Cyclic hexapeptide compound as well as preparation method and application thereof

Abstract

The invention provides a cyclic hexapeptide compound and a preparation method and application thereof.The cyclic hexapeptide compound is of a structure shown in the formula (I), has higher stability and bioavailability, can remarkably promote secretion of collagen, especially I-type collagen, III-type collagen and IV-type collagen, has the tightening effect, and can be used for preparing a skin care product. The composition can be applied to cosmetics to maintain the structure and elasticity of the skin and promote the health of the skin. # imgabs0 #

Description

[0001] This application is a divisional application with the application date of December 12, 2024, application number 202411831485.9, and the name of the invention being “Cyclohexapeptide compounds, preparation methods and applications thereof”. Technical Field

[0002] The present invention relates to the technical field of daily chemical industry, and in particular to a cyclohexapeptide compound, a preparation method thereof and application thereof. Background Art

[0003] Skin is the largest organ in the human body, covering the entire body surface, and has multiple functions such as protection, regulation, sensation, secretion and excretion, immunity and respiration. As we age, the collagen in the human body will gradually decrease, causing the skin to show signs of sagging. Peptides can improve the skin condition and keep the skin healthy and youthful by promoting collagen production, anti-oxidation, scavenging free radicals, promoting skin cell proliferation, repairing skin trauma, and anti-inflammatory mechanisms.

[0004] Peptides are non-toxic and easily absorbed by the skin. They can improve the condition of the epidermis and dermis after use. In recent years, peptide ingredients have received widespread attention due to their significant firming effects, especially cyclic peptides, which have shown great potential in improving skin firmness. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a cyclic hexapeptide compound, a preparation method thereof and an application thereof. The cyclic hexapeptide compound provided in the present application promotes the secretion of collagen, especially type III collagen and type IV collagen, and has a good firming effect.

[0006] The present application provides a cyclohexapeptide compound represented by formula (I):

[0007]

[0008] In formula (I), x and y are independently selected from integers of 1 to 5;

[0009] R 1 , R 2 , R 3 and R 4 Independently selected from H, hydroxy, halogen or C1-C6 alkoxy.

[0010] In some specific implementations, x and y are independently 1, 2, 3, 4, or 5, preferably 1 or 2.

[0011] In some specific implementations, R 1 , R 2 , R 3 and R4 At least one of them is hydroxyl, halogen or C1-C6 alkoxy. Among them, halogen is selected from F, Cl, Br or I, preferably F. C1-C6 alkoxy is preferably methoxy, ethoxy, propoxy, and more preferably methoxy. In some specific implementations, R 1 , R 2 , R 3 and R 4 At least one of them is a hydroxyl group.

[0012] In some specific implementations, R 4 is hydroxyl or H. In some specific implementations, R 4 is hydroxyl or H, and R 2 is H, hydroxyl, halogen or C1-C6 alkoxy. In some specific implementations, R 4 is hydroxyl or H, R 2 is H, hydroxyl, halogen or C1-C6 alkoxy, and R 1 is H or hydroxyl; R 3 For H.

[0013] In some specific implementations, the cyclohexapeptide compound has a structure of formula I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-10, I-11, I-12, I-13, I-14, I-15, I-16, I-17, I-18, I-19, I-20, I-21, I-22, I-23 or I-24:

[0014]

[0015]

[0016] The present application also provides a method for preparing the cyclohexapeptide compound described in the above technical solution, comprising the following steps:

[0017] a) using solid phase synthesis to obtain a linear hexapeptide, wherein the amino acid sequence of the linear hexapeptide is: the amino C2-C6 alkyl acid-4 position is replaced by R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 Replaced Pro;

[0018] Or: 4 bits are R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3The Pro-4 position was replaced by R 4 Substituted Pro-amino C2~C6 alkyl acid;

[0019] Or: 4 bits are R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 1 Replaced Pro;

[0020] b) cyclizing the linear hexapeptide to obtain a cyclic hexapeptide compound.

[0021] The present application firstly adopts solid phase synthesis to prepare a linear hexapeptide, and then cyclizes and cuts it to obtain a cyclic hexapeptide compound. Since cyclization is required, the present application has no special restrictions on the starting amino acid when preparing the linear hexapeptide. For example, the amino acid sequence of the linear hexapeptide can be: the amino acid C2 to C6 alkyl acid -4 position is replaced by R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 Pro replaced; or: 4 digits replaced by R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 Substituted Pro-amino C2-C6 alkyl acid; or: R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 1 The following is a linear hexapeptide with amino C2-C6 alkyl acid-4 position replaced by R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 The replaced Pro is used as an example for explanation.

[0022] In some specific implementations, the linear hexapeptide is prepared according to the following method:

[0023] a) Sequentially connect 4 R 4 Pro replaced, 4th position replaced by R 3Substituted Pro, amino C2-C6 alkyl acid, R 2 Pro replaced, 4th position replaced by R 1 Substituted Pro and amino C2-C6 alkyl acid to obtain a resin containing a peptide chain;

[0024] b) Removal of the resin to obtain a linear hexapeptide.

[0025] The present invention adopts a solid phase synthesis method to sequentially connect 4 R 4 Pro replaced, 4th position replaced by R 3 Substituted Pro, amino C2-C6 alkyl acid, R 2 Pro replaced, 4th position replaced by R 1 Substituted Pro and amino C2~C6 alkyl acid to obtain a resin containing a peptide chain.

[0026] The present invention uses solid phase synthetic resin as the starting point for synthesis, firstly reacts with 4-position R 4 The substituted proline raw material reacts and the 4-position R is attached to the resin 4 Specifically, the present invention comprises a solid phase synthesis resin, a proline substituted by R 4 The substituted proline raw material, the reaction solvent and the base are mixed for the first reaction, and then mixed with the alcohol raw material for the second reaction. After deprotection, the 4-position R 4 Replaced Pro-Resin.

[0027] In some specific implementations, the solid phase synthetic resin includes but is not limited to Wang-resin, CTC-resin, etc., preferably CTC-resin. 4 The substituted proline raw material is preferably Fmoc-Pro (4-R 4 )-OH. The reaction solvent includes but is not limited to dichloromethane (DCM), N,N-dimethylformamide (DMF), dichloromethane (DCM), dimethyl sulfoxide (DMSO), etc., preferably DCM. The base includes but is not limited to N,N-diisopropylethylamine (DIEA), triethylamine (TEA), pyridine (Py), etc., preferably DIEA. In some specific implementations, the solid phase synthesis resin, the 4-position R 4 The molar ratio of the substituted proline raw material to the base is 1-10:5-20:0.005-0.1, preferably 5-8:10-15:0.01-0.05. In some specific implementations, the temperature of the first reaction is preferably 10°C-30°C, preferably 15°C-25°C, and the time is preferably 2h-5h, more preferably 2.5h-4.5h.

