Cycloheptapeptide-4, process for its preparation and use thereof

Abstract

The application discloses cyclic heptapeptide-4 as well as a preparation method and application thereof, belongs to the technical field of synthesis and application of cyclic peptides, and specifically relates to the preparation of a linear peptide containing a protecting group from an amino acid reagent after activation, and then the preparation of a functional cyclic peptide with a cyclic structure through cyclization treatment. The functional cyclic peptide prepared by the application has good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good inhibition effect on release of neurotransmitters, and good whitening effect. After the linear peptide is prepared into the functional cyclic peptide, the permeability is increased, the stability is improved, the activity is improved, and the irritation is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of synthesis and application of cyclic peptides, and particularly relates to a cyclic heptapeptide-4 and a preparation method and application thereof. BACKGROUND

[0002] Polypeptides are composed of amino acids through dehydration condensation, and the amino acids themselves have carboxyl and amino groups. Generally, the polypeptides are hydrophilic compounds, and the polypeptides generally have obvious hydrophilicity and ionicity. Especially, some small molecule polypeptides have special physiological activity effects after being absorbed by the skin, can improve some problems of the skin from the surface to the inside, and have been widely used in cosmetic formulations. At present, there are various cosmetics or skin care products on the market, but most of the skin care products only add polypeptides of a certain type as the active material of the skin care product, so that the effect obtained by the skin is single and slow. Moreover, most of the skin care products with polypeptides as the active material on the market directly add polypeptides or polypeptide stock solution to the conventional skin care product matrix, and the addition amount of the polypeptides is limited based on the consideration of the entire formulation and stability.

[0003] In conventional polypeptide modification, palmitoyl modification or myristic modification is generally used. The polypeptides modified by palmitoyl or myristic generally have hydrophilicity and lipophilicity, and the solvent consumption is greatly increased in the process of separation and purification, thereby increasing the cost. SUMMARY

[0004] The present application aims to provide a functional cyclic peptide with good moisturizing effect, good anti-wrinkle effect, good tightening effect, good soothing effect, good inhibition of neurotransmitter release effect, and good whitening effect, and a preparation method and application thereof.

[0005] The linear polypeptide is cyclized by adopting the polypeptide cyclization method, so as to obtain the cyclized polypeptide. The cyclization of the polypeptide forms a new amide bond through dehydration condensation of the carboxyl and amino groups of the polypeptide, thereby increasing the liposolubility of the polypeptide without significantly increasing the solvent consumption. The cyclized polypeptide has better stability than the linear polypeptide, and the polypeptide obtained by condensation of the carboxyl and amino groups or hydroxyl groups has better liposolubility and better skin permeability.

[0006] The technical scheme adopted by the present application to achieve the above-mentioned purpose is as follows:

[0007] The use of the functional cyclic peptide in moisturizing products and / or anti-wrinkle products and / or tightening products and / or soothing products and / or neurotransmitter release inhibition products and / or whitening products and / or anti-glycation products and / or antioxidant products, the functional cyclic peptide has the following formula structure:

[0008] wherein, 1≤n, R m the number of R is n, R1, R2, R3or R m is selected from H, aliphatic hydrocarbon, substituted aliphatic hydrocarbon, aryl, substituted aryl, imidazole group, substituted imidazole group, indole group, substituted indole group, guanidine group, substituted guanidine group, metal alkyl or alkyl forming a cyclic structure with the adjacent nitrogen atom.

[0009] Preferably, n≤6; or, 1≤n≤6; or, n=2; or, n=3; or, n=4; or, n=5.

[0010] Preferably, R1, R2, R3and R m are the same; or, R1, R2, R3and R m are the same; or, R1, R2, R3or R m is selected from -CH2-CH2-CH2- group, R1, R2, R3or R m forms a ring with the adjacent N; or, R1, R2, R3or R m is selected from H, methyl, -CH2-CH2-CH2- group, propyl, butyl, hydroxymethyl, hydroxyethyl, acetylamide, propionamide, CH3-S-CH2-CH2- group, HS-CH2- group, Ph-CH2- group, p-hydroxybenzyl, acetic acid group, propionic acid group, Se-CH2- group, butylamine group, indole group, imidazole methyl and substituted guanidine group.

[0011] Preferably, the functional cyclic peptide is any one of the following:

[0012] .

[0013] A method for preparing a functional cyclic peptide, comprising: mixing a linear peptide with a cyclization reagent in a solvent to perform a cyclization reaction, and post-treatment to prepare the functional cyclic peptide; the functional cyclic peptide has the following structure:

[0014] wherein, 1≤n, R m the number of R is n, R1, R2, R3or R m is selected from H, aliphatic hydrocarbon, substituted aliphatic hydrocarbon, aryl, substituted aryl, imidazole group, substituted imidazole group, indole group, substituted indole group, guanidine group, substituted guanidine group, metal alkyl or alkyl forming a cyclic structure with the adjacent nitrogen atom.

[0015] Preferably, the functional cyclic peptide is any one of the following:

[0016] .

[0017] Preferably, the linear peptide is prepared by solid phase synthesis; or, the cyclization reagent is HBTU; or, the solvent is at least one of DMF, DCM and DIEA; or, the post-treatment comprises cyclization post-treatment, cleavage treatment and cleavage post-treatment.

[0018] More preferably, in the cyclization post-treatment, ice water or DCM is added to the test solution after the cyclization reaction is completed, and the cyclized peptide with the protective group is separated; or, in the cleavage treatment, the cleavage solution comprises E solution or F solution, the E solution is prepared by mixing TFA, thioanisole, EDT, PhOH and water in a volume ratio of 75-95:2-10:1-5:1-5:1-5, and the F solution is prepared by mixing TFA, TIS and water in a volume ratio of 80-96:2-10:2-10; or, in the cleavage post-treatment, the test solution after the cleavage treatment is added to ice ethyl ether, and the functional cyclic peptide is separated and purified.

[0019] More preferably, in the post-treatment, the functional cyclic peptide is purified by liquid chromatography; or, in the cyclization reaction, the linear peptide is mixed with the solvent to obtain a linear peptide solution, and then the linear peptide solution is mixed with the cyclization reagent, and the cyclization reaction is carried out at 30-50℃; or, in the post-treatment, after the cyclization reaction is completed, the cyclization post-treatment is carried out to obtain the cyclized peptide with the protective group, the cleavage solution is added for treatment, and finally the cleavage post-treatment is carried out to obtain the cyclic peptide.

[0020] More preferably, in the cyclization post-treatment, ice water is added to the test solution after the cyclization reaction is completed, and the solid is separated, stirred and filtered, the solid is dissolved in EA, and then the saturated sodium bicarbonate solution and the saturated brine are used for washing in sequence, anhydrous sodium sulfate is used for drying, filtration is carried out, and evaporation is performed to obtain the cyclized peptide with the protective group; or, in the cyclization post-treatment, DCM is added to the test solution after the cyclization reaction is completed, and then the solid-liquid separation is carried out, the saturated sodium bicarbonate solution and the saturated brine are used for washing in sequence, anhydrous sodium sulfate is used for drying, filtration is carried out, and evaporation is performed to obtain the cyclized peptide with the protective group; or, in the cleavage post-treatment, the test solution after the cleavage treatment is added to ice ethyl ether, the solid is separated, centrifugal washing is carried out, the solid is evaporated, and purification is carried out to obtain the cyclic peptide; or, in the cyclization reaction, LC-MS is used for monitoring; or, the solvent is at least one of DMF, DCM and DIEA, and the solvent is taken as the measurement reference of DMF, and the relationship between the use amount of the linear peptide and DMF is 0.1-30 mg / mL.

[0021] Preferably, in the preparation of the amino acid activation solution, the amino acid reagent and HOBt are mixed, DMF and DIC are added at a temperature of 2-8℃, and standing reaction is carried out for 10-30 min to obtain the amino acid activation solution.

[0022] More preferably, in the preparation of the amino acid activation solution, the molar amount of HOBt used is 50-200% of the molar amount of the amino acid reagent used.

[0023] More preferably, in the preparation of the amino acid activation solution, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used, and the relationship between the amount of DMF used and the amount of the amino acid reagent used is 0.1-4.5 mL / mmol.

[0024] More preferably, in the preparation of the amino acid activation solution, the amino acid reagent comprises any one of the following reagents: Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Asp(otBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH, Fmoc-Glu(otBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Phe-OH.

