Cyclic peptide derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, in particular to a cyclic peptide derivative and a preparation method and application thereof. The cyclopeptide derivative provided by the invention simultaneously comprises a hydrophobic cyclopeptide structure and a hydrophilic pantothenic acid structure which are combined through a covalent ester bond; due to the structure, excellent stability is achieved, and the problems that pantothenic acid is unstable and irritant are effectively solved; after entering the skin, the derivative can be hydrolyzed into pantothenic acid under the action of esterase, and can play a role without oxidative conversion of panthenol. Secondly, the cyclic peptide derivative disclosed by the invention has good biological safety; in an inflammatory cell model, the derivative can more effectively reduce expression of inflammatory factors, and antioxidant activity research shows that the derivative has a strong free radical inhibition effect and can exert excellent anti-inflammatory and antioxidant effects.

Description

Technical Field

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

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Citrusin XI is a cyclic heptapeptide derived from Wenzhou mandarin oranges. It has good cell compatibility and safety, and has potential effects such as anti-oxidation, anti-aging, whitening and anti-inflammatory soothing. Specifically, it can fight against free radical damage to cells, protect cell health, reduce the adverse effects of oxidative stress, slow down the aging process of the skin, reduce wrinkles and sagging, and add it to anti-aging skin care products to help users delay skin aging and maintain firmness and elasticity. Citrusin XI showed significant anti-inflammatory activity in RAW264.7 macrophages stimulated by lipopolysaccharide (LPS). It can reduce the production of nitric oxide (NO) in a dose-dependent manner, and its half inhibitory concentration (IC50 value) is 70μM. This mechanism of reducing NO production is achieved by reducing the expression of inducible nitric oxide synthase (iNOS). Citrusin XI can also inhibit the activation of NF-κB by blocking the degradation of IκBα and the phosphorylation of NF-κB, thereby further reducing the production of NO. By inhibiting the release of inflammatory mediators, Citrusin XI helps reduce cellular inflammatory responses, which has a positive effect on alleviating the adverse effects of accelerated aging caused by chronic inflammation. In sensitive skin care products, it can have a soothing and calming effect, reducing skin redness, swelling and itching.

[0004] Pantothenic acid plays an important role in cosmetics. First, it has a moisturizing effect, which can enhance the skin's moisturizing ability and make the skin hydrated. For example, adding pantothenic acid to moisturizing lotions can improve the moisturizing effect of the product and keep the skin soft and smooth for a long time. Second, it can repair the skin barrier, promote the health of the skin's stratum corneum, enhance the barrier function, and reduce external stimulation damage. For people with damaged skin barriers, skin care products containing pantothenic acid can help repair and improve sensitive conditions. Third, it has a soothing and anti-allergic effect, which can reduce skin inflammation and relieve discomfort. In cosmetics for sensitive skin, it can reduce the incidence of allergies and make the skin more stable. Fourth, it can promote cell regeneration, stimulate cell metabolism and regeneration, make the skin more vibrant and lustrous, and in anti-aging cosmetics, it can promote the renewal of aging cells and reduce the appearance of fine lines and wrinkles.

[0005] However, both of the above active substances have certain disadvantages that affect their application. At present, the main method for obtaining Citrusin XI is extraction. The literature Anti-inflammatory activity of a new cyclic peptide, citrusin XI, isolated from the fruits of Citrus unshiu reported its extraction method, and the extraction yield was extremely low, about 0.0007%, and the process was cumbersome. Pantothenic acid is unstable under certain conditions and is easily inactivated under the influence of various factors, such as temperature, pH, light and oxidation. At present, panthenol is mostly used on the market instead of pantothenic acid, which needs to be converted into pantothenic acid after entering the skin to exert the biological function of pantothenic acid. Summary of the invention

[0006] In order to overcome the above problems, the present invention provides cyclic peptide derivatives and preparation methods and applications thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a cyclic peptide derivative, the structural formula of which is shown in formula (I):

[0009]

[0010] The second aspect of the present invention provides a method for preparing the cyclic peptide derivative described in the first aspect, comprising:

[0011] Compound 1 and compound 2 are coupled to obtain intermediate 1, and intermediate 1 is deprotected to obtain the cyclic peptide derivative;

[0012] Wherein, the structural formula of compound 1 is shown as formula (IV):

[0013]

[0014] The structural formula of compound 2 is shown in formula (III):

[0015]

[0016] The structural formula of intermediate 1 is shown in formula (II):

[0017]

[0018] In one or more embodiments, the method for coupling compound 1 and compound 2 to obtain intermediate 1 includes: in an alkaline environment, dispersing compound 1 and compound 2 in a solvent, stirring until dissolved, adding a coupling agent, and stirring the reaction to obtain intermediate 1.

