WO2024078588A1 - 肽类化合物在制备用于皮肤延衰修复的组合物中的新用途 - Google Patents

肽类化合物在制备用于皮肤延衰修复的组合物中的新用途 Download PDF

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WO2024078588A1
WO2024078588A1 PCT/CN2023/124288 CN2023124288W WO2024078588A1 WO 2024078588 A1 WO2024078588 A1 WO 2024078588A1 CN 2023124288 W CN2023124288 W CN 2023124288W WO 2024078588 A1 WO2024078588 A1 WO 2024078588A1
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ser
tyr
acid
compound
optionally
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PCT/CN2023/124288
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English (en)
French (fr)
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丁文锋
孙新林
赵文豪
观富宜
陈雪
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深圳市维琪科技股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/004—Aftersun preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention belongs to the technical field of active polypeptides, and in particular relates to a new use of peptide compounds in preparing a composition for delaying and repairing skin aging.
  • Skin is the largest and most important organ in the human body, covering the entire surface of the human body. It is composed of the epidermis, dermis and subcutaneous tissue, of which the epidermis and dermis play important roles, such as preventing water loss, forming a protective barrier and providing structural support. However, over time, affected by factors such as aging, internal and external environment, the expression levels of the epidermis and dermis will also change.
  • the proliferation rate and differentiation of epidermal keratinocytes change, the expression of molecules involved in the adhesion of basal cells decreases, and the skin barrier function weakens; the composition of the extracellular matrix of the dermis also undergoes degenerative changes with the aging process, with a large loss of collagen, weakened fibroblast activity, and a gradual decrease in the synthesized collagen and collagen fiber area, which leads to a loose reticular structure of the dermis, reduced moisture retention and toughness of skin tissue, and subsequent dryness, roughness, wrinkles, sagging, and damage to the skin.
  • repair is a precise, complex, and time-phased dynamic process involving network regulation of multiple cells such as cell proliferation, migration, extracellular matrix degradation, angiogenesis, and epithelial tissue remodeling. Therefore, it is of great significance to find compounds with delayed aging and repair effects based on the mechanism of skin aging and repair.
  • the present inventors conducted a large number of experimental studies and found that a peptide compound has the effect of delaying aging and repairing the skin, thereby completing the present invention.
  • the present invention aims to provide a use of a peptide compound or a salt thereof in the preparation of a composition for delaying and repairing skin aging, wherein the skin delaying and repairing includes one or more of increasing fibroblast activity, promoting keratinocyte proliferation and migration, promoting re-epithelialization or healing of skin or mucous membranes, repairing skin barriers, preventing or repairing photoaging damage, promoting collagen production, increasing skin elasticity or improving skin firmness.
  • the peptide compound or its salt described in the present invention has the following general formula (I):
  • R 1 is selected from: H or R 3 -CO-, wherein R 3 is selected from: substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl;
  • R 2 is selected from: -OH, -NH 2 , -Leu-NH 2 , -Leu-OH, -Val-NH 2 or -Val-OH;
  • the alkyl group refers to a saturated aliphatic straight or branched chain 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); and may be 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 group refers to a straight or branched alkenyl group having 2-24 carbon atoms (optionally having 2-16 carbon atoms; optionally having 2-14 carbon atoms; optionally having 2-12 carbon atoms; optionally having 2, 3, 4, 5, or 6 carbon atoms); the alkenyl group has one or more carbon-carbon double bonds, and optionally has 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds; the alkenyl group The group is bound to the rest of the molecule by a single bond; it can be selected from: vinyl, oleyl, or linoleyl;
  • R 1 is selected from: H, acetyl, tert-butyryl, hexanoyl, 2-methylhexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, linoleoyl or phenylacetyl;
  • R1 is H, myristoyl or palmitoyl.
  • the compound is selected from (1)-(24):
  • Palm-Tyr-Ser-NH 2 Palm-Tyr-Ser-NH 2 ;
  • the peptide compounds of the present invention may exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids comprising them may have the L-, D-configuration, or be racemic independently of each other.
  • isomeric mixtures as well as racemic mixtures or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbons and what isomers or isomeric mixtures are present.
  • Preferred structures of the peptide compounds of the present invention are pure isomers, i.e., enantiomers or diastereomers.
  • -Ser- is selected from -L-Ser-, -D-Ser-, or a mixture of the two, and is racemic or non-racemic.
  • the preparation method described in this document enables a person skilled in the art to obtain each stereoisomer of the peptide compound of the present invention by selecting amino acids with the correct configuration.
  • the present invention also includes all suitable isotopic variants of the peptide compounds represented by the general formula (I).
  • Isotopic variants of these peptide compounds of the present invention are understood here to mean compounds in which at least one atom in the peptide compounds of the present invention is replaced by another atom of the same atomic number, but the atomic mass of the other atom is different from the atomic mass usually or predominantly present in nature.
  • Examples of isotopes that can be incorporated into the peptide compounds of the present invention are: those of hydrogen, carbon, nitrogen or oxygen, such as 2H (deuterium), 3H (tritium), 13C , 14C , 15N , 17O or 18O .
  • isotopic variants of the peptide compounds of the present invention may be useful, for example, for examining the mechanism of action or the distribution of active compounds in vivo; due to relatively simple preparability and detectability, especially with Compounds labeled with 3 H or 14 C isotopes are suitable for this purpose.
  • isotopes e.g., deuterium
  • the incorporation of isotopes can produce specific therapeutic benefits, such as an extension of the half-life in vivo or a reduction in the required active dose; therefore, in certain cases, such modifications of the peptide compounds of the invention may also constitute a preferred embodiment of the invention.
  • Isotopic variants of the peptide compounds of the invention can be prepared by methods known to those skilled in the art, for example by the methods further described below and by the methods described in the examples, by using the respective reagents and/or corresponding isotopic modifications of the starting materials.
  • the present invention also includes prodrugs of the peptide compounds of the present invention.
  • prodrug herein means such compounds: they themselves can be biologically active or inactive, but during their residence time in the body, they react (e.g., metabolize or hydrolyze) to generate the compounds of the present invention.
  • the composition comprises a compound represented by general formula (I) or a salt thereof in a mass percentage concentration of 0.0001% to 5%;
  • the composition comprises a compound represented by general formula (I) or a salt thereof in a mass percentage concentration of 0.0005% to 1%;
  • the composition comprises a compound represented by general formula (I) or a salt thereof in a mass percentage concentration of 0.001% to 0.1%;
  • the composition contains the compound represented by general formula (I) or a salt thereof in a mass percentage concentration of 0.005%-0.01%.
  • the compound represented by the above general formula (I) can increase the activity of fibroblasts, promote the proliferation and migration of keratinocytes, promote the re-epithelialization or healing of skin or mucous membranes, repair the skin barrier, prevent or repair photoaging damage, promote collagen production, increase skin elasticity or improve skin firmness.
  • the salt of the compound represented by the general formula (I) includes a metal salt of the compound represented by the general formula (I), and the metal includes: lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum;
  • the salt of the compound represented by the general formula (I) includes a salt formed by the compound represented by the general formula (I) and an organic base, and the organic base includes: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine;
  • the salt of the compound represented by the general formula (I) includes a salt formed by the compound represented by the general formula (I) and an inorganic acid or an organic acid, and the organic acid includes: 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 or gluconic acid;
  • the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid or carbonic acid.
  • the compound represented by the above general formula (I) or its salt can be prepared by conventional methods known in the prior art, such as solid phase synthesis, liquid phase synthesis or a combination of solid phase and liquid phase synthesis. They are produced by targeted biotechnological processes or by controlled hydrolysis of proteins of animal, fungal, or vegetable origin.
  • a method for obtaining a compound represented by general formula (I) comprises the following steps:
  • the C-terminus is bound to a solid support and the method is carried out on a solid phase, comprising 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 C-terminus bound to a polymer support; eliminating the group protecting the N-terminus; and repeating this sequence as many times as required so as to thereby obtain a peptide compound having a desired length, followed by cleavage of the synthesized peptide compound from the initial polymer support.
  • the functional groups of the amino acid side chains remain fully protected with temporary or permanent protecting groups throughout the synthesis and can be deprotected simultaneously or orthogonally to the process of cleavage of the peptide from the polymer support.
  • the method may include the following additional steps: deprotection of the N-terminus and C-terminus and/or cleavage of the peptide from the polymer support in an undefined order using standard conditions and methods known in the art, and the functional groups at the terminals may then be modified.
  • the optional modification of the N-terminus and C-terminus may be performed on the peptide compound of general formula (I) bound to the polymer support, or after the peptide has been cleaved from the polymer support.
  • the compound represented by the above general formula (I) or its salt can be incorporated into a cosmetically or pharmaceutically acceptable delivery system or sustained-release system to achieve better penetration of the active ingredient and/or improve its pharmacokinetic and pharmacodynamic properties.
  • the compound represented by the above general formula (I) or its salt or the above composition can be applied to the skin and/or mucous membrane, or orally or parenterally as needed to treat and/or care for a condition, disorder and/or disease.
  • the frequency of administration or dosing can vary widely, depending on the needs of each subject, with suggested administration or dosing ranging from once a month to 10 times a day, preferably from once a week to 4 times a day, more preferably from 3 times a week to 3 times a day, and even more preferably once or twice a day.
  • delivery system refers to a diluent, adjuvant, excipient or carrier administered with the compounds of the invention, selected from: water, oil or surfactant, including those of petroleum origin, animal origin, plant origin, or synthetic origin, such as and not limited to peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glucosides, maltosides, fatty alcohols, nonoxynol ethers, poloxamers, polyoxyethylenes, polyethylene glycols, dextrose, glycerol, digitonin and the like. It is known to those of ordinary skill in the art that in the delivery system, the compound of the invention can be administered. Diluents that can be used in various delivery systems for the compounds of the present invention.
  • sustained release is used in its conventional sense to refer to a delivery system that provides for gradual release of a compound over a period of time, and preferably, but not necessarily, with a relatively constant level of compound released over the entire period of time.
  • Examples of delivery systems or sustained release systems are liposomes, oleosomes, nonionic surfactant liposome vesicles, ethosomes, millicapsules, microcapsules, nanocapsules, nanostructured lipid carriers, sponges, cyclodextrins, lipid vesicles, micelles, millispheres, microspheres, nanospheres, lipid spheres, microemulsions, nanoemulsions, milliparticles, microparticles or nanoparticles.
  • Preferred delivery systems or sustained release systems are liposomes and microemulsions, more preferably water-in-oil microemulsions with an internal structure of reverse micelles.
  • Sustained release system can be prepared by methods known in the prior art, and can be given, for example, in the following manner: by topical or transdermal administration, including adhesive patch, non-adhesive patch, closure patch and microelectronic patch; or by systemic administration, for example and not limited to, oral or parenteral route, including nose, rectum, subcutaneous implantation or injection or directly implanted or injected into a specific body part, and preferably these compounds of the present invention of a relatively constant amount should be released.
  • the amount of the compound included in the sustained release system will depend on the release kinetics and duration of the position, compound of the present invention, and the condition to be treated and/or the property of care, illness and/or disease, at which the composition will be given, for example.
  • the peptide derivative represented by the general formula (I) or its salt may also be adsorbed on a solid organic polymer or a solid inorganic support, such as but not limited to talc, bentonite, silicon dioxide, starch, or maltodextrin.
  • a solid organic polymer or a solid inorganic support such as but not limited to talc, bentonite, silicon dioxide, starch, or maltodextrin.
  • the composition is a cosmetic composition or a pharmaceutical composition.
  • composition is formulated to be selected from the group consisting of cream, oil, balm, foam, lotion, gel, liniment, slurry, ointment, mousse, powder, stick, pen, spray, aerosol, capsule, tablet, granule, chewing gum, solution, suspension, emulsion, elixir, polysaccharide film, jelly or gelatin.
  • the composition further comprises at least one other active agent for enhancing the skin aging delay and repair effect of the present invention, wherein the other active agent is selected from one or more of peptides, natural plant ingredients, vitamin C and its derivatives, and retinoids.
