Cyclic heptapeptide-4 as well as preparation method and application thereof

Abstract

The invention 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 particularly relates to a functional cyclic peptide with a cyclic structure, which is prepared by activating an amino acid reagent to obtain a linear peptide containing a protecting group and then cyclizing the linear peptide. The functional cyclic peptide prepared by the invention has the advantages of good moisturizing effect, good anti-wrinkle effect, good tightening effect, good soothing effect, good neurotransmitter release inhibition effect and good whitening effect. After the linear peptide is prepared into the functional cyclic peptide, the permeability, the stability and the activity are improved, and the irritation is reduced.

Description

Technical Field

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

[0002] Peptides are composed of amino acids through dehydration condensation. These amino acids themselves have carboxyl and amino groups and are generally hydrophilic compounds. The peptides composed of them generally have obvious hydrophilicity and ionicity. In particular, some small molecule peptides are non-toxic, easily absorbed by the skin, and have special physiological activities after absorption. They can improve certain skin problems from the inside out and are now widely used in beauty cosmetics formulas. There are various cosmetics or skin care products on the market, but most skin care products only add a certain type of peptide as the active ingredient of the skin care product, so the effect obtained by the skin is relatively simple and slow to take effect; moreover, most of the skin care products on the market that use peptides as active ingredients directly add peptides or peptide concentrates to conventional skin care product matrices. Considering the preparation and stability of the entire formula, the amount of peptide added will be limited.

[0003] In conventional peptide modification, palmitoyl modification or nutmeg modification methods are usually used. Such peptides modified with palmitoyl or nutmeg generally have hydrophilicity and lipophilicity, which often greatly increase the consumption of solvents and increase 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] The linear peptide is cyclized by peptide cyclization to obtain a cyclized peptide. The cyclization of the peptide dehydrates and condenses the carboxyl and amino groups of the peptide to form a new amide bond, which increases the lipid solubility of the peptide without significantly increasing the solvent consumption. The cyclized peptide has better stability than the linear peptide. At the same time, the carboxyl and amino or hydroxyl groups are condensed to obtain a peptide with better lipid solubility and better skin permeability.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: 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 for inhibiting neurotransmitter release and / or whitening products and / or anti-glycation products and / or antioxidant products, the functional cyclic peptide has the following structure: , where 1≤n, R mThe number of R is n. 1 , R 2 , R 3 or R m 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.

[0007] Preferably, n≤6; or, 1≤n≤6; or, n=2; or, n=3; or, n=4; or, n=5.

[0008] Preferably, R 1 , R 2 , R 3 and R m Any two or more groups in are the same; or, R 1 , R 2 , R 3 and R m Any adjacent groups in are the same; when R 1 , R 2 , R 3 or R m Selected from -CH 2 -CH 2 -CH 2 - group, R 1 , R 2 , R 3 or R m Connect with adjacent N to form a ring; or, R 1 , R 2 , R 3 or R m Selected from hydrogen atom groups, methyl groups, -CH 2 -CH 2 -CH 2 -group, propyl, butyl, hydroxymethyl, hydroxyethyl, acetamido, propionamido, CH 3 -S-CH 2 -CH 2 - group, HS-CH 2 -group, Ph-CH 2 -group, p-hydroxybenzyl, acetate, propionate, Se-CH 2 -group, butylamino group, indolyl group, imidazolylmethyl group and substituted guanidine group.

[0009] Preferably, the functional cyclic peptide is any of the following: .

[0010] 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: , where 1≤n, R m The number of R is n. 1 , R 2 , R 3 or R m 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.

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

[0012] 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.

[0013] 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 protecting group; or, the cutting solution in the cleavage treatment includes E solution or F solution, E solution 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 F solution 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.

[0014] More preferably, the post-treatment includes purifying the functional cyclic peptide by liquid chromatography; 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 cutting post-treatment is carried out to obtain the cyclic peptide.

[0015] More preferably, in the post-cyclization treatment, ice water is added to the test solution after the cyclization reaction is completed, solids are precipitated, stirred and filtered, the solids are dissolved with EA, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried with anhydrous sodium sulfate, filtered, and evaporated 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 with anhydrous sodium sulfate, filtered, and evaporated 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, solids are precipitated, centrifuged and washed, the solids are evaporated to dryness, and purified to obtain a cyclic peptide; or, LC-MS is used for monitoring in the cyclization reaction; or, the solvent is at least one of DMF, DCM and DIEA, the solvent is based on DMF as the measurement basis, and the relationship between the amount of linear peptide and DMF used is 0.1-30 mg / mL.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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 peptides, 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, a cutting solution is added to obtain a linear peptide.

[0021] More preferably, in the preparation of linear peptides, 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] More preferably, in the preparation of the linear peptide, the first amino acid reagent includes any one of the following: 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 cutting solution is a 30% TFE / DCM solution.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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, and then the deprotection solution is added and stirred for 10-60 minutes. After the treatment is completed, the deprotection solution is removed by suction filtration, washed with DMF, and dried. The deprotection solution is used in an appropriate amount.

[0031] More preferably, in the preparation of the linear peptide, during the treatment with the cutting solution, the cutting solution is added to the CTC resin bonded with the amino acid reagent, and the treatment is carried out at 20-40° C. for 0.5-5 h. After the treatment is completed, the resin is removed by filtration to obtain a filtrate, and the filtrate is dried to obtain the linear peptide. The cutting solution is used in an appropriate amount.

