CN114315968B - Nonapeptide, and cosmetic composition or medicinal composition and application thereof - Google Patents

Abstract

A peptide of formula (I), a stereoisomer, a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a cosmetically or pharmaceutically acceptable composition thereof, and their use in the manufacture of a cosmetic or pharmaceutical composition for inhibiting melanogenesis, lightening skin colour, depigmenting or eliminating uneven skin tone.

Description

Nonapeptide, and cosmetic composition or medicinal composition and application thereof

Technical Field

The present invention relates to nonapeptides, as well as cosmetic or pharmaceutical compositions comprising these peptides and uses thereof.

Background

The color of human skin is influenced by the content and distribution of epidermal chromoplasts, the blood circulation of dermis and the thickness of stratum corneum, wherein the content and distribution of melanin are the main factors for determining the color of skin. When a human body is irradiated by proper ultraviolet rays, melanin cells positioned in the basal layer of the epidermis of the skin generate melanin and are transported into surrounding stratum corneum cells through the dendrites, so that the damage of ultraviolet rays to cell chromosomes and the sunburn of the skin are avoided. However, with the aging, the fast pace of life and the aggravation of environmental pollution, the metabolism of the skin is slowed down, and excessive melanin is accumulated in the human body, so that the skin becomes dark and dark, and even the melanin on the surface of the skin is unevenly distributed and plaques appear. Therefore, pigmentation and stain formation of melanin become hot problems of modern medical cosmetology, and the development of whitening and freckle removing products and functional raw materials thereof has been in a situation of being active and rapidly growing.

In human body, the formation of melanin is regulated by various exogenous factors (e.g., ultraviolet rays, emotion and stress, etc.) or endogenous factors (e.g., protease, cytokine, hormone, etc.), among which Tyrosinase (TYR), DHICA oxidase (TRP-1) and dopachrome isomerase (TRP-2) are key enzymes in the process of melanin production. When the skin is subjected to external stimuli or ultraviolet irradiation, the production of alpha-melanocyte stimulating hormone (alpha-MSH) is increased. alpha-MSH acts on melanocortin receptor 1 (MC 1-R), activates adenylate cyclase, increases cyclic adenosine monophosphate (cAMP), promotes expression of microphthalmia-associated transcription factor (MITF), increases synthesis of Tyrosinase (TYR), tyrosine in melanocytes generates Dopaquinone (DQ) under oxidation catalysis of TYR, DQ generates white dopachrome through intramolecular reaction, and the latter generates dopachrome through further reaction. Most of the generated dopachrome is subjected to dehydroxylation to generate 5, 6-Dihydroxyindole (DHI), the other part of the generated dopachrome is subjected to catalytic action of TRP-2 to generate 5, 6-dihydroxyindole-2-carboxylic acid (DHICA), and finally the DHI and the DHICA are oxidized to generate eumelanin. In addition, DQ can be converted into 5-S-cysteine dopa (5-S-CD) and a small amount of 2-S-cysteine dopa (2-S-CD) by glutathione or cysteine, and then reacted to produce glutathione dopa, which is further oxidized to cystathiazine (HBTA) to produce brown pigment. After melanin is generated, melanin is transferred to a keratin cell through a melanocyte dendritic projection, and melanin granules transferred to the keratin cell ascend to a horny layer along with an epidermal cell, thereby influencing the color of skin or forming color spots.

Currently, various methods for maintaining skin whitening have been developed in order to keep skin white, prevent skin from darkening and forming color spots. For example, scrub cream, fruit acid, hydrogen peroxide, mercuric chloride or various phenol derivatives and the like promote the skin epidermal keratinocytes to be rapidly exfoliated or the melanin tissues to be rapidly disintegrated through physical or chemical stimulation, thereby achieving the effects of whitening and changing the skin; arbutin, kojic acid, L-ascorbic acid, ellagic acid or tranexamic acid, etc. can inhibit the activity of tyrosinase, and specifically block melanin generation, thereby preventing spot formation or lightening plaque; the nicotinamide, the green tea extract or the glycoprotein can effectively inhibit the transfer of melanin granules from melanocytes to keratinocytes, thereby achieving the effects of whitening and brightening the skin. However, these products often have greater cytotoxicity, skin irritation, instability, etc., or poor single treatment efficacy. Therefore, it is necessary to develop a new green, safe, efficient and stable active whitening material aiming at the mechanism of melanin generation.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a nonapeptide capable of stably and efficiently inhibiting melanin generation. These peptides, cosmetic or pharmaceutical compositions containing them, can be used for lightening the skin, removing stains or eliminating unevenness of the complexion, and can also be used for the preparation of sunscreens.

