Application and synthesis method of cyclic peptide

Abstract

The invention relates to the technical field of cosmetics, in particular to application and a synthesis method of cyclic peptide. The inhibition objects of the cyclic peptide comprise IL-1beta, IL-6 and TNF-alpha (Tumor Necrosis Factor-alpha); when the concentration is less than or equal to 10ppm, the inhibition rate on IL-1beta is greater than or equal to 40%, the inhibition rate on IL-6 is greater than or equal to 50%, and the inhibition rate on TNF-alpha is greater than or equal to 40%. The cyclopeptide has a barrier repairing effect and can improve FLG expression; meanwhile, the moisturizing effect is achieved, and percutaneous moisture loss of the skin can be reduced. The invention provides a group of cyclic peptides, which has excellent anti-inflammatory, moisturizing, barrier repairing and soothing effects, and compared with the existing straight-chain peptides in the market, the group of cyclic peptides has more obvious anti-inflammatory and soothing effects, and has a certain skin barrier repairing effect. The group of cyclic peptides can be applied to the fields of cosmetics and skin care products, and the product efficacy is improved; and the synthetic method of the cyclic peptide is easy to implement and wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an application of a cyclic peptide and a synthesis method thereof. Background Art

[0002] Peptides are one of the most important biomolecules in nature. They are widely used in the cosmetics field due to their diverse structures and functions and outstanding biological activities. Peptides are usually composed of short chains of 2-50 amino acids connected together by peptide bonds. Compared with other cosmetic ingredients, peptides have many desirable properties, such as high safety, hypoallergenicity and low cost. In particular, peptides from various natural sources such as plants, animals and microorganisms have been shown to have a variety of physiological activities, including antioxidant, anti-aging, moisturizing, stimulating collagen production and promoting wound healing. In higher organisms, peptides can be flexibly expressed in local areas and have a relatively short half-life. After completing their physiological functions, these peptides can be rapidly degraded and cleared.

[0003] Cyclic peptides are cyclic peptide chains formed by two or more amino acids through amide bonds, and have been reported to have a variety of biological activities, such as anti-tumor, antiviral, antibacterial, inhibition of cyclooxygenase, inhibition of lipid peroxidase and inhibition of tyrosinase. Cyclic peptides play a key role in many life processes and show great potential as therapeutic drugs. Currently, cyclic peptide compounds have been widely used in a variety of therapeutic drugs, including antibiotics such as vancomycin, hormone analogs such as oxytocin, and immunosuppressants such as cyclosporine. Cyclic peptides have become ideal candidates for the development of therapeutic drugs, cosmetics and skin care products due to their low toxicity, high affinity and target specificity. Compared with linear peptides (also known as straight-chain peptides), cyclic peptides not only have higher stability and binding affinity and specificity to target proteins, but also can enhance the efficacy of peptide chains by increasing the surface area of ​​protein-protein interactions. Cyclic peptides not only show superiority in structure, but their pharmacokinetic properties, passive diffusion capacity and active transport capacity are also generally better than linear peptides. In addition, the unique structure of cyclopeptides makes them resistant to hydrolysis by exopeptidases and show some resistance to endopeptidases.

[0004] Cosmetics and beauty products have become an indispensable part of daily life, and the market demand for natural, safe and effective cosmetic raw materials shows a continuous growth trend. Therefore, it is urgent to conduct in-depth research and development of cyclic peptides with high safety and significant biological activity to expand their application potential in the field of beauty and skin care. However, there are currently few natural cyclic peptides that can be used in cosmetics and beauty products, and there are still the following problems in the artificial synthesis of cyclic peptides: (1) There are few straight-chain polypeptides that can be cyclized, and most straight-chain polypeptides do not have sites that can be cyclized; (2) The cyclic peptides formed by cyclization may lose some key active sites, thereby weakening or losing the biological activity of the original straight-chain polypeptide; (3) Although some artificially synthesized cyclic peptides have appeared on the market, the cost of synthetic cyclic peptides is high, the efficiency is low, and it is easy to derive other products that are irrelevant or even have side effects.

[0005] Chinese patent application CN119020439A discloses a method for preparing an in vitro biosynthetic Pseudostellariae cyclopeptide B, which converts linear Pseudostellariae cyclopeptide B into a cyclic Pseudostellariae cyclopeptide B by an in vitro enzymatic reaction. This method solves the technical problems of the complicated preparation steps, low purity, and low yield of the cyclic Pseudostellariae cyclopeptide B in the prior art. Chinese patent CN117903232B discloses a method for synthesizing a diaryl bridged peptide, which uses resin as a raw material, undergoes acid cutting and deprotection to obtain a diaryl bridged peptide. However, none of these prior arts solve the above technical problems at the same time.

[0006] In this context, there is an urgent need to provide a cyclic peptide that is practical, feasible, highly safe and has significant biological activity. Summary of the invention

[0007] Natural cyclopeptides have a wide range of biological activities, such as anti-tumor, anti-viral, antibacterial, inhibition of cyclooxygenase, inhibition of lipid peroxidase and inhibition of tyrosinase. Due to their low toxicity, high affinity and target specificity, cyclopeptides have become ideal candidates for raw materials of cosmetics and skin care products. However, there are currently few natural cyclopeptides that can be used in cosmetics and beauty skin care products, and there is an urgent need to conduct in-depth research and development of a cyclopeptide with high safety and significant biological activity.

[0008] In order to solve the above technical problems, the first aspect of the present invention provides an application of a cyclic peptide, wherein the cyclic peptide is applied to a skin care product; the skin care product comprises at least one of a toner, a facial mask, an essence, an emulsion, a cream, a freeze-dried powder, a spray, and a gel.

[0009] Preferably, the cyclic peptide has anti-inflammatory and soothing effects, and its inhibitory targets include inflammation-related factors IL-1β, IL-6 and TNF-α.

[0010] Preferably, when the concentration of the cyclic peptide is ≤10ppm, the inhibition rate of IL-1β is ≥40%, the inhibition rate of IL-6 is ≥50%, and the inhibition rate of TNF-α is ≥40%.

[0011] More preferably, the cyclic peptide has an inhibition rate of IL-1β of more than 44% at a concentration of 5 ppm.

