Cyclooctapeptide derivatives for increasing expression of alpha-crystallin and methods of making and using the same

Abstract

This invention relates to a cyclic octapeptide derivative having the structure shown in formula (I) or a salt thereof or a stereoisomer thereof, a method for its preparation, and its applications, wherein each R is independently selected from: C5~C 13 Alkyl, C5~C 13 Unsaturated chain hydrocarbon group. The cyclic octapeptide derivatives or their salts or stereoisomers provided by this invention have good skin permeability, can significantly increase the expression of α-crystallin, filaggrin and hyaluronic acid in epidermal cells, and the effect is significantly better than cyclic tetrapeptide derivatives, and far superior to commercially available linear peptide palmitoyl tetrapeptide-10, which can more effectively repair and enhance the skin barrier, and improve skin smoothness and translucency. Furthermore, the cyclic octapeptide derivatives of this invention achieve high bioactivity while having good stability and safety. (I)

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology and relates to cyclic peptide derivatives, specifically to a cyclic octapeptide derivative that enhances the expression of α-crystal protein, its preparation method, and its application. Background Technology

[0002] The skin is the body's first line of defense against external environmental factors such as ultraviolet radiation and pollutants. A healthy skin barrier relies on the synergistic action of various key proteins and molecules. Alpha-crystallin, a small-molecule heat shock protein, plays a crucial role in maintaining cellular homeostasis and resisting stress damage. Increased expression of alpha-crystallin is significant for strengthening the skin barrier and delaying skin aging.

[0003] Peptide components are widely used in functional cosmetics due to their high bioactivity. To overcome the drawbacks of linear peptides, such as easy degradation by proteases and poor stability in biological environments, the inventors of this invention previously proposed cyclizing tetrapeptides with specific sequences and introducing hydrophobic side chains of a certain length, resulting in a series of cyclic tetrapeptide derivatives modified with hydrophobic side chains. These cyclic tetrapeptide derivatives significantly improve the structural rigidity and enzyme stability of peptide molecules by forming a cyclic structure, while the introduction of hydrophobic side chains of a certain length improves their skin permeability, thus achieving good results in enhancing the skin barrier (CN119735635A).

[0004] However, as the market's demands for the efficacy of cosmetics continue to increase, those skilled in the art have been pursuing a new generation of active ingredients with higher activity and more significant effects. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide a cosmetic efficacy ingredient that can highly enhance the expression of α-crystal protein.

[0006] The technical solutions for achieving the above objectives include the following.

[0007] In a first aspect, the present invention provides a cyclic octapeptide derivative having the structure shown in formula (I) or a salt thereof or a stereoisomer thereof.

[0008]

[0009] (I)

[0010] Each R is independently selected from: C5~C 13 Alkyl, C5~C 13 Unsaturated chain hydrocarbon group.

[0011] Secondly, the present invention provides the application of the aforementioned cyclic octapeptide derivative or its salt or its stereoisomer as an active ingredient in the preparation of cosmetics, wherein the cosmetics can repair or improve skin condition, for example, the cosmetics can enhance the skin barrier and / or delay skin aging.

[0012] Thirdly, the present invention provides a cosmetic product in which the active ingredient contains the cyclic octapeptide derivative or its salt or its stereoisomer described in the present invention.

[0013] Fourthly, the present invention provides a method for preparing the cyclic octapeptide derivative or its salt according to the present invention, characterized by comprising the following steps:

[0014] 1) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, a linear octapeptide RCO-N was synthesized. 6 -[RCO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH; where each R is independently selected from C5~C6. 13 Alkyl, C5~C 13 Unsaturated chain hydrocarbon groups;

[0015] 2) The linear octapeptide RCO-N 6 Cyclization of -[RCO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH followed by deprotection yields the cyclic octapeptide derivative or its salt.