In view of this, the present invention provides a peptide of formula (I), or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof,

in the formula (I), the compound is shown in the specification,

R ₁ selected from: r is ₁₂ -CO-, wherein R ₁₂ Selected from the group consisting of: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ and R ₁₀ Independently of one another, selected from: H. deuterium or tritium;

R ₁₁ selected from the group consisting of: -NR ₁₃ R ₁₄ OR-OR ₁₃ Wherein each R is ₁₃ And R ₁₄ Independently of one another, are selected from: H. substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;

the alkyl group refers to a saturated aliphatic linear or branched alkyl group having 1 to 24 carbon atoms (optionally having 1 to 16 carbon atoms; optionally having 1 to 14 carbon atoms; optionally having 1 to 12 carbon atoms; optionally having 1,2, 3, 4, 5, or 6 carbon atoms); optionally selected from: methyl, ethyl, isopropyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, 2-ethylhexyl, 2-methylbutyl, or 5-methylhexyl;

the alkenyl refers to a straight or branched chain alkenyl group having 2 to 24 carbon atoms (optionally having 2 to 16 carbon atoms; optionally having 2 to 14 carbon atoms; optionally having 2 to 12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); said alkenyl having one or more carbon-carbon double bonds, optionally 1,2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group is bonded to the remainder of the molecule by a single bond; optionally selected from: vinyl, oleyl, or linoleyl;

alternatively, the substituents in the "substituted alkyl", "substituted alkenyl" are selected from C ₁ -C ₄ An alkyl group; a hydroxyl group; c ₁ -C ₄ An alkoxy group; an amino group; c ₁ -C ₄ An aminoalkyl group; c ₁ -C ₄ A carbonyloxy group; c ₁ -C ₄ An oxycarbonyl group; halogen (e.g., fluorine, chlorine, bromine, and iodine); a cyano group; a nitro group; an azide; c ₁ -C ₄ An alkylsulfonyl group; a thiol; c ₁ -C ₄ An alkylthio group; c ₆ -C ₃₀ Aryloxy groups such as phenoxy; -NR _b (C＝NR _b )NR _b R _c Wherein R is _b And R _c Is independently selected from: H. c ₁ -C ₄ Alkyl radical, C ₂ -C ₄ Alkenyl radical, C ₂ -C ₄ Alkynyl, C ₃ -C ₁₀ Cycloalkyl radical, C ₆ -C ₁₈ Aryl radical, C ₇ -C ₁₇ Aralkyl radicalA group, a heterocyclic group having three to ten members, or a protecting group of an amino group.

Alternatively, R ₁ Selected from: H. acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl or linoleoyl;

alternatively, R ₁ Selected from H, acetyl, lauroyl, myristoyl or palmitoyl;

alternatively, R ₁ Is H, acetyl or palmitoyl.

Alternatively, R ₁₃ 、R ₁₄ Independently of one another, selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;

alternatively, R ₁₃ Is H and R ₁₄ Selected from: H. methyl, ethyl, hexyl, dodecyl or hexadecyl;

alternatively, R ₁₁ is-OH or-NH ₂ 。

A peptide represented by the formula (I), or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, selected from the following peptides (1) to (12):

(1)H-L-Arg-L-Lys-L-Met-L-Phe-L-Phe-L-Pro-L-Pro-L-Trp-L-Val-OH；

(2)H-L-Arg-L-Lys-L-Met-L-Phe-L-Phe-L-Pro-L-Pro-L-Trp-L-Val-NH ₂ ；

(3)H-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-OH；

(4)H-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ ；

(5)Ac-L-Arg-L-Lys-L-Met-L-Phe-L-Phe-L-Pro-L-Pro-L-Trp-L-Val-OH；

(6)Ac-L-Arg-L-Lys-L-Met-L-Phe-L-Phe-L-Pro-L-Pro-L-Trp-L-Val-NH ₂ ；

(7)Ac-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-OH；

(8)Ac-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ ；

(9)Palm-L-Arg-L-Lys-L-Met-L-Phe-L-Phe-L-Pro-L-Pro-L-Trp-L-Val-OH；

(10)Palm-L-Arg-L-Lys-L-Met-L-Phe-L-Phe-L-Pro-L-Pro-L-Trp-L-Val-NH ₂ ；

(11)Palm-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-OH；

(12)Palm-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ 。

the peptide of formula (I) of the present invention may exist as a stereoisomer or a mixture of stereoisomers; for example, the amino acids which they comprise may have the L-, D-configuration, or, independently of one another, be racemic. It is thus possible to obtain isomeric mixtures, as well as racemic or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and what isomer or isomeric mixture is present. The preferred structure of the peptide of formula (I) of the present invention is a pure isomer, i.e., an enantiomer or diastereomer.

For example, when said-Phe-of the present invention, it is understood that-Phe-is selected from-L-Phe-, -D-Phe-, or a mixture of both, is racemic or non-racemic. The preparation methods described in this document enable one of ordinary skill in the art to obtain each stereoisomer of the peptides of the invention by selecting amino acids with the correct configuration.