[0012] More preferably, the cyclic peptide has an inhibition rate of IL-6 of more than 53% at a concentration of 5 ppm.

[0013] More preferably, the cyclic peptide has an inhibition rate of TNF-α of more than 48% at a concentration of 5 ppm.

[0014] Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) are important inflammation-related cytokines that play a key role in the inflammatory response. TNF-α is one of the earliest and most important mediators of the inflammatory response, which can activate neutrophils and lymphocytes, increase endothelial permeability, regulate metabolism, and promote the synthesis and release of other cytokines. IL-1β is one of the main mediators of skin inflammatory response. It is produced by keratinocytes, macrophages and other immune cells, and can promote the inflammatory cascade, attracting and activating other immune cells to reach damaged or infected skin areas. IL-6 is a polypeptide with a hydrophobic end, which is secreted after the inflammatory pathway is activated and participates in the inflammatory response. The present invention prepares the cyclic peptide through a specific synthesis method, and through the cyclic peptide acting on the inflammation-related factors IL-1β, IL-6 and TNF-α, the secretion of the inflammatory factors is compared, the anti-inflammatory effect of the cyclic peptide is studied, and finally the experiment verifies that the cyclic peptide has excellent anti-inflammatory and soothing effects.

[0015] Preferably, the cyclic peptide has a barrier repair effect and can enhance the expression of FLG.

[0016] More preferably, when the concentration of the cyclic peptide is ≤2%, the enhancement rate of FLG expression is above 100%.

[0017] More preferably, at a concentration of 0.5%, the cyclic peptide has an enhancement rate of FLG expression of more than 113%.

[0018] More preferably, at a concentration of 1%, the cyclic peptide has an enhancement rate of FLG expression of more than 179%.

[0019] Filaggrin (FLG) is a key structural protein in the stratum corneum of the skin. It maintains the structural integrity of the stratum corneum by binding to keratin fibers, enhances the skin barrier function, and prevents water loss and the invasion of harmful external substances. After degradation, FLG protein generates a natural moisturizing factor to help maintain skin moisture and elasticity. Its degradation products also maintain the acidic environment of the skin, help inhibit the growth of pathogens and maintain the balance of the skin microbiome. The present invention prepares cyclic peptides through a specific synthesis method, acts on FLG, compares the expression of FLG, studies the barrier repair effect of cyclic peptides, and finally verifies that the barrier repair effect of cyclic peptides is significant.

[0020] Preferably, the cyclic peptide has moisturizing effect and can reduce transepidermal water loss of the skin.

[0021] More preferably, when the concentration of the cyclic peptide is ≤2%, the TWEL value of the skin is reduced by more than 10%.

[0022] Further preferably, the cyclic peptide at a concentration of 0.5% reduces the TWEL value of the skin by more than 16%;

[0023] Further preferably, at a concentration of 1%, the cyclic peptide reduces the TWEL value of the skin by more than 39%.

[0024] Trans Epidermal Water Loss (TEWL) refers to the amount of water that diffuses and evaporates through the skin, and is an important indicator for evaluating the skin barrier function. A higher TEWL value usually indicates that the skin barrier is damaged, which may be caused by stratum corneum damage, skin lesions or other factors. Healthy skin usually has a lower TEWL value, which can effectively prevent excessive water loss. Monitoring TEWL can evaluate the health of the skin and is used to evaluate the moisturizing effect of skin care products. The present invention prepares a cyclic peptide through a specific synthesis method, acts on a skin model, tests the change in its TEWL value, studies the moisturizing effect of the cyclic peptide, and finally experimentally verifies that the moisturizing effect of the cyclic peptide is significant.

[0025] In some preferred embodiments, the cyclic peptide comprises at least one of the compounds shown in the following formulae 1 to 22:

[0026]

[0027]

[0028]

[0029] More preferably, the cyclic peptide comprises at least one of the compounds having structures shown in Formula 1, Formula 14 and Formula 15.

[0030] The second aspect of the present invention provides a method for synthesizing the cyclic peptide, wherein the cyclic peptide is prepared by cyclizing a single or multiple linear peptides.

[0031] Preferably, the cyclic peptide is prepared by cyclizing a linear peptide, and the preparation steps include:

[0032] S1. Preparation of linear peptides by amino acid dehydration condensation reaction;

[0033] S2. taking a linear peptide and performing a cyclization reaction to prepare a cyclic peptide to obtain a crude product;

[0034] S3. Purify the crude product to obtain pure cyclopeptide.

[0035] More preferably, the step S1 adopts a solid phase synthesis method, a liquid phase synthesis method or a solid phase-liquid phase combination method to carry out a dehydration condensation reaction.

[0036] More preferably, the step S2 is cyclized by lactamization, lactonization or thiolactonization to prepare a cyclic peptide. The linear peptide is cyclized into a cyclic peptide by head-to-tail connection, and then purified to obtain a cyclic peptide product with high biological activity, significant efficacy and high safety. At present, no similar cyclic peptide products have been applied to cosmetics and beauty skin care products. The group of cyclic peptides provided by the present invention can be put into use as a cosmetic efficacy raw material, and has broad application prospects.

[0037] Preferably, the amino acid includes at least one of Fmoc-L-Abu-OH, Fmoc-β-Phe-OH, Fmoc-L-Ser-OH, Boc-L-Leu-OH, Boc-L-Val-OH, Boc-L-lle-OH, Boc-D-Ala-OH, Boc-D-Phe-OH, Boc-Gly-OH, Fmoc-Tyr-O-tBu, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Pro-OH.

[0038] The present invention uses Fmoc (9-fluorenylmethoxycarbonyl) or Boc (tert-butyloxycarbonyl) as an amino protecting group to modify the amino acid, which is beneficial to maintaining the stability and integrity of the peptide chain. At the same time, it cooperates with the effects of other raw materials in the system to minimize the occurrence of side reactions and ensure the generation of the target cyclic peptide.

[0039] Preferably, a condensing agent may be added in steps S1 to S3, and the condensing agent includes at least one of HOBt and DIC.

