Cyclic tetrapeptide derivative as well as preparation method and application thereof

Abstract

The invention relates to a cyclic tetrapeptide derivative as well as a preparation method and application thereof. The structural formula of the cyclic tetrapeptide derivative is shown as a formula (I), and R is selected from C7-C19 alkyl and C7-C19 unsaturated chain alkyl. The cyclic tetrapeptide derivative or the salt or the stereoisomer thereof provided by the invention can improve the contents of hyaluronic acid, silk protein and alpha-crystal protein of epidermal cells, so that the skin barrier can be effectively enhanced, the obvious repairing and improving effects on the skin are achieved, and compared with palmitic acid tetrapeptide-10, the cyclic tetrapeptide derivative or the salt or the stereoisomer thereof has better stability and safety, and can be used for preparing a medicine for treating skin diseases. And when being used in cosmetics, the composition can play a better role in repairing and enhancing the skin barrier, and has a better improvement effect on the skin. # imgabs0 # (I)

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics and relates to cyclic peptide compounds, in particular to a cyclic tetrapeptide derivative and a preparation method thereof and application in cosmetics. Background Art

[0002] Filaggrin is an important structural protein in the stratum corneum, which can gather keratin fibers together to form a dense stratum corneum structure; hyaluronic acid has a strong water-retaining capacity and is an important component for maintaining skin moisture; α-crystallin is a heat shock protein that can protect cells from stress damage, protect keratinocytes from damage by external environmental factors such as ultraviolet rays, and maintain the stability of the skin barrier function. The improvement of these components in the skin is conducive to strengthening the skin barrier, making the skin better resistant to damage from the external environment, delaying skin aging, and reducing the occurrence of wrinkles, spots and other problems.

[0003] Peptides are organic compounds composed of multiple amino acids connected by peptide bonds and have the characteristics of high biological activity. Sederma has developed a cosmetic raw material CRYSTALIDE, whose main active ingredient is palmitoyl tetrapeptide-10 (palm-Lys-Thr-Phe-Lys-OH), which has the effects of improving and / or enhancing the epidermal barrier. Palmitoyl tetrapeptide-10 is a linear peptide. Studies have shown that the molecular structure of linear peptides is relatively loose and easily attacked by proteases and degraded. Therefore, it has the defects of poor stability in the cell environment and relatively short action time. Summary of the invention

[0004] Based on this, the present invention provides a new cyclic tetrapeptide derivative, which can increase the content of hyaluronic acid, filaggrin and α-crystallin in epidermal cells and enhance the skin barrier.

[0005] The present invention includes the following technical solutions.

[0006] In the first aspect, the present invention provides a cyclic tetrapeptide derivative or a salt thereof or a stereoisomer thereof, wherein the structural formula of the cyclic tetrapeptide derivative is shown in formula (I): ; (I) Wherein, R is selected from: C7~C 19 Alkyl, C7~C 19 Unsaturated chain hydrocarbon group.

[0007] In a second aspect, the present invention provides the use of the cyclic tetrapeptide derivative or its salt or its stereoisomer as an effective ingredient in the preparation of cosmetics, wherein the cosmetics can maintain, repair or improve the condition of the skin.

[0008] In a third aspect, the present invention provides a cosmetic comprising the cyclic tetrapeptide derivative or its salt or stereoisomer thereof according to the present invention.

[0009] In a fourth aspect, the present invention provides a method for preparing the cyclic tetrapeptide derivative or a salt thereof, comprising the following steps: 1) H-Lys(Fmoc)-OH reacts with acyl chloride to obtain RCO-Lys(Fmoc)-OH. The reaction formula is as follows:

[0010] Wherein, R is selected from: C7~C 19 Alkyl, C7~C 19 Unsaturated chain hydrocarbon group; 2) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH was synthesized; 3) Cyclizing the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH and then deprotecting it to obtain the cyclic tetrapeptide derivative or its salt.

[0011] The present invention has the following beneficial effects: The cyclic tetrapeptide derivative or its salt or its stereoisomer provided by the present invention can increase the content of hyaluronic acid, filaggrin and α-crystallin in epidermal cells, thereby effectively enhancing the skin barrier and having a significant repair and improvement effect on the skin, for example, it has a significant effect on reducing skin roughness and shrinking skin pores, and can effectively improve skin smoothness.

[0012] The cyclic tetrapeptide derivative or its salt or its stereoisomer provided by the present invention can effectively penetrate into the living epidermis, has good skin permeability, and has better stability and safety than palmitic acid tetrapeptide-10. When used in cosmetics, it can play a better role in repairing and enhancing the skin barrier and has a better effect on improving the skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure is a bar graph showing the skin penetration and skin retention of cyclic tetrapeptides CP4-02 and CP4-03 and the linear tetrapeptides LP4-02 and LP4-03 prepared in comparative examples 2 and 3. DETAILED DESCRIPTION

[0014] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0015] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0016] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0017] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in..." includes "in..." and "on...".