[0028] After the first reaction is completed, an alcohol raw material is directly added thereto to carry out a second reaction. In some specific implementations, the time of the second reaction is preferably 1 min to 10 min, more preferably 3 min to 8 min. In some specific implementations, the alcohol raw material includes but is not limited to methanol, ethanol, isopropanol, etc., preferably methanol. After the second reaction is completed, the reaction product is filtered, and the obtained resin is washed twice with a solvent such as DCM, washed twice with methanol, and then washed twice with DMF, and then deprotected. The present application has no special restrictions on the deprotection method. The deprotection method can be selected according to the type of protecting group. For example, when the solid phase synthetic resin is CTC-resin and the 4-position is replaced by R 4 The substituted proline raw material is preferably Fmoc-Pro (4-R 4 )-OH, preferably piperidine is used for deprotection, specifically comprising: mixing the obtained resin with a piperidine solution for reaction. In some specific implementations, the piperidine solution is preferably a DMF solution of piperidine, wherein the mass concentration of piperidine is preferably 10% to 30%, more preferably 15% to 25%. The reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, and the reaction time is preferably 20min to 50min, more preferably 25min to 45min. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably washed 6 times, and the resin is drained.

[0029] Get 4 bits by R 4 After the Pro-resin is substituted, the 4-position is connected to R 3 Pro replaced. This application first replaces the 4th position with R 3 The substituted Pro raw material, condensation agent and solvent are mixed for reaction, and the obtained reaction solution is mixed with the 4-position R 4 The substituted Pro-resin was mixed and reacted, and after deprotection, the 4-position R 3 The Pro-4 position was replaced by R 4 In some specific implementations, the 4 position is replaced by R 3 The substituted Pro raw material is Fmoc-Pro (4-R 3 )-OH; the condensing agent is a mixture of HOBt / DIC; and the solvent is DMF. Specifically, the present invention firstly 3 )-OH and HOBt are mixed and dissolved with DMF, and then mixed with DIC for reaction. In some specific implementations, the Fmoc-Pro (4-R 3The molar ratio of )-OH, HOBt and DIC is preferably 5-30:5-30:5-30, more preferably 8-25:8-25:8-25. In some specific implementations, the reaction is carried out under stirring, the reaction temperature is preferably 2°C to 8°C, and the reaction time is preferably 10min to 20min. After the reaction is completed, the obtained reaction product is mixed with the 4-positioned R 4 The substituted Pro-resin is mixed for reaction, the reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, the time is preferably 1h to 3h, more preferably 1.5h to 2.5h. After the reaction is completed, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably three times, and then deprotected. The present application has no special restrictions on the deprotection method, and the deprotection method can be selected according to the type of protecting group. For example, when the proline raw material is R- 3 When Pro is substituted, piperidine is preferably used for deprotection, specifically comprising: mixing the obtained resin with a piperidine solution for reaction. In some specific implementations, the piperidine solution is preferably a DMF solution of piperidine, wherein the mass concentration of piperidine is preferably 10% to 30%, more preferably 15% to 25%. The reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, and the time is preferably 20min to 40min, more preferably 25min to 35min. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably washed 6 times, and the resin is drained.

[0030] Get 4 bits by R 3 The Pro-4 position was replaced by R 4 After the Pro-resin is substituted, an amino C2-C6 alkyl acid is connected thereto. In the present application, the amino C2-C6 alkyl acid raw material, a condensing agent and a solvent are mixed for reaction, and then the obtained reaction solution is mixed with R 3 The Pro-4 position was replaced by R 4 The substituted Pro-resin is mixed and reacted, and after deprotection, amino C2-C6 alkyl acid-R 3 The Pro-4 position was replaced by R 4Substituted Pro-resin. In some specific implementations, the amino C2-C6 alkyl acid raw material is Fmoc-amino C2-C6 alkyl acid, such as aminoacetic acid (glycine, Gly) or β-aminopropionic acid (β-Ala); the condensing agent is a mixture of HOBt / DIC; the solvent is DMF. Specifically, the present application first mixes and dissolves the amino C2-C6 alkyl acid raw material and HOBt with DMF, and then mixes with DIC for reaction. In some specific implementations, the molar ratio of the amino C2-C6 alkyl acid raw material, HOBt and DIC is preferably 5-30:5-30:5-30, and more preferably 8-25:8-25:8-25. In some specific implementations, the reaction is carried out under stirring, the reaction temperature is preferably 2°C to 8°C, and the reaction time is preferably 10min to 20min. After the reaction is completed, the obtained reaction product is mixed with R at the 4 position. 3 The Pro-4 position was replaced by R 4 The substituted Pro-resin is mixed for reaction, and the reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, and the time is preferably 1h to 3h, and more preferably 1.5h to 2.5h. After the reaction is completed, the reaction product is filtered, and the obtained resin is washed with a reaction solvent such as DMF, preferably washed three times, and then deprotected. The present application has no special restrictions on the deprotection method, and the deprotection method can be selected according to the type of protecting group. For example, when the amino C2 to C6 alkyl acid raw material is Fmoc-amino C2 to C6 alkyl acid, piperidine is preferably used for deprotection, specifically including: mixing the obtained resin with a piperidine solution for reaction. In some specific implementations, the piperidine solution is preferably a DMF solution of piperidine, wherein the mass concentration of piperidine is preferably 10% to 30%, and more preferably 15% to 25%. The reaction temperature is preferably 10°C to 30°C, preferably 15°C to 25°C, and the time is preferably 20min to 40min, and more preferably 25min to 40min. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably 6 times, and the resin is drained to obtain amino C2-C6 alkyl acid-R 3 The Pro-4 position was replaced by R 4 Replaced Pro-Resin.

[0031] Obtain amino C2~C6 alkyl acid-R 3 The Pro-4 position was replaced by R 4 The Pro-resin substituted by R 2 Pro replaced, 4th position replaced by R 1 Substituted Pro and amino C2-C6 alkyl acid to obtain amino C2-C6 alkyl acid -4 position R 1The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 The substituted Pro-CTC resin is not described in detail in this application.

[0032] The amino C2-C6 alkyl acid -4 position is R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 After replacing the Pro-CTC resin, remove the resin thereon. In this application, trifluoroacetic acid is preferably used to remove the resin. The specific steps are as follows:

[0033] The amino C2-C6 alkyl acid -4 position is replaced by R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 The substituted Pro-CTC resin was mixed with trifluoroacetic acid / DCM solution and reacted, and then filtered to remove the resin.

[0034] In some specific implementations, the mass concentration of trifluoroacetic acid in the trifluoroacetic acid / DCM is 0.5% to 2%, preferably 1% to 1.5%. In some specific implementations, the reaction temperature is 20°C to 40°C, preferably 25°C to 35°C, and the reaction time is 20min to 40min, preferably 25min to 35min. In some specific implementations, the reaction is preferably carried out under stirring.

[0035] After removing the resin, the sequence is amino C2~C6 alkyl acid -4 position is replaced by R 1 The Pro-4 position was replaced by R 2 The substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 The Pro-4 position was replaced by R 4 A linear hexapeptide of substituted Pro, cyclizing the linear hexapeptide to obtain a cyclic hexapeptide compound. Specifically, the present application reacts the linear hexapeptide under the action of a condensing agent to cyclize the hexapeptide. In some specific implementations, the condensing agent is a composition of DIC, HOBt and DIEA, and the molar ratio of DIC, HOBt and DIEA is 1 to 2:1 to 2:1 to 2, preferably 1:1:1. The reaction temperature is 20°C to 40°C, preferably 25°C to 35°C, and the reaction time is 10h to 20h, preferably 12h to 16h.

