Cyclic pentapeptide-4 and its preparation method and application

Abstract

The present invention discloses a cyclic pentapeptide-4, a preparation method thereof, and an application thereof, belonging to the technical field of synthesis and application of cyclic peptides, and specifically relates to a linear peptide containing a protective group prepared by activating an amino acid reagent, and then preparing a functional cyclic peptide having a cyclic structure by cyclization. The functional cyclic peptide prepared by the present invention has good moisturizing effect, good anti-wrinkle effect, good firming effect, good soothing effect, good neurotransmitter release inhibition effect, and good whitening effect. After the linear peptide is prepared into a functional cyclic peptide, the present invention increases permeability, improves stability, improves activity, and reduces irritation.

Description

Technical Field

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

[0002] Peptides are composed of amino acids through dehydration condensation. These amino acids themselves contain carboxyl and amino groups and are generally hydrophilic compounds. The peptides composed of these amino acids generally have significant hydrophilicity and ionic properties. Small molecule peptides are particularly non-toxic, easily absorbed by the skin, and possess special physiological activities after absorption. They can fundamentally improve certain skin problems from the inside out, and are now widely used in beauty and cosmetic formulas. Currently, there are a variety of cosmetics and skin care products on the market, but most skin care products only add peptides with a certain type of efficacy as the active ingredient of the skin care product. As a result, the effects achieved on the skin are relatively simple and slow to take effect. Furthermore, most skin care products on the market that use peptides as active ingredients directly add peptides or peptide concentrates to conventional skin care product matrices. Due to considerations for the preparation and stability of the entire formula, the amount of peptide added is limited.

[0003] Conventional peptide modifications are usually performed using palmitoyl or nutmeg-modified methods. Peptides modified with palmitoyl or nutmeg generally have hydrophilicity and lipophilicity, which often greatly increase solvent consumption and costs during the separation and purification process. Summary of the Invention

[0004] The purpose of the present invention is to provide 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, as well as a preparation method and application thereof.

[0005] Using peptide cyclization, linear peptides are cyclized to produce cyclized peptides. This cyclization dehydrates and condenses the carboxyl and amino groups of the peptide to form new amide bonds, increasing the lipid solubility of the peptide without significantly increasing solvent consumption. Cyclized peptides are more stable than linear peptides. Furthermore, the condensation of carboxyl and amino or hydroxyl groups results in peptides with improved lipid solubility and skin permeability.

[0006] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

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

[0008] , where 1≤n, R m The number of R1, R2, R3 or R m is selected from H, aliphatic hydrocarbon, substituted aliphatic hydrocarbon, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidinyl, substituted guanidinyl, metal alkyl or alkyl forming a ring 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, R3 and R m Any two or more groups are the same; or, R1, R2, R3 and R m Any adjacent groups are the same; when R1, R2, R3 or R m When selected from -CH2-CH2-CH2-group, R1, R2, R3 or R m Connected with adjacent N to form a ring; or, R1, R2, R3 or R m Any one selected from a hydrogen atom group, a methyl group, a -CH2-CH2-CH2-group, a propyl group, a butyl group, a hydroxymethyl group, a hydroxyethyl group, an acetamido group, a propionamido group, a CH3-S-CH2-CH2-group, a HS-CH2-group, a Ph-CH2-group, a p-hydroxybenzyl group, an acetyl group, a propionic acid group, a Se-CH2-group, a butylamino group, an indolyl group, an imidazolemethyl group and a substituted guanidine group.

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

[0012] .

[0013] The method for preparing a functional cyclic peptide comprises: mixing a linear peptide and a cyclization reagent in a solvent to carry out a cyclization reaction, and post-processing to prepare a functional cyclic peptide; the functional cyclic peptide has the following structure:

[0014] , where 1≤n, R m The number of R1, R2, R3 or R m is selected from H, aliphatic hydrocarbon, substituted aliphatic hydrocarbon, aryl, substituted aryl, imidazolyl, substituted imidazolyl, indolyl, substituted indolyl, guanidinyl, substituted guanidinyl, metal alkyl or alkyl forming a ring structure with the adjacent nitrogen atom.

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

[0016] .

[0017] Preferably, the preparation method of the linear peptide is 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 includes cyclization post-treatment, cleavage treatment and cleavage post-treatment.

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

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

[0020] More preferably, in the post-cyclization treatment, ice water is added to the test solution after the cyclization reaction is completed, a solid is precipitated, stirred and filtered, the solid is dissolved with EA, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with a protective group; or, in the post-cyclization treatment, DCM is added to the test solution after the cyclization reaction is completed, layered extraction is performed, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with a protective group; or, in the post-cleavage treatment, the test solution after the cleavage treatment is added to ice ether, a solid is precipitated, centrifuged and washed, the solid is evaporated to dryness, and purified to obtain a cyclic peptide; or, LC-MS is used for monitoring the cyclization reaction; or, the solvent is at least one of DMF, DCM and DIEA, the solvent is based on DMF as the measurement standard, and the relationship between the amount of linear peptide and DMF used 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° C., and the mixture is allowed to react for 10-30 minutes 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 ratio of the amount of DMF to 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 includes 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, and Fmoc-D-Phe-OH.

[0025] Preferably, in the preparation of linear peptides, the first amino acid reagent is mixed with CTC resin, and then dichloromethane (DCM) and DIEA are added, and the reaction is carried out at 20-40°C for 2-5 hours, and then methanol is added and the reaction is carried out for 3-30 minutes. After the reaction is completed, it is filtered, washed, and treated with a deprotection solution; the amino acid activation solution is mixed with the CTC resin bonded with the first amino acid in the order of the linear peptide, and the reaction is carried out for 0.5-3 hours. After each reaction of the amino acid activation solution, the deprotection solution is treated. After the reaction of the last amino acid reagent is completed, the cutting solution is added to obtain a linear peptide.

