Cyclic tetrapeptide derivatives, their preparation methods and applications

Abstract

The present invention relates to a cyclic tetrapeptide derivative, a preparation method and an application thereof. The structural formula of the cyclic tetrapeptide derivative is shown in formula (I), wherein R is selected from: C7-C 19 alkyl, C7-C 19 unsaturated hydrocarbon radical. The cyclic tetrapeptide derivative, its salt or its stereoisomer provided by the present invention can increase the contents of hyaluronic acid, filaggrin and α-crystallin in epidermal cells, thereby effectively enhancing the skin barrier, having an obvious repair and improvement effect on the skin, and having better stability and safety compared with palmitoyl tetrapeptide-10. When used in cosmetics, it can achieve a better effect of repairing and enhancing the skin barrier and has a better improvement effect on the skin. #imgabs0# (I).

Description

Technical Field

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

[0002] Filaggrin is an important structural protein in the stratum corneum, which can aggregate keratin fibers together to form a dense stratum corneum structure; hyaluronic acid has extremely strong water retention ability and is an important component for maintaining skin moisture; α-crystallin is a heat shock protein, which 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 increase of these components in the skin is beneficial to enhancing the skin barrier, enabling the skin to better resist the damage of the external environment, delaying skin aging, and reducing the occurrence of problems such as wrinkles and age spots.

[0003] A polypeptide is an organic compound formed by connecting multiple amino acids through peptide bonds and has the characteristic of high biological activity. Sederma company has developed a cosmetic raw material CRYSTALIDE, whose main active ingredient is palmitoyl tetrapeptide-10 (palm-Lys-Thr-Phe-Lys-OH), which has effects such as improving and / or enhancing the epidermal barrier on the epidermis. Palmitoyl tetrapeptide-10 is a linear peptide. Studies have shown that the molecular structure of the linear peptide is relatively loose and is easily attacked by proteases and degraded. Therefore, it has the defects of poor stability in the cellular 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 contents 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 or a stereoisomer thereof, and the structural formula of the cyclic tetrapeptide derivative is shown as formula (I):

[0007]

[0008] (I)

[0009] Wherein, R is selected from: C7~C 19 alkyl, C7~C 19 unsaturated hydrocarbon radical.

[0010] In a second aspect, the present invention provides the use of the cyclic tetrapeptide derivative or its salt or its stereoisomer as an active ingredient in the preparation of a cosmetic, which can maintain, repair or improve the condition of the skin.

[0011] In a third aspect, the present invention provides a cosmetic containing the cyclic tetrapeptide derivative or its salt or its stereoisomer of the present invention.

[0012] In a fourth aspect, the present invention provides a method for preparing the cyclic tetrapeptide derivative or its salt, comprising the following steps:

[0013] 1) React H-Lys(Fmoc)-OH with an acyl chloride to obtain RCO-Lys(Fmoc)-OH, and the reaction formula is as follows:

[0014]

[0015] Among them, R is selected from: C7~C 19 alkyl, C7~C 19 unsaturated hydrocarbon radical;

[0016] 2) Use Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials to synthesize a linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH;

[0017] 3) Cyclize and deprotect the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH to obtain the cyclic tetrapeptide derivative or its salt.

[0018] The present invention has the following beneficial effects:

[0019] The cyclic tetrapeptide derivative or its salt or its stereoisomer provided by the present invention can increase the contents of hyaluronic acid, filaggrin and α-crystallin in epidermal cells, thereby effectively enhancing the skin barrier and having an obvious 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.

[0020] The cyclic tetrapeptide derivative or its salt or its stereoisomer provided by the present invention can effectively penetrate into the living epidermis layer, has good skin permeability, and has better stability and safety compared with tetrapeptide-10 palmitate. When used in cosmetics, it can play a better role in repairing and enhancing the skin barrier and has a better improvement effect on the skin. Description of the Drawings

[0021] Figure 1Bar charts showing the skin permeation and skin retention of cyclic tetrapeptides CP4-02 and CP4-03 and linear tetrapeptides LP4-02 and LP4-03 prepared in Comparative Examples 2 and 3. Detailed implementation manners

[0022] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0023] For the experimental methods without specific conditions indicated in the following examples, they are generally in accordance with conventional conditions or the conditions recommended by the manufacturers. All kinds of common chemical reagents used in the examples are commercially available products.

[0024] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field 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 used 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.

[0025] 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 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...".

