A class of bicyclic peptide compounds and their applications

Abstract

The present invention discloses a class of bicyclic peptide compounds and their applications, belonging to the technical field of bicyclic peptide compounds. Specifically, the invention relates to the preparation of a fully protected peptide resin using solid-phase synthesis; cleavage and monocyclic cyclization of the fully protected peptide resin to obtain a monocyclic polypeptide; and dicyclic cyclization of the monocyclic polypeptide to obtain a bicyclic peptide compound, wherein the dicyclic cyclization is carried out under the action of iodine methanol and ascorbic acid. The bicyclic peptide compound prepared by the present invention has the following beneficial effects: good safety, low cytotoxicity, good stability, slow degradation rate, good effect on enhancing collagen expression, good effect on enhancing elastin expression, and good effect on inhibiting neurotransmitter release.

Description

Technical Field

[0001] The present invention belongs to the technical field of bicyclic peptide compounds, and in particular relates to a class of bicyclic peptide compounds and applications thereof. Background Art

[0002] In recent years, peptide compounds have been widely used in medicine and cosmetics due to their biological activity and targeted properties. GHK and acetyl hexapeptide-1 are two classic peptide molecules with important applications in anti-aging and skin repair.

[0003] GHK (Glycyl-L-Histidyl-L-Lysine) is a tripeptide that activates fibroblasts and promotes collagen synthesis. Collagen is a key component of skin elasticity, so GHK plays a significant role in anti-aging and skin repair, and is widely used in products that reduce fine lines and delay skin aging. Acetyl Hexapeptide-1 is a classic peptide that relaxes facial muscles by inhibiting the release of neurotransmitters, thereby reducing the formation of dynamic wrinkles. Its mechanism of action relies primarily on blocking calcium channels, making it a widely recognized anti-wrinkle active ingredient.

[0004] While GHK and acetyl hexapeptide-1 exhibit significant biological activity in their respective fields, traditional peptide compounds often suffer from the following issues: existing peptide molecules typically target only a single target or biological pathway, such as collagen production or neurotransmitter release, and cannot simultaneously address the dual needs of anti-aging treatment for both dynamic and static wrinkles. Due to the limitations of a single function, traditional peptide compounds are often less than ideal when used alone as cosmetic ingredients, requiring combination with other active ingredients, increasing the complexity and cost of the formulation. Furthermore, combination therapy cannot change the characteristic of linear peptide molecules that are easily degraded under physiological conditions, resulting in a short duration of action and low bioavailability, limiting their clinical application and efficacy. Summary of the Invention

[0005] The purpose of the present invention is to provide a class of bicyclic peptide compounds and their applications that have good safety, low cytotoxicity, good stability, slow degradation rate, good effect on enhancing collagen expression, good effect on enhancing elastin expression and good effect on inhibiting neurotransmitter release.

[0006] To address these issues, the present invention discloses a dual-target, dual-action bicyclic peptide compound. This compound can simultaneously promote collagen production and inhibit neurotransmitter release, addressing both dynamic and static wrinkle treatments. Compared to using GHK or acetyl hexapeptide-1 alone, this compound addresses the practical limitations of peptide molecules due to their stability.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are:

[0008] The method for preparing a monocyclic polypeptide comprises: mixing a synthetic resin with an amino acid reagent, preparing a fully protected peptide resin by solid phase synthesis, and cleaving and monocyclizing the fully protected peptide resin to obtain a monocyclic polypeptide. The structural formula of the monocyclic polypeptide is as follows:

[0009] ,in,

[0010] X1 and X2 are independently selected from any one of Gly, D-Gly, Lys and D-Lys;

[0011] Y1 and Y2 are independently selected from any one of Trp, D-Trp, Arg and D-Arg;

[0012] R1 and R2 are independently selected from any one of Cys, D-Cys, Cys derivatives and D-Cys derivatives.

[0013] Preferably, X1 and X2 are independently selected from any one of Gly and Lys; or,

[0014] Y1 and Y2 are independently selected from any one of Trp and Arg; or,

[0015] R1 and R2 are independently selected from Cys.

[0016] The invention discloses the monocyclic polypeptide prepared by the method.

[0017] Preferably, the monocyclic polypeptide comprises any one of the following:

[0018] Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-);

[0019] Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-);

[0020] Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-);

[0021] Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-).

[0022] The present invention discloses a bicyclic peptide compound, the structural formula of which is as follows:

[0023] ,in,

[0024] X1 and X2 are independently selected from any one of Gly, D-Gly, Lys and D-Lys;

[0025] Y1 and Y2 are independently selected from any one of Trp, D-Trp, Arg and D-Arg;

[0026] R1 and R2 are independently selected from any one of Cys, D-Cys, Cys derivatives and D-Cys derivatives;

[0027] L1 is formed by bonding R1 and R2.