  • the other active agent is selected from one or more of peptides, natural plant ingredients, vitamin C and its derivatives, and retinoids.
  • the peptide compounds of the present invention have variable solubility in water, depending on the nature of their sequence or any possible modifications in the N-terminus and/or C-terminus.
  • the peptide compounds of the present invention can therefore be incorporated into the composition via an aqueous solution, and those that are insoluble in water can be dissolved in conventional cosmetically or pharmaceutically acceptable solvents, such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol or polyethylene glycol or any combination thereof.
  • the term “skin” is understood to mean the multiple layers that make it up, from the uppermost layer or stratum corneum to the lowermost layer or epidermis. The lower tissues, both ends are included. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and/or adipocytes, etc. In the present invention, the term “skin” includes the scalp.
  • treatment refers to administering a peptide compound according to the present invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with the disease or condition.
  • treatment also encompasses the ability to alleviate or eliminate the physiological consequences of the disease or condition.
  • the term "care” includes the prevention of diseases and/or conditions.
  • prevent refers to the ability of the peptide compounds of the present invention to prevent, delay, or hinder the onset or development of a disease or condition before it occurs.
  • repair refers to the ability of the peptide compounds of the present invention to improve, alleviate or restore a disease or condition to its original state after it occurs.
  • photoaging refers to the premature aging of the skin due to long-term exposure to ultraviolet radiation, which presents the same physiological characteristics as natural aging, such as but not limited to: loosening, sagging, color changes or irregular pigmentation, abnormalities and/or excessive keratinization.
  • amino acid abbreviations used in the present invention follow the rules specified by the IUPAC-IUB Biochemical Nomenclature Commission in the European Journal of Biochemistry (Eur. J. Biochem. 1984, 138: 9-37).
  • Val represents NH2 -CH(CH( CH3 ) 2 )-COOH
  • Val- represents NH2 -CH(CH( CH3 ) 2 )-CO-
  • -Val represents -NH-CH(CH( CH3 ) 2 )-COOH
  • -Val- represents -NH-CH(CH( CH3 ) 2 )-CO-.
  • Ac- is used in the present invention to represent an acetyl group (CH 3 -CO-)
  • Palm- is used in the present invention to represent a palmitoyl group
  • Myr- is used in the present invention to represent a myristoyl group.
  • the compound described in the present invention is obtained through artificial design, is easy to synthesize, is safe and non-irritating to the human body, and can be applied to the field of cosmetics.
  • the compounds described in the present invention can increase fibroblast activity, promote keratinocyte proliferation and migration, promote skin or mucosal re-epithelialization or healing, repair skin barrier, prevent or repair photoaging damage, promote collagen production, increase skin elasticity or improve skin firmness, thereby having a good skin delaying and repairing effect, and can be used in skin delaying and repairing products.
  • FIG1 is a mass spectrum of compound (9) H-Tyr-Ser-Leu-OH (molecular formula C 18 H 27 N 3 O 6 ).
  • the mass-to-charge ratio (m/z) of the [M+H] + quasi-molecular ion peak is 382.2392, and the molecular weight measured by mass spectrometry is 381.24.
  • FIG2 is a mass spectrum of compound (13) Palm-Tyr-Ser-Leu-OH (molecular formula C 34 H 57 N 3 O 7 ), the mass-to-charge ratio (m/z) of the [M+Na] + adduct ion peak is 642.4174, and the molecular weight measured by mass spectrometry is 619.42.
  • FIG3 is a mass spectrum of compound (15) Myr-Tyr-Ser-Leu-OH (molecular formula C 32 H 53 N 3 O 7 ).
  • the mass-to-charge ratio (m/z) of the [M+Na] + adduct ion peak is 614.3734, and the molecular weight measured by mass spectrometry is 591.37.
  • FIG4 is a mass spectrum of compound (17) H-Tyr-Ser-Val-OH (molecular formula C 17 H 25 N 3 O 6 ), the mass-to-charge ratio (m/z) of the [M+H] + quasi-molecular ion peak is 368.2215, and the molecular weight measured by mass spectrometry is 367.22.
  • FIG5 is a mass spectrum of compound (23) Myr-Tyr-Ser-Val-OH (molecular formula C 31 H 51 N 3 O 7 ).
  • the mass-to-charge ratio (m/z) of the [M+Na] + adduct ion peak is 600.3671, and the molecular weight measured by mass spectrometry is 577.37.
  • FIG. 6 is a graph showing the effects of the test samples on the proliferation activity of NIH3T3 cells.
  • FIG. 7 is a graph showing the effects of the test samples on the proliferation activity of HaCaT cells.
  • FIG. 8 is a graph showing the effects of the test samples on HaCaT cell adhesion.
  • FIG. 9 is a diagram showing the repairing effect of the test samples on light-damaged NIH3T3 cells.
  • FIG. 10 is a diagram showing the results of a HaCaT cell scratch test observed under a 100x microscope.
  • FIG. 11 is a graph showing the effect of the test samples on the collagen content.
  • FIG. 12 is a diagram showing the results of the HSF cell collagen fiber experiment observed under a 100x microscope.
  • Wang Resin Wang Resin; DMF: N,N-dimethylformamide; DCM: dichloromethane; DIC: diisopropylcarbodiimide; Ac 2 O: acetic anhydride; DIPEA: diisopropylethylamine; Fmoc: 9-fluorenylmethoxycarbonyl; piperidine: piperidine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; TIS: triisopropylsilane; Palm-OH: palmitic acid; Myr-OH: myristic acid; Ac-: acetyl; Palm-: palmitoyl; Myr-: myristoyl; Tyr: tyrosine; Ser: serine; Leu: leucine; Val: valine; tBu: tert-butyl.
  • the N-terminal Fmoc group was deprotected and 2.6 g of activated Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 0.837 g HOBt and 0.781 mL DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the next amino acid.
  • 3.1 g of activated Fmoc-Tyr(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 0.837 g HOBt and 0.781 mL DIC for 2 h. After the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptide resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 minutes, and the sample was taken for K test, and the color was dark blue.
  • the resin was washed with DMF for 6 times, the solvent was removed, and Tyr(tBu)-Ser(tBu)-Leu-Wang Resin was obtained after shrinkage and drying.
  • the N-terminal Fmoc group was deprotected and 5.3 g of activated Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 2.24 g HOBt and 2.8 mL DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated to couple the next amino acid. In the case of HOBt and 2.8 mL DIC, 6.3 g of Fmoc-Tyr(tBu)-OH was coupled using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptidyl resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 min, and the sample was taken for K test, and the color was dark blue. The resin was washed with DMF for 6 times, and the solvent was removed.
  • Palm-OH was coupled to the peptidyl resin using DMF as solvent.
  • the reaction was continued for 1.5 h.
  • the resin was washed, the solvent was removed, and Palm-Tyr(tBu)-Ser(tBu)-Leu-Wang Resin was obtained after shrinkage and drying.
  • Palm-Tyr(tBu)-Ser(tBu)-Leu-WangResin add it to a round-bottom flask, add the frozen lysate, and stir to react for 2.5 hours. Filter by suction, collect the filtrate and concentrate it to 1/4 of the volume, then add isopropyl ether, stir and centrifuge and wash 6 times until the pH value is 3-4, and vacuum dry to obtain 4.5 g of Palm-Tyr-Ser-Leu-OH crude peptide.
  • the N-terminal Fmoc group was deprotected and 4.9 g of activated Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 2.07 g HOBt and 2.6 mL DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the next amino acid. 5.9 g of Fmoc-Tyr(tBu)-OH was coupled in the presence of 2.07 g HOBt and 2.6 mL DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptidyl resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 min, and the sample was taken for K test, and the color was dark blue. The resin was washed with DMF for 6 times, and the solvent was removed.
  • the N-terminal Fmoc group was deprotected and 2.9 g of activated Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 0.837 g HOBt and 0.781 mL DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the next amino acid.
  • 3.4 g of activated Fmoc-Tyr(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 0.837 g HOBt and 0.781 mL DIC for 2 h. After the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptidyl resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 minutes, and the sample was taken for K inspection, and the color was dark blue.
  • the resin was washed with DMF for 6 times, the solvent was removed, and 3.1g of Tyr(tBu)-Ser(tBu)-Val-WangResin was obtained after shrinkage and drying.
  • the N-terminal Fmoc group was deprotected and 4.9 g of activated Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin using DMF as solvent in the presence of 2.07 g HOBt and 2.6 mL DIC for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated for the next amino acid. 5.9 g of Fmoc-Tyr(tBu)-OH was coupled in the presence of 2.07 g HOBt and 2.6 mL DIC using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was removed.
  • the N-terminal Fmoc group of the peptidyl resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 min, and the sample was taken for K test, and the color was dark blue. The resin was washed with DMF for 6 times, and the solvent was removed.
  • Palm-OH was coupled to the peptidyl resin using DMF as solvent.
  • the reaction was continued for 1.5 h.
  • the resin was washed, the solvent was removed, and the resin was shrunk and dried to obtain 10.8 g of Palm-Tyr(tBu)-Ser(tBu)-Val-Wang Resin.
  • Palm-Tyr(tBu)-Ser(tBu)-Val-Wang Resin add it to a round-bottom flask, add the frozen lysate, and stir to react for 2.5 h. Filter, collect the filtrate and concentrate it to 1/4 of the volume, then add isopropyl ether, stir and centrifuge and wash 6 times until the pH value is 3-4, and vacuum dry to obtain 1.8 g of Palm-Tyr-Ser-Val-OH crude peptide.
  • the N-terminal Fmoc group was deprotected and 4.9 g of activated Fmoc-Ser(tBu)-OH was coupled to the peptidyl resin in the presence of 2.07 g HOBt and 2.6 mL DIC using DMF as solvent for 2 h. The resins were then washed and the deprotection of the Fmoc group was repeated to couple the next amino acid. In the presence of 2.07 g of HOBt and 2.6 mL of DIC, 5.9 g of Fmoc-Tyr(tBu)-OH was coupled using DMF as solvent; after the reaction was complete, the resin was washed and the solvent was pumped off.
  • the N-terminal Fmoc group of the peptidyl resin was deprotected by using 20% piperidine/DMF for two times, each time for 10 min, and the sample was taken for K test, and the color was dark blue. The resin was washed with DMF for 6 times, and the solvent was removed.
  • MTT dimethyl sulfoxide (DMSO), DMEM (deoxynucleotidylserine medium), fetal bovine serum, and PBS.
  • DMSO dimethyl sulfoxide
  • DMEM deoxynucleotidylserine medium
  • fetal bovine serum and PBS.
  • Mouse skin fibroblasts (NIH3T3) were purchased from Shanghai Cell Bank of Type Culture Collection of Chinese Academy of Sciences, and human keratinocytes (HaCaT) were purchased from Kunming Cell Bank of Type Culture Collection of Chinese Academy of Sciences.
  • Test concentrations of the above samples were 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm.
  • Blank control group PBS.
  • NIH3T3 cells and HaCaT keratinocytes in good exponential growth phase were taken, and 0.25% trypsin digestion solution was added to digest the adherent cells to make them fall off, and 1 to 4 ⁇ 10 5 cells/mL were counted to prepare a cell suspension.
  • the cell suspension was inoculated on a 96-well plate at 200 ⁇ L/well and cultured in a constant temperature CO 2 incubator for 24 h.
  • the medium was changed, and samples of the drug-treated group and the control group were added, 20 ⁇ L/well, respectively, and the cells were incubated in a 37°C, 5% CO 2 incubator for 72 h.
  • the MTT assay is a method for detecting cell survival and growth.
  • the measured OD value is proportional to the cell activity.
  • the results of the effects of the test samples on the proliferation activity of NIH3T3 cells are shown in FIG6 .
