A class of cyclic peptide compounds and their preparation method and application

Abstract

The present invention discloses a class of cyclic peptide compounds and their preparation method and application, belong to the field of polypeptide synthesis and application, and specifically relate to the preparation of fully protected peptide resins using solid phase synthesis; fully protected peptide resins are cut and monocyclized to obtain monocyclic peptides; monocyclic peptides are subjected to binary cyclization to obtain cyclic peptide compounds Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bridged Cys & Cys), which are cyclized under the action of iodine methanol and ascorbic acid. The cyclic peptide compounds prepared by the present invention have low toxicity, good stability, can improve type I collagen expression, good antioxidant effect, and good anti-inflammatory effect. Therefore, the present invention is a kind of effect with low toxicity, good stability, can improve type I collagen expression, good antioxidant effect, and good anti-inflammatory effect.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide synthesis and application, and specifically relates to a class of cyclic peptide compounds and a preparation method and application thereof. Background Art

[0002] The oligopeptide GHK (tripeptide-1, glycyl-L-histidyl-L-lysine) is a natural tripeptide first isolated from human plasma by Pickart et al. in 1973. During extracellular matrix degradation, GHK is cleaved and released by the secreted protein acidic and rich in cysteine ​​(SPARC) protein, which is secreted by stromal cells. This protein helps with tissue remodeling by regulating angiogenesis. Studies have found that the average blood level of GHK in 20-year-olds is 200 ng / ml, but it drops to an average of 80 ng / ml by age 60, showing a clear downward regulation with aging.

[0003] During wound healing, GHK stimulates the proliferation and migration of fibroblasts, promotes the synthesis of collagen and elastin fibers, increases skin toughness and elasticity, and accelerates wound repair and regeneration. GHK also possesses antioxidant properties: it scavenges free radicals, reduces oxidative stress-induced cell damage, protects cell integrity and function, and delays skin aging. GHK also exhibits anti-inflammatory effects: it regulates inflammatory responses, inhibits the release of inflammatory factors, and reduces tissue damage caused by inflammation. It has a therapeutic effect on inflammatory skin diseases such as acne and eczema.

[0004] Based on these functions, GHK possesses certain anti-wrinkle and repairing properties as a cosmetic: it can activate the synthesis of the extracellular matrix, increase collagen and elastin content, reduce wrinkles, and make the skin firmer and smoother. For damaged skin, such as sunburn and sensitive skin, GHK has a good repairing effect, relieving discomfort, promoting the skin's self-repair ability, and enhancing the skin's barrier function. However, GHK has a reported half-life of only 30 minutes under physiological conditions, and its high degradation rate limits its continued effectiveness. Summary of the Invention

[0005] Based on the above understanding, the present invention hopes to construct a class of compounds with anti-wrinkle and repair effects and a long half-life through the rational design of polypeptide structure and modification of chemical bonds.

[0006] The purpose of the present invention is to provide a class of cyclic peptide compounds with low toxicity, good stability, the ability to enhance type I collagen expression, good antioxidant effect, and good anti-inflammatory effect, as well as a preparation method and application thereof.

[0007] Peptide cyclization and side-chain stapling are important tools in the development of peptide drugs. The cyclic structure reduces the exposure of terminal groups and the possibility of unwanted chemical reactions, thereby improving overall chemical stability. It also reduces the recognition and degradation of terminal amino acids by some proteases and enhances resistance to enzymatic degradation. Due to this increased stability and resistance to enzymatic degradation, peptides that have undergone cyclization and side-chain stapling can maintain effective concentrations in the body for longer periods, reducing the frequency of dosing.

[0008] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0009] A method for preparing a fully protected peptide resin comprises:

[0010] After mixing Fmoc-Gly-OH, DIEA, and DCM, add CTC resin for coupling to obtain the first amino acid coupling resin; the molar amount of Fmoc-Gly-OH used is 100-300% of the reaction sites on the CTC resin, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used;

[0011] The first amino acid coupling resin is deprotected, and then the activated amino acid reagents are coupled in sequence according to the peptide sequence to obtain a fully protected peptide resin. The structure of the fully protected peptide resin is: H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0012] The present invention discloses a method for preparing a fully protected peptide resin, comprising the following steps:

[0013] S1, pretreatment of CTC resin: CTC resin was mixed with DCM, and nitrogen was passed through at 20-40° C. for 5-30 min, and the liquid was removed to obtain the pretreated CTC resin.

[0014] S2, preparation of H-Gly-CTC resin: Fmoc-Gly-OH and DCM were mixed with the pretreated CTC resin, and DIEA was added at 10-20°C under a nitrogen atmosphere, followed by reaction at 25-30°C for 1-5 hours. After the reaction, methanol was added for end-capping for 10-60 minutes. After end-capping, the liquid was removed and the resin was washed with DMF to obtain Fmoc-Gly-CTC resin; deprotection solution was then added for deprotection treatment, washed with DMF, and dried to obtain H-Gly-CTC resin.

[0015] S3, preparation of H-Cys(Trt)-Gly-CTC resin: Fmoc-Cys(Trt)-OH, HOBT and DMF were mixed and dissolved, DIC was added at 0-5°C, and activation was carried out for 1-5 minutes to obtain an activation solution, the activation solution was mixed with the H-Gly-CTC resin in S2, and the mixture was reacted at 25-30°C under a nitrogen atmosphere for 20-60 minutes. After the reaction was completed, the liquid was removed by filtration and washed with DMF to obtain Fmoc-Cys(Trt)-Gly-CTC resin; then a deprotection solution was added for deprotection treatment, washed with DMF, and dried to obtain H-Cys(Trt)-Gly-CTC resin.