[0040] Preferably, a deprotecting agent is added in the step S1, and the deprotecting agent can be listed as Pip, TFA, HF, DBU, HOBt, etc.; preferably Pip, TFA or HOBt.

[0041] Preferably, a catalyst is added in the step S1, and the catalyst can be exemplified by DMAP.

[0042] Preferably, an organic acid is added in step S3, and the organic acid can be exemplified by TFA.

[0043] Preferably, an organic base may be added in steps S1 to S3, and the organic base may be at least one of DIPEA, DIPEA, and NMM.

[0044] Preferably, a peptide coupling agent may be added in step S1 or S2, and the peptide coupling agent includes at least one of HATU, EDCI, HOAT, and PyBoP.

[0045] Preferably, an organic solvent may be added in steps S1 to S3; the solvent includes at least one of DMF, MeOH, DCM and TH.

[0046] Note: Information on the above-listed substances is shown in Table 1 below.

[0047] Table 1

[0048]

[0049]

[0050] In some embodiments, the linear polypeptides of the present invention can be prepared by solid phase synthesis, liquid phase synthesis, solid phase and liquid phase combined methods, and can also be synthesized by carboxylic acid method, combinatorial chemistry method or natural coupling method. They can also be synthesized by biosynthesis methods aimed at producing the desired sequence, including but not limited to enzymatic hydrolysis, genetic engineering, fermentation and enzyme catalysis.

[0051] In some embodiments, the linear peptide cyclization method can also use disulfide bond formation or chemical selective ligation-mediated methods to synthesize cyclic peptides.

[0052] In some embodiments, the cyclic peptide can be prepared by first preparing at least two linear peptides and then cyclizing the multiple linear peptides.

[0053] In some embodiments, in addition to the cyclic peptides of the structures shown in Formulae 1 to 22, stereoisomers and mixtures of stereoisomers, or salts thereof, or combinations thereof may also be synthesized.

[0054] In some embodiments, the cyclic peptide can be used alone in various product forms of cosmetics and skin care products, and can also be compounded with other active ingredients with skin care or hair care effects to exert anti-aging, moisturizing, anti-inflammatory, skin care and other effects.

[0055] In some embodiments, the cyclic peptide can also be applied to hair care products to exert anti-inflammatory, anti-hair loss and other effects.

[0056] Beneficial effects:

[0057] The present invention provides an application and a synthesis method of a cyclic peptide, which has the following advantages:

[0058] (1) The present invention provides a group of cyclic peptides, which have excellent anti-inflammatory, moisturizing, barrier repair and soothing effects. Compared with the linear peptides available on the market, this group of cyclic peptides has more significant anti-inflammatory and soothing effects, and has certain skin barrier repair effects; this group of cyclic peptides can be used in the field of cosmetics and skin care products to enhance product efficacy.

[0059] (2) The cyclic peptide provided by the present invention has a wide range of biological activities, a stable structure and high safety. When added to products as a cosmetic raw material, it can not only inhibit the secretion of inflammatory-related factors IL-1β, IL-6 and TNF-α, but also increase the expression of FLG, and at the same time reduce the transepidermal water loss of the skin, with significant efficacy.

[0060] (3) The synthesis method of the cyclic peptide of the present invention is simple. It uses common amino acids as raw materials. First, a linear peptide is synthesized by an amino acid dehydration condensation reaction, and then a cyclic peptide is synthesized by a head-to-tail cyclization method. The synthesis steps are simple and have obvious cost advantages.

[0061] (4) The cyclic peptide of the present invention has flexible application and good formula compatibility in the cosmetic system. It can be used in a variety of product forms such as toner, facial mask, essence, lotion, cream, freeze-dried powder, etc.; and it can be used alone or compounded with other active ingredients to exert their efficacy together.

[0062] (5) The present invention effectively solves the technical problems in the existing cyclic peptide synthesis technology, that is, there are few straight-chain polypeptides that can be cyclized, and that the cyclic peptides formed by cyclization may lose certain key active sites, thereby causing the weakening or loss of the biological activity of the original straight-chain polypeptide. It provides a new idea for the preparation of cyclic peptides and their application in skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 . Cytotoxicity test results of the cyclic peptide of formula (1);

[0064] Figure 2 . Anti-inflammatory test results of the cyclic peptide of formula (1) (from left to right are the test results of IL-1β, IL-6 and TNF-α);

[0065] Figure 3 . Test results of barrier repair efficacy of the cyclic peptide of formula (14);

[0066] Figure 4 . The in vitro moisturizing efficacy test results of the cyclic peptide of formula (15);

[0067] Figure 5 .The soothing test results of the cyclic peptide of formula (7) (from left to right are the test results of IL-1β, IL-6 and TNF-α). DETAILED DESCRIPTION

[0068] The resins in the examples are all PEG resins.

[0069] Note: Unless otherwise specified, the solvent of the solution involved in the present invention is water; the solution concentration involved is mass concentration; the drying involved is drying with anhydrous sodium sulfate; and the raw materials used are all commercially available.

[0070] Example

[0071] For information on some of the raw materials used in the examples of the present invention, please refer to Table 1.

[0072] Example 1

[0073] An application of a cyclic peptide, wherein the cyclic peptide is applied in skin care products.

[0074] The cyclic peptide has anti-inflammatory, repairing, moisturizing and soothing effects. The test results are shown in the performance test section.

[0075] The structural formula of the cyclic peptide is The preparation steps include:

[0076] S1. The linear peptide is prepared by solid phase synthesis through amino acid dehydration condensation reaction, specifically comprising:

[0077] Step 1: Take Fmoc-L-Abu-OH (4.88 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 4 ° C, add DMF 25 mL, DIC (2.3 mL, 15 mmol) and let stand for 20 min, then add to the solid phase synthesizer (125 mL) containing the resin and stir for 1.5 h. Wash the resin with DMF solution (wash 3 times, 60 mL each time), then add 65 mL of DMF solution containing 20 wt% Pip, stir for 30 min, filter to remove the deprotecting agent (Pip), then wash with DMF solution (wash 6 times, 60 mL each time), and spin dry.