[0026] The term "alkyl" in the present invention refers to saturated aliphatic hydrocarbon groups including branched and straight-chain ones with a specific number of carbon atoms. For example: the definition of "C1-C6 alkyl" for "C1-C6" includes groups with 1, 2, 3, 4, 5 or 6 carbon atoms arranged in a straight-chain or branched manner. For example: "C1-C6 alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl.

[0027] The term "unsaturated hydrocarbon group" of the present invention refers to branched and straight-chain unsaturated aliphatic hydrocarbon groups with 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)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.

[0028] In some of these 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 as shown in formula (I):

[0029]

[0030] (I)

[0031] Wherein, R is selected from: C7~C 19 alkyl, C7~C 19 unsaturated hydrocarbon group.

[0032] 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.

[0033] In some of these 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 19Alkyl group, C7 unsaturated hydrocarbon group with one or two carbon-carbon double bonds, C8 unsaturated hydrocarbon group with one or two carbon-carbon double bonds, C9 unsaturated hydrocarbon group with one or two carbon-carbon double bonds, C 10 unsaturated hydrocarbon group, C 11 unsaturated hydrocarbon group, C 12 unsaturated hydrocarbon group, C 13 unsaturated hydrocarbon group, C 14 unsaturated hydrocarbon group, C 15 unsaturated hydrocarbon group, C 16 unsaturated hydrocarbon group, C 17 unsaturated hydrocarbon group, C 18 unsaturated hydrocarbon group, C 19 unsaturated hydrocarbon group; preferably, the unsaturated hydrocarbon group contains one carbon-carbon double bond.

[0034] In some preferred embodiments, R is selected from: C7 alkyl group, C9 alkyl group, C 11 alkyl group, C 13 alkyl group, C 15 alkyl group, C 15 unsaturated hydrocarbon group, C with one or two carbon-carbon double bonds 17 unsaturated hydrocarbon group, C with one or two carbon-carbon double bonds 19 unsaturated hydrocarbon group.

[0035] In some preferred embodiments, R is selected from: C9 - C 11 linear alkyl group.

[0036] In some preferred embodiments, the cyclic tetrapeptide derivative is selected from the following compounds:

[0037] .

[0038] In some embodiments, the salts of the cyclic tetrapeptide derivative are selected from its acetate, trifluoroacetate, hydrochloride, sulfate, citrate, ascorbate, lactate, maleate, fumarate, succinate, gluconate, salicylate.

[0039] Based on linear peptides, the present invention cyclizes linear peptides through peptide bonds to obtain cyclic tetrapeptide derivatives. The cyclic structure can make the intramolecular hydrogen bonding more compact, thereby reducing the external hydrogen bonding ability of the molecule and enhancing the structural rigidity and protein resistance. This structural feature makes the cyclic peptide more stable in the cellular environment and less susceptible to protease hydrolysis. Therefore, the cyclic tetrapeptide derivatives provided by the present invention have better stability than palmitoyl tetrapeptide-10, with 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 introducing carbon chains of different lengths to improve its skin permeability, thereby obtaining a series of cyclic tetrapeptide derivatives with good skin permeability and stability, which have a more significant repair and improvement effect on the skin.

[0040] The present invention also finds that the cyclic tetrapeptide derivatives of the present invention have lower cytotoxicity than palmitoyl tetrapeptide-10, with better biosafety, and can be added to cosmetics at a higher concentration, thereby obtaining a more excellent effect of repairing and improving the skin.

[0041] The cyclic tetrapeptide derivatives or their salts or their stereoisomers provided by the present invention can increase the contents of hyaluronic acid, filaggrin, and α-crystallin in epidermal cells, thereby effectively enhancing the skin barrier and having an obvious 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. And its permeability and stability are good, and it is safe and non-toxic. Therefore, it can be applied to cosmetics to achieve a good effect of repairing and enhancing the skin barrier, and has a better improvement effect on the skin than palmitoyl tetrapeptide-10.

[0042] Therefore, in some embodiments of the present invention, it relates to the use of the cyclic tetrapeptide derivatives or their salts or their stereoisomers described in the present invention as active ingredients in the preparation of cosmetics.

[0043] In some embodiments of the present invention, it relates to a cosmetic containing the cyclic tetrapeptide derivatives or their salts or their stereoisomers described in the present invention.

[0044] Among them, the cosmetic can maintain, repair or improve the skin condition; for example, the cosmetic can increase the contents of filaggrin, hyaluronic acid and / or α-crystallin in cells, thereby enhancing the skin barrier; it can reduce skin roughness and / or shrink pores, thereby improving skin smoothness.