[0016] S4, preparation of H-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Lys(Boc)-OH, HOBT and DMF are mixed and dissolved, DIC is added at 0-5°C, and activation is carried out for 1-5 minutes to obtain an activation solution, the activation solution is mixed with the H-Cys(Trt)-Gly-CTC resin in S3, and the mixture is reacted at 25-30°C under a nitrogen atmosphere for 20-60 minutes. After the reaction is completed, the liquid is removed by filtration and washed with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then a deprotection solution is added for deprotection treatment, washed with DMF, and dried to obtain H-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0017] S5, preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-His(Trt)-OH, HOBT and DMF are mixed and dissolved, DIC is added at 0-5°C, and activation is carried out for 1-5 minutes to obtain an activation solution, the activation solution is mixed with the H-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S4, and the mixture is reacted at 25-30°C under a nitrogen atmosphere for 20-60 minutes. After the reaction is completed, the liquid is filtered off and the mixture is washed with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then a deprotection solution is added for deprotection treatment, the mixture is washed with DMF, and the mixture is dried to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0018] S6, preparation of H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Gly-OH, HOBT and DMF were mixed and dissolved, DIC was added at 0-5°C, and activation was carried out for 1-5 minutes to obtain an activation solution, the activation solution was mixed with the H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S5, and the mixture was reacted at 25-30°C under a nitrogen atmosphere for 20-60 minutes. After the reaction was completed, the liquid was filtered off and washed with DMF to obtain Fmoc-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then a deprotection solution was added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0019] S7, preparation of H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Cys(Trt)-OH, HOBT and DMF were mixed and dissolved, and DIC was added at 0-5°C for 1-5 minutes to obtain an activation solution, which was mixed with the H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S6 and stirred under nitrogen atmosphere. The mixture was reacted at 25-30° C. for 20-60 min. After the reaction was completed, the liquid was removed by filtration and washed with DMF to obtain Fmoc-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. A deprotection solution was then added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0020] S8, preparation of H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Lys(Boc)-OH, HOBT and DMF were mixed and dissolved, and DIC was added at 0-5°C and activated for 1-5 minutes to obtain an activation solution. The activation solution was mixed with the H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S7 and the mixture was stirred under nitrogen atmosphere. The mixture was reacted at 25-30° C. for 20-60 min. After the reaction was completed, the liquid was removed by filtration and washed with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. A deprotection solution was then added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin.

[0021] S9, preparation of crude H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-His(Trt)-OH, HOBT and DMF were mixed and dissolved, and DIC was added at 0-5°C for 1-5 minutes to obtain an activation solution. The activation solution was mixed with the H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S8 and the mixture was stirred under nitrogen atmosphere. The mixture was reacted at 25-30° C. for 20-60 min. After the reaction was completed, the liquid was removed by filtration and washed with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. A deprotection solution was then added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain a crude H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin product.

[0022] S10, Preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: The crude H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin is washed sequentially with tertiary methyl ether, tetrahydrofuran, and tertiary methyl ether, and dried under vacuum to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin, i.e., the fully protected peptide resin. Tertiary methyl ether and tetrahydrofuran are used in appropriate amounts during the washing process.

[0023] Preferably, in S1, the amount of CTC resin and DCM used is 1 g: 5-10 mL. The active sites of the CTC resin are 1-1.6 mmol / g.

[0024] Preferably, in S2, the molar amount of Fmoc-Gly-OH is 100-300% of the active sites on the CTC resin; the amount of Fmoc-Gly-OH and DCM is 1 mmol:1-5 mL; the molar amount of DIEA is 100-500% of the molar amount of Fmoc-Gly-OH; and the amount of Fmoc-Gly-OH and methanol is 1 mmol:0.1-0.8 mL. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0025] Preferably, in S3, the molar amount of Fmoc-Cys(Trt)-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-Cys(Trt)-OH, the amount of Fmoc-Cys(Trt)-OH and DMF is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-Cys(Trt)-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0026] Preferably, in S4, the molar amount of Fmoc-Lys(Boc)-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH, the amount of Fmoc-Lys(Boc)-OH and DMF is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0027] Preferably, in S5, the molar amount of Fmoc-His(Trt)-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-His(Trt)-OH, the amount of Fmoc-His(Trt)-OH and DMF is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-His(Trt)-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0028] Preferably, in S6, the molar amount of Fmoc-Gly-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-Gly-OH, the amount of Fmoc-Gly-OH and DMF used is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-Gly-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0029] Preferably, in S7, the molar amount of Fmoc-Cys(Trt)-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-Cys(Trt)-OH, the amount of Fmoc-Cys(Trt)-OH and DMF is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-Cys(Trt)-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0030] Preferably, in S8, the molar amount of Fmoc-Lys(Boc)-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH, the amount of Fmoc-Lys(Boc)-OH and DMF is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-Lys(Boc)-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0031] Preferably, in S9, the molar amount of Fmoc-His(Trt)-OH is 100-150% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT is 80-120% of the molar amount of Fmoc-His(Trt)-OH, the amount of Fmoc-His(Trt)-OH and DMF is 1 mmol:0.5-1.5 mL, and the molar amount of DIC is 80-120% of the molar amount of Fmoc-His(Trt)-OH. The deprotection solution is a DMF solution containing 10-30 vol% Pip, and the amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0032] The invention discloses a preparation method for a fully protected polypeptide. The fully protected peptide resin is mixed with a cutting liquid, reacted at 20-40° C. for 20-60 minutes, filtered, and the filtrate is precipitated with petroleum ether to precipitate a solid, which is vacuum dried to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, i.e., the fully protected polypeptide.

[0033] Preferably, the cutting solution is a DCM solution containing TFA, wherein the cutting solution contains 1-3 vol% TFA, and the cutting solution is used to submerge the fully protected peptide resin. Petroleum ether is used in an appropriate amount.

[0034] The invention discloses a preparation method of a fully protected polypeptide. The fully protected peptide resin is mixed with a cutting liquid, reacted at 20-40° C. for 20-60 minutes, filtered, and the filtrate is concentrated and rotary evaporated to dryness to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, i.e., the fully protected polypeptide.

[0035] Preferably, the cutting solution is a DCM solution containing HFIP, wherein the cutting solution contains 20-40 vol% HFIP, and the cutting solution submerges the fully protected peptide resin during use.

[0036] The invention discloses a preparation method for a fully protected cyclic peptide. The fully protected peptide is mixed with DMF to obtain a fully protected peptide liquid; HATU, DIEA and DMF are mixed to obtain a cyclization liquid; the fully protected peptide liquid is added to the cyclization liquid at 20-40 DEG C for reaction for 0.5-3 hours, and the reaction is completed after HPLC monitoring; water and ethyl acetate are added to the reaction liquid for extraction; the reaction liquid is washed with a saturated sodium bicarbonate aqueous solution, water and a saturated sodium chloride aqueous solution in sequence, dried with anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), i.e., the fully protected cyclic peptide.