  • the results show that compared with the blank control group, the drug-treated group had no toxic effects on NIH3T3 cells within the range of 100 ppm, and could also improve their cell activity, showing different effects on promoting the proliferation of NIH3T3 fibroblasts at different concentrations; among them, compound (17) and compound (21) could significantly improve the activity of NIH3T3 fibroblasts within the low concentration range of 12.5-50 ppm, compound (9) was more effective in improving the activity of NIH3T3 fibroblasts at 100 ppm, and compound (17) was the best in improving the activity of NIH3T3 fibroblasts at 25 ppm, significantly promoting the proliferation of NIH3T3 fibroblasts.
  • the results of the effects of the test samples on the proliferation activity of HaCaT cells are shown in FIG7 .
  • the results show that, compared with the blank control group, the drug-treated group had no toxic effect on HaCaT cells within the range of 100 ppm, and could also improve its cell activity, showing different effects on promoting the proliferation of HaCaT keratinocytes at different concentrations; among them, compound (9), compound (17), compound (21) and compound (23) were better at improving the activity of HaCaT keratinocytes and significantly promoted the proliferation of HaCaT keratinocytes; and compound (17) and compound (21) could significantly promote the proliferation of HaCaT keratinocytes at a low concentration of 12.5 ppm.
  • the compounds of the present invention can improve the activity of NIH3T3 fibroblasts and HaCaT keratinocytes and promote their proliferation.
  • compound (9) and compound (17) can significantly improve the activity of fibroblasts and promote the proliferation of keratinocytes;
  • the new structure compounds (13), compound (15), compound (21), and compound (23) obtained by covalently connecting palmitic acid and myristic acid to the N-terminus of compound (9) and compound (17) respectively can also significantly improve cell activity at different concentrations, and the new structure compound (21) obtained by covalently connecting palmitic acid to the N-terminus of compound (17) can improve cell activity and promote cell proliferation at low concentrations.
  • the peptide compounds of the present invention not only have no toxic effect on fibroblasts and keratinocytes, but can also promote cell proliferation, increase fibroblast activity, and promote keratinocyte proliferation, indicating that the peptide compounds of the present invention have a skin repair effect.
  • Fetal bovine serum Fetal bovine serum, DMEM culture medium, penicillin, streptomycin, MTT.
  • HaCaT Human keratinocytes
  • Blank control group PBS.
  • Cryopreserved HaCaT human keratinocytes were cultured and passaged at a ratio of 1:2 until about the fifth generation, and cells with better growth were selected as experimental subjects.
  • the sample to be tested was added to a 96-well plate at 20 ⁇ L/well and dried in a constant temperature oven at 37°C overnight.
  • the HaCaT cells with good growth were digested and plated at a density of 10,000 HaCaT cells/well, and the culture medium was supplemented to 200 ⁇ L, and incubated in a 37°C, 5% CO 2 incubator for 3 hours.
  • the culture plate was removed and the culture medium was continued to be supplemented until the liquid surface just overflowed, and sealed with a sealing film to ensure that there were no bubbles.
  • Discard the original culture medium add 90 ⁇ L of fresh culture medium and 10 ⁇ L of 5 mg/mL MTT to each well, and incubate in a 37°C, 5% CO 2 incubator for 3 hours. Discard the solution and add 150 ⁇ L of DMSO. Use an enzyme reader to read the reference OD values at wavelengths of 490nm and 630nm.
  • HaCaT keratinocytes were selected, plated in a drug-coated 96-well plate, incubated, and after three-dimensional force, cells with strong adhesion could be maintained on the 96-well plate. MTT quantitative analysis of live cells on the plate could reflect the adhesion of the cells, evaluate the effect of the drug on cell adhesion, and determine whether the compound of the present invention can improve cell elasticity. The more live cells maintained on the 96-well plate, the greater the measured OD value, indicating that the cell adhesion is stronger.
  • the results of the effects of the test samples on HaCaT cell adhesion are shown in Figure 8.
  • the drug administration groups can improve the adhesion ability of HaCaT keratinocytes to varying degrees at different concentrations.
  • compound (9), compound (15), compound (17) and compound (23) can significantly improve the adhesion ability of HaCaT keratinocytes, thereby helping to improve cell elasticity.
  • compound (15) can also significantly improve the adhesion ability of cells at a low concentration of 25 ppm.
  • the overall technical effects of compound (9) and compound (17) are better.
  • the peptide compounds of the present invention can improve the adhesion ability of cells, significantly increase intercellular adhesion and cell-extracellular matrix adhesion, thereby increasing skin elasticity and skin firmness, and can be used to prevent or even treat skin sagging.
  • Fetal bovine serum, high glucose medium (DMEM), penicillin, streptomycin, MTT Fetal bovine serum, high glucose medium (DMEM), penicillin, streptomycin, MTT.
  • Dosing group Compound (9), Compound (13), Compound (15), Compound (17), Compound (21), Compound (23), the test concentrations were 12.5 ppm, 25 ppm, 50 ppm, and 100 ppm.
  • Control group PBS.
  • UV group PBS+UV.
  • Appropriate dilution was taken and 10,000 cells/well of cell suspension were inoculated on a 96-well plate. When the cells grew to about 80%, a UV photoaging model was established. The control group was added with 50 ⁇ L PBS, and the culture medium was supplemented to 200 ⁇ L, and no UV irradiation was performed; the UV group and the drug-treated group were added with an appropriate amount of PBS and washed repeatedly until colorless, and then 50 ⁇ L PBS was added. The cells were irradiated under a 80mJ/cm 2 UV lamp, and the distance between the lamp source and the culture bottle was 15 cm.
  • the PBS was discarded, and the UV group was added with PBS solution and culture medium to 200 ⁇ L, and the drug-treated group was added with culture medium and multiple dilution drugs to 200 ⁇ L.
  • the control group, UV group, and drug-treated group continued to be incubated in a 37°C, 5% CO 2 incubator for 24 hours.
  • Skin aging is affected by endogenous and exogenous factors, such as genetics, environmental exposure, ultraviolet radiation, hormonal changes and metabolism. The accumulation of these factors leads to changes in skin structure, function and appearance, especially ultraviolet radiation.
  • 80mJ/ cm2 UV energy was selected for radiation to establish a skin photoaging model.
  • HaCaT Human keratinocytes
  • Blank control group PBS.
  • Positive control group 50 U/mL of EGF.
  • the scratch test was performed on the basis of HaCaT cells growing all over the culture dish, and the cells were observed under an optical microscope 24 hours after administration.
  • the effects of the drugs on cell proliferation and migration are evaluated to determine whether the compounds of the present invention can promote skin or mucosal re-epithelialization or healing.
  • the scratch migration experiment of cells can reflect the proliferation and repair ability of cells.
  • the experimental results are shown in Figure 10.
  • the cell scratches of the blank control group are still obvious, and the scratch spacing is normal; compared with the blank control group, the cells of the EGF positive control group proliferate and migrate significantly, and the spacing basically disappears, forming a continuous piece; the scratch spacing of the compound (9), compound (17) and compound (21) groups is shorter than that of the blank control group, and cell migration tracks appear; among them, the scratch spacing of the compound (9) and compound (17) groups is comparable to the scratch spacing and cell proliferation and migration effects of the EGF positive control group, indicating that the cell repair ability of the compound (9) and compound (17) of the present invention is comparable to the effect of EGF.
  • the peptide compounds of the present invention can promote the proliferation and migration of HaCaT keratinocytes, have a skin repair effect, can repair the skin barrier, and can promote the re-epithelialization or healing of the skin or mucous membranes.
  • HSF Human skin fibroblasts
  • Drug administration group Compound (9) and Compound (17), the test concentrations were 12.5 ppm and 50 ppm;
  • Blank control group PBS
  • UV group UV radiation, plus PBS.
  • HSF cells in the logarithmic growth phase were taken, and after enzymatic dilution, they were inoculated on a 6-well plate at 10 5 cells/well until the cells grew to about 80% to establish a UV photoaging model.
  • the blank control group was added with 200 ⁇ L PBS, and the culture medium was supplemented to 800 ⁇ L, and no UV irradiation was performed; the UV group and the drug-treated group were repeatedly washed with an appropriate amount of PBS until colorless, and then 200 ⁇ L PBS was added, and the cells were placed under an 80mJ/cm 2 UV lamp for irradiation, with a distance of 15 cm between the lamp source and the culture bottle.
  • the PBS was discarded, and the UV group was added with PBS solution and culture medium to 800 ⁇ L, and the drug-treated group was added with culture medium and multiple dilution drugs to 800 ⁇ L.
  • the blank control group, UV group, and drug-treated group continued to be incubated in a 37°C, 5% CO 2 incubator for 48 h.
  • the cells in the first well were digested and counted, and diluted to 0.5 ⁇ 10 6 cells/mL.
  • the cells in the remaining wells were scraped off with a cell scraper, resuspended in 500 ⁇ L, and 50 ⁇ L was taken from all wells for 30 seconds of ultrasound.
  • the total protein was determined using the BCA method.
  • the other wells were diluted according to the protein concentration of the first well to make the concentration of all cell suspensions 0.5 ⁇ 10 6 cells/mL.
  • the cell suspension after the concentration was adjusted was ultrasonically disrupted for 30 seconds, centrifuged at 1500xg for 15 minutes, and the cell supernatant was collected to obtain the sample solution, and the operation was carried out according to the collagen I ELISA operating instructions.
  • the OD value of each well was measured in sequence at 450nm using an enzyme reader within 15 minutes.
  • Collagen is the most abundant protein found in connective tissue. Collagenase is synthesized and secreted by fibroblasts, which can degrade collagen in the skin and cause skin aging. Therefore, inhibiting the expression of collagenase in cells and increasing the collagen content are important for preventing aging and increasing the fullness and firmness of the skin. In an environment with excessive ultraviolet rays, the activity of collagenase and elastase increases significantly, elastin is hydrolyzed, and the synthesis of collagen is also inhibited. This experiment uses test samples to treat cells irradiated with ultraviolet rays, and detects the content of collagen I in the corresponding cells to determine whether the peptide compounds of the present invention can promote the synthesis of collagen.
  • the peptide compounds of the present invention can promote collagen production, increase collagen content, thereby increasing skin elasticity and/or skin firmness, and can be used to prevent or even treat skin sagging, treat, prevent or repair skin aging or photoaging, and resist skin aging.
  • Fetal bovine serum, DMEM culture medium, phosphate buffer, trypsin, and biological Sirius red staining kit Fetal bovine serum, DMEM culture medium, phosphate buffer, trypsin, and biological Sirius red staining kit.
  • HSF Human skin fibroblasts
  • Drug administration group Compound (9) and Compound (17), the test concentrations were 12.5 ppm and 50 ppm;
  • Blank control group PBS
  • UV group UV radiation, plus PBS.
  • HSF cells in the logarithmic growth phase were taken, and after enzymatic dilution, they were inoculated on a 6-well plate at 10 5 cells/well until the cells grew to about 80% to establish a UV photoaging model.
  • the blank control group was added with 200 ⁇ L PBS, and the culture medium was supplemented to 800 ⁇ L, and no UV irradiation was performed; the UV group and the drug-treated group were repeatedly washed with an appropriate amount of PBS until colorless, and then 200 ⁇ L PBS was added, and they were placed under an 80mJ/cm 2 UV lamp for irradiation, with a distance of 15 cm between the lamp source and the culture bottle.
  • the PBS was discarded, and the UV group was added with PBS solution and culture medium to 800 ⁇ L, and the drug-treated group was added with culture medium and multiple dilution drugs to 800 ⁇ L.
  • the blank control group, UV group, and drug-treated group continued to be incubated in a 37°C, 5% CO 2 incubator for 48 h.
  • the cells were fixed with 4% paraformaldehyde for 15 min, stained according to the instructions of the Sirius red staining kit, and observed under an optical microscope.
  • test sample is used to treat cells irradiated with ultraviolet light, the density of the corresponding cells and the area of collagen fibers in the same unit are detected, and the collagen fibers in the cells are qualitatively analyzed to determine whether the peptide compounds of the present invention can promote collagen expression.
