Ester cyclic peptide as well as preparation method and application thereof

Abstract

The invention discloses an alicyclic peptide and a preparation method and application thereof, the structural formula of the alicyclic peptide is as shown in formula (I): # imgabs0. The alicyclic peptide provided by the invention simultaneously comprises a hydrophobic palmitic acid chain and a hydrophilic cyclic peptide structure, and the hydrophobic palmitic acid chain and the hydrophilic cyclic peptide structure are combined through a covalent bond; the structure endows the polypeptide with excellent stability, and the problems that the two tetrapeptides GPKG and LSVD need to be added separately, are unstable and are not easy to transdermal are effectively improved; after entering the skin, under the action of esterase, the alicyclic peptide can be hydrolyzed into glycoloyl GPKG and palmitoyl LSVD to synergistically play a role in promoting collagen synthesis. And secondly, the alicyclic peptide provided by the invention has good biological safety.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly relates to a cycloester peptide, a preparation method thereof, and an application thereof. Background Art

[0002] The statements herein only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] Synthesized tetrapeptides GPKG and LSVD can effectively promote the natural repair of the skin. The super peptide mixture can enhance the expression of more than 50 important proteins (such as collagen, fibrillin, etc.), enhance the skin's self-repair ability, and is expected to become an efficient and well-tolerated skin care ingredient in the cosmetics market, especially suitable for people with sensitive skin.

[0004] However, there are problems that the above two tetrapeptides need to be added separately, used synergistically, and are not easily absorbed through the skin. Moreover, both of the above two tetrapeptides are linear peptides, and the linear peptides have poor structural stability and are easily degraded by proteases in the body. In addition, the molecular flexibility of linear peptides is relatively large, and conformational changes may lead to a decrease in the binding strength and selectivity with receptors, resulting in a decrease in receptor fit. Finally, the biological activity and metabolic stability of linear peptides are relatively weak. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a cycloester peptide, a preparation method thereof, and an application thereof.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] In the first aspect, the present invention provides a cycloester peptide, the structural formula of which is shown in formula (Ⅰ):

[0008]

[0009] The cyclic peptide loses free amino and carboxyl groups by connecting the head and tail or forming an intramolecular small ring, thus avoiding being degraded by proteases in the body. Secondly, the biological activity of cyclic peptides is usually significantly higher than that of linear peptides. Research shows that the biological activity of cyclized peptide compounds can be increased by dozens of times to tens of thousands of times.

[0010] Cyclic peptides have a fixed conformation, which enables them to better fit with receptors; the metabolic stability and bioavailability of cyclic peptides are much higher than those of linear peptides, which enables cyclic peptides to exist stably in the body for a longer time.

[0011] In the second aspect, the present invention provides a preparation method of the cycloester peptide, including the following steps:

[0012] Synthesize intermediate 1 by solid-phase synthesis method, and the structural formula of intermediate 1 is shown in formula II;

[0013]

[0014] After hydrogenating and debenzylating Intermediate 1, Intermediate 2 is obtained.

[0015] Intermediate 2 is cyclized to obtain Intermediate 3, and the structural formula of Intermediate 3 is shown in Formula (III):

[0016]

[0017] Deprotect Intermediate 3 to obtain the ester cyclic peptide of Formula (I).

[0018] The structural formula of Intermediate 2 is shown in Formula (IV):

[0019]

[0020] In some embodiments, using CTC resin as a solid-phase synthesis carrier, Fmoc-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH and palmitic acid are sequentially condensed from the C-terminus to the N-terminus to obtain a peptide resin. After cleaving the peptide resin, a crude product of Intermediate 1 is obtained.

[0021] Preferably, the method for purifying the crude product of Intermediate 1 is: purification by high performance liquid chromatography, the chromatographic column is a C18 column, mobile phase A is an aqueous solution containing 0.1% by mass of trifluoroacetic acid, and mobile phase B is an acetonitrile solution containing 0.1% by mass of trifluoroacetic acid.

