Peptide derivatives and preparation methods and applications thereof

Abstract

The present invention discloses a peptide derivative and its preparation method and application. The peptide derivative has a structure as shown in formula (I). The peptide derivative or its salt of the present invention has better performance in improving cell activity, promoting cell proliferation, and promoting collagen synthesis, and has good anti-aging and repair effects. #imgabs0# (I)

Description

Technical Field

[0001] The invention relates to a peptide derivative and a preparation method and application thereof. Background Art

[0002] Consumers' demand for skin care products is constantly changing, from the initial demand for moisturizing and hydrating to the demand for improving skin problems to a certain extent.

[0003] The development of functional cosmetics has gradually become the theme of the development of the cosmetics industry. The research and development of effective active ingredients can help companies develop unique formulas and launch skin care products with innovative and differentiated advantages. The research and development of new active ingredients for skin care products can help meet market demand, enhance product competitiveness, and adapt to market trends.

[0004] At present, the beauty peptides used in cosmetics are mainly small molecule oligopeptides composed of two to ten amino acids, which can promote cell growth and differentiation, repair skin damage, and have significant wrinkle removal and anti-aging effects. They have become an important component of functional cosmetics and have extremely high safety. For example, palmitoyl tripeptide-1 and palmitoyl tripeptide-5 can promote the production of more skin repair proteins during the skin repair process, thereby restoring cell vitality and achieving the effect of repairing and removing wrinkles. They are widely used beauty small molecule peptides.

[0005] CN112716826A discloses a polypeptide composition for long-term wrinkle removal and skin elasticity enhancement, with acetyl hexapeptide-8, tripeptide-1, palmitoyl tetrapeptide-7, and palmitoyl pentapeptide-4 as main active ingredients, supplemented with PEG hydrogenated castor oil, glycerol, and propylene glycol. The resulting essence has long-term wrinkle removal and anti-wrinkle effects.

[0006] CN114796003A discloses an anti-aging oligopeptide composition suitable for sensitive skin, comprising the following raw materials, measured by weight: 50-1000 parts of palmitoyl tripeptide-1, 50-1000 parts of palmitoyl tripeptide-5, 50-1000 parts of palmitoyl tripeptide-8, 50-1000 parts of palmitoyl tetrapeptide-7, 50-1000 parts of palmitoyl pentapeptide-450-1000 parts, 50-1000 parts of palmitoyl hexapeptide-12, 10000-100000 parts of deionized water, 10000-200000 parts of emulsifier, 50-100000 parts of rose extract, 50-100000 parts of lemon extract, 50-100000 parts of vitamin E, 50-100000 parts of licorice extract, 50-100000 parts of fig extract and 50-1000 parts of soybean lecithin.

[0007] CN113264984A discloses a peptide compound, a composition containing them, the compound and use thereof, the peptide compound at least comprising an amino acid, which is lysine, ornithine, diaminopropionic acid or diaminobutyric acid, or a derivative of these amino acids, on which a carboxylic acid is grafted through a peptide bound to a side chain nitrogen, the carboxylic acid comprising a (poly) ring or a (poly) heterocycle. Preferably, the grafted carboxylic acid is an amino acid or a derivative, in particular selected from proline, hydroxyproline or pyroglutamic acid. The peptide compound can stimulate the synthesis of the main molecules constituting the extracellular matrix of the skin, in particular collagen 1 and 4 and elastin.

[0008] The problems of small molecule oligopeptides at present include: the synthesis method has obvious scale limitations, a large amount of amino acid raw materials are required in the production process, the synthesis cost is high, a lot of waste is generated, and the cost-effectiveness ratio is high. In addition, it is also expected that active raw materials for skin care products with better efficacy can be obtained. The better efficacy includes better promotion of cell growth, wound healing, collagen and elastin production, so as to achieve the anti-aging effect of smoothing skin fine lines and enhancing skin barrier function. Summary of the invention

[0009] In view of this, an object of the present invention is to provide a peptide derivative or a salt thereof, which peptide derivative or a salt thereof exhibits better performance in improving cell activity, promoting cell proliferation, promoting collagen synthesis, increasing skin elasticity, and improving skin firmness, and has better anti-aging and repair effects.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned peptide derivative or its salt, which has simpler operation and lower cost.

[0011] Another object of the present invention is to provide the use of the above peptide derivatives or their salts in the preparation of anti-aging and anti-wrinkle cosmetics.

[0012] Another object of the present invention is to provide a cosmetic having anti-aging and anti-wrinkle effects, comprising the peptide derivative or a salt thereof as described above.

[0013] In one aspect, the present invention provides a peptide derivative as represented by formula (I) or a salt thereof,

[0014] (I),

[0015] In formula (I), when R2 is a hydroxyl group, R1 is selected from one of the following substituents:

[0016] (A), (B), (C), (D), (G);

[0017] In formula (A), R3 and R4 are independently selected from C1-C3 alkyl; R5 is a substituent at any position on the benzene ring, and R5 is selected from hydrogen or C1-C3 alkyl; in formula (B), R7 and R8 are independently selected from hydrogen or C1-C3 alkyl; in formula (C), R9 to R 12 are independently selected from hydrogen, methyl or ethyl; in formula (D), R 13 , R 14 are independently selected from hydrogen, C1-C3 alkyl or C1-C3 alkoxy; in formula (G), R 19 Selected from hydrogen or C1-C3 alkyl;

[0018] In formula (I), when R1 is hydrogen, R2 is selected from the substituents of the following structures:

[0019] (E);

[0020] In formula (E), R 15 To R 18 are independently selected from hydrogen, methyl or ethyl.

[0021] According to the peptide derivative or salt thereof of the present invention, preferably, R3 and R4 are independently selected from methyl or ethyl; R5 is a substituent at any position on the benzene ring, and R5 is selected from hydrogen or methyl; R7 and R8 are independently selected from hydrogen or methyl; R9 to R 12 are independently selected from hydrogen or methyl; R 13 , R 14 R are independently selected from hydrogen, methyl or ethyl; 15 To R 18 are each independently selected from hydrogen or methyl.

[0022] According to the peptide derivative or salt thereof of the present invention, preferably, in formula (I), when R2 is a hydroxyl group, R1 is selected from one of the following substituents:

[0023] , , , , ,

[0024] In formula (I), when R1 is hydrogen, R2 is a substituent having the following structure:

[0025] .

[0026] On the other hand, the present invention also provides a method for preparing the peptide derivative or its salt according to the above. When R2 is a hydroxyl group, the method for preparing the peptide derivative represented by formula (I) comprises the following steps:

[0027] 1) reacting the compound represented by formula (M6) with one of the compounds represented by formula (A'), (B'), (C'), (D'), and (G') to obtain an amidation product;

[0028] (M6); (A'), (B'), (C'), (D'), (G');

[0029] In formula (A'), R3 and R4 are independently selected from C1-C3 alkyl; R5 is a substituent at any position on the benzene ring, and R5 is selected from hydrogen or C1-C3 alkyl; in formula (B'), R7 and R8 are independently selected from hydrogen or C1-C3 alkyl; in formula (C'), R9 to R 12 are independently selected from hydrogen, methyl or ethyl; in formula (D'), R 13 , R 14 are independently selected from hydrogen, C1-C3 alkyl or C1-C3 alkoxy; in formula (G'), R 19 Selected from hydrogen or C1-C3 alkyl;

[0030] 2) subjecting the amidation products obtained in step 1) to hydrogenation reduction to obtain debenzylated products;

[0031] 3) The debenzylated products are subjected to acid hydrolysis treatment to obtain peptide derivatives as shown in formula (I).