[0025] Preferably, in the preparation of the linear peptide, the first amino acid reagent is mixed with the CTC resin, dichloromethane (DCM) and DIEA are then added, and the mixture is reacted at 20-40°C for 2-5 h, methanol is then added, and the mixture is reacted for 3-30 min, after the reaction is completed, the mixture is filtered and washed, and a deprotection solution is added for treatment; the amino acid activation solution is mixed with the CTC resin to which the first amino acid is bonded in the order of the linear peptide, and the mixture is reacted for 0.5-3 h, the deprotection solution is added for treatment after each reaction of the amino acid activation solution, and after the reaction of the last amino acid reagent is completed, a cleavage solution is added for treatment, thereby obtaining the linear peptide.

[0026] More preferably, in the preparation of the linear peptide, the total substitution degree of the CTC resin is the product of the substitution degree and the mass of the CTC resin, i.e., the total substitution degree is the molar amount of the total active reaction sites of the CTC resin, and the molar amount of the first amino acid reagent used is 50-250% of the total substitution degree of the CTC resin.

[0027] More preferably, in the preparation of the linear peptide, the relationship between the amount of dichloromethane used and the amount of the first amino acid reagent used is 1-65 mL / mmol.

[0028] More preferably, in the preparation of the linear peptide, the relationship between the amount of DIEA used and the amount of the first amino acid reagent used is 0.1-5 mL / mmol.

[0029] More preferably, in the preparation of the linear peptide, the relationship between the amount of methanol used and the amount of the first amino acid reagent used is 0.1-6.5 mL / mmol.

[0030] More preferably, in the preparation of the linear peptide, the amount of the amino acid activation solution used is such that the molar amount of the amino acid reagent is 100-350% of the total degree of substitution in the CTC resin.

[0031] More preferably, in the preparation of the linear peptide, the first amino acid reagent comprises any one of Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Met-OH. The deprotection solution is a 10-30% Pip / DMF solution. The cleavage solution is a 30% TFE / DCM solution.

[0032] The linear peptide comprises any one of H-Phe-Phe-Tyr(tBu)-Pro-OH, H-Asp(otBu)-Val-Lys(Boc)-Tyr(tBu)-OH, H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-OH, H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-OH, H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH, H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-OH, H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH, H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(otBu)-Glu(otBu)-Glu(otBu)-Met-OH, and H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH.

[0033] More preferably, in the preparation of the linear peptide, in the washing after the reaction of the first amino acid with the CTC resin, dichloromethane (DCM), methanol and DMF are used in sequence.

[0034] More preferably, in the preparation of the linear peptide, in the washing after the reaction of the first amino acid with the CTC resin, dichloromethane (DCM), methanol and DMF are used in sequence.

[0035] More preferably, in the preparation of the linear peptide, in the treatment with the deprotection solution, the CTC resin after bonding the amino acid reagent is washed with DMF, then the deprotection solution is added, stirred for 10-60 min, after the treatment is completed, the deprotection solution is removed by suction filtration, washed with DMF, and suction dried. The deprotection solution is used in an appropriate amount.

[0036] More preferably, in the preparation of the linear peptide, in the treatment with the cleavage solution, the cleavage solution is added to the CTC resin after bonding the amino acid reagent, treated at 20-40℃ for 0.5-5h, after the treatment is completed, the resin is removed by filtration, and the filtrate is obtained, and the linear peptide is obtained by drying the filtrate. The cleavage solution is used in an appropriate amount.

[0037] Preferably, in the preparation of the cyclic peptide, the linear peptide is mixed with a solvent to obtain a linear peptide solution, then the linear peptide solution is mixed with a cyclization reagent, reacted at 30-50℃, the reaction is monitored by LC-MS, after the reaction is completed, cyclization post-treatment is performed to obtain a cyclic peptide with a protecting group, a cleavage solution is added for treatment, and finally cleavage post-treatment is performed to obtain the cyclic peptide.

[0038] More preferably, in the preparation of the cyclic peptide, the solvent contains DMF, and can also contain at least one of DCM and DIEA, the solvent is taken as a measurement basis with DMF, the relationship between the use amount of the linear peptide and DMF is 0.1-30mg / mL, the relationship between the use amount of DCM and DMF is 0.1-10mL / mL, the relationship between the use amount of DIEA and DMF is 0.1-5mg / mL, the cleavage solution is E solution or F solution, and the cleavage solution is used in an appropriate amount. The cyclization reagent is HBTU, and the relationship between the use amount of HBTU and DMF is 0.1-5mg / mL.

[0039] More preferably, in the preparation of the cyclic peptide, the E solution is mixed from TFA, anisole, EDT, PhOH and water, and the TFA, anisole, EDT, PhOH and water are mixed in a volume ratio of 87.5:5:2.5:2.5:2.5.

[0040] More preferably, in the preparation of the cyclic peptide, the F solution is mixed from TFA, TIS and water, and the TFA, TIS and water are mixed in a volume ratio of 90:5:5; PR100,

[0041] Preferably, in the cyclization post-treatment, DMF is dried, ice water is added to the reaction solution after the cyclization reaction is completed, a solid is precipitated, stirred and filtered, the solid is dissolved with EA, then sequentially washed with saturated sodium bicarbonate solution and saturated brine, dried with anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain the cyclic peptide with a protecting group.

[0042] Preferably, in the post-cyclization treatment, DMF is drained, DCM is added to the test solution in which the cyclization reaction is completed, the layers are extracted, and then the saturated sodium bicarbonate solution and saturated brine are used for washing in sequence, anhydrous sodium sulfate is used for drying, filtration is performed, and evaporation is performed to obtain the cyclic peptide with the protective group.

[0043] Preferably, in the post-cleavage treatment, the test solution in which the cleavage treatment is completed is added to ice ethyl ether, the solid is precipitated, centrifugal washing is performed, the solid is evaporated to dryness, and purification is performed to obtain the cyclic peptide.

[0044] The purification is performed by using liquid chromatography.

[0045] The functional cyclic peptide prepared by the method has good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good neurotransmitter release inhibition effect, good whitening effect, good anti-glycation effect, and good antioxidant effect. Therefore, the present application is a functional cyclic peptide with good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good neurotransmitter release inhibition effect, and good whitening effect, and a preparation method and application thereof. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 is a PR126 chromatogram;

[0047] Figure 2 is a PR126 mass spectrum;

[0048] Figure 3 is a PR140 chromatogram;

[0049] Figure 4 is a PR140 mass spectrum;

[0050] Figure 5 is a PR100 chromatogram;

[0051] Figure 6 is a PR100 mass spectrum;

[0052] Figure 7 is a PR132 chromatogram;

[0053] Figure 8 is a PR132 mass spectrum;

[0054] Figure 9 is a PR120 chromatogram;

[0055] Figure 10 is a PR120 mass spectrum;

[0056] Figure 11 is a PR102 chromatogram;

[0057] Figure 12 PR102 mass spectrum;

[0058] Figure 13 PR139 chromatogram;

[0059] Figure 14 PR139 mass spectrum;

[0060] Figure 15 PR146 chromatogram;

[0061] Figure 16 PR146 mass spectrum;

[0062] Figure 17 PR116 chromatogram;

[0063] Figure 18 PR116 mass spectrum. DETAILED DESCRIPTION

[0064] The technical solutions of the present application are described in further detail below in combination with the specific embodiments and the accompanying drawings:

[0065] Example 1: Preparation method of Cyclo(Tyr-Pro-Phe-Phe)

[0066] S1, the synthesis steps of the linear peptide are as follows:

[0067] S11, place CTC resin (2.23 g, 2.5 mmol) in a 100 mL solid-phase synthesis reactor, add amino acid Fmoc-Pro-OH (1.68 g, 5 mmol), add dichloromethane (DCM) 20 mL, add DIEA (2.0 mL), and react at 25°C for 3 hours, add methanol 3 mL, and react for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 20 mL for 2 times, methanol 20 mL for 2 times, and DMF 20 mL for 2 times. Add 20% Pip / DMF solution 20 mL, stir for 30 min, filter, remove the deprotection solution, and then wash with DMF solution 20 mL for 6 times, and dry by suction for standby.

[0068] S12, take Fmoc-Tyr (tBu) -OH (2.76g, 6mmol), HOBt (0.81g, 6mmol) in a 50mL beaker, cooling to 5℃, add 5mL of DMF solution, DIC (0.93mL, 6mmol) and stand for 15 minutes, and add the solution in a 100mL beaker to a 100mL solid phase synthesis reactor, stir for 1.5 hours, and the reaction is complete. The resin is washed with DMF solution three times, 20mL each time. After washing, the next step is carried out. Add 20% Pip / DMF solution 20mL, stir for 30min, filter, remove the deprotection solution, then wash with DMF solution 20mL for 6 times, and dry for use.

