Cyclopeptide compound with anti-inflammatory function and application thereof

Abstract

The invention discloses a cyclic peptide compound with an anti-inflammatory function and application thereof, belongs to the technical field of polypeptide compounds, and particularly relates to H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-CTC resin prepared by adopting a solid-phase synthesis method, namely full-protection peptide resin. Carrying out cutting treatment and cyclization treatment on the full-protection peptide resin to obtain a cyclic peptide compound, wherein the structure of the cyclic peptide compound is Cyclo (Phe-Lys-Tyr-Pro-Phe-); the cyclization treatment further comprises deprotection cutting, and a cutting fluid in the deprotection cutting is formed by mixing TFA, Tis, EDT, PhOH and H2O. The cyclopeptide compound with the anti-inflammatory function and the application of the cyclopeptide compound are low in cytotoxicity, good in safety and capable of inhibiting inflammatory factors and inhibiting gene expression of inflammatory mediators.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptide compounds, and particularly relates to a cyclic peptide compound with anti-inflammatory function and application thereof. BACKGROUND

[0002] In recent years, polypeptide compounds are widely used in the fields of medicine and cosmetics due to their biological activity and targeting. However, traditional polypeptide compounds usually have the following problems: existing polypeptide molecules are usually only directed to a single target or a single biological pathway. Due to the limitation of single function, the effect of traditional polypeptide compounds as cosmetic raw materials is often not ideal when used alone, and the polypeptide compounds need to be used in combination with other active ingredients, which increases the complexity and cost of the formula. SUMMARY

[0003] The application aims to provide a cyclic peptide compound with anti-inflammatory function which has small cytotoxicity, good safety, can inhibit inflammatory factors and can inhibit the gene expression of inflammatory mediators, and application thereof.

[0004] The technical scheme adopted by the application to achieve the above-mentioned purpose is as follows: A fully protected peptide resin, which has the structure of H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-CTC resin.

[0005] The application discloses a fully protected polypeptide, which has the structure of H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH.

[0006] The application discloses a fully protected cyclic peptide, which has the structure of Cyclo (Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-).

[0007] The application discloses a cyclic peptide compound, which has the structure of Cyclo (Phe-Lys-Tyr-Pro-Phe-).

[0008] The application discloses a preparation method of a cyclic peptide compound, which comprises the following steps: a fully protected peptide resin H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-CTC resin is prepared by using a solid-phase synthesis method. The fully protected peptide resin is subjected to cutting treatment and cyclization treatment to obtain the cyclic peptide compound, and the cyclic peptide compound has the structure of Cyclo (Phe-Lys-Tyr-Pro-Phe-).

[0009] Preferably, in the preparation of the fully protected peptide resin, the first amino acid coupling resin is synthesized first, and Fmoc-Phe-OH is coupled to the CTC resin.

[0010] Preferably, the activated amino acid reagent used in the preparation of the fully protected peptide resin comprises an amino acid reagent, HOBT and DIC, and the amino acid reagent comprises Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH or Fmoc-Phe-OH.

[0011] The present application discloses a method for preparing a fully protected peptide resin, comprising: synthesizing a first amino acid coupled resin, activating an amino acid reagent and synthesizing a fully protected peptide resin.

[0012] Preferably, in the synthesis of the first amino acid coupled resin, Fmoc-Phe-OH is coupled to the CTC resin. The coupling is performed by solid phase synthesis. The CTC resin is swelled in DCM and then coupled with Fmoc-Phe-OH in a DIEA-DCM solution.

[0013] Preferably, in the synthesis of the first amino acid coupled resin, Fmoc-Phe-OH is coupled to the CTC resin in a DCM solution containing DIEA.

[0014] Preferably, in the synthesis of the first amino acid coupled resin, the CTC resin is swelled in DCM at 20-40℃ for 5-30 min under a nitrogen atmosphere, the DCM is removed by suction filtration, Fmoc-Phe-OH is added, then a DIEA-DCM solution is added at 10-20℃, and the reaction is carried out at 20-30℃ for 2-5 h. After the reaction is completed, methanol is added to cap the end for 10-60 min, and then the product, Fmoc-Phe-CTC resin, is obtained by suction filtration and washing.

[0015] More preferably, in the synthesis of the first amino acid coupled resin, the molar amount of Fmoc-Phe-OH used is 100-300% of the reactive sites on the CTC resin.