[0028] Preferably, L1 is a disulfide bond or other bonding structure.

[0029] Preferably, the bicyclic peptide compound includes any one of the following:

[0030] Cyclo (Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys);

[0031] Cyclo (Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys);

[0032] Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys);

[0033] Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys).

[0034] The present invention discloses a method for preparing a bicyclic peptide compound, comprising: the method for preparing the monocyclic polypeptide.

[0035] Preferably, the monocyclic polypeptide is subjected to a dicyclic cyclization treatment to obtain a bicyclic peptide compound, and the dicyclic cyclization is carried out under the action of iodine methanol and ascorbic acid.

[0036] The present invention discloses the use of the monocyclic polypeptide in preparing a bicyclic peptide compound.

[0037] The present invention discloses the use of the bicyclic peptide compound in preparing a product for increasing collagen expression and / or increasing elastin expression and / or inhibiting neurotransmitter release.

[0038] The present invention discloses use of the bicyclic peptide compound in preparing cosmetics and / or skin care products and / or beauty products.

[0039] The present invention discloses a method for preparing a fully protected peptide resin, comprising:

[0040] After mixing Fmoc-Cys(Trt)-OH, DIEA and DCM, add CTC resin and couple to obtain the first amino acid coupling resin; the molar amount of Fmoc-Cys(Trt)-OH used is 100-300% of the reaction sites on the CTC resin, and the molar amount of DIEA used is 100-200% of the molar amount of Fmoc-Cys(Trt)-OH used;

[0041] The first amino acid coupling resin is deprotected, and then the activated amino acid reagent is coupled in sequence according to the peptide sequence to obtain a fully protected peptide resin.

[0042] The invention discloses a fully protected peptide resin prepared by the method.

[0043] The invention discloses a preparation method of a fully protected peptide resin, which comprises the following steps: synthesizing a first amino acid coupling resin, activating an amino acid reagent and synthesizing the fully protected peptide resin.

[0044] Preferably, during the synthesis of the first amino acid coupling resin, Fmoc-Gly-OH is coupled to CTC resin using a solid-phase synthesis method. After swelling the CTC resin with DCM, the Fmoc-Cys(Trt)-OH is coupled in a DCM solution containing DIEA.

[0045] Preferably, in the synthesis of the first amino acid coupled resin, Fmoc-Cys(Trt)-OH is coupled to CTC resin in a DCM solution containing DIEA.

[0046] Preferably, in the synthesis of the first amino acid coupling resin, CTC resin is swelled in DCM at 20-40°C for 5-30 min under a nitrogen atmosphere, DCM is removed by filtration, Fmoc-Cys (Trt) -OH and DCM are added, and then DIEA is added at 10-20°C, and the reaction is carried out at 20-30°C for 2-5 h. After the reaction is completed, methanol is added for end-capping for 10-60 min, filtered, and washed to obtain Fmoc-Cys (Trt) -CTC resin, i.e., the first amino acid coupling resin.

[0047] More preferably, in the synthesis of the first amino acid coupling resin, the molar amount of Fmoc-Cys(Trt)-OH used is 100-300% of the reactive sites on the CTC resin.

[0048] More preferably, in the synthesis of the first amino acid coupling resin, the usage ratio of Fmoc-Cys(Trt)-OH and DCM is 1 mol: 2000-4000 mL.

[0049] More preferably, in the synthesis of the first amino acid coupling resin, the molar amount of DIEA used is 100-200% of the molar amount of Fmoc-Cys(Trt)-OH used. Methanol end-capping can be used in an appropriate amount.

[0050] Preferably, in the activation of the amino acid reagent, the amino acid reagent is mixed with HOBT and DIC in DMF to obtain an activated amino acid reagent.

[0051] Preferably, in the activation of the amino acid reagent, the amino acid reagent and HOBT are added to DMF, and DIC is added at 0-10° C. and activated for 3-20 min to obtain the activated amino acid reagent.

[0052] More preferably, in the activation of the amino acid reagent, the usage ratio of the amino acid reagent to DMF is 0.1-10 mmol / mL.

[0053] More preferably, in the activation of the amino acid reagent, the molar amount of HOBT used is 50-200% of the molar amount of the amino acid reagent used.

[0054] More preferably, in the activation of the amino acid reagent, the molar amount of DIC used is 50-200% of the molar amount of the amino acid reagent used.

[0055] More preferably, in the activation of the amino acid reagent, the amino acid reagent includes Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH and FmocGly-OH.