[0032] According to the preparation method of the present invention, preferably:

[0033] In step 1), the reaction is carried out in the presence of HATU and DIPEA; in step 2), palladium carbon is used as a catalyst for hydrogenation reaction; in step 3), acid hydrolysis is carried out in the presence of trifluoroacetic acid.

[0034] According to the preparation method of the present invention, preferably, when R1 is hydrogen, the preparation method of the peptide derivative represented by formula (I) comprises the following steps:

[0035] 1') hydrogenating and reducing the compound represented by formula (M5) to remove the benzyl group, thereby obtaining the compound represented by formula (M15);

[0036] 2') reacting the compound represented by formula (M15) with the compound represented by formula (E') to obtain the compound represented by formula (M16);

[0037] 3') removing the Fmoc protecting group from the compound represented by formula (M16) to obtain the compound represented by formula (M17);

[0038] 4') subjecting the compound represented by formula (M17) to acid hydrolysis to obtain a peptide derivative represented by formula (I);

[0039] (M5), (M15), (E'), (M16),

[0040] (M17);

[0041] In formula (E'), formula (M16) and formula (M17), R 15 To R 18 are independently selected from hydrogen, methyl or ethyl.

[0042] According to the preparation method of the present invention, preferably, the compound represented by formula (M6) is prepared by the following steps:

[0043] (1) reacting a compound represented by formula (M1) with a compound represented by formula (M2) to obtain a compound represented by formula (M3);

[0044] (2) removing the Fmoc protecting group from the compound represented by formula (M3) to obtain the compound represented by formula (M4);

[0045] (3) reacting the compound represented by formula (M4) with the compound represented by formula (F) to obtain the compound represented by formula (M5);

[0046] (4) removing the Fmoc protecting group from the compound represented by formula (M5) to obtain the compound represented by formula (M6);

[0047] (M1), (M2), (M3),

[0048] (M4), (F),

[0049] (M5).

[0050] According to the preparation method of the present invention, preferably, in step (1), the reaction is carried out in the presence of HATU and DIPEA; in step (2), the reaction is carried out in the presence of DBU and piperidine; in step (3), the reaction is carried out in the presence of HATU and DIPEA; in step (4), the reaction is carried out in the presence of DBU and piperidine.

[0051] In another aspect, the present invention also provides use of the peptide derivative or its salt as described above in the preparation of anti-aging and anti-wrinkle cosmetics.

[0052] In another aspect, the present invention also provides a cosmetic having anti-aging and anti-wrinkle effects, comprising the peptide derivative or a salt thereof as described above.

[0053] The peptide derivatives or salts thereof of the present invention are applied to cosmetics and beauty products, which can significantly enhance the use effects of functional cosmetics or beauty products, such as skin barrier repair and skin wrinkle smoothing. The peptide derivatives or salts thereof of the present invention show more excellent performance in promoting cell growth, wound healing, collagen, and elastin production, so as to achieve the anti-aging effect of smoothing skin fine lines and enhancing skin barrier function. Compared with existing polypeptide active substances, the peptide derivatives or salts thereof of the present invention have a lower effective concentration, and the anti-aging and repair effects are better at the same addition amount. The preparation method of the peptide derivatives or salts thereof of the present invention is easier to operate, easier to achieve large-scale production, and lower cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 , Figure 2 , Figure 3 This is the result of cell scratch experiment.

[0055] Figure 4 The quantitative data of cell migration level calculated by software are summarized as the average scratch width before and after sample treatment. DETAILED DESCRIPTION

[0056] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0057] Explanation of terms

[0058] In the present invention, Cm-Cn represents a group having m to n carbon atoms; for example, C1-C3 alkyl represents an alkyl group having 1 to 3 carbon atoms.

[0059] Peptide derivative or its salt

[0060] The peptide derivatives of the present invention represent a class of substances. The peptide derivatives of the present invention have a structure shown in formula (I):

[0061] (I).

[0062] The corresponding salts include salts formed by peptide derivatives and inorganic or organic acids. The organic acids include acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid or gluconic acid; the inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid, boric acid or carbonic acid. Alternatively, the salts include metal salts of peptide derivatives, and the metals include lithium, sodium, potassium, calcium, magnesium, manganese, copper, iron, zinc or aluminum. Alternatively, the salts include salts formed by peptide derivatives and organic bases, and the organic bases include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, etc. Trifluoroacetate is preferred.

[0063] The peptide derivatives or salts thereof of the present invention are applied to cosmetics and beauty products, and can significantly enhance the use effects of functional cosmetics or beauty products, such as skin barrier repair and skin wrinkle smoothing. The peptide derivatives or salts thereof of the present invention show more excellent effects in promoting cell growth, wound healing, collagen and elastin production, so as to achieve the anti-aging effect of smoothing skin fine lines and enhancing skin barrier function.

[0064] In the present invention, in certain embodiments, R2 is hydroxyl. When R2 is hydroxyl, R1 is selected from one of the following substituents:

[0065] (A), (B), (C),

[0066] (D), (G);

[0067] In formula (A), R3 and R4 are independently selected from C1-C3 alkyl, preferably, R3 and R4 are independently selected from methyl or ethyl, more preferably, R3 and R4 are methyl. R5 is a substituent at any position on the benzene ring, R5 is selected from hydrogen or C1-C3 alkyl, preferably selected from hydrogen or methyl, more preferably hydrogen.

[0068] In formula (B), R7 and R8 are independently selected from hydrogen or C1-C3 alkyl, preferably, R7 and R8 are independently selected from hydrogen or methyl, more preferably, R7 and R8 are hydrogen.

[0069] In formula (C), R9 to R 12 are independently selected from hydrogen, methyl or ethyl, preferably, R9 to R 12 are independently selected from hydrogen or methyl, more preferably, R9 to R 12 are hydrogen respectively.

[0070] In formula (D), R 13 , R14 are independently selected from hydrogen, C1-C3 alkyl or C1-C3 alkoxy, preferably, R 13 , R 14 are independently selected from hydrogen, methyl or ethyl, more preferably, R 13 , R 14 are hydrogen respectively.

[0071] In formula (G), R 19 It is selected from hydrogen or C1-C3 alkyl, preferably selected from hydrogen or methyl, more preferably hydrogen.

[0072] In the present invention, examples of C1-C3 alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, etc. Examples of C1-C3 alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0073] According to a specific embodiment of the present invention, the peptide derivative of the present invention has the structure shown below:

[0074] ; recorded as SL-PP-012.

[0075] According to another specific embodiment of the present invention, the peptide derivative of the present invention has the structure shown below:

[0076] ; recorded as SL-PP-013.

[0077] According to another specific embodiment of the present invention, the peptide derivative of the present invention has the structure shown below:

[0078] ; Recorded as SL-PP-003.

[0079] According to another specific embodiment of the present invention, the peptide derivative of the present invention has the structure shown below:

[0080] ; recorded as SL-PP-015.