[0018] The term "alkyl" of the present invention refers to a saturated aliphatic hydrocarbon group including branched and straight chains having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes groups having 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight or branched chain. For example, "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0019] The term "unsaturated chain hydrocarbon group" in the present invention refers to branched and straight-chain unsaturated aliphatic hydrocarbon groups having a specific number of carbon atoms, that is, non-cyclic chain hydrocarbon groups, and the carbon chain contains one or more carbon-carbon double bonds, or carbon-carbon triple bonds, such as: -(CH2)7(CH=CH)(CH2)7CH3, -(CH2)8(CH=CH)(CH2)7CH3, -(CH2)8(CH=CH)(CH2)5CH3, -(CH2)8(CH=CH)(CH2)7CH3, -(CH2)8(CH=CH)(CH2)6CH3, -(CH2)8(CH=CH)CH2(CH=CH)(CH2)2CH3, -(CH2)8(CH=CH)CH2(CH=CH)(CH2)4CH3, -(CH2)8(CH=CH)CH2(CH=CH)(CH2)6CH3, etc.

[0020] In some embodiments, a cyclic tetrapeptide derivative or a salt thereof or a stereoisomer thereof is involved, and the structural formula of the cyclic tetrapeptide derivative is shown in formula (I): ; (I) Among them, R is selected from: C7~C 19 Alkyl, C7~C 19 Unsaturated chain hydrocarbon group.

[0021] For example, R can be selected from: n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, 1-hexyl-nonyl, 1-butyl-heptyl, -(CH2)8(CH=CH)(CH2)6CH3, -(CH2)7(CH=CH)(CH2)7CH3, -(CH2)9(CH=CH)(CH2)5CH3, -(CH2)6(CH=CH)(CH2)8CH3, etc.

[0022] In some preferred embodiments, R is selected from: C7 alkyl, C8 alkyl, C9 alkyl, C 10 Alkyl, C 11 Alkyl, C 12 Alkyl, C 13 Alkyl, C 14 Alkyl, C 15 Alkyl, C 16 Alkyl, C 17 Alkyl, C 18 Alkyl, C 19 Alkyl, C7 unsaturated chain hydrocarbon group containing one or two carbon-carbon double bonds, C8 unsaturated chain hydrocarbon group containing one or two carbon-carbon double bonds, C9 unsaturated chain hydrocarbon group containing one or two carbon-carbon double bonds, C 10Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 11 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 12 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 13 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 14 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 15 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 16 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 17 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 18 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 19 Unsaturated chain hydrocarbon group; the unsaturated chain hydrocarbon group preferably contains one carbon-carbon double bond.

[0023] In some preferred embodiments, R is selected from: C7 alkyl, C9 alkyl, C 11 Alkyl, C 13 Alkyl, C 15 Alkyl, C 15 Unsaturated chain hydrocarbon group, containing one or two carbon-carbon double bonds 17 Unsaturated chain hydrocarbon group, C containing one or two carbon-carbon double bonds 19 Unsaturated chain hydrocarbon group.

[0024] In some preferred embodiments, R is selected from: C9~C 11 Straight chain alkyl.

[0025] In some preferred embodiments, the cyclic tetrapeptide derivative is selected from the following compounds: .

[0026] In some embodiments, the salt of the cyclic tetrapeptide derivative is selected from acetate, trifluoroacetate, hydrochloride, sulfate, citrate, ascorbate, lactate, maleate, fumarate, succinate, gluconate, and salicylate.

[0027] The present invention cyclizes the linear peptide through a peptide bond on the basis of the linear peptide to obtain a tetrapeptide derivative with a cyclic structure. The cyclic structure can make the hydrogen bond in the molecule more tightly bound, thereby reducing the external hydrogen bond ability of the molecule and improving the structural rigidity and protein resistance. This structural characteristic makes the cyclic peptide more stable in the cell environment and not easily hydrolyzed by proteases. Therefore, the cyclic tetrapeptide derivative provided by the present invention has better stability than palmitic acid tetrapeptide-10, and has a longer half-life and action time. After the peptide chain is cyclized, its oil-water partition coefficient will change. The present invention adjusts the oil-water partition coefficient of the cyclic peptide by accessing carbon chains of different lengths to improve its skin permeability, thereby obtaining a series of cyclic tetrapeptide derivatives with good skin permeability and stability, and having more significant repair and improvement effects on the skin.

[0028] The present invention also found that the cyclic tetrapeptide derivative of the present invention has lower cytotoxicity and better biosafety than palmitic acid tetrapeptide-10, and can be added to cosmetics at a higher concentration, thereby obtaining a more excellent effect of repairing and improving the skin.

[0029] The cyclic tetrapeptide derivative or its salt or its stereoisomer provided by the present invention can increase the content of hyaluronic acid, filaggrin and α-crystallin in epidermal cells, thereby effectively enhancing the skin barrier, and has a significant effect on repairing and improving the skin, for example, it has a significant effect on reducing skin roughness and shrinking skin pores, and can effectively improve skin smoothness. And it has good permeability and stability, is safe and non-toxic, so it can be used in cosmetics, has a good effect on repairing and enhancing the skin barrier, and has a better improvement effect on the skin than palmitic acid tetrapeptide-10.

[0030] Herein, some embodiments of the present invention relate to the use of the cyclic tetrapeptide derivative or its salt or its stereoisomer as an effective ingredient in the preparation of cosmetics.

[0031] In some embodiments of the present invention, there is provided a cosmetic comprising the cyclic tetrapeptide derivative or its salt or stereoisomer thereof according to the present invention.

[0032] Among them, the cosmetics can maintain, repair or improve the condition of the skin; for example, the cosmetics can increase the content of filaggrin, hyaluronic acid and / or α-crystallin in cells, thereby enhancing the skin barrier; can reduce skin roughness and / or shrink pores, thereby improving skin smoothness.