[0069] Repeat the above S12 step, replace Fmoc-Tyr (tBu) -OH with the following amino acid reagents in order: Fmoc-Phe-OH and Fmoc-Phe-OH; after the deprotection treatment is complete, then wash with methanol 20mL twice, DCM solution 20mL twice, methanol 20mL twice, vacuum dry, to obtain H-Phe-Phe-Tyr (tBu) -Pro-CTC-resin. Stir the above peptide resin with 30% TFE / DCM solution 30mL at 30℃ for 30 minutes, filter, remove the resin, and obtain the filtrate. Dry the filtrate to obtain the fully protected peptide H-Phe-Phe-Tyr (tBu) -Pro-OH 1.10g, yield 90%, purity 97.5%.

[0070] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0071] S21, weigh H-Phe-Phe-Tyr (tBu) -Pro-OH (1.02g), dissolve in DMF (1000mL), add DIEA (1.674g) and reserve as A; weigh HBTU (1.84g) and add to A, stir for 2 hours after dropwise addition, and directly perform the control; control: LC-MS detects that the raw material reaction is complete.

[0072] S22, post-treatment: dry most of the reaction liquid by pulling, then add ice water (30mL) while stirring, precipitate the solid, stir for 10min, then filter, dissolve the solid in EA (20mL), wash with saturated NaHCO3 aqueous solution twice, wash with saturated brine once, dry with anhydrous sodium sulfate, filter and evaporate to obtain Cyclo (Tyr (tBu) -Pro-Phe-Phe) 0.9g.

[0073] S3, the synthesis steps of the cyclic peptide are as follows:

[0074] S31, cleavage: take 0.9 g of Cyclo(Tyr(tBu)-Pro-Phe-Phe) and add cleavage E solution, control temperature at 30°C and stir the reaction. Take sample and test MS, the reaction is substantially complete.

[0075] S32, post-treatment: add the reaction solution dropwise into ice-ethanol and shake, centrifuge the solid 3 times, and evaporate the solid to obtain 0.68 g of Cyclo(Tyr-Pro-Phe-Phe). Test LC-MS and send for purification.

[0076] The purification conditions are as follows:

[0077] Dissolution: take 0.68 g of the crude product and add 30 mL of acetic acid, 30 mL of acetonitrile, and 100 mL of water, and ultrasonically dissolve;

[0078] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0079] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0080] Equilibrium: A:B=95:5, equilibrate for 10 min, flow rate: 60 mL / min;

[0081] Loading: flow rate: 60 mL / min;

[0082] Elution: 30-50-80%B, 0-60-90 min;

[0083] Column cleaning: clean with 80% acetonitrile until baseline equilibrium;

[0084] Collect the purified product and freeze-dry to obtain 55 mg. The LC chart of the purified product is shown in Figure 1 , and the MS chart of the product after purification is shown in Figure 2 .

[0085] Example 2: Preparation method of Cyclo(Asp-Val-Lys-Tyr)

[0086] S1, the synthesis steps of the linear peptide are as follows:

[0087] S11, CTC resin (5.58 g, 6.25 mmol) was placed in a 250 mL solid phase synthesis reactor, amino acid Fmoc-Tyr(tBu)-OH (0.459 g, 12.5 mmol) was added, dichloromethane (DCM) 120 mL was added, DIEA (8.7 mL) was added, and the reaction was carried out at 25°C for 3 hours, methanol 12.5 mL was added, and the reaction was carried out for 5 minutes. Filtration, the resin was washed with dichloromethane (DCM) 75 mL twice, methanol 75 mL twice, and DMF 75 mL twice. 20% Pip / DMF solution 65 mL was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by filtration, and then the resin was washed with DMF solution 120 mL for 6 times, and then dried by suction.

[0088] S12, Fmoc-Lys(Boc)-OH (7.03 g, 15 mmol) and HOBt (2.03 g, 15 mmol) were taken in a 100 mL beaker, cooled to 5°C, DMF solution 14 mL was added, DIC (1.89 mL, 15 mmol) was added and the solution was allowed to react for 15 min, and then the solution in the 100 mL beaker was added to the 250 mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours, and the reaction was completed. The resin was washed with DMF solution three times, each time 200 mL. After the washing was completed, the next step was carried out. 20% Pip / DMF solution 65 mL was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by filtration, and then the resin was washed with DMF solution 120 mL for 6 times, and then dried by suction.

[0089] The above S12 step was repeated, and Fmoc-Lys(Boc)-OH was replaced by the following amino acid reagents in order: Fmoc-Val-OH and Fmoc-Asp(OtBu)-OH; after the deprotection treatment was completed, then washed with methanol 75 mL twice, DCM solution 75 mL twice, methanol 75 mL twice, and dried in vacuum to obtain H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-CTC-resin. The above peptide resin was taken with 3.95 g, 30% TFE / DCM cleavage solution 40 mL, and the reaction was stirred at 30°C for 2.5 hours, and then filtered to remove the resin to obtain the filtrate. The filtrate was dried to obtain the crude peptide H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-OH. 1.67 g, yield 113%, purity 70.7%.

[0090] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0091] S21, weigh H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-OH (2 g), dissolve in DMF (2000 mL), add DIEA (2.36 g), and reserve as sample A; weigh HBTU (2.6 g), add to sample A, and after dropwise addition, stir for 2 hours, and then perform a control test; control test: LC-MS to detect complete reaction of raw materials.

[0092] S22, post-treatment: remove most of the DMF in the reaction liquid, then add ice water (30 mL) while stirring, precipitate the solid, stir for 10 min, then filter, dissolve the solid in EA (20 mL), wash twice with saturated NaHCO3 aqueous solution, wash once with saturated brine, dry over anhydrous sodium sulfate, filter, and evaporate to obtain Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)) 1.22 g, with a yield of 62.5%.

[0093] S3, the synthesis of the cyclic peptide is as follows:

[0094] S31, weigh Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)) 1.22 g, add cleavage E liquid, and stir at 30°C. Control test: take a sample and detect MS, and the reaction is basically complete.

[0095] S32, post-treatment: add the reaction liquid dropwise to ice ether, shake while adding, centrifuge and wash the solid three times, evaporate the solid to obtain Cyclo(Asp-Val-Lys-Tyr) 0.95 g, with a yield of 90%, and perform LC-MS detection and send for purification.

[0096] The purification conditions are as follows:

[0097] Dissolution: take 0.95 g of the crude product, dilute with 100 mL of water;

[0098] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0099] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0100] Equilibration: A:B=100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0101] Loading: flow rate: 60 mL / min;

[0102] Elution: 0-20% B for 60 min;

[0103] Column cleaning: clean with 80% acetonitrile until baseline equilibrium;

[0104] The collected product was lyophilized to obtain 202 mg. The LC chart of the product purification is shown in Figure 3 and the MS chart of the product after purification is shown in Figure 4 .

[0105] Example 3: Preparation method of Cyclo (Lys-Thr-Thr-Lys-Ser)

[0106] S1, the synthesis steps of the linear peptide are as follows:

[0107] S11, the CTC resin (6.25 g, 7 mmol) was placed in a 250 mL solid-phase synthesis reactor, and the amino acid Fmoc-Ser(tBu)-OH (8.42 g, 7 mmol) was added, dichloromethane (DCM) 75 mL, DIEA (8.7 mL) was added, and the reaction was carried out at 25°C for 3 hours, 12.5 mL of methanol was added, and the reaction was carried out for 5 minutes. Filtration, the resin was washed with dichloromethane (DCM) 75 mL for 2 times, methanol 75 mL for 2 times, and DMF 75 mL for 2 times. 40 mL of 20% Pip / DMF solution was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with 75 mL of DMF solution for 6 times, and then dried by suction.

[0108] S12, Fmoc-Lys(Boc)-OH (9.84 g, 21 mmol) and HOBt (2.84 g, 21 mmol) were taken in a 100 mL beaker, cooled to 5°C, 50 mL of DMF solution was added, DIC (3.2 mL, 21 mmol) was added and the solution was allowed to react for 15 minutes, and then the solution in the 100 mL beaker was added to the 250 mL solid-phase synthesis reactor, and the reaction was stirred for 1.5 hours, and the reaction was completed. The resin was washed with DMF solution for three times, each time 75 mL. After the washing was completed, the next reaction was carried out. 40 mL of 20% Pip / DMF solution was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with 65 mL of DMF solution for 6 times, and then dried by suction.