[0081] According to another specific embodiment of the present invention, the peptide derivative of the present invention has the structure shown below:

[0082] ; Recorded as SL-PP-002.

[0083] In other embodiments, R1 is hydrogen. When R1 is hydrogen, R2 is selected from the substituents of the following structures:

[0084] (E);

[0085] In formula (E), R15 To R 18 are independently selected from hydrogen, methyl or ethyl, preferably, R 15 To R 18 are each independently selected from hydrogen or methyl.

[0086] According to a specific embodiment of the present invention, the peptide derivative of the present invention has the structure shown below:

[0087] ; recorded as SL-PP-014.

[0088] The peptide derivative having the structure of formula (I) of the present invention has better anti-wrinkle activity than the existing palmitoyl tripeptide-5 and the naked peptide SL-PP-001.

[0089] The structural formula of SL-PP-001 is as follows:

[0090] .

[0091] The structural formula of palmitoyl tripeptide-5 (denoted as C1-X) is as follows:

[0092] .

[0093] Preparation method

[0094] In the prior art, polypeptides of specific structures are generally prepared by solid phase synthesis: an amino acid whose N-terminus is protected by a specific protecting group is immobilized on a resin of a specific structure at its C-terminus, and the N-terminal protecting group is removed and connected to the next amino acid fragment by amide condensation. The above process is repeated until the synthesis of the target peptide is completed, and the resulting polypeptide is separated from the immobilization, and the target polypeptide molecule is obtained by preparative chromatography purification. This synthesis method has obvious scale limitations, and a very large amount of amino acid raw materials and organic chemical reagents are required in the production process, resulting in high synthesis costs and a lot of waste.

[0095] Due to the high cost of existing synthetic peptide preparation methods, the current price of synthetic peptides is relatively high. Limited by the cost control of cosmetic production, the amount of such synthetic peptides added to cosmetics is generally low.

[0096] The preparation method of the invention does not require solid phase synthesis, the operation steps are simpler, and the synthesis cost is reduced.

[0097] In certain embodiments of the present invention, when R2 is a hydroxyl group, the method for preparing the peptide derivative represented by formula (I) comprises the following steps: 1) reacting the compound represented by formula (M6) with one of the compounds represented by formula (A'), (B'), (C'), (D'), and (G') to obtain an amidated product; 2) a hydrogenation reduction debenzylation step; and 3) an acidolysis treatment step.

[0098] The preparation method of the compound represented by formula (M6) comprises the following steps: (1) reacting the compound represented by formula (M1) with the compound represented by formula (M2) to obtain the compound represented by formula (M3); (2) removing the Fmoc protecting group from the compound represented by formula (M3) to obtain the compound represented by formula (M4); (3) reacting the compound represented by formula (M4) with the compound represented by formula (F) to obtain the compound represented by formula (M5); (4) removing the Fmoc protecting group from the compound represented by formula (M5) to obtain the compound represented by formula (M6). The following is a detailed description.

[0099] The compound represented by formula (M1) is reacted with the compound represented by formula (M2) to obtain a compound represented by formula (M3).

[0100] (M1), (M2), (M3).

[0101] The reaction step is carried out in the presence of HATU and DIPEA. HATU is a peptide condensation reagent, and its system name is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate. DIPEA is N,N-diisopropylethylamine.

[0102] The molar ratio of the compound represented by formula (M1) to the compound represented by formula (M2) is 1:1. The molar ratio of HATU, DIPEA and the compound represented by formula (M1) is 1.15-1.22:1.8-2.2:1.05, preferably 1.18-1.22:1.9-2.2:1.05, and more preferably 1.2-1.21:2.0-2.1:1.05. The reaction solvent can be dimethylformamide (DMF) or dimethylacetamide. The temperature during the addition is -10-0°C, and the temperature can be raised to 20-40°C after the addition. The reaction process can be monitored by TLC. After the reaction is completed, the reaction is quenched with water, filtered, and the filter cake is washed with water.

[0103] The Fmoc protecting group is removed from the compound represented by formula (M3) to obtain the compound represented by formula (M4).

[0104] (M4),

[0105] This reaction step can be carried out in the presence of DBU and piperidine. The chemical name of DBU is 1,8-diazabicyclo(5,4,0)-7-undecene.

[0106] The molar ratio of DBU, piperidine and the compound represented by formula (M3) is 4.0-5.0:0.5-0.7:1, preferably 4.5-4.6:0.6-0.65:1. The reaction solvent can be dimethylformamide (DMF), dimethylacetamide, dimethyl sulfoxide. The reaction temperature can be 20-40°C. The reaction process can be monitored by TLC. After the reaction is completed, the reaction is quenched with a saturated ammonium chloride solution and filtered. The filtrate is extracted with dichloromethane or chloroform, and the organic phases are combined, concentrated, and purified by column chromatography. The elution is gradient elution, and the eluent is 20% EA / PE (referring to the volume ratio of EA to PE of 20:80), 5% MeOH / DCM 0.5% triethylamine (referring to the volume ratio of MeOH to DCM of 5:95, and 0.5vol% triethylamine is added to the mixed solvent of MeOH and DCM). DCM is dichloromethane.

[0107] The compound represented by formula (M4) is reacted with the compound represented by formula (F) to obtain the compound represented by formula (M5).

[0108] (F), (M5),

[0109] The reaction step is carried out in the presence of HATU and DIPEA. The molar ratio of HATU, DIPEA and the compound represented by formula (M4) is 1.15-1.22:1.8-2.2:1.05, preferably 1.18-1.22:1.9-2.2:1.05, and more preferably 1.2-1.21:2.0-2.1:1.05.

[0110] The reaction solvent may be dimethylformamide (DMF) or dimethylacetamide. The reaction temperature is -10 to 0°C. The reaction process may be monitored by TLC. After the reaction is completed, the reaction is quenched with water, filtered, and the filter cake is washed with water. The crude product may be purified by column chromatography. The eluent may be 2 vol% MeOH / DCM (MeOH:DCM 2:98 v / v, the same below).

[0111] The Fmoc protecting group of the compound represented by formula (M5) is removed to obtain the compound represented by formula (M6).

[0112] (M6);

[0113] The reaction step is carried out in the presence of DBU and piperidine. The molar ratio of DBU, piperidine and the compound represented by formula (M5) is 4.0-5.0:0.5-0.7:1, preferably 4.3-4.6:0.55-0.65:1, and more preferably 4.3-4.5:0.6-0.65:1. The reaction solvent may be dimethylformamide (DMF), dimethylacetamide, or dimethyl sulfoxide. The reaction temperature may be -10-0°C. The reaction process may be monitored by TLC. After the reaction is completed, the reaction is quenched with ice water and filtered. The filtrate is extracted with dichloromethane or chloroform, and the organic phase and the filter cake are combined and washed with saturated ammonium chloride solution and saturated brine in sequence. The filtrate is dried with a desiccant (e.g., sodium sulfate), and the filtered filtrate is concentrated. The crude product obtained by concentration is purified by column chromatography. The eluent is 2% MeOH / DCM.

[0114] The compound represented by formula (M6) is reacted with one of the compounds represented by formula (A'), (B'), (C'), and (D') to obtain an amidation product.