[0033] The cosmetics described in the present invention include but are not limited to daily cleaning or skin care products such as cleansers, lotions, emulsions, facial masks, facial creams and essences.

[0034] For example, some embodiments of the present invention relate to an emulsion, which contains the cyclotetrapeptide derivative or its salt or stereoisomer described in the present invention, and conventional excipients in the emulsion. The emulsion can effectively reduce skin roughness and shrink pores, thereby effectively improving skin smoothness.

[0035] The amount of the cyclic tetrapeptide derivative or its salt or its stereoisomer added to the cosmetic is based on the effect of maintaining, repairing or improving the skin without producing obvious toxicity. Since the cyclic tetrapeptide derivative of the present invention has a significant effect on improving the skin and has very low cytotoxicity, its addition amount can be a larger range. In some embodiments, its addition amount is preferably 0.00001% to 1% by mass in the cosmetic, more preferably 0.0001% to 1%, more preferably 0.0001% to 0.1%, more preferably 0.0001% to 0.01%, more preferably 0.0001% to 0.001%, more preferably 0.0001% to 0.001%, more preferably 0.0005% to 0.001%.

[0036] The present invention has no particular restrictions on the auxiliary materials in cosmetics (such as emulsions), and conventional auxiliary materials in cosmetics can be used in the present invention to prepare cosmetics that have a repairing or improving effect on the skin. For example, in some embodiments of the present invention, an emulsion is provided, and the emulsion is prepared from raw materials including the following components in terms of mass percentage: water Add to 100% Trehalose 0.8%~1.2% Carbomer 0.4%~0.5% glycerin 3%~5% Emulsifier A165 0.4%~0.6% M68 emulsifier 0.6%~1.0% Squalane 1.5%~2.5% Silicone oil 0.8%~1.2% Caprylic Capric Triglyceride 0.8%~1.2% Behenyl alcohol 1.2%~1.6% Arginine 0.2%~0.4% Xinxian Ketone 0.4%~0.6% Hexanediol 0.4%~0.6% The cyclic tetrapeptide derivative or its salt or stereoisomer 0.0001%~0.001% Citric acid 0.001%~0.002% Dipropylene glycol 2%~4% .

[0037] The cyclotetrapeptide derivatives or salts thereof of the present invention can be prepared by conventional solid phase synthesis or liquid phase synthesis methods in the art, wherein the amino acid raw material with hydrocarbon side chain modification can be obtained by reacting the corresponding amino acid with an acyl halide compound.

[0038] For example, the cyclic tetrapeptide derivative or its salt according to the present invention can be prepared by the following steps: 1) H-Lys(Fmoc)-OH reacts with acyl chloride to obtain RCO-Lys(Fmoc)-OH. The reaction formula is as follows:

[0039] Among them, R is selected from C7~C 19 Alkyl, C7~C 19 Unsaturated chain hydrocarbon group; further as described above; 2) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH was synthesized; 3) Cyclizing the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH and then deprotecting it to obtain the cyclic tetrapeptide derivative or its salt.

[0040] Among them, the linear tetrapeptide can be prepared by conventional solid phase synthesis or liquid phase synthesis methods in the art, preferably the EMPHASES liquid phase method; the cyclization is completed by dehydration under the action of a condensation reagent, and the condensation reagent is selected from EDCI, DCC, HBTU, HATU, BOP, HOBt, etc.; the deprotection can use conventional reagents in the art, such as trifluoroacetic acid and hydrogen chloride, etc. The final product can also be further purified by methods such as crystallization or high-efficiency preparative chromatography.

[0041] The compounds corresponding to the abbreviations and abbreviations involved in the present invention are described as follows: DMF: N,N-dimethylformamide; ZT-Cl: 1-phenyl-1-(2-chlorophenyl)-1-(N-α-tocopheryloxy-ethyl-N-benzyl-4-benzamido)-methyl chloride; DMT-MM: 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride; TFA: trifluoroacetic acid; HOBt: 1-hydroxybenzotriazole; EDCI: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; DCC: dicyclohexylcarbodiimide; HBTU: O-benzotriazole-tetramethyluronium hexafluorophosphate; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; BOP: benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate; Fmoc: fluorenylmethoxycarbonyl; Boc: tert-butyloxycarbonyl; Lys: lysine; Phe: phenylalanine; Thr: threonine; tBu: tert-butyl; In the following examples, room temperature or normal temperature refers to 20°C-25°C.

[0042] The present invention is further described in detail below with reference to specific embodiments.

[0043] Examples 1a-1f Synthesis of RCO-Lys(Fmoc)-OH

[0044] Example 1a n-C7H 15 Preparation of CO-Lys(Fmoc)-OH

[0045] H-Lys(Fmoc)-OH (36.8 g, 0.1 mol) was dissolved in DMF (500 mL), cooled to 5°C-10°C, N,N-diisopropylethylamine (25.8 g, 0.2 mol) was added, stirred for half an hour after addition, caprylyl chloride (16.2 g, 0.1 mol) was added dropwise, kept at 5°C-10°C for 1 hour after addition, the reaction solution was poured into 10% citric acid aqueous solution, the precipitated solid was filtered, washed with purified water, and dried to obtain n-C7H 15 CO-Lys(Fmoc)-OH 44.5g, yield 90%. MS: 495.28 (M+1) + , HPLC purity>98%.