[0032] 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, reacted 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, a cutting solution is added for treatment, and finally a cleavage post-treatment is performed to obtain the cyclic peptide.

[0033] More preferably, in the preparation of the cyclic peptide, the solvent contains DMF, and may also contain at least one of DCM and DIEA, the solvent is based on DMF, the relationship between the amount of linear peptide and DMF is 0.1-30 mg / mL, the relationship between the amount of DCM and DMF is 0.1-10 mL / mL, the relationship between the amount of DIEA and DMF is 0.1-5 mg / mL, the cutting liquid is E liquid or F liquid, and the cutting liquid is used in an appropriate amount. The cyclization reagent is HBTU, and the relationship between the amount of HBTU and DMF is 0.1-5 mg / mL.

[0034] More preferably, in the preparation of the cyclopeptide, solution E is a mixture of 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.

[0035] More preferably, in the preparation of the cyclopeptide, the F solution is composed of TFA, TIS and water, which are mixed in a volume ratio of 90:5:5; PR100, Preferably, in the post-cyclization treatment, DMF is dried, and ice water is added to the test solution after the cyclization reaction is completed, solids are precipitated, stirred and filtered, the solids are dissolved with EA, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried with anhydrous sodium sulfate, filtered, and evaporated to obtain a cyclic peptide with a protective group.

[0036] 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 extracted in layers, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to obtain a cyclic peptide with a protective group.

[0037] Preferably, in the post-cleavage treatment, the test solution after the cleavage treatment is added to ice ether to precipitate a solid, which is washed by centrifugation, evaporated to dryness, and purified to obtain the cyclic peptide.

[0038] Purification was performed by liquid chromatography.

[0039] The present invention uses an amino acid reagent to prepare a linear peptide containing a protective group after activation, and then prepares a functional cyclic peptide with a cyclic structure through a cyclization treatment and a cutting treatment, so 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, and its preparation method and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 is the chromatogram of PR126; Figure 2 is the mass spectrum of PR126; Figure 3 is the chromatogram of PR140; Figure 4 is the mass spectrum of PR140; Figure 5 is the chromatogram of PR100; Figure 6 is the mass spectrum of PR100; Figure 7 is the chromatogram of PR132; Figure 8 is the mass spectrum of PR132; Fig. 9 is the PR120 chromatogram; Fig.10 is the mass spectrum of PR120; Fig.11 is the chromatogram of PR102; Fig.12 is the mass spectrum of PR102; Fig.13 is the chromatogram of PR139; Fig.14 is the mass spectrum of PR139; Fig.15 is the chromatogram of PR146; Fig.16 is the mass spectrum of PR146; Fig.17 is the chromatogram of PR116; Fig.18 This is the mass spectrum of PR116. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described in detail below in conjunction with specific implementations and drawings: Example 1: Preparation of Cyclo (Tyr-Pro-Phe-Phe) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (2.23 g, 2.5 mmol) was placed in a 100 mL solid phase synthesis reactor, amino acid Fmoc-Pro-OH (1.68 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, methanol 3 mL was added, and the reaction was carried out for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 20 mL twice, methanol 20 mL twice, and DMF 20 mL twice. Add 20% Pip / DMF solution 20 mL, stir and react for 30 min, filter to remove the deprotection solution, and then wash with DMF solution 20 mL 6 times, and drain for use.

[0042] S12, take Fmoc-Tyr (tBu) -OH (2.76g, 6mmol), HOBt (0.81g, 6mmol) in a 50mL beaker, cool to 5℃, add 5mL of DMF solution, DIC (0.93mL, 6mmol) and let stand for 15 minutes, and add the solution in the 100mL beaker to the 100mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin with DMF solution three times, 20mL each time. After washing, proceed to the next step. Add 20mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 20mL of DMF solution, and drain for standby use.

[0043] Repeat the above S12 step, and 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 completed, wash it with 20 mL of methanol twice, 20 mL of DCM solution twice, and 20 mL of methanol twice, and vacuum dry it to obtain H-Phe-Phe-Tyr (tBu) -Pro-CTC-resin. The above peptide resin is stirred at 30°C for 30 minutes with 30 mL of 30% TFE / DCM solution, filtered, and the resin is removed to obtain a filtrate. The filtrate is 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%.

[0044] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 detects whether the raw material reaction is complete.

[0045] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (30 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered and dissolved with EA (20 mL). Saturated NaHCO 3 The product was washed twice with 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).

[0046] S3, the synthesis steps of cyclic peptide are as follows: S31, cutting: weigh 0.9g of Cyclo (Tyr (tBu) -Pro-Phe-Phe) and use cutting solution E, stir and react at 30℃. Mid-control: take samples for MS detection, and the reaction is basically complete.

[0047] S32, post-treatment: the reaction solution was added dropwise into ice ether to settle, shaking while adding dropwise, and the solid was washed by centrifugation for 3 times. The solid was evaporated to dryness to obtain 0.68 g of Cyclo (Tyr-Pro-Phe-Phe), which was detected by LC-MS and sent for purification.