The peptides of the invention may be compounds in which at least one hydrogen atom is replaced by deuterium or tritium.

The term "cosmetically or pharmaceutically acceptable salt" refers to a salt approved for use in animals, and more specifically in humans, including metal salts of the peptides of formula (I), including, but not limited to: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, aluminum, or the like; comprising a salt of a peptide of formula (I) with an organic base, including, but not limited to: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, piperazine, or the like; comprising a salt of a peptide of formula (I) with an inorganic or organic acid, including, but not limited to: acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid (pamoate), gluconic acid, or the like; such inorganic acids include, but are not limited to: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

The synthesis of the peptide of formula (I) of the present invention, or a stereoisomer thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, can be carried out according to conventional methods known in the art, such as solid phase synthesis, liquid phase synthesis, or a method combining solid phase and liquid, and can also be prepared by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins having animal, fungal, or plant origin.

For example, a method of obtaining a peptide of formula (I) comprises the steps of:

-coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a protected or solid support bound C-terminus;

-elimination of the group protecting the N-terminus;

-repeating the coupling sequence and eliminating the group protecting the N-terminus until the desired peptide sequence is obtained;

-elimination of the group protecting the C-terminus or cleavage from the solid support.

Preferably, the C-terminus is bound to a solid support and the method is carried out on a solid phase comprising coupling the amino acid having a protected N-terminus and a free C-terminus with the amino acid having a free N-terminus and a C-terminus bound to a polymeric support; elimination of the group protecting the N-terminus; and repeating this sequence as many times as necessary to thereby obtain a peptide having a desired length, followed by cleavage of the synthesized peptide from the original polymer support.

The functional groups of the side chains of these amino acids remain sufficiently protected throughout the synthesis with temporary or permanent protecting groups and can be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric support.

Alternatively, solid phase synthesis may be performed by a pooling strategy (convergent strategy) of coupling di-or tripeptides to a polymeric support or to di-or amino acids previously bound to a polymeric support.

The functional groups at the N-terminus and C-terminus can be subsequently modified by deprotecting the termini and/or cleaving the peptide from the polymeric support in a non-defined order using standard conditions and methods known in the art. The peptide of formula (I) bound to the polymeric support may be optionally modified at the N-terminus and C-terminus, or after the peptide has been cleaved from the polymeric support.

In another aspect of the present invention, there is provided a cosmetic or pharmaceutical composition comprising an effective amount of a peptide of formula (I) as described above, or a stereoisomer, or a mixture of stereoisomers, or a cosmetically or pharmaceutically acceptable salt thereof, together with at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant.

Optionally, the adjuvant is selected from: <xnotran> , PGC-1 α , PPAR γ , , , , , , , , , , , , , , , , NO- ,5 α - , / , , / , , , , , , , , , , , , , α , β , , , , , , , , , , , , , , , , , , , , , , , , , / , , , , </xnotran> <xnotran> , , cAMP , , HSP70 , , , , , , , , , , , , , , , , , , , , , , , , DNA , DNA , , , / , , , , , , , , , , , , , , , , / , , , , , - , , , , , , , , , , </xnotran> A cell extract, a sunscreen agent, and an organic or inorganic photoprotective agent effective against a and/or B ultraviolet rays, or mixtures thereof.

Optionally, the formulation of the cosmetic or pharmaceutical composition is selected from: creams, oils, milks, balms, foams, lotions, gels, liniments, serums, soaps, shampoos, hair-tonics, serums, ointments, mousses, pomades, powders, sticks, pens, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, jellies or gelatins;

optionally, the capsule comprises: soft capsule, hard capsule, optionally gelatin capsule;

optionally, the tablet comprises: sugar-coated tablets.

The peptides of the invention have variable solubility in water, depending on the nature of their sequence or any possible modification in the N-and/or C-terminus. The peptides of the invention may thus be incorporated into the composition by aqueous solution and those which are insoluble in water may be dissolved in conventional cosmetically or pharmaceutically acceptable solvents such as, and without limitation, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol or polyethylene glycol or any combination thereof.

The cosmetically or pharmaceutically effective amount of the peptides of this invention to be administered and their dosage will depend on a number of factors, including age, the state of the patient, the severity of the condition or disease, the route and frequency of administration, and the specific nature of the peptide to be used.

By "cosmetically or pharmaceutically effective amount" is meant a non-toxic but sufficient amount of one or more peptides of the invention to provide the desired effect. Using the peptide of the invention in a cosmetically or pharmaceutically effective concentration to obtain the desired effect in the cosmetic or pharmaceutical composition of the invention; in a preferred form, it is between 0.00000001% (by weight) and 20% (by weight), preferably between 0.000001% (by weight) and 15% (by weight), more preferably between 0.0001% (by weight) and 10% (by weight), and even more preferably between 0.0001% (by weight) and 5% (by weight), relative to the total weight of the composition.