[0045] The cosmetics of the present invention include but are not limited to daily common cleaning or skin care products such as facial cleanser, lotion, emulsion, facial mask, cream, and essence.

[0046] For example, in some embodiments of the present invention, there is involved an emulsion which contains the cyclic tetrapeptide derivative or its salt or its stereoisomer as described in the present invention, as well as conventional excipients in the emulsion. This emulsion can effectively reduce skin roughness and shrink pores, thereby effectively improving skin smoothness.

[0047] The addition amount of the cyclic tetrapeptide derivative or its salt or its stereoisomer in the cosmetic is based on the criterion that it can produce the effect of maintaining, repairing or improving the skin and does not produce obvious toxicity. Since the cyclic tetrapeptide derivative of the present invention has a significant improvement effect on the skin and very low cytotoxicity, its addition amount can be in a relatively large range. In some of these embodiments, its addition amount is preferably 0.00001% - 1% by mass percentage in the cosmetic, more preferably 0.0001% - 1%, more preferably 0.0001% - 0.1%, more preferably 0.0001% - 0.01%, more preferably 0.0001% - 0.001%, and more preferably 0.0005% - 0.001%.

[0048] The present invention has no particular limitation on the excipients in the cosmetic (such as emulsion), and conventional excipients in the cosmetic can be used in the present invention to prepare a cosmetic having a repairing or improving effect on the skin. For example, in some embodiments of the present invention, there is provided an emulsion which is prepared from raw materials including the following components by mass percentage:

[0049] Water Up to 100% Trehalose 0.8%~1.2% Carbomer 0.4%~0.5% Glycerin 3%~5% Emulsifier A165 0.4%~0.6% Emulsifier M68 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% Triclosan 0.4%~0.6% Hexylene glycol 0.4%~0.6% The cyclic tetrapeptide derivative or its salt or its stereoisomer 0.0001%~0.001% Citric acid 0.001%~0.002% Dipropylene glycol 2%~4%

[0050] The cyclic tetrapeptide derivative or its salt of the present invention can be prepared by conventional solid-phase synthesis or liquid-phase synthesis methods in the art, and the amino acid raw material with hydrocarbon side chain modification can be obtained by reacting the corresponding amino acid with an acyl halide compound.

[0051] For example, the cyclic tetrapeptide derivative or its salt as described in the present invention can be prepared by the following steps:

[0052] 1) H-Lys(Fmoc)-OH reacts with acyl chloride to obtain RCO-Lys(Fmoc)-OH, and the reaction formula is as follows:

[0053]

[0054] Among them, R is selected from C7 - C 19 alkyl, C7 - C 19 unsaturated hydrocarbon radical; further as described above;

[0055] 2) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, synthesize the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH;

[0056] 3) Cyclize and deprotect the linear tetrapeptide RCO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH to obtain the cyclic tetrapeptide derivative or its salt.

[0057] Among them, the linear tetrapeptide can be prepared by conventional solid-phase synthesis or liquid-phase synthesis methods in the art, preferably prepared by 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 finally obtained product can also be further purified by methods such as crystallization or high-performance preparative chromatography.

[0058] The compounds corresponding to the abbreviations and acronyms involved in the present invention are described as follows:

[0059] DMF: N,N-dimethylformamide;

[0060] ZT-Cl: 1-phenyl-1-(2-chlorophenyl)-1-(N-α-tocopheroxyl-ethyl-N-benzyl-4-benzamidino)-methyl chloride;

[0061] DMT-MM: 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride;

[0062] TFA: Trifluoroacetic acid;

[0063] HOBt: 1-Hydroxybenzotriazole;

[0064] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0065] DCC: Dicyclohexylcarbodiimide;

[0066] HBTU: O-Benzotriazole-tetramethyluronium hexafluorophosphate;

[0067] HATU: 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0068] BOP: Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate;

[0069] Fmoc: 9-Fluorenylmethoxycarbonyl;

[0070] Boc: tert-Butyloxycarbonyl;

[0071] Lys: Lysine;

[0072] Phe: Phenylalanine;

[0073] Thr: Threonine;

[0074] tBu: tert-Butyl;

[0075] In the following examples, room temperature or normal temperature refers to 20°C - 25°C.

[0076] The present invention will be further described in detail below with reference to specific examples.