[0115] (M6); (A'), (B'), (C'), (D'); (G');

[0116] In formula (A'), R3 and R4 are independently selected from C1-C3 alkyl, preferably, R3 and R4 are independently selected from methyl or ethyl, more preferably, R3 and R4 are methyl. R5 is a substituent at any position on the benzene ring, R5 is selected from hydrogen or C1-C3 alkyl, preferably selected from hydrogen or methyl, more preferably hydrogen.

[0117] In formula (B'), R7 and R8 are independently selected from hydrogen or C1-C3 alkyl; preferably, R7 and R8 are independently selected from hydrogen or methyl, and more preferably, R7 and R8 are hydrogen.

[0118] In formula (C'), R9 to R 12 are independently selected from hydrogen, methyl or ethyl; preferably, R9 to R 12 are independently selected from hydrogen or methyl, more preferably, R9 to R 12 are hydrogen respectively.

[0119] In formula (D'), R 13 , R 14 Each independently selected from hydrogen, C1-C3 alkyl or C1-C3 alkoxy. Preferably, R 13 , R 14 are independently selected from hydrogen, methyl or ethyl, more preferably, R13 , R 14 are hydrogen respectively.

[0120] In formula (G'), R 19 It is selected from hydrogen or C1-C3 alkyl, preferably selected from hydrogen or methyl, more preferably hydrogen.

[0121] In the present invention, examples of C1-C3 alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, etc. Examples of C1-C3 alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, etc.

[0122] The reaction step is carried out in the presence of HATU and DIPEA. The molar ratio of HATU, DIPEA and the compound represented by formula (M6) is 1.15-1.22:1.8-2.2:1.0, preferably 1.18-1.21:1.9-2.2:1.0, and more preferably 1.2-1.21:2.0-2.1:1.0. The reaction solvent can be dimethylformamide (DMF) or dimethylacetamide. The reaction temperature is -10-0°C. The reaction process can be monitored by TLC. After the reaction is completed, the reaction is quenched with water, filtered, the filter cake is dissolved in dichloromethane or chloroform, dried with a desiccant (e.g., sodium sulfate), filtered, and the filtrate is concentrated by rotation to obtain a crude product. The crude product is purified by column chromatography. The eluent used is 2%-4% MeOH / DCM (MeOH:DCM=2:98-4:96 v / v).

[0123] In the present invention, the structural formula of the amidation product obtained by reacting the compound represented by formula (M6) with the compound represented by formula (A') is as follows:

[0124] .

[0125] The structural formula of the amidation product generated by the reaction of the compound represented by formula (M6) with formula (B'), (C'), (D'), (G') is not listed in detail and can be known to those skilled in the art.

[0126] The obtained amidation products are respectively subjected to hydrogenation reduction to obtain debenzylated products.

[0127] In this step, palladium on carbon is used as a catalyst for hydrogenation reaction to remove the benzyl group.

[0128] The content of palladium in palladium carbon is 5 to 15 wt % wet., for example, it can be 10 wt % wet. (the content of palladium is 10 wt % of the wet weight of palladium carbon).

[0129] The debenzylated products are subjected to acid hydrolysis treatment to obtain peptide derivatives as shown in formula (I).

[0130] In this step, the Boc protecting group is removed by acid hydrolysis.

[0131] A solution containing trifluoroacetic acid is used for acid hydrolysis treatment, specifically a mixed solution of TFA, EDT, TIPS, and H2O is used for acid hydrolysis treatment. The volume ratio of TFA (trifluoroacetic acid), EDT (1,2-ethanedithiol), TIPS (triisopropylsilane), and H2O is 95:1:2:2.

[0132] The compound represented by the raw material formula (M6) and the compound represented by the formula (A') are subjected to amidation, hydrogenation debenzylation and acid hydrolysis to remove Boc, and the structure of the peptide derivative represented by the formula (I) is as follows:

[0133] .

[0134] The compound represented by the raw material formula (M6) reacts with the compounds represented by the formulas (B'), (C'), (D'), and (G'), respectively, and then the Boc is removed by amidation, hydrogenation debenzylation, and acid hydrolysis. The structural formulas of the peptide derivatives represented by the formula (I) are not listed in detail here, and can be known to those skilled in the art.

[0135] In other embodiments of the present invention, when R1 is hydrogen, the preparation method of the peptide derivative represented by formula (I) comprises the following steps: 1') hydrogenating and reducing the compound represented by formula (M5) to remove the benzyl group to obtain the compound represented by formula (M15); 2') reacting the compound represented by formula (M15) with the compound represented by formula (E') to obtain the compound represented by formula (M16); 3') removing the Fmoc protecting group from the compound represented by formula (M16) to obtain the compound represented by formula (M17); 4') subjecting the compound represented by formula (M17) to acid hydrolysis to obtain the peptide derivative represented by formula (I). The following is a detailed description.

[0136] The compound represented by formula (M5) is subjected to hydrogenation reduction to remove the benzyl group to obtain the compound represented by formula (M15).

[0137] (M5), (M15),

[0138] The preparation method of the compound represented by formula (M5) is described above and will not be repeated here.

[0139] In this step, palladium carbon is used as a catalyst for hydrogenation reaction to remove the benzyl group. The content of palladium in the palladium carbon is 5 to 15 wt % wet., for example, 10 wt % wet.

[0140] The compound represented by formula (M15) is reacted with the compound represented by formula (E') to obtain the compound represented by formula (M16).

[0141] (E'), (M16),

[0142] The reaction step is carried out in the presence of HATU and DIPEA. The molar ratio of HATU, DIPEA and the compound represented by formula (M15) is 1.15-1.22:1.8-2.2:1.0, preferably 1.18-1.21:1.9-2.2:1.0, and more preferably 1.2-1.21:2.0-2.1:1.0. The reaction solvent may be dimethylformamide (DMF) or dimethylacetamide. The reaction temperature is -10-0°C. The reaction process may be monitored by TLC. After the reaction is completed, the reaction is quenched with water, filtered, the filter cake is dissolved in dichloromethane or chloroform, dried with a desiccant (e.g., sodium sulfate), filtered, and the filtrate is spin-dried to obtain a crude product. The crude product is purified by column chromatography. The eluent may be 2%-4% MeOH / DCM.

[0143] The Fmoc protecting group of the compound represented by formula (M16) is removed to obtain the compound represented by formula (M17).

[0144] (M17);

[0145] The reaction step is carried out in the presence of DBU and piperidine. The molar ratio of DBU, piperidine and the compound represented by formula (M16) is 4.0-5.0:0.5-0.7:1, preferably 4.3-4.6:0.55-0.65:1, and more preferably 4.3-4.5:0.6-0.65:1. The reaction solvent can be dimethylformamide (DMF), dimethylacetamide, and dimethyl sulfoxide. The reaction temperature can be -10-0°C. The reaction process can be monitored by TLC. After the reaction is completed, the reaction is quenched with ice water, and solids are precipitated. The filtrate is extracted with dichloromethane or chloroform. The organic phase and the filter cake are combined, washed with saturated ammonium chloride solution and saturated brine, the organic phase is dried with a desiccant, filtered, and the filtrate is concentrated to obtain a crude product. The crude product is purified by column chromatography. The eluent is 2% MeOH / DCM.

[0146] The compound represented by formula (M17) is subjected to acid hydrolysis to obtain a peptide derivative represented by formula (I).