[0048] The purification conditions were as follows: Dissolution: Take 0.68g of crude product, add 30mL of acetic acid, 30mL of acetonitrile, and 100mL of water and dissolve by ultrasonication; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=95:5, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 30-50-80%B, 0-60-90min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 55 mg. The LC chart of the purified product is as follows Figure 1 The MS spectrum of the purified product is shown in Figure 2 shown.

[0049] Example 2: Preparation of Cyclo (Asp-Val-Lys-Tyr) S1, the synthesis steps of linear peptide are as follows: 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, and dichloromethane (DCM) 120 mL was added, and DIEA (8.7 mL) was added, and the reaction was carried out at 25 ° C for 3 hours, and methanol 12.5 mL was added and reacted for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 75 mL twice, methanol 75 mL twice, and DMF 75 mL twice. Add 20% Pip / DMF solution 65 mL, stir and react for 30 minutes, filter to remove the deprotection solution, and then wash with DMF solution 120 mL 6 times, and drain for use.

[0050] S12, take Fmoc-Lys (Boc) -OH (7.03g, 15mmol), HOBt (2.03g, 15mmol) in a 100mL beaker, cool to 5℃, add 14mL of DMF solution, DIC (1.89mL, 15mmol), let stand for 15 minutes, and add the solution in the 100mL beaker to a 250mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is complete. Wash the resin with DMF solution three times, 200mL each time. After washing, proceed to the next step. Add 65mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 120mL of DMF solution, and drain for standby use.

[0051] Repeat the above S12 step, and replace Fmoc-Lys (Boc) -OH with the following amino acid reagents in order: Fmoc-Val-OH and Fmoc-Asp (OtBu) -OH; after the deprotection treatment is completed, wash with 75 mL of methanol twice, 75 mL of DCM solution twice, and 75 mL of methanol twice, and vacuum dry to obtain H-Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) -CTC-resin. Take 3.95 g of the above peptide resin, 40 mL of 30% TFE / DCM cutting solution, stir and react at 30 ° C for 2.5 hours, filter, remove the resin, and obtain a filtrate. The filtrate is dried to obtain the crude peptide H-Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu) -OH. 1.67 g, yield 113%, purity 70.7%.

[0052] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 name it A; weigh HBTU (2.6 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform intermediate control; intermediate control: LC-MS detects that the raw material reaction is complete.

[0053] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (30 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered and dissolved with EA (20 mL). Saturated NaHCO 3 The product was washed twice with 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%.

[0054] S3, the synthesis steps of cyclic peptide are as follows: S31, weigh 1.22g of Cyclo (Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu)) and use cutting solution E, control the temperature at 30℃ and stir to react. Mid-control: take samples for MS detection, and the reaction is basically complete.

[0055] S32, post-treatment: the reaction solution was added dropwise into ice ether to settle, shaking while adding dropwise, and the solid was washed by centrifugation for 3 times. The solid was 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.

[0056] The purification conditions were as follows: Dissolution: dilute 0.95 g of crude product with 100 mL of water; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 0-20% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 202 mg. The LC chart of the purified product is as follows Figure 3 The MS spectrum of the purified product is shown in Figure 4 shown.

[0057] Example 3: Preparation of Cyclo (Lys-Thr-Thr-Lys-Ser) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (6.25 g, 7 mmol) was placed in a 250 mL solid phase synthesis reactor, amino acid Fmoc-Ser (tBu) -OH (8.42 g, 7 mmol) was added, dichloromethane (DCM) 75 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. Filter, wash the resin with dichloromethane (DCM) 75 mL twice, methanol 75 mL twice, and DMF 75 mL twice. Add 20% Pip / DMF solution 40 mL, stir and react for 30 minutes, filter to remove the deprotection solution, and then wash with DMF solution 75 mL 6 times, and drain for use.

[0058] S12, take Fmoc-Lys (Boc) -OH (9.84g, 21mmol), HOBt (2.84g, 21mmol) in a 100mL beaker, cool to 5℃, add 50mL of DMF solution, DIC (3.2mL, 21mmol), let stand for 15 minutes, and add the solution in the 100mL beaker to a 250mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is complete. Wash the resin with DMF solution three times, 75mL each time. After washing, proceed to the next step. Add 40mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 65mL of DMF solution, and drain for standby use.

[0059] Repeat the above step S12, and replace Fmoc-Lys (Boc) -OH with the following amino acid reagents in order: Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -OH, 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 vacuum dry to obtain H-Lys (Boc) -Thr (tBu) -Thr (tBu) -Lys (Boc) -Ser (tBu) -CTC-resin. The above peptide resin is stirred for 2.5 hours at 30°C using 3.5 g of cutting solution 30% TFE / DCM, 40 mL, and filtered to remove the resin to obtain a filtrate. The filtrate was dried 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%.

[0060] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 set aside 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 intermediate control; intermediate control: LC-MS detects whether the raw material reaction is complete.

[0061] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (30 mL) was added while stirring. No solid was precipitated. DCM (20 mL) was added to the reaction solution, and the mixture was separated and extracted. Then saturated NaHCO 3 The product was washed twice with aqueous solution and once with saturated brine, 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%.

[0062] S3, the synthesis steps of cyclic peptide are as follows: S31, feeding: weigh 1.22g of Cyclo (Asp (OtBu) -Val-Lys (Boc) -Tyr (tBu)) and use cutting liquid E, stir and react at 30℃. Mid-control: take samples for MS detection, and the reaction is basically complete.

