Long-acting tocopherol succinate modified tripeptide-1 derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polypeptide derivative synthesis, and particularly relates to a long-acting tocopherol succinate modified tripeptide-1 derivative as well as a preparation method and application thereof. In the long-acting tocopherol succinate modified tripeptide-1 derivative provided by the invention, tocopherol is connected with an amino terminal of tripeptide-1 through amidation by taking succinic acid as a connecting arm, and a carboxyl terminal of the tripeptide-1 is free carboxyl, ester group or acylamino. The long-acting tocopherol succinate modified tripeptide-1 derivative provided by the invention has good solubility in both a water phase and an oil phase, is beneficial to formula design in an application process, has good antioxidant activity and skin cell injury protection activity, further reduces expression of matrix metalloproteinase and inflammatory factors increased due to ROS (Reactive Oxygen Species), and can be used for preparing the long-acting tocopherol succinate modified tripeptide-1 derivative. The collagen essence has excellent cell matrix stability, can promote collagen expression for a long time, and has good anti-oxidation and wrinkle fading effects when being used for cosmetics or skin care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide derivative synthesis, and particularly relates to a class of long-acting tocopherol succinate modified tripeptide-1 derivatives, their preparation methods and applications. Background Art

[0002] The amino acid sequence of tripeptide-1 or oligopeptide-1 is H-Gly-His-Lys-OH, CAS No. 49557-75-7. According to research reports, tripeptide-1 stimulates blood vessel and nerve growth, increases the synthesis of collagen, elastin and glycosaminoglycans, and supports the function of dermal fibroblasts, and has the ability to improve skin tissue repair. In addition, tripeptide-1 also has a strong cytoprotective effect, can inhibit signal pathways of accelerated aging diseases such as NFκB, and promotes DNA repair and maintains intracellular homeostasis by activating the proteasome system. In beauty products, tripeptide-1 can tighten loose skin, increase skin elasticity, density and firmness, reduce fine lines and wrinkles, weaken photoaging and pigmentation, and increase the proliferation of keratinocytes.

[0003] Tripeptide-1 and its derivatives (copper peptide, biotin tripeptide-1, palmitoyl tripeptide-1, etc.) have a relatively large molecular polarity and usually exist in the form of aqueous solutions in formulations. However, peptide molecules have poor stability in aqueous solutions, the compounds will undergo hydrolysis and the structure will be damaged. Usually, they need to be stored at -20°C and avoid repeated freezing and thawing. Current skin care products cannot meet the storage conditions for maintaining the stability of peptide molecules. Coupled with the extremely low addition amount of peptide molecules, the skin care effect of skin care products fails to meet expectations. Summary of the Invention

[0004] The purpose of the present invention is to provide a class of long-acting tocopherol succinate modified tripeptide-1 derivatives, their preparation methods and applications. The long-acting tocopherol succinate modified tripeptide-1 derivatives provided by the present invention have good stability in aqueous solutions.

[0005] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a class of long-acting tocopherol succinate modified tripeptide-1 derivatives, and the structure is shown in Formula I:

[0007]

[0008] In Formula I, R1 and R2 are independently -H or C1-C6 alkyl, X is -O- or -NH-, and R3 is -H, a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group or a phenethyl group.

[0009] Preferably, R1 and R2 are independently C1-C6 alkyl groups, and the C1-C6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, n-hexyl, and cyclohexyl.

[0010] Preferably, the saturated hydrocarbon group in R3 is a C1-C18 alkyl group; the unsaturated hydrocarbon group includes butenyl, pentenyl, hexenyl, octenyl, nonadienyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl, or octadecenyl.

[0011] Preferably, the C1-C18 alkyl group includes methyl, ethyl, propyl, isopropyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl.

[0012] The present invention also provides a method for preparing the long-acting tocopherol succinate-modified tripeptide-1 derivative described in the above solution, which includes the following steps:

[0013] (1) In the presence of a basic substance and a condensing agent, tocopherol succinate and glycine are subjected to a first condensation reaction to obtain a compound of formula a;

[0014]

[0015] (2) In the presence of a basic substance and a condensing agent, the compound of formula a is subjected to a second condensation reaction with histidine to obtain a compound of formula b;

[0016]

[0017] (3) In the presence of a basic substance and a condensing agent, the compound of formula b is subjected to a third condensation reaction with H-Lys(Boc)-OH to obtain a compound of formula c;

[0018]

[0019] (4) In the presence of a condensing agent, the compound of formula c is subjected to a fourth condensation reaction with a compound of R3 group; the compound of R3 group is an alcohol or an amine;

[0020]

[0021] In formula a, formula b, formula c, and formula d, the meanings of R1, R2, or R3 are the same as those in formula I;

[0022] (5) The compound of formula d is mixed with trifluoroacetic acid and an organic solvent, and a protecting agent removal reaction is carried out to obtain the long-acting tocopherol succinate-modified tripeptide-1 derivative, and the structure is as shown in formula I.

[0023] Preferably, the tocopherol succinate includes one or more of α-tocopherol succinate, β-tocopherol succinate, Υ-tocopherol succinate, and δ-tocopherol succinate; the mass ratio of the tocopherol succinate to glycine is 20:3 to 6.

[0024] Preferably, the mass ratio of the tocopherol succinate to histidine is 20:6 to 12.

[0025] Preferably, the mass ratio of the compound of formula b to H-Lys(Boc)-OH is 1:0.5 to 1.5.

[0026] Preferably, when R3 is methyl, ethyl, propyl, or isopropyl, the mass ratio of the compound of formula c to the R3 group compound is 1:0.1 to 10; when R3 is octyl, decyl, dodecyl, tetradecyl, hexadecyl, or octadecyl, the mass ratio of the compound of formula c to the R3 group compound is 1:0.1 to 0.5.

[0027] The present invention also provides the use of the long-acting tocopherol succinate-modified tripeptide-1 derivative described in the above solution or the long-acting tocopherol succinate-modified tripeptide-1 derivative obtained by the preparation method described in the above solution in cosmetics.

[0028] The present invention provides a class of long-acting tocopherol succinate-modified tripeptide-1 derivatives, the structure of which is shown in formula I. In the long-acting tocopherol succinate-modified tripeptide-1 derivative provided by the present invention, tocopherol is connected to the amino terminus of tripeptide-1 through amide formation with succinic acid as the linking arm, and the carboxyl terminus of tripeptide-1 is a free carboxyl group, an ester group, or an amide group. The long-acting tocopherol succinate-modified tripeptide-1 derivative provided by the present invention has good solubility in both aqueous and oily phases, which is beneficial to the formulation design during the application process, has good antioxidant activity and skin cell damage protection activity, further reduces the expression of matrix metalloproteinase (MMP2) and inflammatory factor (TNFα) increased by reactive oxygen species (ROS), has excellent cell matrix stability, can promote collagen expression for a long time, and has good antioxidant and wrinkle-reducing effects when used in cosmetics or skin care products.

[0029] The present invention also provides a preparation method of the long-acting tocopherol succinate-modified tripeptide-1 derivative described in the above solution. The preparation method provided by the present invention has simple steps, convenient operation, low cost, and is easy to realize industrial mass production.