[0147] The compound represented by formula (M17) is subjected to acid hydrolysis to remove the Boc group, thereby obtaining a peptide derivative represented by formula (I).

[0148] .

[0149] A solution containing trifluoroacetic acid is used for acid hydrolysis treatment, specifically a mixed solution of TFA, EDT, TIPS, and H2O is used for acid hydrolysis treatment. The volume ratio of TFA (trifluoroacetic acid), EDT (1,2-ethanedithiol), TIPS (triisopropylsilane), and H2O is 95:1:2:2.

[0150] application

[0151] The present invention also provides the use of the peptide derivative or its salt as described above in the preparation of anti-aging and anti-wrinkle cosmetics. The peptide derivative or its salt of the present invention shows a more excellent effect of promoting cell proliferation, promoting collagen synthesis, and reducing the area or degree of skin wrinkles compared to the prototype peptide; and has a smaller effective concentration. It can be used in cosmetics or skin care products as a main active ingredient. Such ingredients are used in cosmetics or skin care products, and have the advantages of high ingredient stability, good formula compatibility, and higher cost-effectiveness compared to commercially available products.

[0152] Cosmetics with anti-aging and anti-wrinkle effects

[0153] The present invention also provides a cosmetic with anti-aging and anti-wrinkle effect, which comprises the peptide derivative or its salt as described above. The peptide derivative or its salt of the present invention can significantly enhance the use effect of functional cosmetics or beauty products, such as skin barrier repair and skin wrinkle smoothing.

[0154] Test Method

[0155] 1 H NMR and 13 C NMR was measured using a Bruker AVANCE III HD 500 MHz nuclear magnetic resonance spectrometer.

[0156] Preparation Example 1 - Preparation of compounds represented by formula (M5) and (M6)

[0157] Reaction equation:

[0158]

[0159] Under argon protection, the compound represented by formula (M2) (11.30 g, 33.6 mmol, 1.05 eq) was dissolved in 225 mL of dry DMF and cooled to -5 °C. Fmoc-L-valine (i.e., the compound represented by M1, 12.01 g, 35.3 mmol, 1.05 eq) and HATU (15.33 g, 40.3 mmol, 1.20 eq) were added to the reaction system in sequence; DIPEA (8.70 g, 67.2 mmol, 2.00 eq) was then added. The temperature was slowly raised to room temperature and stirred for 3 h. The reaction was monitored by TLC (Rf = 0.7, 5% MeOH / DCM). The reaction solution was poured into water (500 mL) for quenching. A large amount of solid precipitated, which was filtered, and the filter cake was washed with water (50 mL × 2), and the solid was dried. The compound represented by formula (M3) was obtained (22.00 g, light yellow solid, 100% yield).

[0160] The compound represented by formula (M3) (22.00 g, 39.4 mmol, 1.0 eq) was added to 126 mL of dry DMF and dissolved. Under argon protection and room temperature, piperidine (3.6 g, 23.6 mmol, 0.6 eq) and DBU (6 mL, 177.2 mmol, 4.5 eq) were added and stirred for 2 h. The reaction was monitored by TLC (Rf = 0.15, 5% MeOH / DCM). The reaction solution was poured into a saturated ammonium chloride solution (900 mL) for quenching. A large amount of solid precipitated and was filtered. The filtrate was extracted with DCM (300 mL × 3), the organic phase was combined, and washed twice with saturated brine (300 mL × 2), dried, and spin-dried. The crude product was purified by column chromatography (100-200 mesh, eluent: 20% EA / PE-5% MeOH / DCM 0.5% triethylamine) to obtain the compound represented by formula (M4) (12.60 g, light yellow oil, 95% yield).

[0161] Under argon protection, the compound represented by formula (M4) (12.50 g, 28.7 mmol, 1.05 eq) was dissolved in 190 mL of dry DMF and cooled to -5 °C. N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butyloxycarbonyl)-L-lysine (13.70 g, 29.3 mmol, 1.02 eq) and HATU (13.10 g, 34.4 mmol, 1.20 eq) were added to the reaction in sequence; DIPEA (7.42 g, 57.4 mmol, 2.00 eq) was then added. Stir at -5 °C for 2 h. The reaction was monitored to be complete by TLC (Rf = 0.5, 5% MeOH / DCM). The reaction solution was poured into water (900 mL) for quenching. A large amount of solid precipitated, which was filtered and the filter cake was washed with water (50 mL × 2) twice, and the solid was dried. The crude product was purified by column chromatography (100-200 mesh, eluent 2% MeOH / DCM). The compound represented by formula (M5) was obtained (23.00 g, light yellow solid, 100% yield).

[0162] The compound represented by formula (M5) (21.90 g, 24.7 mmol, 1.0 eq) was mixed and dissolved with 250 mL of dry DMF and cooled to -5 °C. Under argon atmosphere, piperidine (2.80 g, 14.9 mmol, 0.6 eq) and DBU (9.10 g, 106.7 mmol, 4.3 eq) were added thereto and stirred for 1.5 h. The reaction was monitored to be complete by TLC (Rf = 0.3, 7% MeOH / DCM). The reaction solution was poured into ice water (800 mL) for quenching, and a large amount of solid precipitated. The filtrate was extracted with DCM (400 mL × 3). The organic phase and the filter cake were combined and washed twice with saturated ammonium chloride solution (300 mL × 2) and once with saturated brine (400 mL), then dried over sodium sulfate, filtered, and the filtrate was spin-dried. The crude product was purified by column chromatography (100-200 mesh, eluent 2% MeOH / DCM) to obtain the compound represented by formula (M6) (14.40 g, light yellow oil, 87% yield).

[0163] Among them, the NMR results of the compound represented by formula (M4) are as follows:

[0164] 1H NMR: (500 MHz, chloroform-d) δ 7.85 (d, J = 8.3 Hz, 1H), 7.41 -7.28 (m,5H), 5.23-5.11 (m, 2H), 4.71-4.60 (m, 2H), 3.27 (d, J = 3.3 Hz, 1H), 0.86-080 (d, J = 6.9 Hz, 3H).

[0165] 13 C NMR: (126 MHz, CHLOROFORM-d) δ 174.39, 172.35, 156.01, 135.40, 128.64,128.35, 67.07, 60.08, 53.46, 51.59, 40.21, 32.24, 30.81, 29.33, 28.43, 22.54,19.69, 16.15.

[0166] Example 1 - Preparation of peptide derivatives represented by formula (I)

[0167] Reaction equation:

[0168]

[0169] Under argon protection, the compound represented by formula (M6) (1.51 g, 2.27 mmol, 1.00 eq) was dissolved in 12 mL of dry DMF and cooled to -5°C. Trans-4-(tert-butyloxycarbonylaminomethyl)cyclohexylcarboxylic acid (compound represented by formula (C'), 0.64 g, 2.49 mmol, 1.10 eq) and HATU (1.03 g, 2.70 mmol, 1.20 eq) were added to the reaction system in sequence; DIPEA (592.0 mg, 4.52 mmol, 2.00 eq) was then added. Stir at -5°C for 48 h. Monitor by TLC (Rf = 0.5, 5% MeOH / DCM). The reaction solution was slowly poured into water (300 mL) to quench, and solid precipitated and filtered. The solid was dissolved in DCM (200 mL) again, dried over sodium sulfate, filtered, and the filtrate was spin-dried to obtain a crude product. The crude product was purified by column chromatography (100-200 mesh, 2%-4% MeOH / DCM) to obtain the compound represented by formula (M7) (1.60 g, light yellow solid, 78% yield).