[0026] More preferably, in the preparation of the linear peptide, the total degree of substitution in the CTC resin is the product of the degree of substitution and the mass of the CTC resin, that is, the total degree of substitution is the molar amount of the total active reaction sites in the CTC resin, and the molar amount of the first amino acid reagent used is 50-250% of the total degree of substitution in 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 based on the molar amount of the amino acid reagent therein, and the molar amount of the amino acid reagent used is 100-350% of the total degree of substitution in the CTC resin.

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

[0032] Linear peptides include 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)-Ar Any one of g(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, the washing after the first amino acid reacts with the CTC resin is carried out using dichloromethane (DCM), methanol and DMF in sequence.

[0034] More preferably, in the preparation of the linear peptide, when the amino acid activation solution is added in the order of the linear peptide of the cyclic peptide for reaction, DMF is used for washing after each amino acid activation solution reaction.

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

[0036] More preferably, in the preparation of the linear peptide, during the cleavage solution treatment, the cleavage solution is added to the CTC resin to which the amino acid reagent has been bonded, and the resin is treated at 20-40°C for 0.5-5 hours. After the treatment is complete, the resin is removed by filtration to obtain a filtrate, which is then dried to obtain the linear peptide. The cleavage solution is used in an appropriate amount.

[0037] Preferably, in the preparation of the cyclic peptide, a linear peptide is added to a solvent and mixed to obtain a linear peptide solution, and then the linear peptide solution is mixed with a cyclization reagent, and the reaction is carried out at 30-50°C, and the reaction is monitored by LC-MS. After the reaction is completed, a cyclization post-treatment is performed to obtain a cyclic peptide with a protective group, and a cutting solution is added for treatment, and finally a 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 may further contain at least one of DCM and DIEA, with DMF being used as the solvent basis. The amount of the linear peptide to DMF is 0.1-30 mg / mL, the amount of DCM to DMF is 0.1-10 mL / mL, and the amount of DIEA to DMF is 0.1-5 mg / mL. The cutting fluid is liquid E or liquid F, and the cutting fluid is used in an appropriate amount. The cyclization reagent is HBTU, and the amount of HBTU to DMF is 0.1-5 mg / mL.

[0039] More preferably, in the preparation of the cyclic peptide, solution E is prepared by mixing TFA, thioanisole, EDT, PhOH and water, and TFA, thioanisole, 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 composed of TFA, TIS and water, which are mixed in a volume ratio of 90:5:5; PR100,

[0041] Preferably, in the post-cyclization treatment, DMF is dried, and ice water is added to the test solution after the cyclization reaction is completed to precipitate a solid, stir and filter, dissolve the solid with EA, and then wash with saturated sodium bicarbonate solution and saturated brine in sequence, dry with anhydrous sodium sulfate, filter, and evaporate to dryness to obtain a cyclic peptide with a protective group.

[0042] Preferably, in the post-cyclization treatment, DMF is dried, DCM is added to the test solution after the cyclization reaction is completed, and the mixture is separated and extracted, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a cyclic peptide with a protective group.

[0043] Preferably, in the post-cleavage treatment, the test solution after the cleavage treatment is added to ice ether to precipitate the solid, which is washed by centrifugation, evaporated to dryness, and purified to obtain the cyclic peptide.

[0044] Purification was performed by liquid chromatography.

[0045] The present invention uses an amino acid reagent to activate a linear peptide containing a protective group, and then prepares a functional cyclic peptide with a cyclic structure through a cyclization process and a cleavage process. Therefore, it has the following beneficial effects: the functional cyclic peptide prepared by the present invention 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 invention 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, as well as its preparation method and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the PR126 chromatogram;

[0047] Figure 2 is the mass spectrum of PR126;

[0048] Figure 3 is the PR140 chromatogram;

[0049] Figure 4 is the mass spectrum of PR140;

[0050] Figure 5 is the PR100 chromatogram;

[0051] Figure 6 is the mass spectrum of PR100;

[0052] Figure 7 is the PR132 chromatogram;

[0053] Figure 8 is the mass spectrum of PR132;

[0054] Figure 9 is the PR120 chromatogram;

[0055] Figure 10 is the mass spectrum of PR120;

[0056] Figure 11 is the PR102 chromatogram;

[0057] Figure 12 is the mass spectrum of PR102;

[0058] Figure 13 is the PR139 chromatogram;

[0059] Figure 14 is the mass spectrum of PR139;

[0060] Figure 15 is the PR146 chromatogram;

[0061] Figure 16 is the mass spectrum of PR146;

[0062] Figure 17 is the PR116 chromatogram;

[0063] Figure 18 This is the mass spectrum of PR116. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described in detail below with reference to the specific embodiments and the accompanying drawings:

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

[0066] S1, the synthesis steps of 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 the amino acid Fmoc-Pro-OH (1.68 g, 5 mmol), and add 20 mL of dichloromethane (DCM). Add DIEA (2.0 mL) and react at 25°C for 3 hours. Add 3 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of a 20% Pip / DMF solution, stir, and react for 30 minutes. Filter to remove the deprotection solution, then wash the resin six times with 20 mL of DMF solution and drain.

[0068] In step S12, Fmoc-Tyr(tBu)-OH (2.76 g, 6 mmol) and HOBt (0.81 g, 6 mmol) were placed in a 50 mL beaker and cooled to 5°C. 5 mL of DMF solution and DIC (0.93 mL, 6 mmol) were added and allowed to react for 15 minutes. The solution in the 100 mL beaker was then added to a 100 mL solid-phase synthesis reactor and stirred for 1.5 hours. The reaction was complete. The resin was washed three times with 20 mL of DMF solution each time. After washing, the resin was processed for the next step. 20 mL of a 20% Pip / DMF solution was added and stirred for 30 minutes. The deprotection solution was removed by filtration. The resin was then washed six times with 20 mL of DMF solution and dried.