  • the collagen fiber experiment of cells can reflect the collagen expression ability of cells.
  • the experimental results are shown in Figure 12.
  • the cell density of the UV group was reduced, and the collagen fiber area in the same unit was also reduced;
  • compounds (9) and (17) in the range of 12.5-50ppm were able to increase the cell density, increase the collagen fiber area in the same unit, promote collagen expression, and improve the symptoms of reduced collagen content in cells caused by UV irradiation.
  • the peptide compounds of the present invention can promote collagen expression, thereby increasing skin elasticity and/or skin firmness, and can be used to prevent or even treat skin sagging, treat, prevent or repair skin aging or photoaging, and resist skin aging.
  • Preparation method Weigh phosphatidylcholine and dissolve it in chloroform. Evaporate the solvent under vacuum until a thin layer of phospholipids is obtained. This layer is treated with an aqueous solution of the compound at the desired concentration at 55°C to hydrate and obtain multilamellar liposomes. The multilamellar liposomes are treated by high-pressure homogenization to obtain smaller and more uniform unilamellar liposomes.
  • Example 15 Microemulsion composition containing compound (13)
  • Preparation method According to the prescribed amount, weigh the ingredients of phase B and add them to a container. Next, add phase D to phase B and homogenize under continuous stirring. Then add phase A to the mixture. Finally, add phase C and stir well.
  • Preparation method Stir and heat purified water to 85°C and keep warm for 30 minutes; pre-dissolve sodium hyaluronate and xanthan gum in butylene glycol, add to water, and stir to dissolve completely; stir and cool to 35°C, add remaining ingredients, and stir evenly.
  • Preparation method dissolve allantoin and glycerol in water, heat to 85°C, and keep warm for 30 minutes; dissolve PEG-7 glyceryl cocoate and compound (21) in water; mix the above solutions after cooling, stir evenly to obtain a mixed solution; add propylene glycol, preservatives, and flavors to the above mixed solution in sequence, add water and stir evenly to obtain.

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Abstract

通式(I)的化合物或其盐在制备用于皮肤延衰修复的组合物中的用途，所述皮肤延衰修复包括提高成纤维细胞活性、促进角质形成细胞增殖和迁移、促进皮肤或粘膜的再上皮化或愈合、修复皮肤屏障、预防或修复光老化损伤、促进胶原蛋白生成、增加皮肤弹性或提高皮肤紧致度中的一种或多种。

Description

肽类化合物在制备用于皮肤延衰修复的组合物中的新用途 技术领域
本发明属于活性多肽技术领域，具体涉及肽类化合物在制备用于皮肤延衰修复的组合物中的新用途。
背景技术
皮肤是人体最大和最重要的器官，覆盖于整个人体的表面，由表皮、真皮和皮下组织构成，其中表皮层和真皮层发挥着重要的作用，例如防止水分流失、形成防护屏障和提供结构支撑等。然而，随着时间推移，受年龄增长、内外界环境等多方面因素的影响，皮肤表皮层和真皮层的表达水平也会发生变化，如经历长时间的暴晒或受紫外线辐射后，表皮层角质形成细胞的增殖速度和分化发生改变，参与基底层细胞黏附的分子表达下降，皮肤屏障功能减弱；真皮层细胞外基质的构成也随老化的进程发生退行性改变，胶原蛋白大量流失，成纤维细胞活性减弱，合成的胶原蛋白也逐渐减少，胶原纤维面积也降低，从而导致皮肤真皮层网状结构疏松，皮肤组织的保湿度和强韧性降低，皮肤随即出现干燥、粗糙、皱纹、松弛、损伤等问题。
在皮肤自然老化过程中，成纤维细胞功能下降，胶原纤维和弹性纤维受损、断裂，皮肤中胶原等重要生物分子的产生和降解之间的平衡随着年龄而偏向于降解过程，从而导致真皮层进行性地变薄和组织破坏，引起真皮松弛，随后形成皱纹；而且皮肤光老化时基质金属蛋白酶分泌增加，从而加速胶原蛋白降解，造成皮肤胶原蛋白流失，导致真皮完整性被破坏，皮肤强度和弹性降低，甚至出现色斑；此外，皮肤老化时胶原蛋白、弹性蛋白、纤连蛋白的减少亦会导致细胞外基质减少，不利于皮肤修复，然而修复是一个精确复杂、分时相的动态过程，涉及细胞增殖、迁移、胞外基质降解、血管新生和上皮组织重塑等多种细胞的网络调控。故针对皮肤衰老及修复的作用机制寻找具有延衰修复作用的化合物具有重要意义。
近年来，为了修复皮肤以及延缓皮肤衰老，越来越多的功能性护肤产品应运而生。然而，市售的延衰修复护肤品大多数都是通过简单添加外源性活性物(如保湿剂、自由基清除剂、防晒剂等)来改善皮肤衰老的状态，真正的抗衰老效果并不尽如人意。此外，该类护肤品的成分在很大程度上只能被皮肤表层吸收，并不能真正进入皮肤的深层，以达到持久更新及补充胶原蛋白的目的，因此无法较好地发挥延衰修复的作用。目前，人们已经发现了几种用于延衰修复的方法，例如，通过使用视黄醇促进胶原蛋白生成和增加皮肤弹性，并刺激细胞更新、调节表皮及角质层新陈代谢以达到延衰修复的效果，但大量长期使用易出现皮肤黏膜干 燥、脱皮、瘙痒、损伤、对光敏感等副作用，还可能使皮肤变得敏感而薄弱，刺激皮肤黑色素细胞的分泌，导致皮肤变黑。目前市场尚缺少能够安全高效发挥延衰修复作用的化合物。
因此，仍然需要开发更多安全高效的化合物来促进细胞增殖、提高细胞活性、促进胶原蛋白、弹性蛋白等成分的合成和分泌，从而改善随着老化或暴露于阳光和/或暴露于环境污染物而损失的柔韧性、弹性和紧实度等皮肤机械性质，修复受损皮肤，以满足消费者日益增长的治疗或护理皮肤的需求。
发明内容
本发明人通过大量实验研究，结果发现，一种肽类化合物具有延衰修复皮肤的功效，由此完成了本发明。
因此，本发明旨在提供一种肽类化合物或其盐在制备用于皮肤延衰修复的组合物中的用途，所述皮肤延衰修复包括提高成纤维细胞活性、促进角质形成细胞增殖和迁移、促进皮肤或粘膜的再上皮化或愈合、修复皮肤屏障、预防或修复光老化损伤、促进胶原蛋白生成、增加皮肤弹性或提高皮肤紧致度中的一种或多种。
本发明所述肽类化合物或其盐具有以下通式(I)，
通式(I)中，
R1选自：H或R3-CO-，其中R3选自：取代的或未取代的烷基、取代的或未取代的烯基；
R2选自：-OH、-NH2、-Leu-NH2、-Leu-OH、-Val-NH2或-Val-OH；
所述烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
所述烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子；可选具有2、3、4、5、或6个碳原子)的直链或支链烯基；所述烯基具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述烯 基是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C1-C4烷基；羟基；C1-C4烷氧基；氨基；C1-C4氨基烷基；C1-C4羰氧基；C1-C4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C1-C4烷基磺酰基；硫醇；C1-C4烷硫基；C6-C30芳氧基如苯氧基；-NRb(C＝NRb)NRbRc，其中Rb和Rc是独立地选自：H、C1-C4烷基、C2-C4烯基、C2-C4炔基、C3-C10环烷基、C6-C18芳基、C7-C17芳烷基、具有三至十元的杂环基、或氨基的保护基。
可选地，R1选自：H、乙酰基、叔-丁酰基、己酰基、2-甲基己酰基、辛酰基、癸酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基、亚油酰基或苯乙酰基；
可选地，R1为H、肉豆蔻酰基或棕榈酰基。
可选地，所述化合物选自(1)-(24)：
(1)H-Tyr-Ser-OH；
(2)H-Tyr-Ser-NH2；
(3)Ac-Tyr-Ser-OH；
(4)Ac-Tyr-Ser-NH2；
(5)Palm-Tyr-Ser-OH；
(6)Palm-Tyr-Ser-NH2；
(7)Myr-Tyr-Ser-OH；
(8)Myr-Tyr-Ser-NH2；
(9)H-Tyr-Ser-Leu-OH；
(10)H-Tyr-Ser-Leu-NH2；
(11)Ac-Tyr-Ser-Leu-OH；
(12)Ac-Tyr-Ser-Leu-NH2；
(13)Palm-Tyr-Ser-Leu-OH；
(14)Palm-Tyr-Ser-Leu-NH2；
(15)Myr-Tyr-Ser-Leu-OH；
(16)Myr-Tyr-Ser-Leu-NH2；
(17)H-Tyr-Ser-Val-OH；
(18)H-Tyr-Ser-Val-NH2；
(19)Ac-Tyr-Ser-Val-OH；
(20)Ac-Tyr-Ser-Val-NH2；
(21)Palm-Tyr-Ser-Val-OH；
(22)Palm-Tyr-Ser-Val-NH2；
(23)Myr-Tyr-Ser-Val-OH；
(24)Myr-Tyr-Ser-Val-NH2。
可选地，选自化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)和化合物(23)；具体地，
(9)H-Tyr-Ser-Leu-OH；
(13)Palm-Tyr-Ser-Leu-OH；
(15)Myr-Tyr-Ser-Leu-OH；
(17)H-Tyr-Ser-Val-OH；
(21)Palm-Tyr-Ser-Val-OH；
(23)Myr-Tyr-Ser-Val-OH。
本发明的这些肽类化合物可以作为立体异构体或立体异构体的混合物存在；例如，包含它们的这些氨基酸可以具有L-、D-的构型、或彼此独立地是外消旋的。因此，有可能获得同分异构混合物以及外消旋混合物或非对映混合物、或纯的非对映异构体或对映异构体，这取决于不对称碳的数量和存在什么同分异构体或同分异构混合物。本发明的这些肽类化合物的优选的结构是纯的同分异构体，即，对映异构体或非对映异构体。
例如，当本发明所述-Ser-时，应理解-Ser-选自-L-Ser-、-D-Ser-、或两者的混合物，是外消旋的或非外消旋的。在本文件中描述的制备方法使本领域的普通技术人员能够通过选择具有正确构型的氨基酸来获得本发明的肽类化合物的每种立体异构体。