[0022] Preferably, the cleavage reagent for the cleavage is a mixed solution of trifluoroethanol and dichloromethane, and the volume ratio of trifluoroethanol to dichloromethane is 1:3 - 5, preferably 1:4.

[0023] More preferably, the cleavage temperature is 20 - 30 °C and the cleavage time is 2 - 4 h.

[0024] In some embodiments, the synthesis method of Intermediate 2 is: mixing Intermediate 1, methanol and palladium carbon in proportion, and performing a hydrogenation reaction at 20 - 30 °C for 15 - 20 h to obtain Intermediate 2.

[0025] In some embodiments, the synthesis method of Intermediate 3 is: mixing Intermediate 2, HOBt, DMAP and N,N-dimethylformamide in proportion, stirring until clear, cooling to 0 - 10 °C, adding DIC, and then reacting at 20 - 30 °C for 15 - 20 h.

[0026] In a third aspect, the present invention provides the use of the cycloester peptide in the preparation of drugs, foods, food additives or cosmetics for promoting collagen synthesis.

[0027] In some embodiments, the food includes health foods or ordinary foods.

[0028] In some embodiments, the dosage form of the drug includes but is not limited to injections, ointments, powder injections, liniments, dressings or liquid preparations.

[0029] In some embodiments, the cosmetics include but are not limited to lotion, emulsion, essence, gel, foundation, cream or facial mask.

[0030] The application scope of the cosmetics includes but is not limited to facial washing, care and maintenance, body washing, care and maintenance, and head washing, care and maintenance, etc.

[0031] The beneficial effects obtained by one or more of the above embodiments of the present invention are as follows:

[0032] (1) The cycloester peptide provided by the present invention simultaneously contains a hydrophobic palmitic acid chain and a hydrophilic cyclic peptide structure, which are covalently bonded; this structure endows it with excellent stability, effectively improving the problems that the two tetrapeptides GPKG and LSVD need to be added separately, are unstable and not easily permeable through the skin; after entering the skin, under the action of esterase, the cycloester peptide can be hydrolyzed into glycolyl GPKG and palmitoyl LSVD, which synergistically play a role in promoting collagen synthesis. Secondly, the cycloester peptide in the present invention has good biosafety.

[0033] (2) The present invention also provides a synthesis method of the cycloester peptide. This process has high synthesis efficiency, the total yield of the synthesized cycloester peptide is 31% to 40%, and the operation is simple, which is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0035] Figure 1 Mass spectrum of intermediate 3 prepared in Example 1;

[0036] Figure 2 Mass spectrum of the cycloester peptide of formula (I) prepared in Example 4;

[0037] Figure 3 Bio-safety test chart of the cycloester peptide of Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0038] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0040] Unless otherwise specified, the percentage content in the present invention is the mass percentage content.

[0041] The corresponding Chinese meanings of the substance English abbreviations appearing in the claims and the specification of the present invention are shown in Table 1:

[0042] Table 1

[0043] DCM Dichloromethane DMF N,N - Dimethylformamide PIP Piperidine DIEA Diisopropylethylamine HOBt 1 - Hydroxybenzotriazole DMAP 4 - Dimethylaminopyridine Fmoc 9 - Fluorenylmethyloxycarbonyl Gly Glycine Pro Proline Lys Lysine Leu Leucine Asp Aspartic acid Val Valine Ser Serine Boc tert - Butyloxycarbonyl OtBu tert - Butyloxy OBn Benzyloxy TFA Trifluoroacetic acid TFE Trifluoroethanol CTC Chloro(2 - chlorophenyl)diphenylmethane DIC Diisopropylcarbodiimide TLC Thin - layer chromatography

[0044] Example 1

[0045] A preparation method of the cycloalkane peptide of formula (I) includes the following steps:

[0046] (1) Swelling of the resin: Take 3.16 g (5 mmol) of CTC resin (substitution degree is 1.58 mmol / g), add 40 mL of dichloromethane to swell the resin for 1.5 hours.