[0030] The present invention also provides the use of the long-acting tocopherol succinate-modified tripeptide-1 derivative described in the above solution or the long-acting tocopherol succinate-modified tripeptide-1 derivative obtained by the preparation method described in the above solution in cosmetics. The long-acting tocopherol succinate-modified tripeptide-1 derivative provided by the present invention has good antioxidant activity and skin cell damage protection activity, and has good antioxidant and wrinkle-reducing effects when used in cosmetics or skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0032] Figure 1 It is the synthetic route of the long-acting tocopherol succinate-modified tripeptide-1 derivative of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] The present invention provides a class of long-acting tocopherol succinate-modified tripeptide-1 derivatives, and the structure is shown in Formula I:

[0034]

[0035] In Formula I, R1 and R2 are independently -H or C1-C6 alkyl, X is -O- or -NH-, and R3 is -H, a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group or a phenethyl group.

[0036] In the present invention, R1 and R2 are preferably C1-C6 alkyl; the C1-C6 alkyl preferably includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, n-hexyl or cyclohexyl; the C1-C6 alkyl is preferably C1-C2 alkyl; the C1-C2 alkyl is preferably methyl.

[0037] In the present invention, X is preferably -O-.

[0038] In the present invention, the saturated aliphatic hydrocarbon group in R3 is preferably C1-C18 alkyl; the C1-C18 alkyl preferably includes methyl, ethyl, propyl, isopropyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl or octadecyl.

[0039] In the present invention, the unsaturated aliphatic hydrocarbon group in R3 preferably includes butenyl, pentenyl, hexenyl, octenyl, nonadienyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl or octadecenyl.

[0040] The present invention also provides a method for preparing the long-acting tocopherol succinate-modified tripeptide-1 derivative described in the above solution, comprising the following steps:

[0041] (1) In the presence of a basic substance and a condensing agent, tocopherol succinate and glycine are subjected to a first condensation reaction to obtain a compound of formula a;

[0042]

[0043] (2) In the presence of a basic substance and a condensing agent, the compound of formula a is subjected to a second condensation reaction with histidine to obtain a compound of formula b;

[0044]

[0045] (3) In the presence of a basic substance and a condensing agent, the compound of formula b is subjected to a third condensation reaction with H-Lys(Boc)-OH to obtain a compound of formula c;

[0046]

[0047] (4) In the presence of a condensing agent, the compound of formula c is subjected to a fourth condensation reaction with a compound of R3 group to obtain a compound of formula d; the compound of R3 group is an alcohol or an amine;

[0048]

[0049] In formula a, formula b, formula c and formula d, the meanings of R1, R2 or R3 are the same as those in formula I;

[0050] (5) The compound of formula d is mixed with trifluoroacetic acid and an organic solvent for a protecting agent removal reaction to obtain the long-acting tocopherol succinate-modified tripeptide-1 derivative, the structure of which is shown in formula I.

[0051] The synthetic route of the long-acting tocopherol succinate-modified tripeptide-1 derivative provided by the present invention is as Figure 1 shown.

[0052] In the presence of an alkaline substance and a condensing agent, the present invention performs a first condensation reaction on tocopherol succinate and glycine to obtain a compound of formula a. In the present invention, the tocopherol succinate preferably includes one or more of α-tocopherol succinate, β-tocopherol succinate, Υ-tocopherol succinate, and δ-tocopherol succinate; the mass ratio of the tocopherol succinate to glycine is preferably 20:3 to 6, specifically 20:3, 20:3.5, 20:4, 20:4.5, 20:5, 20:5.5, or 20:6; the glycine is preferably an aqueous solution of glycine; the mass concentration of the aqueous solution of glycine is preferably 1% to 50%, specifically 1%, 10%, 15%, 25%, 35%, 45%, or 50%.

[0053] In the present invention, the alkaline substance preferably includes one or more of alkali metal hydroxides, alkali metal salts, N-methylmorpholine, and triethylamine, more preferably an alkali metal salt; the alkali metal hydroxide preferably includes one or more of sodium hydroxide and potassium hydroxide; the alkali metal salt preferably includes one or more of alkali metal carbonates and alkali metal bicarbonates, more preferably an alkali metal bicarbonate; the alkali metal carbonate preferably includes one or more of sodium carbonate and potassium carbonate; the alkali metal bicarbonate preferably includes one or more of sodium bicarbonate and potassium bicarbonate, more preferably sodium bicarbonate.

[0054] In the present invention, the mass ratio of the tocopherol succinate to the alkaline substance is preferably 20:5 to 15, specifically 20:5, 20:6, 20:7, 20:8, 20:9, 20:9.5, 20:10, 20:11, 20:12, 20:13, 20:14, or 20:15; the alkaline substance is preferably an aqueous solution of the alkaline substance (the mass ratio of the tocopherol succinate to the alkaline substance is based on the mass of the alkaline substance in the aqueous solution of the alkaline substance); the mass concentration of the aqueous solution of the alkaline substance is preferably 1 to 50%, specifically 1%, 8%, 15%, 25%, 35%, 45%, or 50%.

[0055] In the present invention, the condensing agent preferably includes N-hydroxysuccinimide and carbodiimide series condensing agents; the carbodiimide series condensing agents preferably include one or two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide; when the carbodiimide series condensing agents are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 1,3-dicyclohexylcarbodiimide is preferably 1:1.

[0056] In the present invention, the molar ratio of the tocopherol succinate to the condensing agent is preferably 1:2 to 3, and specifically may be 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8 or 1:3.

[0057] In the present invention, the temperature of the first condensation reaction is preferably 0 to 35°C, and specifically may be 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C or 35°C. The heat preservation reaction time is preferably 0.5 to 72 h, and specifically may be 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 30 h, 50 h, 64 h or 72 h.

[0058] In the present invention, the first condensation reaction is preferably carried out in the presence of an organic solvent; the organic solvent is preferably a furan solvent; the furan solvent is preferably tetrahydrofuran.

[0059] In the present invention, after the first condensation reaction, it is preferably further included to perform a first post-treatment on the obtained first condensation reaction solution; the first post-treatment is preferably: removing the solvent from the first condensation reaction solution in turn (denoted as removing solvent A), adjusting the pH value to 2, redissolving the residue, washing and removing the solvent (denoted as removing solvent B).

[0060] In the present invention, the removing solvent A is preferably reduced pressure concentration.

[0061] In the present invention, the reagent used for adjusting the pH value is preferably an aqueous solution of potassium bisulfate; the mass concentration of the aqueous solution of potassium bisulfate is preferably 5%.

[0062] In the present invention, the reagent used for redissolving the residue is preferably ethyl acetate.

[0063] In the present invention, the number of times of washing is preferably more than 3 times; the washing is preferably washing with saturated brine and washing with an aqueous solution of potassium bisulfate in turn; the mass concentration of the aqueous solution of potassium bisulfate is preferably 5%.

[0064] In the present invention, the removing solvent B is preferably reduced pressure concentration.

[0065] After obtaining the compound of formula a, in the present invention, in the presence of a basic substance and a condensing agent, the compound of formula a is subjected to a second condensation reaction with histidine to obtain the compound of formula b. In the present invention, the mass ratio of the tocopherol succinate to histidine is preferably 20:6 to 12, specifically it can be 20:6, 20:7, 20:8, 20:8.5, 20:9, 20:10, 20:11 or 20:12; the histidine is preferably an aqueous solution of histidine (the mass ratio of the tocopherol succinate to histidine is based on the mass of histidine in the aqueous solution of histidine); the mass concentration of the aqueous solution of histidine is preferably 1 to 50%, specifically it can be 1%, 8%, 15%, 25%, 35%, 45% or 50%.