[0109] Repeat the above S12 step, in order to replace Fmoc-Lys (Boc) -OH with the following amino acid reagents: Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Lys (Boc) -OH; after the completion of the deprotection treatment, then washed with methanol 130 mL 2 times, DCM solution 130 mL 2 times, methanol 130 mL 2 times, vacuum drying, to obtain H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -CTC-resin. The above peptide resin is cut using 3.5 g of cleavage fluid 30% TFE / DCM, 40 mL, 30°C, stirring for 2.5 hours, filtering, removing the resin, to obtain the filtrate. The filtrate is pulled dry to obtain the crude peptide H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -OH 1.42 g. Yield 50.9%, purity 88.5%.

[0110] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0111] S21, feeding: weigh H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -OH (1.42 g), dissolve in DMF (750 mL) + DCM (750 mL), add DIEA (2.94 g), and reserve for use, named A; weigh HBTU (1.73 g), add to A, and after dropwise addition is complete, stir for 2 hours, and directly perform the intermediate control; intermediate control: LC-MS detects that the raw material is completely reacted.

[0112] S22, post-treatment: pull dry to remove most of the DMF in the reaction solution, then add ice water (30 mL) while stirring, and no solid can be precipitated. The reaction solution is added with DCM (20 mL), separated into layers, washed with saturated NaHCO3 aqueous solution twice, and saturated brine once. Dry over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain Cyclo (Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) ) 1.3 g, with a yield of 93%.

[0113] S3, the synthesis steps of the cyclic peptide are as follows:

[0114] S31, feeding: weigh Cyclo (Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) ) 1.22 g, use cleavage E liquid, and stir at a temperature of 30°C. Intermediate control: take a sample for MS detection, and the reaction is basically complete.

[0115] S32, post-treatment: the reaction solution was added dropwise into ice-ether for precipitation, shaking while adding, centrifuged for washing the solid 3 times, and the solid was evaporated to dryness to obtain Cyclo(Lys-Thr-Thr-Lys-Ser) 0.83 g, yield 75.5%, LC-MS detection, and sent for purification.

[0116] Purification conditions:

[0117] Dissolution: 0.83 g of the crude product was diluted with 100 mL of water;

[0118] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0119] Mobile phase: A: water; B: acetonitrile;

[0120] Equilibrium: A:B=100:0, equilibrium for 10 min, flow rate: 60 mL / min;

[0121] Loading: flow rate: 60 mL / min;

[0122] Elution: 0-10% B for 60 min;

[0123] Column cleaning: 80% acetonitrile was used to clean to the baseline equilibrium;

[0124] The purified LC chart of the product is shown in Figure 5 , and the MS chart of the product after purification is shown in Figure 6 .

[0125] Example 4: Preparation method of Cyclo(Arg-Lys-Asp-Val-Tyr)

[0126] S1, the synthesis steps of the linear peptide are as follows:

[0127] S11, CTC resin (5.58 g, 6.25 mmol) was placed in a 100 mL solid-phase synthesis reactor, amino acid Fmoc-Tyr(tBu)-OH (4.21 g, 12.5 mmol) was added, dichloromethane (DCM) 20 mL was added, DIEA (5.0 mL) was added, and the reaction was carried out at 25°C for 3 hours, 6 mL of methanol was added, and the reaction was carried out for 5 minutes. Filtration, the resin was washed with dichloromethane (DCM) 50 mL for 2 times, methanol 50 mL for 2 times, and DMF 50 mL for 2 times. 20% Pip / DMF solution 50 mL was added, and the reaction was stirred for 30 min, suction filtration, and the deprotection solution was removed, and then the resin was washed with DMF solution 50 mL for 6 times, and suction dried for use.

[0128] S12, Fmoc-Val-OH (5.09 g, 15 mmol), HOBt (2.03 g, 15 mmol) were taken in a 50 mL beaker, cooled to 5°C, 5 mL of DMF solution was added, DIC (2.3 mL, 7.5 mmol) was added and the solution was allowed to react for 15 minutes, and the solution in the 100 mL beaker was added to the 100 mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours, and the reaction was complete. The resin was washed with DMF solution three times, 20 mL each time. After washing was complete, the next step was carried out. 50 mL of 20% Pip / DMF solution was added, and the reaction was stirred for 30 minutes, filtered, and the deprotection solution was removed, and then the resin was washed with 50 mL of DMF solution six times, and then dried under suction.

[0129] The above S12 step was repeated, and Fmoc-Val-OH was replaced with the following amino acid reagents in order: Fmoc-Asp (otBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH; after the deprotection treatment was complete, then washed with 50 mL of methanol twice, 20 mL of DCM solution twice, and 50 mL of methanol twice, and dried under vacuum to obtain H-Arg (Pbf)-Lys (Boc)-Asp (otBu)-Val-Tyr (tBu)-CTC-resin. The above peptide resin was stirred with 120 mL of 30% TFE / DCM solution at 30°C for 30 minutes, filtered, and the resin was removed to obtain a filtrate. The filtrate was dried to obtain the fully protected peptide H-Arg (Pbf)-Lys (Boc)-Asp (otBu)-Val-Tyr (tBu)-OH 4.2 g, with a yield of 74% and a purity of 92.5%.

[0130] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0131] S21, the following were weighed: H-Arg (Pbf)-Lys (Boc)-Asp (otBu)-Val-Tyr (tBu)-OH (1.5 g) was dissolved in DMF (1500 mL), DIEA (1.35 g) was added, and the mixture was named A; HBTU (1.49 g) was weighed and added to A, and the mixture was stirred for 2 hours after the dropwise addition was complete, and then the mixture was subjected to a control test; control test: LC-MS was used to detect the completion of the reaction of the raw material

[0132] S22, post-treatment: remove most of the reaction liquid DMF by suction, then add ice water (80 mL) while stirring, precipitate the solid, after stirring for 10 min, filter it, dissolve the solid with EA (40 mL), wash twice with saturated NaHC03aqueous solution, wash once with saturated brine, dry with anhydrous sodium sulfate, filter and evaporate to dryness to obtain Cyclo (Arg (Pbf) -Lys (Boc) -Asp (OtBu) -Val-Tyr (tBu) ) 1 g, yield 67.7%.

[0133] S3, the synthesis of the cyclic peptide is as follows:

[0134] S31, dosing: weigh Cyclo (Arg (Pbf) -Lys (Boc) -Asp (OtBu) -Val-Tyr (tBu) ) 1 g with cutting E liquid, control the temperature at 30°C and stir the reaction.

[0135] S32, post-treatment: add the reaction liquid dropwise into ice ether to precipitate, shake while adding, centrifuge and wash the solid 3 times, evaporate the solid to dryness to obtain Cyclo (Arg-Lys-Asp-Val-Tyr) 0.7 g, yield 87.5%, detect LC-MS, and send for purification.

[0136] Purification conditions:

[0137] Dissolution: take 0.7 g of the crude product and dilute with 100 mL of water;

[0138] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0139] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0140] Equilibrium: A: B = 100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0141] Loading: flow rate: 60 mL / min;

[0142] Elution: 0-20% B for 60 min;

[0143] Column cleaning: clean with 80% acetonitrile to the baseline equilibrium;

[0144] Collect the qualified product and freeze-dry to obtain 156 mg. The LC chart of the purified product is shown in Figure 7 , and the MS chart of the product after purification is shown in Figure 8 .

[0145] Example 5: Preparation method of Cyclo (Ser-Val-Val-Val-Arg-Thr)

[0146] S1, the synthesis steps of linear peptide are as follows:

[0147] S11, the CTC resin (5.58 g, 6.25 mmol) is placed in a 100 mL solid-phase synthesis reactor, the amino acid Fmoc-Arg (Pbf)-OH (8.11 g, 12.5 mmol) is added, dichloromethane (DCM) 20 mL is added, DIEA (5.0 mL) is added, and the reaction is carried out at 25°C for 3 hours, 6 mL of methanol is added, and the reaction is carried out for 5 minutes. Filtration, the resin is washed with dichloromethane (DCM) 50 mL twice, methanol 50 mL twice, and DMF 50 mL twice. 20% Pip / DMF solution 50 mL is added, stirred for 30 min, filtered, and the deprotection solution is removed, then washed with DMF solution 50 mL for 6 times, and dried for standby.

