Cyclohexapeptide compounds, methods of making and using the same

Abstract

The application provides a cyclic hexapeptide compound, a preparation method and application thereof. The cyclic hexapeptide compound has the structure of formula (I), 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 tightening effect, and can be applied to cosmetics to maintain the structure and elasticity of skin and promote skin health.

Description

[0001] This application is a divisional application of the application with the application date of December 12, 2024, the application number of 202411831485.9, and the invention title of "Cyclohexapeptide compound, preparation method thereof and application thereof". TECHNICAL FIELD

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

[0003] Skin is the largest organ of the human body, covering the surface of the whole body, and has multiple functions such as protection, regulation, sensation, secretion and excretion, immunity and respiration. With the growth of age, the collagen in the human body will gradually decrease, resulting in the appearance of skin relaxation. Polypeptides can improve the skin condition, maintain the health and youth of the skin through multiple mechanisms such as promoting collagen production, antioxidant, free radical scavenging, promoting skin cell proliferation and repairing skin wounds, and anti-inflammatory.

[0004] Peptides have the effects of non-toxicity and easy absorption by the skin, and can improve the state of the epidermis and dermis after use. In recent years, polypeptide ingredients have attracted widespread attention due to their significant firming effect, especially cyclic polypeptides, which have shown great potential in improving skin firmness. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a cyclohexapeptide compound, a preparation method thereof and application thereof. The cyclohexapeptide 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 from 1 to 5;

[0009] R 1 , R 2 , R 3 and R 4 are independently selected from H, hydroxyl, 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 a hydroxyl group, a halogen, or a C1-C6 alkoxy group. The halogen is selected from F, Cl, Br, or I, preferably F. The C1-C6 alkoxy group is preferably methoxy, ethoxy, or propoxy, 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 It is a hydroxyl group or H. In some specific implementations, R 4 It is a hydroxyl group or H, and at the same time, R 2 It can be H, hydroxyl, halogen, or a C1-C6 alkoxy group. In some specific implementations, R... 4 Hydroxyl group or H, R 2 It is H, hydroxyl, halogen, or C1-C6 alkoxy group, while R 1 H or hydroxyl; R 3 For H.

[0013] In some specific implementations, the cyclic hexapeptide compound has the 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] This application also provides a method for preparing the cyclic hexapeptide compound described in the above technical solution, comprising the following steps:

[0017] a) A linear hexapeptide was obtained by solid-phase synthesis. The amino acid sequence of the linear hexapeptide is as follows: amino group C2-C6 alkyl acid -4 position is R 1 The Pro-4 bit that was replaced was R 2 The substituted Pro-amino C2-C6 alkyl acid is R at the -4 position. 3 The Pro-4 bit that was replaced was R 4 Replacement Pro;

[0018] Or: 4 bits were R 1 The Pro-4 bit that was replaced was R 2 The substituted Pro-amino C2-C6 alkyl acid is R at the -4 position. 3substituted Pro-4 position is substituted by R 4 substituted Pro-amino C2-C6 alkyl acid

[0019] or is: 4 position is substituted by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 3 substituted Pro-4 position is substituted by R 4 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 1 substituted Pro

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

[0021] The present application first adopts solid phase synthesis to prepare a linear hexapeptide, and then circularizes and cuts the linear hexapeptide to obtain a cyclic hexapeptide compound. Since circularization is needed, the present application does not have special restrictions on the starting amino acid when preparing the linear hexapeptide. For example, the amino acid sequence of the linear hexapeptide can be: amino C2-C6 alkyl acid-4 position is substituted by R 1 substituted Pro-4 position is substituted by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 3 substituted Pro-4 position is substituted by R 4 substituted Pro; or is: 4 position is substituted by R 1 substituted Pro-4 position is substituted by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 3 substituted Pro-4 position is substituted by R 4 substituted Pro-amino C2-C6 alkyl acid; or is: 4 position is substituted by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 3 substituted Pro-4 position is substituted by R 4 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 1 substituted Pro. The following takes amino C2-C6 alkyl acid-4 position is substituted by R 1 substituted Pro-4 position is substituted by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is substituted by R 3 substituted Pro-4 position is substituted by R 4 substituted Pro as an example for illustration.

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

[0023] a) sequentially connecting 4 position is substituted by R 4 substituted Pro, 4 position is substituted by R 3substituted Pro, amino C2-C6 alkyl acid, R 2 substituted Pro, R 1 substituted Pro and amino C2-C6 alkyl acid, to obtain a resin containing a peptide chain.

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

[0025] The present application uses solid phase synthesis method, sequentially connects 4-position R 4 substituted Pro, R 3 substituted Pro, amino C2-C6 alkyl acid, R 2 substituted Pro, R 1 substituted Pro and amino C2-C6 alkyl acid, to obtain a resin containing a peptide chain.

[0026] The present application takes solid phase synthesis resin as the starting point of synthesis, first reacts with 4-position R 4 substituted proline raw material, and connects 4-position R 4 substituted proline on the resin. Specifically, the present application mixes solid phase synthesis resin, 4-position R 4 substituted proline raw material, reaction solvent and base to carry out the first reaction, and then mixes with alcohol raw material to carry out the second reaction, and after deprotection, 4-position R 4 substituted Pro-resin is obtained.

[0027] In some specific implementations, the solid phase synthesis resin includes but is not limited to Wang-resin, CTC-resin, etc., and is preferably CTC-resin. The 4-position R 4 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., and is preferably DCM. The base includes but is not limited to N,N-diisopropyl ethylamine (DIEA), triethylamine (TEA), pyridine (Py), etc., and is preferably DIEA. In some specific implementations, the molar ratio of the solid phase synthesis resin, 4-position R 4 substituted proline raw material and base is 1-10:5-20:0.005-0.1, and is 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, and is preferably 15°C-25°C, and the time is preferably 2h-5h, and is more preferably 2.5h-4.5h.