[0046] 1 H-NMR (500MHz, CDCl3): δ8.10-8.05 (d, 1H), 7.85-7.80 (d, 2H), 7.75-7.7 0 (d, 2H), 7.65-7.60 (m, 2H), 7.45-7.40 (m, 2H), 5.73-5.65 (t, 1H), 4.50-4 .45 (d, 2H), 4.27-4.24 (t, 1H), 4.20-4.13 (m, 1H), 3.20-3.15 (m, 1H), 2.8 5-2.75 (m, 1H), 2.22-2.15 (t, 2H), 1.60-1.25 (m, 16H), 0.92-0.85 (t, 3H).

[0047] Example 1b n-C9H 19 Preparation of CO-Lys(Fmoc)-OH

[0048] Using decanoyl chloride instead of octanoyl chloride, the method of Example 1a was used to prepare n-C9H 19 CO-Lys(Fmoc)-OH, yield 85%. MS: 523.20(M+1) + , HPLC purity>98%.

[0049] 1H-NMR (500MHz, CDCl3): δ8.10-8.05 (d, 1H), 7.85-7.80 (d, 2H), 7.75-7.7 0 (d, 2H), 7.65-7.60 (m, 2H), 7.45-7.40 (m, 2H), 5.73-5.65 (t, 1H), 4.50-4 .45 (d, 2H), 4.27-4.24 (t, 1H), 4.20-4.13 (m, 1H), 3.20-3.15 (m, 1H), 2.8 5-2.75 (m, 1H), 2.22-2.15 (t, 2H), 1.60-1.25 (m, 20H), 0.92-0.85 (t, 3H).

[0050] Example 1c nC 11 H 23 Preparation of CO-Lys(Fmoc)-OH

[0051] nC was prepared by the method of Example 1a using lauroyl chloride instead of octanoyl chloride. 11 H 23 CO-Lys(Fmoc)-OH, yield 91%. MS: 551.38(M+1) + , HPLC purity>98%.

[0052] 1 H-NMR (500MHz, CDCl3): δ8.10-8.05 (d, 1H), 7.85-7.80 (d, 2H), 7.75-7.7 0 (d, 2H), 7.65-7.60 (m, 2H), 7.45-7.40 (m, 2H), 5.73-5.65 (t, 1H), 4.50-4 .45 (d, 2H), 4.27-4.24 (t, 1H), 4.20-4.13 (m, 1H), 3.20-3.15 (m, 1H), 2.8 5-2.75 (m, 1H), 2.22-2.15 (t, 2H), 1.60-1.25 (m, 24H), 0.92-0.85 (t, 3H).

[0053] Example 1d nC 15 H 31 Preparation of CO-Lys(Fmoc)-OH

[0054] Using palmitoyl chloride instead of octanoyl chloride, nC 15 H 31 CO-Lys(Fmoc)-OH, yield 88%. MS: 607.41(M+1)+ , HPLC purity>98%.

[0055] 1 H-NMR (500MHz, CDCl3): δ8.10-8.05 (d, 1H), 7.85-7.80 (d, 2H), 7.75-7.7 0 (d, 2H), 7.65-7.60 (m, 2H), 7.45-7.40 (m, 2H), 5.73-5.65 (t, 1H), 4.50-4 .45 (d, 2H), 4.27-4.24 (t, 1H), 4.20-4.13 (m, 1H), 3.20-3.15 (m, 1H), 2.8 5-2.75 (m, 1H), 2.22-2.15 (t, 2H), 1.60-1.25 (m, 32H), 0.92-0.85 (t, 3H).

[0056] Example 1e nC 11 H 23 Preparation of 5-CO-Lys(Fmoc)-OH

[0057] The product was prepared by the method of Example 1a using 2-butyloctanoyl chloride instead of octanoyl chloride. 11 H 23 -5-CO-Lys(Fmoc)-OH, yield 85%. MS:551.36(M+1) + , HPLC purity>98%.

[0058] 1 H-NMR (500MHz, CDCl3): δ8.10-8.05 (d, 1H), 7.85-7.80 (d, 2H), 7.75-7.7 0 (d, 2H), 7.65-7.60 (m, 2H), 7.45-7.40 (m, 2H), 5.73-5.65 (t, 1H), 4.50-4 .45 (d, 2H), 4.27-4.24 (t, 1H), 4.20-4.13 (m, 1H), 3.20-3.15 (m, 1H), 2.8 5-2.75 (m, 1H), 2.45-2.40 (m, 1H), 1.60-1.25 (m, 21H), 0.92-0.85 (t, 6H).

[0059] Example 1f Preparation of Oleoyl-Lys(Fmoc)-OH

[0060] Oleoyl-Lys(Fmoc)-OH was prepared by the method of Example 1a using oleoyl chloride instead of octanoyl chloride, with a yield of 92%. MS: 633.40 (M+1) + , HPLC purity>98%.

[0061] 1 H-NMR (500MHz, CDCl3): δ8.10-8.05 (d, 1H), 7.85-7.80 (d, 2H), 7.75-7.70 (d, 2H), 7.65-7.60 (m, 2H), 7.45-7.40 (m, 2H), 5.73-5.65 (t, 1H), 5.35-5.30 (t, 2H), 4.50-4 .45 (d, 2H), 4.27-4.24 (t, 1H), 4.20-4.13 (m, 1H), 3.20-3.15 (m, 1H), 2.85-2.75 (m , 1H), 2.22-2.15 (t, 2H), 2.00-1.85 (m, 3H), 1.65-1.25 (m, 28H), 0.92-0.85 (t, 3H).