In another aspect of the present invention, there is provided a cosmetically or pharmaceutically acceptable delivery system or sustained release system for achieving better penetration of the active principle and/or for improving its pharmacokinetic and pharmacodynamic properties, comprising an effective amount of a peptide of formula (I) as defined above, or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetically or pharmaceutically acceptable composition as defined above.

The term "delivery system" refers to a diluent, adjuvant, excipient or carrier to be administered with the peptide of the invention, selected from: water, oil or surfactant, including those of petroleum, animal, vegetable, or synthetic origin, such as, and not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan esters, ether sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol, poloxamers, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, and the like. Diluents which can be used in different delivery systems in which the peptides of the invention can be administered are known to the person skilled in the art.

The term "sustained release" is used in the conventional sense to refer to a delivery system of a compound that provides for gradual release of the compound over a period of time, and preferably, but not necessarily, has a relatively constant level of compound release over the entire period of time.

Examples of delivery systems or sustained release systems are liposomes, oleosomes, non-ionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, liposomes, micelles, nanospheres, microspheres, nanospheres, lipid globules, microemulsions, nanoemulsions, nanoparticles, microparticles or nanoparticles. Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions having an internal structure of reverse micelles.

Sustained release systems may be prepared by methods known in the art and may be administered, for example, by: by topical or transdermal administration, including adhesive patches, non-adhesive patches, occlusive patches, and microelectronic patches; or by systemic administration such as, but not limited to, oral or parenteral routes, including nasal, rectal, subcutaneous implantation or injection, or direct implantation or injection into a specific body site, and preferably should release relatively constant amounts of the peptides of the invention. The amount of peptide included in the sustained release system will depend on, for example, the site to which the composition is to be administered, the kinetics and duration of release of the peptide of the invention, and the nature of the condition, disorder and/or disease to be treated and/or cared for.

In another aspect of the present invention, there is provided a use of a peptide represented by formula (I) above, or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically acceptable salt thereof, or a pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, for the preparation of a cosmetic or pharmaceutical composition for inhibiting melanogenesis, lightening skin colour, depigmenting spots or eliminating uneven skin tone.

In another aspect of the present invention there is provided the use of a peptide of formula (I) as described above, or a stereoisomer or a mixture of stereoisomers thereof, or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition as described above, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system as described above, in the manufacture of a sunscreen.

To facilitate an understanding of the invention, some terms and expressions used in the present invention are explained in the following:

in the present invention, the term "skin" is understood to be the layers that make up it, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, both endpoints being included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes, among others.

In the present specification, the abbreviations used for amino acids follow the rules specified in the European journal of biochemistry (Eur.J.Biochem.1984, 138: 9-37) by the IUPAC-IUB Commission on Biochemical Nomenclature.

Thus, for example, val represents NH ₂ -CH(CH(CH ₃ ) ₂ ) -COOH, val-represents NH ₂ -CH(CH(CH ₃ ) ₂ ) -CO-, val represents-NH-CH (CH) ₃ ) ₂ ) -COOH, and-Val-represents-NH-CH (CH) ₃ ) ₂ ) -CO-. Thus, the hyphen representing the peptide bond eliminates the OH in the 1-carboxyl group of the amino acid (represented herein in the conventional non-ionized form) when located to the right of the symbol, and eliminates the H in the 2-amino group of the amino acid when located to the left of the symbol; both modifications can be applied to the same symbol (see table 1).

TABLE 1 Structure of amino acid residues and their one-letter and three-letter abbreviations

The abbreviation "Ac-" is used herein to denote acetyl (CH) ₃ -CO-), and the abbreviation "Palm-" is used to denote palmitoyl (CH) ₃ -(CH ₂ ) ₁₄ -CO-)。

Compared with the prior art, the invention has the advantages that:

the peptide is obtained by artificial design, is convenient to synthesize, is safe and non-irritant to human bodies, can inhibit melanin generation, is used for brightening skin color, removing color spots or eliminating uneven skin color, and can also be used for preparing an opacifier.

Drawings

FIG. 1 is the peptide (4) H-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ (formula C) ₆₁ H ₈₇ N ₁₅ O ₉ S) mass spectrum, [ M + H [ ]] ⁺ m/z 1206.6453 is the molecular ion peak of the compound.

FIG. 2 is a graph showing the results of the inhibition of melanin production by B16F10 cells by the test samples. The # # indicates that the α -MSH group was significantly different compared to the PBS group, p <0.001 (n = 3). * Indicating that the dosed group had statistical differences compared to the α -MSH group, p <0.05 (n = 3).

FIG. 3 is a graph showing the results of the inhibition of tyrosinase activity by the test samples. The # # indicates that the α -MSH group was significantly different compared to the PBS group, p <0.001 (n = 3). * Indicating that the dosed group had statistical differences compared to the α -MSH group, p <0.05 (n = 3).