[0016] More preferably, in the synthesis of the first amino acid coupled resin, the DIEA-DCM solution is prepared by mixing DIEA and DCM, the molar volume ratio of DIEA to DCM in the DIEA-DCM solution is 0.1-1 mol:0.1-1 L, and the amount of the DIEA-DCM solution is measured by the amount of DIEA, and the molar amount of DIEA used is 200-300% of the molar amount of Fmoc-Phe-OH used.

[0017] More preferably, in the synthesis of the first amino acid coupled resin, the capping is performed by using an appropriate amount of methanol. DMF is used for washing.

[0018] Preferably, in the activation of the amino acid reagent, the amino acid reagent and HOBT are added to DMF, DIC is added at 0-10℃, and the activation is carried out for 3-20 min to obtain the activated amino acid reagent.

[0019] More preferably, in the activation of the amino acid reagent, the molar volume ratio of the amino acid reagent to DMF is 0.1-0.5 mol:0.1-0.5 L.

[0020] More preferably, in the activation of the amino acid reagent, the molar amount of HOBT used is 50-200% of the molar amount of the amino acid reagent used.

[0021] More preferably, in the activation of the amino acid reagent, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used. In the activation of the amino acid reagent, the amino acid reagent includes Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, or Fmoc-Phe-OH.

[0022] Preferably, in the synthesis of the fully protected peptide resin, the first amino acid coupled resin is mixed with a deprotection solution for deprotection treatment, liquid is removed by suction filtration, an activated amino acid reagent is added, and the reaction is carried out under a nitrogen atmosphere for 10-60 min. After the reaction is completed, suction filtration is performed, and then washing is performed. Then the above-mentioned coupling of the activated amino acid reagent is repeated, and after the coupling is completed, washing and drying are performed, and finally H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., the fully protected peptide resin, is obtained.

[0023] More preferably, in the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagent is: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH.

[0024] More preferably, in the synthesis of the fully protected peptide resin, in the deprotection treatment, the resin to which the amino acid reagent is coupled is first immersed in a 20% Pip / DMF solution, and deprotection treatment is carried out at 20-30°C for 10-60 min.

[0025] More preferably, in the synthesis of the fully protected peptide resin, when the coupling of the activated amino acid reagent is repeated, deprotection treatment is carried out, and after the coupling is completed, suction filtration and washing are performed. Washing is performed using DMF. In the washing after the coupling of the last amino acid reagent, methyl tert-butyl ether, tetrahydrofuran, and methyl tert-butyl ether are used in sequence.

[0026] More preferably, in the synthesis of the fully protected peptide resin, the molar amount of the amino acid reagent used in the activated amino acid reagent is 100-300% of the reaction site on the CTC resin.

[0027] Preferably, in the cleavage treatment, a cleavage solution is used, and the cleavage solution is a mixture of TFA and DCM; or, the cleavage solution is a mixture of HFIP and DCM.

[0028] The present application discloses a method for preparing a fully-protected polypeptide, comprising: subjecting a fully-protected peptide resin to a full-protection cleavage treatment to obtain a fully-protected polypeptide.

[0029] Preferably, the full-protection cleavage treatment uses a full-protection cleavage solution, and the full-protection cleavage solution comprises a TFA-containing DCM solution or a HFIP-containing DCM solution.

[0030] Preferably, in the preparation of the fully-protected polypeptide, the fully-protected peptide resin is mixed with the cleavage solution, and the mixture is treated at 20-40°C for 10-60 min, the resin is removed by filtration, petroleum ether is added to the filtrate for sedimentation, the supernatant is removed by centrifugation, the petroleum ether is washed by centrifugation, and the fully-protected polypeptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH is obtained by vacuum drying.

[0031] More preferably, in the preparation of the fully-protected polypeptide, the cleavage solution is prepared by mixing TFA and DCM, and the content of TFA in the cleavage solution is 0.1-5 vol %.

[0032] More preferably, in the preparation of the fully-protected polypeptide, the cleavage solution is used to immerse the fully-protected peptide resin, and the sedimentation and washing of petroleum ether are appropriately used.

[0033] Preferably, in the preparation of the fully-protected polypeptide, the fully-protected peptide resin is mixed with the cleavage solution, and the mixture is treated at 20-40°C for 10-60 min, the resin is removed by filtration, and the fully-protected polypeptide H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH is obtained by concentrating and rotary-evaporating the filtrate to dryness.