[0037] Preferably, the amount of fully protected polypeptide to DMF in the fully protected polypeptide solution is 1 g:5-15 mL; the amount of HATU to DMF in the cyclization solution is 1 g:3-12 mL; and the volume ratio of DIEA to DMF in the cyclization solution is 1:5-10. In the mixture of the fully protected polypeptide solution and the cyclization solution, the amount of HATU is 40-60 wt % of the fully protected polypeptide. In the water-ethyl acetate separation extraction, water and ethyl acetate are used in a volume ratio of 1:0.5-2. For washing, saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution are used in appropriate amounts, and anhydrous sodium sulfate is used in appropriate amounts.

[0038] The invention discloses a preparation method for a fully protected cyclic peptide. The fully protected peptide is mixed with DMF to obtain a fully protected peptide liquid; HOOBT is added to dissolve the mixture, and then NMM and DIC are added at 0-5 DEG C. The mixture is reacted at 20-40 DEG C for 8-24 hours. The reaction is monitored by HPLC to ensure that the reaction is complete. Water is added to the reaction liquid to precipitate a solid, which is dissolved in ethyl acetate and then washed in sequence with a saturated sodium bicarbonate aqueous solution, water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and subjected to rotary evaporation to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), i.e., the fully protected cyclic peptide.

[0039] Preferably, the amount of fully protected polypeptide and DMF used in the fully protected polypeptide solution is 1 g: 0.5-1.5 L, the amount of HOOBT used is 3-12 wt % of the fully protected polypeptide, the amount of NMM used is 2-10 wt % of the fully protected polypeptide, and the amount of DIC used is 3-9 wt % of the fully protected polypeptide. Water is used in an appropriate amount for precipitating the solid. Ethyl acetate is used in an appropriate amount for dissolving the solid. Saturated aqueous sodium bicarbonate solution, water, and saturated aqueous sodium chloride solution are used in appropriate amounts for washing. Anhydrous sodium sulfate is used in an appropriate amount.

[0040] The invention discloses H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin prepared by the method.

[0041] The invention discloses a monocyclic peptide having the structure of Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

[0042] The invention discloses a method for preparing a monocyclic peptide, comprising: the method for preparing the above-mentioned fully protected peptide resin.

[0043] Preferably, the fully protected peptide resin is cleaved and cyclized to obtain a monocyclic peptide, and the structure of the monocyclic peptide is: Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

[0044] Preferably, in the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with the deprotection cleavage solution and cleaved for 1-4 hours. After the cleavage is completed, it is precipitated with ice ether, washed, centrifuged, and rotary evaporated to dryness to obtain a crude monocyclic peptide. The crude monocyclic peptide is purified by reverse phase C18 liquid chromatography and lyophilized to obtain Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-), that is, a monocyclic peptide.

[0045] More preferably, in the preparation of a single cyclic peptide, the deprotection cleavage solution is a mixture of TFA, TIS, EDT, PhOH, and water. The volume ratio of TFA, TIS, EDT, PhOH, and water in the deprotection cleavage solution is 1:0.01-0.1:0.01-0.05:0.01-0.05:0.01-0.05. The amount of fully protected cyclic peptide and deprotection cleavage solution used is 1 g:5-15 mL. Glacial ether is used in an appropriate amount during precipitation. Glacial ether is used in an appropriate amount for washing.

[0046] The single-ring peptide in the present invention is numbered PR2924B, and its structural formula is: The single-ring peptide exhibits the functions of inducing extracellular matrix production, anti-inflammatory and antioxidant effects. In cell function tests, the compound has excellent efficacy.

[0047] The invention discloses a cyclic peptide compound having the structure of Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bond-bridging Cys and Cys).

[0048] The present invention discloses a method for preparing a cyclic peptide compound, comprising: the method for preparing the above-mentioned single-cyclic peptide.

[0049] Preferably, the monocyclic peptide is subjected to a dicyclization treatment to obtain a cyclic peptide compound, and the dicyclization is carried out under the action of iodine methanol and ascorbic acid.

[0050] Preferably, in the preparation of the cyclic peptide compound, a monocyclic peptide is mixed and dissolved with an acetic acid solution, an iodine-methanol solution is added at 20-40° C., and the reaction solution is stirred until the reaction solution turns yellow and does not fade, and the reaction is judged to be complete. Then, under stirring, an ascorbic acid solution is added for reduction, and the ascorbic acid solution is added until the reaction solution changes from yellow to milky white. After stirring for 5-30 minutes, the color does not change. A sample is taken and tested by HPLC, and then the reaction solution is filtered through a filter membrane to obtain a crude cyclic peptide compound. The crude cyclic peptide compound is purified by reverse phase C18 liquid chromatography and lyophilized to obtain Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bond bridged Cys & Cys), that is, a cyclic peptide compound.

[0051] More preferably, in the preparation of the cyclic peptide compound, acetic acid and water are mixed in the acetic acid solution at a volume ratio of 1:20-30, the amount of the monocyclic peptide to the acetic acid solution is 1 g:100-300 mL, the concentration of the iodine-methanol solution is 0.05-0.5 mol / L, the amount of the monocyclic peptide to the iodine-methanol solution is 1 g:0.5-5 mL, and the ascorbic acid content in the ascorbic acid solution is 0.05-5 wt %. The pore size of the filter membrane is 0.45 μm.

[0052] The cyclic peptide compound disclosed in this application is numbered PR2924, and its structural formula is: The cyclic peptide compound exhibited the functions of inducing extracellular matrix production, anti-inflammatory and antioxidant effects, and had excellent efficacy in cell function tests.

[0053] The present invention discloses the use of the above-mentioned single cyclic peptide in preparing cyclic peptide compounds and / or products for improving type I collagen expression and / or antioxidant products and / or anti-inflammatory products.

[0054] The present invention discloses use of the above-mentioned cyclic peptide compound in preparing products for improving type I collagen expression and / or antioxidant products and / or anti-inflammatory products.