[0047] (2) Preparation of Fmoc-Gly-CTC resin: Dissolve Fmoc-Gly-OH (3 equivalents, 15 mmol) and DIEA (6 equivalents, 30 mmol) in 40 mL of dichloromethane, add it to the resin (1 equivalent), react at 25 °C for 2 hours. After the reaction is completed, wash with 20 mL of dimethylformamide three times and drain the solvent to obtain Fmoc-Gly-CTC resin.

[0048] (3) Removal of the Fmoc protecting group: Add 40 mL of 20% PIP / DMF solution to remove the Fmoc protecting group twice at 25 °C, the reaction times are 5 minutes and 10 minutes respectively, then wash the resin with dimethylformamide until the pH reaches about 7, and drain the solvent to obtain NH2-Gly-CTC resin.

[0049] (4)Activation of amino acids and carboxylic acids to be coupled: 15 mmol each of Fmoc-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH and palmitic acid were added to 40 mL of dimethylformamide together with DIC (15 mmol) / HOBt (15 mmol) and activated at 25 °C for 5 minutes. For the activation of Fmoc-glycolic acid, DMAP (5 mmol) was added for catalysis.

[0050] (5)Coupling of amino acids and carboxylic acids to be coupled with NH2-Gly-CTC resin: The activated Fmoc-L-Lys(Boc)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed, and then Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH were successively condensed. After each condensation, the operation of removing the Fmoc protecting group was carried out, and finally palmitic acid was condensed. The condensation conditions were: the condensation reaction was carried out at 25 °C for 2 hours, and the reaction process was monitored by ninhydrin color reaction to obtain intermediate 1-resin.

[0051] (6)Cleavage of intermediate 1-resin: 50 mL of cleavage reagent (trifluoroethanol:dichloromethane = 1:4) was added to the peptide resin, and the reaction was carried out at 25 °C for 3 hours. After suction filtration, the filtrate was concentrated under reduced pressure to dryness to obtain 5.2 g of crude intermediate 1.

[0052] (7)Purification of intermediate 1: Intermediate 1 was purified by reverse-phase high performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. 82% mobile phase A and 18% mobile phase B were used for elution for 6 min, and then 45% mobile phase A and 55% mobile phase B were used for elution for 24 min. The flow rate was 6 ml / min, and the column temperature was 30±1 °C. After concentration and lyophilization, intermediate 1 (3.3 g) was obtained, with a yield of 50.3% and a purity of 97.2%. The mass spectrum is as Figure 1 shown.

[0053] (8) Synthesis of Intermediate 2: Add 3.3 g (2.5 mmol) of Intermediate 1, 30 mL of methanol and 0.3 g of palladium on carbon to a 100 mL reaction flask, and carry out a hydrogenation reaction at 25 °C for 16 hours. Detect the completion of the reaction by TLC. Filter with diatomaceous earth as a filter aid, and concentrate the filtrate to dryness to obtain Intermediate 2 (3.2 g), which is a white solid and is directly used for the synthesis of Intermediate 3 without purification.

[0054] (9) Synthesis of Intermediate 3: Add 3.2 g (2.5 mmol, calculated based on Intermediate 1), 0.41 g of HOBt (3.0 mmol, 1.2 equivalents relative to Intermediate 1), 0.15 g of DMAP (1.2 mmol, 0.5 equivalents relative to Intermediate 1) and 30 mL of N,N-dimethylformamide to a 100 mL reaction flask, stir until clear, cool to 0 - 10 °C, and dropwise add 0.63 g of DIC (5.0 mmol, 2.0 equivalents relative to Intermediate 1) while controlling the temperature below 20 °C. Then raise the temperature to 25 °C and react at this temperature for 16 hours. Detect the completion of the reaction by TLC.