[0034] More preferably, in the preparation of the fully-protected polypeptide, the cleavage solution is prepared by mixing HFIP and DCM, and the content of HFIP in the cleavage solution is 20-40 vol %.

[0035] More preferably, in the preparation of the fully-protected polypeptide, the cleavage solution is used to immerse the fully-protected peptide resin.

[0036] Preferably, the cyclization treatment comprises a full-protection cyclization treatment, and the full-protection cyclization treatment uses HATU and DIEA for cyclization, or the full-protection cyclization treatment uses HOOBT, NMM and DIC for cyclization.

[0037] Preferably, the cyclization treatment further comprises a deprotection cleavage, and the cleavage solution used in the deprotection cleavage is prepared by mixing TFA, Tis, EDT, PhOH and H2O.

[0038] The present application discloses a method for preparing a fully-protected polypeptide, comprising: subjecting a fully-protected peptide resin to a full-protection cleavage treatment to obtain a fully-protected polypeptide.

[0039] Preferably, the full-protected polypeptide is subjected to full-protected cyclization in the full-protected cyclization process by using a HATU-DIEA solution; or the full-protected cyclization in the full-protected cyclization process is performed by using a DMF solution containing HOBT, DIC and NMM.

[0040] Preferably, in the preparation of the full-protected cyclic peptide, the full-protected polypeptide is mixed with DMF to obtain a full-protected polypeptide DMF solution; HATU, DIEA and DMF are mixed to obtain a HATU-DIEA solution, the temperature of the HATU-DIEA solution is controlled at 20-30°C, then the full-protected polypeptide DMF solution is added dropwise into the HATU-DIEA solution, and the reaction is performed for 20-120 min, the reaction is monitored by HPLC, after the reaction is completed, water and ethyl acetate are added for extraction, the water phase is extracted again with ethyl acetate, the organic phases are combined, then the combined organic phases are sequentially washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the full-protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-).

[0041] More preferably, in the preparation of the full-protected cyclic peptide, the full-protected polypeptide in the full-protected polypeptide DMF solution is mixed with DMF at a mass-volume ratio of 1-10 g: 50-200 mL.

[0042] More preferably, in the preparation of the full-protected cyclic peptide, HATU, DIEA and DMF in the HATU-DIEA solution are mixed at a mass-volume ratio of 5-20 g: 5-20 mL: 20-40 mL.

[0043] More preferably, in the preparation of the full-protected cyclic peptide, the amount of the full-protected polypeptide DMF solution is measured based on the full-protected polypeptide, and the amount of HATU in the HATU-DIEA solution is measured based on HATU, and the amount of HATU used is 100-200 wt% of the full-protected polypeptide.

[0044] Preferably, in the preparation of the full-protected cyclic peptide, the full-protected polypeptide is mixed with DMF to obtain a full-protected polypeptide DMF solution; then HOBT is added, the temperature of the reaction solution is controlled at 0-10°C, NMM and DIC are added, then the reaction is stirred at 20-40°C for 8-24 h, the reaction is monitored by HPLC, after the reaction is completed, water is added to precipitate the solid, the solid is dissolved in ethyl acetate, then the solution is sequentially washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the full-protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-).

[0045] More preferably, in the preparation of the full-protected cyclic peptide, the full-protected polypeptide in the full-protected polypeptide DMF solution is mixed with DMF at a mass-volume ratio of 1-10 g: 1-10 L.

[0046] More preferably, in the preparation of the full-protected cyclic peptide, the amount of HOOBT used is 10-30wt% of the full-protected polypeptide.

[0047] More preferably, in the preparation of the full-protected cyclic peptide, the amount of NMM used is 5-20wt% of the full-protected polypeptide.

[0048] More preferably, in the preparation of the full-protected cyclic peptide, the amount of DIC used is 5-20wt% of the full-protected polypeptide.

[0049] More preferably, in the preparation of the full-protected cyclic peptide, the amount of water added when the solid is precipitated is appropriate, the amount of ethyl acetate used for dissolving the solid is appropriate, and the amount of saturated sodium bicarbonate solution, water and saturated sodium chloride solution used for washing is appropriate.

[0050] Preferably, in the preparation of the cyclic peptide compound, the full-protected cyclic peptide is mixed with the cleavage solution for 1-4h, concentrated, precipitated with ice ethyl acetate, washed, centrifuged, spin-dried, and the cyclic peptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) is obtained.