[0028] After the first reaction is complete, an alcoholic raw material is directly added to initiate a second reaction. In some specific implementations, the second reaction time is preferably 1 min to 10 min, more preferably 3 min to 8 min. In some specific implementations, the alcoholic raw material includes, but is not limited to, methanol, ethanol, isopropanol, etc., with methanol being preferred. After the second reaction is complete, the reaction product is filtered, and the resulting resin is washed twice with a solvent such as DCM, twice with methanol, and twice with DMF, and then deprotected. This application does not impose any special limitations 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 a CTC-resin, and the 4-position is protected by R... 4 The preferred proline substitute is Fmoc-Pro(4-R) 4 For the OH group, piperidine is preferably used for deprotection, specifically involving 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%–30%, more preferably 15%–25%. The reaction temperature is preferably 10°C–30°C, more preferably 15°C–25°C, and the reaction time is preferably 20 min–50 min, more preferably 25 min–45 min. After the reaction is complete, the obtained product is washed with a reaction solvent such as DMF, preferably six times, and the resin is dried.

[0029] 4 were obtained by R 4 After replacing the Pro-resin, four R-type connections are made on it. 3 The replacement Pro. This application first uses 4 bits that are R 3 The substitute Pro raw material, condensing agent, and solvent are mixed and reacted, and then the resulting reaction solution is reacted with the 4-position R. 4 The substituted Pro-resin mixture was reacted, and after deprotection, 4-position R-protected was obtained. 3 The Pro-4 bit that was replaced was R 4 Replaced Pro-resin. In some specific implementations, the 4 bits are R 3 The replacement Pro material is Fmoc-Pro(4-R) 3 )-OH; the condensing agent is a mixture of HOBt / DIC; the solvent is DMF. Specifically, this application firstly uses Fmoc-Pro(4-R 3 Fmoc-Pro(4-R)-OH and HOBt are mixed and dissolved with DMF, and then reacted with DIC. In some specific implementations, the Fmoc-Pro(4-R)-OH and HOBt are mixed and dissolved with DMF, and then reacted with DIC. 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 embodiments, the reaction is carried out under stirring, and the temperature of the reaction is preferably 2-8°C, and the time is preferably 10-20 min. After the reaction is completed, the obtained reaction product is mixed with the R 4 substituted Pro-resin, and the temperature of the reaction is preferably 10-30°C, preferably 15-25°C, and the time is preferably 1-3 h, more preferably 1.5-2.5 h. After the reaction is completed, the reaction product is filtered, the obtained resin is washed with a reaction solvent such as DMF, preferably three times, and then deprotected. The method of deprotection is not particularly limited in the present application, and the method of deprotection is selected according to the type of the protecting group, for example, when the proline raw material is 4-R 3 substituted Pro, piperidine is preferably used for deprotection, which specifically includes: the obtained resin is mixed with a piperidine solution for reaction. In some specific embodiments, the piperidine solution is preferably a DMF solution of piperidine, and the mass concentration of piperidine is preferably 10%-30%, more preferably 15%-25%. The temperature of the reaction is preferably 10-30°C, preferably 15-25°C, and the time is preferably 20-40 min, more preferably 25-35 min. After the reaction is completed, the obtained product is washed with a reaction solvent such as DMF, preferably six times, and the resin is dried.

[0030] substituted Pro-4-R 3 substituted Pro-4-R 4 substituted Pro-resin, and then an amino C2-C6 alkyl acid is connected thereto. The present application first mixes the amino C2-C6 alkyl acid raw material, a condensing agent and a solvent for reaction, and then mixes the obtained reaction solution with the R 3 substituted Pro-4-R 4 substituted Pro-resin, and then deprotects to obtain an amino C2-C6 alkyl acid-R 3 substituted Pro-4-R 4A substituted Pro-resin. In some specific implementations, the aminoC2-C6 alkyl acid raw material is Fmoc-aminoC2-C6 alkyl acid, such as glycine (Gly) or β-aminopropionic acid (β-Ala); the condensing agent is a mixture of HOBt / DIC; and the solvent is DMF. Specifically, this application first mixes and dissolves the aminoC2-C6 alkyl acid raw material and HOBt with DMF, and then reacts it with DIC. In some specific implementations, the molar ratio of the aminoC2-C6 alkyl acid raw material, 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 conditions, the reaction temperature is preferably 2°C-8°C, and the reaction time is preferably 10-20 min. After the reaction is complete, the obtained reaction product is reacted with the R-coated product at the 4-position. 3 The Pro-4 bit that was replaced was R 4 The substituted Pro-resin is reacted with a mixture of resins. The reaction temperature is preferably 10°C to 30°C, more preferably 15°C to 25°C, and the reaction time is preferably 1h to 3h, more preferably 1.5h to 2.5h. After the reaction is complete, the reaction product is filtered, and the resulting resin is washed with a reaction solvent such as DMF, preferably three times, and then deprotected. This application does not impose any particular limitation on the deprotection method; the method can be selected according to the type of protecting group. For example, when the amino C2-C6 alkyl acid raw material is Fmoc-amino C2-C6 alkyl acid, piperidine is preferably used for deprotection, specifically including: mixing the obtained resin with a piperidine solution and reacting. 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, more preferably 15°C to 25°C, and the reaction time is preferably 20min to 40min, more preferably 25min to 40min. After the reaction is complete, the product is washed with a reaction solvent such as DMF, preferably six times. The resin is then dried to obtain amino C2-C6 alkyl acid-R. 3 The Pro-4 bit that was replaced was R 4 Replacement Pro-resin.

[0031] To obtain amino C2-C6 alkyl acids -R 3 The Pro-4 bit that was replaced was R 4 The replacement Pro-resin is sequentially connected to 4 R-terminals using the same method as described above. 2 The Pro and 4-bit versions were replaced by R. 1 Substitution of Pro and amino C2-C6 alkyl acids yields amino C2-C6 alkyl acids with R at the -4 position. 1substituted Pro-4 position is replaced by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 substituted Pro-4 position is replaced by R 4 substituted Pro-CTC resin, which is not described herein again.