[0019] Preferably, the solvent comprises N,N-dimethylformamide (DMF).

[0020] Preferably, the molar ratio of compound 1 to compound 2 is 1:2-3.

[0021] Preferably, the coupling agent includes one of 1-propylphosphoric acid cyclic anhydride and O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU).

[0022] Preferably, the molar ratio of the compound 1 to the coupling agent is 1:2-3.

[0023] Preferably, the alkaline substance providing the alkaline environment includes one of N-methylmorpholine and triethylamine.

[0024] More preferably, the molar ratio of the compound 1 to the alkaline substance is 1:2-3.

[0025] Preferably, the stirring reaction time is 15 to 20 hours, preferably 16 hours.

[0026] In one or more embodiments, the method for deprotecting the intermediate 1 to obtain the cyclic peptide derivative comprises: reacting the intermediate 1 with an acid, and stirring the reaction to obtain the cyclic peptide derivative.

[0027] Preferably, the acid comprises acetic acid.

[0028] Preferably, the stirring reaction time is 6 to 10 hours, preferably 8 hours.

[0029] In one or more embodiments, Compound 1 is synthesized using solid phase synthesis.

[0030] Preferably, CTC resin is used as a solid phase synthesis carrier, and Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Pro-OH and Fmoc-D-Ser(tBu)-OH are condensed from the C-terminus to the N-terminus in sequence, and the fully protected linear crude peptide NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH is obtained after cleavage and concentration to dryness. The fully protected linear crude peptide is then cyclized, deprotected, purified and lyophilized to obtain compound 1.

[0031] The third aspect of the present invention provides the use of the cyclic peptide derivatives described in the first aspect and / or the cyclic peptide derivatives prepared by the preparation method described in the second aspect in the preparation of drugs and pharmaceutical compositions, foods, food additives or cosmetics with anti-inflammatory function.

[0032] In one or more embodiments, the food includes health food and ordinary food.

[0033] The fourth aspect of the present invention provides the use of the cyclic peptide derivative described in the first aspect and / or the cyclic peptide derivative prepared by the preparation method described in the second aspect in the preparation of drugs and pharmaceutical compositions, foods, food additives or cosmetics with antioxidant function.

[0034] In one or more embodiments, the food includes health food and ordinary food.

[0035] The fifth aspect of the present invention provides an anti-inflammatory product, comprising the cyclic peptide derivative described in the first aspect and / or the cyclic peptide derivative prepared by the preparation method described in the second aspect; the anti-inflammatory product comprises an anti-inflammatory drug, an anti-inflammatory drug composition, an anti-inflammatory food, an anti-inflammatory food additive or an anti-inflammatory cosmetic.

[0036] In one or more embodiments, the food includes health food and ordinary food.

[0037] The sixth aspect of the present invention provides an antioxidant product, comprising the cyclic peptide derivative described in the first aspect and / or the cyclic peptide derivative prepared by the preparation method described in the second aspect; the antioxidant product comprises an antioxidant drug, an antioxidant drug composition, an antioxidant food, an antioxidant food additive or an antioxidant cosmetic.

[0038] In one or more embodiments, the food includes health food and ordinary food.

[0039] Unless otherwise specified, the anti-inflammatory product or antioxidant product of the present invention can be taken orally, applied externally or injected. A person skilled in the art can select and add suitable adjuvants according to conventional methods to prepare the anti-inflammatory product or antioxidant product of the present invention into a suitable dosage form or form of use. Such dosage forms include injections, ointments, powder injections, liniments, dressings, liquid preparations, and the like. When used as cosmetics, the anti-inflammatory product or antioxidant product may include but is not limited to lotions, emulsions, essences, gels, foundations, creams, and masks, and the scope of application of the cosmetics includes but is not limited to facial washing and maintenance, body washing and maintenance, and head washing and maintenance, and the like. The cyclic peptide derivatives of the present invention can be used to configure cosmetics with soothing, anti-inflammatory, antioxidant, and skin repairing functions.