[0062] Examples 2a-2f Preparation of Cyclotetrapeptide Derivatives ;

[0063] Example 2a Synthesis of CP4-01

[0064] 1) Dissolve ZT-Cl (9.0 g, 0.01 mol) in methyl tert-butyl ether (150 mL), add Fmoc-Lys(Boc)-OH (7.0 g, 0.015 mol) and N,N-diisopropylethylamine (2.6 g, 0.02 mol), heat to 55°C-60°C for reflux reaction for 8 hours, and cool to room temperature; add diethylenetriamine (6.2 g, 0.06 mol) and mercaptopropionic acid (4.26 g, 0.04 mol) in DMF (40 mL), and heat to 40°C-50°C for reaction; after 2 hours, add water (20 mL) and separate the water layer; wash the organic layer with water until neutral and use it directly in the next step.

[0065] 2) Add Fmoc-Phe-OH (3.8 g, 0.01 mol) and N-methylmorpholine (1.11 g, 0.011 mol) in DMF (40 mL) and DMT-MM (3.1 g, 0.011 mol) in water (20 mL) to the methyl tert-butyl ether solution obtained in the previous step for reaction. After half an hour, separate the water layer; add diethylenetriamine (6.2 g, 0.06 mol) and mercaptopropionic acid (4.25 g, 0.04 mol) in DMF (40 mL) to the methyl tert-butyl ether layer solution, and heat to 40°C-50°C for reaction; after 2 hours, add water (20 mL), separate the water layer, wash the organic layer with water until neutral, and directly proceed to the next step of reaction.

[0066] 3) Follow the method in step 2 to sequentially access Fmoc-Thr(tBu)-OH and n-C7H 15 CO-Lys(Fmoc)-OH to obtain n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-O-ZT.

[0067] 4) n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-O-ZT was dissolved in dichloromethane (200 mL), and 3% TFA-dichloromethane solution (100 mL) was added dropwise. After the addition, the mixture was reacted at room temperature for 1 hour, washed with water to remove TFA, concentrated, and slurried with heptane. The protected tetrapeptide n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH, 7.2 g, yield 90%, HPLC purity>98%. MS ESI: 805.53[M+H] + .

[0068] 5) n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH was dissolved in DMF (700 mL), cooled to 5°C-10°C, and N,N-diisopropylethylamine (2.6 g, 0.02 mol), HOBt (1.35 g, 0.01 mol) and EDCI (1.9 g, 0.01 mol) were added. The mixture was kept at 5°C-10°C for 1 hour, and then heated to room temperature for 12 hours. Water was added to precipitate the solid, which was filtered. The filter cake was slurried with water, filtered, and dried to obtain 6.3 g of protected cyclic tetrapeptide, with a yield of 90%. The HPLC purity was about 95%, and MS ESI: 787.40 [M+H] + .

[0069] 6) The protected cyclotetrapeptide obtained in step 5) was dissolved in dichloromethane (40 mL), trifluoroacetic acid (20 mL) was added, and the mixture was reacted at room temperature for 2 hours. Methyl tert-butyl ether (400 mL) was added to precipitate a solid, which was collected by filtration and dried to obtain 6.0 g of a crude trifluoroacetate salt of the cyclotetrapeptide, with a yield of 100%, a HPLC purity of 90%, and MS ESI: 631.30 [M+H] + .

[0070] 7) The crude product obtained in step 6) was purified and converted to salt using high performance preparative chromatography to obtain 4.4 g of cyclotetrapeptide acetate, with HPLC purity greater than 98%, purification yield of 80%, MS ESI: 631.30 [M+H] + .

[0071] Example 2b Synthesis of CP4-02

[0072] According to the synthesis method of Example 2a, n-C9H 19 CO-Lys(Fmoc)-OH replaces n-C7H 15 CO-Lys(Fmoc)-OH gave 4.74 g of CP4-02 acetate, HPLC purity greater than 98%, total yield 66%, MS ESI: 659.36[M+H] + .

[0073] Example 2c Synthesis of CP4-03

[0074] According to the synthesis method of Example 2a, nC 11 H 23 CO-Lys(Fmoc)-OH replaces n-C7H 15 CO-Lys(Fmoc)-OH gave 4.48 g of acetate of CP4-03, HPLC purity greater than 98%, total yield 60%, MS ESI: 687.34[M+H] + .

[0075] Example 2d Synthesis of CP4-04

[0076] According to the synthesis method of Example 2a, nC 15 H 31 CO-Lys(Fmoc)-OH replaces n-C7H 15CO-Lys(Fmoc)-OH gave 4.41 g of CP4-04 acetate, HPLC purity greater than 98%, total yield 55%, MS ESI: 743.50[M+H] + .

[0077] Example 2e Synthesis of CP4-05

[0078] According to the synthesis method of Example 2a, nC 11 H 23 -5-CO-Lys(Fmoc)-OH replaces n-C7H 15 CO-Lys(Fmoc)-OH gave 4.70 g of CP4-05 acetate, HPLC purity greater than 98%, total yield 63%, MS ESI: 687.40 [M+H] + .

[0079] Example 2f Synthesis of CP4-06

[0080] According to the synthesis method of Example 2a, Oleoyl-Lys(Fmoc)-OH was used to replace n-C7H 15 CO-Lys(Fmoc)-OH gave 5.40 g of acetate of CP4-06, HPLC purity greater than 98%, total yield 65%, MS ESI: 769.54[M+H] + .