[0066] In the present invention, the basic substance preferably includes one or more of alkali metal hydroxides, alkali metal salts, N-methylmorpholine and triethylamine, more preferably an alkali metal salt; the alkali metal hydroxide preferably includes one or more of sodium hydroxide and potassium hydroxide; the alkali metal salt preferably includes one or more of alkali metal carbonates and alkali metal bicarbonates, more preferably an alkali metal bicarbonate; the alkali metal carbonate preferably includes one or more of sodium carbonate and potassium carbonate; the alkali metal bicarbonate preferably includes one or more of sodium bicarbonate and potassium bicarbonate, more preferably sodium bicarbonate.

[0067] In the present invention, the mass ratio of the tocopherol succinate to the basic substance is preferably 20:5 to 15, specifically it can be 20:5, 20:6, 20:7, 20:7.5, 20:8, 20:8.5, 20:9, 20:10, 20:11, 20:12, 20:13, 20:14 or 20:15; the basic substance is preferably an aqueous solution of the basic substance; the mass concentration of the aqueous solution of the basic substance is preferably 1 to 50%, specifically it can be 1%, 8%, 15%, 25%, 35%, 45% or 50%.

[0068] In the present invention, the condensing agent preferably includes N-hydroxysuccinimide and carbodiimide series condensing agents; the carbodiimide series condensing agents preferably include one or two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide; when the carbodiimide series condensing agents are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 1,3-dicyclohexylcarbodiimide is preferably 1:1.

[0069] In the present invention, the molar ratio of the tocopherol succinate to the condensing agent is preferably 1:2 to 3, specifically it can be 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8 or 1:3.

[0070] In the present invention, the temperature of the second condensation reaction is preferably 0 to 35 °C, specifically it can be 0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C or 35 °C, and the holding reaction time is preferably 0.5 to 72 h, specifically it can be 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 30 h, 50 h, 64 h or 72 h.

[0071] In the present invention, the second condensation reaction is preferably carried out in the presence of an organic solvent; the organic solvent is preferably a furan solvent; the furan solvent is preferably tetrahydrofuran.

[0072] In the present invention, after the second condensation reaction, it is preferably further included to perform a second post-treatment on the obtained second condensation reaction solution; the second post-treatment is preferably: solid-liquid separating the second condensation reaction solution, then adding water to the obtained liquid phase to precipitate the product, and then performing filtration and drying in sequence.

[0073] After obtaining the compound of formula b, in the present invention, in the presence of a basic substance and a condensing agent, the compound of formula b is subjected to a third condensation reaction with H-Lys(Boc)-OH to obtain the compound of formula c. In the present invention, the structure of the H-Lys(Boc)-OH is as shown in formula e:

[0074]

[0075] In the present invention, the mass ratio of the compound of formula b to H-Lys(Boc)-OH is preferably 1:0.5 to 1.5, specifically it can be 1:0.5, 1:0.7, 1:0.9, 1:1, 1:1.1, 1:1.3 or 1:1.5.

[0076] In the present invention, the basic substance preferably includes one or more of alkali metal hydroxides, alkali metal salts, N-methylmorpholine and triethylamine, more preferably an alkali metal salt; the alkali metal hydroxide preferably includes one or more of sodium hydroxide and potassium hydroxide; the alkali metal salt preferably includes one or more of alkali metal carbonates and alkali metal bicarbonates, more preferably an alkali metal bicarbonate; the alkali metal carbonate preferably includes one or more of sodium carbonate and potassium carbonate; the alkali metal bicarbonate preferably includes one or more of sodium bicarbonate and potassium bicarbonate, more preferably sodium bicarbonate.

[0077] In the present invention, the mass ratio of the tocopherol succinate to the basic substance is preferably 20:5 to 15, specifically it can be 20:5, 20:6, 20:7, 20:7.5, 20:8, 20:8.5, 20:9, 20:10, 20:11, 20:12, 20:13, 20:14 or 20:15; the basic substance is preferably an aqueous solution of the basic substance; the mass concentration of the aqueous solution of the basic substance is preferably 1 to 50%, specifically it can be 1%, 8%, 15%, 25%, 35%, 45% or 50%.

[0078] In the present invention, the condensing agent preferably includes N-hydroxysuccinimide and carbodiimide series condensing agents; the carbodiimide series condensing agents preferably include one or two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide; when the carbodiimide series condensing agents are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 1,3-dicyclohexylcarbodiimide is preferably 1:1.

[0079] In the present invention, the molar ratio of the tocopherol succinate to the condensing agent is preferably 1:2 to 3, specifically it can be 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8 or 1:3.

[0080] In the present invention, the temperature of the third condensation reaction is preferably 0 to 35 °C, specifically it can be 0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C or 35 °C, and the heat preservation reaction time is preferably 0.5 to 72 h, specifically it can be 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 30 h, 50 h, 64 h or 72 h.

[0081] In the present invention, the third condensation reaction is preferably carried out in the presence of an organic solvent; the organic solvent is preferably a furan solvent; the furan solvent is preferably tetrahydrofuran.

[0082] In the present invention, after the third condensation reaction, it preferably further includes performing a third post-treatment on the obtained third condensation reaction solution, and the third post-treatment is preferably: concentrating the third condensation reaction solution under reduced pressure to obtain a residue, then dissolving the residue to obtain an organic phase, and then sequentially washing, drying and removing the solvent of the organic phase.

[0083] In the present invention, the reagent used for redissolving the residue is preferably ethyl acetate.

[0084] In the present invention, the number of washings is preferably more than 3 times; the washing preferably includes washing successively with an aqueous potassium bisulfate solution and saturated brine; the mass concentration of the aqueous potassium bisulfate solution is preferably 5%.

[0085] In the present invention, the removal of the solvent is preferably by concentration under reduced pressure.

[0086] After obtaining compound c, in the present invention, in the presence of a condensing agent, the compound of formula c is subjected to a fourth condensation reaction with a compound of R3 group to obtain a compound of formula d; the compound of R3 group is an alcohol or an amine. In the present invention, the structure of the alcohol is as shown in formula f:

[0087] R3-OH formula f;

[0088] In formula f, the meaning of R3 is the same as that in formula I.

[0089] In the present invention, the alcohol can specifically be ethanol, propanol, isopropanol, octanol, decanol, dodecanol, tetradecanol, hexadecanol, octadecanol or phenethyl alcohol.

[0090] In the present invention, the structure of the amine is as shown in formula g:

[0091] R3-NH2 formula g;

[0092] In formula g, the meaning of R3 is the same as that in formula I.

[0093] In the present invention, when R3 is methyl, ethyl, propyl or isopropyl, the mass ratio of the compound of formula c to the compound of R3 group is preferably 1:0.1 to 10, and specifically can be 1:0.1, 1:0.3, 1:0.5, 1:0.7, 1:1, 1:3, 1:5, 1:7 or 1:10; when R3 is octyl, decyl, dodecyl, tetradecyl, hexadecyl or octadecyl, the mass ratio of the compound of formula c to the compound of R3 group is preferably 1:0.1 to 0.5, and specifically can be 1:0.1, 1:0.2, 1:0.3, 1:3.5, 1:0.4 or 1:0.5.