[0016] The present invention has the following beneficial effects:

[0017] The cyclic octapeptide derivative, its salt, or its stereoisomer provided by this invention optimizes the oil-water partition coefficient of the cyclic octapeptide by optimizing its size and introducing a hydrophobic side chain of a specific length. This results in excellent skin permeability, significantly increasing the expression of α-crystallin, filaggrin, and hyaluronic acid in epidermal cells. The effect is significantly superior to cyclic tetrapeptide derivatives and far superior to the commercially available linear peptide palmitoyl tetrapeptide-10. It more effectively repairs and strengthens the skin barrier, protects the skin from oxidative damage, and improves skin smoothness and translucency. Furthermore, the cyclic octapeptide derivative of this invention achieves high bioactivity while exhibiting excellent stability and safety. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0020] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0021] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0022] The term "alkyl" in this invention refers to a saturated aliphatic hydrocarbon group, including both branched and straight-chain groups 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. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0023] The term "unsaturated chain hydrocarbon group" in this invention refers to a branched or straight-chain unsaturated aliphatic hydrocarbon group with a specific number of carbon atoms, i.e., a non-cyclic chain hydrocarbon group, 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.

[0024] Some embodiments of the present invention relate to a cyclic octapeptide derivative having the structure shown in formula (I) or a salt thereof or a stereoisomer thereof.

[0025]

[0026] (I)

[0027] Each R is independently selected from: C5~C 13 Alkyl, C5~C 13 Unsaturated chain hydrocarbon group.

[0028] In some implementations, each R is independently selected from: C9~C 11 Alkyl group, C9~C 11 Unsaturated chain hydrocarbon group.

[0029] In some of these embodiments, each R is independently selected from: C5 alkyl, C5 unsaturated chain hydrocarbon.

[0030] In some of these embodiments, each R is independently selected from: C6 alkyl, C6 unsaturated chain hydrocarbon.

[0031] In some embodiments, each R is independently selected from: C7 alkyl, C7 unsaturated chain hydrocarbon.

[0032] In some of these embodiments, each R is independently selected from: C8 alkyl, C8 unsaturated chain hydrocarbon.

[0033] In some of these embodiments, each R is independently selected from: C9 alkyl, C9 unsaturated chain hydrocarbon.

[0034] In some implementations, each R is independently selected from: C 10 Alkyl, C 10 Unsaturated chain hydrocarbon group.

[0035] In some implementations, each R is independently selected from: C 11 Alkyl, C 11 Unsaturated chain hydrocarbon group.

[0036] In some implementations, each R is independently selected from: C 12 Alkyl, C 12 Unsaturated chain hydrocarbon group.

[0037] In some implementations, each R is independently selected from: C 13 Alkyl, C 13 Unsaturated chain hydrocarbon group.

[0038] In some embodiments, each R is independently selected from: n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and n-tridecyl.

[0039] In some embodiments, the cyclic octapeptide derivative is selected from the following compounds:

[0040] .

[0041] In some embodiments, the salt of the cyclic octapeptide derivative is selected from its acetate, trifluoroacetate, hydrochloride, sulfate, citrate, ascorbate, lactate, maleate, fumarate, succinate, gluconate, and salicylate.

[0042] Some embodiments of the present invention also relate to the use of the cyclic peptide derivatives thereof, or their salts thereof, or their stereoisomers, as active ingredients in the preparation of cosmetics capable of repairing the skin.

[0043] Some embodiments of the present invention also relate to the use of the aforementioned cyclic octapeptide derivatives or their salts or stereoisomers as active ingredients in the preparation of cosmetics that can enhance the skin barrier and / or delay skin aging.

[0044] Some embodiments of the present invention also relate to the use of the aforementioned cyclic octapeptide derivatives or their salts or stereoisomers as active ingredients in the preparation of antioxidant cosmetics.

[0045] In some embodiments, the cosmetic can increase the content of filaggrin, hyaluronic acid and / or α-crystallin in cells.

[0046] Some embodiments of the present invention also relate to a cosmetic product in which the active ingredient contains the cyclic octapeptide derivative or its salt or its stereoisomer described in the present invention.

[0047] The cosmetics described in this invention include, but are not limited to, daily cleansing or skincare products such as facial cleansers, toners, lotions, masks, creams, and serums.

[0048] The amount of the cyclic octapeptide derivative, its salt, or its stereoisomer added to the cosmetic is determined by its ability to maintain, repair, or improve the skin without producing significant toxicity. Because the cyclic octapeptide derivative of this invention has significant skin-improving effects and very low cytotoxicity, its addition amount can be within a wide range.

[0049] This invention does not impose any particular restrictions on excipients in cosmetics. Conventional excipients in cosmetics can be used in this invention to prepare cosmetics that have a repairing or improving effect on the skin.

[0050] The cyclic octapeptide derivatives or their salts of the present invention can be prepared by conventional solid-phase or liquid-phase synthesis methods in the art, wherein the amino acid raw materials with hydrocarbon side chain modifications can be obtained by reacting the corresponding amino acids with acyl halide compounds.