[0063] S32, post-treatment: the reaction solution was added dropwise into ice ether for precipitation while shaking, the solid was washed by centrifugation for 3 times, and the solid was evaporated to dryness to obtain 0.83 g of Cyclo (Lys-Thr-Thr-Lys-Ser) with a yield of 75.5%, detected by LC-MS, and sent for purification.

[0064] Purification conditions: Dissolution: Take 0.83g of crude product and dilute it with 100mL of water; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: water; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 0-10% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 49 mg. The LC chart of the purified product is as follows Figure 5The MS spectrum of the purified product is shown in Figure 6 shown.

[0065] Example 4: Preparation of Cyclo (Arg-Lys-Asp-Val-Tyr) S1, the synthesis steps of linear peptide are as follows: 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, methanol 6 mL was added, and the reaction was carried out for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 50 mL twice, methanol 50 mL twice, and DMF 50 mL twice. Add 20% Pip / DMF solution 50 mL, stir and react for 30 minutes, filter to remove the deprotection solution, and then wash with DMF solution 50 mL 6 times, and drain for use.

[0066] S12, take Fmoc-Val-OH (5.09g, 15mmol), HOBt (2.03g, 15mmol) in a 50mL beaker, cool to 5℃, add 5mL of DMF solution, DIC (2.3mL, 7.5mmol) and let stand for 15 minutes, and add the solution in the 100mL beaker to the 100mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin with DMF solution three times, 20mL each time. After washing, proceed to the next step. Add 50mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 50mL of DMF solution, and drain for standby use.

[0067] Repeat the above step S12, and replace Fmoc-Val-OH with the following amino acid reagents in order: Fmoc-Asp(otBu)-OH, Fmoc-Lys(Boc)-OH, 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 vacuum dry to obtain H-Arg(Pbf)-Lys(Boc)-Asp(otBu)-Val-Tyr(tBu)-CTC-resin. The above peptide resin is stirred at 30°C for 30 minutes with 120 mL of 30% TFE / DCM solution, filtered, and the resin is removed to obtain a filtrate. The filtrate was dried to obtain 4.2 g of 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%.

[0068] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 name it A; weigh HBTU (1.49 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform intermediate control; intermediate control: LC-MS detects that the raw material reaction is complete S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (80 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered, and the solids were dissolved with EA (40 mL). Saturated NaHCO 3 The product was washed twice with aqueous solution and once with saturated brine, 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%.

[0069] S3, the synthesis steps of cyclic peptide are as follows: S31, feeding: weigh 1g of Cyclo (Arg (Pbf) -Lys (Boc) -Asp (OtBu) -Val-Tyr (tBu)) and use cutting liquid E, control the temperature at 30℃ and stir to react; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0070] S32, post-treatment: the reaction solution was added dropwise into ice ether to settle, shaking while adding dropwise, and the solid was washed by centrifugation for 3 times. The solid was evaporated to dryness to obtain 0.7 g of Cyclo (Arg-Lys-Asp-Val-Tyr) with a yield of 87.5%. Detected by LC-MS and sent for purification.

[0071] Purification conditions: Dissolution: Take 0.7g of crude product and dilute it with 100mL of water; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 0-20% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 156 mg. The LC chart of the purified product is as follows Figure 7The MS spectrum of the purified product is shown in Figure 8 shown.

[0072] Example 5: Preparation of Cyclo (Ser-Val-Val-Val-Arg-Thr) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (5.58 g, 6.25 mmol) was placed in a 100 mL solid phase synthesis reactor, amino acid Fmoc-Arg (Pbf) -OH (8.11 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, methanol 6 mL was added, and the reaction was carried out for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 50 mL twice, methanol 50 mL twice, and DMF 50 mL twice. Add 20% Pip / DMF solution 50 mL, stir and react for 30 minutes, filter to remove the deprotection solution, and then wash with DMF solution 50 mL 6 times, and drain for use.

[0073] S12, take Fmoc-Val-OH (5.09g, 15mmol), HOBt (2.03g, 15mmol) in a 50mL beaker, cool to 5℃, add 5mL of DMF solution, DIC (2.3mL, 7.5mmol) and let stand for 15 minutes, and add the solution in the 100mL beaker to the 100mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin with DMF solution three times, 20mL each time. After washing, proceed to the next step. Add 50mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 50mL of DMF solution, and drain for standby use.

[0074] Repeat the above S12 step, and replace Fmoc-Val-OH with 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, wash with 50 mL of methanol twice, 20 mL of DCM solution twice, and 50 mL of methanol twice, and vacuum dry to obtain H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -CTC-resin. The above peptide resin is stirred at 30 ° C for 30 minutes with 100 mL of 30% TFE / DCM solution, filtered, and the resin is removed to obtain a filtrate. The filtrate is dried to obtain 3.96 g of the fully protected polypeptide H-Thr (tBu) -Ser (tBu) -Val-Val-Val-Arg (Pbf) -OH, with a yield of 79% and a purity of 92.1%.

[0075] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: S21, feeding: weigh H-Thr(tBu)-Ser(tBu)-Val-Val-Val-Arg(Pbf)-OH (1 g), dissolve it in DMF (1000 mL), add DIEA (1.48 g) and DCM (10 mL), set aside and name it A; weigh HBTU (1.12 g), add it to A, stir the reaction for 2 hours after the addition is complete, and directly perform intermediate control; intermediate control: LC-MS detects whether the raw material reaction is complete.