[0032] substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 1 substituted Pro-4 position is replaced by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 substituted Pro-4 position is replaced by R 4 substituted Pro-CTC resin, and then the resin on the substituted Pro-CTC resin is removed, and trifluoroacetic acid is preferably used to remove the resin, and the specific steps are as follows:

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

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

[0035] substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 1 substituted Pro-4 position is replaced by R 2 substituted Pro-amino C2-C6 alkyl acid-4 position is replaced by R 3 substituted Pro-4 position is replaced by R 4 substituted Pro, and the linear hexapeptide is cyclized to obtain a cyclic hexapeptide compound. Specifically, the linear hexapeptide is reacted under the action of a condensing agent to form a ring. In some specific implementations, the condensing agent is a combination of DIC, HOBt, and DIEA, and the molar ratio of the DIC, HOBt, and DIEA is 1 to 2: 1 to 2: 1 to 2, and is preferably 1: 1: 1. The temperature of the reaction is 20°C to 40°C, and is preferably 25°C to 35°C, and the time of the reaction is 10 h to 20 h, and is preferably 12 h to 16 h.

[0036] As described 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 are independently selected from H, the corresponding amino acid is proline (Pro) and no protection is needed for the substituent at position 4; when R 1 , R 2 , R 3 and R 4 are independently selected from halogen or C1-C6 alkoxy, no protection is needed for the substituent at position 4 and the corresponding cyclic hexapeptide compound can be obtained after the completion of the cyclization reaction. When R 1 , R 2 , R 3 and R 4 are independently selected from hydroxyl, the corresponding amino acid is hydroxyproline (Hyp) and the hydroxyl group at position 4 needs to be protected, and the protecting group is not particularly limited in the present application, and a tert-butyl protecting group is preferred. When the substituent at position 4 is protected by a protecting group, the deprotection is preferably performed after the completion of the cyclization reaction. The deprotection is preferably performed by using trifluoroacetic acid, and the specific steps are as follows:

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

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

[0039] After the cyclization reaction to obtain a cyclic hexapeptide compound or the deprotection and crystallization to obtain a crude peptide, the crude peptide is preferably purified by high performance liquid chromatography (HPLC), and then freeze-dried to obtain a cyclic hexapeptide compound of formula (I). In some specific embodiments, the HPLC column is a reversed-phase C18 column.

[0040] The present application also provides a cosmetic product comprising the cyclic hexapeptide compound described in the above technical solution and an excipient.

[0041] In the present application, according to the classification principle of cosmetics, the cosmetics can be divided into: cleaning type cosmetics, care type cosmetics and beauty / ornament type cosmetics.

[0042] Among them, the cleaning type cosmetics refer to the cosmetics applied to the human body surface (such as the epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods, which play a role in cleaning and sanitation or eliminating bad smell. The care type cosmetics refer to the cosmetics applied to the human body surface (such as the epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods, which play a role in care. The beauty / ornament type cosmetics refer to the cosmetics applied to the human body surface (such as the epidermis, hair, nails, lips, etc.) by smearing, spraying or other similar methods, which play a role in beauty, ornament and increasing the charm of the human body.

[0043] The skin applicable cleaning type cosmetics include but are not limited to facial cleanser, makeup remover (milk), cleansing cream (honey), facial mask, floral water, zits powder, body powder or bath liquid; the skin applicable care type cosmetics include but are not limited to skin cream, emulsion or cosmetic water; the skin applicable beauty / ornament type cosmetics include but are not limited to powder, rouge, eye shadow, eyeliner (liquid), eyebrow pencil, perfume or cologne; the hair applicable cleaning type cosmetics include but are not limited to shampoo, hair wash or shaving cream; the hair applicable care type cosmetics include but are not limited to hair conditioner, hair cream, hair oil / wax, etc.; the hair applicable beauty / ornament type cosmetics include but are not limited to styling mousse / hair gel, hair dye, hair relaxer, mascara (cream), hair restorer or hair removal agent; the nail applicable cleaning type cosmetics include but are not limited to nail polish remover; the nail applicable care type cosmetics include but are not limited to nail care water (cream), nail hardener; the nail applicable beauty / ornament type cosmetics include but are not limited to nail polish; the lip applicable cleaning type cosmetics include but are not limited to lip makeup remover; the lip applicable care type cosmetics include but are not limited to lip balm; the lip applicable beauty / ornament type cosmetics include but are not limited to lipstick, lip gloss or lip liner.

[0044] The cyclic hexapeptide 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, and can be applied to cosmetics to maintain the structure and elasticity of the skin and promote skin health. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Results of the effect of the cyclic hexapeptide prepared in Example 1 on human keratinocytes;

[0046] Figure 2 Type I collagen expression amount of the cyclic hexapeptide experimental group prepared in Example 1;

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

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

[0049] The present application provides a cyclic hexapeptide compound, a preparation method thereof and an application thereof, and those skilled in the art can refer to the content herein, and appropriately improve the process parameters to realize. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0050] The present application is further illustrated below in combination with examples: Example 1

[0051] A preparation method of a cyclic hexapeptide compound 1, comprising:

[0052] Step S11: CTC resin (6.0 g, 6 mmol) was placed in a 100 mL solid-phase synthesis reactor, amino acid Fmoc-Hyp(tBu)-OH (4.9 g, 12 mmol) was added, dichloromethane (DCM) 60 mL was added, N,N-diisopropyl ethylamine (DIEA) (4.0 mL) was added, and the reaction was carried out at 25°C for 3h, 6.0 mL of methanol was added, and the reaction was carried out for 5min, then the resin was washed with dichloromethane (DCM) 60mL for 2 times, methanol 60mL for 2 times, and DMF 60mL for 2 times; then 20% piperidine (Pip) / DMF solution 60mL was added to the solid-phase synthesis reactor, and the reaction was carried out under stirring for 30min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with DMF solution 60mL for 6 times, and then it was dried by suction.