[0078] Step 2: The operation is the same as step 1, except that Fmoc-L-Abu-OH is replaced with Fmoc-β-Phe-OH (5.81 g, 15 mmol); the resin used is the resin after spin drying in step 1;

[0079] Step 3: The operation is the same as step 1, except that Fmoc-L-Abu-OH is replaced with Fmoc-L-Ser-OH (4.91 g, 15 mmol); the resin used is the resin after spin drying in step 2;

[0080] Step 4. Dissolve Fmoc-L-Pro-OH (6.75 g, 20 mmol) in carbon tetrachloride (500 mL), add triphenylphosphine (21.4 g, 81.7 mmol) at room temperature, and stir. Then heat the mixture at 80-90°C for 12 h, concentrate the solution under reduced pressure, and purify by column chromatography, eluting with 5% EtOAc / petroleum ether to obtain Fmoc-cis-3,4-dichloro-L-Pro-OH (yield 74%) as colorless crystals.

[0081] Step 5: The operation is the same as step 1, except that Fmoc-L-Abu-OH is replaced with Fmoc-cis-3,4-dichloro-L-Pro-OH (6.09 g, 15 mmol); the resin used is the resin after spin drying in step 3.

[0082] Step 6: Take the resin after spin drying in step 5 (i.e., the last step of connecting amino acids is completed), wash the resin with DMF and MeOH 3 times (60 mL each time), and dry it with N2 bubbling overnight. Add DCM (100 mL) containing 1% TFA to the flask containing the resin, shake it on a shaker for 2 hours, filter out the resin, add ice anhydrous ether (50 mL) to precipitate the filtrate, place the precipitate in a vacuum drying kettle, dry it at room temperature for 24 hours, and collect the linear peptide released from the resin.

[0083] S2. Cyclization: Take the linear peptide obtained in step 5 of S1, add HATU (0.711 g, 1.87 mmol) and DIPEA (0.24 g, 1.87 mmol), stir and react at 30°C for 14 h to complete the cyclization and obtain a crude cyclic peptide product; the cyclization process is monitored by LC-MS (liquid chromatography-mass spectrometry).

[0084] S3. Purification: Take the crude cyclopeptide product from step S2, remove the solvent by rotary evaporation under reduced pressure, and purify the crude cyclopeptide by Prep-HPLC (preparative high performance liquid chromatography) to obtain a pure cyclopeptide product (Formula 1).

[0085] The purification conditions are:

[0086] Chromatographic column: ACE Excel 5C18-Amide, 250×4.6mm;

[0087] Elution conditions: 5%-80% MeCN / H2O gradient elution for 60 min, peak time t=18.3 min;

[0088] Sample concentration: 1 mg / mL;

[0089] Injection volume: 50 μL;

[0090] Detection wavelength: 244nm.

[0091] The reaction formula of the above reaction is as follows:

[0092]

[0093] Example 2

[0094] An application of a cyclic peptide, wherein the cyclic peptide is applied in skin care products.

[0095] The cyclic peptide has anti-inflammatory, repairing, moisturizing and soothing effects. The test results are shown in the performance test section.

[0096] The structural formula of the cyclic peptide is The preparation steps include:

[0097] S1. Prepare linear peptides by dehydration condensation reaction of amino acids using solid phase-liquid phase combination method, specifically including:

[0098] Step 1: Take Boc-L-Leu-OH (2.54 g, 11.00 mmol) and 2-trimethylsilylethanol (1.00 g, 8.46 mmol) and dissolve them in DCM (30 mL) in ice bath in advance, stir and dissolve, then weigh and add EDCI (2.60 g, 13.56 mmol) and DMAP (1.60 g, 13.10 mmol), react for 15 min, slowly drop DIPEA (2.73 g, 21.12 mmol), continue to react at 0°C for 30 min after the addition is complete, and react at room temperature overnight. Evaporate the system to obtain a concentrate, dissolve the concentrate in 50 mL of ethyl acetate, collect the organic phase, wash with 0.5 M HCl aqueous solution, saturated NaHCO3 aqueous solution and saturated brine in sequence, and then dry over anhydrous sodium sulfate. Evaporate the ethyl acetate and purify by silica gel column chromatography to obtain 2.67 g of product 1 (colorless transparent oily liquid).

[0099] Step 2: Dissolve the product 1 (1.50 g, 4.24 mmol) obtained in step 1 in dichloromethane (9 mL) precooled to 0°C in an ice-water bath. After complete dissolution, increase the stirring speed to 200 rpm, slowly drop trifluoroacetic acid (3 mL) into the system, reduce the stirring speed to 100 rpm after the addition, and warm to room temperature for 3 hours. Evaporate the solvent and excess trifluoroacetic acid, take the residue and dissolve it in 10 mL acetonitrile and 10 mL dichloromethane in turn, spin dry, repeat 2-4 times, and finally use a high vacuum oil pump to dry to obtain product 2.

[0100] Step 3: Take Boc-L-Val-OH (1.27 g, 5.84 mmol) and dissolve it in dichloromethane (45 mL) at 0°C, add EDCl (1.13 g, 5.89 mmol), HOBt (0.79 g, 5.84 mmol), NMM (1.14 g, 11.27 mmol) in sequence, react for 15 min, slowly drop the dichloromethane solution containing the pre-cooled product II obtained in step 2 (1 g dissolved in 10 mL), stir at 0°C for 1 h under ice-water bath conditions, remove the ice-water bath, and react at room temperature overnight. Evaporate the system, dissolve the residue in ethyl acetate (80 mL), and wash the ethyl acetate organic phase with 0.5 M hydrochloric acid aqueous solution, saturated sodium bicarbonate aqueous solution and saturated brine in sequence (each washing 3 times, 15 mL each time), and dry the organic phase. Evaporate the ethyl acetate, and then separate and purify it by silica gel column chromatography to obtain 1.66 g of product III (white solid).