[0069] Repeat step S12 above, replacing Fmoc-Tyr(tBu)-OH with the following amino acid reagents in sequence: Fmoc-Phe-OH and Fmoc-Phe-OH. After deprotection, wash the resin twice with 20 mL of methanol, twice with 20 mL of DCM solution, and twice with 20 mL of methanol. Dry the resin under vacuum to obtain H-Phe-Phe-Tyr(tBu)-Pro-CTC-resin. The peptide resin was reacted with 30 mL of 30% TFE / DCM solution at 30°C with stirring for 30 minutes. The resin was then filtered and removed to obtain a filtrate. The filtrate was dried to obtain 1.10 g of the fully protected peptide H-Phe-Phe-Tyr(tBu)-Pro-OH, with a yield of 90% and a purity of 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 (1.02 g), dissolve it in DMF (1000 mL), add DIEA (1.674 g) and name it A. Weigh HBTU (1.84 g) and add it to A. After the addition is complete, stir the reaction for 2 hours and directly perform in-process control. In-process control: LC-MS detection shows that the raw material reaction is complete.

[0072] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry, and then ice water (30 mL) was added with stirring to precipitate a solid. After stirring for 10 min, the solid was filtered and dissolved with EA (20 mL). The solid was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 0.9 g of Cyclo(Tyr(tBu)-Pro-Phe-Phe).

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

[0074] S31, cleavage: Weigh 0.9 g of Cyclo(Tyr(tBu)-Pro-Phe-Phe) and add cleavage solution E. Stir and react at 30°C. In-process control: Samples were taken for MS analysis, indicating that the reaction was essentially complete.

[0075] S32, post-treatment: The reaction solution was added dropwise to icy ether for precipitation while shaking. The solid was washed by centrifugation three times and evaporated to dryness to obtain 0.68 g of Cyclo (Tyr-Pro-Phe-Phe). The product was detected by LC-MS and sent for purification.

[0076] The purification conditions are as follows:

[0077] Dissolution: Take 0.68g of crude product, add 30mL of acetic acid, 30mL of acetonitrile, and 100mL of water and dissolve by ultrasonication;

[0078] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0081] Sample loading: flow rate: 60 mL / min;

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

[0083] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

[0084] The qualified product was collected and freeze-dried to obtain 55 mg. The LC diagram of the product purification is as follows Figure 1 The MS pattern of the purified product is shown in Figure 2 shown.

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

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

[0087] S11: Place CTC resin (5.58 g, 6.25 mmol) in a 250 mL solid-phase synthesis reactor. Add the amino acid Fmoc-Tyr(tBu)-OH (0.459 g, 12.5 mmol) and 120 mL of dichloromethane (DCM). Add DIEA (8.7 mL) and react at 25°C for 3 hours. Add 12.5 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 75 mL of dichloromethane (DCM), twice with 75 mL of methanol, and twice with 75 mL of DMF. Add 65 mL of a 20% Pip / DMF solution, stir, and react for 30 minutes. Filter to remove the deprotection solution, then wash the resin six times with 120 mL of DMF and drain.

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

[0089] Repeat step S12 above, replacing Fmoc-Lys(Boc)-OH with the following amino acid reagents, Fmoc-Val-OH and Fmoc-Asp(OtBu)-OH, in order. After deprotection, wash the resin twice with 75 mL of methanol, twice with 75 mL of DCM, and twice with 75 mL of methanol. Dry the resin under vacuum to obtain H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-CTC-resin. Add 3.95 g of the peptide resin to 40 mL of 30% TFE / DCM cutting solution and stir at 30°C for 2.5 hours. Filter and remove the resin to obtain a filtrate. Drain the filtrate to obtain the crude peptide H-Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)-OH. The yield is 113% and the purity is 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 it in DMF (2000 mL), add DIEA (2.36 g), and reserve it as A. Weigh HBTU (2.6 g), add it to A, stir and react for 2 hours after the addition is complete, and then directly perform in-process control. In-process control: LC-MS detection shows that the raw material reaction is complete.

[0092] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry, and then ice water (30 mL) was added with stirring to precipitate a solid. After stirring for 10 min, the solid was filtered and dissolved with EA (20 mL). The solid was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 1.22 g of Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)), with a yield of 62.5%.

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

[0094] In step S31, weigh 1.22 g of Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)) and react with stirring in Cutting Solution E at 30°C. In-process control: Samples were taken for MS analysis, indicating that the reaction was essentially complete.

[0095] S32, post-treatment: The reaction solution was added dropwise to icy ether for precipitation while shaking. The solid was washed by centrifugation three times and evaporated to dryness to obtain 0.95 g of Cyclo(Asp-Val-Lys-Tyr) with a yield of 90%. The product was detected by LC-MS and sent for purification.

[0096] The purification conditions are as follows:

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

[0098] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0101] Sample loading: flow rate: 60 mL / min;

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

[0103] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

[0104] The qualified product was collected and freeze-dried to obtain 202 mg. The LC diagram of the product purification is as follows Figure 3 The MS pattern of the purified product is shown in Figure 4 shown.