[0040] The beneficial effects of the present invention are:

[0041] (1) The cyclic peptide derivative provided by the present invention contains both a hydrophobic cyclic peptide structure and a hydrophilic pantothenic acid structure, which are combined by a covalent ester bond; this structure gives it excellent stability, effectively improving the problem that pantothenic acid is unstable and irritating; after entering the skin, under the action of esterase, the derivative can be hydrolyzed into pantothenic acid, and can exert its effect without undergoing oxidative conversion of panthenol. Secondly, the cyclic peptide derivative in the present invention has good biosafety; in the inflammatory cell model, the derivative can more effectively reduce the expression of inflammatory factors, and the antioxidant activity study shows that it has a strong free radical inhibition effect and can exert excellent anti-inflammatory and antioxidant effects.

[0042] (2) The present invention also provides a method for synthesizing a cyclopeptide derivative. The process has high synthesis efficiency, the yield of the synthesized compound 1 is 60% to 66%, and the operation is simple and suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0044] Figure 1 is the mass spectrum of compound 1 prepared in Example 1;

[0045] Figure 2 The mass spectrum of the cyclic peptide derivative prepared in Example 4;

[0046] Figure 3 The survival rate of RAW 264.7 cells after being treated with the cyclic peptide derivatives prepared in Example 6 at different concentrations;

[0047] Figure 4 is the amount of NO produced in LPS-induced RAW264.7 cells after intervention in experimental groups 1 to 4;

[0048] Figure 5 is the expression of IL-6 in LPS-stimulated macrophages after intervention in experimental groups 1 to 4;

[0049] Figure 6 is the expression level of TNF-α in LPS-stimulated macrophages after intervention in experimental groups 1 to 4. DETAILED DESCRIPTION

[0050] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0051] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0052] Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. The reagents or raw materials used in this application can be purchased through conventional channels. If there is no special explanation, the reagents or raw materials used in this application are used in a conventional manner in this field or in accordance with the product instructions. In addition, any method and material similar to or equal to the recorded content can be applied to the method of this application. The preferred implementation methods and materials described in the text are for demonstration purposes only. If there is no special explanation, the percentages described in the present invention are all percentages by mass.

[0053] The corresponding Chinese meanings of the English abbreviations of substances appearing in the claims and specification of the present invention are shown in the following table:

[0054]

[0055]

[0056] 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.

[0057] Example 1

[0058] Preparation of compound 1 from Satsuma mandarin:

[0059] (1) Resin swelling: 3.16 g (5 mmol) of CTC resin (substitution degree: 1.58 mmol / g) was added to 40 mL of dichloromethane and the resin was swollen for 1.5 h.

[0060] (2) Preparation of Fmoc-Gly-CTC resin: Fmoc-Gly-OH (3 equivalents, 15 mmol) and DIEA (6 equivalents, 30 mmol) were dissolved in 40 mL of dichloromethane, added to the resin (1 equivalent), and reacted at 25°C for 2 hours. After the reaction was completed, it was washed three times with 20 mL of dimethylformamide, and the solvent was drained to obtain Fmoc-Gly-CTC resin.

[0061] (3) Removal of Fmoc protecting group: 40 mL of 20% PIP / DMF solution was added at 25°C to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. The resin was then washed with dimethylformamide until the pH reached about 7, and the solvent was drained to obtain NH2-Gly-CTC resin.

[0062] (4) Activation of amino acids: 15 mmol each of Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Pro-OH and Fmoc-D-Ser(tBu)-OH and DIC (15 mmol) / HOBt (15 mmol) were added to 40 mL of dimethylformamide and activated at 25°C for 5 min.

[0063] (5) Coupling of amino acids with NH2-Gly-CTC resin: The activated Fmoc-D-Tyr(tBu)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed. Then, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, and Fmoc-L-Pro-OH were condensed in sequence. After each condensation of an amino acid, the Fmoc protecting group was removed. Finally, Fmoc-D-Ser(tBu)-OH was condensed. The amino acid condensation reaction was carried out at 25°C for 2 hours. The reaction progress was monitored by ninhydrin colorimetric reaction. After removing Fmoc, NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH-resin was obtained.