[0053] Step S12: Fmoc-Pro-OH (5.1 g, 15 mmol) and HOBt (2.03 g, 15 mmol) were taken in a 100 mL beaker, cooled to 2-8°C, 25 mL of DMF solution was added, DIC (2.3 mL, 15 mmol) was added and reacted for 10-20min, and then the solution in the 100 mL beaker was added to the 100 mL solid-phase synthesis reactor, and the reaction was carried out under stirring for 1.5h, and then the resin was washed with DMF solution for 3 times, each time 60mL, and then 60mL of 20% Pip / DMF solution was added after the washing was completed, and the reaction was carried out under stirring for 30min, and then the deprotection solution was removed by suction filtration, and then the resin was washed with DMF solution 60mL for 6 times, and then it was dried by suction.

[0054] Step S13: Take Fmoc-Gly-OH (4.5 g, 15 mmol), HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2-8 °C, add 25 mL of DMF solution, DIC (2.3 mL, 15 mmol) and stand for 10-20 min, and then add the solution in the 100 mL beaker to the 100 mL solid-phase synthesis reactor, stir for 1.5 h, and then wash the resin with 60 mL of DMF solution three times, after the washing is complete, add 60 mL of 20% Pip / DMF solution, stir for 30 min, filter, remove the deprotection solution, and then wash with 60 mL of DMF solution six times, and then dry by suction.

[0055] Step S14: Take Fmoc-Hyp(tBu)-OH (6.1 g, 15 mmol), HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2-8 °C, add 25 mL of DMF solution, DIC (2.3 mL, 15 mmol) and stand for 10-20 min, and then add the solution in the 100 mL beaker to the 100 mL solid-phase synthesis reactor, stir for 1.5 h, and then wash the resin with 60 mL of DMF solution three times, after the washing is complete, add 60 mL of 20% Pip / DMF solution, stir for 30 min, filter, remove the deprotection solution, and then wash with 60 mL of DMF solution six times, and then dry by suction.

[0056] Step S15: Take Fmoc-Pro-OH (5.1 g, 15 mmol), HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 2-8 °C, add 25 mL of DMF solution, DIC (2.3 mL, 15 mmol) and stand for 10-20 min, and then add the solution in the 100 mL beaker to the 100 mL solid-phase synthesis reactor, stir for 1.5 h, and then wash the resin with 60 mL of DMF solution three times, after the washing is complete, add 60 mL of 20% Pip / DMF solution, stir for 30 min, filter, remove the deprotection solution, and then wash with 60 mL of DMF solution six times, and then dry by suction.

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

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

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

[0060] Step S4: Cut the Cyclo(Gly-Pro-Hyp(tBu)-Gly-Pro-Hyp(tBu)) solid with TFA / TIS / H2O = 90 / 5 / 5 (50 mL) for 2.5 h, add the cutting solution to 500 mL of tert-butyl methyl ether (2-8 °C) solution, precipitate white solid, centrifuge to obtain white solid crude peptide, and dry the white solid crude peptide under vacuum to obtain the 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 the obtained product was lyophilized to obtain the cyclohexapeptide compound 1: Cyclo(Gly-Pro-Hyp-Gly-Pro-Hyp), i.e., the cyclohexapeptide compound shown in Formula I-1. The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.64-4.60 (m, 2H), 4.40-4.35 (m, 2H), 4.32 (ddd, J = 7.0, 5.3, 0.7 Hz, 2H), 3.99 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.8, 6.0 Hz, 2H), 3.71-3.59 (m, 6H), 3.48 (d, J = 5.7 Hz, 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 starting 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 the cyclohexapeptide compound 2 shown in Formula I-2: Cyclo(b-Ala-Pro-Hyp-Gly-Pro-Hyp). The nuclear magnetic resonance data thereof are as follows: 1H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 5.9 Hz, 1H), 4.64 - 4.57 (m, 2H), 4.41 - 4.34 (m, 2H), 4.31 (dtd, J = 6.5, 5.6, 0.9 Hz, 2H), 3.99 (dd, J = 15.7, 6.0 Hz, 1H), 3.88 (dd, J = 15.7, 6.0 Hz, 1H), 3.70 - 3.59 (m, 5H), 3.55 (dt, J = 12.3, 4.2 Hz, 1H), 3.47 (dd, J = 15.5, 5.8 Hz, 3H), 3.44 - 3.37 (m, 3H), 2.56 (dt, J = 14.8, 6.1 Hz, 1H), 2.48 (dt, J = 14.8, 6.1 Hz, 1H), 2.17 - 2.09 (m, 2H), 2.09 - 1.92 (m, 5H), 1.89 (dtt, J = 6.5, 5.0, 3.4 Hz, 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 prepare a cyclohexapeptide compound 3 represented by formula I-3: Cyclo(Gly-Hyp-Hyp-Gly-Pro-Hyp). The nuclear magnetic resonance data are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.65 - 4.60 (m, 1H), 4.57 (ddd, J = 7.7, 5.9, 0.7 Hz, 1H), 4.45 - 4.35 (m, 3H), 4.32 (ddt, J = 7.1, 5.3, 0.9 Hz, 2H), 3.97 (ddd, J = 17.8, 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.9, 5.9 Hz, 2H), 3.67 - 3.59 (m, 7H), 3.48 (d, J = 5.7 Hz, 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 prepare a cyclohexapeptide compound 4 represented by Formula I-4: Cyclo(Gly-Pro-Pro(4-F)-Gly-Pro-Hyp). The nuclear magnetic data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.91-4.77 (m, 1H), 4.66-4.59 (m, 2H), 4.51 (ddd, J = 6.6, 4.8, 0.8 Hz, 1H), 4.41-4.34 (m, 1H), 4.34-4.28 (m, 1H), 3.99 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.8, 6.0 Hz, 2H), 3.80-3.71 (m, 1H), 3.71-3.68 (m, 1H), 3.68-3.59 (m, 4H), 3.48 (d, J = 5.7 Hz, 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 prepare a cyclohexapeptide compound 5 represented by Formula I-5: Cyclo(Gly-Pro-Pro(4-OMe)-Gly-Pro-Hyp). The nuclear magnetic data thereof are as follows: 1H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.65-4.59 (m, 2H), 4.41-4.34 (m, 2H), 4.32 (ddd, J = 7.0, 5.3, 0.7 Hz, 1H), 4.26-4.20 (m, 1H), 3.99 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.8, 6.0 Hz, 2H), 3.75-3.60 (m, 6H), 3.48 (d, J = 5.7 Hz, 1H), 3.45-3.38 (m, 2H), 3.19 (d, J = 1.5 Hz, 3H), 2.25 (ddd, J = 12.4, 5.8, 4.9 Hz, 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 prepare a cyclohexapeptide compound 6 represented by Formula I-6: Cyclo(Gly-Pro-Pro-Gly-Pro-Hyp). The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.65-4.59 (m, 2H), 4.41-4.34 (m, 2H), 4.32 (ddd, J = 7.0, 5.3, 0.7 Hz, 1H), 4.26-4.20 (m, 1H), 3.99 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.8, 6.0 Hz, 2H), 3.75-3.60 (m, 6H), 3.48 (d, J = 5.7 Hz, 1H), 3.45-3.38 (m, 2H), 3.19 (d, J = 1.5 Hz, 3H), 2.25 (ddd, J = 12.4, 5.8, 4.9 Hz, 1H), 2.10-1.95 (m, 5H), 1.95-1.86 (m, 6H).C 24 H 34 N6O7, [M+H] + 519.25.