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

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

[0107] In step S11, CTC resin (6.25 g, 7 mmol) was placed in a 250 mL solid-phase synthesis reactor. The amino acid Fmoc-Ser(tBu)-OH (8.42 g, 7 mmol) was added, along with 75 mL of dichloromethane (DCM). The mixture was reacted at 25°C for 3 hours, followed by DIEA (8.7 mL). 12.5 mL of methanol was added and allowed to react for 5 minutes. The mixture was filtered, and the resin was washed twice with 75 mL of dichloromethane (DCM), twice with 75 mL of methanol, and twice with 75 mL of DMF. 40 mL of a 20% Pip / DMF solution was added, and the reaction was stirred for 30 minutes. The deprotection solution was removed by filtration, and the mixture was then washed six times with 75 mL of DMF solution and dried.

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

[0109] Repeat the above S12 step, replacing Fmoc-Lys(Boc)-OH with the following amino acid reagents in order: Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, and Fmoc-Lys(Boc)-OH. After the deprotection treatment is completed, wash with 130 mL of methanol twice, 130 mL of DCM solution twice, and 130 mL of methanol twice, and dry in vacuo to obtain H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-CTC-resin. The above peptide resin is treated with 3.5 g of cleavage solution 30% TFE / DCM, 40 mL, and stirred at 30°C for 2.5 hours. The resin is then removed by filtration to obtain a filtrate. The filtrate was drained to obtain 1.42 g of crude peptide H-Lys(Boc)-Thr(tBu)-Thr(tBu)-Lys(Boc)-Ser(tBu)-OH, with a yield of 50.9% and a purity of 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 it with DMF (750 mL) + DCM (750 mL), add DIEA (2.94 g), and reserve it and name it A; weigh HBTU (1.73 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform in-process control; In-process control: LC-MS detects that the raw material reaction is complete.

[0112] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry, and then ice water (30 mL) was added with stirring. No solid was precipitated. DCM (20 mL) was added to the reaction solution, and the mixture was separated and extracted. The product was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine. The product was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 1.3 g of Cyclo (Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu)), with a yield of 93%.

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

[0114] S31, Feed: Weigh 1.22 g of Cyclo(Asp(OtBu)-Val-Lys(Boc)-Tyr(tBu)) and stir with Cutting Solution E at 30°C. In-process control: Samples were taken for MS analysis, indicating that the reaction was essentially complete.

[0115] S32, post-treatment: The reaction solution was added dropwise to glacial ether for precipitation with shaking during the addition. The solid was washed by centrifugation three times and evaporated to dryness to obtain 0.83 g of Cyclo (Lys-Thr-Thr-Lys-Ser) with a yield of 75.5%. The product was detected by LC-MS and sent for purification.

[0116] Purification conditions:

[0117] Dissolution: dilute 0.83 g of crude product with 100 mL of water;

[0118] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0121] Sample loading: flow rate: 60 mL / min;

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

[0123] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

[0124] The qualified product was collected and freeze-dried to obtain 49 mg. The LC diagram of the purified product is shown in FIG. Figure 5 The MS pattern of the purified product is shown in Figure 6 shown.

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

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

[0127] S11: Place CTC resin (5.58 g, 6.25 mmol) in a 100 mL solid-phase synthesis reactor. Add the amino acid Fmoc-Tyr(tBu)-OH (4.21 g, 12.5 mmol) and 20 mL of dichloromethane (DCM). Add DIEA (5.0 mL) and react at 25°C for 3 hours. Add 6 mL of methanol and react for 5 minutes. Filter and wash the resin twice with 50 mL of dichloromethane (DCM), twice with 50 mL of methanol, and twice with 50 mL of DMF. Add 50 mL of a 20% Pip / DMF solution and stir for 30 minutes. Filter to remove the deprotection solution, then wash the resin six times with 50 mL of DMF solution and drain.

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

[0129] Repeat the above S12 step, replacing Fmoc-Val-OH with the following amino acid reagents in order: Fmoc-Asp(otBu)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-Arg(Pbf)-OH. After the deprotection treatment is completed, wash with 50 mL of methanol twice, 20 mL of DCM solution twice, and 50 mL of methanol twice, and dry in vacuo to obtain H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-CTC-resin. The above peptide resin is treated with 120 mL of 30% TFE / DCM solution and stirred at 30°C for 30 minutes. The resin is then removed by filtration to obtain a filtrate. The filtrate was dried to obtain 4.2 g of the fully protected peptide H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-OH 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, feeding: weigh H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-OH (1.5 g), dissolve it in DMF (1500 mL), add DIEA (1.35 g), and reserve it as A; weigh HBTU (1.49 g), add it to A, stir and react for 2 hours after the addition is complete, and then directly perform in-process control; In-process control: LC-MS detection of raw material reaction completion

[0132] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry. Ice water (80 mL) was then added with stirring to precipitate a solid. After stirring for 10 min, the solid was filtered and dissolved with EA (40 mL). The solid was washed twice with saturated aqueous NaHCO3 solution and once with saturated brine. The product was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 1 g of Cyclo(Arg(Pbf)-Lys(Boc)-Asp(OtBu)-Val-Tyr(tBu)), with a yield of 67.7%.

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

[0134] S31, feeding: weigh 1 g of Cyclo (Arg (Pbf) -Lys (Boc) -Asp (OtBu) -Val-Tyr (tBu)) and use cutting solution E, control the temperature at 30 ° C and stir to react; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0135] S32, post-treatment: The reaction solution was added dropwise to glacial ether for precipitation while shaking. The solid was washed by centrifugation three times and evaporated to dryness to obtain 0.7 g of Cyclo(Arg-Lys-Asp-Val-Tyr) with a yield of 87.5%. The product was detected by LC-MS and sent for purification.

[0136] Purification conditions:

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

[0138] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0141] Sample loading: flow rate: 60 mL / min;

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

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

[0144] The qualified product was collected and freeze-dried to obtain 156 mg. The LC diagram of the product purification is as follows Figure 7 The MS pattern of the purified product is shown in Figure 8 shown.