[0076] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (30 mL) was added while stirring. No solid was precipitated. DCM (20 mL) was added to the reaction solution, and the mixture was separated and extracted. Then saturated NaHCO 3 The product was washed twice with aqueous solution and once with saturated brine, 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)).

[0077] S3, the synthesis steps of cyclic peptide are as follows: S31, feeding: weigh 0.66 g of Cyclo (Ser (tBu) -Val-Val-Val-Arg (Pbf) -Thr (tBu)) 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.

[0078] S32, post-treatment: the reaction solution was added dropwise into icy ether to precipitate, shaking while adding dropwise, and the solid was washed by centrifugation 3 times. The solid was evaporated to dryness to obtain 0.28 g of Cyclo (Ser-Val-Val-Val-Arg-Thr), detected by LC-MS, and sent for purification.

[0079] Purification conditions: Dissolution: dilute 0.28 g of crude product with 100 mL of water; Packing: 21.2*250mm, 10-120, C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 10 mL / min; Sample loading: Flow rate: 10 mL / min; Elution: 4-24% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 28 mg. The LC chart of the purified product is as follows Fig. 9 The MS spectrum of the purified product is shown in Fig.10 shown.

[0080] Example 6: Preparation of Cyclo (Glu-Glu-Met-Gln-Arg-Arg) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (2.23 g, 2.5 mmol) was placed in 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, methanol 3 mL was added, and the reaction was carried out for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 20 mL twice, methanol 20 mL twice, and DMF 20 mL twice. Add 20% Pip / DMF solution 20 mL, stir and react for 30 min, filter to remove the deprotection solution, and then wash with DMF solution 20 mL 6 times, and drain for use.

[0081] S12, take Fmoc-Glu (otBu) -OH (2.55g, 6mmol), HOBt (0.81g, 6mmol) in a 50mL beaker, cool to 5℃, add 5mL of DMF solution, DIC (0.93mL, 6mmol) and let stand for 15 minutes, and add the solution in the 100mL beaker to the 100mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin with DMF solution three times, 20mL each time. After washing, proceed to the next step. Add 20mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 20mL of DMF solution, and drain for standby use.

[0082] 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, 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, 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. Add 40 mL of 30% TFE / DCM solution to the above peptide resin, stir at 30°C for 30 minutes, filter, remove the resin, and obtain a filtrate. The filtrate was dried to obtain 2.0 g of the fully protected polypeptide 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%.

[0083] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 name it A; weigh HBTU (1.08 g), add it to A, stir the reaction for 2 hours after the addition is complete, and directly perform intermediate control; intermediate control: LC-MS detects whether the raw material reaction is complete.

[0084] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (30 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered, and the solids were dissolved with EA (15 mL). Saturated NaHCO 3The product was washed twice with aqueous solution and once with saturated brine, 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%.

[0085] S3, the synthesis steps of cyclic peptide are as follows: S31, feeding: weigh 1.57g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf)) and use cutting liquid E, control the temperature at 30℃ and stir to react for 2 hours; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0086] S32, post-treatment: the reaction solution was added dropwise into icy ether for precipitation, and the solid was shaken while adding dropwise. The solid was washed by centrifugation for 3 times and dried in a vacuum 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 solid was detected by LC-MS and sent for purification.

[0087] Purification conditions: Dissolution: dilute 1.5 g of crude product with 200 mL of water; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 0-20% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 200 mg. The LC chart of the purified product is as follows Fig.11 The MS spectrum of the purified product is shown in Fig.12 shown.

[0088] Example 7: Preparation of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (2.23 g, 2.5 mmol) was placed in In a 100mL solid phase synthesis reactor, add amino acid Fmoc-Met-OH (1.85g, 5mmol), add dichloromethane (DCM) 20mL, add DIEA (2.0mL), react at 25℃ for 3 hours, add methanol 3mL, react for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 20mL twice, methanol 20mL twice, and DMF 20mL twice. Add 20mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with DMF solution 20mL, and drain for use.

[0089] S12, take Fmoc-Glu (otBu) -OH (2.55g, 6mmol), HOBt (0.81g, 6mmol) in a 50mL beaker, cool to 5℃, add 5mL of DMF solution, DIC (0.93mL, 6mmol) and let stand for 15 minutes, and add the solution in the 100mL beaker to the 100mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin with DMF solution three times, 20mL each time. After washing, proceed to the next step. Add 20mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 20mL of DMF solution, and drain for standby use.

[0090] Repeat the above step S12, and replace 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, 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 vacuum dry to obtain H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin. The above peptide resin is stirred at 30°C for 30 minutes with 40 mL of 30% TFE / DCM solution, filtered, and the resin is removed 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%.

[0091] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 name it A; weigh HBTU (0.99 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform intermediate control; intermediate control: LC-MS detects whether the raw material reaction is complete.

[0092] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (36 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered, and the solids were dissolved with EA (18 mL). Saturated NaHCO 3 The product was washed twice with aqueous solution and once with saturated brine, 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%.

[0093] S3, the synthesis steps of cyclic peptide are as follows: S31, feeding: weigh 1.36g of Cyclo (Glu (OtBu) -Glu (OtBu) -Met-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Ala) and use cutting liquid E, control the temperature at 30℃ and stir to react for 2 hours; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0094] S32, post-treatment: the reaction solution was added dropwise into icy ether for precipitation, and the mixture was shaken while being added dropwise. The solid was washed by centrifugation for 3 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 sent for purification.