[0077] Example 1a - 1f Synthesis of RCO-Lys(Fmoc)-OH

[0078]

[0079] Example 1a n-C7H 15 Preparation of CO-Lys(Fmoc)-OH

[0080]

[0081] Dissolve H-Lys(Fmoc)-OH (36.8 g, 0.1 mol) in DMF (500 mL), cool down to 5°C - 10°C, add N,N-diisopropylethylamine (25.8 g, 0.2 mol), stir for half an hour after addition, dropwise add octanoyl chloride (16.2 g, 0.1 mol), keep the temperature at 5°C - 10°C for 1 hour after addition, pour the reaction solution into 10% aqueous citric acid solution, filter the precipitated solid, wash with purified water, and dry to obtain n-C7H 15 CO-Lys(Fmoc)-OH 44.5 g, with a yield of 90%. MS: 495.28 (M+1) + , HPLC purity > 98%.

[0082] 11H-NMR (500 MHz, CDCl3): δ 8.10 - 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), 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), 1.60 - 1.25 (m, 16H), 0.92 - 0.85 (t, 3H).

[0083] Example 1b n-C9H 19 Preparation of CO-Lys(Fmoc)-OH

[0084]

[0085] n-C9H-CO-Lys(Fmoc)-OH was prepared by the method of Example 1a using decanoyl chloride instead of octanoyl chloride 19 CO-Lys(Fmoc)-OH, yield 85%. MS: 523.20 (M+1) + , HPLC purity > 98%.

[0086] 1 1H-NMR (500 MHz, CDCl3): δ 8.10 - 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), 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), 1.60 - 1.25 (m, 20H), 0.92 - 0.85 (t, 3H).

[0087] Example 1c n-C 11 H 23 Preparation of CO-Lys(Fmoc)-OH

[0088]

[0089] n-C-H-CO-Lys(Fmoc)-OH 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%.

[0090] 1 1H-NMR (500 MHz, CDCl3): δ 8.10 - 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), 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), 1.60 - 1.25 (m, 24H), 0.92 - 0.85 (t, 3H).

[0091] Example 1d n-C 15 H 31 Preparation of n-C

[0092]

[0093] Using palmitoyl chloride instead of octanoyl chloride, n-C 15 H 31 CO-Lys(Fmoc)-OH was prepared with a yield of 88%. MS: 607.41 (M+1) + , HPLC purity > 98%.

[0094] 1 1H-NMR (500 MHz, CDCl3): δ 8.10 - 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), 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), 1.60 - 1.25 (m, 32H), 0.92 - 0.85 (t, 3H).

[0095] Example 1e n-C 11 H 23 -5-CO-Lys(Fmoc)-OH Preparation

[0096]

[0097] Prepared by the method of Example 1a using 2-butyl octanoyl chloride instead of octanoyl chloride, n-C 11 H 23 -5-CO-Lys(Fmoc)-OH, with a yield of 85%. MS: 551.36 (M+1) + , HPLC purity > 98%.

[0098] 1 H-NMR(500 MHz, 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), 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), 2.45 - 2.40 (m, 1H), 1.60 - 1.25 (m, 21H), 0.92 - 0.85 (t, 6H).

[0099] Example 1f Preparation of Oleoyl-Lys(Fmoc)-OH

[0100]

[0101] 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%.

[0102] 1 H-NMR(500 MHz, 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).

[0103] Example 2a - 2f Preparation of Cyclic Tetrapeptide Derivatives

[0104]

[0105]

[0106] Synthesis of Example 2a CP4-01

[0107]

[0108] 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 reflux at 55 °C - 60 °C for 8 hours, and cool to room temperature; add a DMF (40 mL) solution of diethylenetriamine (6.2 g, 0.06 mol) and mercaptopropionic acid (4.26 g, 0.04 mol), and raise the temperature to 40 °C - 50 °C for reaction; after 2 hours, add water (20 mL), separate the aqueous layer; wash the organic layer with water until neutral, and directly use it for the next step of reaction.

[0109] 2) Add a DMF (40 mL) solution of Fmoc-Phe-OH (3.8 g, 0.01 mol) and N-methylmorpholine (1.11 g, 0.011 mol) and a water (20 mL) solution of DMT-MM (3.1 g, 0.011 mol) to the methyl tert-butyl ether solution obtained in the previous step for reaction. After half an hour, separate the aqueous layer; add a DMF (40 mL) solution of diethylenetriamine (6.2 g, 0.06 mol) and mercaptopropionic acid (4.25 g, 0.04 mol) to the methyl tert-butyl ether layer solution, and raise the temperature to 40 °C - 50 °C for reaction; after 2 hours, add water (20 mL), separate the aqueous layer, and wash the organic layer with water until neutral, and directly proceed to the next step of reaction.