[0055] The present invention adopts solid-phase synthesis to prepare H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin, i.e., peptide resin; the peptide resin is cut and monocyclized to obtain a monocyclic peptide, and the monocyclic peptide structure is: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-); the monocyclic peptide is subjected to a binary cyclization treatment to obtain a cyclic peptide compound Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-, with disulfide bond-bridged Cys and Cys), and the binary cyclization is carried out under the action of iodine methanol and ascorbic acid, so that the present invention has the following beneficial effects: low toxicity, good stability, ability to enhance type I collagen expression, good antioxidant effect, and good anti-inflammatory effect. Therefore, the present invention provides a cyclic peptide compound with low toxicity, good stability, the ability to enhance type I collagen expression, good antioxidant effect, and good anti-inflammatory effect, as well as a preparation method and application thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is the liquid chromatogram of PR2924B.

[0057] Figure 2 This is the mass spectrum of PR2924B.

[0058] Figure 3 This is the liquid chromatogram of PR2924.

[0059] Figure 4 This is the mass spectrum of PR2924.

[0060] Figure 5 Figure 2 is the result of cytotoxicity test.

[0061] Figure 6 This is the result of serum stability test.

[0062] Figure 7 This is a graph showing the results of the COL1A expression test.

[0063] Figure 8 This is the SOD expression level test result diagram.

[0064] Figure 9 This is a graph showing the GSH level test results.

[0065] Figure 10 Figure 2 is a graph of TNF-α expression levels.

[0066] Figure 11 Graph showing IL-6 expression levels.

[0067] Figure 12 Graph showing IL-8 expression levels. DETAILED DESCRIPTION

[0068] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0069] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0070] Example 1: Preparation of a fully protected peptide resin

[0071] S1. Pretreatment of CTC resin: Mix CTC resin with DCM and purge with nitrogen at 25°C for 10 minutes. Remove the liquid to obtain pretreated CTC resin. The amount of CTC resin and DCM used is 1 g: 8.77 mL. The active sites of CTC resin are 40 mmol, and the amount of CTC resin used is 28.5 g.

[0072] S2, Preparation of H-Gly-CTC Resin: Fmoc-Gly-OH and DCM were mixed with pretreated CTC resin. DIEA was added at 10°C under a nitrogen atmosphere, followed by reaction at 25°C for 2.5 h. Methanol was added for end-capping for 30 min. The end-capping solution was removed and the resin was washed with DMF to obtain Fmoc-Gly-CTC resin. Deprotection was then performed with a deprotection solution, followed by washing with DMF and drying to obtain H-Gly-CTC resin. The molar amount of Fmoc-Gly-OH was 200% of the active sites on the CTC resin. The molar amounts of Fmoc-Gly-OH and DCM were 1 mmol:2.5 mL, the molar amount of DIEA was 250% of the molar amount of Fmoc-Gly-OH, and the molar amount of Fmoc-Gly-OH and methanol was 1 mmol:0.38 mL. The deprotection solution was a DMF solution containing 20 vol% Pip. The amount of deprotection solution was sufficient to submerge the resin. An appropriate amount was used for the DMF wash.

[0073] S3, preparation of H-Cys(Trt)-Gly-CTC resin: Fmoc-Cys(Trt)-OH, HOBT and DMF were mixed and dissolved, DIC was added at 0°C, and activation was carried out for 3 minutes to obtain an activation solution, the activation solution was mixed with the H-Gly-CTC resin in S2, and the mixture was reacted at 25°C under a nitrogen atmosphere for 40 minutes. After the reaction was completed, the liquid was filtered off and washed with DMF to obtain Fmoc-Cys(Trt)-Gly-CTC resin; then a deprotection solution was added for deprotection treatment, washed with DMF, and dried to obtain H-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Cys(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Cys(Trt)-OH used, the ratio of Fmoc-Cys(Trt)-OH to DMF is 1 mmol:1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Cys(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. Use an appropriate amount for DMF washing.

[0074] S4, preparation of H-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Lys(Boc)-OH, HOBT and DMF were mixed and dissolved, DIC was added at 0°C, and activation was carried out for 3 minutes to obtain an activation solution, the activation solution was mixed with the H-Cys(Trt)-Gly-CTC resin in S3, and the mixture was reacted at 25°C under a nitrogen atmosphere for 40 minutes. After the reaction was completed, the liquid was filtered off and washed with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then a deprotection solution was added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain H-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Lys(Boc)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used, the ratio of Fmoc-Lys(Boc)-OH to DMF used is 1 mmol:1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. Use an appropriate amount for DMF washes.

[0075] S5, preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-His(Trt)-OH, HOBT and DMF were mixed and dissolved, DIC was added at 0°C, and activation was carried out for 3 minutes to obtain an activation solution, the activation solution was mixed with the H-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S4, and the mixture was reacted at 25°C under a nitrogen atmosphere for 40 minutes. After the reaction was completed, the liquid was filtered off and washed with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then a deprotection solution was added for deprotection treatment, washed with DMF, and dried to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-His(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-His(Trt)-OH used, the ratio of Fmoc-His(Trt)-OH to DMF used is 1 mmol:1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-His(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. Use an appropriate amount for DMF washing.

[0076] S6, preparation of H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Gly-OH, HOBT and DMF were mixed and dissolved, DIC was added at 0°C, and activation was carried out for 3 minutes to obtain an activation solution, which was mixed with the H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S5, and the mixture was reacted at 25°C under a nitrogen atmosphere for 40 minutes. After the reaction was completed, the liquid was filtered off and washed with DMF to obtain Fmoc-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; then a deprotection solution was added for deprotection treatment, and the mixture was washed with DMF and dried to obtain H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Gly-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Gly-OH used, the ratio of Fmoc-Gly-OH to DMF used is 1 mmol:1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Gly-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. Use an appropriate amount for DMF washing.