[0094] In the present invention, the condensing agent preferably includes N-hydroxysuccinimide and carbodiimide series condensing agents; the carbodiimide series condensing agents preferably include one or both of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide; when the carbodiimide series condensing agents are 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1,3-dicyclohexylcarbodiimide, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 1,3-dicyclohexylcarbodiimide is preferably 1:1.

[0095] In the present invention, the molar ratio of the compound of formula c to the condensing agent is preferably 1:2 to 3, and specifically may be 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8 or 1:3.

[0096] In the present invention, the temperature of the fourth condensation reaction is preferably 0 to 35 °C, and specifically may be 0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C or 35 °C. The heat preservation reaction time is preferably 0.5 to 72 h, and specifically may be 0.5 h, 1 h, 2 h, 4 h, 8 h, 16 h, 30 h, 50 h, 64 h or 72 h.

[0097] In the present invention, the fourth condensation reaction is preferably carried out in the presence of an organic solvent; the organic solvent is preferably a furan solvent; the furan solvent is preferably tetrahydrofuran.

[0098] In the present invention, after the fourth condensation reaction, it is preferably further included to perform a fourth post-treatment on the obtained fourth condensation reaction solution. The fourth post-treatment is preferably: concentrating the fourth condensation reaction solution under reduced pressure to obtain a residue, redissolving the residue to obtain an organic phase, and sequentially washing, drying and removing the solvent of the organic phase.

[0099] In the present invention, the reagent used for redissolving the residue is preferably ethyl acetate.

[0100] In the present invention, the number of times of washing is preferably more than 3 times; the washing preferably includes sequentially washing with an aqueous potassium bisulfate solution and saturated brine; the mass concentration of the aqueous potassium bisulfate solution is preferably 5%.

[0101] In the present invention, the removal of the solvent is preferably concentration under reduced pressure.

[0102] After obtaining the compound of formula d, the present invention mixes the compound of formula d with trifluoroacetic acid and an organic solvent to carry out a protecting group removal reaction to obtain the long-acting tocopherol succinate-modified tripeptide-1 derivative, and the structure is as shown in formula I. In the present invention, the molar ratio of the compound of formula d to trifluoroacetic acid is preferably 1:10 to 200, and specifically may be 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, 1:130, 1:170 or 1:200.

[0103] In the present invention, the organic solvent is preferably a substituted alkane; the substituted alkane is preferably a halogenated alkane; the halogenated alkane is preferably dichloromethane; the volume ratio of trifluoroacetic acid to the organic solvent is preferably 1:0.2 to 5, and specifically may be 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:4 or 1:5.

[0104] In the present invention, the temperature of the protecting agent removal reaction is preferably 0 to 35 °C, specifically it can be 0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C or 35 °C, and the heat preservation reaction time is preferably 0.5 to 24 h, specifically it can be 0.5 h, 1 h, 2 h, 5 h, 8 h, 12 h, 18 h or 24 h.

[0105] In the present invention, after the protecting agent removal reaction, it is preferably further included to perform a fifth post-treatment on the obtained protecting agent removal reaction solution. The fifth post-treatment is preferably: removing the solvent from the protecting agent removal reaction solution and then redissolving it with dichloromethane until a colorless oily product is obtained, redissolving the colorless oily product with dichloromethane again and then washing, drying and concentrating under reduced pressure in sequence.

[0106] In the present invention, the solvent removal is preferably suction filtration; the number of washings is preferably more than 3 times; the washing is preferably sequentially with saturated brine, saturated sodium bicarbonate aqueous solution and saturated brine.

[0107] The present invention also provides the application of the long-acting tocopherol succinate-modified tripeptide-1 derivative described in the above scheme or the long-acting tocopherol succinate-modified tripeptide-1 derivative obtained by the preparation method described in the above scheme in cosmetics.

[0108] In the present invention, the cosmetics preferably include one or more of antioxidant cosmetics, anti-wrinkle cosmetics and skin care products; the skin care products preferably include one or more of antioxidant skin care products and anti-wrinkle skin care products.

[0109] To further illustrate the present invention, the solutions of the present invention will be described in detail below in conjunction with the drawings and examples, but they cannot be construed as limiting the protection scope of the present invention.

[0110] Example 1

[0111] This example prepares an α-tocopherol succinate-modified tripeptide-1 derivative (α-tocopherol succinate acyl GHK phenethyl ester), and the specific steps are as follows:

[0112] (1) The synthesis of the compound of formula a, and the reaction route is shown as follows:

[0113]

[0114] Under stirring in an ice-water bath, 400 mL of tetrahydrofuran was added to a 1-L eggplant-shaped flask. According to the molar ratio of α-tocopherol succinate (VES), N-hydroxysuccinimide (HOSu), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) of 1:1.1:1.1, 40 g of VES, 10.8 g of HOSu, and 16.8 g of EDC were successively added to the reaction flask; after 30 min, the ice-water bath was removed, and stirring was continued for 3 h; 6.6 g of glycine (Gly) and 14.0 g of sodium bicarbonate (NaHCO3) were weighed into the eggplant-shaped flask, and 100 mL of distilled water was added thereto to dissolve the raw materials; after filtration, under stirring in an ice-water bath, the filtrate was added dropwise to the aqueous solution prepared from glycine and sodium bicarbonate; after the addition was complete, stirring was continued for 4 h; the reaction solution was concentrated under reduced pressure to remove tetrahydrofuran; the pH value was adjusted to 2 with a 5% aqueous solution of potassium bisulfate; the residue was dissolved in ethyl acetate and washed 3 times with a 5% aqueous solution of potassium bisulfate and saturated brine respectively; ethyl acetate was removed by concentration under reduced pressure to obtain 43.4 g of the compound of formula a, which was a colorless oily product with a yield of 98.1%.

[0115] (2) Synthesis of the compound of formula b, and the reaction route is as follows:

[0116]

[0117] The compound of formula a obtained in step (1) was dissolved in 300 mL of tetrahydrofuran. Under stirring in an ice-water bath, calculated based on the feeding amount of α-tocopherol succinate (VES) in step (1), according to the molar ratio of VES, HOSu, and EDC of 1:1:1.3, 10.8 g of HOSu and 16.8 g of EDC were successively added to the reaction flask; after 30 min, the ice-water bath was removed, and stirring was continued for 3 h; 11.2 g of histidine (His) and 14 g of sodium bicarbonate (NaHCO3) were weighed into the eggplant-shaped flask, and 100 mL of distilled water was added thereto to dissolve the raw materials; after filtration, under stirring in an ice-water bath, the filtrate was added dropwise to the aqueous solution prepared from histidine and sodium bicarbonate; after the addition was complete, stirring was continued for 4 h; after the reaction was completed, a small amount of precipitate was formed, which was an impurity and was removed by filtration; under stirring, distilled water was added to the filtrate in batches to precipitate the product, and after filtration, it was dried to obtain 42.2 g of the compound of formula b with a yield of 77.1%.