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

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

[0147] In step S11, CTC resin (5.58 g, 6.25 mmol) was placed in a 100 mL solid-phase synthesis reactor. The amino acid Fmoc-Arg(Pbf)-OH (8.11 g, 12.5 mmol) was added, along with 20 mL of dichloromethane (DCM). The mixture was reacted at 25°C for 3 hours, followed by 5.0 mL of DIEA. 6 mL of methanol was then added and allowed to react for 5 minutes. The mixture was filtered, and the resin was washed twice with 50 mL of dichloromethane (DCM), twice with 50 mL of methanol, and twice with 50 mL of DMF. 50 mL of a 20% Pip / DMF solution was added, and the reaction was stirred for 30 minutes. The mixture was then filtered to remove the deprotection solution, and then washed six times with 50 mL of DMF solution. The mixture was then dried and used.

[0148] In step S12, Fmoc-Val-OH (5.09 g, 15 mmol) and HOBt (2.03 g, 15 mmol) were placed in a 50 mL beaker and cooled to 5°C. 5 mL of DMF solution and DIC (2.3 mL, 7.5 mmol) were added and allowed to react for 15 minutes. The solution in the 100 mL beaker was then added to a 100 mL solid-phase synthesis reactor and stirred for 1.5 hours. The reaction was complete. The resin was washed three times with 20 mL of DMF solution each time. After washing, the resin was processed for the next step. 50 mL of a 20% Pip / DMF solution was added and stirred for 30 minutes. The deprotection solution was removed by filtration. The resin was then washed six times with 50 mL of DMF solution and dried.

[0149] Repeat step S12 above, replacing Fmoc-Val-OH with the following amino acid reagents in order: Fmoc-Val-OH, Fmoc-Val-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Thr(tBu)-OH. After deprotection, the resin was washed twice with 50 mL of methanol, twice with 20 mL of DCM, and twice with 50 mL of methanol. The resin was then dried under vacuum to obtain H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-CTC-resin. The peptide resin was reacted with 100 mL of 30% TFE / DCM solution at 30°C for 30 minutes with stirring. The resin was removed by filtration to obtain a filtrate. The filtrate was then drained to obtain 3.96 g of the fully protected peptide H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH, 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: weigh H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH (1 g), dissolve it with DMF (1000 mL), add DIEA (1.48 g) and DCM (10 mL), and set aside and name it A; weigh HBTU (1.12 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform in-process control; In-process control: LC-MS detects that the raw material reaction is complete.

[0152] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry, and then ice water (30 mL) was added with stirring. No solid could be precipitated. DCM (20 mL) was added to the reaction solution, and the mixture was separated and extracted. The product was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine. The product was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 0.66 g of Cyclo(Ser(tBu)-Val-Val-Val-Arg(Pbf)-Thr(tBu)).

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

[0154] S31, feeding: weigh 0.66 g of Cyclo (Ser (tBu) -Val-Val-Val-Arg (Pbf) -Thr (tBu)) and use cutting solution E, control the temperature at 30 ° C and stir to react; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0155] S32, post-treatment: The reaction solution was added dropwise into icy ether to settle with shaking during the addition. The solid was washed by centrifugation three times and evaporated to dryness to obtain 0.28 g of Cyclo (Ser-Val-Val-Val-Arg-Thr), which was detected by LC-MS and sent for purification.

[0156] Purification conditions:

[0157] Dissolution: dilute 0.28 g of crude product with 100 mL of water;

[0158] Packing: 21.2*250mm, 10-120, C18; Flow rate: 60mL / min; Wavelength: 220nm;

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

[0160] Balance: A:B=100:0, balance for 10 min, flow rate: 10 mL / min;

[0161] Sample loading: flow rate: 10 mL / min;

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

[0163] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

[0164] The qualified product was collected and freeze-dried to obtain 28 mg. The LC diagram of the product purification is as follows Figure 9 The MS pattern of the purified product is shown in Figure 10 shown.

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

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

[0167] S11: Place CTC resin (2.23 g, 2.5 mmol) in a 100 mL solid-phase synthesis reactor, add the amino acid Fmoc-Met-OH (1.85 g, 5 mmol), and add 20 mL of dichloromethane (DCM). Add DIEA (2.0 mL) and react at 25°C for 3 hours. Add 3 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of a 20% Pip / DMF solution, stir, and react for 30 minutes. Filter to remove the deprotection solution, then wash the resin six times with 20 mL of DMF solution and drain.

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

[0169] Repeat the above S12 step and replace Fmoc-Glu (otBu) -OH with the following amino acid reagents in sequence: Fmoc-Glu (otBu) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Arg (Pbf) -OH, and Fmoc-Gln (Trt) -OH; after the deprotection treatment is completed, wash with 20 mL of methanol twice, wash with 20 mL of DCM solution twice, and wash with 20 mL of methanol twice, and vacuum dry to obtain H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin. The above peptide resin is treated with 40 mL of 30% TFE / DCM solution, stirred at 30 ° C for 30 minutes, filtered, and the resin is removed to obtain a filtrate. The filtrate was dried to obtain 2.0 g of the fully protected peptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-OH with a yield of 58.8% and a purity of 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 it with DMF (1620 mL), add DIEA (0.98 g), and reserve it and name it A; weigh HBTU (1.08 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform in-process control; In-process control: LC-MS detects that the raw material reaction is complete.

[0172] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry, and then ice water (30 mL) was added with stirring to precipitate a solid. After stirring for 10 min, the solid was filtered and dissolved with EA (15 mL). The solid was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine. The product was dried over anhydrous sodium sulfate, filtered and evaporated to dryness to obtain 1.57 g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf)), with a yield of 98%.