[0036] As mentioned above, R 1 , R 2 , R 3 and R 4 are independently selected from H, hydroxyl, halogen or C1-C6 alkoxy, when R 1 , R 2 , R 3 and R 4 When R is independently selected from H, the corresponding amino acid is proline (Pro), and there is no need to protect the 4-position substituent; 1 , R 2 , R 3 and R 4 When R is independently selected from halogen or C1-C6 alkoxy, there is no need to protect the 4-substituent, and the corresponding cyclohexapeptide compound can be obtained after the cyclization reaction is completed. 1 , R 2 , R 3 and R 4 When independently selected from hydroxyl, the corresponding amino acid is hydroxyproline (Hyp), which needs to protect the hydroxyl group of the 4-position substituent. The present application has no special restrictions on the protecting group, and is preferably a tert-butyl protecting group. When the 4-position substituent is protected by a protecting group, it is preferably deprotected after the cyclization reaction is completed. The present application preferably uses trifluoroacetic acid for deprotection, and the specific steps are as follows:

[0037] The cyclohexapeptide compound containing a protecting group is mixed with a mixture of trifluoroacetic acid / triisopropylsilane / water, reacted, filtered, and the obtained filtrate is crystallized in a solvent to obtain a crude peptide.

[0038] In some specific implementations, the volume ratio of trifluoroacetic acid / triisopropylsilane / water is 80-90:5-10:5-10, more preferably 90:5:5. In some specific implementations, the reaction temperature is 0-5°C, and the reaction time is 1h-3h, preferably 1.5h-2.5h. In some specific implementations, the solvent includes but is not limited to tert-butyl methyl ether (MTBE), ether, isopropyl ether, etc., preferably MTBE. In some specific implementations, the crystallization temperature is preferably 2°C-8. The crystallized solid is centrifuged and vacuum dried to obtain a crude peptide.

[0039] After the cyclohexapeptide compound is obtained by cyclization reaction or the crude peptide is obtained by deprotection and crystallization, it is preferably purified by high performance liquid chromatography (HPLC), and the cyclohexapeptide compound represented by formula (I) can be obtained after freeze-drying. In some specific implementations, the HPLC column is a reverse phase C18 column.

[0040] The present application also provides a cosmetic comprising the cyclohexapeptide compound and auxiliary materials described in the above technical solution.

[0041] In this application, according to the cosmetics classification principle, cosmetics can be divided into: cleaning cosmetics, care cosmetics and beauty / decorative cosmetics.

[0042] Among them, the cleaning cosmetics refer to cosmetics that are applied to the human body surface (such as epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods to play a role in cleaning and hygiene or eliminating bad odors. The nursing cosmetics refer to cosmetics that are applied to the human body surface (such as epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods to play a role in maintenance. The beauty / modification cosmetics refer to cosmetics that are applied to the human body surface (such as epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods to play a role in beautification, modification, and increasing the charm of the human body.

[0043] Skin-applicable cleansing cosmetics include but are not limited to cleansers, makeup removers (lotions), cleansing creams (honeys), facial masks, floral water, prickly heat powder, talcum powder or bath gel; skin-care cosmetics include but are not limited to skin care creams, lotions or toners; skin-beautification / modification cosmetics include but are not limited to pressed powder, rouge, eye shadow, eyeliner (liquid), eyebrow pencil, perfume or cologne; hair-applicable cleansing cosmetics include but are not limited to shampoos, shampoo or shaving creams; hair-care cosmetics include but are not limited to conditioners, hair creams, hair oils / waxes, etc.; hair-beautification / modification cosmetics include but are not limited to powder, rouge, eye shadow, eyeliner (liquid), eyebrow pencils, perfume or cologne; hair-applicable cleansing cosmetics include but are not limited to shampoos, shampoo or shaving creams; hair-care cosmetics include but are not limited to conditioners, hair creams, hair oils / waxes, etc.; hair-beautification / modification cosmetics include but are not limited to Makeup / decoration cosmetics include but are not limited to styling mousse / hair spray, hair dye, perm, mascara (cream), hair growth agent or depilatory agent; cleaning cosmetics for nails include but are not limited to nail polish remover; care cosmetics for nails include but are not limited to nail water (cream), nail hardener; beauty / decoration cosmetics for nails include but are not limited to nail polish; cleaning cosmetics for lips include but are not limited to lip makeup remover; care cosmetics for lips include but are not limited to lip balm; beauty / decoration cosmetics for lips include but are not limited to lipstick, lip gloss or lip liner.

[0044] The cyclohexapeptide compound has higher stability and bioavailability, can significantly promote the secretion of collagen, especially type I collagen, type III collagen and type IV collagen, has a firming effect, can be used in cosmetics to maintain the structure and elasticity of the skin and promote skin health. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The results of the effect of the cyclohexapeptide prepared in Example 1 on human keratinocytes;

[0046] Figure 2 The expression level of type I collagen in the cyclic hexapeptide experimental group prepared in Example 1;

[0047] Figure 3 The expression level of type III collagen in the cyclic hexapeptide experimental group prepared in Example 1;

[0048] Figure 4 The expression level of type IV collagen in the cyclic hexapeptide experimental group prepared in Example 1. DETAILED DESCRIPTION

[0049] The present invention provides a cyclohexapeptide compound, a preparation method thereof and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0050] The present invention is further described below in conjunction with the embodiments:

[0051] A method for preparing a cyclohexapeptide compound 1, comprising:

[0052] Step S11: CTC resin (6.0 g, 6 mmol) was placed in a 100 mL solid phase synthesis reactor, and amino acid Fmoc-Hyp(tBu)-OH (4.9 g, 12 mmol) was added, and 60 mL of dichloromethane (DCM) was added, and then N,N-diisopropylethylamine (DIEA) (4.0 mL) was added, and the reaction was carried out at 25°C for 3 h, and 6.0 mL of methanol was added, and the reaction was carried out for 5 min. The resin was filtered, and the resin was washed twice with 60 mL of dichloromethane (DCM), 60 mL of methanol, and 60 mL of DMF. Then, 60 mL of 20% piperidine (Pip) / DMF solution was added to the solid phase synthesis reactor, and the reaction was stirred for 30 min. The deprotection solution was removed by suction filtration, and then the resin was washed 6 times with 60 mL of DMF solution, and dried for use.

[0053] Step S12: Take Fmoc-Pro-OH (5.1 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2°C to 8°C, add 25 mL of DMF solution and DIC (2.3 mL, 15 mmol) and let it react for 10 min to 20 min. Add the solution in the 100 mL beaker into a 100 mL solid phase synthesis reactor and stir to react for 1.5 h. After the reaction is completed, wash the resin three times with 60 mL of DMF solution each time. After washing, add 60 mL of 20% Pip / DMF solution and stir to react for 30 min. Filter to remove the deprotection solution, then wash 6 times with 60 mL of DMF solution and drain for use.

