A cyclic peptide derivative, a preparation method and application thereof

Abstract

The present application relates to the technical field of biological materials, in particular to a cyclic peptide derivative, a preparation method and application thereof.The cyclic peptide derivative provided by the present application simultaneously comprises a hydrophobic cyclic peptide structure and a hydrophilic pantothenic acid structure, which are combined by a covalent ester bond; this structure endows the cyclic peptide derivative with excellent stability, effectively improving the problems of instability and irritation of pantothenic acid; after entering the skin, the derivative can be hydrolyzed into pantothenic acid under the action of esterase, and can play a role without undergoing pantoic acid oxidation conversion.Secondly, the cyclic peptide derivative in the present application has good biological safety; in an inflammatory cell model, the derivative can more effectively reduce the expression of inflammatory factors, and antioxidant activity research shows that it has strong free radical inhibition effect, and can play superior anti-inflammatory and antioxidant effects.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a cyclic peptide derivative, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

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

[0004] Pantothenic acid plays a vital role in cosmetics. Firstly, it has moisturizing properties, enhancing the skin's ability to retain moisture and leaving it hydrated. Adding pantothenic acid to moisturizing lotions can improve the product's hydrating effect, keeping the skin soft and smooth for a long time. Secondly, it can repair the skin barrier, promote a healthy stratum corneum, strengthen barrier function, and reduce damage from external stimuli. For those with damaged skin barriers, skincare products containing pantothenic acid can help repair them and improve sensitivity. Thirdly, it has soothing and anti-allergic effects, reducing skin inflammation and discomfort. In cosmetics for sensitive skin, it can lower the incidence of allergies and make the skin more stable. Fourthly, it can promote cell regeneration, stimulating cell metabolism and regeneration, making the skin more vibrant and radiant. 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 these active substances have certain drawbacks that affect their application. Currently, 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" reports its extraction method, but the yield is extremely low, approximately 0.0007%, and the process is cumbersome. Pantothenic acid is unstable under certain conditions and is easily inactivated by various factors, such as temperature, pH, light, and oxidation. Currently, panthenol is more commonly used on the market than pantothenic acid, as it needs to be converted into pantothenic acid after entering the skin to exert its biological functions. Summary of the Invention

[0006] To overcome the above problems, the present invention provides cyclic peptide derivatives, their preparation methods, and applications.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a cyclic peptide derivative having the structural formula shown in formula (I):

[0009]

[0010] A 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] The structural formula of compound 1 is shown in 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 of coupling compound 1 and compound 2 to obtain intermediate 1 includes: dispersing compound 1 and compound 2 in a solvent in an alkaline environment, stirring until dissolved, adding a coupling agent, and stirring the reaction to obtain intermediate 1.

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

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

[0021] Preferably, the coupling agent comprises one of 1-propyl cyclic phosphate anhydride and O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU).

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

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

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

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

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

[0027] Preferably, the acid includes acetic acid.

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

[0029] In one or more embodiments, compound 1 is synthesized using a solid-phase synthesis method.

[0030] Preferably, CTC resin is used as the solid-phase synthesis support, 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 sequentially from the C-terminus to the N-terminus. 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 is obtained. The fully protected linear crude peptide is then cyclized, deprotected, purified and lyophilized to obtain compound 1.

[0031] A 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 pharmaceuticals and pharmaceutical compositions having anti-inflammatory functions, foods, food additives, or cosmetics.

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

[0033] A fourth 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 pharmaceuticals and pharmaceutical compositions having antioxidant functions, foods, food additives, or cosmetics.

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

[0035] A fifth aspect of the present invention provides an anti-inflammatory product comprising 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; the anti-inflammatory product includes 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] A sixth aspect of the present invention provides an antioxidant product comprising 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; the antioxidant product includes antioxidant drugs, antioxidant drug compositions, antioxidant foods, antioxidant food additives, or antioxidant cosmetics.

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

[0039] Unless otherwise specified, the anti-inflammatory or antioxidant products described in this invention can be taken orally, applied topically, or injected. Those skilled in the art can select appropriate excipients according to conventional methods to prepare the anti-inflammatory or antioxidant products of this invention into suitable dosage forms or usage formats. Such dosage forms include injections, ointments, powder injections, liniments, dressings, liquid preparations, etc. When used as cosmetics, the anti-inflammatory or antioxidant products may include, but are not limited to, toners, lotions, serums, gels, foundations, creams, and masks, etc. The applications of these cosmetics include, but are not limited to, facial cleansing and care, body cleansing and care, and scalp cleansing and care, etc. The cyclic peptide derivatives described in this invention can be used to formulate cosmetics with soothing, anti-inflammatory, antioxidant, and skin-repairing functions.