[0055] (10) Synthesis of the crude cyclic ester peptide of formula (I): Add 5 mL of trifluoroacetic acid to the reaction system, stir at 20 °C for 8 hours, and detect the completion of the reaction by TLC. Concentrate the reaction solution to a volume of about 2 ml, release the reaction solution into 20 ml of methyl tert-butyl ether, stir for 2 hours, then centrifuge to obtain a white solid, and dry it under reduced pressure at 40 °C for 12 hours to obtain the crude peptide.

[0056] (11) Purification of the crude cyclic ester peptide of formula (I): Use a C18 preparative column (50×250 mm, 10 μm) to purify the crude peptide by reverse-phase high-performance liquid chromatography. Mobile phase A is 0.1% trifluoroacetic acid / aqueous solution, and mobile phase B is 0.1% trifluoroacetic acid / acetonitrile solution. Elute with 82% mobile phase A and 18% mobile phase B for 6 min, and then continue to elute with 45% mobile phase A and 55% mobile phase B for 24 min, with a flow rate of 6 ml / min and a column temperature of 30 ± 1 °C. After concentration and lyophilization, 1.67 g of the cyclic ester peptide of formula (I) is obtained, with a yield of 63.3% (calculated based on Intermediate 3), a purity of 97.3%, and the mass spectrum is as Figure 2 shown.

[0057] Calculated based on the resin, the total yield of the synthesized cyclic ester peptide is 31.8%.

[0058] Example 2

[0059] The preparation method of the cyclic ester peptide of formula (I) comprises the following steps:

[0060] (1) Swelling of the resin: Take 31.6 g (50 mmol) of CTC resin (substitution degree of 1.58 mmol / g) and add 400 mL of dichloromethane to swell the resin for 2 hours.

[0061] (2) Preparation of Fmoc-Gly-CTC resin: Dissolve Fmoc-Gly-OH (3 equivalents, 150 mmol) and DIEA (6 equivalents, 300 mmol) in 400 mL of dichloromethane, add it to the resin (1 equivalent), and react at 25 °C for 2.5 hours. After the reaction is completed, wash it three times with 200 mL of dimethylformamide, and drain the solvent to obtain Fmoc-Gly-CTC resin.

[0062] (3) Removal of Fmoc protecting group: Add 400 mL of 20% PIP / DMF solution to remove the Fmoc protecting group twice at 25 °C, and the reaction times are 5 minutes and 10 minutes respectively. Then wash the resin with dimethylformamide until the pH reaches about 7, and drain the solvent to obtain NH2-Gly-CTC resin.

[0063] (4) Activation of amino acids and carboxylic acids to be coupled: Add 15 mmol of each of Fmoc-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH and palmitic acid and DIC (15 mmol) / HOBt (150 mmol) to 400 mL of dimethylformamide, and activate at 25 °C for 5 minutes. The activation of Fmoc-glycolic acid requires the addition of DMAP (50 mmol) for catalysis.

[0064] (5) Coupling of amino acids and carboxylic acids to be coupled with NH2-Gly-CTC resin: Add the activated Fmoc-L-Lys(Boc)-OH to NH2-Gly-CTC resin for condensation. After the condensation is completed, remove the Fmoc protecting group, and then continue to condense Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH in turn. After each condensation is completed, the operation of removing the Fmoc protecting group is carried out, and finally palmitic acid is condensed. The condensation conditions are: carry out the condensation reaction at 25 °C for 2 hours, and monitor the reaction process by ninhydrin color reaction to obtain the intermediate 1-resin.

[0065] (6) Cleavage of intermediate 1-resin: Add 500 mL of cleavage reagent (trifluoroethanol:dichloromethane = 1:4) to the peptide resin, react at 25 °C for 4 hours, filter by suction, and concentrate the filtrate under reduced pressure to dryness to obtain 54.3 g of crude intermediate 1.