[0054] Step S13: Take Fmoc-Gly-OH (4.5 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2°C to 8°C, add 25 mL of DMF solution and DIC (2.3 mL, 15 mmol) and let it react for 10 min to 20 min. Add the solution in the 100 mL beaker into a 100 mL solid phase synthesis reactor and stir to react for 1.5 h. After the reaction is completed, wash the resin three times with 60 mL of DMF solution each time. After washing, add 60 mL of 20% Pip / DMF solution and stir to react for 30 min. Filter to remove the deprotection solution, then wash 6 times with 60 mL of DMF solution and drain for use.

[0055] Step S14: Take Fmoc-Hyp(tBu)-OH (6.1 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2°C to 8°C, add 25 mL of DMF solution and DIC (2.3 mL, 15 mmol) and let it react for 10 min to 20 min. Add the solution in the 100 mL beaker into a 100 mL solid phase synthesis reactor and stir to react for 1.5 h. After the reaction is completed, wash the resin three times with 60 mL of DMF solution each time. After washing, add 60 mL of 20% Pip / DMF solution and stir to react for 30 min. Filter to remove the deprotection solution, then wash 6 times with 60 mL of DMF solution and drain for standby use.

[0056] Step S15: Take Fmoc-Pro-OH (5.1 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2°C to 8°C, add 25 mL of DMF solution and DIC (2.3 mL, 15 mmol) and let it react for 10 min to 20 min. Add the solution in the 100 mL beaker into a 100 mL solid phase synthesis reactor and stir to react for 1.5 h. After the reaction is completed, wash the resin three times with 60 mL of DMF solution each time. After washing, add 60 mL of 20% Pip / DMF solution and stir to react for 30 min. Filter to remove the deprotection solution, then wash 6 times with 60 mL of DMF solution and drain for use.

[0057] Step S16: Take Fmoc-Gly-OH (4.5g, 15mmol), HOBt (2.03g, 15mmol) in a 100mL beaker, cool to 2°C to 8°C, add 25mL of DMF solution, DIC (2.3mL, 15mmol) and let stand for 10min to 20min, and add the solution in the 100mL beaker to a 100mL solid phase synthesis reactor, stir and react for 1.5h, the reaction is complete, the resin is washed three times with DMF solution, 60mL each time, after washing, add 60mL of 20% Pip / DMF solution, stir and react for 30min, filter to remove the deprotection solution, and then wash 6 times with 60mL of DMF solution, 2 times with 60mL of methanol, 2 times with 60mL of DCM solution, and 2 times with 60mL of methanol. Vacuum drying to obtain the peptide resin of H-Gly-Pro-Hyp(tBu) Gly-Pro-Hyp(tBu)-CTC resin.

[0058] Step S2: Add 200 mL of 1% trifluoroacetic acid (TFA) / DCM solution to the peptide resin of H-Gly-Pro-Hyp(tBu)-Gly-Pro-Hyp(tBu)-CTC resin, stir and react at 30°C for 30 minutes, filter, remove the resin, and obtain a filtrate. The filtrate is drained to obtain the fully protected peptide H-Gly-Pro-Hyp(tBu) Gly-Pro-Hyp(tBu)-OH.

[0059] Step S3: Dissolve the fully protected polypeptide H-Gly-Pro-Hyp(tBu)-Gly-Pro-Hyp(tBu)-OH in 1.0 L of dichloromethane (DCM), add DIC (1.5 mL, 10 mmol), HOBt (1.4 g, 10 mmol), and DIEA (1.7 mL, 10 mmol), and stir the reaction at 30°C for 12 to 16 hours to form Cyclo(Gly-Pro-Hyp(tBu)-Gly-Pro-Hyp(tBu)).

[0060] Step S4: The Cyclo(Gly-Pro-Hyp(tBu)-Gly-Pro-Hyp(tBu)) solid was cut with TFA / TIS / H2O=90 / 5 / 5 (50 mL) for 2.5 hours, the cutting solution was added to 500 mL of tert-butyl methyl ether (2°C~8°C) solution, a white solid was precipitated, and the mixture was centrifuged to obtain a white solid crude peptide. The white solid crude peptide was dried under vacuum to obtain a crude peptide powder Cyclo(Gly-Pro-Hyp-Gly-Pro-Hyp).

[0061] Step S5: The crude peptide powder Cyclo (Gly-Pro-Hyp Gly-Pro-Hyp) was purified by reverse phase C18 preparative chromatography and freeze-dried to obtain cyclohexapeptide compound 1: Cyclo (Gly-Pro-Hyp-Gly-Pro-Hyp), i.e., the cyclohexapeptide compound shown in formula I-1. Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.64-4.60(m,2H),4.40-4.35(m,2H),4.32(ddd,J=7.0,5.3,0.7Hz,2H),3.99(dd,J=15.8,6.0Hz,2H), 3.88(dd,J=15.8,6.0Hz,2H),3.71-3.59(m,6H),3.48(d,J=5.7Hz,2H),3. 45-3.38(m,2H),2.10-2.03(m,2H),2.03-1.93(m,4H),1.93-1.86(m,6H).C 24 H 34 N6O8,[M+H] + 535.25.

[0062]

[0063] Example 2

[0064] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to obtain cyclohexapeptide compound 2 shown in formula I-2: Cyclo (b-Ala-Pro-Hyp-Gly-Pro-Hyp). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=5.9Hz,1H),4.64-4.57(m,2H),4.41-4.34(m,2H), 4.31(dtd,J=6.5,5.6,0.9Hz,2H),3.99(dd,J=15.7,6.0Hz,1H),3.88(dd,J=15.7,6.0Hz,1H),3.70-3.59(m, 5H),3.55(dt,J=12.3,4.2Hz,1H),3.47(dd,J=15.5,5.8Hz,3H),3.44-3.37(m,3H),2.56(dt,J=14.8,6.1Hz, 1H),2.48(dt,J=14.8,6.1Hz,1H),2.17-2.09(m,2H),2.09-1.92(m,5H),1.89(dtt,J=6.5,5.0,3.4Hz,5H).C 25 H 36 N6O8,[M+H] + 549.26.

[0065]

[0066] Example 3

[0067] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH to obtain cyclohexapeptide compound 3 shown in formula I-3: Cyclo(Gly-Hyp-Hyp-Gly-Pro-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.65-4.60(m,1H),4.57(ddd,J=7 .7,5.9,0.7Hz,1H),4.45-4.35(m,3H),4.32(ddt,J=7.1,5.3,0.9Hz,2H),3.97( ddd,J=17.8,15.8,6.0Hz,2H),3.88(dd,J=15.9,5.9Hz,2H),3.67-3.59(m,7H), 3.48(d,J=5.7Hz,3H),3.45-3.38(m,1H),2.13-1.93(m,7H),1.93-1.85(m,3H).C 24 H 34 N6O9,[M+H] + 551.25.