本发明还包括通式(I)所示的肽类化合物的所有合适的同位素变体。本发明的这些肽类化合物的同位素变体此处理解为是指这样的化合物：其中在本发明的肽类化合物内至少一个原子被替换为相同原子序数的另一个原子，但所述另一原子的原子质量不同于自然界中通常或主要存在的原子质量。可掺入本发明的肽类化合物中的同位素的实例是：氢、碳、氮或氧的那些，例如2H(氘)、3H(氚)、13C、14C、15N、17O或18O。本发明的肽类化合物的特定的同位素变体(特别是其中已经掺入一种或多种放射性同位素的那些)可能有利于，例如检查在体内的作用机理或活性化合物的分布；由于相对简单的可制备性和可检测性，尤其是用 3H或14C同位素标记的化合物适用于该目的。另外，由于化合物的更强的代谢稳定性，同位素(例如氘)的掺入可以产生特定的治疗益处，例如体内半衰期的延长或所需活性剂量的降低；因此，在某些情况下，本发明的肽类化合物的这种改性还可构成本发明的优选实施方案。通过本领域技术人员已知的方法，例如通过在下文中进一步描述的方法和在实施例中所述的方法，通过使用各自的试剂和/或起始物质的相应的同位素改性物，可制备本发明的肽类化合物的同位素变体。
此外，本发明还包括本发明的肽类化合物的前药。术语“前药”在本文中意指这样的化合物：其本身可以是生物学上有活性的或无活性的，但是在它们在身体内的停留时间期间，其反应(例如代谢或水解)生成本发明的化合物。
可选地，所述组合物包含质量百分浓度为0.0001％-5％的通式(I)所示的化合物或其盐；
可选地，所述组合物包含质量百分浓度为0.0005％-1％的通式(I)所示的化合物或其盐；
可选地，所述组合物包含质量百分浓度为0.001％-0.1％的通式(I)所示的化合物或其盐；
可选地，所述组合物包含质量百分浓度为0.005％-0.01％的通式(I)所示的化合物或其盐。
上述通式(I)所示的化合物，能够提高成纤维细胞活性、促进角质形成细胞增殖和迁移、促进皮肤或粘膜再上皮化或愈合、修复皮肤屏障、预防或修复光老化损伤、促进胶原蛋白生成、增加皮肤弹性或提高皮肤紧致度。
可选地，所述通式(I)所示的化合物的盐包括通式(I)所示的化合物的金属盐，所述金属包括：锂、钠、钾、钙、镁、锰、铜、锌或铝；
可选地，所述通式(I)所示的化合物的盐包括通式(I)所示的化合物与有机碱形成的盐，所述有机碱包括：乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨酸或哌嗪；
可选地，所述通式(I)所示的化合物的盐包括通式(I)所示的化合物与无机酸或有机酸形成的盐，所述有机酸包括：乙酸、柠檬酸、乳酸、丙二酸、马来酸、酒石酸、延胡索酸、苯甲酸、天冬氨酸、谷氨酸、琥珀酸、油酸、三氟乙酸、草酸、扑酸或葡萄糖酸；
可选地，所述无机酸包括：盐酸、硫酸、硼酸或碳酸。
本发明上述通式(I)所示的化合物或其盐，可以根据现有技术中已知的常规方法来进行，例如固相合成法、液相合成法或固相与液相结合的方法，还可以通过以产生所希望的序 列为目标的生物技术方法、或通过具有动物、真菌、或植物来源的蛋白质的控制水解来制备。
例如，一种获得通式(I)所示的化合物的方法包括以下步骤：
-将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和受保护的或与固体载体结合的C-末端的氨基酸偶联；
-消除保护N-末端的基团；
-重复该偶联顺序和消除保护N-末端的基团，直到获得所希望的肽序列；
-消除保护C-末端的基团或从该固体载体裂解。
优选地，C-末端与一种固体载体结合并且该方法是在固相上进行，包括将具有受保护的N-末端和自由的C-末端的氨基酸与具有自由的N-末端和与一种聚合物载体结合的C-末端的氨基酸偶联；消除保护N-末端的基团；并且重复此顺序所需要的次数以便因此获得具有所希望的长度的肽类化合物，接着从最初的聚合物载体裂解所合成的肽类化合物。
在整个合成中这些氨基酸的侧链的官能团用临时或永久的保护基团保持充分地保护，并且可以与从该聚合物载体裂解肽的过程同时地或正交地脱保护。
该方法可以包括如下另外的步骤：使用本领域已知的标准条件和方法对N-末端和C-末端脱保护和/或以非确定的次序从聚合物支持体裂解肽，随后可以修饰所述末端的官能团。可以对与聚合物支持体结合的通式(I)的肽类化合物进行N-末端和C-末端的任选的修饰，或在肽已从聚合物支持体裂解后进行N-末端和C-末端的任选的修饰。
上述通式(I)所示的化合物或其盐，可以掺入到美容上或药学上可接受的递送系统或缓释系统中，以便实现有效成分的更好渗透和/或改进它的药物代谢动力学和药效动力学特性，上述通式(I)所示的化合物或其盐或上述组合物可以被施用至皮肤和/或粘膜、或根据需要被口服地或胃肠外给予以便治疗和/或护理一种病况、病症和/或疾病。
施用或给药频率可以变化很大，这取决于每个受试者的需要，其中建议的施用或给药范围从每月1次至每天10次，优选从每周1次至每天4次，更优选从每周3次至每天3次，甚至更优选每天1次或2次。
术语“递送系统”是指与本发明的化合物一起施用的稀释剂、佐剂、赋形剂或载体，它们选自：水、油或表面活性剂、包括石油来源、动物来源、植物来源、或合成来源的那些，例如并且不限于花生油、大豆油、矿物油、芝麻油、蓖麻油、聚山梨醇酯、脱水山梨糖醇酯、醚硫酸酯、硫酸酯、甜菜碱、葡萄糖苷、麦芽糖苷、脂肪醇、壬苯醇醚、泊洛沙姆、聚氧乙烯、聚乙二醇、右旋糖、甘油、毛地黄皂苷和类似物。本领域的普通技术人员已知在可以给 予本发明的化合物的不同递送系统中可以使用的稀释剂。
术语“缓释”以常规含义使用，指提供化合物在一段时间内逐渐释放的化合物的递送系统，且优选地但不是必须地，在整个时间段内具有相对恒定的化合物释放水平。
递送系统或缓释系统的实例是脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子或纳米粒子。优选的递送系统或缓释系统是脂质体和微米乳液，更优选具有反胶束的内部结构的油包水型微米乳液。
缓释系统可以通过现有技术中已知的方法来制备，并且可以例如通过以下方式来给予：通过局部或经皮给药，包括粘附贴剂、非粘附贴剂、封闭贴剂、以及微电子贴剂；或通过全身给药例如并且不局限于，口服或胃肠外途径，包括鼻、直肠、皮下植入或注射、或直接植入或注射至特定身体部位中，并且优选地应该释放相对恒定量的本发明的这些化合物。在该缓释系统中包含的化合物的量将取决于例如该组合物将被给予的部位、本发明的化合物的释放动力学和持续时间、以及有待治疗和/或护理的病状、病症和/或疾病的性质。
上述通式(I)所示的肽衍生物或其盐，还可以吸附在固体有机聚合物或固体无机支撑体上，例如并且不局限于，滑石、膨润土、二氧化硅、淀粉、或麦芽糖糊精等。
可选地，所述组合物是美容组合物或药物组合物。
所述组合物的制剂选自：霜剂、油、香膏、泡沫、洗剂、凝胶、擦剂、浆液、软膏、摩丝、粉末、杆剂、笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、酏剂、多糖薄膜、胶冻或明胶。
可选地，所述组合物还包含至少一种用于增强本发明所述皮肤延衰修复作用的其他活性剂，所述其他活性剂选自肽类、天然植物成分、维生素C及其衍生物和类视黄醇中的一种或多种。
本发明的肽类化合物根据它们的序列的性质或N-末端和/或C-末端中的任何可能的修饰，在水中具有可变的溶解度。因此本发明的肽类化合物可以通过水溶液掺入组合物中，且不溶于水的那些可溶解于美容上或药学上可接受的常规溶剂中，所述溶剂例如并且不限于乙醇、丙醇、异丙醇、丙二醇、甘油、丁二醇或聚乙二醇或其任何组合。
为了便于理解本发明，对在本发明所使用的一些术语和表述的含义说明如下：
在本发明中，术语“皮肤”应理解为是构成它的多个层，从最上层或角质层至最下层或皮 下组织，两个端点都包括在内。这些层由不同类型的细胞组成，如角质形成细胞、成纤维细胞、黑色素细胞、和/或脂肪细胞等。在本发明中，术语“皮肤”包括头皮。
术语“治疗”，指的是给予根据本发明的肽类化合物以减轻或消除一种疾病或病症、或减少或消除与这种疾病或病症相关的一种或多种症状。术语“治疗”还涵盖了减轻或消除该疾病或病症的生理后果的能力。
术语“护理”包括疾病和/或病症的预防。
术语“预防”，指的是本发明的肽类化合物在一种疾病或病症出现前防止、延迟、或阻碍其出现或发展的能力。
术语“修复”，指的是本发明的肽类化合物在一种疾病或病症出现后改善、缓解或恢复其原状的能力。
术语“光老化”指的是由于皮肤长期暴露于紫外线辐射而导致的皮肤过早老化，它呈现出与自然老化相同的生理特征，例如并且不局限于：松弛、下垂、颜色改变或色素沉着不规则、异常和/或过度角质化。
本发明中所使用的氨基酸缩写遵循IUPAC-IUB生化命名委员会在欧洲生物化学杂志(Eur.J.Biochem.1984,138:9-37)中所指定的规则。
因此，例如，Val表示NH2-CH(CH(CH3)2)-COOH，Val-表示NH2-CH(CH(CH3)2)-CO-，-Val表示-NH-CH(CH(CH3)2)-COOH，并且-Val-表示-NH-CH(CH(CH3)2)-CO-。因此，表示肽键的连字符消除了当位于该符号的右侧时的氨基酸(在此用常规非离子化形式来表示)1-羧基中的OH，并且消除了当位于该符号的左侧时的氨基酸2-氨基中的H；两种修饰可以应用于同一个符号(见表1)。
表1氨基酸残基的结构以及它们的单字母和三字母缩写符号

缩写“Ac-”在本发明中用来表示乙酰基(CH3-CO-)，缩写“Palm-”在本发明中用来表示棕榈酰基，缩写“Myr-”在本发明中用来表示肉豆蔻酰基。
本发明相对于现有技术所取得的有益效果包括：
1、本发明所述的化合物通过人工设计得到，合成方便，且对人体安全无刺激，可以应用于化妆品领域。
2、本发明所述的化合物能够提高成纤维细胞活性、促进角质形成细胞增殖和迁移、促进皮肤或粘膜再上皮化或愈合、修复皮肤屏障、预防或修复光老化损伤、促进胶原蛋白生成、增加皮肤弹性或提高皮肤紧致度，从而具有良好的皮肤延衰修复效果，可应用于皮肤延衰修复的产品中。
附图说明
图1是化合物(9)H-Tyr-Ser-Leu-OH(分子式C18H27N3O6)质谱图，[M+H]+准分子离子峰的质荷比(m/z)为382.2392，质谱测得的分子量为381.24。
图2是化合物(13)Palm-Tyr-Ser-Leu-OH(分子式C34H57N3O7)质谱图，[M+Na]+加和离子峰的质荷比(m/z)为642.4174，质谱测得的分子量为619.42。
图3是化合物(15)Myr-Tyr-Ser-Leu-OH(分子式C32H53N3O7)质谱图，[M+Na]+加和离子峰的质荷比(m/z)为614.3734，质谱测得的分子量为591.37。
图4是化合物(17)H-Tyr-Ser-Val-OH(分子式C17H25N3O6)质谱图，[M+H]+准分子离子峰的质荷比(m/z)为368.2215，质谱测得的分子量为367.22。
图5是化合物(23)Myr-Tyr-Ser-Val-OH(分子式C31H51N3O7)质谱图，[M+Na]+加和离子峰的质荷比(m/z)为600.3671，质谱测得的分子量为577.37。
图6是测试样品对NIH3T3细胞增殖活性的影响图。
图7是测试样品对HaCaT细胞增殖活性的影响图。
图8是测试样品对HaCaT细胞黏附的影响图。
图9是测试样品对光损伤NIH3T3细胞的修复作用图。
图10是100倍镜下观察的HaCaT细胞划痕实验结果图。
图11是测试样品对胶原蛋白含量的影响结果图。
图12是100倍镜下观察的HSF细胞胶原纤维实验结果图。
具体实施方式
为了更好地理解本发明，下面结合实施例及附图对发明作详细的说明，然而，应当理解的是，这些实施例及附图仅用作说明目的，并且不旨在限制本发明的范围。
缩写
用于氨基酸的缩写遵循IUPAC-IUB的生物化学命名委员会在Eur J.Biochem.(1984)138：9-37和J.Chem(1989)264：633-673中指定的规则。
Wang Resin：王树脂；DMF：N,N-二甲基甲酰胺；DCM：二氯甲烷；DIC：二异丙基碳二亚胺；Ac2O：乙酸酐；DIPEA：二异丙基乙胺；Fmoc：9-芴基甲氧羰基；piperidine：哌啶；HOBt：1-羟基苯并三氮唑；TFA：三氟乙酸；TIS：三异丙基硅烷；Palm-OH：棕榈酸；Myr-OH：肉豆蔻酸；Ac-：乙酰基；Palm-：棕榈酰基；Myr-：肉豆蔻酰基；Tyr：酪氨酸；Ser：丝氨酸；Leu：亮氨酸；Val：缬氨酸；tBu：叔丁基。
实施例1 H-Tyr-Ser-Leu-OH的制备
1.1树脂的溶胀
称取5g的Wang树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
1.2投料反应