[0064] (6) Cleavage of peptide resin

[0065] Add 50 mL of cleavage reagent (trifluoroethanol: dichloromethane = 1:4) to the peptide resin, react at 25°C for 3 hours, filter, and concentrate the filtrate to dryness under reduced pressure to obtain 3.4 g of fully protected linear crude peptide NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH.

[0066] (7) Cyclization and purification of fully protected linear peptides

[0067] The fully protected linear crude peptide and DIC (10 mmol) / HOBt (10 mmol) were added to 600 mL of dichloromethane, and reacted at 25 ° C for 5 hours. After TLC detection, the reaction was completed, and the mixture was evaporated to 200 mL of liquid, washed twice with saturated sodium bicarbonate aqueous solution, and anhydrous magnesium sulfate was added to the reaction solution for 3 hours. After filtering, the mixture was evaporated to dryness using a rotary evaporator, and 120 mL of a deprotection reagent (trifluoroacetic acid: dichloromethane = 1:5) was added, and the mixture was reacted at 25 ° C for 2 hours. After concentration, the mixture was precipitated in ice MTBE, centrifuged, washed, and dried to obtain a white solid.

[0068] The crude peptide was purified by reverse phase HPLC using a C18 preparative column (50×250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 2.2 g of compound 1 was obtained, with a yield of 60.3% and a purity of 97.2%. The mass spectrum is shown in FIG. Figure 1 shown.

[0069] Example 2

[0070] Preparation of compound 1 from Satsuma mandarin:

[0071] (1) Swelling of the resin: 31.6 g (50 mmol) of CTC resin (substitution degree: 1.58 mmol / g) was added to 400 mL of dichloromethane to swell the resin for 2 h.

[0072] (2) Preparation of Fmoc-Gly-CTC resin: Fmoc-Gly-OH (3 equivalents, 150 mmol) and DIEA (6 equivalents, 300 mmol) were dissolved in 400 mL of dichloromethane, added to the resin (1 equivalent), and reacted at 25°C for 2 hours. After the reaction was completed, it was washed three times with 200 mL of dimethylformamide, and the solvent was drained to obtain Fmoc-Gly-CTC resin.

[0073] (3) Removal of Fmoc protecting group: 400 mL of 20% PIP / DMF solution was added at 25°C to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. The resin was then washed with dimethylformamide until the pH reached about 7, and the solvent was drained to obtain NH2-Gly-CTC resin.

[0074] (4) Activation of amino acids: 15 mmol each of Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Pro-OH and Fmoc-D-Ser(tBu)-OH and DIC (15 mmol) / HOBt (15 mmol) were added to 400 mL of dimethylformamide and activated at 25°C for 5 min.

[0075] (5) Coupling of amino acids with NH2-Gly-CTC resin: The activated Fmoc-D-Tyr(tBu)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed. Then, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, and Fmoc-L-Pro-OH were condensed in sequence. After each condensation of an amino acid, the Fmoc protecting group was removed. Finally, Fmoc-D-Ser(tBu)-OH was condensed. The amino acid condensation reaction was carried out at 25°C for 2.5 hours. The reaction progress was monitored by ninhydrin colorimetric reaction. After removing Fmoc, NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH-resin was obtained.

[0076] (6) Cleavage of peptide resin

[0077] Add 50 mL of cleavage reagent (trifluoroethanol: dichloromethane = 1:4) to the peptide resin, react at 25°C for 5 hours, filter, and concentrate the filtrate to dryness under reduced pressure to obtain 35 g of fully protected linear crude peptide NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH.

[0078] (7) Cyclization and purification of fully protected linear peptides

[0079] The fully protected linear crude peptide and DIC (75 mmol) / HOBt (75 mmol) were added to 6 L of dichloromethane, and the mixture was reacted at 25 ° C for 6 hours. The reaction was completed by TLC detection, and the mixture was vortexed to 2000 mL of liquid, washed twice with saturated sodium bicarbonate aqueous solution, and anhydrous magnesium sulfate was added to the reaction solution for 3 hours to remove water. After filtering, the mixture was vortexed to dryness using a rotary evaporator, and 1200 mL of a deprotection reagent (trifluoroacetic acid: dichloromethane = 1:5) was added, and the mixture was reacted at 25 ° C for 2 hours. After concentration, the mixture was precipitated in ice MTBE, centrifuged, washed, and dried to obtain a white solid.