Detailed Description

For a better understanding of the present invention, the following detailed description of the invention is made with reference to examples and the accompanying drawings, however, it should be understood that these examples and drawings are for illustrative purposes only and are not intended to limit the scope of the present invention.

Abbreviations

Abbreviations for amino acids follow the committee on biochemical nomenclature of IUPAC-IUB in Eur j. Biochem. (1984) 138: chem (1989) 264: rules specified in 633-673.

Amide Resin: a starting resin for polypeptide synthesis (degree of crosslinking 1%, degree of substitution 1.72 mmol/g); fmoc-Linker:4- [ (2, 4-Dimethoxyphenyl) (Fmoc-amino) methyl]Phenoxyacetic acid; ac ₂ O: acetic anhydride; DMF: n, N-dimethylformamide; DIPEA: diisopropylethylamine; DIC: diisopropylcarbodiimide; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; and (3) TIS: triisopropylsilane; EDT (electro-thermal transfer coating): 1, 2-ethanedithiol; val: valine; trp: tryptophan; pro: (ii) proline; phe: phenylalanine; met: methionine; lys: lysine; arg: arginine; fmoc: 9-fluorenylmethoxycarbonyl; boc: a tert-butoxycarbonyl group; pbf:2, 4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl.

EXAMPLE 1 preparation of Fmoc-L-Arg (Pbf) -L-Lys (Boc) -L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp (Boc) -L-Val-Linker-Amide Resin

1.1 Preparation of Fmoc-Linker-Amide Resin

Weighing 5g of Amide Resin in a solid phase synthesis reaction column, swelling the solid phase synthesis reaction column with DMF, washing the Resin, and pumping away the solvent.

Weighing 7.0g of Fmoc-Linker and 2.10g of HOBt in a drying triangular flask, dissolving the Fmoc-Linker and the HOBt in 40mL of DMF, placing the mixture in an ice-water bath, cooling for 10min, and adding DIC to activate for 10min to avoid water vapor.

Adding the activated Fmoc-Linker into the swelled resin for reaction for 2.5h, pumping reaction liquid, washing the resin, and using Ac ₂ And treating for 1.5h by using O and DIPEA, and blocking the residual amino group. The resin was washed and the solvent was removed.

1.2 DeFmoc

Fmoc-Linker-Amide Resin was Fmoc-removed twice with 20% piperidine/DMF for 10min each time, sampled for K test, and developed dark blue. The resin was washed repeatedly with DMF 7-8 times and the solvent was removed.

1.3 Charge reaction

3.7g of Fmoc-L-Val-OH and 1.8g of HOBt were weighed into a dry flask. Adding DMF to dissolve, sealing, and placing in a refrigerator at-18 deg.C for 30min. 2.5mL DIC was added for 3min to avoid water vapor. Adding the activated amino acid into the deprotected resin to react for 2h, and pumping the reaction solution. The K detection resin is colorless and transparent, which indicates that the reaction is complete.

The N-terminal Fmoc group was deprotected and 5.8g of Fmoc-D-Trp (Boc) -OH after activation was coupled to the peptidyl resin in the presence of 1.8g of HOBt and 2.5mL of IC using DMF as solvent for 2h. The resins were then washed and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. In each coupling, 3.7g of Fmoc-L-Pro-OH were coupled sequentially in the presence of 1.8g of HOBt and 2.5ml of IC using DMF as solvent; 3.7g of Fmoc-L-Pro-OH.

6.1g of Fmoc-L-Phe-OH were coupled sequentially in the presence of 2.5g of HOBt and 3.6ml of IC using DMF as solvent; 6.1g of Fmoc-D-Phe-OH;5.8g of Fmoc-L-Met-OH;7.3g Fmoc-L-Lys (Boc) -OH. 13.0g of Fmoc-L-Arg (Pbf) -OH was then coupled in the presence of 3.2g of HOBt and 4.6ml of IC using DMF as solvent. After the reaction was complete, the resin was washed and the solvent was pumped away.

EXAMPLE 2 removal of the N-terminal Fmoc protecting group

The N-terminal Fmoc group of these peptidyl resins obtained in example 1 was deprotected.

500mL of piperidine/DMF 20%, bubbling with nitrogen gas for 10min, and draining for 3min. Then 500mL of piperidine/DMF 20%, bubbling with nitrogen for 10min, and draining for 3min. Then adding 500mL of DMF, introducing nitrogen, bubbling for 1-2 min, pumping for 3min, repeatedly washing the resin for 7-8 times, and pumping away the solvent.

Example 3 reaction of R ₁ Acetyl group introduction to the peptidyl resin obtained in example 2

Using 5mL of DMF as solvent in the presence of 25 equivalents of DIPEA, 25 equivalents of Ac ₂ O-treating 1mmol of the peptidyl resin obtained in example 2. They were allowed to react for 30min, after which the resin was washed 7-8 times with 600mL of DMF, 2min each time, and the solvent was pumped away.