[0066] (7) Purification of Intermediate 1: Intermediate 1 was purified by reverse-phase high performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm). The mobile phase A was 0.1% trifluoroacetic acid / water solution, and the mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. 82% mobile phase A and 18% mobile phase B were used for elution for 6 min, and then 45% mobile phase A and 55% mobile phase B were used for elution for 24 min. The flow rate was 6 ml / min, and the column temperature was 30±1 °C. After concentration and lyophilization, 35.6 g of Intermediate 1 was obtained, with a yield of 54.3% and a purity of 97.6%.

[0067] (8) Synthesis of Intermediate 2: 35.6 g (27.0 mmol) of Intermediate 1, 350 mL of methanol and 3 g of palladium on carbon were added to a 1000 mL reaction flask, and hydrogenation reaction was carried out at 30 °C for 16 hours. The reaction was detected by TLC to be completed. The filtrate was filtered with diatomaceous earth and concentrated to dryness to obtain Intermediate 2 (36.0 g), which was a white solid and was directly used for the synthesis of Intermediate 3 without purification.

[0068] (9) Synthesis of Intermediate 3: 36.0 g (27.0 mmol, calculated according to Intermediate 1), 4.37 g of HOBt (32.4 mmol, 1.2 equivalents relative to Intermediate 1), 1.65 g of DMAP (13.5 mmol, 0.5 equivalent relative to Intermediate 1) and 300 mL of N,N-dimethylformamide were added to a 1000 mL reaction flask, and the mixture was stirred until clear. The temperature was cooled to 0 - 10 °C, and 6.81 g of DIC (54.0 mmol, 2.0 equivalents relative to Intermediate 1) was added dropwise while controlling the temperature below 20 °C. The temperature was raised to 30 °C and the reaction was carried out at this temperature for 16 hours. The reaction was detected by TLC to be completed.

[0069] (10) Synthesis of the crude cyclic peptide of formula (I): 50 mL of trifluoroacetic acid was added to the reaction system, and the mixture was stirred at 30 °C for 8 hours. The reaction was detected by TLC to be completed. The reaction solution was concentrated to a volume of 20 ml, and the reaction solution was released into 200 ml of methyl tert-butyl ether. After stirring for 2 hours, centrifugation was carried out to obtain a white solid, which was dried under reduced pressure at 40 °C for 10 hours to obtain the crude peptide.

[0070] (11) Purification of the crude cyclic peptide of formula (I): The crude peptide was purified by reverse-phase high performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm). The mobile phase A was 0.1% trifluoroacetic acid / water solution, and the mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. 82% mobile phase A and 18% mobile phase B were used for elution for 6 min, and then 45% mobile phase A and 55% mobile phase B were used for elution for 24 min. The flow rate was 6 ml / min, and the column temperature was 30±1 °C. After concentration and lyophilization, 18.8 g of the cyclic peptide of formula (I) was obtained, with a yield of 66.3% (calculated according to Intermediate 3) and a purity of 98.4%.

[0071] Based on the resin calculation, the total yield of the synthetic cyclic peptide ester is 36.0%.

[0072] Example 3

[0073] The preparation method of the cyclic peptide ester of formula (I) comprises the following steps:

[0074] (1) Swelling of the resin: Take 63.3 g (100 mmol) of CTC resin (substitution degree 1.58 mmol / g), add 800 mL of dichloromethane, and swell the resin for 2 hours.

[0075] (2) Preparation of Fmoc-Gly-CTC resin: Dissolve Fmoc-Gly-OH (3 equivalents, 300 mmol) and DIEA (6 equivalents, 600 mmol) in 40 mL of dichloromethane, add to the resin (1 equivalent), react at 25 °C for 3 hours. After the reaction is completed, wash three times with 400 mL of dimethylformamide, and dry the solvent to obtain Fmoc-Gly-CTC resin.