[0040] The beneficial effects of this invention are as follows:

[0041] (1) The cyclic peptide derivative provided by this invention contains both a hydrophobic cyclic peptide structure and a hydrophilic pantothenic acid structure, which are linked by a covalent ester bond. This structure endows it with excellent stability and effectively improves the problem of instability and irritation caused by pantothenic acid. After entering the skin, the derivative can be hydrolyzed into pantothenic acid under the action of esterase, and can exert its effect without undergoing panthenol oxidation conversion. Secondly, the cyclic peptide derivative in this invention has good biocompatibility. In the inflammatory cell model, the derivative can more effectively reduce the expression of inflammatory factors. Antioxidant activity studies show that it has a strong free radical inhibition effect and can exert superior anti-inflammatory and antioxidant effects.

[0042] (2) The present invention also provides a method for synthesizing cyclic peptide derivatives. The process has high synthesis efficiency, the yield of compound 1 is 60% to 66%, and the operation is simple and suitable for industrial production. Attached Figure Description

[0043] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0044] Figure 1 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 RAW 264.7 cell viability after treatment with cyclic peptide derivatives prepared in Example 6 at different concentrations;

[0047] Figure 4 The amount of NO produced in RAW264.7 cells induced by LPS after intervention in experimental groups 1–4;

[0048] Figure 5 The expression level of IL-6 in LPS-stimulated macrophages after intervention in experimental groups 1–4;

[0049] Figure 6 The expression level of TNF-α in macrophages stimulated by LPS after intervention in experimental groups 1–4. Detailed Implementation

[0050] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

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

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this application are readily available through conventional means, and unless otherwise specified, they are used in accordance with conventional methods or product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this application. The preferred embodiments and materials described herein are for illustrative purposes only. Unless otherwise specified, all percentages mentioned in this invention are by weight.

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

[0054]

[0055]

[0056] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0057] Example 1

[0058] Preparation of compound 1 derived from Wenzhou mandarin oranges:

[0059] (1) Swelling of resin: Take 3.16g (5mmol) of CTC resin (substitution degree of 1.58mmol / g) and add 40mL of dichloromethane to swell the resin for 1.5h.

[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 and added to the resin (1 equivalent). The reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the resin was washed three times with 20 mL of dimethylformamide and the solvent was dried 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 removed 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 minutes.

[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 condensation, 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 sequentially. After each amino acid was condensed, 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 process 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) Peptide resin cleavage

[0065] Add 50 mL of lysis 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) Fully protected linear peptide cyclization and purification

[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 mixture was concentrated to 200 mL by rotary evaporation. The solution was washed twice with saturated sodium bicarbonate aqueous solution. Anhydrous magnesium sulfate was added to the reaction mixture to remove water for 3 hours. After filtration, the mixture was evaporated to dryness using a rotary evaporator. 120 mL of 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-cold MTBE, centrifuged, washed, and dried to obtain a white solid.

[0068] The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 preparative column (50 × 250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / water solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and lyophilization, compound 1 was obtained as 2.2 g, with a yield of 60.3% and a purity of 97.2%. The mass spectrum is shown below. Figure 1 As shown.

[0069] Example 2

[0070] Preparation of compound 1 derived from Wenzhou mandarin oranges:

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

[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 and added to the resin (1 equivalent). The reaction was carried out at 25 °C for 2 hours. After the reaction was completed, the resin was washed three times with 200 mL of dimethylformamide and the solvent was dried 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 removed 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 minutes.

[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 condensation, the Fmoc protecting group was removed, and then Fmoc-L-Leu-OH, Fmoc-L-Leu-OH, Fmoc-L-Pro-OH, and Fmoc-L-Pro-OH were condensed sequentially. After each amino acid was condensed, 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 process 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) Peptide resin cleavage

[0077] Add 50 mL of lysis 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) Fully protected linear peptide cyclization and purification

[0079] The fully protected linear crude peptide and DIC (75 mmol) / HOBt (75 mmol) were added to 6 L of dichloromethane and reacted at 25 °C for 6 hours. The reaction was monitored by TLC until complete. The solution was evaporated to 2000 mL using a rotary evaporator, washed twice with saturated sodium bicarbonate solution, and dehydrated by adding anhydrous magnesium sulfate for 3 hours. After filtration, the solution was evaporated to dryness using a rotary evaporator. 1200 mL of 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-cold MTBE, centrifuged, washed, and dried to obtain a white solid.