称取6.1g的Fmoc-Leu-OH，2.322g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min，加2.7mL DIC活化3min，避免水汽。将活化后的氨基酸加入溶胀后的树脂中反应3.5h，抽走反应液，继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在0.837gHOBt和0.781mL DIC的情况下，使用DMF作为溶剂，将活化后的2.6g的Fmoc-Ser(tBu)-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在0.837g HOBt和0.781mL DIC的情况下，使用DMF作为溶剂，将活化后的3.1g的Fmoc-Tyr(tBu)-OH偶联至肽基树脂上，持续反应2h；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂，收缩干燥后得到Tyr(tBu)-Ser(tBu)-Leu-Wang Resin。
1.3裂解
量取14.25mL的TFA、0.375mL的TIS和0.375mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取5g的Tyr(tBu)-Ser(tBu)-Leu-Wang Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩到体积的1/4后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到3.3g的H-Tyr-Ser-Leu-OH粗肽。
1.4纯化
称取3.3g粗肽溶于50mL纯水中，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，pH值为5，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度98.35％的化合物(9)H-Tyr-Ser-Leu-OH。
实施例2 Palm-Tyr-Ser-Leu-OH的制备
2.1树脂的溶胀
称取9g的Wang树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
2.2投料反应
称取10.8g的Fmoc-Leu-OH，4.95g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min，加6.24mLDIC活化3min，避免水汽。将活化后的氨基酸加入溶胀后的树脂中反应3.5h，抽走反应液，继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在2.24g HOBt和2.8mL DIC的情况下，使用DMF作为溶剂，将活化后的5.3g的Fmoc-Ser(tBu)-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在2.24g  HOBt和2.8mLDIC的情况下，使用DMF作为溶剂，偶联6.3g的Fmoc-Tyr(tBu)-OH；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂。
在存在DIPEA的情况下，使用DMF作为溶剂，将5.6g的Palm-OH偶联至肽基树脂上，持续反应1.5h，洗涤树脂，抽走溶剂，收缩干燥后得到Palm-Tyr(tBu)-Ser(tBu)-Leu-Wang Resin。
2.3裂解
量取70mL的TFA、1.8mL的TIS和1.8mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取13.9g的Palm-Tyr(tBu)-Ser(tBu)-Leu-WangResin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩到体积的1/4后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到4.5g的Palm-Tyr-Ser-Leu-OH粗肽。
2.4纯化
称取4.5g粗肽溶于30mL醋酸溶液中，再加入60mL纯水使其完全溶解，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度95.37％的化合物(13)Palm-Tyr-Ser-Leu-OH。
实施例3 Myr-Tyr-Ser-Leu-OH的制备
3.1树脂的溶胀
称取8g的Wang树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
3.2投料反应
称取8g的Fmoc-Leu-OH，3.82g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min，加4.8mLDIC活化3min，避免水汽。将活化后的氨基酸加入溶胀后的树脂中反应3.5h，抽走反应液，继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在2.07g HOBt和2.6mL DIC的情况下，使用DMF作为溶剂，将活化后的4.9g的Fmoc-Ser(tBu)-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在2.07g HOBt和2.6mLDIC的情况下，使用DMF作为溶剂，偶联5.9g的Fmoc-Tyr(tBu)-OH；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂。
在存在DIPEA的情况下，使用DMF作为溶剂，将4.7g的Myr-OH偶联至肽基树脂上，持续反应1.5h，洗涤树脂，抽走溶剂，收缩干燥后得到Myr-Tyr(tBu)-Ser(tBu)-Leu-Wang Resin。3.3裂解
量取61.6mL的TFA、1.6mL的TIS和1.6mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取10.8g的Myr-Tyr(tBu)-Ser(tBu)-Leu-Wang Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩到体积的1/4后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到1.5g的Myr-Tyr-Ser-Leu-OH粗肽。
3.4纯化
称取1.5g粗肽溶于醋酸：甲醇(V：V＝1：2)中，再加入15mL纯水使其完全溶解，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：

将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度96.45％的化合物(15)Myr-Tyr-Ser-Leu-OH。
实施例4 H-Tyr-Ser-Val-OH的制备
4.1树脂的溶胀
称取5g的Wang树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
4.2投料反应
称取5.9g的Fmoc-Val-OH，2.322g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min，加2.7mLDIC活化3min，避免水汽。将活化后的氨基酸加入溶胀后的树脂中反应3.5h，抽走反应液，继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在0.837gHOBt和0.781mL DIC的情况下，使用DMF作为溶剂，将活化后的2.9g的Fmoc-Ser(tBu)-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在0.837g HOBt和0.781mL DIC的情况下，使用DMF作为溶剂，将活化后的3.4g的Fmoc-Tyr(tBu)-OH偶联至肽基树脂上，持续反应2h；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂，收缩干燥后得到3.1g的Tyr(tBu)-Ser(tBu)-Val-WangResin。
4.3裂解
量取23.75mL的TFA、0.625mL的TIS和0.625mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取3.1g的Tyr(tBu)-Ser(tBu)-Val-WangResin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩到体积的1/4后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到3.3g的H-Tyr-Ser-Val-OH粗肽。
4.4纯化
称取3.1g粗肽溶于50mL纯水中，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液， pH值为5，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度98.37％的化合物(17)H-Tyr-Ser-Val-OH。
实施例5 Palm-Tyr-Ser-Val-OH的制备
5.1树脂的溶胀
称取8g的Wang树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
5.2投料反应
称取8g的Fmoc-Val-OH，3.82g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min，加4.8mLDIC活化3min，避免水汽。将活化后的氨基酸加入溶胀后的树脂中反应3.5h，抽走反应液，继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在2.07g HOBt和2.6mL DIC的情况下，使用DMF作为溶剂，将活化后的4.9g的Fmoc-Ser(tBu)-OH偶联至肽基树脂上，持续反应2h。然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在2.07g HOBt和2.6mLDIC的情况下，使用DMF作为溶剂，偶联5.9g的Fmoc-Tyr(tBu)-OH；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂。
在存在DIPEA的情况下，使用DMF作为溶剂，将4.7g的Palm-OH偶联至肽基树脂上，持续反应1.5h，洗涤树脂，抽走溶剂，收缩干燥后得到10.8g的Palm-Tyr(tBu)-Ser(tBu)-Val-Wang Resin。
5.3裂解
量取61.6mL的TFA、1.6mL的TIS和1.6mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取10.8g的Palm-Tyr(tBu)-Ser(tBu)-Val-Wang Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩到体积的1/4后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到1.8g的Palm-Tyr-Ser-Val-OH粗肽。
5.4纯化
称取1.8g粗肽溶于115mL醋酸：甲醇：水(V：V：V＝2：4：1)溶液中，使其完全溶解，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度95.52％的化合物(21)Palm-Tyr-Ser-Val-OH。
实施例6 Myr-Tyr-Ser-Val-OH的制备
6.1树脂的溶胀
称取8g的Wang树脂于固相合成反应柱中，用DMF溶胀，洗涤树脂，抽走溶剂。
6.2投料反应
称取8g的Fmoc-Val-OH，3.82g的HOBt加入干燥三角瓶中，加入DMF使其溶解，密封置于-18℃冰箱30min，加4.8mLDIC活化3min，避免水汽。将活化后的氨基酸加入溶胀后的树脂中反应3.5h，抽走反应液，继续加入Ac2O与DIPEA封端处理1.5h。洗涤树脂，抽走溶剂。K检树脂无色透明说明反应完全。
对N-末端Fmoc基团进行脱保护，并且在存在2.07g HOBt和2.6mL DIC的情况下，使用DMF作为溶剂，将活化后的4.9g的Fmoc-Ser(tBu)-OH偶联至肽基树脂上，持续反应2h。 然后洗涤这些树脂并且重复Fmoc基团的脱保护处理以便偶联下一个氨基酸。在存在2.07g HOBt和2.6mL DIC的情况下，使用DMF作为溶剂，偶联5.9g的Fmoc-Tyr(tBu)-OH；反应完全之后，洗涤树脂，抽走溶剂。
对肽基树脂的N-末端Fmoc基团脱保护，用20％piperidine/DMF脱Fmoc二次，每次10min，取样K检，显色深蓝。用DMF洗涤树脂6次，抽走溶剂。
在存在DIPEA的情况下，使用DMF作为溶剂，将4.7g的Myr-OH偶联至肽基树脂上，持续反应1.5h，洗涤树脂，抽走溶剂，收缩干燥后得到10.8g的Myr-Tyr(tBu)-Ser(tBu)-Val-Wang Resin。