[0076] (3) Removal of the Fmoc protecting group: Add 800 mL of 20% PIP / DMF solution at 25 °C to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. Then wash the resin with dimethylformamide until the pH reaches about 7, and dry the solvent to obtain NH2-Gly-CTC resin.

[0077] (4) Activation of the amino acids and carboxylic acids to be coupled: Dissolve 300 mmol each of Fmoc-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH and palmitic acid together with DIC (300 mmol) / HOBt (300 mmol) in 40 mL of dimethylformamide, and activate at 25 °C for 5 minutes. The activation of Fmoc-glycolic acid requires the addition of DMAP (100 mmol) for catalysis.

[0078] (5) Coupling of the amino acid and carboxylic acid to be coupled with NH2-Gly-CTC resin: The activated Fmoc-L-Lys(Boc)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed, and then Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, and Fmoc-L-Leu-OH were successively condensed. After each condensation, the operation of removing the Fmoc protecting group was carried out, and finally palmitic acid was condensed. The condensation conditions were as follows: The condensation reaction was carried out at 25 °C for 2 hours, and the reaction progress was monitored by ninhydrin color reaction to obtain intermediate 1-resin.

[0079] (6) Cleavage of intermediate 1-resin: 1000 mL of cleavage reagent (trifluoroethanol:dichloromethane = 1:4) was added to the peptide resin, and the reaction was carried out at 25 °C for 4 hours. After suction filtration, the filtrate was concentrated under reduced pressure to dryness to obtain 108.3 g of crude intermediate 1.

[0080] (7) Purification of intermediate 1: Intermediate 1 was purified by reversed-phase high-performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. 82% of mobile phase A and 18% of mobile phase B were used for elution for 6 min, and then 45% of mobile phase A and 55% of mobile phase B were used for elution for 24 min. The flow rate was 6 ml / min, and the column temperature was 30 ± 1 °C. After concentration and lyophilization, 79.7 g of intermediate 1 was obtained, with a yield of 60.6% and a purity of 95.1%.

[0081] (8) Synthesis of intermediate 2: 79.7 g (60.6 mmol) of intermediate 1, 800 mL of methanol, and 8 g of palladium carbon were added to a 2000 mL reaction flask, and the hydrogenation reaction was carried out at 30 °C for 16 hours. The reaction was detected by TLC to be completed. The filtrate was filtered with diatomaceous earth and concentrated to dryness to obtain 80.0 g of intermediate 2, which was a white solid and was directly used for the synthesis of intermediate 3 without purification.

[0082] (9) Synthesis of Intermediate 3: Add 80.0 g (60.6 mmol, calculated based on Intermediate 1), Intermediate 2, 9.82 g of HOBt (72.7 mmol, 1.2 equivalents relative to Intermediate 1), 3.70 g of DMAP (30.3 mmol, 0.5 equivalent relative to Intermediate 1), and 800 mL of N,N-dimethylformamide to a 2000 mL reaction flask. Stir until clear, cool to 0 - 10 °C, and add 15.3 g of DIC (121.2 mmol, 2.0 equivalents relative to Intermediate 1) dropwise while controlling the temperature below 20 °C. Heat to 20 °C and react at this temperature for 16 hours. Detect the completion of the reaction by TLC.

[0083] (10) Synthesis of the crude cyclic peptide of formula (I): Add 120 mL of trifluoroacetic acid to the reaction system, stir at 30 °C for 8 hours, and detect the completion of the reaction by TLC. Concentrate the reaction solution to a volume of 60 mL, release the reaction solution into 600 mL of methyl tert-butyl ether, stir for 2 hours, then centrifuge to obtain a white solid. Control the temperature at 35 °C and dry under reduced pressure for 12 hours to obtain the crude peptide.