[0056] Preferably, in the synthesis of the fully protected peptide resin, the first amino acid coupling resin is mixed with the deprotection solution for deprotection treatment, the liquid is removed by filtration, an activated amino acid reagent and NMM are added, and the reaction is carried out under a nitrogen atmosphere for 10-60 minutes. After the reaction is completed, the resin is filtered and washed; then the coupling of the activated amino acid reagent is repeated. After the coupling is completed, the resin is washed and dried to finally obtain the fully protected peptide resin.

[0057] More preferably, in the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagent is: Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH.

[0058] More preferably, in the synthesis of the fully protected peptide resin, the molar amount of NMM used is 30-60% of the molar amount of the amino acid reagent used in the activated amino acid reagent.

[0059] More preferably, in the synthesis of the fully protected peptide resin, during the deprotection treatment, the resin coupled with the amino acid reagent is first immersed in a 20 vol% Pip / DMF solution and subjected to a deprotection treatment at 20-30° C. for 10-60 min.

[0060] More preferably, in the synthesis of the fully protected peptide resin, when the coupling of the activated amino acid reagent is repeated, the resin is subjected to deprotection treatment. After the coupling is completed, the resin is filtered and washed with DMF.

[0061] More preferably, in the synthesis of the fully protected peptide resin, the washing after coupling is carried out in sequence with methanol, dichloromethane and methanol.

[0062] The present invention discloses a fully protected polypeptide, including any one of the following:

[0063] H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH;

[0064] H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH;

[0065] H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH;

[0066] H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH.

[0067] The present invention discloses a method for preparing a fully protected polypeptide, comprising: subjecting a fully protected peptide resin to a fully protected cleavage treatment to obtain the fully protected polypeptide.

[0068] Preferably, the full protective cutting treatment uses a full protective cutting solution, which includes a DCM solution containing TFA.

[0069] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with a cutting solution, treated at 20-40° C. for 10-60 min, the resin is removed by filtration, petroleum ether is added to the filtrate for sedimentation, the supernatant is removed by centrifugation, the petroleum ether is washed and centrifuged, and vacuum dried to obtain the fully protected polypeptide.

[0070] More preferably, in the preparation of a fully protected peptide, the cutting solution is a mixture of TFA and DCM, and the TFA content in the cutting solution is 0.1-5 vol%. The cutting solution is immersed in the fully protected peptide resin, and petroleum ether is used in an appropriate amount for precipitation and washing.

[0071] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with a cutting solution, treated at 20-40° C. for 10-60 min, the resin is removed by filtration, the filtrate is washed with a saturated sodium bicarbonate solution and water in sequence, and the organic phase is finally dried over anhydrous sodium sulfate, filtered, concentrated and dried to obtain the fully protected polypeptide.

[0072] The present invention discloses a fully protected cyclic peptide, including any one of the following:

[0073] Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-);

[0074] Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-);

[0075] Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-);

[0076] Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-).

[0077] The invention discloses a method for preparing a fully protected cyclic peptide, which comprises: subjecting a fully protected polypeptide to a fully protected cyclization treatment to obtain the fully protected cyclic peptide.

[0078] Preferably, the fully protective cyclization treatment is performed using a DMF solution containing HOOBT, DIC, and NMM.

[0079] Preferably, in the preparation of the fully protected cyclic peptide, the fully protected polypeptide is mixed with DMF to obtain a fully protected polypeptide DMF solution; then HOOBT is added, the reaction liquid temperature is controlled to 0-10°C, NMM and DIC are added, and then the reaction is stirred at 20-40°C for 8-24 hours, and the reaction is monitored by HPLC. After the reaction is completed, water is added to precipitate a solid, and the solid is dissolved with ethyl acetate, and then washed with a saturated sodium bicarbonate solution, water and a saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide.

[0080] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected peptide and DMF are mixed in a DMF solution of the fully protected peptide at a mass volume ratio of 1 g:0.2-1 L.

[0081] More preferably, in the preparation of the fully protected cyclic peptide, the amount of HOOBT used is 3-15 wt % of the fully protected peptide.

[0082] More preferably, in the preparation of the fully protected cyclic peptide, the usage ratio of the fully protected peptide and NMM is 1 g: 0.01-0.5 mL.

[0083] More preferably, in the preparation of the fully protected cyclic peptide, the usage ratio of the fully protected peptide and DIC is 1 g: 0.02-0.8 mL.

[0084] More preferably, in the preparation of the fully protected cyclic peptide, an appropriate amount of water is added when the solid is precipitated, an appropriate amount of ethyl acetate is used to dissolve the solid, and an appropriate amount of saturated sodium bicarbonate solution, water and saturated sodium chloride solution are used in washing.