[0148] S12, Fmoc-Val-OH (5.09 g, 15 mmol) and HOBt (2.03 g, 15 mmol) are taken in a 50 mL beaker, cooled to 5°C, 5 mL of DMF solution is added, DIC (2.3 mL, 7.5 mmol) is added, and the solution in the 100 mL beaker is added to the 100 mL solid-phase synthesis reactor, stirred for 1.5 hours, and the reaction is completed. The resin is washed with DMF solution three times, each time 20 mL. After washing, the next step is carried out. 20% Pip / DMF solution 50 mL is added, stirred for 30 min, filtered, and the deprotection solution is removed, then washed with DMF solution 50 mL for 6 times, and dried for standby.

[0149] The above S12 step is repeated, and the Fmoc-Val-OH is replaced by the following amino acid reagents in order: Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Ser (tBu)-OH, Fmoc-Thr (tBu)-OH; After the deprotection treatment is completed, then washed with methanol 50 mL twice, DCM solution 20 mL twice, methanol 50 mL twice, and vacuum dried to obtain H-Thr (tBu)-Ser (tBu)-Val-Val-Val-Arg (Pbf)-CTC-resin. The above peptide resin is stirred with 30% TFE / DCM solution 100 mL at 30°C for 30 minutes, filtered, and the resin is removed to obtain the filtrate. The filtrate is dried to obtain the fully protected polypeptide H-Thr (tBu)-Ser (tBu)-Val-Val-Val-Arg (Pbf)-OH 3.96 g, with a yield of 79% and a purity of 92.1%.

[0150] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0151] S21, feeding: H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -OH (1 g) was weighed and dissolved in DMF (1000 mL), DIEA (1.48 g) and DCM (10 mL) were added, and the mixture was named A; HBTU (1.12 g) was added to A, and after dropwise addition was completed, the mixture was stirred for 2 hours, and then subjected to intermediate control; intermediate control: LC-MS was used to detect the completion of the reaction of the raw material.

[0152] S22, post-treatment: most of the reaction liquid was removed by drying, then ice water (30 mL) was added while stirring, but no solid was precipitated, the reaction liquid was added to DCM (20 mL), and then the mixture was separated by extraction, and then the mixture was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine, and then the mixture was dried over anhydrous sodium sulfate, filtered, and evaporated to obtain Cyclo (Ser (tBu) -Val-Val-Val-Arg (Pbf) -Thr (tBu) ) 0.66 g.

[0153] S3, the synthesis of the cyclic peptide is as follows:

[0154] S31, feeding: Cyclo (Ser (tBu) -Val-Val-Val-Arg (Pbf) -Thr (tBu) ) 0.66 g was weighed and dissolved in cleavage E solution, and the mixture was stirred at 30°C; intermediate control: sample detection MS, and the reaction was basically complete.

[0155] S32, post-treatment: the reaction liquid was added dropwise to ice ether and settled, and the solid was washed three times by centrifugation, and then the solid was evaporated to obtain Cyclo (Ser-Val-Val-Val-Arg-Thr) 0.28 g, which was detected by LC-MS and sent for purification.

[0156] Purification conditions:

[0157] Dissolution: 0.28 g of the crude product was diluted with 100 mL of water;

[0158] Filler: 21.2*250mm, 10-120, C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0159] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0160] Equilibrium: A: B = 100:0, equilibrium 10 min, flow rate: 10 mL / min;

[0161] Loading: flow rate: 10 mL / min;

[0162] Elution: 4-24% B 60 min;

[0163] Column cleaning: 80% acetonitrile was used to clean the column to the baseline equilibrium;

[0164] The collected product was lyophilized to give 28 mg. The LC chart of the product purification is shown in Figure 9 and the MS chart of the product after purification is shown in Figure 10 .

[0165] Example 6: Preparation method of Cyclo (Glu-Glu-Met-Gln-Arg-Arg)

[0166] S1, the synthesis steps of linear peptide are as follows:

[0167] S11, the CTC resin (2.23 g, 2.5 mmol) was placed in a 100 mL solid-phase synthesis reactor, and the amino acid Fmoc-Met-OH (1.85 g, 5 mmol) was added, 20 mL of dichloromethane (DCM) was added, 2.0 mL of DIEA was added, and the reaction was carried out at 25°C for 3 hours, 3 mL of methanol was added, and the reaction was carried out for 5 minutes. Filtration, the resin was washed with dichloromethane (DCM) 20 mL for 2 times, methanol 20 mL for 2 times, and DMF 20 mL for 2 times. 20% Pip / DMF solution 20 mL was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with DMF solution 20 mL for 6 times, and then dried by suction.

[0168] S12, Fmoc-Glu (otBu)-OH (2.55 g, 6 mmol) and HOBt (0.81 g, 6 mmol) were taken in a 50 mL beaker, cooled to 5°C, 5 mL of DMF solution was added, DIC (0.93 mL, 6 mmol) was added and the solution was allowed to react for 15 minutes, and then the solution in the 100 mL beaker was added to the 100 mL solid-phase synthesis reactor, and the reaction was stirred for 1.5 hours, and the reaction was completed. The resin was washed with DMF solution for three times, each time 20 mL. After washing, the next step reaction was carried out. 20% Pip / DMF solution 20 mL was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with DMF solution 20 mL for 6 times, and then dried by suction.

[0169] Repeat the above S12 step, in order to replace Fmoc-Glu (otBu)-OH with the following amino acid reagents: Fmoc-Glu (otBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Gln (Trt)-OH; after the deprotection treatment is complete, then washed with methanol 20 mL twice, DCM solution 20 mL twice, methanol 20 mL twice, vacuum drying, to obtain H-Gln (Trt)-Arg (Pbf)-Arg (Pbf)-Glu (otBu)-Glu (otBu)-Met-CTC-resin. The above peptide resin is stirred with 30% TFE / DCM solution 40 mL at 30°C for 30 minutes, filtered, remove the resin, to obtain the filtrate. The filtrate is pulled dry to obtain the fully protected polypeptide H-Gln (Trt)-Arg (Pbf)-Arg (Pbf)-Glu (otBu)-Glu (otBu)-Met-OH 2.0 g, yield 58.8%, purity 95.6%.

[0170] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0171] S21, feeding: weigh H-Gln (Trt)-Arg (Pbf)-Arg (Pbf)-Glu (otBu)-Glu (otBu)-Met-OH (1.62 g), dissolve in DMF (1620 mL), add DIEA (0.98 g), and reserve for use, named A; weigh HBTU (1.08 g) and add to A, after dropwise addition is complete, stir for 2 hours, and directly perform a control; control: LC-MS detects that the raw material is completely reacted.

[0172] S22, post-treatment: pull dry to remove most of the DMF in the reaction liquid, then add ice water (30 mL) while stirring, precipitate the solid, stir for 10 min, then filter, dissolve the solid in EA (15 mL), wash with saturated NaHCO3 aqueous solution twice, and saturated brine once, dry over anhydrous sodium sulfate, filter, and evaporate to dryness to obtain Cyclo (Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (Pbf)-Arg (Pbf)) 1.57 g, yield 98%.

[0173] S3, the synthesis steps of the cyclic peptide are as follows:

[0174] S31, feeding: weigh Cyclo (Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (Pbf)-Arg (Pbf)) 1.57 g, stir at 30°C for 2 hours with cleavage E liquid; control: take a sample for MS detection, and the reaction is basically complete.

[0175] S32, post-treatment: the reaction solution was added dropwise into ice-ether for precipitation, shaking while adding, centrifuged for 3 times, and dried in a vacuum drying oven for 16 hours to obtain Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala) 0.98 g, with a yield of 99.9%, and LC-MS detection, sent for purification.

[0176] Purification conditions:

[0177] Dissolution: 1.5 g of the crude product was diluted with 200 mL of water;

[0178] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0179] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0180] Equilibrium: A:B=100:0, equilibrated for 10 min, flow rate: 60 mL / min;

[0181] Loading: flow rate: 60 mL / min;

[0182] Elution: 0-20% B for 60 min;

[0183] Column cleaning: 80% acetonitrile was used to clean to the baseline equilibrium;

[0184] The purified product was collected and freeze-dried to obtain 200 mg. The LC chart of the purified product is shown in Figure 11 , and the MS chart of the product after purification is shown in Figure 12 .