[0080] The crude peptide was purified by reverse phase HPLC using a C18 preparative column (50×250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 23 g of compound 1 was obtained, with a yield of 63.0% and a purity of 96.8%.

[0081] Example 3

[0082] Preparation of compound 1 from Satsuma mandarin:

[0083] (1) Resin swelling: 63.3 g (100 mmol) of CTC resin (substitution degree: 1.58 mmol / g) was added to 800 mL of dichloromethane and the resin was swollen for 2 hours.

[0084] (2) Preparation of Fmoc-Gly-CTC resin: Fmoc-Gly-OH (3 equivalents, 300 mmol) and DIEA (6 equivalents, 600 mmol) were dissolved in 700 mL of dichloromethane, added to the resin (1 equivalent), and reacted at 25°C for 3 hours. After the reaction was completed, it was washed three times with 300 mL of dimethylformamide, and the solvent was drained to obtain Fmoc-Gly-CTC resin.

[0085] (3) Removal of Fmoc protecting group: 700 mL of 20% PIP / DMF solution was added at 25°C to remove the Fmoc protecting group twice, with reaction times of 5 minutes and 10 minutes respectively. The resin was then washed with dimethylformamide until the pH reached about 7, and the solvent was drained to obtain NH2-Gly-CTC resin.

[0086] (4) Activation of amino acids: 15 mmol each of Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Pro-OH and Fmoc-D-Ser(tBu)-OH and DIC (15 mmol) / HOBt (15 mmol) were added to 700 mL of dimethylformamide and activated at 25°C for 5 min.

[0087] (5) Coupling of amino acids with NH2-Gly-CTC resin: The activated Fmoc-D-Tyr(tBu)-OH was added to NH2-Gly-CTC resin for condensation. After the condensation was completed, the Fmoc protecting group was removed. Then, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, and Fmoc-L-Pro-OH were condensed in sequence. After each condensation of an amino acid, the Fmoc protecting group was removed. Finally, Fmoc-D-Ser(tBu)-OH was condensed. The amino acid condensation reaction was carried out at 25°C for 3 hours. The reaction progress was monitored by ninhydrin colorimetric reaction. After removing Fmoc, NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH-resin was obtained.

[0088] (6) Cleavage of peptide resin

[0089] Add 1.0 L of cleavage reagent (trifluoroethanol: dichloromethane = 1:4) to the peptide resin, react at 25 ° C for 5 hours, filter, and concentrate the filtrate to dryness under reduced pressure to obtain 68 g of fully protected linear crude peptide NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH.

[0090] (7) Cyclization and purification of fully protected linear peptides

[0091] The fully protected linear crude peptide and DIC (10 mmol) / HOBt (10 mmol) were added to 12 L of dichloromethane, reacted at 25 ° C for 6 hours, and the reaction was completed by TLC detection. The reaction was spun to 4000 mL of liquid using a rotary evaporator, washed twice with a saturated sodium bicarbonate aqueous solution, anhydrous magnesium sulfate was added to the reaction solution to remove water for 3 hours, filtered and spun to dryness using a rotary evaporator, 2.4 L of a deprotection reagent (trifluoroacetic acid: dichloromethane = 1:5) was added, and the reaction was carried out at 25 ° C for 2 hours. After concentration, the solution was precipitated in ice MTBE, centrifuged, washed, and dried to obtain a white solid.

[0092] The crude peptide was purified by reverse phase HPLC using a C18 preparative column (50×250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 145.3 g of the compound was obtained, with a yield of 62% and a purity of 98.6%.

[0093] Example 4

[0094] Preparation of cyclic peptide derivatives:

[0095] To a 100 mL reaction bottle, 2.0 g (2.8 mmol) of compound 1 prepared in Example 3, 1.6 g of compound 2 (6.0 mmol, 2.2 equivalents relative to compound 1), 0.6 g of N-methylmorpholine (6.0 mmol, 2.2 equivalents relative to compound 1) and 20 mL of N,N-dimethylformamide were added, stirred until dissolved, cooled to 0-10°C, and 1.9 g of 1-propylphosphoric acid cyclic anhydride (6.0 mmol, 2.2 equivalents relative to compound 1) was added dropwise while controlling the temperature below 20°C. The temperature was raised to 25°C and reacted at this temperature for 16 hours. The reaction was completed by TLC detection.