[0170] At room temperature, the compound represented by formula (M7) (1.60 g, 1.77 mmol, 1.00 eq) was dissolved in 30 mL of methanol. Palladium carbon (0.30 g, palladium content of 10 wt% wet.) was added to the reaction system; hydrogen was used for replacement three times and stirred for 2 h under a hydrogen atmosphere. The reaction was monitored by mass spectrometry. The reaction solution was filtered and the filtrate was dried to obtain the compound represented by formula (M8) (1.45 g, light yellow colloid, 100% yield).

[0171] At room temperature, the compound represented by formula (M8) (780.0 mg, 0.96 mmol, 1.00 eq) was dissolved in a prepared TFA:EDT:TIPS:H2O 95:1:2:2 (v / v%) (10 mL) solution. Stir at room temperature for 2 h. The reaction was completed by mass spectrometry monitoring. The reaction solution was poured into ether at about 0°C, and solid precipitated and filtered. The solid was dissolved in pure water and then freeze-dried to obtain a peptide derivative represented by formula (I), recorded as SL-PP-003 (500.0 mg, light yellow powder, 100% yield).

[0172] The hydrogen spectrum of SL-PP-003 is as follows:

[0173] 1 H NMR (500 MHz, methanol- d 4) δ 4.38 (dd, J = 9.4, 4.8 Hz, 1H), 4.32 (dd, J=8.7, 5.8 Hz, 1H), 4.17 (d, J = 7.3 Hz, 1H), 2.93 (td, J = 7.6, 5.4 Hz, 4H), 2.79(d, J = 7.0 Hz, 2H), 2.30 (tt, J = 12.2, 3.3 Hz, 1H), 2.13 – 2.04 (m, J = 6.8 Hz,1H), 1.97 – 1.86 (m, 5H), 1.84 – 1.72 (m, 2H), 1.72 – 1.60 (m, 6H), 1.56 –1.38 (m, 6H), 1.15 – 1.03 (m, 2H), 0.98 (dd, J = 13.3, 6.8 Hz, 6H).

[0174] Example 2

[0175] The difference from Example 1 is that the R1 substituent is different. The obtained peptide derivative represented by formula (I) is recorded as SL-PP-012. The reaction equation is as follows:

[0176]

[0177] The specific preparation steps are shown in Example 1 and will not be described in detail here.

[0178] The hydrogen spectrum of SL-PP-012 is as follows:

[0179] 1 H NMR (500 MHz, methanol- d 4) δ 7.55 (s, 1H), 7.27 (s, 1H), 7.17 (d, J = 8.1Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 15.7 Hz, 1H), 4.51 (d, J = 14.3 Hz,1H), 4.39 (dd, J = 9.3, 4.9 Hz, 1H), 4.17 (d, J = 7.3 Hz, 1H), 3.86 (s, 3H), 2.94 (s, 4H), 2.27 (s, 3H), 2.10 (h, J= 6.7 Hz, 1H), 1.98 – 1.84 (m, 2H), 1.81 –1.61 (m, 6H), 1.52 – 1.48 (m, 4H), 1.01 (dd, J = 6.8, 5.0 Hz, 6H).

[0180] Example 3

[0181] The difference from Example 1 is that the R1 substituent is different. The obtained peptide derivative represented by formula (I) is recorded as SL-PP-013. The reaction equation is as follows:

[0182]

[0183] The specific preparation steps are shown in Example 1 and will not be described in detail here.

[0184] The hydrogen spectrum of SL-PP-013 is as follows:

[0185] 1 H NMR (500 MHz, methanol- d 4) δ 4.40 (ddd, J = 11.1, 9.4, 4.8 Hz, 1H), 4.32(ddd, J = 8.7, 5.9, 4.3 Hz, 1H), 4.27 (dt, J = 9.1, 4.8 Hz, 1H), 4.17 (dd, J = 7.5,2.4 Hz, 1H), 2.94 (td, J = 8.5, 8.0, 3.6 Hz, 4H), 2.52 – 2.26 (m, 3H), 2.19 –2.03 (m, 2H), 1.98 – 1.89 (m, 1H), 1.85 (ddd, J = 13.6, 6.5, 3.7 Hz, 1H), 1.79– 1.63 (m, 6H), 1.55 – 1.42 (m, 4H), 0.99 (dd, J = 13.5, 6.7 Hz, 6H).

[0186] Example 4

[0187] The difference from Example 1 is that the R1 substituent is different. The obtained peptide derivative represented by formula (I) is recorded as SL-PP-015. The reaction equation is as follows:

[0188]

[0189] The specific preparation steps are shown in Example 1 and will not be described in detail here.

[0190] The hydrogen spectrum data of SL-PP-015 are as follows:

[0191] 1 H NMR (500 MHz, methanol- d 4) δ 7.13 (dd, J = 15.1, 10.6 Hz, 1H), 6.28 –6.19 (m, 1H), 6.18 – 6.08 (m, 1H), 5.97 (d, J = 15.1 Hz, 1H), 4.43 (dd, J = 8.7,5.8 Hz, 1H), 4.37 (dd, J = 9.3, 4.8 Hz, 1H), 4.15 (d, J = 7.2 Hz, 1H), 2.92 (td, J = 7.6, 2.6 Hz, 5H), 2.08 (h, J = 6.9 Hz, 1H), 1.95 – 1.89 (m, 1H), 1.83 (d, J =6.6 Hz, 3H), 1.67 (q, J = 7.3 Hz, 6H), 1.51 – 1.44 (m, 4H), 0.98 (t, J = 7.4 Hz,6H).

[0192] Example 5

[0193] The difference from Example 1 is that the R1 substituent is different. The obtained peptide derivative represented by formula (I) is recorded as SL-PP-002. The reaction equation is as follows:

[0194]

[0195] The specific preparation steps are referred to Example 1 and will not be described in detail here. The hydrogen spectrum data of SL-PP-002 are as follows:

[0196] 1 H NMR (500 MHz, methanol- d 4) δ 8.99 (d, J = 2.3 Hz, 1H), 8.71 (dd, J= 4.9,1.6 Hz, 1H), 8.30 (dt, J = 8.1, 2.0 Hz, 1H), 7.59 (dd, J = 8.0, 4.9 Hz, 1H), 4.67(t, J = 7.3 Hz, 1H), 4.23 (dd, J = 7.6, 5.8 Hz, 2H), 3.05 – 2.92 (m, 5H), 2.26 – 2.14 (m, 1H), 2.08 – 1.97 (m, 1H), 1.95 – 1.81 (m, 2H), 1.81 – 1.67 (m, 3H), 1.65 – 1.50 (m, 2H), 1.50 – 1.40 (m, 3H), 1.00 (t, J = 6.4 Hz, 6H).

[0197] Example 6

[0198] The reaction equation is as follows:

[0199]

[0200] At room temperature, the compound represented by formula (M5) (2.20 g, 2.43 mmol, 1.00 eq) was dissolved in 30 mL of methanol. Palladium carbon (0.40 g, palladium content of 10 wt% wet.) was added to the reaction system. Then, hydrogen was used for replacement three times and stirred for 2 h under a hydrogen atmosphere. The reaction was monitored by mass spectrometry. The reaction solution was filtered and the filtrate was dried to obtain the compound represented by formula (M15) (1.92 g, light yellow colloid, 97% yield).