[0077] S7, preparation of H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Cys(Trt)-OH, HOBT and DMF were mixed and dissolved, and DIC was added at 0°C and activated for 3 minutes to obtain an activation solution. The activation solution was mixed with the H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S6 and the mixture was stirred under nitrogen atmosphere. The reaction was carried out at 25°C for 40 minutes. After the reaction was completed, the liquid was filtered off and washed with DMF to obtain Fmoc-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. Deprotection treatment was then carried out by adding a deprotection solution, washing with DMF, and drying to obtain H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Cys(Trt)-OH was 120% of the molar amount of Fmoc-Gly-OH in S2, the molar amount of HOBT was 100% of the molar amount of Fmoc-Cys(Trt)-OH, the amount of Fmoc-Cys(Trt)-OH and DMF was 1 mmol:1.04 mL, and the molar amount of DIC was 100% of the molar amount of Fmoc-Cys(Trt)-OH. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. An appropriate amount is used during DMF washing.

[0078] S8, preparation of H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-Lys(Boc)-OH, HOBT and DMF were mixed and dissolved, and DIC was added at 0°C and activated for 3 minutes to obtain an activation solution. The activation solution was mixed with the H-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S7 and the mixture was stirred under nitrogen atmosphere. The mixture was reacted at 25°C for 40 min. After the reaction was completed, the liquid was removed by filtration and washed with DMF to obtain Fmoc-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. A deprotection solution was then added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-Lys(Boc)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used, the ratio of Fmoc-Lys(Boc)-OH to DMF used is 1 mmol:1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-Lys(Boc)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. Use an appropriate amount for DMF washes.

[0079] S9, preparation of crude H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: Fmoc-His(Trt)-OH, HOBT and DMF were mixed and dissolved, and DIC was added at 0°C and activated for 3 minutes to obtain an activation solution. The activation solution was mixed with the H-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin in S8 and the mixture was stirred under nitrogen atmosphere. The mixture was reacted at 25°C for 40 min under ambient conditions. After the reaction was completed, the liquid was removed by filtration and washed with DMF to obtain Fmoc-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. A deprotection solution was then added for deprotection treatment, the mixture was washed with DMF, and the mixture was dried to obtain a crude product of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin. The molar amount of Fmoc-His(Trt)-OH used is 120% of the molar amount of Fmoc-Gly-OH used in S2, the molar amount of HOBT used is 100% of the molar amount of Fmoc-His(Trt)-OH used, the ratio of Fmoc-His(Trt)-OH to DMF used is 1 mmol:1.04 mL, and the molar amount of DIC used is 100% of the molar amount of Fmoc-His(Trt)-OH used. The deprotection solution is a DMF solution containing 20 vol% Pip. The amount of deprotection solution used is sufficient to immerse the resin. Use an appropriate amount for DMF washing.

[0080] S10, Preparation of H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin: The crude H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin is washed sequentially with tertiary methyl ether, tetrahydrofuran, and tertiary methyl ether, and dried under vacuum to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin, i.e., the fully protected peptide resin. Tertiary methyl ether and tetrahydrofuran are used in appropriate amounts during the washing process.

[0081] Example 2: A method for preparing a fully protected polypeptide

[0082] Preparation of a fully protected peptide: A fully protected peptide resin was mixed with a cutting solution, reacted at 30°C for 30 min, filtered, and the filtrate was precipitated with petroleum ether to obtain a solid, which was then dried under vacuum to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, the fully protected peptide. The cutting solution was a DCM solution containing TFA, containing 1 vol% TFA, and the cutting solution was used to submerge the fully protected peptide resin. Petroleum ether was used in an appropriate amount. The yield of the fully protected peptide was 107.7%, and the purity was 90.9%. The fully protected peptide resin was obtained from Example 1.

[0083] Example 3: A method for preparing a fully protected polypeptide

[0084] Preparation of a fully protected peptide: A fully protected peptide resin was mixed with a cutting solution, reacted at 30°C for 30 minutes, filtered, and the filtrate concentrated and evaporated to dryness to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, the fully protected peptide. The cutting solution was a DCM solution containing HFIP (33 vol%), which submerged the fully protected peptide resin. The yield of the fully protected peptide was 120.5%, with a purity of 84.7%. The fully protected peptide resin was obtained from Example 1.

[0085] Example 4: Preparation of a fully protected cyclic peptide

[0086] Preparation of fully protected cyclic peptide: a fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide liquid; HATU, DIEA and DMF are mixed to obtain a cyclization liquid; the fully protected polypeptide liquid is added to the cyclization liquid to react for 1 hour at 25°C, and the reaction is completed after HPLC monitoring, water and ethyl acetate are added to the reaction liquid for extraction, and the reaction liquid is washed with a saturated sodium bicarbonate aqueous solution, water and a saturated sodium chloride aqueous solution in sequence, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), i.e., a fully protected cyclic peptide. The fully protected peptide solution was prepared with a ratio of 1 g of fully protected peptide to 10 mL of DMF. The cyclization solution was prepared with a ratio of 1 g of HATU to 6.08 mL of DMF, and a volume ratio of DIEA to DMF of 1:7.03. In the mixture of the fully protected peptide solution and the cyclization solution, the amount of HATU was 49.33 wt % of the fully protected peptide. For the water / ethyl acetate extraction, water and ethyl acetate were used in a 1:1 volume ratio. For the wash, saturated sodium bicarbonate solution, water, and saturated sodium chloride solution were used in appropriate amounts, along with anhydrous sodium sulfate. The yield of the fully protected cyclic peptide was 87.5%, and the purity was 91.4%. The fully protected peptide was obtained from Example 2.

[0087] Example 5: Preparation of a fully protected cyclic peptide

[0088] Preparation of a fully protected cyclic peptide: A fully protected peptide was mixed with DMF to obtain a fully protected peptide solution. HOOBT was added to dissolve the peptide, followed by the addition of NMM and DIC at 0°C. The reaction was allowed to proceed at 25°C for 16 h. The reaction was monitored for completion by HPLC. Water was added to the reaction solution to precipitate a solid, which was dissolved in ethyl acetate and washed sequentially with saturated sodium bicarbonate, water, and saturated sodium chloride. The solid was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), the fully protected cyclic peptide. The ratio of fully protected peptide to DMF in the fully protected peptide solution was 1 g:1 L. The amount of HOOBT, NMM, and DIC was 7.33 wt% of the fully protected peptide, 4.67 wt% of the fully protected peptide, and 6 wt% of the fully protected peptide. Water was used in an appropriate amount for the precipitated solid. Ethyl acetate was used in an appropriate amount for dissolving the solid. For washing, appropriate amounts of saturated sodium bicarbonate aqueous solution, water, and saturated sodium chloride aqueous solution were used. Anhydrous sodium sulfate was also used in appropriate amounts. The yield of the fully protected cyclic peptide was 69.5%, with a purity of 80.3%. The fully protected peptide was obtained from Example 3.