[0095] Purification conditions: Dissolution: dilute 1.5 g of crude product with 200 mL of water; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 0-20% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 154 mg. The LC chart of the purified product is as follows Fig.13 The MS spectrum of the purified product is shown in Fig.14 shown.

[0096] Example 8: Preparation of Cyclo (Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (2.23 g, 2.5 mmol) was placed in 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, methanol 3 mL was added, and the reaction was carried out for 5 minutes. Filter, wash the resin with dichloromethane (DCM) 20 mL twice, methanol 20 mL twice, and DMF 20 mL twice. Add 20% Pip / DMF solution 20 mL, stir and react for 30 min, filter to remove the deprotection solution, and then wash with DMF solution 20 mL 6 times, and drain for use.

[0097] S12, take Fmoc-Glu (otBu) -OH (2.55g, 6mmol), HOBt (0.81g, 6mmol) in a 50mL beaker, cool to 5℃, add 5mL of DMF solution, DIC (0.93mL, 6mmol) and let stand for 15 minutes, and add the solution in the 100mL beaker to the 100mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is completed. Wash the resin with DMF solution three times, 20mL each time. After washing, proceed to the next step. Add 20mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 20mL of DMF solution, and drain for standby use.

[0098] Repeat the above step S12, and replace 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, 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 vacuum dry to obtain H-Gln (Trt) -Arg (Pbf) -Arg (Pbf) -Glu (otBu) -Glu (otBu) -Met-CTC-resin. The above peptide resin is stirred at 30°C for 30 minutes with 40 mL of 30% TFE / DCM solution, filtered, and the resin is removed 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%.

[0099] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: 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 set aside 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 intermediate control; intermediate control: LC-MS detects whether the raw material reaction is complete.

[0100] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (40 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered, and the solids were dissolved with EA (20 mL). Saturated NaHCO 3 The product was washed twice with aqueous solution and once with saturated brine, 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%.

[0101] S3, the synthesis steps of cyclic peptide are as follows: 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℃ and stir to react; intermediate control: take samples for MS detection, and the reaction is basically complete.

[0102] S32, post-treatment: the reaction solution was added dropwise into icy ether to settle, shaking while adding dropwise, and the solid was washed by centrifugation 3 times. The solid was 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.

[0103] Purification conditions: Dissolution: Take 0.52g of crude product and dilute it with 100mL of water; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=100:0, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 0-20% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 170 mg. The LC chart of the purified product is as follows Fig.15 The MS spectrum of the purified product is shown in Fig.16 shown.

[0104] Example 9: Preparation of Cyclo (Met-Pro-{D-Phe}-Arg-{D-Trp}-Phe-Lys-Pro-Val) S1, the synthesis steps of linear peptide are as follows: S11, CTC resin (2.77 g, 2 mmol) was placed in a 250 mL solid phase synthesis reactor, amino acid Fmoc-Pro-OH (0.674 g, 2 mmol) was added, 120 mL of dichloromethane (DCM) was added, DIEA (8.7 mL) was added, and the reaction was carried out at 25 °C for 3 hours, and 12.5 mL of methanol was added and the reaction was carried out for 5 minutes. After filtering, the resin was washed twice with 75 mL of dichloromethane (DCM), 75 mL of methanol, and 75 mL of DMF. 25 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 washed 6 times with 130 mL of DMF solution, and dried for use.

[0105] S12, take Fmoc-Lys (Boc) -OH (2.814g, 6mmol), HOBt (0.81g, 6mmol) in a 100mL beaker, cool to 5℃, add 25mL of DMF solution, DIC (3.2mL, 21mmol), let stand for 15 minutes, and add the solution in the 100mL beaker to a 250mL solid phase synthesis reactor, stir and react for 1.5 hours, and the reaction is complete. Wash the resin with DMF solution three times, 25mL each time. After washing, proceed to the next step. Add 25mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, then wash 6 times with 25mL of DMF solution, and drain for standby use.

[0106] Repeat the above step S12, and replace 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, Fmoc-Val-OH; after the deprotection treatment is completed, wash with 50 mL of methanol twice, 50 mL of DCM solution twice, and 50 mL of methanol twice, and vacuum dry 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, stirred at 30°C for 2.5 hours, filtered, and the resin is removed to obtain a filtrate. The filtrate was dried 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%.

[0107] S2, the synthesis steps of the cyclic peptide with a protecting group are as follows: S21, feeding: weigh H-Val-Met-Pro-{D-Phe}-Arg(Pbf)-{D-Trp}(Boc)-Phe-Lys(Boc)-Pro-OH (1 g), dissolve it in DMF (1000 mL), add DIEA (0.64 g), and set aside and name it A; weigh HBTU (0.65 g), add it to A, stir and react for 2 hours after the addition is complete, and directly perform intermediate control; intermediate control: LC-MS detects whether the raw material reaction is complete.

[0108] S22, post-treatment: DMF in the reaction solution was removed by drying, and then ice water (30 mL) was added while stirring to precipitate solids. After stirring for 10 min, the solids were filtered and dissolved with EA (20 mL). Saturated NaHCO 3 The product was washed twice with aqueous solution and once with saturated brine, 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%.