[0081] Comparative Example 1

[0082] Commercially available acetate of palmitoyl tetrapeptide-10, purity ≥95%.

[0083] Comparative Example 2

[0084] According to the synthesis method of Example 2a, n-C9H 19 CO-Lys(Fmoc)-OH replaces n-C7H 15 CO-Lys(Fmoc)-OH, and no cyclization reaction was performed, i.e., step 5 in Example 2a was omitted). The remaining steps were the same as in Example 2a, and 5.74 g of acetate of decanoyl tetrapeptide-10 (LP4-02) was obtained, with HPLC purity greater than 98%, a total yield of 78.0%, and MS ESI: 677.40 [M+H] + .

[0085] Comparative Example 3

[0086] According to the method of Example 2a, nC 11 H 23 CO-Lys(Fmoc)-OH replaces n-C7H 15 CO-Lys(Fmoc)-OH, and no cyclization reaction was performed, i.e. step 5 in Example 2a was omitted). The remaining steps were the same as in Example 2a, and 6.27 g of acetate of lauroyl tetrapeptide-10 (LP4-03) was obtained, with HPLC purity greater than 98%, and a total yield of 82.0%, MS ESI: 705.30 [M+H] + .

[0087] Example 3. Stability test The acetate of the cyclotetrapeptide derivative prepared in Examples 2a to 2f was dissolved in an aqueous solution containing 20% ​​ethanol to prepare a cyclotetrapeptide derivative solution with a concentration of 0.5%. The resulting solutions were stored at room temperature of 25° C. and 40±2° C., respectively. The content of the cyclotetrapeptide derivative in each solution was detected at 0 day, 30 days and 60 days. The test results are shown in Table 1.

[0088] Table 1. Stability test results of cyclic tetrapeptide derivatives under different conditions

[0089] As shown in Table 1, the content of the cyclotetrapeptide derivatives did not decrease significantly when stored at room temperature and 40°C for 60 days, indicating that they have high stability and meet the needs of practical applications.

[0090] Example 4. Cytotoxicity assay The acetate salts of the cyclic tetrapeptide derivatives prepared in Examples 2a to 2f and the acetate salts of the linear tetrapeptides in Comparative Examples 1 to 3 were prepared into 5 mM stock solutions using 50% DMSO-water solution, and then diluted with DMEM complete culture medium to prepare a series of peptide solutions.

[0091] Human immortalized keratinocytes (Hacat) were cultured at 2 × 10 4 / well were inoculated in a 96-well plate and cultured in a 37℃, 5% CO2 incubator for 24h. After the culture was completed, the medium in the plate was discarded, and the plate was washed twice with PBS. 100μL of peptide solution was added to each well of the sample group, and 100μL of DMEM complete medium containing equimolar concentration of solvent (50% DMSO-water) was added to each well of the blank control group. The plate was placed in a 37℃, 5% CO2 incubator for 24h. After the culture was completed, the medium in the plate was discarded, and the plate was washed twice with PBS. 100μL of CCK8 working solution was added to each well, and incubated in a 37℃, 5% CO2 incubator for 30min. The absorbance of each well at a wavelength of 450nm was detected using an ELISA reader, and three biological replicates were set for each concentration. The relative cell activity (%) of each group was calculated according to formula (1). The results are shown in Table 2 and expressed as mean ± deviation.

[0092] Relative cell viability (%) = (1) In formula (1): V e —OD value of sample group; V c —OD value of blank control group.

[0093] Table 2 Relative activity of cells in each group (%)

[0094] As shown in Table 2, the straight-chain tetrapeptide has obvious cytotoxicity at 80 μM, and the relative cell activity is reduced to about 55%. The cytotoxicity of the tetrapeptide after cyclization is significantly reduced, and there is no significant cytotoxicity at a concentration of 80 μM, indicating that the cyclic tetrapeptide derivatives of the present invention have good biosafety.

[0095] Example 5 Effects of Cyclic Tetrapeptide on Filaggrin, Hyaluronic Acid and α-Crystallin In this example, the content of filaggrin, hyaluronic acid and α-crystallin was detected by ELISA method, and the specific steps were as follows: The acetate salts of the cyclic tetrapeptide derivatives prepared in Examples 2a to 2f and the acetate salts of the linear tetrapeptides in Comparative Examples 1 to 3 were prepared into 5 mM stock solutions using 50% DMSO-water solution, and then diluted with DMEM complete culture medium to prepare a series of peptide solutions.

[0096] Hacat cells were collected at 2×10 4 / well were inoculated in a 96-well plate and cultured in a 37℃, 5% CO2 incubator for 24h. After the culture, the culture medium in the plate was discarded and washed twice with PBS. 100 μL of peptide solution was added to each well of the sample group, and 100 μL of DMEM complete culture medium containing equimolar solvent (50% DMSO-water) was added to each well of the blank control group. The plate was cultured in a 37℃, 5% CO2 incubator for 24h. After the culture, the supernatant was collected and centrifuged at 4℃, 3000 rpm for 20 min. According to the instructions of the ELISA kit, the supernatant was taken for the detection of filaggrin, hyaluronic acid and α-crystallin content. The expression upregulation rate (%) of filaggrin, hyaluronic acid and α-crystallin in each group was calculated according to formula (2). The results are shown in Table 3 and expressed as mean ± deviation.