[0096] 20 mL of purified water and 20 mL of acetic acid were added to the reaction system. After stirring at 25°C for 8 hours, the reaction was completed by TLC detection. After concentration to dryness, 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. After concentration and freeze drying, 2.2 g of the cyclopeptide derivative was obtained, with a yield of 72.3% and a purity of 97.3%. The mass spectrum is shown in FIG. Figure 2 shown.

[0097] Example 5

[0098] Preparation of cyclic peptide derivatives:

[0099] To a 100 mL reaction bottle, 2.0 g (2.8 mmol) of compound 1 prepared in Example 3, 1.6 g of compound 2 (6.0 mmol, 2.2 equivalents relative to compound 1), 0.6 g of triethylamine (6.0 mmol, 2.2 equivalents relative to compound 1) and 20 mL of N,N-dimethylformamide were added, stirred until dissolved, cooled to 0-10°C, and 2.3 g of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 6.0 mmol, 2.2 equivalents relative to compound 1) was added in batches while controlling the temperature below 20°C. The temperature was raised to 25°C and reacted at this temperature for 16 hours. The reaction was completed after TLC detection.

[0100] 20 mL of purified water and 20 mL of acetic acid were added to the reaction system, and after stirring at 25°C for 8 hours, the reaction was completed by TLC detection. After concentration to dryness, the crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 1.7 g of the cyclopeptide derivative was obtained, with a yield of 55.4% and a purity of 96.9%.

[0101] Example 6

[0102] Preparation of cyclic peptide derivatives:

[0103] To a 2L reaction bottle, 40.0 g (56.0 mmol) of compound 1 prepared in Example 3, 32 g of compound 2 (120.0 mmol, 2.1 equivalents relative to compound 1), 12.0 g of N-methylmorpholine (120.0 mmol, 2.1 equivalents relative to compound 1) and 400 mL of N,N-dimethylformamide were added, stirred until dissolved, cooled to 0-10°C, and 38 g of 1-propylphosphoric acid cyclic anhydride (120.0 mmol, 2.1 equivalents relative to compound 1) was added dropwise while controlling the temperature below 20°C. The temperature was raised to 25°C and reacted at this temperature for 16 hours. The reaction was completed by TLC detection.

[0104] 400 mL of purified water and 400 mL of acetic acid were added to the reaction system, and after stirring at 25°C for 8 hours, the reaction was completed by TLC detection. After concentration to dryness, the crude peptide was purified by reverse phase high performance liquid chromatography using a C18 preparative column (50×250 mm, 10 μm), with mobile phase A being 0.1% trifluoroacetic acid / water solution and mobile phase B being 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and freeze drying, 44.7 g of the cyclopeptide derivative was obtained, with a yield of 73.5% and a purity of 98.1%.

[0105] Example 7

[0106] This experimental example detects the cytotoxicity, anti-inflammatory, soothing and antioxidant effects of the test samples.

[0107] 7.1 Cytotoxicity of Cyclic Peptide Derivatives:

[0108] 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 Model Culture Institute (Manassas, VA, USA). The revived RAW264.7 cells were inoculated into a cell culture dish and high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin double antibody was added. The cells were transferred to a constant temperature incubator at 37°C and 5% CO2 for overnight culture. The cell growth status was observed the next day and the medium was changed or subcultured.

[0109] 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, with 5000 cells per well. Under constant temperature conditions of 37°C, the cells were exposed to different concentrations (0-256 μM) of the cyclic peptide derivative solution prepared in Example 6 and incubated for 24 hours. At the end of the incubation, 20 μL of MTT solution (5 μg / mL) was added to each well. Incubate at 37°C for another 4 hours, then centrifuge to remove the supernatant. Add 200 μL of dimethyl sulfoxide (DMSO) to each well in the dark to dissolve the formamide crystals. The absorbance value was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage relative to the untreated control value.

[0110] The MTT method was used to detect the biosafety of the cyclic peptide derivatives prepared in Example 6 on RAW264.7 cells. Figure 3 As shown, when the concentration is less than 256 μM, the cyclic peptide derivative has no effect on cytotoxicity. Even at a concentration of 256 μmol / L, the cell survival rate is as high as more than 95%, showing good biosafety.