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

[0174] S31, feeding: weigh 1.57 g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met -Gln (Trt) -Arg (Pbf) -Arg (Pbf)) and use cutting liquid E, control the temperature at 30 ° C and stir to react for 2 hours; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0175] S32, post-treatment: The reaction solution was added dropwise to glacial ether for precipitation with shaking during the addition. The solid was washed by centrifugation three times and dried in a vacuum drying oven for 16 hours to obtain 0.98 g of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala) with a yield of 99.9%. The product was detected by LC-MS and sent for purification.

[0176] Purification conditions:

[0177] Dissolution: dilute 1.5 g of crude product with 200 mL of water;

[0178] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0181] Sample loading: flow rate: 60 mL / min;

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

[0183] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

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

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

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

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

[0188] To a 100 mL solid-phase synthesis reactor, add the amino acid Fmoc-Met-OH (1.85 g, 5 mmol) and 20 mL of dichloromethane (DCM). Add DIEA (2.0 mL) and react at 25°C for 3 hours. Then add 3 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of a 20% Pip / DMF solution, stir, and react for 30 minutes. Filter to remove the deprotection solution, then wash six times with 20 mL of DMF solution and drain.

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

[0190] Repeat the above S12 step, replacing Fmoc-Glu(otBu)-OH with the following amino acid reagents in order: Fmoc-Glu(otBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Gln(Trt)-OH. After the deprotection treatment is completed, wash with 20 mL of methanol twice, 20 mL of DCM solution twice, and 20 mL of methanol twice, and dry in vacuo to obtain H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-CTC-resin. The above peptide resin is treated with 40 mL of 30% TFE / DCM solution, stirred at 30°C for 30 minutes, and filtered to remove the resin to obtain a filtrate. The filtrate was dried to obtain 1.79 g of the fully protected polypeptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH 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, feeding: weigh H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Glu(otBu)-Glu(otBu)-Met-OH (1.56 g), dissolve it with DMF (1500 mL), add DIEA (0.9 g), and reserve it and name it A; weigh HBTU (0.99 g), add it to A, stir the reaction for 2 hours after the addition is complete, and directly perform in-process control; In-process control: LC-MS detects that the raw material reaction is complete.

[0193] S22, post-treatment: DMF was removed from most of the reaction solution by pulling it dry, and then ice water (36 mL) was added with stirring to precipitate a solid. After stirring for 10 min, the solid was filtered and dissolved with EA (18 mL). The solid was washed twice with saturated NaHCO3 aqueous solution and once with saturated brine. The product was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain 1.36 g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala), with a yield of 88%.

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

[0195] S31, feeding: weigh 1.36 g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met -Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala) and use cutting liquid E, control the temperature at 30 ° C and stir to react for 2 hours; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0196] S32, post-treatment: The reaction solution was added dropwise to glacial ether for precipitation with shaking during the addition. The solid was washed by centrifugation three times and dried in a vacuum drying oven for 16 hours to obtain 1.15 g of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala) with a yield of 132%, which was then sent for purification.

[0197] Purification conditions:

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

[0199] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0202] Sample loading: flow rate: 60 mL / min;

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

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

[0205] The qualified product was collected and freeze-dried to obtain 154 mg. The LC diagram of the purified product is shown in FIG. Figure 13 The MS pattern of the purified product is shown in Figure 14 shown.

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

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

[0208] S11: Place CTC resin (2.23 g, 2.5 mmol) in a 100 mL solid-phase synthesis reactor, add the amino acid Fmoc-Met-OH (1.85 g, 5 mmol), and add 20 mL of dichloromethane (DCM). Add DIEA (2.0 mL) and react at 25°C for 3 hours. Add 3 mL of methanol and react for 5 minutes. Filter, wash the resin twice with 20 mL of dichloromethane (DCM), twice with 20 mL of methanol, and twice with 20 mL of DMF. Add 20 mL of a 20% Pip / DMF solution, stir, and react for 30 minutes. Filter to remove the deprotection solution, then wash the resin six times with 20 mL of DMF solution and drain.

[0209] In step S12, Fmoc-Glu(otBu)-OH (2.55 g, 6 mmol) and HOBt (0.81 g, 6 mmol) were placed in a 50 mL beaker and cooled to 5°C. 5 mL of DMF solution and DIC (0.93 mL, 6 mmol) were added and allowed to react for 15 minutes. The solution in the 100 mL beaker was then added to a 100 mL solid-phase synthesis reactor and stirred for 1.5 hours. The reaction was complete. The resin was washed three times with 20 mL of DMF solution each time. After washing, the resin was processed for the next step. 20 mL of a 20% Pip / DMF solution was added and stirred for 30 minutes. The deprotection solution was removed by filtration. The resin was then washed six times with 20 mL of DMF solution and dried.

[0210] Repeat the above step S12, replacing Fmoc-Lys(Boc)-OH with the following amino acid reagents in order: Fmoc-Glu(otBu)-OH, Fmoc-Asp(otBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Gln(Trt)-OH. After deprotection, wash with 20 mL of methanol twice, 20 mL of DCM solution twice, and 20 mL of methanol twice, and dry under vacuum to obtain H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Glu(otBu)-Glu(otBu)-Met-CTC-resin. The above peptide resin was treated with 40 mL of 30% TFE / DCM solution and stirred at 30°C for 30 minutes. The resin was removed by filtration to obtain a filtrate. The filtrate was dried to obtain 2.83 g of the fully protected polypeptide H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(otBu)-Glu(otBu)-Glu(otBu)-Met-OH with a yield of 73.1% and a purity of 93.3%.