[0118] (3) Synthesis of the compound of formula c, and the reaction route is as follows:

[0119]

[0120] Under stirring in an ice-water bath, 300 mL of tetrahydrofuran was added to a 1-L eggplant-shaped flask. The compounds of formula b (VES-GH-OH), N-hydroxysuccinimide (HOSu), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were fed in a molar ratio of 1:1:1.3. 42.2 g of VES-GH-OH, 11.4 g of HOSu, and 19.6 g of EDC were successively added to the reaction flask. After 30 min, the ice-water bath was removed, and stirring was continued for 3 h. 15.8 g of H-Lys(Boc)-OH and 15 g of sodium bicarbonate (NaHCO3) were weighed into a 500-mL beaker, 100 mL of distilled water was added thereto to dissolve the raw materials, and then it was transferred to the 1-L eggplant-shaped flask containing the reaction solution, and stirring was continued for 4 h. It was filtered, and the reaction solution was concentrated under reduced pressure. 300 mL of ethyl acetate was added to the residue obtained by concentration under reduced pressure, and after being dispersed evenly, it was transferred to a 1-L separatory funnel and washed 3 times each with 5% aqueous potassium bisulfate solution and saturated brine. The ethyl acetate solution was dried with anhydrous sodium sulfate. After 30 min, anhydrous sodium sulfate was filtered off, and ethyl acetate was removed by concentration under reduced pressure to obtain 46.3 g of the compound of formula c, which was a pale yellow solid with a purity of 98.7% and a yield of 83.3%; ESI-MS (m / z): 953.74 [M+H] + 。

[0121] (4) Synthesis of α-tocopherol succinate acyl GHK phenethyl ester (VESGHK-OPe), and the reaction route is as follows:

[0122]

[0123] Under stirring in an ice-water bath, 30 mL of tetrahydrofuran was added to a 100-mL eggplant-shaped flask. The compounds of formula c (VES-GHK(Boc)-OH), N-hydroxysuccinimide (HOSu), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were fed in a molar ratio of 1:1:1.3. 1.0 g of VES-GHK(Boc)-OH, 0.13 g of HOSu, and 0.26 g of EDC were successively added to the reaction flask. After 30 min, the ice-water bath was removed, and stirring was continued for 3 h. 0.25 g of phenethyl alcohol was weighed into a 50-mL beaker, 10 mL of tetrahydrofuran was added thereto to dissolve it, and then it was transferred to the 100-mL eggplant-shaped flask containing the reaction solution, and stirring was continued for 4 h. It was filtered, and the reaction solution was concentrated under reduced pressure. 50 mL of ethyl acetate was added to the residue obtained by concentration under reduced pressure, and after being dispersed evenly, it was transferred to a separatory funnel and washed 3 times each with 5% aqueous sodium bicarbonate solution and saturated brine. The ethyl acetate solution was dried with anhydrous sodium sulfate. After 30 min, anhydrous sodium sulfate was filtered off, and ethyl acetate was removed by concentration under reduced pressure to obtain 1.07 g of the compound of formula d.

[0124] Transfer the obtained compound of formula d to a 50 mL eggplant-shaped flask. Under stirring in an ice-water bath, add 5 mL of dichloromethane and 5 mL of trifluoroacetic acid thereto, and react on the ice-water bath for 2 h. Pump dry the solvent with a water pump, then add 10 mL of dichloromethane to redissolve, and pump dry the solvent again. Repeat this 3 times to obtain a colorless oily product; the colorless oily product is redissolved with 100 mL of dichloromethane, and then successively washed 3 times each with a small amount of saturated brine, saturated sodium bicarbonate aqueous solution, and saturated brine. Collect the dichloromethane solution and dry it with anhydrous sodium sulfate; after 30 min, filter off the anhydrous sodium sulfate, and concentrate under reduced pressure to remove dichloromethane to obtain 1.05 g of α-tocopherol succinate acyl GHK phenethyl ester (VESGHK-OPe), a pale yellow oily substance, with a purity of 96.1% and a yield of 98.2%; ESI-MS (m / z): 958.62 [M+H] + 。

[0125] Example 2

[0126] Synthesis of α-tocopherol succinate acyl GHK ethyl ester (VESGHK-OEt)

[0127] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 1.0 g of absolute ethanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, to obtain 0.91 g of α-tocopherol succinate acyl GHK ethyl ester (VESGHK-OEt), a pale yellow oily substance, with a purity of 95.9% and a yield of 91.3%; ESI-MS (m / z): 882.54 [M+H] + 。

[0128] Example 3

[0129] Synthesis of α-tocopherol succinate acyl GHK propyl ester (VESGHK-OPr)

[0130] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 1.0 g of propanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, to obtain 0.89 g of α-tocopherol succinate acyl GHK propyl ester (VESGHK-OPr), a pale yellow oily substance, with a purity of 97.7% and a yield of 84.1%; ESI-MS (m / z): 896.43 [M+H] + 。

[0131] Example 4

[0132] Synthesis of α-tocopherol succinate acyl GHK isopropyl ester (VESGHK-OiPr)

[0133] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 1.0 g of isopropanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.92 g of α-tocopherol succinate acyl GHK isopropyl ester (VESGHK-OiPr), a pale yellow oily substance, with a purity of 99.1% and a yield of 86.9%; ESI-MS (m / z): 896.43 [M+H] + 。

[0134] Example 5

[0135] Synthesis of α-tocopherol succinate acyl GHK octyl ester (VESGHK-OC8)

[0136] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 0.25 g of n-octanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.84 g of α-tocopherol succinate acyl GHK octyl ester (VESGHK-OC8), a pale yellow oily substance, with a purity of 97.7% and a yield of 74.2%; ESI-MS (m / z): 966.57 [M+H] + 。

[0137] Example 6

[0138] Synthesis of α-tocopherol succinate acyl GHK decyl ester (VESGHK-OC10)

[0139] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 0.25 g of n-decanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.77 g of α-tocopherol succinate acyl GHK decyl ester (VESGHK-OC10), a pale yellow oily substance, with a purity of 98.1% and a yield of 66.3%; ESI-MS (m / z): 994.61 [M+H] + 。

[0140] Example 7

[0141] Synthesis of α-tocopherol succinate acyl GHK-dodecyl ester (VESGHK-OC12)

[0142] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 0.25 g of n-dodecanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.71 g of α-tocopherol succinate acyl GHK-dodecyl ester (VESGHK-OC12), a colorless wax, with a purity of 95.5% and a yield of 59.6%; ESI-MS (m / z): 1022.31 [M+H] + 。

[0143] Example 8

[0144] Synthesis of α - tocopherol succinate acyl GHK - tetradecyl ester (VESGHK - OC14)

[0145] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 0.30 g of n - tetradecanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.81 g of α - tocopherol succinate acyl GHK - tetradecyl ester (VESGHK - OC14), which is a colorless wax, with a purity of 98.5% and a yield of 66.4%; ESI - MS (m / z): 1050.28[M + H] + .

[0146] Example 9

[0147] Synthesis of α - tocopherol succinate acyl GHK - hexadecyl ester (VESGHK - OC16)

[0148] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 0.35 g of n - hexadecanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.94 g of α - tocopherol succinate acyl GHK - hexadecyl ester (VESGHK - OC16), which is a colorless wax, with a purity of 96.6% and a yield of 75.2%; ESI - MS (m / z): 1078.45[M + H] + .

