because they have the property of being easily degraded by protease, one of the most enzymes in the human body. Since these proteases are also present in the skin, they have the disadvantage of degrading before their efficacy when used externally for cosmetic use.

In order to overcome the shortcomings of these peptides, the most approach is to wrap and protect the peptide material in capsule form. However, these methods are not only costly, but also difficult to select the appropriate capsule material and the development of the technology that the capsule must be broken at the right time is a difficult step in the cosmetic industry. Another method is to make some modifications to the peptide itself so that the protease is not easily recognized. The most representative method is to introduce an alkyl group into the amide bond in the middle of the peptide to form a modified peptoid. However, since amide bonds are very stable, they are also difficult to use in commercially available materials. Thus, the easiest method to be used is a method of modifying a part of the peptide by introducing an organic material. Although it may be weak in terms of effectiveness compared to the above-mentioned method, it is advantageous in terms of applicability and cost, and many commercially available peptide materials are of this type.

In the present invention, it was intended to solve the above problems by introducing coumaric acid to melanostin, a novel mechanism of peptide expression inhibition of a-MSH. That is, by introducing an organic substance at the N-terminal of the peptide to ensure long-lasting efficacy, and after degradation, the coaric acid inhibits tyrosinase to maintain whitening efficacy. In addition, to facilitate the commercialization, the terminal amino acid Pro was replaced with another amino acid consisting of alkyl side chain group, and the efficacy was checked.

Introduction of the basic peptide structure and organics of the present invention proceeded to a solid phase synthesis method. Solid phase synthesis method is a method widely used in the synthesis of peptides are shown in Schemes 1 and 2.

[Reaction Scheme 1]

[Reaction Scheme 2]

When X represents a covalent bond in the above scheme, the process of introducing X is omitted and the coumaric acid is directly bonded to Leu. When X represents an amino acid in the above scheme, the amino acid is typically Gly, Leu, Ile, Pro, Hyp or Val. To prevent side reactions in the synthesis, amino acids protected with amines were used as Fmoc, and polystyrene-based resin (Scheme 2) having 2-CTC resin (Scheme 1) or Rink amide linker as a support for solid phase synthesis was used. Used. BOP and HOBt were used as binding reagents to generate amide bonds, 20% peperidine / NMP was used for the removal of the Fmoc protecting group, and reagent K (Trifuloroacetic acid) was used for the reaction that favored the product in the resin.

Hereinafter, the present invention will be described in more detail by way of examples. The following examples are only for the description of the present invention, and thus the scope of the present invention is not limited.

< Example  1> coumaric acid - Pro - Leu - Gly - Of NH2  Produce

1 g of Rink amid resin (substitution rate: 0.5 mmol / g) was added to the reactor, swollen in NMP solvent for 2 hours, and then reacted for 30 minutes by adding 20% piperidine / NMP to remove the Fmoc protecting group of the resin. After the solution was removed, 20% piperidine / NMP was added to the reactor containing the resin and allowed to react for 1 hour. After the solution was removed again, the resin was washed three times with NMP, MeOH and DCM, respectively, and finally with NMP again to remove excess reaction solution.

297 mg of Fmoc-Gly-OH (2 equivalents of substituent), 442 mg of BOP reagent (equivalent to amino acid reagent), 135 mg of HOBt (equivalent to amino acid reagent), 142 mg of DIEA (1.1 equivalent of amino acid reagent) were added, and 10 ml of NMP was added thereto. After that, it reacts for 3 hours. Termination of the reaction was confirmed using the kaiser's ninhydrin test ( Anal . Biochem . , 1981, 117, 147). After removing all the solutions in the reactor, the resin was washed three times with NMP, MeOH and DCM, respectively, and finally with NMP again. 20% piperidine / NMP was added to the finished resin and the Fmoc protecting group was removed by the same reaction as described above. Subsequently, Fmoc-Leu-OH and Fmoc-Pro-OH are introduced into the resin in the same manner.

Into the resin into which Pro-Leu-Gly was introduced, 164 mg of coumaric acid ((2 equivalents of substituent), 442 mg of BOP reagent (equivalent to amino acid reagent), HOBt 135 mg (equivalent to amino acid reagent), DIEA 142 mg (1.1 equivalent of amino acid reagent) After addition of 10 ml of NMP, the reaction was performed for 24 hours.The completion of the reaction was confirmed using kaiser's ninhydrin test.