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

[0212] S21, feeding: weigh H-Gln(Trt)-Arg(Pbf)-Arg(Pbf)-Ala-Asp(otBu)-Glu(otBu)-Glu(otBu)-Met-OH (1.5 g), dissolve it with DMF (1500 mL), add DIEA (1 mL), and reserve it and name it A; weigh HBTU (0.87 g), add it to A, stir the reaction for 2 hours after the addition is complete, and directly perform in-process control; In-process control: LC-MS detects that the reaction of the raw materials is complete.

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

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

[0215] S31, feeding: weigh 0.94 g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala-Asp (OtBu)) and use cutting liquid E, control the temperature at 30 ° C and stir to react; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0216] S32, post-treatment: The reaction solution was added dropwise to glacial ether for precipitation with shaking during the addition. The solid was washed by centrifugation three times and evaporated to dryness to obtain 0.52 g of Cyclo (Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val) with a yield of 86%. The product was detected by LC-MS and sent for purification.

[0217] Purification conditions:

[0218] Dissolution: dilute 0.52 g of crude product with 100 mL of water;

[0219] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0222] Sample loading: flow rate: 60 mL / min;

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

[0224] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

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

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

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

[0228] In step S11, CTC resin (2.77 g, 2 mmol) was placed in a 250 mL solid-phase synthesis reactor. The amino acid Fmoc-Pro-OH (0.674 g, 2 mmol) was added, along with 120 mL of dichloromethane (DCM). The mixture was reacted at 25°C for 3 hours, followed by 8.7 mL of DIEA. 12.5 mL of methanol was then added and allowed to react for 5 minutes. The mixture was filtered, and the resin was washed twice with 75 mL of dichloromethane (DCM), twice with 75 mL of methanol, and twice with 75 mL of DMF. 25 mL of a 20% Pip / DMF solution was added, and the reaction was stirred for 30 minutes. The mixture was then filtered to remove the deprotection solution, and then washed six times with 130 mL of DMF solution. The mixture was then dried and used.

[0229] In step S12, Fmoc-Lys(Boc)-OH (2.814 g, 6 mmol) and HOBt (0.81 g, 6 mmol) were placed in a 100 mL beaker and cooled to 5°C. 25 mL of DMF solution and DIC (3.2 mL, 21 mmol) were added and allowed to react for 15 minutes. The solution in the 100 mL beaker was then added to a 250 mL solid-phase synthesis reactor and stirred for 1.5 hours. The reaction was complete. The resin was washed three times with 25 mL of DMF solution each time. After washing, the resin was processed for the next step. 25 mL of a 20% Pip / DMF solution was added and stirred for 30 minutes. The deprotection solution was removed by filtration. The resin was then washed six times with 25 mL of DMF solution and dried.

[0230] Repeat the above step S12, replacing Fmoc-Lys(Boc)-OH with 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, and Fmoc-Val-OH. After the deprotection treatment is completed, the resin is washed twice with 50 mL of methanol, twice with 50 mL of DCM solution, and twice with 50 mL of methanol, and then dried under vacuum to obtain H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-CTC-resin. The above peptide resin is treated with 40 mL of 30% TFE / DCM cutting solution and stirred at 30°C for 2.5 hours. The resin is then filtered to remove the resin and obtain a filtrate. The filtrate was drained to obtain 2.1 g of crude peptide H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH, with a yield of 64.02% and a purity of 95.3%.

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

[0232] S21, feeding: weigh H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH (1 g), dissolve it with DMF (1000 mL), add DIEA (0.64 g), and reserve it and name it A; weigh HBTU (0.65 g), add it to A, stir the reaction for 2 hours after the addition is complete, and directly perform in-process control; In-process control: LC-MS detects that the reaction of the raw materials is complete.

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

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

[0235] S31, feeding: weigh 0.85 g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met -Gln (Trt) -Arg (Pbf) -Arg (Pbf)) and use cutting liquid E, control the temperature at 30 ° C and stir to react; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0236] S32, post-treatment: The reaction solution was added dropwise to glacial ether with shaking during the addition. The solid was washed three times by centrifugation and dried in a vacuum oven for 16 h to obtain 0.62 g of Cyclo (Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val) with a yield of 84.6%. The product was detected by LC-MS and sent for purification.

[0237] Purification conditions:

[0238] Dissolution: Take 0.62g of crude product, add 160mL of water and 10mL of acetic acid and dissolve by ultrasonication;

[0239] Packing: 50DAC10-100C18; Flow rate: 60 mL / min; Wavelength: 220 nm;

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

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

[0242] Sample loading: flow rate: 60 mL / min;

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

[0244] Column cleaning: 80% acetonitrile cleaning to baseline equilibrium;

[0245] The qualified product was collected and freeze-dried to obtain 160 mg. The LC chart of the product purification is as follows Figure 17 The MS pattern of the purified product is shown in Figure 18 shown.

[0246] Test example:

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

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

[0249] (2) Liquid preparation: Prepare the working solution of the test substance according to the experimental design.

[0250] Table 1 AQP3 experimental design table

[0251]

[0252] The diluent in the AQP3 experiment was cell culture medium, such as DMEM. The sample concentrations were 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group was selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157.

[0253] (3) Add the test substance: After culturing in an incubator (37°C, 5% CO2) for 24 hours, add the test substance as shown in the table and continue culturing for another 24 hours.

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

[0255] (5) Immunofluorescence staining:

[0256] a. Add methanol to fix the cells, rinse three times with PBS, and then add 1 mL of BSA to each well to block for 1 hour.

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

[0258] c. Add secondary antibody to each well and allow to react for 2 hours. Discard the secondary antibody and rinse three times with PBS.

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

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

[0261] Table 2 AQP3 content test results

[0262]

[0263] The present invention has been tested and concluded that the PR095 sample has a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR100 sample has a moisturizing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. The PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, and PR157 sample all have a moisturizing effect at 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 24-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[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 used in the experiment was cell culture medium, such as DMEM. The concentrations of the sample groups were 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group was selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157.