[0051] More preferably, in the preparation of the cyclic peptide compound, the cleavage solution is a mixture of TFA, Tis, EDT, PhOH and H2O, and the volume ratio of TFA, Tis, EDT, PhOH and H2O in the cleavage solution is 80-90:2-10:1-5:1-5:1-5.

[0052] More preferably, in the preparation of the cyclic peptide compound, the amount of full-protected cyclic peptide and cleavage solution used is 1-10g:20-100mL.

[0053] More preferably, in the preparation of the cyclic peptide compound, the amount of ice ethyl acetate used for precipitation is appropriate.

[0054] The application discloses the application of the above-mentioned cyclic peptide compound in the preparation of anti-inflammatory products and / or cosmetics and / or drugs.

[0055] This invention utilizes a solid-phase synthesis method to prepare H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., a fully protected peptide resin. The fully protected peptide resin is then cleaved and cyclized to obtain a cyclic peptide compound with the structure Cyclo(Phe-Lys-Tyr-Pro-Phe-). The cyclization process also includes deprotection cleavage, where the cleavage solution is a mixture of TFA, Tis, EDT, PhOH, and H2O. Therefore, this invention offers the following advantages: Cyclo(Phe-Lys-Tyr-Pro-Phe-) exhibits low cytotoxicity and good safety. Furthermore, Cyclo(Phe-Lys-Tyr-Pro-Phe-) can inhibit inflammatory factors and the gene expression of inflammatory mediators. Thus, this invention provides a cyclic peptide compound with low cytotoxicity, good safety, and the ability to inhibit inflammatory factors and the gene expression of inflammatory mediators, along with its applications. Attached Figure Description

[0056] Figure 1 This is an HPLC chromatogram.

[0057] Figure 2 This is a mass spectrum.

[0058] Figure 3 This is a graph showing the results of the cytotoxicity test.

[0059] Figure 4 This is a graph showing TNF-α levels under LPS-induced inflammatory conditions.

[0060] Figure 5 This is a graph showing IL-6 levels under LPS-induced inflammatory conditions.

[0061] Figure 6 This is a graph showing IL-1β levels under LPS-induced inflammatory conditions.

[0062] Figure 7 This is a graph showing the expression levels of COX-2 and iNOD genes under LPS-induced inflammatory conditions. Detailed Implementation

[0063] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0064] The concepts involved in the present application will be described below in conjunction with the accompanying drawings. It should be noted that the following description of the various concepts is merely intended to make the content of the present application easier to understand, and does not represent a limitation on the scope of protection of the present application; meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0065] Example 1: A method for preparing a fully-protected peptide resin Synthesis of the first amino acid coupling resin: under a nitrogen atmosphere, the CTC resin was added into DCM and treated with stirring at 25°C for 10 min, and then filtered to remove the DCM. Fmoc-Phe-OH was added, followed by the addition of a DIEA-DCM solution at 10°C. The reaction was carried out at 25°C for 2.5 h. After the reaction was completed, methanol was added for capping for 30 min. The mixture was filtered and washed to obtain the Fmoc-Phe-CTC resin, i.e., the first amino acid coupling resin. The molar amount of Fmoc-Phe-OH used was 200% of the reaction sites on the CTC resin. The DIEA-DCM solution was prepared by mixing DIEA and DCM. The molar volume ratio of DIEA to DCM in the DIEA-DCM solution was 0.5 mol:0.5 L. The amount of the DIEA-DCM solution was measured based on the amount of DIEA. The molar amount of DIEA used was 250% of the molar amount of Fmoc-Phe-OH used. The amount of methanol used for capping was appropriate. DMF was used for washing.

[0066] Activation of the amino acid reagent: the amino acid reagent and HOBT were added into DMF, and DIC was added at 2°C. The mixture was activated for 3 min to obtain the activated amino acid reagent. The molar volume ratio of the amino acid reagent to DMF was 0.24 mol:0.25 L. The molar amount of HOBT used was 100% of the molar amount of the amino acid reagent used. The molar amount of DIC used was 100% of the molar amount of the amino acid reagent used. In the activation of the amino acid reagent, the amino acid reagent included Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Phe-OH.