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

[0052] 1) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, a linear octapeptide RCO- was synthesized. N 6 - [RCO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH; where R is independently selected from C5~C6. 13 Alkyl, C5~C 13 Unsaturated chain hydrocarbon groups;

[0053] 2) The linear octapeptide RCO-N 6 Cyclization of -[RCO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH followed by deprotection yields the cyclic octapeptide derivative or its salt.

[0054] The linear octapeptide can be prepared by conventional solid-phase or liquid-phase synthesis methods in the art, preferably by the EMPHASES liquid-phase method. Cyclization is completed by dehydration under the action of a condensing reagent, which is selected from EDCI, DCC, HBTU, HATU, BOP, HOBt, etc. Deprotection can be carried out using conventional reagents in the art, such as trifluoroacetic acid and hydrogen chloride. The final product can be further purified by crystallization or high-performance preparative chromatography.

[0055] The compounds corresponding to the abbreviations and short names involved in this invention are described below:

[0056] DMF: N,N-dimethylformamide;

[0057] ZT-Cl: 1-Phenylacet-1-(2-Chlorophenyl)-1-(N-α-Tocopheryloxy-ethyl-N-benzyl-4-benzamido)-methylchloro;

[0058] DMT-MM: 4-(4,6-dimethoxytriazine)-4-methylmorpholine hydrochloride;

[0059] TFA: Trifluoroacetic acid;

[0060] HOBt: 1-Hydroxybenzotriazole;

[0061] EDCI: 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0062] DCC: Dicyclohexylcarbodiimide;

[0063] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate;

[0064] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate;

[0065] Fmoc: fluorenemethyloxycarbonyl;

[0066] Boc: tert-butyloxycarbonyl;

[0067] Lys: Lysine;

[0068] Phe: Phenylalanine;

[0069] Threonine;

[0070] tBu: tert-butyl.

[0071] In the following examples, room temperature or normal temperature refers to 20℃-25℃.

[0072] The present invention will be further described in detail below with reference to specific embodiments.

[0073] The cyclic octapeptide derivatives or their salts in the following embodiments are prepared by the following steps:

[0074]

[0075]

[0076] The synthesis of the raw material RCO-Lys(Fmoc)-OH used below is based on the method in patent CN119735635A.

[0077]

[0078] H-Lys(Fmoc)-OH (0.1 mol) was dissolved in DMF (500 ml), cooled to 5℃-10℃, and N,N-diisopropylethylamine (0.2 mol) was added. After the addition was complete, the mixture was stirred for half an hour, and then acyl chloride (0.1 mol) was added dropwise. After the addition was complete, the mixture was kept at 5℃-10℃ for 1 hour. The reaction solution was poured into a 10% citric acid aqueous solution, and the precipitated solid was filtered, washed with purified water, and dried to obtain RCO-Lys(Fmoc)-OH.

[0079] n-CH3CO-Lys(Fmoc)-OH: Yield 87.5%, purity >98%.

[0080] 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), 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), 1.92 (s, 3H), 1.80-1.25 (m, 6H).

[0081] n-C3H7CO-Lys(Fmoc)-OH: Yield 87.5%, purity >98%.

[0082] 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. 85-2.75 (m, 1H), 2.22-2.15 (t, 2H), 1.60-1.25 (m, 8H), 0.92-0.85 (t, 3H).

[0083] n-C5H 11 CO-Lys(Fmoc)-OH: Yield 88.5%, purity >98%.

[0084] 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, 12H), 0.92-0.85 (t, 3H).

[0085] n-C6H 13 CO-Lys(Fmoc)-OH: Yield 89.0%, purity >98%.

[0086] 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, 14H), 0.92-0.85 (t, 3H).

[0087] n-C8H17 CO-Lys(Fmoc)-OH: Yield 89.5%, purity >98%

[0088] 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, 18H), 0.92-0.85 (t, 3H).

[0089] nC 13 H 27 CO-Lys(Fmoc)-OH: Yield 88.5%, purity >98%

[0090] 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, 28H), 0.92-0.85 (t, 3H).

[0091] Example 1 Synthesis of hexanoyl cyclic octapeptide (8CP-6)

[0092]

[0093] 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℃-60℃ and reflux for 8 hours, then 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 heat to 40℃-50℃; after 2 hours, add water (20 mL), separate the aqueous layer; wash the organic layer with water until neutral, and use it directly for the next step of the reaction.