[0068]

[0069] Example 4

[0070] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH to obtain cyclohexapeptide compound 4 shown in formula I-4: Cyclo(Gly-Pro-Pro(4-F)-Gly-Pro-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.91-4.77(m,1H),4.66-4.59(m,2H),4.51(dd d,J=6.6,4.8,0.8Hz,1H),4.41-4.34(m,1H),4.34-4.28(m,1H),3.99(dd,J=15.8,6.0Hz,2H), 3.88(dd,J=15.8,6.0Hz,2H),3.80-3.71(m,1H),3.71-3.68(m,1H),3.68-3.59(m,4H),3.48( d,J=5.7Hz,1H),3.45-3.38(m,2H),2.47-2.29(m,2H),2.10-1.95(m,5H),1.95-1.86(m,5H).C 24 H 33 FN6O7,[M+H] + 537.25.

[0071]

[0072] Example 5

[0073] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH to obtain cyclohexapeptide compound 5 shown in formula I-5: Cyclo(Gly-Pro-Pro(4-OMe)-Gly-Pro-Hyp). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.65-4.59(m,2H),4.41-4.34(m,2H),4.3 2(ddd,J=7.0,5.3,0.7Hz,1H),4.26-4.20(m,1H),3.99(dd,J=15.8,6.0Hz,2H),3.88(dd, J=15.8,6.0Hz,2H),3.75-3.60(m,6H),3.48(d,J=5.7Hz,1H),3.45-3.38(m,2H),3.19(d, J=1.5Hz,3H),2.25(ddd,J=12.4,5.8,4.9Hz,1H),2.10-1.95(m,5H),1.95-1.86(m,6H).C 25 H 36 N6O8,[M+H] + 549.27.

[0074]

[0075] Example 6

[0076] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro-OH to obtain cyclohexapeptide compound 6 shown in formula I-6: Cyclo(Gly-Pro-Pro-Gly-Pro-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=5.9Hz,1H),6.73(t,J=6.0Hz,1H),4.61(dt,J =6.1,4.3Hz,2H),4.47(dd,J=5.4,3.6Hz,1H),4.41-4.35(m,1H),4.34-4.30(m, 1H),3.99(dd,J=15.8,6.0Hz,2H),3.88(dd,J=15.8,6.0Hz,2H),3.69-3.56(m,5 H),3.54-3.47(m,2H),3.45-3.38(m,2H),2.10-1.95(m,6H),1.95-1.82(m,8H).C 24 H 34 N6O7,[M+H] + 519.25.

[0077]

[0078] Example 7

[0079] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro-OH, and Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro-OH, and step S4 is not required, to obtain cyclohexapeptide compound 7 shown in formula I-7: Cyclo(Gly-Pro-Pro-Gly-Pro-Pro). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.73(t,J=6.0Hz,2H),4.64-4.58(m,2H),4.47(dd,J=5.4,3.7Hz,2H),3.99(dd,J=15.7,6.0Hz,2H),3.88(dd,J=15.7,6.0H z,2H),3.68-3.56(m,4H),3.56-3.48(m,2H),3.45-3.38(m,2H),2.12-2. 02(m,4H),1.98(dddd,J=11.5,7.8,5.5,3.8Hz,2H),1.94-1.82(m,10H).C 24 H 34 N6O6,[M+H] + 503.26.

[0080]

[0081] Example 8

[0082] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Pro-OH in step S12 is replaced by Fmoc-Hyp(tBu)-OH, and Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH to obtain cyclohexapeptide compound 8 shown in formula I-8: Cyclo(Gly-Hyp-Hyp-Gly-Hyp-Hyp). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.57(ddd,J=7.7,5.9,0.7Hz,2H),4.44-4.35(m,2H),4.32(ddd,J=7.1,5.4,0.8Hz,2H),3.95(dd C 24 H 34 N6O 10 ,[M+H] + 567.24.

[0083]

[0084] Example 9

[0085] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, to obtain cyclohexapeptide compound 9 shown in formula I-9: Cyclo(b-Ala-Hyp-Hyp-Gly-Pro-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=6.0Hz,1H),4.66-4.59(m,1H),4.51( dddd,J=7.9,6.0,0.8Hz,1H),4.46-4.35(m,3H),4.31(dddd,J=7.3,6.5,5.4,0.7Hz,2H),3.99(d d,J=15.8,6.0Hz,1H),3.88(dd,J=15.7,6.0Hz,1H),3.71-3.58(m,7H),3.52-3.35(m,6H),2.5 8(dt,J=14.8,6.1Hz,1H),2.49(dt,J=14.8,6.0Hz,1H),2.14-1.94(m,7H),1.94-1.86(m,3H).C 25 H 36 N6O9,[M+H] + 565.26.

[0086]

[0087] Example 10

[0088] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Pro-OH in step S12 is replaced by Fmoc-Hyp(tBu)-OH, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to obtain cyclohexapeptide compound 10 shown in formula I-10: Cyclo(b-Ala-Hyp-Hyp-Gly-Hyp-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=6.0Hz,1H),4.57(ddd,J=7.7,5.9,0.8Hz,1H),4.51 (dddd,J=7.9,6.0,0.8Hz,1H),4.45-4.35(m,4H),4.31(dddd,J=7.3,6.4,5.4,0.7Hz,2H),3.95(dd,J=15.8,6 .0Hz,1H),3.88(dd,J=15.7,6.0Hz,1H),3.71-3.59(m,8H),3.49(t,J=5.5Hz,4H),3.47-3.36(m,2H),2.58(d t,J=14.8,6.1Hz,1H),2.49(dt,J=14.8,6.0Hz,1H),2.10(dtd,J=12.2,6.1,2.3Hz,2H),2.06-1.93(m,6H).C 25 H 36 N6O 10 ,[M+H] + 581.26.

[0089]

[0090] Embodiment 11

[0091] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to obtain cyclohexapeptide compound 11 shown in formula I-11: Cyclo(b-Ala-Pro-Pro(4-F)-Gly-Pro-Hyp). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=5.9Hz,1H),4.91-4.76(m,1H),4.64-4.58( m,2H),4.51(ddd,J=6.6,4.8,0.8Hz,1H),4.41-4.35(m,1H),4.33-4.28(m,1H),3.99(dd,J=15.7,6.0 Hz,1H),3.88(dd,J=15.7,6.0Hz,1H),3.80-3.67(m,2H),3.67-3.59(m,3H),3.55(dt,J=12.3,4.2Hz, 1H),3.51-3.36(m,5H),2.61-2.45(m,2H),2.45-2.30(m,2H),2.17-2.03(m,3H),2.03-1.85(m,7H).C 25 H 35 FN6O7,[M+H] + 551.26.