[0077]

[0078] Example 7

[0079] The starting 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, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro-OH, and step S4 is not needed, to obtain a cyclohexapeptide compound 7 represented by Formula I-7: Cyclo(Gly-Pro-Pro-Gly-Pro-Pro). The NMR data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.73 (t, J = 6.0 Hz, 2H), 4.64-4.58 (m, 2H), 4.47 (dd, J = 5.4, 3.7 Hz, 2H), 3.99 (dd, J = 15.7, 6.0 Hz, 2H), 3.88 (dd, J = 15.7, 6.0 Hz, 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, 3.8 Hz, 2H), 1.94-1.82 (m, 10H).C 24 H 34 N6O6, [M+H] + 503.26.

[0080]

[0081] Example 8

[0082] The starting 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, to obtain a cyclohexapeptide compound 8 represented by Formula I-8: Cyclo(Gly-Hyp-Hyp-Gly-Hyp-Hyp). The NMR data thereof are as follows: 1H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.57 (ddd, J = 7.7, 5.9, 0.7 Hz, 2H), 4.44-4.35 (m, 2H), 4.32 (ddd, J = 7.1, 5.4, 0.8 Hz, 2H), 3.95 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.7, 6.0 Hz, 2H), 3.70-3.59 (m, 8H), 3.48 (d, J = 5.7 Hz, 4H), 2.10 (dt, J = 12.2, 6.0 Hz, 2H), 2.06-1.93 (m, 8H).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 prepare a cyclohexapeptide compound 9 represented by Formula I-9: Cyclo(b-Ala-Hyp-Hyp-Gly-Pro-Hyp). The nuclear magnetic resonance data is as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 6.0 Hz, 1H), 4.66-4.59 (m, 1H), 4.51 (ddd, J = 7.9, 6.0, 0.8 Hz, 1H), 4.46-4.35 (m, 3H), 4.31 (dddd, J = 7.3, 6.5, 5.4, 0.7 Hz, 2H), 3.99 (dd, J = 15.8, 6.0 Hz, 1H), 3.88 (dd, J = 15.7, 6.0 Hz, 1H), 3.71-3.58 (m, 7H), 3.52-3.35 (m, 6H), 2.58 (dt, J = 14.8, 6.1 Hz, 1H), 2.49 (dt, J = 14.8, 6.0 Hz, 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 starting materials and preparation methods 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 prepare a cyclohexapeptide compound 10 represented by Formula I-10: Cyclo(b-Ala-Hyp-Hyp-Gly-Hyp-Hyp). Its NMR data are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 6.0 Hz, 1H), 4.57 (ddd, J = 7.7, 5.9, 0.8 Hz, 1H), 4.51 (ddd, J = 7.9, 6.0, 0.8 Hz, 1H), 4.45-4.35 (m, 4H), 4.31 (dddd, J = 7.3, 6.4, 5.4, 0.7 Hz, 2H), 3.95 (dd, J = 15.8, 6.0 Hz, 1H), 3.88 (dd, J = 15.7, 6.0 Hz, 1H), 3.71-3.59 (m, 8H), 3.49 (t, J = 5.5 Hz, 4H), 3.47-3.36 (m, 2H), 2.58 (dt, J = 14.8, 6.1 Hz, 1H), 2.49 (dt, J = 14.8, 6.0 Hz, 1H), 2.10 (dtd, J = 12.2, 6.1, 2.3 Hz, 2H), 2.06-1.93 (m, 6H).C 25 H 36 N6O 10 ,[M+H] + 581.26.