[0094] 2) Add a 40 mL solution of Fmoc-Phe-OH (3.8 g, 0.01 mol) and N-methylmorpholine (1.11 g, 0.011 mol) in DMF and a 20 mL solution of DMT-MM (3.1 g, 0.011 mol) in water to the methyl tert-butyl ether solution obtained in the previous step and react. After half an hour, separate the aqueous layer. Add a 40 mL solution of diethylenetriamine (6.2 g, 0.06 mol) and mercaptopropionic acid (4.25 g, 0.04 mol) in DMF to the methyl tert-butyl ether layer solution and heat to 40℃-50℃. After 2 hours, add water (20 mL), separate the aqueous layer, wash the organic layer with water until neutral, and proceed directly to the next step of the reaction.

[0095] 3) Following the method in step 2, sequentially add Fmoc-Thr(tBu)-OH and n-C5H. 11 CO-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and n-C5H 11 CO-Lys(Fmoc)-OH gives n-C5H 11 CO-N 6 - [n-C5H 11 CO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-O-ZT.

[0096] 4) n-C5H 11 CO-N 6 - [n-C5H 11[CO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-O-ZT was dissolved in dichloromethane (400 mL), and 3% TFA-dichloromethane solution (200 mL) was added dropwise. After the addition was complete, the reaction was carried out at room temperature for 1 hour. The TFA was removed by washing with water, the solution was concentrated, heptane was added and stirred, and the mixture was filtered to obtain the protected octapeptide n-C5H 11 CO-N 6 - [n-C5H 11 CO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH 12.3 g, yield 80%, HPLC purity >98%. MS ESI: 1536.11 [M+H] + .

[0097] 5) n-C5H 11 CO-N 6 - [n-C5H 11 [CO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH was dissolved in DMF (1500 mL), cooled to 5-10°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 5-10°C for 1 hour, then allowed to react at 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 10.9 g of the protected cyclic octapeptide, with a yield of 91% and an HPLC purity of approximately 95%. MS ESI: 1517.99 [M+H]. + .

[0098] 6) Dissolve the protected cyclic octapeptide obtained in step 5) in dichloromethane (50 mL), add trifluoroacetic acid (30 mL), react at room temperature for 2 hours, add methyl tert-butyl ether (500 mL) to precipitate a solid, filter and collect the solid, dry to obtain 10.3 g of crude trifluoroacetate of the cyclic octapeptide, yield 100%, HPLC purity 90%, MS ESI: 1205.80 [M+H] + .

[0099] 7) The crude product obtained in step 6) was purified and converted to salt using high-performance preparative chromatography to obtain 7.6 g of 8CP-6 diacetate, with an HPLC purity greater than 98% and a purification yield of 80%. MS ESI: 1205.80 [M+H] + .

[0100] Example 2 Synthesis of heptanyl cyclic octapeptide (8CP-7)

[0101]

[0102] According to the synthesis method of Example 1, n-C6H 13 CO-Lys(Fmoc)-OH replaces n-C5H 11 CO-Lys(Fmoc)-OH yielded 8.1 g of 8CP-7 diacetate with an HPLC purity greater than 98% and an overall yield of 60%. MS ESI: 1233.68 [M+H] + .

[0103] Example 3 Synthesis of octanoyl cyclic octapeptide (8CP-8)

[0104]

[0105] According to the synthesis method of Example 1, n-C7H 15 CO-Lys(Fmoc)-OH (prepared according to the method in patent CN119735635A) replaces n-C5H 11 CO-Lys(Fmoc)-OH yielded 7.5 g of 8CP-8 diacetate with an HPLC purity greater than 98% and an overall yield of 54%. MS ESI: 1261.60 [M+H]. + .

[0106] Example 4 Synthesis of nonanoyl cyclic octapeptide (8CP-9)

[0107]

[0108] According to the synthesis method of Example 1, n-C8H 17 CO-Lys(Fmoc)-OH replaces n-C5H 11 CO-Lys(Fmoc)-OH yielded 7.0 g of 8CP-9 diacetate with HPLC purity greater than 98% and overall yield of 50%. MS ESI: 1289.90 [M+H] + .