[0110] 3) Connect Fmoc-Thr(tBu)-OH and n-C7H 15 CO-Lys(Fmoc)-OH in sequence according to the method of step 2 to obtain n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-O-ZT.

[0111] 4) Dissolve n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-O-ZT in dichloromethane (200 mL), dropwise add a 3% TFA-dichloromethane solution (100 mL), after dropping, react at room temperature for 1 hour, wash with water to remove TFA, concentrate, add heptane for pulping, filter to obtain the protected tetrapeptide n-C7H 15CO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH, 7.2 g, yield 90%, HPLC purity > 98%. MS ESI: 805.53 [M+H] + .

[0112] 5) Dissolve n-C7H 15 CO-Lys-Thr(tBu)-Phe-Lys(Boc)-OH in DMF (700 mL), cool to 5°C - 10°C, add N,N-diisopropylethylamine (2.6 g, 0.02 mol), HOBt (1.35 g, 0.01 mol) and EDCI (1.9 g, 0.01 mol), maintain the temperature at 5°C - 10°C and react for 1 hour, then raise the temperature to room temperature and react for 12 hours. Add water to precipitate a solid, filter, wash the filter cake with water, filter again and dry to obtain 6.3 g of the protected cyclic tetrapeptide, yield 90%, HPLC purity about 95%, MS ESI: 787.40 [M+H] + .

[0113] 6) Dissolve the protected cyclic tetrapeptide obtained in step 5) in dichloromethane (40 mL), add trifluoroacetic acid (20 mL), react at room temperature for 2 hours, add methyl tert-butyl ether (400 mL) to precipitate a solid, filter and collect the solid, dry to obtain 6.0 g of the crude trifluoroacetate salt of the cyclic tetrapeptide, yield 100%, HPLC purity 90%, MS ESI: 631.30 [M+H] + .

[0114] 7) Purify and convert the salt of the crude product obtained in step 6) using high-performance preparative chromatography to obtain 4.4 g of the acetate salt of the cyclic tetrapeptide, HPLC purity > 98%, purification yield 80%, MS ESI: 631.30 [M+H] + .

[0115] Example 2b Synthesis of CP4-02

[0116]

[0117] According to the synthesis method of Example 2a, use n-C9H 19 CO-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH to obtain 4.74 g of the acetate salt of CP4-02, HPLC purity > 98%, total yield 66%, MS ESI: 659.36 [M+H] + .

[0118] Example 2c Synthesis of CP4-03

[0119]

[0120] According to the synthesis method of Example 2a, using n-C 11 H 23 CO-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH, 4.48 g of acetate of CP4-03 was obtained, with HPLC purity greater than 98%, total yield 60%, MS ESI: 687.34[M+H] + .

[0121] Synthesis of Example 2d CP4-04

[0122]

[0123] According to the synthesis method of Example 2a, using n-C 15 H 31 CO-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH, 4.41 g of acetate of CP4-04 was obtained, with HPLC purity greater than 98%, total yield 55%, MS ESI: 743.50[M+H] + .

[0124] Synthesis of Example 2e CP4-05

[0125]

[0126] According to the synthesis method of Example 2a, using n-C 11 H 23 -5-CO-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH, 4.70 g of acetate of CP4-05 was obtained, with HPLC purity greater than 98%, total yield 63%, MS ESI:687.40[M+H] + .

[0127] Synthesis of Example 2f CP4-06

[0128]

[0129] According to the synthesis method of Example 2a, using Oleoyl-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH, 5.40 g of acetate of CP4-06 was obtained, with HPLC purity greater than 98%, total yield 65%, MS ESI: 769.54[M+H] + .

[0130] Comparative Example 1

[0131]

[0132] The commercially available acetate of palmitoyl tetrapeptide-10, with a purity ≥ 95%.

[0133] Comparative Example 2

[0134]

[0135] According to the synthesis method of Example 2a, using n-C9H 19 CO-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH, and without carrying out the cyclization reaction, that is, omitting step 5) in Example 2a, and the remaining steps are the same as those in Example 2a, to obtain 5.74 g of the acetate of decanoyl tetrapeptide-10 (LP4-02), with an HPLC purity greater than 98%, a total yield of 78.0%, MS ESI: 677.40[M+H] + .