6.3裂解
量取61.6mL的TFA、1.6mL的TIS和1.6mL的水混合搅拌均匀后得到裂解液，封口放置-18℃冰箱备用；异丙醚放置于-18℃冰箱冷冻备用。
称取10.8g的Myr-Tyr(tBu)-Ser(tBu)-Leu-Wang Resin，加入圆底烧瓶中，加入上述冷冻好的裂解液，搅拌反应2.5h。抽滤，收集滤液浓缩到体积的1/4后加入异丙醚搅拌离心洗涤6次，直至pH值为3-4，真空干燥，得到1.5g的Myr-Tyr-Ser-Leu-OH粗肽。
6.4纯化
称取1.5g粗肽溶于醋酸：甲醇：水(V：V：V＝2:4:1)中，使其完全溶解，用孔径为0.22μm微孔滤膜过滤得到澄清透明溶液，通过反相HPLC纯化处理，纯化梯度如下表：
将过滤后的样品进样纯化，收集馏分，浓缩冻干，得到纯度96％的化合物(23)Myr-Tyr-Ser-Val-OH。
实施例7
本发明通式(I)中的其他化合物可以通过类似的方法制备。
所获得的这些化合物通过ESI-MS测定其分子量，部分化合物的测试结果见下表2及图1-5。
表2质谱法测定分子量
实施例8细胞增殖实验
8.1试剂与材料
噻唑蓝(MTT)、二甲基亚砜(DMSO)、高糖培养基(DMEM)、胎牛血清、PBS。
8.2仪器
酶标仪、CO2培养箱、超净工作台。
8.3细胞株
小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库，人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库。
8.4待测样品
给药组：化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)、化合物(23)，上述样品测试浓度均为12.5ppm、25ppm、50ppm、100ppm。
空白对照组：PBS。
8.5实验方法
取处于指数生长期状态良好的NIH3T3细胞和HaCaT角质形成细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×105个/mL，制成细胞悬液。
取细胞悬液接种于96孔板上，200μL/孔，置恒温CO2培养箱中培养24h。
换液，分别加入给药组和对照组样品，20μL/孔，置于37℃、5％CO2培养箱中孵育72h。
之后每孔加入20μL 5mg/mL MTT，继续于37℃、5％CO2培养箱中孵育4h。弃去原溶液，加入150μL/孔的DMSO，置于平板摇床上振摇5min后，使用酶标仪在波长570nm处 测定每孔的OD值，并计算细胞活力。
细胞活力＝(给药孔OD-调零孔OD)/(对照孔OD-调零孔OD)×100％
8.6结果
MTT法是一种检测细胞存活和生长的方法，测得的OD值与细胞活性成正比。
测试样品对NIH3T3细胞增殖活性的影响结果见图6，结果显示，与空白对照组相比，给药组在100ppm范围内对NIH3T3细胞均没有毒性作用，而且还能提高其细胞活性，在不同浓度表现出不同的促进NIH3T3成纤维细胞增殖作用；其中，化合物(17)和化合物(21)在12.5-50ppm低浓度范围内即能够明显提高NIH3T3成纤维细胞活性，化合物(9)在100ppm时提高NIH3T3成纤维细胞活性更优，化合物(17)在25ppm时提高NIH3T3成纤维细胞活性最优，显著促进NIH3T3成纤维细胞增殖。
测试样品对HaCaT细胞增殖活性的影响结果见图7，结果显示，与空白对照组相比，给药组在100ppm范围内对HaCaT细胞没有毒性作用，而且还能提高其细胞活性，在不同浓度表现出不同的促进HaCaT角质形成细胞增殖作用；其中，化合物(9)、化合物(17)、化合物(21)和化合物(23)提高HaCaT角质形成细胞活性更优，显著促进HaCaT角质形成细胞增殖；而且化合物(17)和化合物(21)在12.5ppm低浓度即能够明显地促进HaCaT角质形成细胞增殖。
综上所述，本发明所述的化合物可以提高NIH3T3成纤维细胞和HaCaT角质形成细胞的活性，促进其增殖。其中，化合物(9)和化合物(17)可以明显提高成纤维细胞活性，促进角质形成细胞增殖；将棕榈酸、肉豆蔻酸分别与化合物(9)和化合物(17)的N-末端通过共价键相互连接得到的新结构化合物(13)、化合物(15)、化合物(21)、化合物(23)在不同浓度下亦能够明显提高细胞活性，并且棕榈酸与化合物(17)的N-末端通过共价键相互连接得到的新结构化合物(21)在低浓度下即能够提高细胞活性，促进细胞的增殖。
由此可知，本发明的肽类化合物对成纤维细胞和角质形成细胞不仅没有毒性作用，而且还能促进细胞增殖，提高成纤维细胞活性，促进角质形成细胞增殖，表明本发明的肽类化合物具有皮肤修复作用。
实施例9细胞黏附实验
9.1试剂与材料
胎牛血清、DMEM培养基、青霉素、链霉素、MTT。
9.2仪器
酶标仪、CO2培养箱、超净工作台。
9.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库。
9.4待测样品
给药组：化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)、化合物(23)，上述样品均用PBS溶解，测试浓度均为12.5ppm、25ppm、50ppm、100ppm；
空白对照组：PBS。
9.5实验方法
取冻存的HaCaT人角质层细胞培养，按照1：2传代至5代左右，选择长势较好的细胞作为实验对象。
将待测样品按照20μL/孔加入至96孔板中，37℃恒温烘箱干燥过夜。于第二天将长势较好的HaCaT细胞消化后，以HaCaT细胞1万/孔密度种板，并将培养基补至200μL，于37℃、5％CO2培养箱中孵育3h。培养结束后，将培养板取出并继续补充培养基至液面刚好溢出，用封口膜封闭，以保证没有气泡。顺时针翻转20min。弃去原有培养基，每孔加入90μL新鲜培养基和10μL 5mg/mL的MTT，置于37℃、5％CO2培养箱中孵育3h。弃去溶液，加入150μL的DMSO。使用酶标仪读取490nm和630nm波长下的参比OD值。
9.6实验结果
选择HaCaT角质形成细胞，铺板在药物包被好的96孔板中，孵育且在经过三维力作用之后，黏附性强的细胞能保持在96孔板上，通过对板上活细胞进行MTT定量分析，即可反映细胞的黏附作用，评价药物对细胞黏附的影响，以此确定本发明的化合物能否改善细胞弹性。保持在96孔板上的活细胞越多，测得的OD值越大，表明细胞的黏附作用越强。
测试样品对HaCaT细胞黏附影响结果见图8，给药组在不同浓度能够不同程度地提高HaCaT角质形成细胞的黏附能力，与空白对照组相比，化合物(9)、化合物(15)、化合物(17)和化合物(23)均能够明显提高HaCaT角质形成细胞的黏附能力，从而有利于提高细胞弹性。其中，化合物(15)在25ppm低浓度下亦能够明显提高细胞的黏附能力，化合物(9)和化合物(17)的整体技术效果更优。
由此可知，本发明的肽类化合物能够提高细胞的黏附能力，显著增加细胞间黏附和细胞-细胞外基质的黏附，从而增加皮肤弹性，提高皮肤紧致度，可用于预防甚至治疗皮肤松弛。
实施例10光损伤修复实验
10.1试剂与材料
胎牛血清、高糖培养基(DMEM)、青霉素、链霉素、MTT。
10.2仪器
酶标仪、CO2培养箱、超净工作台。
10.3细胞株
小鼠皮肤成纤维细胞(NIH3T3)购买自中国科学院典型培养物保藏委员会上海细胞库。
10.4待测样品
给药组：化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)、化合物(23)，测试浓度均为12.5ppm、25ppm、50ppm、100ppm。
对照组：PBS。
UV组：PBS+UV。
10.5实验方法
取处于指数生长期状态良好的NIH3T3成纤维细胞，加入0.25％胰蛋白酶消化液，消化使贴壁细胞脱落，计数1～4×105个/mL，制成细胞悬液。
适当稀释取10000个/孔细胞悬液接种于96孔板上，待细胞长满至80％左右时，建立UV光老化模型。对照组加50μLPBS，补充培养基至200μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入50μL PBS，以80mJ/cm2UV灯下照射，灯源和培养瓶间距15cm，经照射后，弃去PBS，UV组加入PBS溶液和培养基至200μL，给药组加入培养液和倍比稀释药物至200μL。对照组、UV组、给药组继续于37℃、5％CO2培养箱中孵育24h。
之后每孔加入20μL 5mg/mL MTT，继续于37℃、5％CO2培养箱中孵育4h。弃去原溶液，加入150μL/孔的DMSO，置于平板摇床上振摇5min后，使用酶标仪在波长570nm处测定每孔的OD值，并计算细胞活力。
细胞活力＝(给药孔OD-调零孔OD)/(对照孔OD-调零孔OD)×100％
10.6实验结果
皮肤衰老受到内源性因素和外源性因素的影响，如遗传、环境暴露、紫外线照射、激素变化和新陈代谢。这些因素的累积导致皮肤结构、功能和外观的改变，特别是紫外线的照射。本实验选取80mJ/cm2UV能量进行辐射建立皮肤光老化模型。
实验结果如图9所示，与空白对照组相比，经UV辐射后，成纤维细胞活性显著下降， 表明成功建立了光老化模型；给药组在100ppm范围内均能够不同程度地提高光损伤NIH3T3细胞的活性，从而改善细胞老化，产生明显的抗光老化作用；与UV组相比，化合物(9)、化合物(13)、化合物(17)和化合物(21)在12.5-100ppm范围内均能够明显地提高细胞活性，化合物(13)在12.5ppm低浓度即能明显提高细胞活性，化合物(15)和化合物(23)在50ppm时亦能够明显提高细胞活性。总的来说，本发明的化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)和化合物(23)均产生了明显的抗光老化作用，能够修复光老化受损细胞，具有良好的皮肤修复能力。
由此可知，本发明的肽类化合物能够用于预防或修复光老化损伤，具有皮肤修复作用。实施例11细胞划痕实验
11.1试剂与材料
胎牛血清、DMEM培养基、青霉素、链霉素。
11.2仪器
光学显微镜、CO2培养箱。
11.3细胞株
人角质形成细胞(HaCaT)购买自中国科学院典型培养物保藏委员会昆明细胞库。
11.4待测样品
给药组：化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)、化合物(23)，测试浓度均为50ppm。
空白对照组：PBS。
阳性对照组：50U/mL的EGF。
11.5实验方法
取冻存的HaCaT人角质层细胞培养，按照1：2传代至5代左右，选择长势较好的细胞作为实验对象。将细胞以20万个/孔的密度接种于12孔板，每孔2mL细胞悬浮液，待细胞贴壁长满后，换含0.5％～1％胎牛血清的培养基维持细胞24h使其同步化。用灭菌移液枪头进行划痕，PBS洗去掉落细胞，加入待测样品，继续于37℃、5％CO2培养箱中孵育，24h后置于光学显微镜下分析伤口愈合区域。
11.6实验结果
在HaCaT细胞长满培养皿的基础上进行划痕实验，于给药后24h在光学显微镜下观察 细胞的迁移情况，评价药物对细胞增殖和迁移的影响，以此确定本发明的化合物能否促进皮肤或粘膜再上皮化或愈合。
细胞的划痕迁移实验能反应细胞的增殖修复能力，实验结果如图10所示，空白对照组的细胞划痕仍旧明显，划痕间距正常；与空白对照组相比，EGF阳性对照组的细胞增殖迁移明显，间距基本消失，连成一片；化合物(9)、化合物(17)和化合物(21)组的划痕间距均较空白对照组细胞划痕间距缩短，并出现细胞迁移轨迹；其中，化合物(9)和化合物(17)组的划痕间距与EGF阳性对照组的划痕间距及细胞增殖迁移效果相当，表明本发明的化合物(9)和化合物(17)对细胞的修复能力与EGF的效果相当。
由此可知，本发明的肽类化合物能够促进HaCaT角质形成细胞增殖和迁移，具有皮肤修复作用，能够修复皮肤屏障，可以促进皮肤或粘膜的再上皮化或愈合。
实施例12胶原蛋白含量测试
12.1试剂与材料
胎牛血清、DMEM培养基、磷酸盐缓冲液、胰蛋白酶、胶原蛋白I ELISA试剂盒、BCA蛋白试剂盒。
12.2仪器
酶标仪、CO2培养箱、超净工作台、恒温箱。
12.3细胞株
人皮肤成纤维细胞(HSF)购买自中国科学院典型培养物保藏委员会上海细胞库。
12.4待测样品
给药组：化合物(9)和化合物(17)，测试浓度均为12.5ppm、50ppm；
空白对照组：PBS；
UV组：UV辐射，加PBS。
12.5实验方法
取处于对数生长期的HSF细胞，酶解稀释后，按照105个/孔接种于6孔板上，直至细胞长满至80％左右时建立UV光老化模型。空白对照组加200μLPBS，补充培养基至800μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入200μLPBS，置于80mJ/cm2UV灯下照射，灯源和培养瓶间距15cm。经照射后，弃去PBS，UV组加入PBS溶液和培养基至800μL，给药组加入培养基和倍比稀释药物至800μL。空白对照组、UV组、给药组继续于37℃、5％CO2培养箱中孵育48h。