 Synthetic Peptides: A User's Guide (GA Grant, ed.), WH Freeman and Company, New York, prepared coumaric acid-Pro-Leu-Gly-NH2 prepared on Rink amide resin using reagent K as described above. 1992). The isolated peptide derivatives were precipitated using cold ether and then centrifuged at 0 ° C. to separate them from the solution. The separated peptide derivative was added to 20 ml of cold ether under the same conditions and centrifuged three times to remove impurities. The final product was lyophilized for 24 hours by centrifugation of the peptide derivative to give 161 mg.

The presence of the final product was confirmed by HPLC analysis and MALDI-Tof analysis. Respective data are shown in FIGS. 1 and 2.

< Example  2-16>

In a manner similar to Example 1, through the scheme 1 or 2, the compounds shown in Table 1 below were synthesized.

The presence of the final product was confirmed by HPLC analysis and MALDI-Tof analysis. Each data is shown in FIGS. 3 to 22.

matter mw water Example 2 coumaric acid-GLG-OH 391.4 97.8% Example 3 coumaric acid-VLG-OH 433.5 98.0% Example 4 coumaric acid-ILG-OH 447.5 98.7% Example 5 coumaric acid-PLG-OH 431.5 95.1% Example 6 coumaric acid-LG-OH 334.4 98.1% Example 7 coumaric acid-GLG-NH2 391.0 96.3% Example 8 coumaric acid-VLG-NH2 433.1 94.4% Example 9 coumaric acid-LG-NH2 333.7 94.2% Example 10 coumaric acid-HyP-LG-NH2 446.7 95.7% Example 11 coumaric acid-ILG-NH2 447.0 95.5%

&Lt; Test Example 1 >

The melanogenesis inhibitory effect using B16-F1 melanoma cells was measured. B16-F1 melanoma cells were seeded in 6-well plates at a density of 3 × 10 ⁵ cells in DMEM with 10% FBS and incubated at 37% for 5% CO ₂ for one day. Thereafter, after replacing with fresh DMEM, the samples were incubated for 2 days by treating the samples at a concentration of 50 uM. After 2 days, the cells from which the medium was removed were washed with PBS (phosphate buffered saline) and treated with trypsin. The recovered cells were separated by centrifugation, and the supernatant was discarded. The cells were treated with 0.2 ml of 1N NaOH + 10% DMSO solution to obtain intracellular melanin. The collected cells were transferred to a 96-well plate, and the absorbance was measured at 490 nm. A standard curve was prepared using synthetic melanin as a standard material to determine the intracellular melanin amount, and the amount of protein was measured by using a protein assay reagent. Quantification through The amount of melanin produced was determined by the amount of melanin for a certain protein. The control group was used kojiic acid (Kojic acid) of 50uM concentration, the negative control group was used as the reaction solution without the sample. Table 2 shows the experimental results of the melanin production inhibitory effect on the negative control.

matter Inhibition%
(50 uM) Example 1 coumaric acid-PLG-NH2 47.1% Example 2 coumaric acid-GLG-OH 42.9% Example 3 coumaric acid-VLG-OH 31.3% Example 4 coumaric acid-ILG-OH 41.1% Example 5 coumaric acid-PLG-OH 45.2% Example 6 coumaric acid-LG-OH 44.6% Example 7 coumaric acid-GLG-NH2 33.7% Example 8 coumaric acid-VLG-NH2 42.4% Example 9 coumaric acid-LG-NH2 45.3% Example 10 coumaric acid-HyP-LG-NH2 41.3% Example 11 coumaric acid-ILG-NH2 42.3% PLG-NH2 13.1% coumaric acid 23.2% kojic acid 13.8%

As shown in Table 2, the whitening compound in which the peptide was bound to coumaric acid was shown to more effectively inhibit melanin production.

Claims (4)

A compound for whitening represented by the following formula (1).
[Formula 1]

Wherein X represents a covalent bond or an amino acid,
The amino group of the amino acid binds to the carboxy group of coumaric acid, the carboxy group of the amino acid binds to the amino group of Leu (leucine),
The amino acid is Gly (glycine), Leu, Ile (isoleucine), Pro (proline), Hyp (hydroxyproline) or Val (valine).) A compound for whitening represented by the following formula (2).
(2)

Wherein X represents a covalent bond or an amino acid,
The amino group of the amino acid binds to the carboxy group of coumaric acid, the carboxy group of the amino acid binds to the amino group of Leu,
The amino acid is Gly, Leu, Ile, Pro or Val.) The compound for whitening according to claim 1 or 2, wherein the amino acid is Pro. The compound for whitening according to claim 1 or 2, wherein X is a covalent bond.

Images