[0080] The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) 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. After concentration and lyophilization, 123 g of the compound was obtained, with a yield of 63.0% and a purity of 96.8%.

[0081] Example 3

[0082] Preparation of compound 1 derived from Wenzhou mandarin oranges:

[0083] (1) Swelling of the resin: 63.3 g (100 mmol) of CTC resin (substitution degree of 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 and added to the resin (1 equivalent). The reaction was carried out at 25 °C for 3 hours. After the reaction was completed, the resin was washed three times with 300 mL of dimethylformamide and the solvent was dried 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 removed 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 minutes.

[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 condensation, 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 sequentially. After each amino acid was condensed, 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 process 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) Peptide resin cleavage

[0089] 1.0 L of lysis reagent (trifluoroethanol: dichloromethane = 1:4) was added to the peptide resin, and the reaction was carried out at 25 °C for 5 hours. The mixture was then filtered, and the filtrate was concentrated 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) Fully protected linear peptide cyclization and purification

[0091] The fully protected linear crude peptide and DIC (10 mmol) / HOBt (10 mmol) were added to 12 L of dichloromethane and reacted at 25 °C for 6 hours. The reaction was monitored by TLC until complete. The solution was evaporated to 4000 mL using a rotary evaporator, washed twice with saturated sodium bicarbonate solution, and dehydrated by adding anhydrous magnesium sulfate for 3 hours. After filtration, the solution was evaporated to dryness using a rotary evaporator. 2.4 L of 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-cold MTBE, centrifuged, washed, and dried to obtain a white solid.

[0092] The crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 preparative column (50 × 250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / water solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and lyophilization, 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] Add 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 to a 100 mL reaction flask. Stir until dissolved, cool to 0–10 °C, and add 1.9 g of 1-propylphosphonic anhydride (6.0 mmol, 2.2 equivalents relative to compound 1) dropwise while maintaining the temperature below 20 °C. Raise the temperature to 25 °C and react at this temperature for 16 hours. The reaction is then detected by TLC to indicate completion.

[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 detected by TLC to indicate completion. The crude peptide was concentrated to dryness and then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 preparative column (50 × 250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / water solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and lyophilization, 2.2 g of the cyclic peptide derivative was obtained, with a yield of 72.3% and a purity of 97.3%. The mass spectrum is shown below. Figure 2 As shown.

[0097] Example 5

[0098] Preparation of cyclic peptide derivatives:

[0099] Add 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 to a 100 mL reaction flask. Stir until dissolved, cool to 0–10 °C, and add 2.3 g of O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, 6.0 mmol, 2.2 equivalents relative to Compound 1) in portions while maintaining the temperature below 20 °C. Raise the temperature to 25 °C and react at this temperature for 16 hours. The reaction is complete as detected by TLC.

[0100] 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 detected by TLC to indicate completion. The crude peptide was concentrated to dryness and then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 preparative column (50 × 250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / water solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and lyophilization, 1.7 g of the cyclic peptide 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] Add 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 to a 2 L reaction flask. Stir until dissolved, cool to 0–10 °C, and add 38 g of 1-propylphosphonic anhydride (120.0 mmol, 2.1 equivalents relative to compound 1) dropwise while maintaining the temperature below 20 °C. Raise the temperature to 25 °C and react at this temperature for 16 hours. The reaction is complete as detected by TLC.

[0104] 400 mL of purified water and 400 mL of acetic acid were added to the reaction system. After stirring at 25 °C for 8 hours, the reaction was detected by TLC to indicate completion. The crude peptide was concentrated to dryness and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 preparative column (50 × 250 mm, 10 μm). Mobile phase A was 0.1% trifluoroacetic acid / water solution, and mobile phase B was 0.1% trifluoroacetic acid / acetonitrile solution. After concentration and lyophilization, 44.7 g of the cyclic peptide derivative was obtained, with a yield of 73.5% and a purity of 98.1%.

[0105] Example 7

[0106] This experiment tested the cytotoxicity, anti-inflammatory and soothing effects, 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 hepatic adenocarcinoma cells), and JEG-3 (human placental choriocarcinoma cells) were selected as experimental cell lines and obtained from the Manassas Institute of Model Cultures (Manassas, VA, USA). After resuscitation, RAW264.7 cells were seeded in cell culture dishes and placed in high-glucose DMEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics. The cells were then transferred to a 37°C incubator containing 5% CO2 and cultured overnight. Cell growth was observed the following day, and the medium was changed or the cells were passaged.