[0101] Step 4: Dissolve Boc-L-lle-OH (0.64 g, 2.77 mmol) in 40 mL of dichloromethane, then stir in an ice-water bath and cool to 0°C, slowly add HATU (1.06 g, 2.79 mmol), HOAT (0.38 g, 2.79 mmol), react at 0°C for 30 min, then add DIPEA (1.50 g, 11.60 mmol) and the product 3 obtained in step 3 (i.e., the deprotected product), naturally warm to room temperature, and react overnight. After the reaction is completed, add 80 mL of dichloromethane, wash with 0.5 M aqueous hydrochloric acid solution, saturated aqueous sodium bicarbonate solution and saturated brine in sequence (each washing 3 times, 20 mL each time), collect the organic phase and dry. Concentrate under reduced pressure, purify by column chromatography to obtain 0.99 g of product 4 (white solid).

[0102] Step 5: Take EDCI (2.60 g, 13.56 mmol), DMAP (1.60 g, 13.10 mmol), DIPEA (2.73 g, 21.12 mmol), Boc-D-Ala-OH (2.08 g, 11.00 mmol) and 2-trimethylsilylethanol (1.00 g, 8.46 mmol) and mix them for esterification reaction to obtain 2.28 g of product 5 (transparent oil).

[0103] Step 6: The product five (1.25 g, 4.32 mmol) obtained in step 5 was mixed with trifluoroacetic acid (15 mL), the Boc protecting group was removed, and the product was mixed with Boc-D-Phe-OH (1.38 g, 5.18 mmol), dichloromethane (20 mL), HATU (1.97 g, 5.18 mmol), HOAT (0.71 g, 5.18 mmol), and DIPEA (2.79 g 21.60 mmol) for amide condensation reaction, followed by extraction with ethyl acetate, washing, drying, concentration, and purification (isolation and purification by ethyl acetate and petroleum ether column chromatography) to obtain product six (white solid).

[0104] Step 7: The product VI (0.68 g, 1.56 mmol) after removing the Boc protecting group in step 6 was mixed with Boc-Gly-OH (0.33 g, 1.87 mmol), dichloromethane (20 mL), HATU (0.711 g, 1.87 mmol), HOAT (0.25 g, 1.87 mmol), and DIPEA (1.00 g, 7.80 mmol) to carry out amide condensation reaction. After the reaction, 150 mL of dichloromethane was added for washing, drying, evaporation and concentration, and separation and purification by column chromatography to obtain 0.54 g of product VII (white solid).

[0105] Step 8: The product tetra (0.62 g, 1.14 mmol) obtained in step 4 was mixed with tetrabutylammonium fluoride (0.8 g) for deesterification reaction, the deesterified product and the product seven (0.56 g, 1.14 mmol) obtained in step 7 and trifluoroacetic acid were mixed to remove Boc, the product was mixed with HATU (0.52 g, 1.37 mmol), HOAT (0.19 g, 1.37 mmol), DIPEA (0.74 g, 5.7 mmol) and dichloromethane (20 mL) for condensation reaction, and purified (purified by silica gel column chromatography) to obtain an oily compound. The oily compound was then taken for de-Boc protection reaction to obtain product eight (white solid).

[0106] S2. Cyclization: PyBoP (942 mg, 1.81 mmol), HOAT (246 mg, 1.81 mmol) and NMM (366 mg, 3.62 mmol) were dissolved in DMF (100 mL), and the system was cooled to 0°C. The product eight (224 mg, 0.36 mmol) obtained in step 8 was dissolved in DMF (500 mL), and the mixture was slowly added dropwise under N2 atmosphere. After the addition was completed, the temperature was raised to room temperature and the reaction was carried out for 60 hours to obtain a crude cyclopeptide product.

[0107] S3. Purification: Take the crude cyclopeptide product of step S2, remove DMF by vacuum rotary evaporation, dissolve the residue with 150 mL of ethyl acetate, wash the ethyl acetate with saturated brine, dry, and concentrate to obtain a light yellow oily liquid. Separate and purify by column chromatography to obtain 57 mg of a white solid to obtain a pure cyclopeptide product (Formula 14).

[0108] Example 3

[0109] An application of a cyclic peptide, wherein the cyclic peptide is applied in skin care products.

[0110] The cyclic peptide has anti-inflammatory, repairing, moisturizing and soothing effects. The test results are shown in the performance test section.

[0111] The structural formula of the cyclic peptide is The preparation steps include:

[0112] S1. The linear peptide is prepared by solid phase synthesis through amino acid dehydration condensation reaction, specifically comprising:

[0113] Step 1: Take Fmoc-Tyr-O-tBu (6.89 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 4 ° C, add DMF (25 mL) and DIC (2.3 mL, 15 mmol), let stand for 20 min, then add to a solid phase synthesizer (125 mL) containing resin, and stir for 1.5 h. Wash the resin with DMF solution (wash 3 times, 60 mL each time), then add 65 mL of DMF solution containing 20 wt% Pip, stir for 30 min, filter to remove the deprotection solution (Pip), then wash with DMF solution (wash 6 times, 60 mL each time), and spin dry.

[0114] Step 2: The operation is the same as step 1, except that Fmoc-Tyr-O-tBu is replaced with Fmoc-Ile-OH (5.30 g, 15 mmol); the resin used is the resin after spin drying in step 1;

[0115] Step 3: The operation is the same as step 1, except that Fmoc-Tyr-O-tBu is replaced with Fmoc-Phe-OH (5.81 g, 15 mmol); the resin used is the resin after spin drying in step 2;

[0116] Step 4: The operation is the same as step 1, except that Fmoc-Tyr-O-tBu is replaced with Fmoc-Pro-OH (5.06 g, 15 mmol); the resin used is the resin after spin drying in step 3;

[0117] Step 5: Take the resin after spin drying in step 4 (i.e., the last step of connecting amino acids) and vacuum dry it, add 200 mL of DCM solution containing 1 wt% TFA, stir the reaction at 30°C for 30 minutes, and filter to obtain a filtrate; use a rotary evaporator to reduce pressure and concentrate the filtrate to obtain a straight-chain peptide (i.e., a fully protected linear polypeptide).

[0118] S2. Cyclization: Take the linear peptide obtained in step 5 of S1, dissolve it in 1.4L DCM, add DIC (1.54mL, 10mmol), HOBt (1.35g, 10mmol), and DIPEA (1.74mL, 10mmol), and stir the reaction at 30°C for 14h to form a cyclic peptide compound. Concentrate under reduced pressure to remove DCM to obtain a crude cyclic peptide product.