[0149] Example 10

[0150] Synthesis of α - tocopherol succinate acyl GHK - octadecyl ester (VESGHK - OC18)

[0151] The preparation method of this example is the same as that of Example 1, except for step (4): Weigh 0.4 g of n - octadecanol to replace 0.25 g of phenethyl alcohol in step (4) for reaction, and obtain 0.98 g of α - tocopherol succinate acyl GHK - octadecyl ester (VESGHK - OC18), which is a colorless wax, with a purity of 97.1% and a yield of 76.6%; ESI - MS (m / z): 1106.51[M + H] + .

[0152] Test Example 1 Detection of the solubility of the long - acting tocopherol succinate - modified tripeptide - 1 derivative

[0153] Take 1.5 mL centrifuge tubes and weigh 10 mg of the long-acting tocopherol succinate modified tripeptide-1 derivative into each of them, with 6 portions weighed for each sample. After making marks, divide the centrifuge tubes of each compound into 2 groups. Add 0.1 mL of n-octanol to the three centrifuge tubes in Group 1 and add 0.1 mL of ultrapure water to the three centrifuge tubes in Group 2. Vortex for 3 min at room temperature, then centrifuge at 12,000 rpm for 5 min at room temperature; after centrifugation, take the supernatant and dilute it with absolute ethanol at a ratio of 1:1000 (volume ratio), and then detect the ultraviolet absorption intensity of the ethanol solution in different groups at 292 nm.

[0154] Standard curve drawing: Weigh accurately 50 mg of the target compound, add 1 mL of absolute ethanol to it, and vortex at room temperature to completely dissolve the target compound. Then, further dilute the sample with absolute ethanol step by step to obtain standard solutions with concentrations of 50 mg / mL, 10 mg / mL, 2 mg / mL, 0.4 mg / mL, 0.08 mg / mL, and 0.016 mg / mL respectively. Perform linear regression on the ultraviolet absorption intensity of the standard solutions at 292 nm and their concentrations, and the standard curve equation y = ax + b can be obtained, where y is the absorbance, a is the equation coefficient, x is the compound concentration, and b is the intercept of the equation.

[0155] Solubility = (OD of the test group 292 - b) / a Equation 1.

[0156] The solubility of the long-acting tocopherol succinate modified tripeptide-1 derivatives prepared in Examples 1 to 10 was determined in this test example in the ester phase and the aqueous phase as shown in Table 1.

[0157] Table 1 Solubility of the long-acting tocopherol succinate modified tripeptide-1 derivative in the ester phase and the aqueous phase

[0158] Specimen n-Octanol (mg / mL) Water (mg / mL) VESGHK-OPe >50 >50 VESGHK-OEt >50 >50 VESGHK-OPr >50 >50 VESGHK-OiPr >50 >50 VESGHK-OC8 35.5±2.9 15.1±3.1 VESGHK-OC10 31.9±3.1 5.3±1.2 VESGHK-OC12 30.5±2.8 4.1±1.1 VESGHK-OC14 23.1±1.0 1.3±0.3 VESGHK-OC16 19.7±2.1 0.3±0.01 VESGHK-OC18 16.4±1.3 0.05±0.02

[0159] It can be seen from Table 1 that the long-acting tocopherol succinate modified tripeptide-1 derivatives prepared in the present invention all have good solubility in n-octanol; as the fatty chain extends, the solubility of the long-acting tocopherol succinate modified tripeptide-1 derivative in both the lipid phase and the aqueous phase decreases, and the decrease in solubility in the aqueous phase is more significant.

[0160] Test Example 2 ROS-induced cell damage protection test (MTT method)

[0161] Human keratinocytes (HaCaT) are cultured in a cell culture incubator at 37 °C and 5% CO2. Cells that have been passaged 3 to 10 times can be used for subsequent cell viability test experiments, and the culture medium is DMEM medium containing 10% FBS and 1% P / S.

[0162] When preparing the test sample, the test compound was dissolved in DMSO to form a stock solution with a concentration of 10 mg / mL. 10 μL was pipetted from it and added to 190 μL of the culture medium (5-fold final concentration of the test compound solution). After mixing, it was reserved for use. The IC 50 value of the cell line was detected again by the MTT method, and the measurement was repeated at least 3 times under the same experimental conditions.

[0163] Normal control group: not treated with TBHP; negative control group: culture medium containing 1% DMSO; treatment group: culture medium containing tripeptide-1 (GHK), tocopherol (VE), and the derivative of tripeptide-1 modified with long-acting tocopherol succinate, with the final concentration of the compound being 100 ppm.

[0164] Cell viability = [(average OD value of the negative control group - average OD value of the test compound group) / average OD value of the negative control group - average OD value of the Blank group] × 100% Formula 2.

[0165] The specific method was as follows: 24 hours after inoculating the cells, except for the normal control group, TBHP with a final concentration of 400 μmol / L was added to all wells to induce cell damage by generating ROS; 1 hour after adding TBHP, all the culture media were removed, and then 200 μL of PBS was added to each well to wash the residual TBHP, and the washing was done 3 times in total; after washing, all the PBS was removed, 100 μL of fresh culture medium was added to each well, and then 25 μL of the test sample solution with different concentrations was added. Then, the OD values of each group were obtained according to the above method; the protective effects of the test compounds with different concentrations on the ROS-induced cell damage of each cell line are shown in Table 2.

[0166] Table 2 Protective effects of the compound on the ROS-induced cell damage of each cell line (Mean±SD)

[0167] Specimen HaCaT Normal control group 100.0±2.4 Negative control group 65.1±1.2 VE 88.7±1.1 GHK 64.3±2.7 VESGHK-OPe 97.2±1.7 VESGHK-OEt 89.9±2.3 VESGHK-OPr 86.6±2.6 VESGHK-OiPr 92.8±0.6 VESGHK-OC8 80.7±1.2 VESGHK-OC10 76.6±4.4 VESGHK-OC12 77.1±1.3 VESGHK-OC14 76.3±3.7 VESGHK-OC16 73.1±1.1 VESGHK-OC18 74.6±1.9

[0168] It can be seen from Table 2 that the long-acting tocopherol succinate-modified tripeptide-1 derivative of the present invention has a significant protective effect on the cell damage induced by reactive oxygen species; at the same mass concentration, the cell protection activity of the long-acting tocopherol succinate-modified tripeptide-1 derivative is superior to that of tripeptide-1 (GHK) and tocopherol (VE).

[0169] Test Example 3 Detection of the effects on the expressions of MMP2 and TNFα in human keratinocytes (HaCaT)

[0170] Human keratinocytes (HaCaT) and human immortalized fibroblasts (HSF) were cultured in a cell incubator at 37°C and 5% CO2. Cells passaged 3 - 10 times were used for subsequent cell viability test experiments. The culture medium was DMEM medium containing 10% FBS and 1% P / S.

[0171] When preparing the test sample, the test compound was dissolved in DMSO to form a sample solution with a concentration of 10 mg / mL.

[0172] Normal control group: not stimulated with TBHP; Negative control group: medium containing 1% DMSO.