[0097] Expression upregulation rate (%) = (2) In formula (2): V e —The content of filaggrin, hyaluronic acid or α-crystallin in the sample group; V c —The content of filaggrin, hyaluronic acid or α-crystallin in the blank control group.

[0098] Table 3 Up-regulation rate of filaggrin, hyaluronic acid and α-crystallin expression in each group of cells (%)

[0099] As shown in Table 3, at a concentration of 30 μM, the linear tetrapeptides of Comparative Examples 1 to 3 not only had no up-regulation effect on the filaggrin, hyaluronic acid and α-crystallin of Hacat cells, but also had a down-regulation phenomenon, which may be caused by its toxic side effects. At the same concentration, the cyclic tetrapeptides CP4-01 to CP4-06 all had up-regulation effects on filaggrin, hyaluronic acid and α-crystallin, among which CP4-02 and CP4-03 had more significant up-regulation effects, and could strengthen the skin barrier, resist damage to the skin by external stimuli, and keep the skin healthy and moisturized.

[0100] At a low concentration of 10 μM, the upregulation effects of cyclic tetrapeptides CP4-02 and CP4-03 on filaggrin, hyaluronic acid and α-crystallin were also significantly higher than those of the linear tetrapeptides of Comparative Examples 1 to 3.

[0101] Example 6 Skin penetration test The skin permeability of the acetate salts of the cyclic tetrapeptides CP4-02 and CP4-03 and the acetate salts of the linear tetrapeptides LP4-02 and LP4-03 prepared in Comparative Examples 2 and 3 was tested by the vertical Franz diffusion cell method. Physiological saline was selected as the receiving medium, and piglet skin with a skin thickness of 0.7 to 0.8 mm was selected for the experiment. The specific experimental steps are as follows: The pig skin was fixed between the supply pool and the receiving pool. The test samples were dissolved in an aqueous solution containing 10% butanol. The final concentration of the sample solution was 0.5%. 0.5±0.01 mL of the sample solutions of CP4-02, CP4-03, LP4-02 and LP4-03 were placed in the supply pool. The rotor speed in the receiving pool was adjusted to 300 rpm. The permeation test was carried out at 32°C. The receiving medium was absorbed from the receiving pool at 12 h, filtered through a 0.22 µm filter membrane, and the active content was detected by HPLC. The 12 h skin permeation was calculated.

[0102] Wipe the sample remaining on the surface of the suckling pig skin with absorbent cotton, cut out the part in contact with the sample and cut into pieces, put them into 2 mL grinding tubes respectively, grind them with a tissue grinder, add 1 mL of 50% DMSO-water solution, ultrasonicate for 30 min, centrifuge at 5000 rpm for 15 min, take the supernatant and filter it through a 0.22 µm filter membrane, and detect the active ingredient content by HPLC, and calculate the 12 h skin retention amount.

[0103] The liquid phase detection conditions were as follows: chromatographic column C18 (4.6 mm × 250 mm, 5 μm), flow rate 1.0 mL / min, column temperature 35 °C, mobile phase A: 0.1% TFA water, mobile phase B: 0.1% TFA acetonitrile, gradient elution, and detection wavelength 220 nm.

[0104] Test results such as Figure 1 As shown: compared with the linear tetrapeptides LP4-02 and LP4-03 prepared in Comparative Examples 2 and 3, the cyclic tetrapeptides CP4-02 and CP4-03 have higher skin retention and lower permeation, indicating that the cyclized tetrapeptides have better skin permeability and can better exert their activity.

[0105] Example 7 Human efficacy test - skin smoothness This example uses Antera 3D ® Skin tester tests skin smoothness.

[0106] Volunteers: Volunteers aged 25-45 years old, healthy, with smooth facial test areas, no wounds, thick skin keratin, and dark skin were selected for skin smoothness test, half of them were male and half were female. Those whose test areas were affected by scars, pigments, atrophy, port wine stains or other blemishes that affect the test results; those with highly sensitive constitutions; those who are pregnant or breastfeeding, or have plans to get pregnant in the near future; those who have used products containing clear chemical exfoliants or promoting metabolism or accelerating keratin renewal in the month before the test are excluded from the scope of test volunteers.

[0107] Grouping: Volunteers were randomly divided into 6 groups, 5 experimental groups and a blank control group, with 25 people in each group. The 5 experimental groups tried the emulsion samples added with cyclic tetrapeptide CP4-02, cyclic tetrapeptide CP4-03, palmitoyl tetrapeptide-10, linear tetrapeptide LP4-02 (Comparative Example 2), and linear tetrapeptide LP4-03 (Comparative Example 3) (the formula is shown in Table 4); the difference between the emulsion samples used in the blank control group and the experimental group is that no cyclic tetrapeptide and linear tetrapeptide are added, that is, pure water is used to make up the formula shown in Table 4.

[0108] Method for volunteers to try lotion samples: After the volunteers clean their faces, take about 2 mg of the sample and apply it evenly on the face, massage until absorbed, and use the sample once in the morning and evening. During the test period, the volunteers' other skin care habits can remain unchanged, but the test parts are prohibited from using skin care products that have exfoliating effects, astringent pores, etc. that affect the skin test. During the test period, the volunteers' living, eating, exercise and other habits should not change significantly.