[0119] S3. Purification: trifluoroacetic acid (18 mL) and dichloromethane (20 mL) were mixed, added to the crude cyclopeptide product obtained in step S2, stirred at room temperature for about 1 h, the Fmoc protecting group was removed to obtain a cyclopeptide resin, and the cyclopeptide resin was immersed in THF; treated with THF (200 mL) containing 20 wt% DIPEA in a N2 atmosphere for 24 h, the resin was filtered and washed with THF and dichloromethane 3 times each (25 mL each time), and the filtrate was collected; after evaporating the solvent under reduced pressure, glacial ether (100 mL) was added to precipitate the crude cyclopeptide product, and the precipitate was placed on RPHPLC (reverse phase high performance liquid chromatography) for further purification to obtain a pure cyclopeptide product (Formula 15).

[0120] The purification conditions are:

[0121] Chromatographic column: ACE Excel 5C18-Amide, 250×4.6mm;

[0122] Elution conditions: 30%-100% MeCN / H2O gradient elution for 60 min, peak time t=10.6 min;

[0123] Sample concentration: 1 mg / mL;

[0124] Injection volume: 50 μL;

[0125] Detection wavelength: 220nm.

[0126] Performance Testing

[0127] 1. Cytotoxicity test of the cyclic peptide of formula (1)

[0128] 1) Experimental methods

[0129] (1) Cell inoculation: Macrophages were plated at 8×10 3 The cells were seeded into a 96-well plate at a density of 100 / well and incubated overnight in an incubator (37°C, 5% CO2).

[0130] (2) Experimental grouping: The experiment set up a zero adjustment group, a solvent control group, a positive control group and a sample group. The sample group was set up with 8 concentration gradients, and 3 replicate wells were set up under each concentration gradient.

[0131] (3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 ppm.

[0132] (4) Administration: Administration was performed when the cell plating rate in the 96-well plate reached 40% to 60%. 200 μL of culture medium was added to each well of the solvent control group; 200 μL of culture medium containing 10 wt% DMSO was added to each well of the positive control group; 200 μL of culture medium containing samples of corresponding concentrations (i.e., the above-mentioned sample working solution) was added to each well of the sample group; the zero adjustment group had no cell inoculation and only 200 μL of cell culture medium was added. After administration, the 96-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 hours. The culture medium was DMEM medium.

[0133] (5) Detection: After incubating the cells for 24 h, discard the supernatant, add MTT working solution (0.5 mg / mL), and incubate at 37°C in the dark for 4 h. After the incubation, discard the supernatant, add 150 μL DMSO to each well, and read the OD value at 490 nm.

[0134] (6) Calculation of relative cell viability: The cell viability of the sample was calculated according to the formula: Cell viability (%) = (sample well OD - zeroing well OD / (solvent control well OD - zeroing well OD) × 100%.

[0135] 2) Experimental results

[0136] The cell viability of the cyclic peptide of formula (1) at different concentrations is shown in Figure 1 . Figure 1 The MTT results showed that the cyclic peptide of formula (1) did not show obvious cytotoxicity in the macrophage concentration range of 6.25 ppm. 2. Anti-inflammatory efficacy test of the cyclic peptide of formula (1)

[0137] 1) Reagents and instruments

[0138] Based on the macrophage (LPS-Raw264.7) model, the soothing effect of the cyclic peptide of formula (1) was evaluated by detecting the contents of IL-1β, TNF-α and IL-6 in the culture medium. The main reagents for this test include: high-glucose DMEM (Gibco), dexamethasone (Sigma), lipopolysaccharide LPS (E.Coli.Sigma), PBS (Solaibao), DMSO (Sigma), IL-1β ELISA kit (Abcam), IL-6 ELISA kit (Abcam) and TNF-α ELISA kit (NOVUS). The main instruments for this test include: CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-2F) and microplate reader (BioTek, Epoch).

[0139] 2) Test steps

[0140] 2.1) Cell seeding: 2.2×10 5 Macrophages were inoculated into 6-well plates at an inoculation density of 1 / well, 2 mL of cell suspension was added to each well, and the inoculated cell culture plate was placed in an incubator for further culture for 24 h (5% CO2, 37°C).

[0141] 2.2) Experimental grouping: This test was divided into 4 groups: blank control group (BC, no treatment), negative control group (NC, LPS treatment), positive control group (PC, LPS + dexamethasone treatment), experimental group (LPS + 5ppm sample treatment).

[0142] 2.3) Discard the old cell culture medium, add 1.8mL of culture medium containing the test substance at the corresponding concentration (i.e., the sample working solution) to each well of the sample group; add 1.8mL of solvent control culture medium (i.e., the solvent control group mentioned above) to each well of the negative control group; add 1.8mL of positive control culture medium (i.e., the positive control group mentioned above) to each well of the positive control group; add 1.8mL of normal culture medium (i.e., the zero adjustment group mentioned above) to each well of the blank control group. After the administration is completed, place the 6-well plate in a cell culture incubator (5% CO2, 37°C) and culture for 2h.

[0143] 2.4) LPS stimulation: 2 hours after administration, 200 μL of the prepared LPS-containing working solution was added to each well of each group and the cells were placed in a cell culture incubator (5% CO 2 , 37° C.) and cultured for 22 hours.

[0144] 2.5) Collect the cell culture supernatant and perform detection according to the instructions of the ELISA kit.

[0145] 3) Test results

[0146] The anti-inflammatory efficacy test results of the cyclic peptide of formula (1) are shown in Figure 2 .like Figure 2As shown, compared with the BC group, the IL-1β, IL-6 and TNF-α contents of the NC group increased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the IL-1β, IL-6 and TNF-α contents of the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the content of the inflammation-related factor IL-1β of the cyclic peptide of formula (1) at a concentration of 5ppm was significantly reduced, with an inhibition rate of 44.28%; compared with the NC group, the content of the inflammation-related factor IL-6 of the cyclic peptide of formula (1) at a concentration of 5ppm was significantly reduced, with an inhibition rate of 53.8%; compared with the NC group, the content of the inflammation-related factor TNF-α of the cyclic peptide of formula (1) at a concentration of 5ppm was significantly reduced, with an inhibition rate of 48.82%. The above results show that the cyclic peptide of formula (1) provided by the present invention can significantly inhibit the secretion of inflammation-related factors (IL-1β, IL-6 and TNF-α) at ​​a concentration of 5ppm, and has anti-inflammatory and soothing effects.