[0067] Synthesis of the fully protected peptide resin: the first amino acid coupled resin was mixed with the deprotection solution for deprotection, the liquid was removed by suction filtration, the activated amino acid reagent was added, and the reaction was carried out under a nitrogen atmosphere for 40 min. After the reaction was completed, the resin was suction filtered and washed. Then the above-mentioned coupling of the activated amino acid reagent was repeated, and after the coupling was completed, the resin was washed and dried to obtain the H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-CTC resin, i.e. the fully protected peptide resin. The coupling order of the activated amino acid reagent was: Fmoc-Pro-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Phe-OH. In the deprotection process, the resin coupled with the amino acid reagent was first immersed in a 20% Pip / DMF solution for deprotection at 25°C for 30 min. When repeating the coupling of the activated amino acid reagent, the resin was deprotected, and after the coupling was completed, the resin was suction filtered and washed with DMF. In the washing after the coupling of the last amino acid reagent, methyl tert-butyl ether, tetrahydrofuran and methyl tert-butyl ether were used in sequence. The molar amount of the amino acid reagent used in the activated amino acid reagent was 240% of the reaction sites on the CTC resin.

[0068] Example 2: A method for preparing a fully protected polypeptide Preparation of the fully protected polypeptide: the fully protected peptide resin was mixed with the cleavage solution, and the mixture was treated at 30°C for 30 min. The resin was removed by filtration, and petroleum ether was added to the filtrate for sedimentation. The supernatant was removed by centrifugation, and the residue was washed with petroleum ether and dried under vacuum to obtain the fully protected polypeptide H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH. The cleavage solution was prepared by mixing TFA and DCM, and the content of TFA in the cleavage solution was 1 vol %. The cleavage solution was used to immerse the fully protected peptide resin, and the petroleum ether was used in an appropriate amount for sedimentation and washing. The fully protected peptide resin was obtained from Example 1. The yield of the fully protected polypeptide H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH prepared in this example was 80.4%, and the purity was 83.8%.

[0069] Example 3: A method for preparing a fully protected polypeptide Preparation of the fully protected polypeptide: the fully protected peptide resin was mixed with the cleavage solution, and the mixture was treated at 30°C for 30 min. The resin was removed by filtration, and petroleum ether was added to the filtrate for sedimentation. The supernatant was removed by centrifugation, and the residue was washed with petroleum ether and dried under vacuum to obtain the fully protected polypeptide H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH. The cleavage solution was prepared by mixing TFA and DCM, and the content of TFA in the cleavage solution was 1 vol %. The cleavage solution was used to immerse the fully protected peptide resin, and the petroleum ether was used in an appropriate amount for sedimentation and washing. The fully protected peptide resin was obtained from Example 1. The yield of the fully protected polypeptide H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH prepared in this example was 80.4%, and the purity was 83.8%.

[0069] Example 3: A method for preparing a fully protected polypeptide Preparation of the fully protected polypeptide: the fully protected peptide resin was mixed with the cleavage solution, and the mixture was treated at 30°C for 30 min. The resin was removed by filtration, and petroleum ether was added to the filtrate for sedimentation. The supernatant was removed by centrifugation, and the residue was washed with petroleum ether and dried under vacuum to obtain the fully protected polypeptide H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH. The cleavage solution was prepared by mixing TFA and DCM, and the content of TFA in the cleavage solution was 1 vol %. The cleavage solution was used to immerse the fully protected peptide resin, and the petroleum ether was used in an appropriate amount for sedimentation and washing. The fully protected peptide resin was obtained from Example 1. The yield of the fully protected polypeptide H-Phe-Lys (Boc)-Tyr (tBu)-Pro-Phe-OH prepared in this example was 80.4%, and the purity was 83.8%.

[0070] Example 4: A method for preparing a fully protected cyclic peptide Preparation of a fully protected cyclic peptide: a fully protected polypeptide was mixed with DMF to obtain a fully protected polypeptide DMF solution; HATU, DIEA and DMF were mixed to obtain a HATU-DIEA solution, the temperature of the HATU-DIEA solution was controlled at 25°C, then the fully protected polypeptide DMF solution was added dropwise into the HATU-DIEA solution, and reacted for 60 min, the reaction was monitored by HPLC, after the reaction was completed, water and ethyl acetate were added for extraction, the water phase was extracted again with ethyl acetate, the organic phases were combined, then washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-). The fully protected polypeptide and DMF in the fully protected polypeptide DMF solution were mixed at a mass-volume ratio of 10 g: 100 mL. HATU, DIEA and DMF in the HATU-DIEA solution were mixed at a mass-volume ratio of 11.8 g: 10.2 mL: 30 mL. The amount of the fully protected polypeptide DMF solution was measured based on the amount of the fully protected polypeptide therein, and the amount of the HATU-DIEA solution was measured based on the amount of HATU therein, and the amount of HATU used was 118 wt% of the fully protected polypeptide. The fully protected polypeptide was from Example 2. The yield of the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-) prepared in this example was 69.4%, and the purity was 73.6%.