[0185] Example 7: Preparation method of Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala)

[0186] S1, the synthesis steps of the linear peptide are as follows:

[0187] S11, CTC resin (2.23 g, 2.5 mmol) was placed in

[0188] a 100 mL solid-phase synthesis reactor, amino acid Fmoc-Met-OH (1.85 g, 5 mmol) was added, dichloromethane (DCM) 20 mL was added, DIEA (2.0 mL) was added, and the reaction was carried out at 25°C for 3 hours, 3 mL of methanol was added, and the reaction was carried out for 5 minutes. Filtration, the resin was washed with dichloromethane (DCM) 20 mL for 2 times, methanol 20 mL for 2 times, and DMF 20 mL for 2 times. 20% Pip / DMF solution 20 mL was added, and the reaction was stirred for 30 min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with DMF solution 20 mL for 6 times, and then dried by suction for use.

[0189] S12, Fmoc-Glu(otBu)-OH (2.55 g, 6 mmol), HOBt (0.81 g, 6 mmol) were taken in a 50 mL beaker, cooled to 5°C, 5 mL of DMF solution was added, DIC (0.93 mL, 6 mmol) was added and the reaction was allowed to stand for 15 minutes, and the solution in the 100 mL beaker was added to the 100 mL solid phase synthesis reactor, and the reaction was stirred for 1.5 hours, and the reaction was completed. The resin was washed with DMF solution three times, 20 mL each time. After the washing was completed, the next step was carried out. 20 mL of 20% Pip / DMF solution was added, and the reaction was stirred for 30 min, and the deprotection solution was removed by suction filtration, and then the resin was washed with 20 mL of DMF solution for 6 times, and then suction dried for use.

[0190] The above S12 step was repeated, and the following amino acid reagents were sequentially replaced for Fmoc-Glu(otBu)-OH: Fmoc-Glu(otBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH; after the deprotection treatment was completed, then washed with 20 mL of methanol twice, 20 mL of DCM solution twice, 20 mL of methanol twice, and vacuum dried to obtain H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-CTC-resin. The above peptide resin was stirred with 40 mL of 30% TFE / DCM solution at 30°C for 30 minutes, filtered, and the resin was removed to obtain a filtrate. The filtrate was dried to obtain a fully protected peptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH 1.79 g, with a yield of 50.8% and a purity of 89.8%.

[0191] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0192] S21, the amount of H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH (1.56 g) was weighed, dissolved with DMF (1500 mL), DIEA (0.9 g) was added, and the mixture was named A; HBTU (0.99 g) was weighed and added to A, and the reaction was stirred for 2 hours after the dropwise addition was completed, and then the reaction was directly controlled; control: LC-MS detection of the raw material reaction was complete.

[0193] S22, post-treatment: remove most of the reaction liquid DMF by suction, then add ice water (36 mL) while stirring, precipitate the solid, after stirring for 10 min, filter it, dissolve the solid with EA (18 mL), wash twice with saturated NaHC03aqueous solution, wash once with saturated brine, dry with anhydrous sodium sulfate, filter and evaporate to dryness to obtain Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala) 1.36 g, yield 88%.

[0194] S3, the synthesis of the cyclic peptide is as follows:

[0195] S31, dosing: weigh Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala) 1.36 g, add cleavage E solution, control the temperature at 30°C, stir for 2 hours; control: take a sample for MS detection, the reaction is basically complete.

[0196] S32, post-treatment: add the reaction liquid dropwise into ice ether to precipitate, shake while adding, centrifuge and wash the solid 3 times, dry in a vacuum drying oven for 16 hours to obtain Cyclo(Glu-Glu-Met-Gln-Arg-Arg-Ala) 1.15 g, yield 132%, send for purification.

[0197] Purification conditions:

[0198] Dissolution: take 1.5 g of the crude product, dilute with 200 mL of water;

[0199] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0200] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0201] Equilibrium: A:B=100:0, equilibrate for 10 min, flow rate: 60 mL / min;

[0202] Loading: flow rate: 60 mL / min;

[0203] Elution: 0-20% B for 60 min;

[0204] Column cleaning: clean with 80% acetonitrile to the baseline equilibrium;

[0205] Collect the qualified product and freeze-dry to obtain 154 mg. The LC chart of the purified product is shown in Figure 13 , and the MS chart of the product after purification is shown in Figure 14 .

[0206] Example 8: Preparation method of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp)

[0207] S1, the synthesis steps of linear peptide are as follows:

[0208] S11, the CTC resin (2.23 g, 2.5 mmol) is placed in a 100 mL solid-phase synthesis reactor, and the amino acid Fmoc-Met-OH (1.85 g, 5 mmol) is added, 20 mL of dichloromethane (DCM) is added, 2.0 mL of DIEA is added, and the reaction is carried out at 25°C for 3 hours, 3 mL of methanol is added, and the reaction is carried out for 5 minutes. Filtration, the resin is washed with dichloromethane (DCM) 20 mL for 2 times, methanol 20 mL for 2 times, and DMF 20 mL for 2 times. 20% Pip / DMF solution 20 mL is added, and the reaction is stirred for 30 min, and then the deprotection solution is removed by suction filtration, and then the resin is washed with DMF solution 20 mL for 6 times, and then dried by suction for use.

[0209] S12, Fmoc-Glu (otBu)-OH (2.55 g, 6 mmol) and HOBt (0.81 g, 6 mmol) are taken in a 50 mL beaker, cooled to 5°C, 5 mL of DMF solution is added, DIC (0.93 mL, 6 mmol) is added, and the reaction is carried out for 15 min, and then the solution in the 100 mL beaker is added to the 100 mL solid-phase synthesis reactor, and the reaction is carried out for 1.5 hours, and the reaction is completed. The resin is washed with DMF solution for 3 times, each time 20 mL. After washing, the next step reaction is carried out. 20% Pip / DMF solution 20 mL is added, and the reaction is stirred for 30 min, and then the deprotection solution is removed by suction filtration, and then the resin is washed with DMF solution 20 mL for 6 times, and then dried by suction for use.

[0210] Repeat the above S12 step, in order to replace Fmoc-Lys (Boc) -OH with the following amino acid reagent: Fmoc-Glu (otBu) -OH, Fmoc-Asp (otBu) -OH, Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH; after the completion of the deprotection treatment, then washed with methanol 20 mL 2 times, DCM solution 20 mL 2 times, methanol 20 mL 2 times, vacuum drying, to get H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin. The above peptide resin with 30% TFE / DCM solution 40 mL, 30 ℃ stirring reaction 30 min, filter, remove the resin, to get the filtrate. The filtrate is drawn to get the full protection of polypeptide H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (otBu) -Glu (otBu) -Glu (otBu) -Met-OH 2.83 g, yield 73.1%, purity 93.3%.

[0211] S2, the synthesis of the cyclic peptide with a protecting group is as follows:

[0212] S21, the feeding: H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (otBu) -Glu (otBu) -Glu (otBu) -Met-OH (1.5 g) is weighed and dissolved in DMF (1500 mL), DIEA (1 mL) is added, and the mixture is named A; HBTU (0.87 g) is weighed and added to A, and after dropwise addition, the mixture is stirred for 2 hours, and then subjected to the control; the control: LC-MS is used to detect the completion of the reaction of the raw material.

[0213] S22, the post-treatment: most of the DMF in the reaction solution is removed by drawing, then ice water (40 mL) is added while stirring, and the solid is precipitated, which is filtered after stirring for 10 min, the solid is dissolved in EA (20 mL), washed with saturated NaHCO3 aqueous solution twice, washed with saturated brine once, dried over anhydrous sodium sulfate, filtered and evaporated to dryness to obtain Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (OtBu) ) 0.94 g, yield 63%.

[0214] S3, the synthesis of the cyclic peptide is as follows:

[0215] S31, dosing: Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala- Asp(OtBu)) 0.94 g was weighed and reacted with cleavage E solution at 30 °C with stirring. Mid-control: sample was taken for MS detection, and the reaction was substantially complete.

[0216] S32, post-treatment: the reaction solution was added dropwise to ice ethyl ether and settled, and the solid was washed 3 times by centrifugation and dried by evaporation to obtain Cyclo(Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val) 0.52 g, with a yield of 86%, which was detected by LC-MS and sent for purification.

[0217] Purification conditions:

[0218] Dissolution: 0.52 g of the crude product was diluted with 100 mL of water;

[0219] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0220] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0221] Equilibrium: A:B=100:0, equilibrated for 10 min, flow rate: 60 mL / min;

[0222] Loading: flow rate: 60 mL / min;

[0223] Elution: 0-20% B for 60 min;

[0224] Column cleaning: 80% acetonitrile was used to clean the column to baseline equilibrium;

[0225] The purified product was collected and freeze-dried to obtain 170 mg. The LC chart of the purified product is shown in Figure 15 , and the MS chart of the product after purification is shown in Figure 16 .