[0089] Example 6: Preparation method of a monocyclic peptide

[0090] Preparation of a monocyclic peptide: A fully protected cyclic peptide was mixed with a deprotection cleavage solution and cleaved for 2 hours. After cleavage, the product was settled with glacial ether, washed, centrifuged, and rotary evaporated to dryness to obtain a crude monocyclic peptide. The crude monocyclic peptide was purified by reverse-phase C18 liquid chromatography and lyophilized to obtain Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-), the monocyclic peptide. The deprotection cleavage solution consisted of a mixture of TFA, TIS, EDT, PhOH, and water in a volume ratio of 1:0.06:0.03:0.03:0.03. The amount of fully protected cyclic peptide to deprotection cleavage solution was 1 g:8 mL. Glacial ether was used in appropriate amounts during settling. Glacial ether was used in appropriate amounts for washing. The monocyclic peptide had a purity of 83.2% and a yield of 99.3%. The fully protected cyclic peptide was obtained from Example 4. The single-cyclic peptide prepared in Example 6 is numbered PR2924B.

[0091] The chromatogram of PR2924B reverse phase C18 chromatography purification in Example 6 of the present invention is as follows Figure 1 shown.

[0092] The mass spectrum of PR2924B obtained in Example 6 of the present invention is as follows Figure 2 shown.

[0093] Example 7: Preparation method of a cyclic peptide compound

[0094] Preparation of cyclic peptide compounds: Dissolve a monocyclic peptide in acetic acid solution, add iodine-methanol solution at 25°C, and stir until the reaction solution turns yellow and does not fade, indicating that the reaction is complete. Then, add ascorbic acid solution for reduction while stirring, and add ascorbic acid solution until the reaction solution turns from yellow back to milky white. If the color does not change after stirring for 10 minutes, sample the reaction solution for HPLC analysis, and then filter the reaction solution through a filter membrane to obtain a crude cyclic peptide compound. The crude cyclic peptide compound is purified by reverse-phase C18 liquid chromatography and lyophilized to obtain Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bond-bridged Cys & Cys), i.e., a cyclic peptide compound. The acetic acid solution was prepared by mixing acetic acid and water in a volume ratio of 1:24. The amount of the monocyclic peptide to the acetic acid solution was 1 g:200 mL. The concentration of the iodine-methanol solution was 0.1 mol / L, and the amount of the monocyclic peptide to the iodine-methanol solution was 1 g:1 mL. The ascorbic acid content in the ascorbic acid solution was 1 wt %. The pore size of the filter membrane was 0.45 μm. The purity of the cyclic peptide compound was 98.9%. The monocyclic peptide was obtained from Example 6. The cyclic peptide compound prepared in Example 7 is numbered PR2924.

[0095] The chromatogram of PR2924 reverse phase C18 chromatography purification in Example 7 of the present invention is as follows Figure 3 shown.

[0096] The mass spectrum of PR2924 obtained in Example 7 of the present invention is as follows Figure 4 shown.

[0097] Test example:

[0098] In order to verify the physicochemical properties and physiological functions of the cyclic peptide compound and the monocyclic peptide of the present invention, the following tests were performed. The culture medium or cell culture medium used in the test of the present invention was DMEM culture medium, the cyclic peptide compound was prepared by the method of Example 7, and the cyclic peptide compound was numbered PR2924. The monocyclic peptide was prepared by the method of Example 6, and the monocyclic peptide was numbered PR2924B.

[0099] 1. Cytotoxicity Test

[0100] The present invention evaluates the potential toxicity of PR2924 and PR2924B to cells and determines their possible safe concentration ranges.

[0101] In the cytotoxicity test of the present invention, HFF-1 cells were seeded into 96-well plates, and 200 μL of culture medium was added to each well, containing about 10,000 cells / well, to ensure consistent cell density. Cultured overnight in an incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall and grow. After 24 hours, the cell culture medium was aspirated, 200 μL of DMEM culture medium containing test samples at different concentrations were added, and the 96-well plates were returned to the incubator for continued culture. After 24 hours, the absorbance value was measured at a wavelength of 490 nm using an enzyme reader by the MTT method to determine whether the test sample had cytotoxicity and its safe concentration range. The test sample was PR2924 or PR2924B, and the different concentrations of the test sample included 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM. 0 μM was the negative control group, and another group containing only culture medium but no cells was designed as the blank control group. Cell viability was calculated as follows: cell viability = (absorbance of test group - absorbance of blank control group / absorbance of negative control group - absorbance of blank control group) × 100%.

[0102] The results of cytotoxicity experiments were as follows Figure 5 As shown, the cell viability of epithelial cells treated with different concentrations of PR2924 and PR2924B did not show a significant decrease. When treated with a concentration of 500 μM, the cell viability remained at 95% relative to the control group, confirming that the cytotoxicity of PR2924 and PR2924B was extremely low.

[0103] 2. Serum stability test

[0104] The present invention evaluates the decomposition of PR2924 and PR2924B in a serum environment to predict their metabolism in vivo.

[0105] The lyophilized test sample was directly dissolved in 10 vol% fetal bovine serum to a final concentration of 1 mg / mL. Lyophilized oligopeptide GHK was dissolved under the same conditions and used as a control. The prepared solution was sterile-filtered through a 0.22 μM filter, placed into sterile containers, and incubated in a 37°C incubator. Samples were collected at designated time points. The residual peptide content was determined using analytical HPLC. The degradation percentage was calculated by comparing the peak areas at different time points, with the content at time 0 defined as 100%. The calculation formula was: relative content = (test sample peak area / test sample peak area at time 0) × 100%. The residual percentage was plotted against time to calculate the half-life. The test samples were PR2924 or PR2924B. The designated time points were 0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h.