[0071] Example 5: A method for preparing a fully protected cyclic peptide Preparation of the fully protected cyclic peptide: the fully protected polypeptide was mixed with DMF to obtain a fully protected polypeptide DMF solution; then HOOBT was added, and the temperature of the reaction solution was controlled at 0°C, NMM and DIC were added, and then the reaction was stirred at 25°C for 16h, the reaction was monitored by HPLC, after the reaction was completed, water was added to precipitate the solid, the solid was dissolved in ethyl acetate, and then sequentially washed with saturated sodium bicarbonate solution, water and saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-). The fully protected polypeptide DMF solution was mixed with DMF at a mass-volume ratio of 10g:10L. The amount of HOOBT used was 17wt% of the fully protected polypeptide. The amount of NMM used was 11wt% of the fully protected polypeptide. The amount of DIC used was 13wt% of the fully protected polypeptide. The amount of water added when the solid was precipitated was appropriate, the amount of ethyl acetate used for dissolving the solid was appropriate, and the amounts of saturated sodium bicarbonate solution, water and saturated sodium chloride solution used for washing were all appropriate. The fully protected polypeptide was from Example 2. The yield of the fully protected cyclic peptide Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-) prepared in this example was 87.9%, and the purity was 84.8%.

[0072] Example 6: a method for preparing a cyclic peptide compound Preparation of the cyclic peptide compound: the fully protected cyclic peptide was mixed with a cleavage solution for 1-4h, concentrated, precipitated with ice ethyl acetate, washed, centrifuged, and rotary evaporated to obtain the cyclic peptide compound Cyclo(Phe-Lys-Tyr-Pro-Phe-). The cleavage solution was a mixture of TFA, Tis, EDT, PhOH and H2O, and the volume ratio of TFA, Tis, EDT, PhOH and H2O in the cleavage solution was 87.5:5:2.5:2.5:2.5. The amount of the fully protected cyclic peptide used was 5g, and the amount of the cleavage solution used was 40mL. The amount of ice ethyl acetate used for precipitation was appropriate. The fully protected cyclic peptide was from Example 5. The yield of the cyclic peptide compound Cyclo(Phe-Lys-Tyr-Pro-Phe-) prepared in this example was 78.5%, and the purity was 83.8%.

[0073] The cyclic peptide compound Cyclo(Phe-Lys-Tyr-Pro-Phe-) of Example 6 in the present application was further purified by HPLC using a reversed-phase C18 chromatographic column to obtain the purified cyclic peptide compound Cyclo(Phe-Lys-Tyr-Pro-Phe-), and the purity was 99.%, the HPLC chart is shown in Figure 1 , and the mass spectrum chart is shown in Figure 2The cyclopeptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) in the present application can also be named as cyclopeptide PR2928. The cyclopeptide compound Cyclo (Phe-Lys-Tyr-Pro-Phe-) and the cyclopeptide PR2928 in the present application all refer to the same product.

[0074] The structure of Cyclo (Phe-Lys-Tyr-Pro-Phe-) is shown as follows: .

[0075] Test Example: 1. Cytotoxicity test of cyclopeptide compound The present application tests the potential toxicity of the prepared cyclopeptide PR2928 on cells to determine the safe concentration range of the cyclopeptide PR2928.

[0076] In the present application, fibroblast HFF-1 is inoculated into a 96-well plate, 200 μL of culture medium is added to each well, about 10000 cells / well, and the cell density is ensured to be uniform. The cells are cultured overnight at 37℃ in a culture box with 5% CO2 to allow the cells to adhere and grow. After 24 hours, the cell culture medium is aspirated, 200 μL of DMEM culture medium containing different concentrations of cyclopeptide PR2928 is added, and the 96-well plate is returned to the culture box for continued culture. After 24 hours, the absorbance value is measured at 490 nm wavelength by MTT method using a microplate reader. A group of blank control groups without cells is designed by adding only culture medium. The cell viability is calculated as follows: cell viability = (absorbance of experimental group - absorbance of blank group) / (absorbance of control group - absorbance of blank group) x 100%, to determine the cytotoxicity of cyclopeptide PR2928 on fibroblasts and its safe concentration range. The concentration of cyclopeptide PR2928 is 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM.