[0109] S3, the synthesis steps of cyclic peptide are as follows: 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.

[0110] S32, post-treatment: the reaction solution was added dropwise into icy ether for precipitation, and the mixture was shaken while adding dropwise. The solid was washed by centrifugation for 3 times, and dried in a vacuum oven for 16 hours 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.

[0111] Purification conditions: Dissolution: Take 0.62g crude product, add 160mL water and 10mL acetic acid and dissolve by ultrasonication; Filler: 50DAC10-100C18; Flow rate: 60mL / min; Wavelength: 220nm; Mobile phase: A: 1% acetic acid; B: acetonitrile; Balance: A:B=95:5, balance for 10 min, flow rate: 60 mL / min; Sample loading: Flow rate: 60mL / min; Elution: 16-36% B 60 min; Column cleaning: 80% acetonitrile to baseline equilibrium; The qualified product was collected and freeze-dried to obtain 160 mg. The LC chart of the purified product is as follows Fig.17 The MS spectrum of the purified product is shown in Fig.18 shown.

[0112] Test example: Moisturizing test method: AQP3 (aquaporin 3) content test (1) Cell inoculation: Inoculate cells into 24-well plates and place in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0113] (2) Solution preparation: Prepare the test substance working solution according to the experimental design.

[0114] Table 1 AQP3 experimental design table

[0115] The diluent in the AQP3 experiment is a cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157.

[0116] (3) Add the test substance: In an incubator (37°C, 5% CO 2 ) after culturing for 24 h, add the test substances according to the table and continue culturing for 24 h.

[0117] (4) Sample collection: Discard the supernatant and rinse the cells three times with PBS.

[0118] (5) Immunofluorescence staining: a. Add methanol to fix the cells, rinse with PBS three times, and add 1 mL of BSA to each well to block for 1 hour.

[0119] 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 with PBS three times.

[0120] c. Add secondary antibody to each well and let it act for 2 hours. Discard the secondary antibody and rinse with PBS 3 times.

[0121] d. Add DAPI to each well for nuclear staining for 10 min, discard DAPI, rinse with PBS three times, and then take pictures using a fluorescence microscope.

[0122] (6) Result analysis: ImageProPlus software was used to quantitatively analyze the fluorescence intensity of AQP3.

[0123] Table 2 AQP3 content test results

[0124] The present invention can be tested to obtain the following conclusions: PR095 sample has moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. PR100 sample has moisturizing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample, PR157 sample all have moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL.

[0125] Moisturizing test method: HA (hyaluronic acid) content test (1) Cell inoculation: Inoculate cells into 24-well plates and place in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0126] (2) Solution preparation: Prepare the test substance working solution according to the experimental design.

[0127] Table 3 HA experimental design table

[0128] The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157.

[0129] (3) Add the test substance: In an incubator (37°C, 5% CO 2 ) after culturing for 24 h, add the test substances according to the table and continue culturing for 24 h.

[0130] (4) Sample collection: Collect the supernatant and determine the HA content using an ELISA kit.

[0131] Table 4 HA content test results

[0132] The present invention can be tested to obtain the following conclusions: PR095 sample has moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. PR100 sample, PR102 sample, PR116 sample, PR120 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample and PR157 sample have moisturizing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. PR126 sample has moisturizing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL.

[0133] Anti-wrinkle and firming efficacy test method: Type I collagen and MMP-1 content test (1) Cell inoculation: Inoculate cells into 24-well plates and place in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0134] (2) Solution preparation: Prepare the test substance working solution according to the experimental design.

[0135] Table 5 Experimental design table

[0136] The diluent in the experiment is a cell culture medium, such as DMEM culture medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are 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; VE is vitamin E.

[0137] (3) UVA radiation: After 24 hours of culture, the negative control group, positive control group and sample group were exposed to 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).

[0138] (4) Addition of test substances: According to the experimental design, after irradiation, the test substances were 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% CO). 2 ) for 24 h.

[0139] (5) Collect the supernatant and measure the content of type I collagen and MMP-1.

[0140] (6) Result analysis: The t-test statistical analysis was used for comparison among the groups, and all statistical analyses were two-tailed.

[0141] Table 6 Test results of type Ⅰ collagen content

[0142] The present invention can draw the following conclusions after testing: 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.

[0143] Soothing test method: IL-6 content test (1) Cell inoculation: Inoculate cells into 24-well plates and place in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0144] Table 7 IL-6 synthesis experimental design table

[0145] The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentrations of the sample groups are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample groups are selected from PR126, PR140, PR100, PR132, PR120, PR102, PR139, PR146, PR116, PR095, and PR157. In the test, IL-6 is interleukin-6 (cytokine), and LPS is lipopolysaccharide.

[0146] (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°C, 5% CO 2 ) and incubate for 24 h.

[0147] (3) Detection: After 24 hours of culture, the supernatant was collected and the IL-6 content was determined using an ELISA kit.

[0148] Table 8 IL-6 content test results

[0149] The present invention can draw the following conclusions after testing: PR095 sample has soothing effect at concentrations of 0.063 mg / mL and 0.125 mg / mL. PR100 sample has soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL. PR102 sample, PR116 sample, PR120 sample, PR126 sample, PR132 sample, PR139 sample, PR140 sample, PR146 sample all have soothing effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL. PR157 sample has soothing effect at concentrations of 0.063 mg / mL and 0.25 mg / mL.