[0226] Example 9: Preparation method of Cyclo(Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val)

[0227] S1, the synthesis steps of the linear peptide are as follows:

[0228] S11, CTC resin (2.77 g, 2 mmol) was placed in a 250 mL solid-phase synthesis reactor, and amino acid Fmoc-Pro-OH (0.674 g, 2 mmol) was added, 120 mL of dichloromethane (DCM) was added, 8.7 mL of DIEA was added, and the reaction was carried out at 25°C for 3 hours, 12.5 mL of methanol was added, and the reaction was carried out for 5 minutes. Filtration, the resin was washed with dichloromethane (DCM) 75 mL for 2 times, methanol 75 mL for 2 times, DMF 75 mL for 2 times. 25 mL of 20% Pip / DMF solution was added, and the reaction was stirred for 30 min, and the deprotection solution was removed by filtration, and then washed with DMF solution 130 mL for 6 times, and dried by suction for use.

[0229] S12, Fmoc-Lys (Boc)-OH (2.814 g, 6 mmol), HOBt (0.81 g, 6 mmol) were taken in a 100 mL beaker, cooled to 5°C, 25 mL of DMF solution was added, DIC (3.2 mL, 21 mmol) was added and the solution in the 100 mL beaker was added to the 250 mL solid-phase synthesis reactor, and the reaction was stirred for 1.5 hours, and the reaction was completed. The resin was washed with DMF solution for 3 times, 25 mL each time. After washing, the next step was carried out. 25 mL of 20% Pip / DMF solution was added, and the reaction was stirred for 30 min, and the deprotection solution was removed by filtration, and then washed with DMF solution 25 mL for 6 times, and dried by suction for use.

[0230] The above S12 step was repeated, and Fmoc-Lys (Boc)-OH was replaced by the following amino acid reagents in order: Fmoc-Phe-OH, Fmoc-D-Trp (Boc)-OH, Fmoc-Arg (pbf)-OH, Fmoc-D-Phe-OH, Fmoc-Pro-OH, Fmoc-Met-OH, Fmoc-Val-OH; After the deprotection treatment was completed, then washed with methanol 50 mL for 2 times, DCM solution 50 mL for 2 times, methanol 50 mL for 2 times, vacuum dried, to obtain H-Val-Met-Pro-{D-Phe}-Arg (Pbf)-{D-Trp} (Boc)-Phe-Lys (Boc)-Pro-CTC-resin. The above peptide resin was cut with 30% TFE / DCM cutting solution 40 mL, and the reaction was stirred at 30°C for 2.5 hours, filtered, and the resin was removed to obtain the filtrate. The filtrate was dried to obtain the crude peptide H-Val-Met-Pro-{D-Phe}-Arg (Pbf)-{D-Trp} (Boc)-Phe-Lys (Boc)-Pro-OH 2.1 g. Yield 64.02%, purity 95.3%.

[0231] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows:

[0232] S21, dosing: H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH (1 g) was weighed and dissolved in DMF (1000 mL), DIEA (0.64 g) was added, and the mixture was reserved as sample A; HBTU (0.65 g) was added to sample A, and after dropwise addition was completed, the mixture was stirred for 2 hours, and then subjected to intermediate control; intermediate control: LC-MS was used to detect the completion of the reaction of the raw material.

[0233] S22, post-treatment: most of the DMF in the reaction solution was removed by suction, and then ice water (30 mL) was added while stirring, and a solid was precipitated; after stirring for 10 min, the solid was filtered, and the solid was dissolved in EA (20 mL), washed twice with saturated NaHCO3 aqueous solution, and once with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness to obtain Cyclo(Met-Pro-D-Phe-Arg(Pbf)-D-Trp(Boc)-Phe-Lys(Boc)-Pro-Val) 0.85 g, with a yield of 86%.

[0234] S3, the synthesis of the cyclic peptide was as follows:

[0235] S31, dosing: Cyclo(Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(Pbf)-Arg(Pbf)) 0.85 g was weighed and dissolved in cleavage E solution, and the mixture was stirred at 30°C; intermediate control: sample was taken for MS detection, and the reaction was basically complete.

[0236] S32, post-treatment: the reaction solution was added dropwise to ice ether, and the mixture was shaken while adding dropwise; the solid was washed three times by centrifugation, and then dried in a vacuum drying oven for 16 hours to obtain Cyclo(Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val) 0.62 g, with a yield of 84.6%; LC-MS was used for detection, and the sample was sent for purification.

[0237] Purification conditions:

[0238] Dissolution: 0.62 g of the crude product was dissolved in 160 mL of water and 10 mL of acetic acid by ultrasonic dissolution;

[0239] Filler: 50DAC10-100C18; flow rate: 60 mL / min; wavelength: 220 nm;

[0240] Mobile phase: A: 1% acetic acid; B: acetonitrile;

[0241] Equilibrium: A:B=95:5, equilibrium for 10 min, flow rate: 60 mL / min;

[0242] Load: Flow rate: 60 mL / min;

[0243] Elution: 16-36%B 60 min;

[0244] Column clean-up: 80% acetonitrile clean-up to baseline equilibration;

[0245] The collected product was lyophilized to obtain 160 mg. The LC chart of the purified product is shown in Figure 17 , and the MS chart of the product after purification is shown in Figure 18 .

[0246] Test Example:

[0247] Moisturizing test method: AQP3 (aquaporin 3) content test

[0248] (1) Cell inoculation: inoculate cells into a 24-well plate and incubate in an incubator (37°C, 5% CO2) overnight.

[0249] (2) Solution preparation: prepare the test substance working solution according to the experimental design.

[0250] Table 1 AQP3 experimental design table

[0251]

[0252] The diluent in the AQP3 experiment is a cell culture solution, for example, DMEM medium. The concentration of the sample group is 0.063 mg / mL, 0.125 mg / mL, 0.25 mg / mL; the sample group is selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, PR157.

[0253] (3) Add test substance: after incubation in the incubator (37°C, 5% CO2) for 24 h, add the test substance according to the table and continue to incubate for 24 h.

[0254] (4) Sample collection: discard the supernatant and rinse the cells with PBS 3 times.

[0255] (5) Immunofluorescence staining:

[0256] a. Add methanol to fix the cells, rinse with PBS 3 times, and add 1 mL of BSA per well for blocking for 1 h.

[0257] b. Discard the blocking solution, add the primary antibody per well, and place in a 4°C refrigerator overnight. Discard the primary antibody and rinse with PBS 3 times.

[0258] c. Add the secondary antibody per well and act for 2 h. Discard the secondary antibody and rinse with PBS 3 times.

[0259] d. Add DAPI to each well for nuclear staining, act for 10 min, discard DAPI, rinse with PBS for 3 times, and then take photos by using a fluorescence microscope.

[0260] (6) Result analysis: the AQP3 fluorescence intensity was quantitatively analyzed by using ImageProPlus software.

[0261] Table 2 AQP3 content test results

[0262]

[0263] The present application can obtain the following conclusions through tests: the PR095 sample has a moisturizing effect at the concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. The PR100 sample has a moisturizing effect at the concentrations of 0.063 mg / mL and 0.25 mg / mL. The PR102 sample, the PR116 sample, the PR120 sample, the PR126 sample, the PR132 sample, the PR139 sample, the PR140 sample, the PR146 sample and the PR157 sample all have a moisturizing effect at the concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL.

[0264] Moisturizing test method: HA (hyaluronic acid) content test

[0265] (1) Cell inoculation: inoculate cells into a 24-well plate, and incubate in an incubator (37℃, 5% CO2) overnight.

[0266] (2) Liquid preparation: prepare the working solution of the test substance according to the experimental design.

[0267] Table 3 HA experimental design table

[0268]

[0269] The diluent in the experiment is a cell culture solution, for example, a DMEM culture medium. The concentration of the sample group is 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL; the sample group is selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095 and PR157.

[0270] (3) Add the test substance: after 24 h of culture in an incubator (37℃, 5% CO2), add the test substance according to the table, and continue to culture for 24 h.

[0271] (4) Sample collection: collect the supernatant, and determine the HA content by using an ELISA kit.