[0136] Comparative Example 3

[0137]

[0138] According to the method of Example 2a, using n-C 11 H 23 CO-Lys(Fmoc)-OH to replace n-C7H 15 CO-Lys(Fmoc)-OH, and without carrying out the cyclization reaction, that is, omitting step 5) in Example 2a, and the remaining steps are the same as those in Example 2a, to obtain 6.27 g of the acetate of lauroyl tetrapeptide-10 (LP4-03), with an HPLC purity greater than 98%, a total yield of 82.0%, MS ESI: 705.30[M+H] + .

[0139] Example 3. Stability Test

[0140] Respectively dissolve the acetates of the cyclic tetrapeptide derivatives prepared in Examples 2a - 2f in an aqueous solution containing 20% ethanol to prepare a 0.5% solution of the cyclic tetrapeptide derivative. Store the obtained solutions at 25 °C at room temperature and 40 ± 2 °C respectively, and detect the content of the cyclic tetrapeptide derivative in each solution at 0 day, 30 days and 60 days. The test results are shown in Table 1.

[0141] Table 1. Stability test results of each cyclic tetrapeptide derivative under different conditions

[0142]

[0143] As can be seen from Table 1, the content of the cyclic tetrapeptide derivative did not significantly decrease after being stored at room temperature and 40 °C for 60 days, indicating its high stability and meeting the requirements of practical applications.

[0144] Example 4. Cytotoxicity Detection

[0145] The acetates of the cyclic tetrapeptide derivatives prepared in Examples 2a - 2f and the acetates of the linear tetrapeptides in Comparative Examples 1 - 3 were respectively formulated into stock solutions with a concentration of 5 mM using 50% DMSO - aqueous solution, and then serially diluted with DMEM complete medium to obtain a series of peptide solutions.

[0146] Human immortalized keratinocytes (Hacat) were seeded at 2×10 4 / well in a 96 - well plate and cultured in an incubator at 37 °C and 5% CO2 for 24 h. After the culture, the medium in the well plate was discarded, and the wells were washed twice with PBS. 100 μL of the peptide solution was added to each well in the sample group, and 100 μL of DMEM complete medium containing an equimolar concentration of solvent (50% DMSO - water) was added to each well in the blank control group. The well plate was placed in an incubator at 37 °C and 5% CO2 for 24 h. After the culture, the medium in the well plate was discarded, and the wells were washed twice with PBS. 100 μL of CCK8 working solution was added to each well and incubated in an incubator at 37 °C and 5% CO2 for 30 min. The absorbance of each well at a wavelength of 450 nm was measured using a microplate reader, and 3 biological replicates were set for each concentration. The relative cell viability (%) of each group was calculated according to formula (1), and the results are shown in Table 2, expressed as mean ± deviation.

[0147] Relative cell viability (%) = (1)

[0148] In formula (1):

[0149] V e —OD value of the sample group;

[0150] V c —OD value of the blank control group.

[0151] Table 2 Relative viability of cells in each group (%)

[0152]

[0153] As can be seen from Table 2, the linear tetrapeptide had obvious cytotoxicity at 80 μM, and the relative cell viability decreased to about 55%. The cytotoxicity of the cyclized tetrapeptide to cells was significantly reduced, and there was no significant cytotoxicity at a concentration of 80 μM, indicating that the cyclic tetrapeptide derivative of the present invention has good biosafety.

[0154] Example 5 Effects of Cyclic Tetrapeptide on Filaggrin, Hyaluronic Acid and α - Crystallin in Cells

[0155] In this example, the contents of filaggrin, hyaluronic acid, and α-crystallin in cells were detected by ELISA method, and the specific steps are as follows:

[0156] The acetates of the cyclic tetrapeptide derivatives prepared in Examples 2a - 2f and the acetates of the linear tetrapeptides in Comparative Examples 1 - 3 were respectively prepared into stock solutions with a concentration of 5 mM using 50% DMSO - aqueous solution, and then serially diluted with DMEM complete medium to obtain serial peptide solutions.

[0157] Hacat cells were seeded at 2×10 4 / well in a 96 - well plate and cultured in an incubator at 37°C and 5% CO2 for 24 h. After the culture, the medium in the well plate was discarded, and the cells were washed twice with PBS. 100 μL of peptide solution was added to each well in the sample group, and 100 μL of DMEM complete medium containing an equimolar concentration of solvent (50% DMSO - water) was added to each well in the blank control group. The well plate was then cultured in an incubator at 37°C and 5% CO2 for 24 h. After the culture, the supernatant was collected, centrifuged at 3000 rpm for 20 min at 4°C. According to the operation instructions of the ELISA kit, the supernatant was taken for the detection of the contents of filaggrin, hyaluronic acid, and α - crystallin. The up - regulation rate (%) of the expression of filaggrin, hyaluronic acid, and α - crystallin in each group was calculated according to formula (2), and the results are shown in Table 3, expressed as mean ± deviation.