[0201] Under argon protection, the compound represented by formula (M15) (1.91 g, 2.41 mmol, 1.00 eq) was dissolved in 22 mL of dry DMF and cooled to -5°C. Then, trans-4-aminomethyl-cyclohexylcarboxylic acid tert-butyl ester (i.e., the compound represented by formula (E'), 563 mg, 2.64 mmol, 1.10 eq) and HATU (1.09 g, 2.88 mmol, 1.20 eq) were added to the reaction system in sequence; and DIPEA (620.3 mg, 4.80 mmol, 2.00 eq) was added. Stir at -5°C for 48 h. The reaction was monitored by TLC (Rf = 0.5, 5% MeOH / DCM). The reaction solution was slowly poured into water (300 mL), and solids precipitated and filtered. The filter cake was dissolved in DCM (200 mL) again, then dried over sodium sulfate, filtered, and the filtrate was spin-dried to obtain a crude product. The crude product was purified by column chromatography (100-200 mesh, 2%-4% MeOH / DCM) to obtain the compound represented by formula (M16) (2.12 g, light yellow solid, 89% yield).

[0202] The compound represented by formula (M16) (2.1 g, 2.14 mmol, 1.0 eq) was dissolved in 12 mL of dry DMF and cooled to -5 °C. Under argon atmosphere, piperidine (110 mg, 1.28 mmol, 0.6 eq) and DBU (1.40 g, 9.20 mmol, 4.3 eq) were added and stirred for 1.5 h. The reaction was monitored by TLC (Rf = 0.3, 7% MeOH / DCM). The reaction solution was poured into ice water (800 mL) for quenching, and solids precipitated. The filtrate was extracted with DCM (100 mL × 3). The organic phase and the filter cake were combined and washed twice with saturated ammonium chloride solution (100 mL × 2) and once with saturated brine (100 mL), and then the organic phase was dried over sodium sulfate, filtered, and the filtrate was spin-dried to obtain a crude product. The crude product was purified by column chromatography (100-200 mesh, 2% MeOH / DCM) to obtain the compound represented by formula (M17) (1.35 g, light yellow oil, 82% yield).

[0203] At room temperature, the compound represented by formula (M17) (1.35 g, 0.96 mmol, 1.00 eq) was dissolved in a prepared TFA:EDT:TIPS:H2O 95:1:2:2 (v / v%) solution (10 mL). Stir at room temperature for 2 h. The reaction was monitored by mass spectrometry. The reaction solution was poured into ether at about 0°C, and solids precipitated and filtered. The filter cake was dissolved in pure water and freeze-dried to obtain a peptide derivative represented by formula (I), which was recorded as SL-PP-014 (600.0 mg, white powder, 66% yield).

[0204] The hydrogen spectrum data of SL-PP-014 are as follows:

[0205] 1 H NMR (500 MHz, methanol- d 4) δ 4.27 (ddd, J = 17.3, 9.1, 5.2 Hz, 1H), 4.14(dd, J = 56.9, 7.4 Hz, 1H), 3.99 (td, J = 6.6, 3.0 Hz, 1H), 3.04 (t, J = 7.0 Hz,2H), 2.95 (dt, J = 20.9, 7.6 Hz, 5H), 2.21 (dq, J = 8.5, 4.3 Hz, 1H), 2.15 – 2.03(m, 1H), 1.99 (dd, J = 13.6, 3.8 Hz, 2H), 1.91 – 1.77 (m, 3H), 1.70 (ddd, J =14.7, 8.2, 5.0 Hz, 5H), 1.59 – 1.30 (m, 8H), 1.05 (d, J = 6.7 Hz, 2H), 1.00(dt, J = 6.8, 2.3 Hz, 6H).

[0206] Experimental example

[0207] The obtained products SL-PP-002, SL-PP-003, SL-PP-012, SL-PP-013, SL-PP-014, SL-PP-015, as well as palmitoyl tripeptide-5 (prototype peptide, denoted as C1-X), SL-PP-001 (naked peptide) and active small molecules (niacin, pyroglutamic acid, sorbic acid, tranexamic acid and acetylferulic acid) were respectively tested for performance.

[0208] 1. Test of MMP-1 inhibition rate and type I collagen production:

[0209] The present invention evaluates the anti-wrinkle activity of the peptide derivatives by the inhibitory activity of inhibiting matrix metalloproteinase MMP-1 and the amount of promoting type I collagen production.

[0210] 1. Effect of the derivatives of the present invention on the expression of matrix metalloproteinase MMP-1 in human foreskin fibroblasts (HFF-1). Test method: After HFF-1 cells were revived, they were cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. HFF-1 cells in the logarithmic growth phase were digested and resuspended for counting; 2×10 4 Cells were inoculated into 96-well plates, and DMEM high-glucose medium containing 10% FBS was added to culture overnight at 37°C and 5% CO2 to allow them to adhere to the wall; the old medium was discarded, and the cells were gently rinsed once with 200μL PBS; blank group, model group and test group were set up respectively; the blank group was replaced with 200μL 1% FBS medium at 37°C and 5% CO2 for 24h, and the model group and test group were replaced with 50μL PBS; the model group and test group were irradiated with 30mJ UVB in a UV crosslinker. PBS was discarded, 200μL 1% FBS medium was added to the model group, and 200μL of the test sample solution prepared with the above medium to a final concentration of 0.05mg / mL was added to each well of the test group, and cultured at 37°C and 5% CO2 for 24h; the cell supernatant was collected, and the expression of MMP-1 was detected using a human matrix metalloproteinase 1 (MMP-1) ELISA kit.

[0211] Calculation formula: MMP-1 expression inhibition rate = ((UM) / U) × 100%;

[0212] Wherein: M-the average value of MMP-1 expression in the test sample; U-the average value of MMP-1 expression in the model group.

[0213] Experimental groups: Blank group: cultured in medium containing 1% FBS. Model group: cultured in 30mJ UVB+1% FBS medium. Test group: cultured in 30mJ UVB+test sample with a final concentration of 0.05 mg / mL+1% FBS medium.

[0214] 2. The effect of the derivatives of the present invention on the production of human type I collagen in human foreskin fibroblasts (HFF-1).

[0215] Test method: After HFF-1 cells were revived, they were cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. HFF-1 cells in the logarithmic growth phase were digested, resuspended, and counted; 2×10 4Cells were inoculated into 96-well plates, and DMEM high-glucose medium containing 10% FBS was added to culture overnight at 37°C and 5% CO2 to allow them to adhere to the wall; the old medium was discarded and the cells were gently rinsed once with 200μL PBS; blank group, model group and test group were set up respectively; medium containing the test sample with a final concentration of 0.05mg / mL was added to the wells of the test sample group, and normal cell culture medium was added to the control wells of the blank group, 200μL per well. After the administration, the 96-well plate was placed in a CO2 incubator for 24h; after the incubation, 200μL of cell culture supernatant was collected from each well in a 1.5mL sterile centrifuge tube, and the type I collagen content was detected according to the instructions of the human type I collagen enzyme-linked immunosorbent assay kit;

[0216] Calculation formula: Increase rate = ((TC) / C) × 100%;

[0217] Where: T-average value of type I collagen content in the test sample; C-average value of type I collagen content in the blank control.