[0150] Inhibition of neurotransmitter release test method: Norepinephrine test (1) Cell inoculation: Inoculate cells into 24-well plates and place in an incubator (37°C, 5% CO 2 ) for 6 days.

[0151] (2) Cell treatment: To study 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 sample to be tested dissolved in HBSS for 60 minutes. The supernatant was removed and the induction of norepinephrine release was performed according to the protocol described below.

[0152] The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentrations of the sample group are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group is selected from PR102.

[0153] (3) Inducing human norepinephrine release: Treat with HBSS containing TPA for 8 minutes, remove TPA, and continue incubating in HBSS containing ION and TPA for 5 minutes to induce the release of norepinephrine (NA). This group is the positive control of the test. The basic 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 the incubation is completed, the supernatant containing the released NA is immediately collected and stored at -80°C for further testing and analysis by ELISA.

[0154] (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. First, it was incubated with extraction buffer, then washed with double distilled water, then incubated with acylation reagent under extraction buffer, and finally washed with double distilled water. Finally, after adding release buffer, the sample was quantified for NA using ELISA.

[0155] (5) ELISA was used to determine the NA content.

[0156] (6) Data analysis: The t-test statistical analysis was used for comparison among the groups, and all statistical analyses were two-tailed.

[0157] Table 9 Test results

[0158] 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 could inhibit the release of the neurotransmitter norepinephrine.

[0159] Melanin content test Table 10 Melanin content experimental design table

[0160] The diluent in the experiment is a cell culture medium, such as DMEM medium. The concentrations of the sample group are 0.063 mg / mL, 0.125 mg / mL, and 0.25 mg / mL; the sample group is selected from PR116.

[0161] The cells in the logarithmic growth phase were collected and inoculated into 24-well plates and incubated in an incubator (37°C, 5% CO 2 ) after culturing for 24 h, the test substances were added according to the cytotoxicity results as shown in the table, and untreated cells were used as blank controls. Three parallels were set up for each group.

[0162] After adding the drug, place in an incubator (37°C, 5% CO 2 ) for 24 h, discard the supernatant, add 0.5 mL of 1 M NaOH containing 10% DMSO, incubate at 80 °C for 1 h, use 1 M NaOH containing 10% DMSO as solvent control, read the absorbance on a microplate reader and calculate the relative inhibition rate of cellular melanin.

[0163]

[0164] Table 11 Melanin content test results

[0165] The present invention conducted the above test on PR116, and the PR-116 sample had whitening effect at concentrations of 0.063 mg / mL, 0.125 mg / mL and 0.25 mg / mL, indicating that the PR-116 sample had whitening effect.

[0166] The above embodiments are only used to illustrate the present invention, but not to limit the present invention. A person 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 belong to the scope of the present invention, and the patent protection scope of the present invention shall be defined by the claims.

[0167] 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 are not intended to impose any form of limitation on the implementation methods of the technology of the present invention. Any technical personnel in this field 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. 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 its 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 and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A cycloheptapeptide-4, the structure of which is shown below: 。 2. Use of the cyclic heptapeptide-4 according to claim 1 in the preparation of moisturizing products and / or anti-wrinkle products and / or firming products and / or soothing products and / or products for inhibiting neurotransmitter release and / or whitening products.

3. The method for preparing the cycloheptapeptide-4 according to claim 1, include: The linear peptide and the cyclization reagent are mixed in a solvent for cyclization reaction, and then post-processed to prepare the cycloheptapeptide-4; The structure of cycloheptapeptide-4 is shown below: 。 4. The method for preparing cycloheptapeptide-4 according to claim 3, Its characteristics are: 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.

5. The method for preparing cycloheptapeptide-4 according to claim 3, Its characteristics are: 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 solution in the cleavage treatment includes E solution or F solution, E solution 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 F solution 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 cycloheptapeptide-4 is separated and purified.

6. The method for preparing cycloheptapeptide-4 according to claim 3, Its characteristics are: The post-treatment includes purifying cycloheptapeptide-4 by liquid chromatography; or, in the cyclization reaction, 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 a 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 cyclopeptide with a protective group, a cutting liquid is added for treatment, and finally a cutting post-treatment is carried out to obtain a cyclopeptide.

7. The method for preparing cycloheptapeptide-4 according to claim 3, Its characteristics are: In the post-cyclization treatment, ice water is added to the test solution after the cyclization reaction is completed, solids are precipitated, stirred and filtered, the solids are dissolved with EA, and then washed with saturated sodium bicarbonate solution and saturated brine in sequence, dried with anhydrous sodium sulfate, filtered, evaporated to dryness, and a cyclic peptide with a protective group is obtained; 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 with anhydrous sodium sulfate, filtered, evaporated to dryness, and a cyclic peptide with a protective group is obtained; or, in the post-cleavage treatment, the test solution after the cleavage treatment is added to ice ether, solids are precipitated, centrifuged and washed, the solids are evaporated to dryness, purified, and a cyclic peptide is obtained; or, LC-MS is used for monitoring in the cyclization reaction; Or, the solvent is at least one of DMF, DCM and DIEA, the solvent is based on DMF, and the relationship between the amount of the linear peptide and DMF used is 0.1-30 mg / mL.

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