[0173] The specific method was as follows: 1) Inoculating cells: Cells in good growth condition and in the logarithmic growth phase were diluted with the medium to a cell concentration of 1×10 6 cells / mL, and evenly inoculated into 6-well plates, 2 mL per well; 2) ROS-induced inflammation: Observe the cell growth and adhesion situation. When the adhesion rate reached over 80%, add 10 μL of TBHP solution (80 mmol / L) to each well, gently tap to disperse the sample solution evenly, and incubate in the cell incubator for 1 h. Then remove the medium in the 6-well plate, wash 3 times with PBS at a volume of 2 mL per well, and then add fresh medium; 3) Administration: Add 20 μL of the test sample solution according to the preset multiple wells, gently tap to disperse the sample solution evenly, and incubate in the cell incubator for 6 h; 4) Post-treatment: Remove the medium in the 6-well plate, add 1 mL of TRIzol reagent to each well, gently pipette the bottom of the plate to lyse all cells, and then transfer all the solutions to a 1.5 mL centrifuge tube. Extract total RNA according to the operation steps of the TRIzol reagent instruction manual (Beyotime, R0016), and measure the RNA concentration; 5) Synthesize cDNA: Use BeyoRT TM II cDNA Synthesis Kit (with gDNA Eraser). According to the operation steps in the instruction manual, add gDNA Eraser to 1 μg of RNA, incubate at 37°C for 2 min, and then sequentially add 4 μL of 5x buffer, 1 μL of M-mLV reverse transcriptase, 2 μL of mixed primers, supplement an appropriate amount of DEPC water to a final volume of 20 μL, incubate at 2°C for 60 min for reverse transcription reaction, and then incubate at 80°C for 10 min to inactivate the reverse transcriptase and place on ice; 6) Real-time fluorescence quantitative PCR analysis (qPCR): Use the cDNA in the above steps as a template, and use BeyoFast TM SYBR Green qPCRMix (2X) reagent (Beyotime, D7260). According to the operation steps in the instruction manual, sequentially add 10 μL of BeyoFast TMSYBR Green qPCR Mix (2X), 2 μL of GAPDH, MMP2 or TNFα forward / reverse primer mixture (3 μmol / L), and 6 μL of DEPC water were gently pipetted and mixed well or slightly vortexed, then centrifuged at room temperature for a few seconds to accumulate the liquid at the bottom of the tube. The following PCR program was used:

[0174] a. Pre-denaturation: 95 °C, 2 min;

[0175] b. Denaturation: 95 °C, 15 sec;

[0176] c. Annealing / extension: 60 °C, 15 - 30 sec;

[0177] d. Repeat steps b and c for a total of 40 cycles;

[0178] e. Analyze the results using the software provided by the fluorescence quantitative PCR instrument.

[0179] The effect of the test compound on the expression levels of MMP2 and TNFα was calculated according to Equation 3, and each experiment was repeated at least 3 times.

[0180] Gene expression rate = [2^-(Ct value of the target gene in the test compound group - Ct value of GAPDH in the test compound group)] /

[0181] [2^-(Ct value of the target gene in the negative control group - Ct value of GAPDH in the negative control group)] Equation 3.

[0182] The expression rates of MMP2 and TNFα are shown in Table 3.

[0183] Table 3 Expression rates of MMP2 and TNFα

[0184]

[0185]

[0186] It can be seen from Table 3 that the long-acting tocopherol succinate-modified tripeptide-1 derivative of the present invention can significantly down-regulate the high expression of matrix metalloproteinase (MMP2) and inflammatory factor (TNFα) induced by reactive oxygen species; at the same mass concentration, the long-acting tocopherol succinate-modified tripeptide-1 derivative has better activity in inhibiting the expression of MMP2 and TNFα than tripeptide-1 (GHK) and tocopherol (VE), suggesting that the long-acting tocopherol succinate-modified tripeptide-1 derivative has more excellent anti-inflammatory and anti-wrinkle effects.

[0187] Test Example 4 Effect test on the production of collagen (COL1A1) in human immortalized fibroblasts (HSF)

[0188] Human immortalized fibroblasts (HSF) were cultured in a cell incubator at 37°C and 5% CO2. Cells passaged 3 - 10 times were used for subsequent cell viability test experiments. The culture medium was DMEM medium containing 10% FBS and 1% P / S.

[0189] When preparing the test sample, the test compound was dissolved in DMSO to form a sample solution with a concentration of 10 mg / mL.

[0190] Positive control group: 10 ng / mL TGFβ; Negative control group: medium containing 1% DMSO.

[0191] The specific method was as follows: 1) Cell seeding: Cells in good growth condition and in the logarithmic growth phase were diluted with the medium to a cell concentration of 1×10 6 cells / mL, and evenly seeded in a 6-well plate, 2 mL per well; 2) Drug administration: Observe the cell growth and adhesion situation. When the adhesion rate reached over 80%, add 20 μL of the test sample solution according to the preset replicates, gently tap to disperse the sample solution evenly, and incubate in the cell incubator for 48 h; 3) Post-treatment: Collect the medium in the 6-well plate, centrifuge at 13000 g for 10 min at 4°C, and transfer the supernatant to a new 1.5 mL centrifuge tube; 4) Use the Yeasen HumanPro-Collagen Ialpha 1 ELISA (Enzyme-Linked Immunosorbent Assay) kit (Catalog number: 97044ES96), and according to the operation guide in the kit instructions, detect the content of type I collagen secreted in the cell culture medium. The total amount of collagen secreted into the medium after 48 h of HSF cell culture measured by ELISA is shown in Table 4.

[0192] Table 4 Content of type I collagen secreted in the cell culture medium

[0193]

[0194]

[0195] According to the results in Table 4, the tripeptide-1 derivative has better activity in inducing collagen expression than tripeptide-1 (GHK) and tocopherol (VE).

[0196] Test Example 5 Cell matrix stability test of long-acting tocopherol succinate modified tripeptide-1 derivative

[0197] Human immortalized fibroblasts (HSF) were cultured in a cell incubator at 37°C and 5% CO2. Cells passaged 3 - 10 times were used for subsequent cell matrix stability test experiments. The culture medium was DMEM medium containing 10% FBS and 1% P / S.

[0198] When preparing the test sample, the test compound was dissolved in DMSO to form a sample solution with a concentration of 10 mg / mL.

[0199] Reference compound: DL-α-tocopherol succinate-GHK-benzyl ester (VESGHK-OBzl) dissolved in DMSO to a concentration of 10 mg / mL.

[0200] Specific method: 1) Cell seeding: Cells in good growth condition and in the logarithmic growth phase were diluted with medium to a cell concentration of 1×10 6 cells / mL, and evenly seeded in 10-cm cell culture dishes, 10 mL per dish; 2) Cell protein extraction: Observe the cell growth and adhesion situation. When the adhesion rate reaches more than 90%, collect the cells, and lyse the cells with PBS containing 0.2% dodecyl-β-D-maltoside, centrifuge at 13,000 g for 10 min at 4°C, transfer the supernatant to a new 1.5-mL centrifuge tube, 98 μL per tube; 4) Cell matrix incubation: Add 2 μL of the test sample and the control sample respectively to centrifuge tubes containing 99 μL of cell lysate, and incubate at 37°C for 0 h, 0.5 h, 1 h, 2 h, 4 h, and 8 h. At the corresponding time points, add 300 μL of acetonitrile to the tubes, vortex for 30 s, then centrifuge at 13,000 g for 10 min at 4°C, transfer the supernatant to a new 1.5-mL centrifuge tube, remove the solvent under reduced pressure, add 50 μL of aqueous solution (containing 0.1% TFA) for reconstitution, take the supernatant after ultracentrifugation, and detect the content of the test compound by high performance liquid chromatography; 5) Detection of compound content: The liquid chromatography used Waters 2695, the chromatographic column used Dalian Elite Hypersil ODS (C18, 5 μm, 2.1 mm * 150 mm), the detection wavelength was 292 nm; gradient elution was used, the flow rate was 0.5 mL / min, the mobile phase A was water (containing 0.1% TFA), the mobile phase B was acetonitrile (containing 0.1% TFA), and the elution condition was that from 0 min to 30 min, the proportion of A decreased from 60% to 5%. The content of the compound was calculated by peak area, as shown in Table 5.