[0077] In the present application, macrophage RAW264.7 is also used for testing, and the testing steps are the same as those of HFF-1 described above. The corresponding HFF-1 is replaced by RAW264.7, which is used to determine the cytotoxicity of cyclopeptide PR2928 on macrophages and its safe concentration range.

[0078] The test results of cytotoxicity in the present application are shown as follows: Figure 3 The cell viability of HFF-1 and RAW264.7 treated with different concentrations of cyclopeptide PR2928 does not show a significant decrease in cell viability. The cell viability still remains at 95% relative to the control group at a concentration of 500 μM, which confirms that cyclopeptide PR2928 has very low cytotoxicity.

[0079] 2. Detection of the effect of cyclopeptide PR2928 on the secretion ability of inflammatory factors In the inflammatory response, cytokines (such as TNF-a, IL-6 and IL-1β) are important regulatory molecules. They interact through a complex network, together involved in the regulation of immune response, tissue repair and pathological damage. TNF-a is mainly responsible for activating immune cells, IL-6 promotes the persistence of immune response, while IL-1β is produced by inflammasome activation and is closely related to tissue damage. Under external stimulation, their overexpression can lead to tissue damage and organ dysfunction. Macrophages produce inflammatory stress under lipopolysaccharide (LPS) stimulation, in which case the present application tests the ability of cyclic peptide PR2928 to inhibit the production of inflammatory factors by macrophages under LPS stimulation.

[0080] The present application inoculates the macrophage cell line RAW264.7 into a 6-well plate at 50000 cells / well and cultures for 24 hours. Treatment groups, negative control groups and blank control groups are set up, and the groups are dosed. The treatment group is added with DMEM medium containing cyclic peptide PR2928, and the concentration of cyclic peptide PR2928 is 10 μM. The negative control group is not added with cyclic peptide PR2928, and the blank control group is not added with cyclic peptide PR2928. After 2 hours of dosing, 200 μL of LPS working solution is added to each well of the remaining groups except the blank control group, and the groups are placed in the incubator for continued culture for 22 hours. The cell culture supernatant is collected, and TNF-a, IL-6 and IL-1β are detected and analyzed according to the operating instructions of the ELISA kit.

[0081] The t-test method is used for statistical analysis of comparison between groups. P<0.05 is considered to have a significant difference (marked * in the figure), and P<0.01 is considered to have a very significant difference (marked ** in the figure).

[0082] According to the formula: inhibition rate%=100%- (negative control group-treatment group) / (control group-blank control) x 100%, the inhibition rate is normalized and calculated.

[0083] The detection results of TNF-a are shown in Table 1. Figure 4 As shown in Table 1, compared with the blank control group, the content of TNF-a in the LPS-stimulated negative control group increased significantly, indicating that the test stimulation condition can increase the content of TNF-a. Figure 5 The detection results of IL-6 are shown in Table 2. Figure 6As shown, compared with the blank control group, the IL-1β content of the LPS stimulation negative control group increased significantly, indicating that the test stimulation condition can increase the content of IL-1β. Compared with the negative control group, the inhibition rates of TNF-α, IL-6 and IL-1β after LPS stimulation and addition of 10 μM cyclic peptide PR2928 were 58.2%, 37.9% and 31.9% respectively. It is proved that cyclic peptide PR2928 shows significant inhibition of inflammatory factor production after treatment.

[0084] 3. Effect of cyclic peptide PR2928 on inflammatory mediator gene expression COX-2 (cyclooxygenase-2) and iNOS (inducible nitric oxide synthase) are two extremely important and representative "enzyme" inflammatory mediators, both of which are inducible enzymes, and normal cells do not express in the resting state, but can be strongly induced by LPS. They are not directly cytokines, but are responsible for catalyzing the production of inflammatory factors. COX-2 catalyzes the synthesis of prostaglandins, which can cause local vascular permeability to increase, leading to tissue edema and reducing the pain threshold, causing hyperalgesia. iNOS catalyzes the production of a large amount of NO, which combines with superoxide anions to form more toxic peroxynitroso, leading to protein nitration, DNA damage and cell death, thereby exacerbating tissue damage.