[0111] 7.2 2. Anti-inflammatory and soothing activity of cyclic peptide derivatives:

[0112] Experimental principle: Mouse RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) were used as an in vitro inflammatory model to detect the expression levels of NO, IL-6 and TNF-α related to the inflammatory factors released by them, and the anti-inflammatory and soothing activity of the samples was evaluated by testing the inhibition rate of the test substance on the expression of inflammatory factors. Experimental group 1 (100 μM of cyclic peptide derivative), experimental group 2 (100 μM of compound 1), experimental group 3 (100 μM of pantothenic acid) and experimental group 4 (50 μM of compound 1 + 50 μM of pantothenic acid) were set up respectively.

[0113] NO release determination

[0114] RAW264.7 cells (5×10 5 Each well of the experimental group 1, experimental group 2, experimental group 3 and experimental group 4 were pretreated with 100 μL per well for 2 hours, and then treated with LPS (1 μg / mL) for 24 hours. While setting up a blank control, dexamethasone (100 nM) was used as a positive control. Griess reagent (Sigma-Aldrich, St.) was used to detect NO in the culture medium as an indicator of NO release. Griess reagent was mixed with DMEM culture medium in a 1:1 ratio and incubated at room temperature for 15 minutes. Finally, an ELISA reader was used to measure the absorbance at 540 nm to obtain the absorbance value.

[0115] To determine whether different compounds could modulate LPS-induced inflammatory mediators in RAW264.7 cells, NO production in the culture medium was measured using Griess reagent. Inflammatory stimuli involve high levels of NO and lead to the induction of pro-inflammatory mediators. Figure 4 As shown, experimental group 1 can significantly reduce the NO production of RAW264.7 cells stimulated by LPS compared with other control groups, and has an anti-inflammatory effect similar to that of dexamethasone in the positive control group. The effect of cyclic peptide derivatives in reducing NO release is better than compound 1, pantothenic acid, and the mixed group of compound 1 and pantothenic acid.

[0116] Evaluation of the inhibition rate of inflammatory factors: Mouse RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) were prepared as an in vitro inflammatory model, and the inhibition rate of the test substance on the inflammatory factors serum interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) was tested by adding sample intervention to evaluate the anti-inflammatory activity of the sample. First, RAW264.7 cells in the logarithmic growth phase were selected and incubated at 37°C and 5% CO2 for 24 hours. 100 μL of the sample solution in each well of the above experimental groups 1 to 4 was added, and a blank control group was set up at the same time. The incubation continued for 24 hours, and the expression levels of IL-6 and TNF-α were determined using the Mouse IL-6ValukineTMELISA kit and the Mouse TNF-alphaValukineTMELISA kit, respectively.

[0117] By establishing an LPS-stimulated mouse macrophage model in vitro and adding samples to intervene in the stimulation process, we observed whether the samples could inhibit the secretion of inflammatory factors IL-6 and TNF-α, thus making them have anti-inflammatory effects. Figure 5 and 6 As shown, compared with the blank control group and other parallel experimental groups, the effect of experimental group 1 in inhibiting the secretion of inflammatory factors IL-6 and TNF-α was better than that of other experimental groups.

[0118] The NO release and inflammatory factor inhibition experiments fully demonstrated that the anti-inflammatory effect of the cyclic peptide derivatives prepared in the present invention is not produced by a simple mixture of compound 1 and pantothenic acid (experimental group 4), but is due to the structure.

[0119] 7.3 Evaluation of antioxidant activity:

[0120] The present invention adopts NBT (nitro blue tetrazolium) photochemical reduction method to determine the antioxidant properties of different samples. Prepare 50μM, 2mL of xanthine in a test tube, add appropriate xanthine oxidase to form a mother solution. Weigh 0.01g NBT, 0.35g methionine, measure 100mmol / L EDTA, and then add PBS buffer with a pH of 7.8 to 200mL to prepare NBT solution. Take 3mL of NBT solution and add it to the mother solution, take 1mL of the test solution of experimental groups 1 to 4 respectively, under 37°C water bath conditions, 2×50W fluorescent lamps are irradiated for 20min to terminate the reaction, and the test tube is placed in dark conditions. Use a 1cm thick cuvette to measure at a constant temperature of 25±0.2°C, and compare the I50 value (i.e., the concentration that produces 50% inhibition of NBT reduction).