[0109] Example 5 Synthesis of Decanoyl Cyclic Octapeptide (8CP-10)

[0110]

[0111] According to the synthesis method of Example 1, n-C9H 19 CO-Lys(Fmoc)-OH (prepared according to the method in patent CN119735635A) replaces n-C5H 11CO-Lys(Fmoc)-OH yielded 7.9 g of 8CP-10 diacetate with an HPLC purity greater than 98% and an overall yield of 53%. MS ESI: 1373.58 [M+H]. + .

[0112] Example 6 Synthesis of lauroyl cyclic octapeptide (8CP-12)

[0113]

[0114] According to the synthesis method of Example 1, with nC 11 H 23 CO-Lys(Fmoc)-OH (prepared according to the method in patent CN119735635A) replaces n-C5H 11 CO-Lys(Fmoc)-OH yielded 8.1 g of 8CP-12 diacetate with an HPLC purity greater than 98% and an overall yield of 56%. MS ESI: 1317.50 [M+H]. + .

[0115] Example 7 Synthesis of Myristoyl Cyclic Octapeptide (8CP-14)

[0116]

[0117] According to the synthesis method of Example 1, with nC 13 H 27 CO-Lys(Fmoc)-OH replaces n-C5H 11 CO-Lys(Fmoc)-OH yielded 7.2 g of 8CP-14 diacetate with HPLC purity greater than 98% and overall yield of 46%. MS ESI: 1430.20 [M+H] + .

[0118] Comparative Example 1: Synthesis of Acetyl Cyclic Octapeptide (8CP-2)

[0119]

[0120] Following the synthesis method of Example 1, CH3CO-Lys(Fmoc)-OH was used to replace n-C5H. 11 CO-Lys(Fmoc)-OH yielded 7.4 g of 8CP-2 diacetate with an HPLC purity greater than 98% and an overall yield of 61%. MS ESI: 1093.70 [M+H]. + .

[0121] Comparative Example 2: Synthesis of Butyryl Cyclic Octapeptide (8CP-4)

[0122]

[0123] Following the synthesis method of Example 1, n-C3H7CO-Lys(Fmoc)-OH was used instead of n-C5H. 11 CO-Lys(Fmoc)-OH yielded 7.2 g of 8CP-4 diacetate with an HPLC purity greater than 98% and an overall yield of 57%. MS ESI: 1149.60 [M+H] + .

[0124] Comparative Example 3: Synthesis of Palmitoyl Cyclic Octapeptide (8CP-16)

[0125]

[0126] According to the synthesis method of Example 1, with C 15 H 31 CO-Lys(Fmoc)-OH (prepared according to the method in patent CN119735635A) replaces n-C5H 11 CO-Lys(Fmoc)-OH yielded 7.7 g of 8CP-16 diacetate with an HPLC purity greater than 98% and an overall yield of 48%. MS ESI: 1606.10 [M+H] + .

[0127] Comparative Example 4: Synthesis of Decanoyl Cyclic Tetrapeptide

[0128]

[0129] Decanoyl cyclic tetrapeptide with a purity ≥98% was prepared according to the method in patent CN119735635A.

[0130] Comparative Example 5: Synthesis of Hexanoyl Cyclododecapeptide (12CP-6)

[0131]

[0132] Following the synthesis method of Example 1, amino acids Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, and n-C5H were sequentially added. 11 CO-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, n-C5H 11 CO-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and n-C5H 11CO-Lys(Fmoc)-OH, decarrier, cyclization, deprotection and purification yielded 6.1 g of cyclic dodecapeptide 12CP-6 triacetate, yield 30.1%, MS ESI: 1808.14.

[0133] Comparative Example 6: Synthesis of Decanoyl Cyclododecapeptide (12CP-10)

[0134]

[0135] Following the synthesis method of Example 1, amino acids Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, and n-C9H were sequentially added. 19 CO-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, n-C9H 19 CO-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and n-C9H 19 CO-Lys(Fmoc)-OH, decarrier, cyclization, deprotection and purification yielded 5.9 g of cyclic dodecapeptide 12CP-10 triacetate, yield 27.5%, MS ESI: 1976.33.

[0136] Test Example 1: Stability Test

[0137] According to the components in Table 1, emulsions with a concentration of 0.5 wt% were prepared from the cyclic octapeptide derivatives prepared in Examples 1-7. The preparation method is as follows: Phase A and Phase B were heated and stirred at a temperature of 80℃-85℃ respectively. After they were dispersed evenly, Phase B was added to Phase A. The mixture was homogenized for 3 min-5 min at a temperature of 80℃-85℃ and a stirring speed of 9000 r / min. After cooling to 45℃, Phase C was added and stirred evenly. Then, Phase D and Phase E, which were previously dispersed evenly, were added and stirred until they were completely dispersed.