[0089]

[0090] Example 11

[0091] The starting materials and preparation methods 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 prepare a cyclohexapeptide compound 11 represented by Formula I-11: Cyclo(b-Ala-Pro-Pro(4-F)-Gly-Pro-Hyp). Its NMR data are as follows: 1H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 5.9 Hz, 1H), 4.91 - 4.76 (m, 1H), 4.64 - 4.58 (m, 2H), 4.51 (ddd, J = 6.6, 4.8, 0.8 Hz, 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, 6.0 Hz, 1H), 3.80 - 3.67 (m, 2H), 3.67 - 3.59 (m, 3H), 3.55 (dt, J = 12.3, 4.2 Hz, 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 starting 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 a cyclohexapeptide compound 12 represented by Formula I-12: Cyclo(b-Ala-Pro-Pro(4-OMe)-Gly-Pro-Hyp). The NMR data thereof are as follows: 1H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 6.0 Hz, 1H), 4.64 - 4.57 (m, 2H), 4.37 (dddd, J = 7.7, 7.0, 4.2, 1.3 Hz, 2H), 4.31 (ddd, J = 7.1, 5.5, 0.8 Hz, 1H), 4.26 - 4.20 (m, 1H), 3.99 (dd, J = 15.8, 6.0 Hz, 1H), 3.88 (dd, J = 15.8, 6.0 Hz, 1H), 3.75 - 3.53 (m, 6H), 3.51 - 3.36 (m, 5H), 3.19 (d, J = 1.5 Hz, 3H), 2.56 (dt, J = 14.8, 6.1 Hz, 1H), 2.48 (dt, J = 14.8, 6.1 Hz, 1H), 2.25 (ddd, J = 12.4, 5.8, 4.9 Hz, 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] Example 13

[0097] The starting materials and preparation methods 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 needed, to obtain a cyclohexapeptide compound 13 represented by Formula I-13: Cyclo(b-Ala-Pro-Pro(4-F)-Gly-Pro-Pro(4-F)). Its NMR data are as follows: 1H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 5.9 Hz, 1H), 4.93 - 4.76 (m, 2H), 4.61 (ddd, J = 10.5, 6.1, 4.5 Hz, 2H), 4.54 - 4.46 (m, 2H), 3.99 (dd, J = 15.7, 6.0 Hz, 1H), 3.88 (dd, J = 15.8, 6.0 Hz, 1H), 3.80 - 3.67 (m, 4H), 3.67 - 3.60 (m, 1H), 3.55 (dt, J = 12.3, 4.2 Hz, 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] Example 14

[0100] The starting materials and the 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 needed, to obtain a cyclohexapeptide compound 14 represented by Formula I-14: Cyclo(b-Ala-Pro-Pro(4-OMe)-Gly-Pro-Pro(4-OMe)). The NMR data thereof are as follows: 1H NMR (500 MHz, MeOH-d4) δ 7.38 (t, J = 5.6 Hz, 1H), 6.82 (t, J = 6.0 Hz, 1H), 4.64-4.58 (m, 2H), 4.35 (dddd, J = 7.5, 6.9, 4.9, 0.8 Hz, 2H), 4.23 (tddd, J = 6.7, 4.1, 1.6, 1.0 Hz, 2H), 3.99 (dd, J = 15.8, 6.0 Hz, 1H), 3.88 (dd, J = 15.8, 6.0 Hz, 1H), 3.72 (ddd, J = 12.4, 4.2, 0.7 Hz, 2H), 3.69-3.60 (m, 3H), 3.59-3.52 (m, 1H), 3.51-3.36 (m, 4H), 3.19 (d, J = 1.5 Hz, 6H), 2.59-2.45 (m, 2H), 2.25 (ddd, J = 12.3, 5.9, 5.0 Hz, 2H), 2.16-2.01 (m, 5H), 1.95-1.85 (m, 5H).C 27 H 40 N6O8, [M+H] + 577.30.

[0101]

[0102] Example 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 prepare a cyclohexapeptide compound 15 represented by Formula I-15: Cyclo(b-Ala-Pro-Hyp-b-Ala-Pro-Hyp). The nuclear magnetic resonance data are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.34 (t, J = 5.6 Hz, 2H), 4.60 (dd, J = 5.9, 4.6 Hz, 2H), 4.37 (dddd, J = 7.3, 6.3, 5.4, 3.2, 0.8 Hz, 2H), 4.31 (ddd, J = 7.1, 5.5, 0.8 Hz, 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, 6.1 Hz, 2H), 2.48 (dt, J = 14.8, 6.0 Hz, 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 a cyclohexapeptide compound 16 represented by Formula I-16: Cyclo(b-Ala-Hyp-Hyp-b-Ala-Hyp-Hyp). The nuclear magnetic resonance data are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.34 (t, J = 5.6 Hz, 2H), 4.51 (ddd, J = 7.9, 6.0, 0.8 Hz, 2H), 4.45 - 4.34 (m, 4H), 4.31 (ddd, J = 7.1, 5.5, 0.8 Hz, 2H), 3.67 (ddd, J = 12.4, 5.1, 2.5 Hz, 4H), 3.62 (ddd, J = 12.4, 4.4, 2.2 Hz, 4H), 3.49 (t, J = 5.5 Hz, 4H), 3.47 - 3.36 (m, 4H), 2.58 (dt, J = 14.8, 6.1 Hz, 2H), 2.49 (dt, J = 15.0, 6.1 Hz, 2H), 2.11 (dt, J = 12.4, 6.2 Hz, 2H), 2.05 - 1.94 (m, 6H).C 26 H 38 N6O 10 ,[M+H] + 595.27.

[0107]

[0108] Example 17

[0109] The starting materials and the preparation method of this example are basically the same as those of 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, Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, and step S4 is not needed, to obtain a cyclohexapeptide compound 17 represented by Formula I-17: Cyclo(b-Ala-Pro-Pro(4-F)-b-Ala-Pro-Pro(4-F)). The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.34 (t, J = 5.6 Hz, 2H), 4.91-4.76 (m, 2H), 4.60 (dd, J = 5.9, 4.6 Hz, 2H), 4.49 (ddd, J = 6.4, 5.0, 0.8 Hz, 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] Example 18:

[0112] The starting materials and the preparation method of this example are basically the same as those of 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, Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, and step S4 is not needed, to obtain a cyclohexapeptide compound 17 represented by Formula I-17: Cyclo(b-Ala-Pro-Pro(4-F)-b-Ala-Pro-Pro(4-F)). The nuclear magnetic resonance data thereof are as follows: 1H NMR (500 MHz, MeOH-d4) δ 7.34 (t, J = 5.6 Hz, 2H), 4.60 (dd, J = 5.9, 4.6 Hz, 2H), 4.34 (ddd, J = 7.0, 4.9, 0.8 Hz, 2H), 4.26 - 4.20 (m, 2H), 3.72 (dd, J = 12.2, 4.2 Hz, 2H), 3.66 (dd, J = 12.6, 2.7 Hz, 2H), 3.58 - 3.53 (m, 2H), 3.51 - 3.36 (m, 6H), 3.19 (d, J = 1.5 Hz, 6H), 2.56 (dt, J = 14.8, 6.1 Hz, 2H), 2.48 (dt, J = 14.8, 6.0 Hz, 2H), 2.25 (ddd, J = 12.5, 5.8, 4.9 Hz, 2H), 2.16 - 2.09 (m, 4H), 2.04 (ddd, J = 12.4, 6.8, 4.0 Hz, 2H), 1.92 - 1.86 (m, 4H).C 28 H 42 N6O8, [M+H] + 591.31.

[0113]

[0114] Example 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, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, and step S4 is not required, to obtain a cyclohexapeptide compound 19 represented by Formula I-19: Cyclo(Gly-Pro-Pro(4-F)-Gly-Pro-Pro(4-F)). The nuclear magnetic resonance data are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.91 - 4.76 (m, 2H), 4.65 - 4.59 (m, 2H), 4.51 (ddd, J = 6.6, 4.9, 0.8 Hz, 2H), 3.99 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.8, 6.0 Hz, 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] Example 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, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-OMe)-OH, and step S4 is not required, to obtain a cyclohexapeptide compound 20 represented by Formula I-20: Cyclo(Gly-Pro-Pro(4-OMe)-Gly-Pro-Pro(4-OMe)). The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.65-4.58 (m, 2H), 4.36 (ddd, J = 6.8, 5.0, 0.8 Hz, 2H), 4.23 (dddt, J = 4.9, 4.1, 3.3, 1.5 Hz, 2H), 3.99 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.8, 6.0 Hz, 2H), 3.72 (dd, J = 12.5, 4.2 Hz, 2H), 3.69-3.65 (m, 2H), 3.65-3.61 (m, 2H), 3.44-3.38 (m, 2H), 3.19 (d, J = 1.5 Hz, 6H), 2.25 (ddd, J = 12.5, 5.8, 5.0 Hz, 2H), 2.10-2.00 (m, 4H), 1.95-1.85 (m, 6H).C 26 H 38 N6O8, [M+H] + 563.28.

[0119]

[0120] Example 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, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, to prepare a cyclohexapeptide compound 21 represented by Formula I-21: Cyclo(Gly-Hyp-Pro(4-F)-Gly-Hyp-Pro(4-F)). The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.91-4.77 (m, 2H), 4.57 (ddd, J = 7.7, 5.9, 0.7 Hz, 2H), 4.51 (ddd, J = 6.6, 4.9, 0.8 Hz, 2H), 4.44-4.38 (m, 2H), 3.95 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.7, 6.0 Hz, 2H), 3.78-3.71 (m, 2H), 3.71-3.67 (m, 2H), 3.67-3.61 (m, 4H), 3.48 (d, J = 5.7 Hz, 2H), 2.46-2.29 (m, 4H), 2.10 (dt, J = 12.2, 6.0 Hz, 2H), 2.01 (ddd, J = 12.2, 7.8, 4.5 Hz, 2H). C 24 H 32 F2N6O8, [M+H] + 571.23.

[0122]

[0123] Example 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, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, to prepare a cyclohexapeptide compound 22 represented by Formula I-22: Cyclo(Gly-Hyp-Pro(4-OMe)-Gly-Hyp-Pro(4-OMe)). The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 6.82 (t, J = 6.0 Hz, 2H), 4.57 (ddd, J = 7.7, 5.9, 0.7 Hz, 2H), 4.44-4.38 (m, 2H), 4.35 (ddd, J = 7.0, 5.0, 0.8 Hz, 2H), 4.23 (dtt, J = 5.8, 2.6, 1.6 Hz, 2H), 3.95 (dd, J = 15.8, 6.0 Hz, 2H), 3.88 (dd, J = 15.7, 6.0 Hz, 2H), 3.72 (dd, J = 12.2, 4.2 Hz, 2H), 3.69-3.61 (m, 6H), 3.48 (d, J = 5.7 Hz, 2H), 3.19 (d, J = 1.5 Hz, 6H), 2.25 (ddd, J = 12.5, 5.8, 4.9 Hz, 2H), 2.10 (dt, J = 12.2, 6.0 Hz, 2H), 2.07-1.97 (m, 4H).C 26 H 38 N6O 10 ,[M+H] + 595.27.

[0125]

[0126] Example 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, Fmoc-Hyp(tBu)-OH in step S14 is replaced by Fmoc-Pro(4-F)-OH, Fmoc-Pro-OH in step S15 is replaced by Fmoc-Hyp(tBu)-OH, Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, to prepare a cyclohexapeptide compound 23 represented by Formula I-23: Cyclo(b-Ala-Hyp-Pro(4-F)-b-Ala-Hyp-Pro(4-F)). The nuclear magnetic resonance data thereof are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.34 (t, J = 5.6 Hz, 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, 4.2 Hz, 2H), 3.49 (d, J = 5.7 Hz, 2H), 3.48-3.36 (m, 4H), 2.58 (dt, J = 14.8, 6.1 Hz, 2H), 2.49 (dt, J = 15.0, 6.1 Hz, 2H), 2.45-2.30 (m, 4H), 2.11 (dt, J = 12.3, 6.2 Hz, 2H), 1.99 (ddd, J = 12.2, 7.7, 4.4 Hz, 2H).C 26 H 36 F2N6O8, [M+H] + 599.27.