[0092]

[0093] Example 12

[0094] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to obtain cyclohexapeptide compound 12 shown in formula I-12: Cyclo(b-Ala-Pro-Pro(4-OMe)-Gly-Pro-Hyp). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=6.0Hz,1H),4.64-4.57(m,2H),4.37(dddd,J=7.7,7.0,4 .2,1.3Hz,2H),4.31(ddd,J=7.1,5.5,0.8Hz,1H),4.26-4.20(m,1H),3.99(dd,J=15.8,6.0Hz,1H),3.88(dd,J=15. 8,6.0Hz,1H),3.75-3.53(m,6H),3.51-3.36(m,5H),3.19(d,J=1.5Hz,3H),2.56(dt,J=14.8,6.1Hz,1H),2.48(dt, J=14.8,6.1Hz,1H),2.25(ddd,J=12.4,5.8,4.9Hz,1H),2.16-2.09(m,2H),2.09-1.93(m,4H),1.93-1.86(m,5H).C 26 H 38 N6O8,[M+H] + 563.28.

[0095]

[0096] Embodiment 13

[0097] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, and step S4 is not required, to obtain cyclohexapeptide compound 13 shown in formula I-13: Cyclo(b-Ala-Pro-Pro(4-F)-Gly-Pro-Pro(4-F)). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=5.9Hz,1H),4.93-4.76(m,2H),4.61(dd d,J=10.5,6.1,4.5Hz,2H),4.54-4.46(m,2H),3.99(dd,J=15.7,6.0Hz,1H),3.88(dd,J=15.8,6. 0Hz,1H),3.80-3.67(m,4H),3.67-3.60(m,1H),3.55(dt,J=12.3,4.2Hz,1H),3.51-3.36(m,4H), 2.59-2.45(m,2H),2.45-2.30(m,5H),2.17-2.09(m,2H),2.09-2.02(m,1H),1.94-1.86(m,5H).C 25 H 34 F2N6O6,[M+H] + 553.26.

[0098]

[0099] Embodiment 14

[0100] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-OMe)-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, and step S4 is not required, to obtain cyclohexapeptide compound 14 shown in formula I-14: Cyclo(b-Ala-Pro-Pro(4-OMe)-Gly-Pro-Pro(4-OMe)). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ7.38(t,J=5.6Hz,1H),6.82(t,J=6.0Hz,1H),4.64-4.58(m,2H),4.35(dddd,J=7.5,6. 9,4.9,0.8Hz,2H),4.23(tddd,J=6.7,4.1,1.6,1.0Hz,2H),3.99(dd,J=15.8,6.0Hz,1H),3.88(dd,J=15.8,6. 0Hz,1H),3.72(ddd,J=12.4,4.2,0.7Hz,2H),3.69-3.60(m,3H),3.59-3.52(m,1H),3.51-3.36(m,4H),3.19(d ,J=1.5Hz,6H),2.59-2.45(m,2H),2.25(ddd,J=12.3,5.9,5.0Hz,2H),2.16-2.01(m,5H),1.95-1.85(m,5H).C 27 H 40 N6O8,[M+H] + 577.30.

[0101]

[0102] Embodiment 15

[0103] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that Fmoc-Gly-OH in step S13 is replaced by Fmoc-b-Ala-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to obtain cyclohexapeptide compound 15 shown in formula I-15: Cyclo (b-Ala-Pro-Hyp-b-Ala-Pro-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.34(t,J=5.6Hz,2H),4.60(dd,J=5.9,4.6Hz,2H),4.37(ddddd ,J=7.3,6.3,5.4,3.2,0.8Hz,2H),4.31(ddd,J=7.1,5.5,0.8Hz,2H),3.70-3.59(m,4H), 3.59-3.53(m,2H),3.51-3.44(m,4H),3.44-3.36(m,4H),2.56(dt,J=14.8,6.1Hz,2H), 2.48(dt,J=14.8,6.0Hz,2H),2.16-2.08(m,4H),2.07-1.94(m,4H),1.92-1.85(m,4H).C26 H 38 N6O8,[M+H] + 563.28.

[0104]

[0105] Example 16

[0106] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Pro-OH in step S12 is replaced by Fmoc-Hyp(tBu)-OH, Fmoc-Gly-OH in step S13 is replaced by Fmoc-b-Ala-OH, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to prepare cyclohexapeptide compound 16 shown in formula I-16: Cyclo(b-Ala-Hyp-Hyp-b-Ala-Hyp-Hyp). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.34(t,J=5.6Hz,2H),4.51(ddd,J=7.9,6.0,0.8Hz,2H),4.45-4. 34(m,4H),4.31(ddd,J=7.1,5.5,0.8Hz,2H),3.67(ddd,J=12.4,5.1,2.5Hz,4H),3.62(dd d,J=12.4,4.4,2.2Hz,4H),3.49(t,J=5.5Hz,4H),3.47-3.36(m,4H),2.58(dt,J=14.8,6. 1Hz,2H),2.49(dt,J=15.0,6.1Hz,2H),2.11(dt,J=12.4,6.2Hz,2H),2.05-1.94(m,6H).C 26 H 38 N6O 10 ,[M+H] + 595.27.

[0107]

[0108] Embodiment 17

[0109] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Gly-OH in step S13 is replaced by Fmoc-b-Ala-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, and step S4 is not required, thereby obtaining cyclohexapeptide compound 17 shown in formula I-17: Cyclo(b-Ala-Pro-Pro(4-F)-b-Ala-Pro-Pro(4-F)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.34(t,J=5.6Hz,2H),4.91-4.76(m,2H),4.60(dd,J=5.9,4.6Hz,2H),4.49(ddd,J=6.4,5.0,0.8Hz,2H),3.78-3.72( m,2H),3.72-3.65(m,2H),3.58-3.52(m,2H),3.51-3.36(m,6H),2.60- 2.45(m,4H),2.45-2.30(m,4H),2.18-2.07(m,4H),1.92-1.86(m,4H).C 26 H 36 F2N6O6,[M+H] + 567.27.

[0110]

[0111] Embodiment 18:

[0112] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-OMe)-OH, Fmoc-Gly-OH in step S13 is replaced by Fmoc-b-Ala-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, and step S4 is not required, to obtain cyclohexapeptide compound 18 shown in formula I-18: Cyclo(b-Ala-Pro-Pro(4-OMe)-b-Ala-Pro-Pro(4-OMe)). Its NMR data are as follows: 1H NMR(500MHz,MeOH-d4)δ7.34(t,J=5.6Hz,2H),4.60(dd,J=5.9,4.6Hz,2H),4.34(ddd,J=7.0,4.9,0.8Hz, 2H),4.26-4.20(m,2H),3.72(dd,J=12.2,4.2Hz,2H),3.66(dd,J=12.6,2.7Hz,2H),3.58-3.53(m,2H),3. 51-3.36(m,6H),3.19(d,J=1.5Hz,6H),2.56(dt,J=14.8,6.1Hz,2H),2.48(dt,J=14.8,6.0Hz,2H),2.25( ddd,J=12.5,5.8,4.9Hz,2H),2.16-2.09(m,4H),2.04(ddd,J=12.4,6.8,4.0Hz,2H),1.92-1.86(m,4H).C 28 H 42 N6O8,[M+H] + 591.31.