[0270] (3) Add the test substance: After culturing in an incubator (37°C, 5% CO2) for 24 hours, add the test substance as shown in the table and continue culturing for another 24 hours.

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

[0272] Table 4 HA content test results

[0273]

[0274] The present invention has been tested and concluded that the PR095 sample has a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR100 sample, PR102 sample, PR116 sample, PR120 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, and PR157 sample have a moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR126 sample has a moisturizing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL.

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

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

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

[0278] Table 5 Experimental design table

[0279]

[0280] The diluent used in the experiment was cell culture medium, such as DMEM. The sample concentrations were 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group consisted of PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157. In the test, Collagen I refers to type I collagen; UVA refers to ultraviolet A; VC refers to vitamin C; and VE refers to vitamin E.

[0281] (3) UVA radiation: After 24 hours of culture, the negative control group, positive control group and sample group received UVA radiation with a total dose of 9 J / cm2. At the same time, the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm2).

[0282] (4) Addition of test substances: According to the experimental design, after irradiation, the test substances were added to the cells in groups. 1 mL of cell culture medium was added to each well of the blank control group and the negative control group; 1 mL of cell culture medium containing vitamin C and vitamin E was added to each well of the positive control group; 1 mL of culture medium containing the test substances at the corresponding concentration was added to each well of the sample group; after the addition of the test substances, the 24-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 h.

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

[0284] (6) Result analysis: The comparison between the groups was performed using t-test statistical analysis, and all statistical analyses were two-tailed.

[0285] Table 6 Test results of type Ⅰ collagen content

[0286]

[0287] The present invention has been tested and concluded that PR095 sample, PR100 sample, PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, and PR157 sample have anti-wrinkle and firming effects 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 24-well plates and incubate overnight in an incubator (37°C, 5% CO2).

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

[0291]

[0292] The diluent used in the experiment was cell culture medium, such as DMEM. Sample concentrations were 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group consisted of PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157. IL-6 in the test represents interleukin-6 (a cytokine), and LPS represents lipopolysaccharide.

[0293] (2) Adding the test substance: According to the experimental grouping, when the cell plating rate in the 24-well plate reaches 40%~60%, the test substance is added in groups, with 3 replicate wells in each group. The 24-well plate is placed in an incubator (37℃, 5% CO2) and incubated for 24 hours.

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

[0295] Table 8 IL-6 content test results

[0296]

[0297] The present invention has been tested and concluded that the PR095 sample has a soothing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL. The PR100 sample has a soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. The PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, and PR146 sample all have a soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL. The PR157 sample has a soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL.

[0298] Inhibition of neurotransmitter release test method: Norepinephrine test

[0299] (1) Cell inoculation: Inoculate cells into 24-well plates and culture in an incubator (37°C, 5% CO2) for 6 days.

[0300] (2) Cell treatment: To investigate the inhibitory effect of NA release, the culture medium was discarded and the cells were washed with HBSS. Before inducing exocytosis, the cells were pre-incubated with different concentrations of the test sample dissolved in HBSS for 60 minutes. The supernatant was removed and the protocol for inducing NA release was followed as described below.

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

[0302] (3) Induction of human norepinephrine release: Treat with HBSS containing TPA for 8 minutes, remove TPA, and continue incubation in HBSS containing ION and TPA for 5 minutes to induce the release of norepinephrine (NA). This group serves as the positive control for the test. The basal control group is treated with HBSS containing 100nM TPA for 8+5 minutes, and the rest of the steps are the same as the positive control group. After incubation, the supernatant containing released NA is immediately collected and stored at -80°C for further ELISA analysis.

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

[0304] (5) Determination of NA content by ELISA.

[0305] (6) Data analysis: The comparison between groups was performed using t-test statistical analysis, and all statistical analyses were two-tailed.

[0306] Table 9 Test results

[0307]

[0308] The present invention conducted the above test on PR-102, and the PR-102 sample could inhibit the release of the neurotransmitter norepinephrine at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL, indicating that the PR-102 sample can inhibit the release of the neurotransmitter norepinephrine.

[0309] Melanin content test

[0310] Table 10 Melanin content experimental design table

[0311]

[0312] The diluent in the experiment was a cell culture medium, such as DMEM medium. The concentrations of the sample groups were 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group was selected from PR116.

[0313] Cells in the logarithmic growth phase were collected and inoculated into 24-well plates. After culturing in an incubator (37°C, 5% CO2) for 24 h, the test substances were added according to the table based on the cytotoxicity results. Untreated cells were used as blank controls, and three parallel assays were set up for each group.

[0314] After adding the drug, continue culturing in an incubator (37°C, 5% CO2) for 24 hours, discard the supernatant, add 0.5 mL of 1 M NaOH containing 10% DMSO, and incubate at 80°C for 1 hour. Use 1 M NaOH containing 10% DMSO as a solvent control, read the absorbance value on a microplate reader, and calculate the relative inhibition rate of cytomelanin.

[0315]

[0316] Table 11 Melanin content test results

[0317]

[0318] The present invention conducted the above test on PR116, and the PR-116 sample had whitening efficacy at concentrations of 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL, indicating that the PR-116 sample had whitening efficacy.

[0319] The above embodiments are intended to illustrate the present invention only and are not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also fall within the scope of the present invention, and the scope of patent protection of the present invention shall be defined by the claims.

[0320] 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.

[0321] 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. Use of cyclic pentapeptide-4 in the preparation of moisturizing products and / or anti-wrinkle products and / or firming products and / or soothing products; The structure of cyclopentapeptide-4 is shown below: 。

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