[0109] Cytotoxicity evaluation: The relative cytotoxicity of the drug was determined using the MTT assay. Cells in DMEM medium (supplemented with 10% FBS) were seeded into 96-well plates at a density of 5000 cells per well. Cells were incubated for 24 hours at a constant temperature of 37°C with different concentrations (0–256 μM) of the cyclic peptide derivative solution prepared in Example 6. At the end of incubation, 20 μL of MTT solution (5 μg / mL) was added to each well. The cells were then incubated at 37°C for another 4 hours, followed by centrifugation to remove the supernatant. 200 μL of dimethyl sulfoxide (DMSO) was added to each well in the dark to dissolve the formamide crystals. The absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage of the untreated control.

[0110] The biosafety of the cyclic peptide derivative prepared in Example 6 to RAW264.7 cells was assessed using the MTT assay. Figure 3 As shown, the cyclic peptide derivative had no effect on cytotoxicity at concentrations below 256 μM. Even at a concentration of 256 μmol / L, its cell viability reached over 95%, demonstrating good biocompatibility.

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

[0112] Experimental principle: Lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages were used as an in vitro inflammation model. The expression levels of inflammatory factors NO, IL-6, and TNF-α released from these macrophages were detected. The anti-inflammatory and soothing activity of the samples was evaluated by testing the inhibition rate of the test substances on the expression of inflammatory factors. Experimental groups were set up as follows: Group 1 (100 μM cyclic peptide derivative), Group 2 (100 μM compound 1), Group 3 (100 μM pantothenic acid), and Group 4 (50 μM compound 1 + 50 μM pantothenic acid).

[0113] NO emission measurement

[0114] RAW264.7 cells in the logarithmic growth phase (5 × 10⁻⁶ cells) were selected. 5 Experiments were cultured in 96-well plates (samples / well) for 24 hours, and then pretreated with 100 μL of each of experimental groups 1, 2, 3, and 4 for 2 hours, followed by treatment with LPS (1 μg / mL) for 24 hours. A blank control was provided, and dexamethasone (100 nM) was used as a positive control. The NO release was measured using Griess reagent (Sigma-Aldrich, St.) in the culture medium. Griess reagent was mixed with DMEM medium at a 1:1 ratio and incubated at room temperature for 15 minutes. Finally, the absorbance at 540 nm was measured using an ELISA reader.

[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 stimulation involved high levels of NO and led to the induction of pro-inflammatory mediators. Figure 4 As shown, experimental group 1 significantly reduced NO production in LPS-stimulated RAW264.7 cells compared to other control groups, exhibiting an anti-inflammatory effect similar to that of the positive control group (dexamethasone). The cyclic peptide derivative showed superior NO-reduction effects compared to compound 1, pantothenic acid, and the mixture of compound 1 and pantothenic acid.

[0116] Evaluation of Inflammatory Factor Inhibition Rate: Lipopolysaccharide (LPS)-stimulated mouse RAW264.7 macrophages were used as an in vitro inflammation model. The anti-inflammatory activity of the samples was evaluated by measuring the inhibition rate of the test substances on serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) using the sample intervention process. First, RAW264.7 cells in logarithmic growth phase were selected and incubated at 37℃ and 5% CO2 for 24 hours. 100 μL of the sample solutions from experimental groups 1–4 were added to each well, with a blank control group included. Incubation continued for another 24 hours. The expression levels of IL-6 and TNF-α were measured using the Mouse IL-6Valukine™ ELISA kit and the Mouse TNF-alphaValukine™ ELISA kit, respectively.

[0117] By establishing an in vitro LPS-stimulated mouse macrophage model, and intervening in the stimulation process with added samples, we observed whether the samples could inhibit the secretion of inflammatory factors IL-6 and TNF-α, thereby exhibiting anti-inflammatory effects. Figure 5 and 6 As shown, compared with the blank control group and other parallel experimental groups, experimental group 1 showed better inhibition of the secretion of inflammatory factors IL-6 and TNF-α than other experimental groups.

[0118] The experiments on NO release and inflammatory factor inhibition rate fully demonstrate that the anti-inflammatory effect of the cyclic peptide derivative prepared in this invention is not produced by the simple mixing of compound 1 and pantothenic acid (experimental group 4), but is due to its structure.