[0272] Table 4 HA content test results

[0273]

[0274] The present application can be concluded as follows through tests: the PR095 sample has moisturizing efficacy at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. The PR100 sample, the PR102 sample, the PR116 sample, the PR120 sample, the PR132 sample, the PR139 sample, the PR140 sample, the PR146 sample and the PR157 sample have moisturizing efficacy at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. The PR126 sample has moisturizing efficacy at concentrations of 0.063 mg / mL and 0.125 mg / mL.

[0275] Anti-wrinkle firming efficacy test method: Type I collagen and MMP-1 content test

[0276] (1) Cell inoculation: inoculate cells into a 24-well plate and incubate in an incubator (37°C, 5% CO2) overnight.

[0277] (2) Solution preparation: prepare the working solution of the test substance according to the experimental design.

[0278] Table 5. Experimental design table

[0279]

[0280] The diluent in the experiment is a cell culture solution, for example, a DMEM medium. The concentration of the sample group is 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL; the sample group is selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095 and PR157. In the test, Collagen I is Type I collagen; UVA is ultraviolet A; VC is vitamin C; and VE is vitamin E.

[0281] (3) UVA radiation: after 24 hours of culture, the negative control group, the positive control group and the sample group receive a total dose of 9 J / cm2of UVA radiation, while the blank control group is placed in the same environment (UVA radiation dose is 0 J / cm2).

[0282] (4) Add the test substance: according to the experimental design, after irradiation, add the test substance to the groups, 1 mL of cell culture solution is added to each well of the blank control group and the negative control group; 1 mL of cell culture solution containing vitamin C and vitamin E is added to each well of the positive control group; 1 mL of culture solution containing the corresponding concentration of the test substance is added to each well of the sample group; after adding the test substance, place the 24-well plate in the incubator (37 DEG C, 5% CO2) for 24 h.

[0283] (5) Collect the supernatant for type I collagen and MMP-1 content determination.

[0284] (6) Result analysis: t-test statistical analysis is used for comparison between groups, and statistical analysis is two-tailed.

[0285] Table 6 Type I collagen content test results

[0286]

[0287] The present application can obtain the following conclusions through testing: the PR095 sample, the PR100 sample, the PR102 sample, the PR116 sample, the PR120 sample, the PR126 sample, the PR132 sample, the PR139 sample, the PR140 sample, the PR146 sample and the PR157 sample have anti-wrinkle firming efficacy at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL.

[0288] Soothing test method: IL-6 content test

[0289] (1) Cell inoculation: inoculate cells into a 24-well plate and incubate in an incubator (37 DEG C, 5% CO2) overnight.

[0290] Table 7 IL-6 synthesis experimental design table

[0291]

[0292] The diluent in the experiment is cell culture solution, for example, DMEM culture medium. The concentration of the sample group is 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL; the sample group is selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095 and PR157. IL-6 in the test is interleukin-6 (cytokine), and LPS is lipopolysaccharide.

[0293] (2) Add the test substance: according to the experimental grouping, when the cell plating rate in the 24-well plate reaches 40%~60%, add the test substance to the groups, 3 replicate wells are set for each group, and the 24-well plate is placed in the incubator (37 DEG C, 5% CO2) for 24 h.

[0294] (3) Detection: After 24 hours of incubation, the supernatant was collected and the IL-6 content was determined using an ELISA kit.

[0295] Table 8. Results of IL-6 content test

[0296]

[0297] The present application has been tested and the following conclusions can be drawn: the PR095 sample has soothing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL. The PR100 sample has soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. The PR102 sample, the PR116 sample, the PR120 sample, the PR126 sample, the PR132 sample, the PR139 sample, the PR140 sample, the PR146 sample have soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. The PR157 sample has soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL.

[0298] Test method for inhibiting the release of neurotransmitters: norepinephrine test

[0299] (1) Cell seeding: Cells were seeded into 24-well plates and incubated in an incubator (37°C, 5% CO2) for 6 days.

[0300] (2) Cell treatment: To study the inhibition of NA release, the culture medium was discarded and the cells were washed with HBSS. Before inducing exocytosis, the cells were pre-incubated for 60 minutes with the sample to be tested at different concentrations dissolved in HBSS. The supernatant was removed and the induction of norepinephrine release was carried out according to the protocol described below.

[0301] The diluent in the experiment was cell culture medium, for example DMEM medium. The concentrations of the sample groups were 0.063 mg / mL, 0.125 mg / mL, 0.25 mg / mL; the sample groups were selected from PR102.

[0302] (3) Induction of human norepinephrine release: The induction of norepinephrine (NA) was carried out using an 8-minute treatment with TPA-containing HBSS, removal of TPA and continued incubation for 5 minutes in TPA- and ION-containing HBSS. This group was the positive control of the test. The basal control group was treated with 100 nM TPA for 8+5 minutes, the rest of the steps being the same as in the positive control group. Immediately after incubation, the supernatant containing the released NA was collected and stored at -80°C until further analysis by ELISA.

[0303] (4) Extraction of norepinephrine (NA) from supernatant: On the day of assay, samples were thawed at room temperature for 1 hour, and NA samples were extracted using a 24-well plate coated with a borate gel. The samples were first incubated with extraction buffer, then washed with double distilled water, then incubated with acylation reagent in extraction buffer, and finally washed with double distilled water. Finally, the samples were quantified for NA using ELISA after the addition of release buffer.

[0304] (5) NA content was determined by ELISA.

[0305] (6) Data analysis: t-test statistical analysis was used for comparison between groups, and statistical analysis was two-tailed.

[0306] Table 9 Test results

[0307]

[0308] The present application tested PR-102 as described above, and the PR-102 sample at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL can inhibit the release of neurotransmitter norepinephrine. It is shown that the PR-102 sample can inhibit the release of neurotransmitter norepinephrine.

[0309] Melanin content test

[0310] Table 10 Melanin content experimental design table

[0311]

[0312] The diluent in the experiment is cell culture medium, such as DMEM medium. The concentration of the sample group is 0.063 mg / mL, 0.125 mg / mL, 0.25 mg / mL; the sample group is selected from PR116.

[0313] Logarithmic growth phase cells were collected and seeded into 24-well plates, and after 24 h of incubation in an incubator (37°C, 5% CO2), according to the cytotoxicity results, the test substances were added according to the table, with untreated cells as a blank control, and 3 parallel groups were set for each group.

[0314] After drug addition, continue to incubate in an incubator (37°C, 5% CO2) for 24 h, discard the supernatant, add 0.5 mL of 1M NaOH containing 10% DMSO, and incubate at 80°C for 1 h. 1M NaOH containing 10% DMSO was used as a solvent control, and the absorbance value was read under a microplate reader and the relative inhibition rate of melanin in cells was calculated.

[0315]

[0316] Table 11 Melanin content test results

[0317]

[0318] The present application carried out the above test on PR116, the PR-116 sample has whitening efficacy at 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL concentrations. It is shown that the PR-116 sample has whitening efficacy.

[0319] The above embodiments are only used to illustrate the present application, and not to limit the present application, those skilled in the art, without departing from the spirit and scope of the present application, can also make various changes and modifications. Therefore, all equivalent technical solutions also belong to the scope of the present application, the patent protection scope of the present application should be limited by the claims.

[0320] 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, and any person skilled in the art can make some changes or modifications as other equivalent embodiments without departing from the scope of the technical means disclosed in the present application, but should be considered as the same as the present application.

[0321] The principles and implementations of the present application are described by using specific examples in this paper, and the above embodiment description is only used to help understand the method of the present application and its core idea. The above-mentioned is only the preferred embodiment of the present application, it should be pointed out that, due to the limited nature of the expression, there are infinite specific structures, for those skilled in the art, without departing from the principles of the present application, can make some improvements, decoration or change, also can combine the above technical features in appropriate way; these improvements, decoration, change or combination, or without improvement, the application of the concept and technical scheme to other occasions, should be considered as the protection scope of the present application.

Claims

1. Use of cyclic heptapeptide-4 in the preparation of moisturizing and / or anti-wrinkle and / or firming and / or soothing products; The structure of cyclic heptapeptide-4 is shown below: ； The preparation of the cyclic heptapeptide-4 includes: A linear peptide was mixed with a cyclizing agent in a solvent and cyclized, followed by post-treatment to prepare cyclic heptapeptide-4. The linear peptide is prepared by solid-phase synthesis; or, the cyclizing agent is HBTU; or, the solvent is at least one of DMF, DCM and DIEA; or, the post-processing includes cyclization post-processing, cleavage post-processing and cleavage post-processing.

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