[0109] Test method: Using Antera 3D ® The skin tester tested the change in skin roughness Ra value and pore volume of the cheek area after 4 weeks of trial use of the lotion sample compared to before the trial use. The test results are shown in Table 5 below.

[0110] Table 4 Formula of trial emulsion samples ;

[0111] Preparation method of emulsion sample: heat and stir phase A and phase B at 80℃-85℃ respectively. After each is evenly dispersed, add phase B to phase A and homogenize for 3min-5min at a temperature of 80℃-85℃ and a stirring speed of 9000r / min. After cooling to 45℃, add phase C and stir to disperse evenly. Then add phase D and phase E that have been evenly dispersed in advance and stir until completely dispersed.

[0112] Table 5 Human efficacy test data results

[0113] Note: The change is calculated as (after use - before use) / before use. The smaller the roughness Ra value and pore volume, the smoother the skin. Indicates p < 0.05; Indicates p<0.01.

[0114] As can be seen from Table 5, cyclic tetrapeptides CP4-02 and CP4-03 can significantly reduce skin roughness and shrink pores, and can significantly improve skin smoothness; while the straight-chain tetrapeptides palmitoyl tetrapeptide-10 and LP4-03 can shrink pores to a certain extent, but the effect is not as significant as that of cyclic tetrapeptides CP4-02 and CP4-03, and there is no obvious effect on improving skin roughness; the straight-chain tetrapeptide LP4-02 has no obvious effect on improving skin roughness and shrinking pores, and cannot significantly improve skin smoothness.

[0115] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A cyclic tetrapeptide derivative or a salt thereof or a stereoisomer thereof, characterized in that: The structural formula of the cyclic tetrapeptide derivative is shown in formula (I): ； （I） Among them, R is selected from: C7~C 19 Alkyl, C7~C 19 Unsaturated chain hydrocarbon group.

2. The cyclic tetrapeptide derivative or its salt or its stereoisomer according to claim 1, characterized in that: R is selected from: n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, 1-hexyl-nonyl, 1-butyl-heptyl, -(CH2)8(CH=CH)(CH2)6CH3, -(CH2)7(CH=CH)(CH2)7CH3, -(CH2)9(CH=CH)(CH2)5CH3, -(CH2)6(CH=CH)(CH2)8CH3.

3. The cyclic tetrapeptide derivative or its salt or its stereoisomer according to claim 1, characterized in that: The cyclic tetrapeptide derivative is selected from the following compounds: ； 。 4. The cyclic tetrapeptide derivative or its salt or its stereoisomer according to any one of claims 1 to 3, characterized in that: The salt of the cyclotetrapeptide derivative is selected from acetate, trifluoroacetate, hydrochloride, sulfate, citrate, ascorbate, lactate, maleate, fumarate, succinate, gluconate and salicylate thereof.

5. Use of the cyclic tetrapeptide derivative or its salt or its stereoisomer as claimed in any one of claims 1 to 4 as an effective ingredient in the preparation of cosmetics, characterized in that: The cosmetics can maintain, repair or improve the condition of the skin.

6. The use according to claim 5, characterized in that: The cosmetic can strengthen the skin barrier.

7. The use according to claim 5, characterized in that: The cosmetic can increase the content of filaggrin, hyaluronic acid and / or α-crystallin in cells.

8. The use according to claim 5, characterized in that: The cosmetic can improve the smoothness of the skin.

9. The use according to claim 5, characterized in that: The cosmetic can reduce skin roughness and / or shrink pores.

10. The use according to any one of claims 5 to 9, characterized in that: The cosmetics include cleansing milk, toner, emulsion, facial mask, facial cream and essence.

11. A cosmetic, characterized in that: The cosmetic contains the cyclotetrapeptide derivative or its salt or stereoisomer according to any one of claims 1 to 4.

12. The cosmetic according to claim 11, characterized in that The mass percentage of the cyclotetrapeptide derivative or its salt or its stereoisomer in the cosmetic is 0.00001% to 1%.

13. The cosmetic according to claim 11 or 12, characterized in that: The cosmetics include cleansing milk, toner, emulsion, facial mask, facial cream and essence.

14. The cosmetic according to claim 11 or 12, characterized in that: The cosmetic is an emulsion, which is prepared from raw materials including the following components by mass percentage: 0.8%-1.2% trehalose, 0.4%-0.5% carbomer, 3%-5% glycerol, 0.4%-0.6% emulsifier A165, 0.6%-1.0% M68 emulsifier, 1.5%-2.5% squalane, 0.8%-1.2% silicone oil, 0.8%-1.2% caprylic capric triglyceride, 1.2%-1.6% behenyl alcohol, 0.2%-0.4% arginine, 0.4%-0.6% cinnamone, 0.4%-0.6% hexylene glycol, 0.0001%-0.001% of the cyclotetrapeptide derivative or its salt or its stereoisomer, 0.001%-0.002% citric acid, 2%-4% dipropylene glycol, and water added to 100%.

15. A method for preparing the cyclic tetrapeptide derivative or a salt thereof according to any one of claims 1 to 4, characterized in that: The steps include: 1) H-Lys(Fmoc)-OH reacts with acyl chloride to obtain RCO-Lys(Fmoc)-OH. The reaction formula is as follows: ； Wherein, R is as described in any one of claims 1 to 3; 2) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH was synthesized; 3) Cyclizing the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH and then deprotecting it to obtain the cyclic tetrapeptide derivative or its salt.

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