[0106] Serum stability test results Figure 6 As shown in the figure, the GHK in the control group was almost completely degraded within 4 hours under 10 vol% serum conditions, and its degradation half-life was calculated to be approximately 0.7 hours based on the degradation curve. Under the same conditions, the half-life of PR2924 was approximately 19 hours, and the half-life of PR2924B was approximately 15 hours. The stability was significantly improved after cyclization. Based on PR2924B, the half-life of PR2924 was further improved through a secondary cyclization of the intramolecular disulfide bond.

[0107] 3. Test on the increase rate of type I collagen expression

[0108] The present invention studies the effects of PR2924 and PR2924B on the expression level of COL1A in human fibroblast HFF-1.

[0109] HFF-1 fibroblasts in the logarithmic growth phase were seeded at 50,000 cells / well in a 6-well plate. 2 mL of culture medium was added to each well and the cells were incubated overnight in an incubator to allow the cells to adhere and grow for 24 hours. The cells were divided into groups and treated with DMEM medium containing the test sample. A separate DMEM blank control group and 100 ng / mL TGF-β1 served as a positive control group. The test samples were GHK, PR2924, or PR2924B at concentrations of 0, 10, 20, or 40 μM.

[0110] After 24 hours of treatment, the culture medium was removed and RNA was extracted from each well. The Ct values ​​were measured by qRT-PCR to determine the expression levels of type I collagen mRNA in HFF-1 cells at different concentrations. Expression levels were calculated using the formula: relative RNA expression level = 2ΔΔC(t).

[0111] Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, marked with * in the figures; P < 0.01 was considered an extremely significant difference, marked with ** in the figures.

[0112] The expression upregulation rate was calculated according to the formula: upregulation rate = (test group - blank control group) / blank control group × 100%.

[0113] The experimental results of type I collagen expression are as follows Figure 7 As shown, compared with the blank control group, TGF-β1 treatment in the positive control group significantly increased COL1A expression, demonstrating the functional performance of the cell model and qPCR system. Compared with the blank control group, PR2924B treatment significantly positively regulated COL1A expression, with increases of 29% at 10 μM, 40% at 20 μM, and 44% at 40 μM. PR2924B treatment also significantly positively regulated COL1A expression, with increases of 36% at 10 μM, 50% at 20 μM, and 63% at 40 μM. Both PR2924 and PR2924B promoted type I collagen expression.

[0114] 4. Antioxidant effect detection

[0115] Superoxide dismutase (SOD) is a crucial member of the antioxidant enzyme system in biological systems. As a terminal compound, it effectively scavenges superoxide anion free radicals to prevent excessive cellular damage. Glutathione (GSH) undergoes continuous redox reactions within cells. Through the action of glutathione peroxidase (GPx), glutathione peroxidase reduces superoxide to water and oxygen molecules, while simultaneously being oxidized to glutathione disulfide (GSSG). GSSG is rapidly reduced to GSH by glutathione reductase, maintaining intracellular levels of reduced glutathione and reducing oxidative stress.

[0116] The present invention studies whether PR2924 and PR2924B can enhance the antioxidant capacity of cells, whether they affect the expression levels of antioxidant-related enzymes and the ultimate antioxidant capacity.

[0117] Fibroblasts HFF-1 in the logarithmic growth phase were seeded into 6-well plates at a density of 50,000 cells / well. 2 mL of culture medium was added to each well and placed in an incubator overnight to allow the cells to adhere and grow for 24 hours. The cells were divided into groups and DMEM culture medium containing the test sample was added. A blank control group was also set up. The blank control group did not add polypeptides and was not given oxidative stress. The negative control group did not add polypeptides and 7 μg / mL vitamin E was used as an antioxidant positive control group. Oxidative stress was applied by ultraviolet irradiation UVA, and the blank control group was not irradiated. After 24 hours, the cell supernatant was collected, and the SOD and GSH levels were detected according to the instructions of the SOD detection kit and the GSH quantification kit. The test sample was GHK or PR2924 or PR2924B, and the concentration of the test sample was 10 μM. The unit of ultraviolet irradiation UVA was 30 J / cm 2 .

[0118] Comparisons between groups were analyzed using the t-test. P < 0.05 was considered a significant difference, marked with * in the figures; P < 0.01 was considered an extremely significant difference, marked with ** in the figures.

[0119] According to the formula: improvement rate = (test group - positive control group) / positive control group × 100%, the improvement levels of SOD and GSH in each group were calculated.

[0120] The results of the antioxidant test were as follows Figure 8-9 As shown in the results, compared with the blank control, SOD and GSH levels in the negative control group were significantly downregulated, while those in the positive control group significantly increased. SOD levels in the positive control group increased by 53.7% and GSH levels by 119.7%, demonstrating effective oxidative stress stimulation and the proper function of the antioxidant positive control. GHK treatment demonstrated some antioxidant capacity. Compared with the negative control, SOD levels increased by 36.5% and GSH levels increased by 61.0% at 10 μM. PR2924B increased SOD levels by 61% and GSH levels by 110% compared with the negative control; PR2924 increased SOD levels by 80% and GSH levels by 117% compared with the negative control. Both PR2924 and PR2924B demonstrated greater antioxidant capacity than the positive control. This demonstrates that PR2924 and PR2924B can elevate the expression of the key antioxidant enzyme SOD, ultimately increasing the expression of the key antioxidant substance GSH.

[0121] 5. Anti-inflammatory Effect Detection

[0122] In the inflammatory response, cytokines such as TNF-α, IL-6, and IL-8 are important regulatory molecules. They interact through complex networks and jointly participate in the regulation of immune responses, tissue repair, and pathological damage. TNF-α is mainly responsible for activating immune cells, IL-6 promotes the persistence of immune responses, and IL-8 enhances the infiltration and activation of immune cells. There is a complex synergistic relationship between these three factors, which together constitute the core network of the inflammatory response. Under pathological conditions, such as autoimmune diseases or infections, their overexpression may lead to tissue damage and organ dysfunction. This part of the study will compare the ability of GHK, PR2924, and PR2924B to inhibit the production of inflammatory factors in macrophages under inflammatory pressure caused by lipopolysaccharide (LPS) stimulation.