[0147] 3. In vitro barrier repair efficacy test of the cyclic peptide of formula (14)

[0148] 1) Test method

[0149] 1.1) Reagents and instruments: UVB was used to irradiate the 3D epidermal skin model, and the barrier repair effect of the cyclic peptide (2) was evaluated by detecting the expression of FLG. The main reagents for this test include: EpiGrowth culture medium (Guangdong Boxi Biological), PBS (Solaibao), glycerol (Sinopharm), WY14643 (Sigma), paraformaldehyde (Biosharp), xylene (Sinopharm), and FLG antibody (Abcam). The main instruments for this test include: CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-2F), UVB irradiator (Philips) and fluorescence microscope (Olympus, BX43).

[0150] 1.2) Experimental grouping: This test was divided into 4 groups: blank control group (BC, no UVB radiation), negative control group (NC, UVB radiation), positive control group (PC, UVB radiation + 50 μM WY14643 (PPARα agonist) treatment), experimental group (two concentrations, one is UVB radiation + 0.5% formula (14) cyclic peptide sample, the other is UVB radiation + 1% formula (14) cyclic peptide sample treatment, the solvent is water).

[0151] 1.3) Experimental steps:

[0152] 1.3.1) Grouping: Transfer the models to a 6-well plate (pre-added with 0.9 mL EpiGrowth culture medium) and mark the groups.

[0153] 1.3.2) UVB irradiation: Except for the BC group, the other groups received 600mJ / cm 2 of UVB radiation.

[0154] 1.3.3) Drug treatment: After irradiation, the BC group was not treated, the PC group was added with the corresponding working solution, and the sample group was coated with the sample working solution evenly on the model surface. The 6-well plate was placed in an incubator at 37°C and 5% CO2 for 24 hours.

[0155] 1.3.4) After the incubation, the model was washed with sterile PBS and the residual liquid was removed with a sterile cotton swab. The sample was fixed in 4% paraformaldehyde solution for 24 hours and then immunofluorescence detection was performed. The image was observed under a microscope and analyzed.

[0156] 2) Test results

[0157] The results of the in vitro barrier repair efficacy test of the cyclic peptide of formula (14) are shown in Figure 3 .like Figure 3 As shown, compared with the BC group, the FLG expression level in the NC group was significantly reduced, proving that the stimulation conditions of this test were effective. Compared with the NC group, the FLG expression level in the PC group was significantly increased, proving that the positive control of this test was effective. Compared with the NC group, the FLG expression levels after treatment with 0.5% and 1% concentrations of cyclic peptide (2) were significantly increased, with the increase rates being 113.83% and 179.79%, respectively, indicating that the cyclic peptide (2) provided by the present invention can increase FLG expression at this concentration and has a repairing effect.

[0158] 4. In vitro moisturizing efficacy test of the cyclic peptide of formula (15)

[0159] 1) Test method

[0160] (1) Reagents and instruments: UVB was used to irradiate the 3D epidermal skin model, and the moisturizing effect of the cyclic peptide of formula (15) was evaluated by measuring the TWEL value. The main reagents for this test include: EpiGrowth culture medium (Guangdong Boxi Biological), PBS (Solabao), glycerol (Sinopharm), and WY14643 (Sigma). The main instruments for this test include: CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-2F), UVB irradiator (Philips) and CM 825 Skin Moisture Content Tester (Courage+Khazaka electronic).

[0161] (2) Experimental grouping: This test is divided into 4 groups: blank control group (BC, no UVB radiation), negative control group (NC, UVB radiation), positive control group (PC, UVB radiation + 50 μM WY14643 treatment), experimental group (two concentrations, one is UVB radiation + 0.5% cyclic peptide sample of formula (15), the other is UVB radiation + 1% cyclic peptide sample of formula (15), the solvent is water).

[0162] (3) Experimental steps:

[0163] 1) Grouping: Transfer the model to a 6-well plate (pre-added with 0.9 mL EpiGrowth culture medium) and mark the groups.

[0164] 2) UVB irradiation: Except for the BC group, the other groups received 600mJ / cm 2 of UVB radiation.

[0165] 3) Drug treatment: After irradiation, the BC group was not treated, the PC group was added with the corresponding working solution, and the sample group evenly coated the sample working solution on the surface of the model. Place the 6-well plate in an incubator at 37°C and 5% CO2 for 24 hours. 4) After cleaning, prepare 24-well plates according to the number of models and mark them. Add 0.3mL EpiGrowth culture solution to each well. Place the 24-well plate containing the model in an ultra-clean workbench, open the lid, and let it stand for 30 minutes before measuring. Connect TW24 to the MPA6 adapter, start the measurement software, calibrate the probe to zero, and set the measurement time and parameters. Select TEWL as the measurement parameter and set the measurement time to 2.5 minutes. After the measurement is completed, save the data.

[0166] 2) Test results

[0167] The in vitro moisturizing effect test results of the cyclic peptide of formula (15) are shown in Figure 4 .like Figure 4 As shown, compared with the BC group, the TWEL value of the NC group was significantly increased, proving that the stimulation conditions of this test were effective. Compared with the NC group, the TWEL value of the PC group was significantly reduced, proving that the positive control of this test was effective. Compared with the NC group, the TWEL values ​​after treatment with the 0.5% and 1% concentrations of the cyclic peptide of formula (15) were significantly reduced, with reductions of 16.76% and 39.72%, respectively, indicating that the cyclic peptide of formula (15) provided by the present invention can reduce the TWEL value at this concentration and has a moisturizing effect.

[0168] 5. Soothing effect test of the cyclic peptide of formula (7)

[0169] Note: The cyclic peptide of formula (7) is commercially available with CAS number 55466-29-0.