[0119] 7.3 Evaluation of antioxidant activity:

[0120] This invention uses the NBT (nitroblue tetrazolium) photochemical reduction method to determine the antioxidant properties of different samples. A 50 μM xanthine solution (2 mL) was prepared in a test tube, and an appropriate amount of xanthine oxidase was added to form a stock solution. 0.01 g NBT, 0.35 g methionine, and 100 mmol / L EDTA were weighed and added to a PBS buffer solution (pH 7.8) to a final volume of 200 mL to prepare an NBT solution. 3 mL of the NBT solution was added to the stock solution. 1 mL of each of the experimental groups 1–4 was then taken and irradiated with two 50W fluorescent lamps at 37°C for 20 min, after which the reaction was stopped and the test tubes were placed in darkness. Measurements were performed using a 1 cm thick cuvette at a constant temperature of 25 ± 0.2°C, and the I50 value (i.e., the concentration at which NBT reduction inhibition reaches 50%) was compared.

[0121] Table 1. Evaluation of the efficacy in scavenging reactive oxygen species (ROS)

[0122]

[0123]

[0124] NBT is reduced by CO2 to a blue-violet substance, which has a maximum absorption wavelength in the 530–580 nm range. The NBT reduction reaction may be inhibited when the test solution has antioxidant properties. (I) 50 The smaller the amount of the product, the greater its antioxidant activity. Analysis of the data in Table 1 shows that the I-value of experimental group 1... 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 in this invention is not produced by the simple mixture of compound 1 and pantothenic acid, but is due to its structure.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

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

2. A process for the preparation of a cyclic peptide derivative according to claim 1, characterized in that, The method comprises the following steps: The intermediate 1 is obtained by coupling compound 1 and compound 2, and the ring peptide derivative is obtained by deprotecting the intermediate 1; The structural formula of compound 1 is shown in formula (IV): Formula (IV); The structural formula of compound 2 is shown in formula (III): Formula (III); The structural formula of the intermediate 1 is shown in formula (II): Formula (II).

3. The production method according to claim 2, wherein The method for obtaining the intermediate 1 by coupling compound 1 and compound 2 comprises the following steps: dispersing compound 1 and compound 2 in a solvent in an alkaline environment, stirring until the solution is clear, adding a coupling agent, and stirring to react to obtain the intermediate 1.

4. The production method according to claim 3, wherein The solvent comprises N,N-dimethylformamide; Alternatively, the molar ratio of the compound 1 and the compound 2 is 1:2-3; Alternatively, the coupling agent comprises one of 1-propylphosphonic anhydride and O-benzotriazol-tetramethylurea hexafluorophosphate; Alternatively, the molar ratio of the compound 1 and the coupling agent is 1:2-3; Alternatively, the alkaline substance for providing the alkaline environment comprises one of N-methylmorpholine and triethylamine; Alternatively, the stirring reaction time is 15-20 h.

5. The production method according to claim 4, wherein The stirring reaction time is 16 h.

6. The production method according to claim 2, wherein The method for obtaining the ring peptide derivative by deprotecting the intermediate 1 comprises the following steps: reacting an acid with the intermediate 1, and stirring to react to obtain the ring peptide derivative; The acid comprises acetic acid; The stirring reaction time is 6-10 h.

7. The production method according to claim 6, wherein The stirring reaction time is 8 h.

8. The production method according to claim 2, wherein Compound 1 is synthesized by using a solid-phase synthesis method.

9. The production method according to claim 8, wherein The CTC resin is used 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 are sequentially condensed from the C terminal to the N terminal, and after cleavage and concentration to dryness, a fully-protected linear crude peptide NH2-D-Ser(tBu)-L-Pro-L-Pro-L-Leu-L-Leu-D-Tyr(tBu)-Gly-OH is obtained, and after cyclization, deprotection, purification and freeze-drying, compound 1 is obtained.

10. The use of the ring peptide derivative of claim 1 or the ring peptide derivative prepared by the method of any one of claims 2-9 in the preparation of a drug and a pharmaceutical composition, a health product, a food and a food additive or a cosmetic product with anti-inflammatory function.

11. The use of the ring peptide derivative of claim 1 or the ring peptide derivative prepared by the method of any one of claims 2-9 in the preparation of a drug and a pharmaceutical composition, a health product, a food and a food additive or a cosmetic product with antioxidant function.

12. A product characterized by, The product comprises the ring peptide derivative of claim 1 or the ring peptide derivative prepared by the method of any one of claims 2-9; and the product comprises a drug, a pharmaceutical composition, a food, a food additive or a cosmetic product.

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