Example 4 reaction of R ₁ Introduction of palmitoyl group onto the peptidyl resin obtained in example 2

2.56g of palmitic acid (10 equivalents in 10 mmol) pre-dissolved in 1mL of DMMF was added to 1mmol of the peptidyl resin obtained in example 2 in the presence of 1.53g of HOBt (10 equivalents in 10 mmol) and 1.54mL of DIC (10 equivalents in 10 mmol. They were allowed to react for 15h, after which the resin was washed 7-8 times with 600mL DMF for 2min each time, and the solvent was pumped away.

Example 5 cleavage of peptidyl resins obtained in examples 2, 3 and 4 from a polymeric support

The peptidyl resins obtained in examples 2, 3 and 4 were shrunk three times with 500mL,300mL,200mL of methanol. And (5) vacuum pumping.

1g of each of the obtained dried peptidyl resins of examples 2, 3 and 4 was weighed, and 7mL of a lysis solution (TFA: TIS: EDT: H) was added ₂ O =113.5:3.2:3.2:3.2 Treatment) and stirring the reaction solution for 2 hours. Filtering with a sand core funnel, slowly adding the filtrate into ice isopropyl ether, precipitating, centrifuging, and washing with isopropyl ether for 6 times. The final precipitate was dried under vacuum to give the following crude peptides:

1、H-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ i.e. peptide (4);

2、Ac-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ i.e. peptide (8);

3、Palm-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ i.e. peptide (12).

Example 6 purification of crude peptide by HPLC

The peptide (4) obtained in example 5 was dissolved in an aqueous methanol solution (methanol: pure water =1, volume ratio) and filtered through a 0.22 μm microporous membrane to give a clear and transparent solution, which was purified by reverse phase HPLC with the following purification gradient:

time (min)	Flow rate (mL/min)	A% (acetonitrile)	B% (0.1% acetic acid aqueous solution)
				0	40	10	90
5	40	25	75
				45	40	35	65
65	40	40	60
				75	40	60	40

And (3) injecting and purifying the filtered sample, collecting fractions, concentrating and freeze-drying to obtain the peptide (4) with the purity of 98%. The molecular weight was determined by ESI-MS, the mass spectrometry result is shown in FIG. 1, and the molecular weight of the peptide (4) was found to be 1205.6453.

The other compounds of formula (I) of the present invention can be prepared by analogous methods as described above.

Example 7 Effect on melanogenesis by mouse melanoma cells B16F10

7.1 reagents and materials

DMEM culture medium, fetal bovine serum, trypsin, alpha-MSH, naOH and DMSO.

7.2 Instrument

Enzyme-linked immunosorbent assay (American MD), CO ₂ Incubator (Shanghai-Heng), super clean bench (Suzhou purification).

7.3 cell lines

Mouse B16F10 melanoma cell line, purchased from kunming cell bank, chinese academy of sciences.

7.4 samples to be tested

Administration group: peptide (4), tested at a concentration of 100ppm; alpha-arbutin, test concentration 100ppm.

Control group: PBS blank control.

7.5 Experimental methods

Reference standard: T/SHRH 027-2019 test in vitro B16 cell melanin synthesis inhibition assay.

Taking a bottle of cells in exponential growth phase, adding 0.25% trypsin digestive juice to make adherent cells fall off, and counting 1-4 × 10 ⁵ And (4) preparing cell suspension per mL.

The medium in the wells was aspirated completely and starved for 3-6h with 1mL of PBS.

The sample to be tested was added, and 1. Mu.g/mL of alpha-MSH was added to all wells except the PBS control.

Changing culture medium every 24-48h, culturing for 72h, collecting cells, adjusting cell density to 5 × 10 ⁵ pieces/mL, centrifuged (4000 r/min,5 min) and the supernatant discarded. Adding 450 μ L DMSO with mass fraction of 10% and 1M NaOH solution, and placing in 80 deg.C water bath for 30min to completely dissolve cell mass. The OD of each well was measured at 490nm using a microplate reader.

Melanin content (%) = treatment OD ₄₉₀ Blank control OD ₄₉₀ ×100％

7.6 results

To investigate the effect of the peptides of the present invention on melanogenesis, a model of α -MSH-stimulated melanogenesis was established by α -MSH-stimulated B16F10 melanoma cells, and the inhibitory effect of the test samples on melanogenesis of B16F10 cells was evaluated, the results are shown in fig. 2.

The results show that the melanin content of the melanoma cells is obviously increased after the stimulation of alpha-MSH. After treatment of the administration group, the melanin content can be obviously reduced by 100ppm of the peptide (4), so that the melanin content in the B16F10 melanoma cells of the mice returns to the level close to that of a PBS blank control group, and the effect of the peptide (4) on inhibiting the melanin generation is equivalent to that of alpha-arbutin under the same concentration.