[0201] Relative content of compound = peak area of compound at each time point / average peak area of compound at 0 h × 100%

[0202] Equation 4.

[0203] Table 5 Content of the test compound in the cell matrix at different time points (%)

[0204]

[0205] As can be seen from Table 5, modifying the carboxyl terminus of the long-acting tocopherol succinate-modified tripeptide-1 derivative with long-chain fatty alcohols can better increase the stability of the compound than benzyl alcohol.

[0206] Test Example 6: Expression Test of Collagen (COL1A1) in Human Immortalized Fibroblasts

[0207] Human immortalized fibroblasts (HSF) were cultured in a cell incubator at 37 °C and 5% CO2. Cells passaged 3 - 10 times can be used for subsequent cell viability test experiments. The culture medium was DMEM medium containing 10% FBS and 1% P / S.

[0208] When preparing the test sample, the test compound was dissolved in DMSO to prepare a sample solution with a concentration of 10 mg / mL.

[0209] Reference compound: DL-α-tocopherol succinate-GHK-benzyl ester (VESGHK-OBzl) dissolved in DMSO to a concentration of 10 mg / mL.

[0210] Positive control group: 10 ng / mL TGFβ; Negative control group: Medium containing 1% DMSO.

[0211] The specific method is as follows: 1) Inoculating cells: Cells in good growth condition and in the logarithmic growth phase were diluted with the culture medium to a cell concentration of 1×10 6 cells / mL, and evenly inoculated into 6-well plates, 2 mL per well; 2) Administering drugs: Observe the cell growth and adhesion situation. When the adhesion rate reaches more than 80%, add 20 μL of the test sample solution according to the preset replicate wells, gently tap to disperse the sample solution evenly, place it in the cell incubator and incubate for 24 h, then replace the culture medium with a complete medium without drugs and continue to culture for 0 h, 12 h, and 24 h; 3) Post-treatment: Collect cells at the corresponding time points according to the post-treatment method in Test Example 3, extract RNA, synthesize cDNA, and perform real-time fluorescence quantitative PCR analysis.

[0212] Calculate the effect of the test compound on the expression level of COL1A1 according to Equation 3, and each experiment was repeated at least 3 times. The expression rate of COL1A1 is shown in Table 6.

[0213] Table 6 Expression Rates of COL1A1 at Different Time Points after Withdrawal of Drugs

[0214]

[0215]

[0216] As can be seen from Table 6, when long-chain fatty alcohols are used to modify the carboxyl terminus of the long-acting tocopherol succinate-modified tripeptide-1 derivative, the resulting compound can continuously induce collagen expression even 24 hours after the drug is withdrawn, while the reference compound cannot induce collagen expression 12 hours after the drug is withdrawn.

[0217] As can be seen from the above examples, the long-acting tocopherol succinate-modified tripeptide-1 derivative provided by the present invention has good solubility in both aqueous and oil phases, good antioxidant activity and skin cell damage protection activity, excellent cell matrix stability and long-acting collagen expression-promoting activity, and good antioxidant and wrinkle-reducing effects.

[0218] Although the above examples have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments can be obtained based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A long-acting tocopherol succinate modified tripeptide-1 derivative, characterized in that: The structure is shown in Formula I: In formula I, R1 and R2 are independently -H or C1-C6 alkyl, X is -O- or -NH-, and R3 is -H, a saturated aliphatic hydrocarbon group, an unsaturated aliphatic hydrocarbon group or a phenethyl group.

2. The long-acting tocopherol succinate modified tripeptide-1 derivative according to claim 1, characterized in that: Said R1 and R2 are independently C1-C6 alkyl; The C1-C6 alkyl group includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, n-hexyl and cyclohexyl.

3. The long-acting tocopherol succinate modified tripeptide-1 derivative according to claim 1 or 2, characterized in that: The saturated aliphatic hydrocarbon group in R3 is a C1-C18 alkyl group; The unsaturated aliphatic hydrocarbon group includes butenyl, pentenyl, hexenyl, octenyl, nonadienyl, decenyl, dodecenyl, tetradecenyl, hexadecenyl or octadecenyl.

4. The long-acting tocopherol succinate modified tripeptide-1 derivative according to claim 3, characterized in that: The C1-C18 alkyl group includes methyl, ethyl, propyl, isopropyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl or octadecyl.

5. The method for preparing the long-acting tocopherol succinate modified tripeptide-1 derivative according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) in the presence of an alkaline substance and a condensing agent, subjecting tocopherol succinate and glycine to a first condensation reaction to obtain a compound of formula a; (2) in the presence of an alkaline substance and a condensing agent, subjecting the compound of formula a to a second condensation reaction with histidine to obtain a compound of formula b; (3) in the presence of an alkaline substance and a condensing agent, subjecting the compound of formula b to a third condensation reaction with H-Lys(Boc)-OH to obtain a compound of formula c; (4) in the presence of a condensing agent, subjecting the compound of formula c to a fourth condensation reaction with a compound of group R3 to obtain a compound of formula d; wherein the compound of group R3 is an alcohol or an amine; In formula a, formula b, formula c and formula d, R1, R2 or R3 have the same meaning as in formula I; (5) The compound of formula d is mixed with trifluoroacetic acid and an organic solvent, and a protective agent removal reaction is performed to obtain the long-acting tocopherol succinate modified tripeptide-1 derivative, the structure of which is shown in formula I.

6. The preparation method according to claim 5, characterized in that: The tocopherol succinate includes one or more of α-tocopherol succinate, β-tocopherol succinate, γ-tocopherol succinate and δ-tocopherol succinate; The mass ratio of tocopherol succinate to glycine is 20:3-6.

7. The preparation method according to claim 5 or 6, characterized in that: The mass ratio of tocopherol succinate to histidine is 20:6-12.

8. The preparation method according to claim 5, characterized in that: The mass ratio of the compound of formula b to H-Lys(Boc)-OH is 1:0.5-1.

5.

9. The preparation method according to claim 5 or 8, characterized in that: When the R3 is methyl, ethyl, propyl or isopropyl, the mass ratio of the compound of formula c to the compound of the R3 group is 1:0.1-10; When the R3 is octyl, decyl, dodecyl, tetradecyl, hexadecyl or octadecyl, the mass ratio of the compound of formula c to the compound of the R3 group is 1:0.1-0.

5.

10. Use of the long-acting tocopherol succinate modified tripeptide-1 derivative according to any one of claims 1 to 4 or the long-acting tocopherol succinate modified tripeptide-1 derivative obtained by the preparation method according to any one of claims 5 to 9 in cosmetics.

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