[0158] Up - regulation rate (%) = (2)

[0159] In formula (2):

[0160] V e — the content of filaggrin, hyaluronic acid, or α - crystallin in the sample group;

[0161] V c — the content of filaggrin, hyaluronic acid, or α - crystallin in the blank control group.

[0162] Table 3 Up - regulation rate (%) of the expression of filaggrin, hyaluronic acid, and α - crystallin in cells of each group

[0163]

[0164] As can be seen from Table 3, at a concentration of 30 μM, the linear tetrapeptides of Comparative Examples 1 to 3 not only did not up-regulate filaggrin, hyaluronic acid, and α-crystallin in Hacat cells, but also showed a down-regulation phenomenon, which may be caused by their toxic side effects. At the same concentration, the cyclic tetrapeptides CP4-01 to CP4-06 up-regulated filaggrin, hyaluronic acid, and α-crystallin. Among them, CP4-02 and CP4-03 had more significant up-regulation effects, were more capable of strengthening the skin barrier, resisting the damage of external stimuli to the skin, and keeping the skin healthy, moist, and hydrated.

[0165] At a low concentration of 10 μM, the up-regulation effects of the 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.

[0166] Example 6 Skin Permeation Ability Test

[0167] The vertical Franz diffusion cell method was used to test the skin permeation abilities of the acetates of the cyclic tetrapeptides CP4-02 and CP4-03 and the acetates of the linear tetrapeptides LP4-02 and LP4-03 prepared in Comparative Examples 2 and 3. Normal saline was selected as the receiving medium, and the skin of suckling pigs with a skin thickness of 0.7 - 0.8 mm was selected for the experiment. The specific experimental steps are as follows:

[0168] The pig skin was fixed between the supply cell and the receiving cell. The test samples were dissolved in an aqueous solution containing 10% butanol, and 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 respectively placed in the supply cell. The rotation speed of the rotor in the receiving cell was adjusted to 300 rpm, and the transdermal test was carried out at 32 °C. The receiving medium was aspirated from the receiving cell at 12 h, filtered through a 0.22 µm filter membrane, and the active ingredient content was detected by HPLC to calculate the skin permeation amount at 12 h.

[0169] The sample residues on the surface of the suckling pig skin were wiped clean with absorbent cotton, the part in contact with the sample was cut out and shredded, and they were respectively put into 2 mL grinding tubes. After grinding with a tissue grinder, 1 mL of 50% DMSO-aqueous solution was added, ultrasonicated for 30 min, centrifuged at 5000 rpm for 15 min, and the supernatant liquid was filtered through a 0.22 µm filter membrane and the active ingredient content was detected by HPLC to calculate the skin retention amount at 12 h.

[0170] 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, detection wavelength 220 nm.

[0171] The test results are as follows Figure 1 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 cyclic tetrapeptides have better skin permeability and can better exert their activity.

[0172] Example 7 Human Efficacy Test - Skin Smoothness

[0173] In this example, an Antera 3D ® skin tester was used to test skin smoothness.

[0174] Volunteers: Volunteers aged 25 - 45 years old, with good health, flat test sites on the face, no wounds, thick skin cutin, and dull skin were selected for the skin smoothness test, with an equal number of men and women. Those whose test sites are affected by scars, pigmentation, atrophy, nevus flammeus or other defects and affect the determination of test results; those with highly sensitive constitutions; those who are pregnant or lactating, or have a recent pregnancy plan; those who have used products with functions such as clear chemical exfoliation, promoting metabolism or accelerating cutin renewal within one month before the test were excluded from the scope of test volunteers.

[0175] Grouping: The 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 were respectively given 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 sample used in the blank control group and the experimental groups is that no cyclic tetrapeptide and linear tetrapeptide are added, and the formula shown in Table 4 is made up with pure water.

[0176] Method for volunteers to use the emulsion sample: After the volunteers' faces were cleaned, about 2 mg of the sample was taken and evenly applied to the face, and massaged until absorbed. The sample was used once in the morning and once in the evening. During the test, the other skin care habits of the volunteers could remain unchanged, but skin care products with functions such as exfoliation and pore shrinking that affect skin testing were prohibited on the test sites. During the test period, the living, diet, exercise and other habits of the volunteers should not change significantly.