[0138] The resulting emulsions were stored at room temperature (25℃) and 40±2℃, respectively. The content of cyclic octapeptide derivatives in each emulsion was measured at 0 days, 30 days, and 60 days. The test results are shown in Table 2.

[0139] Table 1 Emulsion Formulation

[0140]

[0141] Table 2. Stability test results of various cyclic octapeptide derivatives under different conditions (content (%))

[0142]

[0143] As shown in Table 2, the content of the cyclic octapeptide derivative did not decrease significantly after 60 days of storage at room temperature and 40℃, indicating that it has high stability and meets the needs of practical applications.

[0144] Test Example 2: Effect of Cyclic Octapeptide Derivatives on α-Crystal Protein Expression After Oxidative Stimulation in Cells

[0145] The cyclic octapeptide derivatives prepared in Examples 1-7 and the cyclic peptide derivatives or mixtures in Comparative Examples 1-6 were respectively prepared into a stock solution with a concentration of 7 mg / mL using 50% DMSO-water solution, and then diluted with DMEM complete medium to obtain a peptide solution with a concentration of 35 μg / mL.

[0146] Hacat cells at 2 × 10 4 Seeds were seeded in 96-well plates and incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, the culture medium in the plates was discarded, and the plates were washed twice with PBS. Except for the blank control group, each well was stimulated with DMEM complete medium containing 200 μM tert-butyl peroxide for 8 h. The culture medium in the plates was discarded, and the plates were washed twice with PBS. 150 μL of peptide solution was added to each well of the sample group, 150 μL of DMEM complete medium containing an equimolar concentration of solvent (50% DMSO-water) was added to each well of the model group, and 150 μL of DMEM complete medium containing an equimolar concentration of solvent (50% DMSO-water) was added to each well of the blank control group. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, the supernatant was collected, centrifuged at 2000 rpm for 20 min at 4°C, and the supernatant was used to detect the α-crystal protein content according to the ELISA kit instructions. The upregulation rate (%) of α-crystal protein expression in each group was calculated according to formula (1), and the results are shown in Table 3, expressed as mean ± deviation.

[0147] Expression upregulation rate (%) = (1)

[0148] In formula (1):

[0149] V e —The content of α-crystal protein in the sample group or model group;

[0150] V c —The content of α-crystallin in the blank control group.

[0151] Table 3. Upregulation rate (%) of α-crystallin expression in cells of each group

[0152]

[0153] Test Example 3: Effects of Cyclic Octapeptide Derivatives on the Expression of Cellular Filament and Hyaluronic Acid

[0154] The cyclic octapeptide derivatives prepared in Examples 1-7 and the cyclic peptide derivatives or mixtures in Comparative Examples 1-6 were respectively prepared into a stock solution with a concentration of 7 mg / mL using 50% DMSO-water solution, and then diluted with DMEM complete medium to obtain a peptide solution with a concentration of 35 μg / mL.

[0155] Hacat cells at 4 × 10 4 The culture medium was seeded in 48-well plates and incubated at 37°C with 5% CO2 for 24 h. After incubation, the culture medium in the plates was discarded, and the plates were washed twice with PBS. Except for the blank control group, each well was stimulated with DMEM complete medium containing 100 μM tert-butyl peroxide for 8 h. After stimulating, the culture medium in the plates was discarded, and the plates were washed twice with PBS. 150 μL of peptide solution was added to each well of the sample group, 150 μL of DMEM complete medium containing an equimolar concentration of solvent (50% DMSO-water) was added to each well of the model group, and 150 μL of DMEM complete medium containing an equimolar concentration of solvent (50% DMSO-water) was added to each well of the blank control group. The plates were incubated at 37°C with 5% CO2 for 24 h. After incubation, the supernatant was collected, centrifuged at 2000 rpm for 20 min at 4°C, and the supernatant was used to detect the content of filaggrin and hyaluronic acid according to the ELISA kit instructions. The upregulation rate (%) of filaggrin and hyaluronic acid expression in each group was calculated according to formula (2). The results are shown in Table 4 and expressed as mean ± deviation.