[0084] (11) Purification of the crude cyclic peptide of formula (I): Purify the crude peptide by reverse-phase high-performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm). Mobile phase A is 0.1% trifluoroacetic acid / aqueous solution, and mobile phase B is 0.1% trifluoroacetic acid / acetonitrile solution. Elute with 82% mobile phase A and 18% mobile phase B for 6 min, and then continue to elute with 45% mobile phase A and 55% mobile phase B for 24 min at a flow rate of 6 mL / min and a column temperature of 30 ± 1 °C. After concentration and lyophilization, 41.6 g of the cyclic peptide of formula (I) is obtained, with a yield of 65.4% (calculated based on Intermediate 1) and a purity of 98.2%.

[0085] Calculated based on the resin, the total yield of the synthesized cyclic peptide is 39.6%.

[0086] Example 4

[0087] This experimental example detects the cytotoxicity of the test sample.

[0088] Cytotoxicity of the cyclic peptide of formula (I):

[0089] Cell culture: Mouse mononuclear-macrophage leukemia cells (RAW264.7 cells), A549 (human lung adenocarcinoma cells), SKOV-3 (human ovarian adenocarcinoma cells), MCF-7 (human breast adenocarcinoma cells), HepG2 (human liver adenocarcinoma cells), and JEG-3 (human placental choriocarcinoma cells) were selected as experimental test cell lines and obtained from the American Type Culture Collection (Manassas, VA, USA). The revived RAW264.7 cells were inoculated into cell culture dishes and added with high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin double antibody. The cells were transferred to a constant temperature incubator at 37°C with 5% CO2 for overnight culture. The next day, the cell growth state was observed and the medium was changed or the cells were passaged.

[0090] Cytotoxicity evaluation: MTT was used to determine the relative cytotoxicity of the drug. Cells in DMEM medium (supplemented with 10% FBS) were inoculated into 96-well plates, 5000 cells per well. Under the constant temperature condition of 37°C, the cells were exposed to the solution of the cycloalkane peptide of formula (I) prepared in Example 3 at different concentrations (0 - 512 μM) and incubated for 24 hours.

[0091] At the end of incubation, 20 μL of MTT solution (5 μg / mL) was added to each well. Then it was incubated at 37°C for 4 hours, and then centrifuged to remove the supernatant. 200 μL of dimethyl sulfoxide (DMSO) was added to each well in the dark to dissolve the formazan crystals. The absorbance value was measured using a microplate reader at 570 nm. Cell viability was calculated as a percentage relative to the untreated control value.

[0092] The MTT method was used to detect the biosafety of the cycloalkane peptide of formula (I) prepared in Example 3 against RAW264.7 cells. As Figure 3 shown, when the concentration was less than 512 μM, the cycloalkane peptide of formula (I) had no effect on cytotoxicity. Even at a concentration of 512 μmol / L, its cell survival rate was as high as over 95%, showing good biosafety.

[0093] Example 5

[0094] This experimental example detected the thermal stability of the test sample.

[0095] The thermal stability experiment was carried out on the cycloalkane peptide of formula (I) prepared in Example 3 above, and compared with the synthetic tetrapeptides GPKG and LSVD. The results all showed significantly better thermal stability than the synthetic tetrapeptides GPKG and LSVD.

[0096] Thermal stability experiment: The synthetic tetrapeptides GPKG and LSVD and the cycloalkane peptide of formula (I) prepared in Example 3 above were simultaneously placed in a vacuum drying oven at 80°C. Samples were taken at 0 h, 24 h, and 48 h respectively, and their content reduction degrees were detected by HPLC. The results are shown in Table 2.

[0097] Table 2

[0098]

[0099] Example 6

[0100] This experimental example detects the transdermal absorbability of the test sample.

[0101] The transdermal absorbability experiment was carried out on the cycloalkane peptide of formula (I) prepared in Example 3 above, and compared with the synthetic tetrapeptides GPKG and LSVD. The results all showed significantly better transdermal absorbability than the synthetic tetrapeptides GPKG and LSVD.