[0177] Test method: Use an Antera 3D ® skin tester to test the change amount of the skin roughness Ra value and pore volume of the cheek area after 4 weeks of using the emulsion sample compared with before using. The test results are shown in Table 5 below.

[0178] Table 4 Formulas of the emulsion samples for trial use

[0179]

[0180]

[0181] Preparation method of emulsion sample: Heat and stir phase A and phase B at a temperature of 80°C - 85°C respectively. After each is uniformly dispersed, add phase B to phase A, and homogenize for 3 min - 5 min under the conditions of a temperature of 80°C - 85°C and a stirring speed of 9000 r / min. After cooling to 45°C, add phase C, stir and disperse uniformly, then add phase D and phase E which have been previously dispersed uniformly, and stir until completely dispersed uniformly.

[0182] Table 5 Results of human efficacy test data

[0183]

[0184] Note: The change amount is calculated as (after use - before use) / before use. The smaller the Ra value of roughness and the pore volume, the higher the skin smoothness; Indicates p < 0.05; Indicates p < 0.01.

[0185] As can be seen from Table 5, the cyclic tetrapeptides CP4 - 02 and CP4 - 03 can significantly reduce skin roughness and shrink pores, and can significantly improve skin smoothness; while the linear tetrapeptides palmitoyl tetrapeptide - 10 and LP4 - 03 can shrink pores to a certain extent, but the effect is not as significant as that of the cyclic tetrapeptides CP4 - 02 and CP4 - 03, and there is no obvious effect on improving skin roughness; the linear tetrapeptide LP4 - 02 has no obvious effect on improving skin roughness and shrinking pores, and cannot significantly improve skin smoothness.

[0186] The above - described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A cyclic tetrapeptide derivative or a salt thereof, characterized in that, The structural formula of the cyclic tetrapeptide derivative is shown in Formula (I): （I） Among them, R is selected from: C9-C 11 linear alkyl group.

2. The cyclic tetrapeptide derivative or a salt thereof according to claim 1, wherein, R is selected from: n-nonyl, n-decyl or n-undecyl.

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

5. Use of the cyclic tetrapeptide derivative or its salt according to any one of claims 1-4 as an active ingredient in the preparation of cosmetics, characterized in that, The cosmetic can repair or improve the skin condition.

6. The application according to claim 5, characterized in that The cosmetic can enhance the skin barrier.

7. The application 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 application according to claim 5, wherein The cosmetic can improve the smoothness of the skin.

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

10. The application according to any one of claims 5-9, characterized in that, The cosmetic includes a facial cleanser, lotion, emulsion, facial mask, cream or essence.

11. A cosmetic, characterized in that, The cosmetic contains the cyclic tetrapeptide derivative or its salt according to any one of claims 1-4.

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

13. The cosmetic according to claim 12, characterized in that, The mass percentage of the cyclic tetrapeptide derivative or its salt in the cosmetic is 0.0001% - 1%.

14. The cosmetic according to claim 13, characterized in that, The mass percentage of the cyclic tetrapeptide derivative or its salt in the cosmetic is 0.0001% - 0.1%.

15. The cosmetic according to claim 14, characterized in that, The mass percentage of the cyclic tetrapeptide derivative or its salt in the cosmetic is 0.0001% - 0.01%.

16. The cosmetic according to claim 15, characterized in that, The mass percentage of the cyclic tetrapeptide derivative or its salt in the cosmetic is 0.0001% - 0.001%.

17. The cosmetic according to claim 16, characterized in that, The mass percentage of the cyclic tetrapeptide derivative or its salt in the cosmetic is 0.0005% - 0.001%.

18. The cosmetic according to any one of claims 11-17, characterized in that, The cosmetic includes a facial cleanser, lotion, emulsion, facial mask, cream or essence.

19. The cosmetic according to any one of claims 11-16, characterized in that, The cosmetic is an emulsion. Calculated by mass percentage, the emulsion is prepared from raw materials including the following components: trehalose 0.8% - 1.2%, carbomer 0.4% - 0.5%, glycerol 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%, sym-xylenol 0.4% - 0.6%, hexylene glycol 0.4% - 0.6%, the cyclic tetrapeptide derivative or its salt 0.0001% - 0.001%, citric acid 0.001% - 0.002%, dipropylene glycol 2% - 4% and water up to 100%.

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

Images