Arbutin is a commonly used whitening component at present, can inhibit tyrosinase activity so as to block melanin formation, wherein alpha-arbutin has a better whitening effect. However, arbutin is also highly irritating to the skin, and cannot be used for a long time, and some patients are prone to have side effects such as local skin redness, itching, facial stabbing pain and the like after use. In addition, arbutin has rather strict requirements on the concentration of use, and the use at high concentration causes white spots on the skin. Compared with the prior art, the micromolecule active polypeptide has higher safety and is mild and non-irritant to the skin, and the peptide can play a whitening effect equivalent to alpha-arbutin under the condition of meeting the requirement of high safety.

Example 8 Effect on B16F10 cell tyrosinase Activity

8.1 reagents and materials

DMEM culture medium, fetal calf serum, trypsin, alpha-MSH, sodium deoxycholate and L-DOPA.

8.2 instruments

Enzyme-linked immunosorbent assay (American MD), CO ₂ Incubator (Shanghai-Heng), super clean bench (Suzhou purification).

8.3 cell lines

Mouse B16F10 melanoma cell line, purchased from kunming cell bank, chinese academy of sciences.

8.4 samples to be tested

Administration group: peptide (4), tested at a concentration of 100ppm; alpha-arbutin, test concentration 100ppm.

Control group: PBS blank control.

8.5 Experimental methods

Taking a bottle of cells in exponential growth phase, adding 0.25% trypsin digestive juice to make adherent cells fall off, and counting 1-4 × 10 ⁵ And (4) preparing cell suspension per mL.

The well was completely aspirated and starved for 3-6h with 1mL PBS.

The sample to be tested was added, and 1. Mu.g/mL of alpha-MSH was added to all wells except the PBS control.

Changing culture medium every 24-48h, culturing for 72h, collecting cells, adjusting cell density to 5 × 10 with PBS buffer solution ⁵ One per mL. 1mL of each cell suspension was pipetted into 3 parallel tubes, centrifuged and the supernatant discarded. Cell lysates containing active tyrosinase were prepared by adding 1ml of 0.5% sodium deoxycholate solution and shaking thoroughly to lyse the cells.

The tube was placed at 0 ℃ for 15min and then in a 37 ℃ water bath for 10min. Adding 0.1% L-DOPA solution, shaking, reading OD value at 490nm wavelength with microplate reader at 0min and 10min, and calculating the difference between the two OD values.

Tyrosinase activity (%) = treatment OD ₄₉₀ Blank control OD ₄₉₀ ×100％

8.6 results

To further explore the effect of the peptides of the invention on the downstream tyrosinase pathway, the effect of test samples on tyrosinase activity was evaluated based on the above-described alpha-MSH stimulated melanin model, and the results are shown in FIG. 3.

The results show that the tyrosinase activity is greatly improved after the stimulation of alpha-MSH. The tyrosinase activity is obviously reduced by using 100ppm of peptide (4) and alpha-arbutin for treatment, the inhibition effect of the peptide (4) and the alpha-arbutin on the tyrosinase activity is equivalent, and the generation of melanin can be reduced by inhibiting the activity of tyrosinase.

In conclusion, the peptide of the invention can inhibit the generation of melanin, is used for lightening the skin color, removing color spots or eliminating uneven skin color, and can also be used for preparing a sunscreen agent.

EXAMPLE 9 preparation of essence containing peptide (4)

Heating purified water to 85 deg.C under stirring, and maintaining the temperature for 30min; pre-dissolving sodium hyaluronate and xanthan gum in butanediol, adding into water, and stirring to dissolve completely; cooling to 35 deg.C under stirring, adding the rest components, and stirring.

The above description is further intended to describe the present invention in detail with reference to specific preferred embodiments, but it is not intended to limit the present invention to the specific embodiments. For those skilled in the art to which the invention pertains, numerous simple deductions or substitutions may be made without departing from the spirit of the invention, which shall be deemed to belong to the scope of the invention.

Claims (10)

1. A peptide of formula (I) or a cosmetically or pharmaceutically acceptable salt thereof,

in the formula (I), the compound is shown in the specification,

R ₁ selected from: h;

R ₂ 、R ₃ 、R ₄ 、R ₅ 、R ₆ 、R ₇ 、R ₈ 、R ₉ and R ₁₀ Independently of one another, selected from: H. deuterium or tritium;

R ₁₁ is-OH or-NH ₂ ；

And the 4 th and 8 th amino acids at the N-terminal are in D configuration, and the rest amino acids are in L configuration.

2. The peptide of formula (I) or a cosmetically or pharmaceutically acceptable salt thereof according to claim 1, selected from the group consisting of:

(4)H-L-Arg-L-Lys-L-Met-D-Phe-L-Phe-L-Pro-L-Pro-D-Trp-L-Val-NH ₂ 。

3. the peptide of formula (I) or a cosmetically or pharmaceutically acceptable salt thereof according to claim 1,

the cosmetically or pharmaceutically acceptable salt comprises a metal salt of a peptide of formula (I), the metal comprising: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum.