[0156] Expression upregulation rate (%) = (2)

[0157] In formula (2):

[0158] V e —The content of filaggrin or hyaluronic acid in the sample group or blank control group;

[0159] V c —The content of filaggrin and hyaluronic acid in the model group.

[0160] Table 4. Upregulation rate (%) of filaggrin and hyaluronic acid expression in cells of each group.

[0161]

[0162] Alpha-crystallin is an important member of the small heat shock protein family. It is expressed in many amorphous cells, such as keratinocytes, and plays a crucial role in molecular chaperone and cell protection. In Test Example 2, a keratinocyte model was constructed through oxidative stimulation, demonstrating that cells initiate a heat shock defense mechanism under stress, and upregulation of alpha-crystallin expression is the core of this mechanism. Meanwhile, in Test Example 3, cells in the model group suffered oxidative damage, leading to a decrease in the expression of filaggrin and hyaluronic acid.

[0163] As shown in Tables 3-4, compared with decanoyl cyclic tetrapeptide, the cyclic octapeptide derivatives with specific hydrophobic side chain modifications exhibit superior ability to upregulate the expression of α-crystallin, filaggrin, and hyaluronic acid. Secondly, a comparison of the examples with Comparative Examples 5-6 reveals that the effect weakens as the cyclic peptide size continues to increase. A comparison of the examples with Comparative Examples 1-3 shows that different side chain modifications also significantly affect the efficacy of the cyclic octapeptide derivatives; both excessively short and excessively long hydrophobic carbon chains reduce the efficacy of the cyclic octapeptide derivatives.

[0164] In summary, this invention has discovered and synthesized cyclic octapeptide derivatives with specific molecular sizes and carbon chain modifications, which can more effectively promote the expression of key protective components of α-crystallin, filaggrin, and hyaluronic acid in keratinocytes under oxidative stress compared to cyclic tetrapeptides or larger cyclic peptides.

[0165] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A cyclic octapeptide derivative or a salt thereof with the structure shown in formula (I), ； in, Each R is independently selected from: C5~C 13 alkyl.

2. The cyclic octapeptide derivative or its salt according to claim 1, characterized in that, Each R is independently selected from: C9~C 11 alkyl.

3. The cyclic octapeptide derivative or its salt according to claim 2, characterized in that, Each R is independently selected from C9 alkyl groups.

4. The cyclic octapeptide derivative or its salt according to claim 1, characterized in that, Each R is independently selected from: n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and n-tridecyl.

5. The cyclic octapeptide derivative or its salt according to claim 1, characterized in that, The cyclic octapeptide derivative is selected from the following compounds: ； ； ； 。 6. The cyclic octapeptide derivative or its salt according to any one of claims 1-5, characterized in that, The salt of the cyclic octapeptide derivative is selected from its acetate, trifluoroacetate, hydrochloride, sulfate, citrate, ascorbate, lactate, maleate, fumarate, succinate, gluconate, or salicylate.

7. The use of the cyclic octapeptide derivative or its salt as an active ingredient in the preparation of cosmetics capable of repairing the skin, according to any one of claims 1-6.

8. The use of the cyclic octapeptide derivative or its salt as an active ingredient in the preparation of cosmetics that can enhance the skin barrier, delay skin aging and / or resist oxidative damage, according to any one of claims 1-6.

9. The application according to claim 7 or 8, characterized in that, The cosmetic product can increase the content of filaggrin, hyaluronic acid and / or α-crystallin in cells.

10. A cosmetic product, characterized in that, The active ingredient in the cosmetic contains the cyclic octapeptide derivative or its salt as described in any one of claims 1-6.

11. The cosmetic product according to claim 10, characterized in that, The cosmetics include facial cleansers, toners, lotions, face masks, face creams, and serums.

12. A method for preparing a cyclic octapeptide derivative or a salt thereof according to any one of claims 1-6, characterized in that, Includes the following steps: 1) Using Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH and RCO-Lys(Fmoc)-OH as raw materials, a linear octapeptide RCO-N was synthesized. 6 -[RCO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH; wherein R is as described in any one of claims 1-5; 2) The linear octapeptide RCO-N 6 Cyclization of -[RCO-Lys-Thr(tBu)-Phe-Lys(Boc)]-Lys-Thr(tBu)-Phe-Lys(Boc)-OH followed by deprotection yields the cyclic octapeptide derivative or its salt.

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