[0123] RAW264.7 macrophage cells were seeded at 50,000 cells / well in 6-well plates and cultured for 24 hours. Groups were assigned for drug administration. Treatment groups received DMEM medium containing the test sample. A blank control group and a negative control group were also established, without the peptide. 0.01% dexamethasone served as an anti-inflammatory positive control. Two hours after drug administration, 200 μL of LPS working solution was added to each well of all groups except the blank control, and the cells were incubated in an incubator for an additional 22 hours. Cell culture supernatants were collected and analyzed for TNF-α, IL-6, and IL-8 according to the ELISA kit's instructions. Comparisons between groups were analyzed using the t-test. P < 0.05 was considered significant, as indicated by *; P < 0.01 was considered extremely significant, as indicated by **. Test samples were GHK, PR2924, or PR2924B at a concentration of 10 μM.

[0124] The inhibition rate was calculated according to the formula: inhibition rate % = (negative control group - test group) / negative control group × 100%.

[0125] The results of anti-inflammatory experiments were as follows Figure 10-12As shown in the figure, TNF refers to TNF-α, IL6 refers to IL-6, and IL8 refers to IL-8. Compared with the blank control group, the TNF-α, IL-6, and IL-8 levels in the negative control group increased significantly, indicating that the stimulation conditions in this test were effective. Compared with the negative control group, the TNF-α, IL-6, and IL-8 levels in the positive control group decreased significantly, indicating that the positive control in this test was effective. Compared with the negative control group, the inhibition rates of TNF-α, IL-6, and IL-8 in the positive control group were 34.7%, 31.8%, and 48.0%, respectively; the inhibition rates of TNF-α, IL-6, and IL-8 in the 10 μM GHK group were 15.1%, 34.2%, and 19.4%, respectively; the inhibition rates of TNF-α, IL-6, and IL-8 in the 10 μM PR2924B group were 28.6%, 51.9%, and 21.8%, respectively; and the inhibition rates of TNF-α, IL-6, and IL-8 in the 10 μM PR2924 group were 29%, 54.5%, and 33.7%, respectively. At the same concentration, both PR2924 and PR2924B exhibited a stronger effect than GHK and demonstrated significant anti-inflammatory potential.

[0126] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0127] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing a monocyclic peptide, comprising: After mixing Fmoc-Gly-OH, DIEA and DCM, CTC resin was added and coupled to obtain the first amino acid coupling resin; The molar amount of Fmoc-Gly-OH used is 100-300% of the reactive sites on the CTC resin, and the molar amount of DIEA used is 100-500% of the molar amount of Fmoc-Gly-OH used; The first amino acid coupling resin is deprotected, and then the activated amino acid reagent is coupled in sequence according to the peptide sequence to obtain a fully protected peptide resin. The structure of the fully protected peptide resin is: H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-CTC resin; The fully protected peptide resin is cleaved and cyclized to obtain a monocyclic peptide, the structure of which is: Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

2. The method for preparing the monocyclic peptide according to claim 1, wherein: The fully protected peptide resin is cleaved to obtain a fully protected polypeptide. Preparation of the fully protected polypeptide: The fully protected peptide resin is mixed with the cutting solution, reacted at 20-40°C for 20-60 minutes, filtered, and the filtrate is precipitated with petroleum ether to obtain a solid, which is then dried under vacuum to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, i.e., the fully protected peptide; or, The fully protected peptide resin is mixed with the cutting solution, reacted at 20-40°C for 20-60 minutes, filtered, and the filtrate is concentrated and evaporated to dryness to obtain H-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-OH, i.e., the fully protected peptide.

3. The method for preparing the monocyclic peptide according to claim 1, wherein: The fully protected peptide resin is subjected to cleavage treatment and monocyclization to obtain a fully protected cyclic peptide. Preparation of the fully protected cyclic peptide: The fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide solution; HATU, DIEA and DMF are mixed to obtain a cyclization solution; the fully protected polypeptide solution is added to the cyclization solution and reacted at 20-40° C. for 0.5-3 h. The reaction is completed after monitoring by HPLC. Water and ethyl acetate are added to the reaction solution for extraction, and the solution is washed with a saturated aqueous sodium bicarbonate solution, water and a saturated aqueous sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), i.e., a fully protected cyclic peptide; or, A fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide solution; HOOBT is added to dissolve the mixture, and then NMM and DIC are added at 0-5°C. The mixture is reacted at 20-40°C for 8-24 hours. The reaction is monitored by HPLC to ensure completion. Water is added to the reaction solution to precipitate a solid, which is dissolved in ethyl acetate and then washed sequentially with a saturated aqueous sodium bicarbonate solution, water, and a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain Cyclo(His(Trt)-Lys(Boc)-Cys(Trt)-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Gly-), i.e., a fully protected cyclic peptide.

4. The method for preparing the monocyclic peptide according to claim 1, wherein: In the preparation of the monocyclic peptide, the fully protected cyclic peptide is mixed with a deprotection cleavage solution and cleaved for 1-4 hours. After the cleavage is completed, the peptide is precipitated with glacial ether, washed, centrifuged, and rotary evaporated to dryness to obtain a crude monocyclic peptide. The crude monocyclic peptide is purified by reverse-phase C18 liquid chromatography and lyophilized to obtain Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-), i.e., a monocyclic peptide.

5. The monocyclic peptide prepared by the method of claim 1 has the structure: Cyclo(His-Lys-Cys-Gly-His-Lys-Cys-Gly-).

6. A cyclic peptide compound having the structure: Cyclo (His-Lys-Cys-Gly-His-Lys-Cys-Gly-, disulfide bond bridged Cys & Cys).

7. A method for preparing a cyclic peptide compound, comprising: A method for preparing a monocyclic peptide according to claim 1.

8. The method for preparing a cyclic peptide compound according to claim 7, wherein: The monocyclic peptide is subjected to a dicyclic treatment to obtain a cyclic peptide compound, and the dicyclic treatment is carried out under the action of iodine methanol and ascorbic acid.

9. Use of the single cyclic peptide according to claim 5 in the preparation of cyclic peptide compounds and / or products for increasing type I collagen expression and / or antioxidant products and / or anti-inflammatory products.

10. Use of the cyclic peptide compound according to claim 6 in the preparation of a product for increasing type I collagen expression and / or an antioxidant product and / or an anti-inflammatory product.

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