4. The peptide of formula (I) or a cosmetically or pharmaceutically acceptable salt thereof according to claim 1,

the cosmetically or pharmaceutically acceptable salt comprises a salt of the peptide of formula (I) with an organic base comprising: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine.

5. The peptide of formula (I) or a cosmetically or pharmaceutically acceptable salt thereof according to claim 1,

the cosmetically or pharmaceutically acceptable salt includes a salt of the peptide represented by formula (I) with an inorganic acid or an organic acid, including: acetic, citric, lactic, malonic, maleic, tartaric, fumaric, benzoic, aspartic, glutamic, succinic, oleic, trifluoroacetic, oxalic, pamoic or gluconic acids; the inorganic acid includes: hydrochloric acid, sulfuric acid, boric acid, or carbonic acid.

6. A cosmetic or pharmaceutical composition comprising an effective amount of a peptide of formula (I) according to any one of claims 1 to 5, or a cosmetically or pharmaceutically acceptable salt thereof, and at least one excipient and optionally a cosmetically or pharmaceutically acceptable adjuvant.

7. Cosmetic or pharmaceutical composition according to claim 6, characterized in that said adjuvant is selected from: <xnotran> , PGC-1 α , PPAR γ , , , , , , , , , , , , , , , , NO- ,5 α - , / , , / , , , , , , , , , , , , α , β , , , , , , , , , , , , , , , , , , , , / , , , , , , cAMP , </xnotran> An agent that stimulates HSP70 synthesis, an agent that stimulates heat shock protein synthesis, an agent that stimulates hyaluronic acid synthesis, an agent that stimulates fibronectin synthesis, an agent that stimulates deacetylase synthesis, an agent that stimulates synthesis of lipids and stratum corneum components, ceramides, fatty acids, an agent that inhibits collagen degradation, an agent that inhibits elastin degradation, an agent that inhibits serine proteases, an agent that stimulates fibroblast proliferation, an agent that stimulates keratinocyte proliferation, an agent that stimulates adipocyte proliferation, an agent that stimulates melanocyte proliferation, an agent that stimulates keratinocyte differentiation, an agent that inhibits acetylcholinesterase, a skin relaxant, an agent that stimulates glycan synthesis, an anti-hyperkeratotic agent, an acne lytic agent, an anti-psoriatic agent, an anti-eczema agent, a DNA repair agent, a DNA protectant, a stabilizer, an agent for the treatment and/or care of sensitive skin, a firming agent, a restructuring agent, anti-stretch agents, adhesives, agents for modulating sebum production, antiperspirants, agents for stimulating healing, agents for assisting healing, agents for stimulating re-epithelialization, agents for assisting re-epithelialization, cytokine growth factors, sedatives, anti-inflammatory agents, anesthetics, agents acting on capillary circulation and/or microcirculation, agents for stimulating angiogenesis, agents for inhibiting vascular permeability, intravenous tensides, agents acting on cellular metabolism, agents for improving dermal-epidermal junction, agents for inducing hair growth, hair growth inhibiting or retarding agents, fragrances, chelating agents, plant extracts, essential oils, marine extracts, agents derived from biological fermentation processes, inorganic salts, cell extracts, sunscreens, and organic or inorganic photoprotective agents effective against a and/or B ultraviolet rays, or mixtures thereof.

8. Cosmetic or pharmaceutical composition, according to claim 6, characterized in that the formulation of said cosmetic or pharmaceutical composition is selected from: creams, oils, balms, foams, lotions, gels, liniments, serums, ointments, mousses, powders, sticks, pencils, sprays, aerosols, capsules, tablets, granules, chewing gums, solutions, suspensions, emulsions, elixirs, polysaccharide films, jellies or gelatins.

9. A cosmetically or pharmaceutically acceptable delivery system or sustained release system comprising an effective amount of a peptide of formula (I) according to any one of claims 1 to 5 or a cosmetically or pharmaceutically acceptable salt thereof, or a cosmetic or pharmaceutical composition according to any one of claims 6 to 8;

the cosmetically or pharmaceutically acceptable delivery system or sustained release system is selected from: liposomes, oleosomes, nonionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, liposomes, micelles, nanospheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, nanoparticles, microparticles, or nanoparticles.

10. Use of a substance selected from the group consisting of: a peptide of formula (I) or a cosmetically or pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 5, or a cosmetic or pharmaceutical composition as defined in any one of claims 6 to 8, or a cosmetically or pharmaceutically acceptable delivery system or sustained release system, as defined in claim 9, for use in inhibiting melanogenesis, lightening skin colour, depigmenting or eliminating uneven skin tone.

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