[0218] Experimental groups: Blank group: cultured in normal cell culture medium. Test group: cultured in culture medium with a final concentration of 0.05 mg / mL test sample.

[0219] Test results: See Table 1 and Table 2. In Table 1, the model group refers to the ultraviolet irradiation model group. As shown in Table 1 and Table 2, the anti-wrinkle activity of the peptide derivatives represented by formula (I) of the present invention is significantly better than that of the naked peptide SL-PP-001 and the prototype peptide C1-X, and is more significantly better than the active small molecules used to modify the polypeptide (nicotinic acid, pyroglutamic acid, sorbic acid, tranexamic acid and acetylferulic acid), achieving unexpected results.

[0220]

[0221]

[0222] (II) Cell scratch assay:

[0223] The cell scratch test was used to evaluate the skin barrier repair effect of the test products.

[0224] Test subject: Human immortalized keratinocytes (HaCat).

[0225] Test method: (1) After HaCat cells are revived, they are cultured in DMEM high-glucose medium containing 10% FBS, placed in a 37°C, 5% CO2 incubator, and passaged at a ratio of 1:3. (2) HaCat cells in the logarithmic growth phase are digested, resuspended, and counted; (3) 6×10 5100 cells / well were inoculated in a 12-well plate, and DMEM high-glucose medium containing 10% FBS was added and cultured overnight at 37℃ and 5% CO2 to allow them to adhere to the wall; (4) After the cells have grown, use a 200μL pipette tip (yellow pipette tip) to scratch the culture wells along the ruler perpendicular to the well plate, and make a "cross" in each well. Wash the cells twice with PBS, wash away the scratched cells, and make the gaps left clearly visible to the naked eye; (5) Set up a blank group, a positive control group, and a test group respectively; (6) Add 1% FBS culture medium to the blank group, add 1% FBS culture medium containing 0.001% EGF to the positive control group, and add 1% FBS culture medium containing 0.001% sample to the test group. Take pictures under a microscope with a 4× objective lens, and record it as 0h. Culture in an incubator at 37℃ and 5% CO2 for 24h. (7) After 24h, discard the old culture medium, wash the cells twice with PBS to wash away dead cells, and take pictures under a microscope with a 4× objective lens, and record it as 24h. (8) ImageJ software was used to measure the width of the scratch area and calculate the cell migration rate.

[0226] Calculation formula: cell migration rate = ((0h scratch width - 24h scratch width) / 0h scratch width) × 100%;

[0227] Experimental groups: Blank group: cultured in 1% FBS medium. Positive control group: cultured in 1% FBS medium containing 0.001% EGF. Test group: cultured in 1% FBS medium containing 0.001% sample.

[0228] Experimental results: see Figure 1 , Figure 2 and Figure 3 . Figure 1 , Figure 2 , Figure 3 Attached are photos of the cell scratch experiment, in which the blank group represents the addition of a blank control without any active ingredients; the EGF group represents the addition of a positive control EGF (EGF refers to recombinant human epidermal growth factor), SL-PP-001 is a naked peptide, and C1-X is a prototype peptide (palmitoyl tripeptide-5).

[0229] Conclusion: The cell layer uniformly grown in the plate wells was damaged by external force in a cross shape. The repair conditions before and after treatment with the peptide derivatives of formula (I) of the present invention or the positive control were different, and both had different degrees of repair promotion effects. The quantitative data of cell migration level calculated by software were compared with the average scratch width before and after sample treatment. Figure 4 .

[0230] (III) Cell migration test:

[0231] Figure 4 The average scratch width before and after sample treatment is a summary of the quantitative data of cell migration level calculated by the software. Figure 4 It can be seen that the peptide derivatives of the present invention have greater cell proliferation promoting activity after derivatization and modification than the prototype peptide compound.

[0232] The present invention is not limited to the above-mentioned embodiments. Without departing from the essential content of the present invention, any deformation, improvement and substitution that can be conceived by those skilled in the art shall fall within the scope of the present invention.

Claims

1. A peptide derivative represented by formula (I) or a salt thereof, （I）， In formula (I), when R2 is a hydroxyl group, R1 is selected from one of the following substituents: ， ， ， ， ， In formula (I), when R1 is hydrogen, R2 is a substituent having the following structure: 。 2. The method for preparing a peptide derivative or a salt thereof according to claim 1, characterized in that: When R2 is a hydroxyl group, the preparation method of the peptide derivative represented by formula (I) comprises the following steps: 1) reacting the compound represented by formula (M6) with one of the compounds represented by formula (A'), (B'), (C'), (D'), and (G') to obtain an amidation product; (M6); (A’), (B’), (C’), (D’), (G’); 2) subjecting the amidation products obtained in step 1) to hydrogenation reduction to obtain debenzylated products; 3) The debenzylated products are subjected to acid hydrolysis treatment to obtain peptide derivatives as shown in formula (I).

3. The preparation method according to claim 2, characterized in that: In step 1), reacting in the presence of HATU and DIPEA; In step 2), palladium carbon is used as a catalyst for hydrogenation reaction; In step 3), acid hydrolysis is carried out in the presence of trifluoroacetic acid.

4. The method for preparing a peptide derivative or a salt thereof according to claim 1, characterized in that: When R1 is hydrogen, the preparation method of the peptide derivative represented by formula (I) comprises the following steps: 1') hydrogenating and reducing the compound represented by formula (M5) to remove the benzyl group, thereby obtaining the compound represented by formula (M15); 2') reacting the compound represented by formula (M15) with the compound represented by formula (E') to obtain the compound represented by formula (M16); 3') removing the Fmoc protecting group from the compound represented by formula (M16) to obtain the compound represented by formula (M17); 4') subjecting the compound represented by formula (M17) to acid hydrolysis to obtain a peptide derivative represented by formula (I); (M5), (M15), (AND'), (M16), （M17）。 5. The preparation method according to claim 2, characterized in that: The compound represented by formula (M6) is prepared by the following steps: (1) reacting a compound represented by formula (M1) with a compound represented by formula (M2) to obtain a compound represented by formula (M3); (2) removing the Fmoc protecting group from the compound represented by formula (M3) to obtain the compound represented by formula (M4); (3) reacting the compound represented by formula (M4) with the compound represented by formula (F) to obtain the compound represented by formula (M5); (4) removing the Fmoc protecting group from the compound represented by formula (M5) to obtain the compound represented by formula (M6); <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (M1)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (M2)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> (M3) (M4), (F), （M5）。 6. The preparation method according to claim 5, characterized in that: In step (1), the reaction is carried out in the presence of HATU and DIPEA; in step (2), the reaction is carried out in the presence of DBU and piperidine; in step (3), the reaction is carried out in the presence of HATU and DIPEA; in step (4), the reaction is carried out in the presence of DBU and piperidine.

7. Use of the peptide derivative or salt thereof according to claim 1 in the preparation of anti-aging and anti-wrinkle cosmetics.

8. A cosmetic with anti-aging and anti-wrinkle effects, characterized in that: It comprises the peptide derivative according to claim 1 or a salt thereof.

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