[0170] 1) Test method

[0171] (1) Reagents and instruments: UVB was used to irradiate the 3D epidermal skin model, and the anti-inflammatory effect of the cyclic peptide of formula (7) was evaluated by detecting the concentrations of IL-1α, IL-6 and TNF-α in the culture medium. The main reagents for this test include: EpiGrowth culture medium (Guangdong Boxi Biological), PBS (Solebo), glycerol (Sinopharm), dexamethasone (China Food and Drug Administration), IL-1α ELISA kit (Abcam), IL-6 ELISA kit (Abcam), TNF-α ELISA kit (NOVUS). The main instruments for this test include: CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-2F), UVB irradiator (Philips) and microplate reader (BioTek, Epoch).

[0172] (2) Experimental grouping: This test is divided into 4 groups: blank control group (BC, no UVB radiation), negative control group (NC, UVB radiation), positive control group (PC, UVB radiation + 50 μM WY14643 treatment), experimental group (two concentrations, one is UVB radiation + 0.5% cyclic peptide sample of formula (7), the other is UVB radiation + 1% cyclic peptide sample of formula (7), the solvent is water).

[0173] (3) Experimental steps:

[0174] 1) Grouping: Transfer the model to a 6-well plate (pre-added with 0.9 mL EpiGrowth culture medium) and mark the groups.

[0175] 2) UVB irradiation: Except for the BC group, the other groups received 600mJ / cm 2 of UVB radiation.

[0176] 3) Drug treatment: After irradiation, the BC group was not treated, the PC group was added with the corresponding working solution, and the sample group evenly coated the sample working solution on the model surface. The 6-well plate was placed in an incubator at 37°C and 5% CO2 for 24 hours. 4) After the incubation, the model was washed with sterile PBS and the residual liquid was removed with a sterile cotton swab. The 3D epidermal skin model culture fluid was collected in a centrifuge tube. After collection, the samples for ELISA detection were placed in a -80°C refrigerator for freezing and stored, and the detection and analysis were performed according to the operating instructions of the ELISA kit.

[0177] 2) Test results

[0178] The soothing effect test results of the cyclic peptide of formula (7) are shown in Figure 5 .like Figure 5As shown, compared with the BC group, the IL-1α, IL-6 and TNF-α contents of the NC group increased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the IL-1α, IL-6 and TNF-α contents of the PC group decreased significantly, indicating that the positive control of this test was effective. Compared with the NC group, the content of the inflammatory factor IL-1α of the cyclic peptide of formula (7) at concentrations of 0.5% and 1% was significantly reduced, with inhibition rates of 27.18 and 42.41%, respectively; compared with the NC group, the content of the inflammatory factor IL-6 of the cyclic peptide of formula (7) at concentrations of 0.5% and 1% was significantly reduced, with inhibition rates of 33.72% and 51.91%, respectively; compared with the NC group, the content of the inflammatory factor TNF-α of the cyclic peptide of formula (7) at concentrations of 0.5% and 1% was significantly reduced, with inhibition rates of 48.96% and 57.09%. The above results indicate that the cyclic peptide of formula (7) provided by the present invention can inhibit the secretion of inflammation-related factors (IL-1α, IL-6 and TNF-α) at ​​concentrations of 0.5% and 1%, and has anti-inflammatory and soothing effects.

Claims

1. An application of a cyclic peptide, characterized in that: The cyclic peptide is used in skin care products; The inhibitory targets of the cyclic peptide include IL-1β, IL-6 and TNF-α; When the concentration of the cyclic peptide is ≤10ppm, the inhibition rate of IL-1β is ≥40%, the inhibition rate of IL-6 is ≥50%, and the inhibition rate of TNF-α is ≥40%.

2. The use of the cyclic peptide according to claim 1, characterized in that: At a concentration of 5 ppm, the cyclic peptide has an inhibition rate of more than 44% on IL-1β, an inhibition rate of more than 53% on IL-6, and an inhibition rate of more than 48% on TNF-α.

3. The use of the cyclic peptide according to claim 1, characterized in that: When the concentration of the cyclic peptide is ≤2%, the enhancement rate of FLG expression is above 100%.

4. The use of the cyclic peptide according to claim 1, characterized in that: When the concentration of the cyclic peptide is ≤2%, the TWEL value of the skin is reduced by more than 10%.

5. The use of the cyclic peptide according to any one of claims 1 to 4, characterized in that: The cyclic peptide includes at least one of the compounds shown in the following formulas 1 to 22: Formula 1 Formula 2: Formula 3: Formula 4: Formula 5: Formula 6: Formula 7: Formula 8: Formula 9: Formula 10: Formula 11: Formula 12: Formula 13: Formula 14: Formula 15: Formula 16: Formula 17: Formula 18: Formula 19: Formula 20: Formula 21: Formula 22:

6. The use of the cyclic peptide according to claim 5, characterized in that: The cyclic peptide includes at least one of the compounds with structures shown in Formula 1, Formula 14 and Formula 15.

7. The use of the cyclic peptide according to claim 1, characterized in that: The skin care product comprises at least one of toner, facial mask, essence, lotion, cream, freeze-dried powder, spray and gel.

8. A method for synthesizing a cyclic peptide, characterized in that: The cyclic peptide of claim 5 or 6 is prepared by cyclizing a single or multiple linear peptides.

9. The method for synthesizing a cyclic peptide according to claim 8, characterized in that: The cyclic peptide is prepared by cyclizing a linear peptide, and the preparation steps include: S1. Preparation of linear peptides by amino acid dehydration condensation reaction; S2. taking a linear peptide and performing a cyclization reaction to prepare a cyclic peptide to obtain a crude product; S3. Purify the crude product to obtain pure cyclopeptide.

10. The method for synthesizing a cyclic peptide according to claim 9, characterized in that: The amino acid includes at least one of Fmoc-L-Abu-OH, Fmoc-β-Phe-OH, Fmoc-L-Ser-OH, Boc-L-Leu-OH, Boc-L-Val-OH, Boc-L-lle-OH, Boc-D-Ala-OH, Boc-D-Phe-OH, Boc-Gly-OH, Fmoc-Tyr-O-tBu, Fmoc-Ile-OH, Fmoc-Phe-OH, and Fmoc-Pro-OH.

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