[0085] The present application tests the mRNA expression levels of COX-2 and iNOS by qPCR.

[0086] The present application inoculates the macrophage cell line RAW264.7 into a 6-well plate at 50000 cells / well, and cultures for 24 hours. Set up the treatment group and the control group, and administer the drugs in groups. The treatment group is added with DMEM medium containing cyclic peptide PR2928, and the concentration of cyclic peptide PR2928 is 10 μM. The control group is treated with DMEM without polypeptide. At the same time of administration, 200 μL of LPS working solution is added to each well, and placed in the incubator for continuous culture for 6 hours. The cells are washed with PBS twice, and the RNA in each well is extracted by TRIZOL method. GAPDH is used as an internal reference for gene expression, and the mRNA expression levels of COX-2 and iNOS are evaluated. The calculation formula is: ; ; .

[0087] The results are statistically analyzed by t-test method. Compared with the control group, the significance is represented by *, P value < 0.05, which has significant difference, and P value < 0.01, which has extremely significant difference.

[0088] The gene expression inhibition rate was normalized and calculated according to the formula: inhibition rate% = (control group - treatment group) / control group x 100%.

[0089] The test results are shown in Table 2. Figure 7 As shown in Table 2, compared with the macrophages treated with LPS alone in the control group, the COX-2 gene expression level did not change significantly after the addition of cyclic peptide PR2928, and the iNOS gene expression level was significantly reduced, with an inhibition rate of 57.84%.

[0090] In summary, the cyclic peptide PR2928 in the method has low cytotoxicity, good safety, can effectively inhibit the expression of key pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in LPS-induced macrophages, and the cyclic peptide PR2928 can effectively inhibit the gene expression of inflammatory mediators such as iNOS in LPS-induced macrophages. It is proved that the cyclic peptide PR2928 of the present application not only significantly inhibits the expression of various pro-inflammatory factors, but also inhibits the expression of inflammatory mediator iNOS, and has good anti-inflammatory ability.

[0091] The above-described embodiments and / or implementations are merely used to illustrate the preferred embodiments and / or implementations of the present application and do not limit the embodiments of the present application in any form. Any person skilled in the art can make some changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the present application, and such changes or modifications should be considered as substantially the same technology or embodiments as the present application.

[0092] The principles and implementations of the present application are described using specific examples. The above example is only used to help understand the method and core idea of the present application. The above description is only the preferred embodiments of the present application. It should be pointed out that due to the limited nature of the language expression, there are infinite specific structures. For ordinary skilled persons in the art, without departing from the principles of the present application, some improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner. These improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be considered as the protection scope of the present application.

Claims

1. A fully protected peptide resin with the structure: H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin.

2. A fully protected polypeptide with the structure: H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-OH.

3. A fully protected cyclic peptide with the structure: Cyclo(Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-).

4. A cyclic peptide compound with the structure: Cyclo(Phe-Lys-Tyr-Pro-Phe-).

5. A method for preparing a cyclic peptide compound, comprising: H-Phe-Lys(Boc)-Tyr(tBu)-Pro-Phe-CTC resin, i.e., fully protected peptide resin, was prepared by solid-phase synthesis. The fully protected peptide resin was cleaved and cyclized to obtain a cyclic peptide compound with the structure: Cyclo(Phe-Lys-Tyr-Pro-Phe-).

6. The method for preparing a cyclic peptide compound according to claim 5, characterized in that, In the preparation of the fully protected peptide resin, the first amino acid coupling resin is synthesized first, and Fmoc-Phe-OH is coupled to CTC resin.

7. The method for preparing a cyclic peptide compound according to claim 5, characterized in that, The preparation of the fully protected peptide resin uses an activated amino acid reagent, which includes an amino acid reagent, HOBT and DIC. The amino acid reagent includes Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH or Fmoc-Phe-OH.

8. The method for preparing a cyclic peptide compound according to claim 5, characterized in that, The cutting process uses a cutting fluid, which is a mixture of TFA and DCM; or, the cutting fluid is a mixture of HFIP and DCM.

9. The method for preparing a cyclic peptide compound according to claim 5, characterized in that, The ringing process includes a fully protected ringing process, in which HATU and DIEA are used for ringing; or, in which HOOBT, NMM and DIC are used for ringing.

10. The use of the cyclic peptide compound of claim 4 in the preparation of anti-inflammatory products and / or cosmetics and / or pharmaceuticals.

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