[0102] Transdermal absorbability experiment: Artificial skin was selected and the skin was fixed in three Franz diffusion cells respectively, ensuring that the epidermis was facing up. The cycloalkane peptide of formula (I) prepared in Example 3 and the synthetic tetrapeptides GPKG and LSVD were dissolved in ethanol at a concentration of 1 mg / mL. 200 μL of each of the three solutions was added to the skin surface. 5 mL of PBS (pH 7.4) was added to the receiving cell, and magnetic stirring was maintained at 37 °C with a stirring speed of 600 rpm. 200 μL of the receiving solution was collected at 0, 1, 2, and 4 hours, and an equal amount of fresh PBS was supplemented. The collected samples were analyzed by HPLC for the sample concentration. The results are shown in Table 3. The transdermal absorbability of the synthetic tetrapeptide GPKG was about 3 times that of LSVD at the same time. When the transdermal absorbability experiment of the cycloalkane peptide of formula (I) was compared with the synthetic tetrapeptides GPKG and LSVD, the cycloalkane peptide of formula (I) was significantly better than the synthetic tetrapeptides GPKG and LSVD in transdermal absorbability, about 4 times that of the synthetic tetrapeptide GPKG, and about 12 times that of the synthetic tetrapeptide LSVD.

[0103] Table 3

[0104]

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An ester cyclic peptide, characterized in that: Its structural formula is shown in formula (I):

2. The method for preparing the ester cyclic peptide according to claim 1, characterized in that: The steps include: Intermediate 1 was synthesized by solid phase synthesis, and the structural formula of intermediate 1 is shown in Formula II; After hydrogenation and debenzylation of intermediate 1, intermediate 2 was obtained; Intermediate 2 is cyclized to obtain intermediate 3, the structural formula of intermediate 3 is shown in formula (III): Intermediate 3 is deprotected to obtain the ester cyclic peptide of formula (I).

3. The preparation method according to claim 2, characterized in that: Using CTC resin as a solid phase synthesis carrier, Fmoc-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Pro-OH, Fmoc-Gly-OH, Fmoc-glycolic acid, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-Ser(OBn)-OH, Fmoc-L-Leu-OH and palmitic acid were condensed in sequence from C-terminus to N-terminus to obtain a peptide resin. After the peptide resin was cleaved, a crude intermediate 1 was obtained.

4. The preparation method according to claim 3, characterized in that: The method for purifying the crude intermediate 1 is: purifying by high performance liquid chromatography, the chromatographic column is a C18 column, the mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid by mass, and the mobile phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid by mass.

5. The preparation method according to claim 3, characterized in that: The cleavage reagent for the cleavage is a mixture of trifluoroethanol and dichloromethane, and the volume ratio of trifluoroethanol to dichloromethane is 1:3-5; Preferably, the pyrolysis temperature is 20-30°C and the pyrolysis time is 2-4h.

6. The preparation method according to claim 2, characterized in that: The synthesis method of intermediate 2 is as follows: intermediate 1, methanol and palladium carbon are mixed in proportion, and then hydrogenated at 20-30° C. for 15-20 hours to obtain intermediate 2.

7. The preparation method according to claim 2, characterized in that: The synthesis method of intermediate 3 is as follows: intermediate 2, HOBt, DMAP and N,N-dimethylformamide are mixed in proportion, stirred until dissolved, cooled to 0-10°C, DIC is added, and then reacted at 20-30°C for 15-20h.

8. Use of the ester cyclic peptide according to claim 1 in the preparation of drugs, foods, food additives or cosmetics for promoting collagen synthesis.

9. The use according to claim 8, characterized in that: The dosage form of the drug includes, but is not limited to, injection, ointment, powder injection, liniment, dressing or liquid preparation.

10. The use according to claim 8, characterized in that: The cosmetics include but are not limited to lotions, emulsions, essences, gels, foundations, creams or masks.

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