[0113]

[0114] Embodiment 19:

[0115] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-F)-OH, and Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, and step S4 is not required, to obtain cyclohexapeptide compound 19 shown in formula I-19: Cyclo(Gly-Pro-Pro(4-F)-Gly-Pro-Pro(4-F)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.91-4.76(m,2H),4.65-4.59(m,2H),4.51(ddd,J=6.6,4.9,0.8Hz,2H),3.99(dd,J=15.8, 6.0Hz,2H),3.88(dd,J=15.8,6.0Hz,2H),3.80-3.60(m,6H),3.46-3.37(m,2H),2.46-2.30(m,4H),2.10-2.01(m,2H),1.94-1.85(m,6H).C 24 H 32 F2N6O6,[M+H] +539.24.

[0116]

[0117] Embodiment 20

[0118] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-OMe)-OH, and Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, and step S4 is not required, to obtain cyclohexapeptide compound 20 shown in formula I-20: Cyclo(Gly-Pro-Pro(4-OMe)-Gly-Pro-Pro(4-OMe)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.65-4.58(m,2H),4.36(ddd,J=6.8,5.0,0.8Hz, 2H), 4.23 (dddt, J=4.9, 4.1, 3.3, 1.5Hz, 2H), 3.99 (dd, J=15.8, 6.0Hz, 2H), 3.88 (dd, J=15.8, 6.0 Hz,2H),3.72(dd,J=12.5,4.2Hz,2H),3.69-3.65(m,2H),3.65-3.61(m,2H),3.44-3.38(m,2H),3 .19(d,J=1.5Hz,6H),2.25(ddd,J=12.5,5.8,5.0Hz,2H),2.10-2.00(m,4H),1.95-1.85(m,6H).C 26 H 38 N6O8,[M+H] + 563.28.

[0119]

[0120] Embodiment 21

[0121] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Pro-OH in step S12 is replaced by Fmoc-Hyp(tBu)-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, and Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH to prepare cyclohexapeptide compound 21 shown in formula I-21: Cyclo(Gly-Hyp-Pro(4-F)-Gly-Hyp-Pro(4-F)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.91-4.77(m,2H),4.57(ddd,J=7.7,5.9,0.7Hz ,2H),4.51(ddd,J=6.6,4.9,0.8Hz,2H),4.44-4.38(m,2H),3.95(dd,J=15.8,6.0Hz,2H),3.88( dd,J=15.7,6.0Hz,2H),3.78-3.71(m,2H),3.71-3.67(m,2H),3.67-3.61(m,4H),3.48(d,J=5. 7Hz,2H),2.46-2.29(m,4H),2.10(dt,J=12.2,6.0Hz,2H),2.01(ddd,J=12.2,7.8,4.5Hz,2H).C 24 H 32 F2N6O8,[M+H] + 571.23.

[0122]

[0123] Embodiment 22

[0124] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-OMe)-OH, Fmoc-Pro-OH in step S12 is replaced by Fmoc-Hyp(tBu)-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, and Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH to prepare cyclohexapeptide compound 22 shown in formula I-22: Cyclo(Gly-Hyp-Pro(4-OMe)-Gly-Hyp-Pro(4-OMe)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ6.82(t,J=6.0Hz,2H),4.57(ddd,J=7.7,5.9,0.7Hz,2H),4.44-4.38(m,2H),4 .35(ddd,J=7.0,5.0,0.8Hz,2H),4.23(dtt,J=5.8,2.6,1.6Hz,2H),3.95(dd,J=15.8,6.0Hz,2H),3.88 (dd,J=15.7,6.0Hz,2H),3.72(dd,J=12.2,4.2Hz,2H),3.69-3.61(m,6H),3.48(d,J=5.7Hz,2H),3.19( d,J=1.5Hz,6H),2.25(ddd,J=12.5,5.8,4.9Hz,2H),2.10(dt,J=12.2,6.0Hz,2H),2.07-1.97(m,4H).C 26 H 38 N6O 10 ,[M+H] + 595.27.

[0125]

[0126] Embodiment 23:

[0127] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Pro-OH in step S12 is replaced by Fmoc-Hyp(tBu)-OH, Fmoc-Gly-OH in step S13 is replaced by Fmoc-b-Ala-OH, and Fmoc-Hyp( In step S14, tBu)-OH is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to obtain cyclohexapeptide compound 23 shown in formula I-23: Cyclo(b-Ala-Hyp-Pro(4-F)-b-Ala-Hyp-Pro(4-F)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.34(t,J=5.6Hz,2H),4.91-4.76(m,2H),4.55-4.46(m,4H),4.45 -4.38(m,2H),3.78-3.71(m,2H),3.71-3.65(m,4H),3.62(dd,J=12.4,4.2Hz,2H),3.49(d ,J=5.7Hz,2H),3.48-3.36(m,4H),2.58(dt,J=14.8,6.1Hz,2H),2.49(dt,J=15.0,6.1Hz, 2H),2.45-2.30(m,4H),2.11(dt,J=12.3,6.2Hz,2H),1.99(ddd,J=12.2,7.7,4.4Hz,2H).C 26 H 36 F2N6O8,[M+H] + 599.27.

[0128]

[0129] Embodiment 24

[0130] The raw materials and preparation method used in this example are basically the same as those in Example 1, except that: Fmoc-Hyp(tBu)-OH in step S11 is replaced by Fmoc-Pro(4-OMe)-OH, Fmoc-Pro-OH in step S12 is replaced by FmocHyp(tBu)-OH, Fmoc-Gly-OH in step S13 is replaced by Fmoc-b-Ala-OH, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, and Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH to prepare cyclohexapeptide compound 24 shown in formula I-24: Cyclo

[0131] (b-Ala-Hyp-Pro(4-OMe)-b-Ala-Hyp-Pro(4-OMe)). Its NMR data are as follows: 1 H NMR(500MHz,MeOH-d4)δ7.34(t,J=5.6Hz,2H),4.51(ddd,J=7.9,6.1,0.8Hz,2H),4.44-4.38(m,2H) ,4.34(ddd,J=7.0,5.0,0.8Hz,2H),4.23(dtt,J=5.8,2.6,1.6Hz,2H),3.74-3.59(m,8H),3.49(d,J= 5.7Hz,2H),3.48-3.36(m,4H),3.19(d,J=1.5Hz,6H),2.58(dt,J=14.8,6.1Hz,2H),2.49(dt,J=15. 0,6.1Hz,2H),2.25(ddd,J=12.5,5.8,4.9Hz,2H),2.11(dt,J=12.4,6.2Hz,2H),2.07-1.95(m,4H).C 28 H 42 N6O 10 ,[M+H] + 623.31.

[0132]

[0133] Test Example 1 Cytotoxicity Detection

[0134] Human keratinocytes (HaCaT) were cultured at 2×10 4 / 96well was cultured in a 96-well cell culture plate and placed in a 37°C, 5% CO2 incubator. After 48 hours, the culture medium was aspirated and discarded, and the cells were washed once with 1×PBS. Bioactive substance treatment was performed, and different concentrations of the cyclohexapeptide compounds prepared in Examples 1 to 7 were added respectively. After 24 hours of treatment, the culture medium was removed, and 100 μL of culture medium containing 10% CCK-8 solution was added. The cells were placed in an incubator for 1 hour, and the absorbance at 450 nm was detected by an enzyme marker. At the same time, the solvent without the sample was used as the control group, and the cell survival rate was calculated according to the following formula: Cell survival rate % = (absorbance of the experimental group / absorbance of the control group) × 100%.