[0128]

[0129] Example 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 Fmoc-Hyp(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, Fmoc-Gly-OH in step S16 is replaced by Fmoc-b-Ala-OH, to prepare a cyclohexapeptide compound 24 represented by Formula I-24: Cyclo

[0131] (b-Ala-Hyp-Pro(4-OMe)-b-Ala-Hyp-Pro(4-OMe)). Its nuclear magnetic data are as follows: 1 H NMR (500 MHz, MeOH-d4) δ 7.34 (t, J = 5.6 Hz, 2H), 4.51 (ddd, J = 7.9, 6.1, 0.8 Hz, 2H), 4.44-4.38 (m, 2H), 4.34 (ddd, J = 7.0, 5.0, 0.8 Hz, 2H), 4.23 (dtt, J = 5.8, 2.6, 1.6 Hz, 2H), 3.74-3.59 (m, 8H), 3.49 (d, J = 5.7 Hz, 2H), 3.48-3.36 (m, 4H), 3.19 (d, J = 1.5 Hz, 6H), 2.58 (dt, J = 14.8, 6.1 Hz, 2H), 2.49 (dt, J = 15.0, 6.1 Hz, 2H), 2.25 (ddd, J = 12.5, 5.8, 4.9 Hz, 2H), 2.11 (dt, J = 12.4, 6.2 Hz, 2H), 2.07-1.95 (m, 4H).C 28 H 42 N6O 10 ,[M+H] + 623.31.

[0132]

[0133] Cytotoxicity test

[0134] Human keratinocytes (HaCaT) were seeded at 2 x 10 4The density culture in 96 well was cultured in 96 well cell culture plate, placed in 37℃, 5% CO2 incubator, 48h after the culture medium was discarded, 1xPBS was washed once, and the biological active substance treatment was carried out, and different concentrations of cyclohexapeptide compounds prepared in examples 1-7 were added, and after 24h treatment, the culture medium was removed, 100μL of culture medium containing 10%cck-8 solution was added, and placed in the incubator for 1h, and the absorbance at 450nm was detected by enzyme-labeled instrument, at the same time, the solvent without sample was used as control group, and the cell survival rate was calculated according to the following formula: cell survival rate%= (experimental group absorbance / control group absorbance) x 100%.

[0135] Results are shown in Figure 1 and Table 1, Figure 1 The results of the effect of cyclohexapeptide prepared in example 1 on human keratinocytes are shown in Table 1, and the results of the effect of cyclohexapeptide prepared in examples 1-7 on keratinocytes are shown in Table 1. Figure 1 As can be seen from Table 1, the cyclohexapeptides prepared in examples 1-7 have no significant cytotoxicity to human keratinocytes.

[0136] Table 1 Effect of cyclohexapeptide prepared in examples 1-7 on keratinocytes

[0137] Cell relative 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 tightening efficacy

[0139] Detection reagent: human type I collagen alpha 1 (COL1 alpha 1) enzyme-linked immunosorbent assay kit (item number: E-EL-H0869, Elabscience, China); human type III collagen (COL3) enzyme-linked immunosorbent assay kit (item number: E-EL-H6049, Elabscience, China); human type IV collagen (COL4) enzyme-linked immunosorbent assay kit (item number: E-EL-H0178, Elabscience, China).

[0140] Detection cell: fibroblast (HFF-1) from Chinese Academy of Sciences Typical Culture Collection Cell Library (Chinese Academy of Sciences Cell Library).

[0141] Detection method: fibroblasts (HFF-1) were cultured in 96 well plates at a density of 5x10 4The density culture of / 6wel was placed in a 37℃, 5% CO2 incubator, and after 48h, the culture medium was aspirated, washed once with 1x PBS, and 500 μg / mL of the cyclic hexapeptide prepared in Examples 1-7 was added to each well to a final volume of 2 mL. Normal control and experimental groups were set up, with three biological replicates in each group. The cells were incubated in the incubator for another 48h. Then the supernatant was aspirated and centrifuged at 1000g, 4℃ for 20min. The supernatant was collected and the precipitate was discarded. The subsequent operation was performed according to the instructions of the collagen ELISA kit.

[0142] The results are shown in Figure 2 、 Figure 3 、 Figure 4 , Table 2, Table 3 and Table 4, Figure 2 The expression of type I collagen in the experimental group of the cyclic hexapeptide prepared in Example 1, Figure 3 The expression of type III collagen in the experimental group of the cyclic hexapeptide prepared in Example 1, Figure 4 The expression of type IV collagen in the experimental group of the cyclic hexapeptide prepared in Example 1, Table 2 shows the expression of type I collagen in the experimental group of the cyclic hexapeptide prepared in Examples 1-7, Table 3 shows the expression of type III collagen in the experimental group of the cyclic hexapeptide prepared in Examples 1-7, and Table 4 shows the expression of type IV collagen in the experimental group of the cyclic hexapeptide prepared in Examples 1-7. It can be seen that the expression of type I, III and IV collagen is significantly increased after treatment with the cyclic hexapeptide prepared in Examples 1-7, which indicates that the cyclic hexapeptide has excellent firming efficacy.

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

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

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

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

[0147] Table 4 Expression of type IV collagen in the experimental group of the cyclic hexapeptide prepared in Examples 1-7

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

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

[0150]

[0151] The above are only preferred embodiments of the present application, and it should be noted that those of ordinary skill in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. Use of a cyclohexapeptide compound as represented by Formula I-3, Formula I-4, Formula I-6 or Formula I-7 in the manufacture of a cosmetic product having a firming efficacy: 。 2. Use of a cyclohexapeptide compound as represented by Formula I-3, Formula I-4, Formula I-6 or Formula I-7 in the manufacture of a cosmetic product for promoting collagen secretion: 。 3. Use according to claim 2, characterized in that, the collagen is collagen type I, collagen type III and / or collagen type IV.

4. A cosmetic product comprising a cyclohexapeptide compound as represented by Formula I-3, Formula I-4, Formula I-6 or Formula I-7: 。 5. The cosmetic product according to claim 4, characterized in that, which is a care cosmetic product.

Images