培养结束后，将第1孔细胞消化计数，并稀释至0.5×106个/mL，其余孔的细胞使用细胞刮刮下来，500μL重悬后，所有孔取50μL超声30s后使用BCA法测定总蛋白，根据第1孔蛋白浓度稀释其他孔，使所有细胞悬液浓度为0.5×106个/mL。将调整浓度后的细胞悬液超声破碎30s，1500xg离心15min，收集细胞上清获得样本液，并按照胶原蛋白I ELISA操作说明书进行操作。15min内用酶标仪在450nm处依序测量各孔的OD值。
12.6实验结果
胶原蛋白是结缔组织中发现的最丰富的蛋白质，胶原蛋白酶由成纤维细胞合成分泌，能够降解皮肤中的胶原蛋白导致皮肤老化。因此抑制细胞中胶原蛋白酶的表达，提升胶原蛋白含量对于预防衰老、增加皮肤饱满和紧致度具有重要作用。在紫外线过暴环境中，胶原蛋白酶和弹性蛋白酶活性大幅增加，弹性蛋白水解，胶原蛋白的合成也受到抑制。本实验采用测试样品处理经紫外线辐射后的细胞，检测相应细胞中的胶原蛋白I含量，以确定本发明的肽类化合物是否能够促进胶原蛋白的合成。
测试样品对胶原蛋白含量的影响结果见图11。结果显示，与空白对照组相比，UV组的胶原蛋白含量大幅降低，表明成功建立了光老化模型；与UV组相比，12.5-50ppm范围内的化合物(9)和化合物(17)均能够提高胶原蛋白含量，促进胶原蛋白表达，且在低浓度12.5ppm条件下，化合物(9)和化合物(17)即能够明显地提高胶原蛋白含量。
由此可知，本发明的肽类化合物能够促进胶原蛋白生成，增加胶原蛋白含量，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛，治疗、预防或修复皮肤老化或光老化，抵抗皮肤衰老。
实施例13胶原纤维实验
13.1试剂与材料
胎牛血清、DMEM培养基、磷酸盐缓冲液、胰蛋白酶、生物天狼星红染色试剂盒。
13.2仪器
光学显微镜、CO2培养箱。
13.3细胞株
人皮肤成纤维细胞(HSF)购买自中国科学院典型培养物保藏委员会上海细胞库。
13.4待测样品
给药组：化合物(9)和化合物(17)，测试浓度均为12.5ppm、50ppm；
空白对照组：PBS；
UV组：UV辐射，加PBS。
13.5实验方法
取处于对数生长期的HSF细胞，酶解稀释后，按照105个/孔接种于6孔板上，直至细胞长满至80％左右时建立UV光老化模型。空白对照组加200μL PBS，补充培养基至800μL，不进行UV照射；UV组和给药组，加入适量PBS反复洗至无色后，加入200μLPBS，置于80mJ/cm2UV灯下照射，灯源和培养瓶间距15cm。经照射后，弃去PBS，UV组加入PBS溶液和培养基至800μL，给药组加入培养基和倍比稀释药物至800μL。空白对照组、UV组、给药组继续于37℃、5％CO2培养箱中孵育48h。
培养结束后，上述细胞使用4％多聚甲醛固定15min后，按照天狼星红染色试剂盒说明书进行染色，置于光学显微镜下观察。
13.6实验结果
本实验采用测试样品处理经紫外线辐射后的细胞，检测相应细胞的密度及同一单位内胶原纤维面积，对细胞中的胶原纤维定性分析以确定本发明的肽类化合物是否能够促进胶原蛋白表达。
细胞的胶原纤维实验能反应细胞的胶原蛋白表达能力，实验结果如图12所示，与空白对照组相比，UV组的细胞密度降低，同一单位内胶原纤维面积也降低；与UV组相比，12.5-50ppm范围内的化合物(9)和化合物(17)均能够提高细胞密度，增加同一单位内胶原纤维面积，促进胶原蛋白表达，改善细胞因UV照射而导致的胶原蛋白含量降低的症状。
由此可知，本发明的肽类化合物能够促进胶原蛋白表达，从而增加皮肤弹性和/或皮肤紧致度，可用于预防甚至治疗皮肤松弛，治疗、预防或修复皮肤老化或光老化，抵抗皮肤衰老。
实施例14含化合物(9)的脂质体的制备
制备方法：将磷脂酰胆碱称重且溶于氯仿。于真空下蒸发溶剂，直至得到磷脂薄层，将此层于55℃以所需浓度的化合物水溶液处理而水化，得到多室脂质体。将多室脂质体通过高压均质处理，得到尺寸更加小而均一的单室脂质体。
实施例15含化合物(13)的微米乳液组合物
制备方法：按照处方用量，称取B相的成分加于容器中。接着，将D相添加至B相且于连续搅拌下均质化。随后将A相添加至混合物。最后，添加C相，搅拌均匀即得。
实施例16含化合物(17)的精华液的制备
制备方法：纯化水搅拌加热至85℃，保温30min；将透明质酸钠、黄原胶预溶于丁二醇，加入水中，搅拌溶解完全；搅拌降温至35℃，加入剩余成分，搅拌均匀即得。
实施例17含化合物(21)的爽肤水的制备

制备方法：将尿囊素、甘油用水溶解，加热至85℃，保温30分钟；将PEG-7甘油椰油酸酯、化合物(21)用水溶解；上述溶液冷却后混合，搅拌均匀，得混合溶液；将丙二醇、防腐剂、香精依次加入上述混合溶液，加水搅拌均匀，即得。
尽管已描述了本发明实施例的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例做出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明实施例范围的所有变更和修改。
最后，还需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者终端设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者终端设备所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者终端设备中还存在另外的相同要素。
以上对本发明所提供的肽类化合物在制备用于皮肤延衰修复的组合物中的新用途，进行了详细介绍，本文中应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想；同时，对于本领域的一般技术人员，依据本发明的思想，在具体实施方式及应用范围上均会有改变之处，综上所述，本说明书内容不应理解为对本发明的限制。

Claims (9)

  1. 通式(I)所示的化合物或其盐在制备用于皮肤延衰修复的组合物中的用途，所述皮肤延衰修复包括提高成纤维细胞活性、促进角质形成细胞增殖和迁移、促进皮肤或粘膜的再上皮化或愈合、修复皮肤屏障、预防或修复光老化损伤、促进胶原蛋白生成、增加皮肤弹性或提高皮肤紧致度中的一种或多种；
    通式(I)中，
    R1选自：H或R3-CO-，其中R3选自：取代的或未取代的烷基、取代的或未取代的烯基；
    R2选自：-OH、-NH2、-Leu-NH2、-Leu-OH、-Val-NH2或-Val-OH；
    所述烷基是指具有1-24个碳原子(可选具有1-16个碳原子；可选具有1-14个碳原子；可选具有1-12个碳原子；可选具有1、2、3、4、5、或6个的碳原子)的饱和脂肪族直链或支链的烷基；可选选自：甲基、乙基、异丙基、异丁基、叔丁基、戊基、己基、庚基、辛基、癸基、十二烷基、十四烷基、十六烷基、十八烷基、2-乙基己基、2-甲基丁基、或5-甲基己基；
    所述烯基是指具有2-24个碳原子(可选具有2-16个碳原子；可选具有2-14个碳原子；可选具有2-12个碳原子；可选具有2、3、4、5、或6个碳原子)的直链或支链烯基；所述烯基具有一个或多个碳-碳双键，可选具有1、2或3个共轭或非共轭的碳-碳双键；所述烯基是通过一个单键而结合至分子的其余部分；可选选自：乙烯基、油烯基、或亚油烯基；
    可选地，所述“取代的烷基”、“取代的烯基”中的取代基选自C1-C4烷基；羟基；C1-C4烷氧基；氨基；C1-C4氨基烷基；C1-C4羰氧基；C1-C4氧基羰基；卤素(如氟、氯、溴、以及碘)；氰基；硝基；叠氮化物；C1-C4烷基磺酰基；硫醇；C1-C4烷硫基；C6-C30芳氧基如苯氧基；-NRb(C＝NRb)NRbRc，其中Rb和Rc是独立地选自：H、C1-C4烷基、C2-C4烯基、C2-C4炔基、C3-C10环烷基、C6-C18芳基、C7-C17芳烷基、具有三至十元的杂环基、或氨基的保护基。
  2. 根据权利要求1所述的用途，其特征在于，R1选自：H、乙酰基、叔-丁酰基、己酰基、2-甲基己酰基、辛酰基、癸酰基、月桂酰基、肉豆蔻酰基、棕榈酰基、硬脂酰基、油酰基、亚油酰基或苯乙酰基；
    可选地，R1为H、肉豆蔻酰基或棕榈酰基。
  3. 根据权利要求1所述的用途，其特征在于，所述化合物选自(1)-(24)：
    (1)H-Tyr-Ser-OH；
    (2)H-Tyr-Ser-NH2；
    (3)Ac-Tyr-Ser-OH；
    (4)Ac-Tyr-Ser-NH2；
    (5)Palm-Tyr-Ser-OH；
    (6)Palm-Tyr-Ser-NH2；
    (7)Myr-Tyr-Ser-OH；
    (8)Myr-Tyr-Ser-NH2；
    (9)H-Tyr-Ser-Leu-OH；
    (10)H-Tyr-Ser-Leu-NH2；
    (11)Ac-Tyr-Ser-Leu-OH；
    (12)Ac-Tyr-Ser-Leu-NH2；
    (13)Palm-Tyr-Ser-Leu-OH；
    (14)Palm-Tyr-Ser-Leu-NH2；
    (15)Myr-Tyr-Ser-Leu-OH；
    (16)Myr-Tyr-Ser-Leu-NH2；
    (17)H-Tyr-Ser-Val-OH；
    (18)H-Tyr-Ser-Val-NH2；
    (19)Ac-Tyr-Ser-Val-OH；
    (20)Ac-Tyr-Ser-Val-NH2；
    (21)Palm-Tyr-Ser-Val-OH；
    (22)Palm-Tyr-Ser-Val-NH2；
    (23)Myr-Tyr-Ser-Val-OH；
    (24)Myr-Tyr-Ser-Val-NH2。
  4. 根据权利要求3所述的用途，其特征在于，所述化合物选自化合物(9)、化合物(13)、化合物(15)、化合物(17)、化合物(21)和化合物(23)；具体地，
    (9)H-Tyr-Ser-Leu-OH；
    (13)Palm-Tyr-Ser-Leu-OH；
    (15)Myr-Tyr-Ser-Leu-OH；
    (17)H-Tyr-Ser-Val-OH；
    (21)Palm-Tyr-Ser-Val-OH；
    (23)Myr-Tyr-Ser-Val-OH。
  5. 根据权利要求1所述的用途，其特征在于，所述组合物包含质量百分浓度为0.0001％-5％的通式(I)所示的化合物或其盐；
    可选地，所述组合物包含质量百分浓度为0.0005％-1％的通式(I)所示的化合物或其盐；
    可选地，所述组合物包含质量百分浓度为0.001％-0.1％的通式(I)所示的化合物或其盐；
    可选地，所述组合物包含质量百分浓度为0.005％-0.01％的通式(I)所示的化合物或其盐。
  6. 根据权利要求1所述的用途，其特征在于，所述通式(I)所示的化合物的盐包括通式(I)所示的化合物的金属盐，所述金属包括：锂、钠、钾、钙、镁、锰、铜、锌或铝；
    可选地，所述通式(I)所示的化合物的盐包括通式(I)所示的化合物与有机碱形成的盐，所述有机碱包括：乙胺、二乙胺、乙二胺、乙醇胺、二乙醇胺、精氨酸、赖氨酸、组氨酸或哌嗪；
    可选地，所述通式(I)所示的化合物的盐包括通式(I)所示的化合物与无机酸或有机酸形成的盐，所述有机酸包括：乙酸、柠檬酸、乳酸、丙二酸、马来酸、酒石酸、延胡索酸、苯甲酸、天冬氨酸、谷氨酸、琥珀酸、油酸、三氟乙酸、草酸、扑酸或葡萄糖酸；
    可选地，所述无机酸包括：盐酸、硫酸、硼酸或碳酸。
  7. 根据权利要求1所述的用途，所述通式(I)所示的化合物或其盐，掺入到美容上或药学上可接受的递送系统或缓释系统，或被吸附到美容上或药学上可接受的固体有机聚合物或固体无机支撑体上；
    所述美容上或药学上可接受的递送系统或缓释系统选自：脂质体、油质体、非离子型表面活性剂脂质体囊泡、醇质体、毫米胶囊、微米胶囊、纳米胶囊、纳米结构的脂质载体、海绵状物、环糊精、类脂囊泡、胶束、毫米球、微米球、纳米球、脂质球、微米乳液、纳米乳液、毫米粒子、微米粒子以及纳米粒子；可选为脂质体或微米乳液，可选具有反胶束的内部结构的油包水型微米乳液；
    所述美容上或药学上可接受的固体有机聚合物或固体无机支撑体选自：滑石、膨润土、二氧化硅、淀粉或麦芽糖糊精。
  8. 根据权利要求1所述的用途，其特征在于，所述组合物是美容组合物或药物组合物，所述组合物的制剂选自：霜剂、油、香膏、泡沫、洗剂、凝胶、擦剂、浆液、软膏、摩丝、粉末、杆剂、笔剂、喷雾剂、气溶胶、胶囊剂、片剂、颗粒剂、口香糖、溶液、混悬液、乳剂、酏剂、多糖薄膜、胶冻或明胶。
  9. 根据权利要求1所述的用途，其特征在于，所述组合物还包含至少一种用于增强本发明所述皮肤延衰修复作用的其他活性剂，所述其他活性剂选自肽类、天然植物成分、维生素C及其衍生物或类视黄醇中的一种或多种。
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