[0121] Table 1 Investigation of the effect of scavenging reactive oxygen free radicals (ROS)

[0122]

[0123]

[0124] NBT is reduced by CO2 to a blue-purple substance with a maximum absorption wavelength of 530-580 nm. When the test solution has an antioxidant effect, the NBT reduction reaction may be inhibited. 50 The smaller the amount of the product with the value, the greater its antioxidant activity. From the data analysis of Table 1, the I 50 The value is only 0.2 μM, indicating that it has a strong free radical inhibition effect at low concentrations, showing high activity, and is significantly better than the effects of other experimental groups, especially significantly better than the simple mixture of compound 1 and pantothenic acid (experimental group 4). This fully proves that the antioxidant effect of the cyclic peptide derivative prepared by the present invention is not produced by a simple mixture of compound 1 and pantothenic acid, but is due to its structure.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cyclic peptide derivative, characterized in that: The structural formula is shown in formula (I):

2. The method for preparing the cyclic peptide derivative according to claim 1, characterized in that: include: Compound 1 and compound 2 are coupled to obtain intermediate 1, and intermediate 1 is deprotected to obtain the cyclic peptide derivative; Wherein, the structural formula of compound 1 is shown as formula (IV): The structural formula of compound 2 is shown in formula (III): The structural formula of intermediate 1 is shown in formula (II):

3. The preparation method according to claim 2, characterized in that: The method for coupling compound 1 and compound 2 to obtain intermediate 1 comprises: in an alkaline environment, dispersing compound 1 and compound 2 in a solvent, stirring until dissolved, adding a coupling agent, and stirring to react to obtain intermediate 1.

4. The preparation method according to claim 3, characterized in that: The solvent includes N,N-dimethylformamide; Or, the molar ratio of compound 1 to compound 2 is 1:2-3; Or, the coupling agent includes one of 1-propylphosphoric acid cyclic anhydride and O-benzotriazole-tetramethyluronium hexafluorophosphate; Or, the molar ratio of the compound 1 to the coupling agent is 1:2-3; Or, the alkaline substance providing the alkaline environment includes one of N-methylmorpholine and triethylamine; preferably, the molar ratio of the compound 1 to the alkaline substance is 1:2-3; Alternatively, the stirring reaction time is 15 to 20 hours, preferably 16 hours.

5. The preparation method according to claim 2, characterized in that: The method for deprotecting the intermediate 1 to obtain the cyclopeptide derivative comprises: reacting the intermediate 1 with an acid, stirring the reaction to obtain the cyclopeptide derivative; Preferably, the acid comprises acetic acid; Preferably, the stirring reaction time is 6 to 10 hours, preferably 8 hours.

6. The preparation method according to claim 2, characterized in that: Compound 1 was synthesized by solid phase synthesis.

7. The preparation method according to claim 6, characterized in that: Using CTC resin as a solid phase synthesis carrier, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, Fmoc-L-Pro-OH and Fmoc-D-Ser(tBu)-OH were condensed from C-terminus to N-terminus in sequence. After cleavage and concentration to dryness, the fully protected linear crude peptide NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH was obtained. The fully protected linear crude peptide was then cyclized, deprotected, purified and lyophilized to obtain compound 1.

8. Use of the cyclic peptide derivative according to claim 1 and / or the cyclic peptide derivative prepared by the preparation method according to any one of claims 2 to 7 in the preparation of drugs and pharmaceutical compositions, health products, foods, food additives or cosmetics with anti-inflammatory function.

9. Use of the cyclic peptide derivative according to claim 1 and / or the cyclic peptide derivative prepared by the preparation method according to any one of claims 2 to 7 in the preparation of drugs and pharmaceutical compositions, health products, foods, food additives or cosmetics with antioxidant function.

10. A product, characterized in that The invention comprises the cyclic peptide derivatives as claimed in claim 1 and / or the cyclic peptide derivatives prepared by the preparation method as claimed in any one of claims 2 to 7; the products comprise medicines, pharmaceutical compositions, foods, food additives or cosmetics.

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