[0135] Results Figure 1 and Table 1, Figure 1 Table 1 shows the effect of the cyclohexapeptide prepared in Example 1 on human keratinocytes, and Table 2 shows the effect of the cyclohexapeptide prepared in Examples 1 to 7 on keratinocytes. Figure 1 As can be seen from Table 1, the cyclohexapeptides prepared in Examples 1 to 7 have no significant cytotoxicity to human keratinocytes.

[0136] Table 1 Effect of cyclohexapeptides prepared in Examples 1 to 7 on keratinocytes

[0137] Relative cell viability (%) 1000 μg / mL 100 μg / mL 10 μg / mL 1 μg / mL Example 1 85.59 96.84 95.73 99.35 Example 2 84.32 95.44 95.21 98.45 Example 3 83.44 94.22 92.46 98.74 Example 4 81.36 92.82 94.54 96.26 Example 5 80.68 95.48 93.86 98.52 Example 6 82.31 93.54 95.12 94.88 Example 7 82.45 93.62 93.54 96.84

[0138] Test Example 2 Firming Effect

[0139] Detection reagents: Human type I collagen α1 (COL1α1) enzyme-linked immunosorbent assay kit (catalog number: E-EL-H0869, Elabscience, China); human type III collagen (COL3) enzyme-linked immunosorbent assay kit (catalog number: E-EL-H6049, Elabscience, China); human type IV collagen (COL4) enzyme-linked immunosorbent assay kit (catalog number: E-EL-H0178, Elabscience, China).

[0140] Test cells: fibroblasts (HFF-1), from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (Cell Bank of the Chinese Academy of Sciences).

[0141] Detection method: Fibroblasts (HFF-1) were cultured at 5×10 4 / 6wel was cultured in a 6-well cell culture plate and placed in a 37°C, 5% CO2 incubator. After 48 hours, the culture medium was discarded, washed once with 1×PBS, and 500μg / mL of the cyclohexapeptide prepared in Examples 1 to 7 was added. The volume of each well was 2mL. A normal control group and an experimental group were set, with three biological replicates for each group. Incubate in a cell culture incubator for 48 hours. Then, the cell supernatant was aspirated, centrifuged at 1000g, 4°C for 20 minutes, the supernatant was collected, and the precipitate was discarded. Follow the instructions of the collagen ELISA kit for subsequent operations.

[0142] Results Figure 2 , Figure 3 , Figure 4 , Table 2, Table 3 and Table 4, Figure 2 The expression level of type I collagen in the cyclohexapeptide experimental group prepared in Example 1, Figure 3 The expression level of type III collagen in the cyclohexapeptide experimental group prepared in Example 1, Figure 4 Table 2 is the expression of type I collagen in the cyclohexapeptide experimental group prepared in Example 1, Table 3 is the expression of type III collagen in the cyclohexapeptide experimental group prepared in Examples 1 to 7, and Table 4 is the expression of type IV collagen in the cyclohexapeptide experimental group prepared in Examples 1 to 7. It can be seen that after the treatment with the cyclohexapeptide prepared in Examples 1 to 7, the expression of type I, type III and type IV collagen increased significantly, which shows that the cyclohexapeptide has an excellent firming effect.

[0143] Table 2 Expression of type I collagen in the cyclic hexapeptide experimental groups prepared in Examples 1 to 7

[0144] Group Type I collagen concentration (ng / mL) P-value Blank control 6.07±0.1 / Example 1 6.82±0.11 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 2 6.37±0.02 <![CDATA[ ＊ P<0.5 <!-- 20 -->]]> Example 3 6.44±0.06 <![CDATA[ ＊ P<0.5]]> Example 4 6.37±0.05 <![CDATA[ ＊ P<0.5]]> Example 5 6.38±0.11 <![CDATA[ ＊ P<0.5]]> Example 6 6.41±0.1 <![CDATA[ ＊ P<0.5]]> Example 7 6.38±0.08 <![CDATA[ ＊ P<0.5]]>

[0145] Table 3 Expression of type III collagen in the cyclic hexapeptide experimental group prepared in Examples 1 to 7

[0146] Group Type III collagen concentration (ng / mL) P-value Blank control 0.86±0.18 / Example 1 2.19±0.05 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 2 1.83±0.05 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 3 1.58±0.2 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 4 1.68±0.03 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 5 1.78±0.03 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 6 1.68±0.03 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 7 1.82±0.02 <![CDATA[ ＊＊＊＊ P<0.0001]]>

[0147] Table 4 Expression of type IV collagen in the cyclohexapeptide experimental group prepared in Examples 1 to 7

[0148] Group Type IV collagen concentration (ng / mL) P-value Blank control 6.97±0.23 / Example 1 15.15±0.36 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 2 13.64±0.32 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 3 12.26±0.41 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 4 11.86±0.48 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 5 13.41±0.15 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 6 12.09±0.63 <![CDATA[ ＊＊＊＊ P<0.0001]]> Example 7 11.88±0.32 <![CDATA[ ＊＊＊＊ P<0.0001]]>

[0149] In the above embodiments, the amino acid sequences are shown in the following table:

[0150]

[0151] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of the cyclohexapeptide compound represented by formula (I) in the preparation of products with firming effect: In formula (I), x and y are independently 1 or 2; R 4 is hydroxyl or H; R 2 is H, hydroxyl or halogen; R 1 is H or hydroxyl; R 3 For H.

2. The use according to claim 1, characterized in that: The cyclohexapeptide compound is shown in Formula I-3, Formula I-4, Formula I-6 or Formula I-7:

3. The use according to claim 1 or 2, characterized in that: The product is a cosmetic.

4. Application of the cyclohexapeptide compound represented by formula (I) in the preparation of products promoting collagen secretion: In formula (I), x and y are independently 1 or 2; R 4 is hydroxyl or H; R 2 is H, hydroxyl or halogen; R 1 is H or hydroxyl; R 3 For H.

5. The use according to claim 4, characterized in that: The cyclohexapeptide compound is shown in Formula I-3, Formula I-4, Formula I-6 or Formula I-7:

6. The use according to claim 4, characterized in that: The collagen is type I collagen, type III collagen and / or type IV collagen.

7. The use according to any one of claims 4 to 6, characterized in that: The product is a cosmetic.

8. A cosmetic comprising the cyclohexapeptide compound according to claim 1: In formula (I), x and y are independently 1 or 2; R 4 is hydroxyl or H; R 2 is H, hydroxyl or halogen; R 1 is H or hydroxyl; R 3 For H.

9. The cosmetic according to claim 8, characterized in that: The cyclohexapeptide compound is shown in Formula I-3, Formula I-4, Formula I-6 or Formula I-7:

10. The cosmetic according to claim 8, characterized in that: It is a care cosmetic.

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