[0085] The present invention discloses a monocyclic polypeptide, including any one of the following:

[0086] Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-);

[0087] Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-);

[0088] Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-);

[0089] Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-).

[0090] The bicyclic peptide compounds of the present invention include any of the following structural formulas:

[0091] 、 、 and .

[0092] The present invention discloses a method for preparing a monocyclic polypeptide, comprising:

[0093] The fully protected peptide resin was prepared by solid phase synthesis;

[0094] The fully protected peptide resin is cleaved and monocyclized to obtain a monocyclic polypeptide.

[0095] Preferably, the polypeptide synthesis method includes solid phase synthesis and / or liquid phase synthesis.

[0096] Preferably, in the preparation of the monocyclic polypeptide, the fully protected cyclic peptide is mixed with the cutting solution for 1-4 hours, concentrated, and then precipitated with ice ether, washed, centrifuged, and dried to obtain the monocyclic polypeptide.

[0097] More preferably, in the preparation of the monocyclic polypeptide, the cutting solution is a mixture of TFA, EDT, Tis, PhOH, and H2O, and the cutting solution contains TFA, EDT, Tis, PhOH, and H2O in a volume ratio of 1:0.01-0.1:0.01-0.1:0.01-0.1:0.01-0.1.

[0098] More preferably, in the preparation of the monocyclic peptide, the ratio of the amount of the fully protected cyclic peptide to the amount of the cutting solution is 1 g: 5-20 mL. During the precipitation, an appropriate amount of glacial ether is used.

[0099] Preferably, in the preparation of the monocyclic polypeptide, the fully protected cyclic peptide is mixed with the cutting solution for 1-4 hours. After unsuccessful cutting, the peptide is precipitated with icy ether, washed by centrifugation, and dried by spin drying to obtain the monocyclic polypeptide.

[0100] The present invention discloses a bicyclic peptide compound, including any one of the following:

[0101] Cyclo (Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys);

[0102] Cyclo (Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys);

[0103] Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys);

[0104] Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys).

[0105] The bicyclic peptide compounds of the present invention include any of the following structural formulas:

[0106] 、 、 and .

[0107] The present invention discloses a method for preparing a bicyclic peptide compound, comprising: a method for preparing the above-mentioned monocyclic polypeptide.

[0108] Preferably, the monocyclic polypeptide is subjected to a dicyclic cyclization treatment to obtain a bicyclic peptide compound, and the dicyclic cyclization is carried out under the action of iodine methanol and ascorbic acid.

[0109] Preferably, in the preparation of the bicyclic peptide compound, the monocyclic polypeptide is mixed with an acetic acid solution, the insoluble matter is removed by filtration, the filtrate is diluted with purified water, and an iodine-methanol solution is added with stirring at 20-40° C. and stirred until the reaction solution turns yellow and does not fade, and the reaction is judged to be complete; then, an ascorbic acid solution is added with stirring for reduction until the reaction solution turns from yellow back to the original milky white, and stirred until the color does not change. The reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45 μm filter membrane to obtain a crude solution of the bicyclic peptide compound, which is purified by reverse-phase C18 chromatography and lyophilized to obtain the bicyclic peptide compound.

[0110] More preferably, in the preparation of the bicyclic peptide compound, the acetic acid solution is prepared by mixing acetic acid and pure water in a volume ratio of 1:0.2-5, and the usage ratio of the monocyclic peptide and the acetic acid solution is 1 g:800-1000 mL.

[0111] More preferably, in the preparation of the bicyclic peptide compound, the content of iodine methanol in the iodine methanol solution is 0.001-0.5 mol / L, the content of ascorbic acid in the ascorbic acid solution is 0.05-5 wt %, and the iodine methanol solution and ascorbic acid solution are used in appropriate amounts.

[0112] The invention discloses the use of a monocyclic polypeptide in preparing a bicyclic peptide compound.

[0113] The present invention discloses the use of a bicyclic peptide compound in preparing a product for improving collagen expression and / or a product for inhibiting neurotransmitter release and / or an anti-aging product and / or an anti-wrinkle product.

[0114] Preferably, the collagen includes at least one of type I collagen, type III collagen and type XVII collagen.

[0115] The present invention also discloses a preparation method of the bicyclic peptide compound, which comprises the following steps: synthesis of a linear polypeptide, cyclization of the linear polypeptide, and secondary cyclization of the monocyclic polypeptide.

[0116] Preferably, the method for preparing the above-mentioned bicyclic peptide compound specifically comprises: preparing a fully protected peptide resin including a pair of cysteines by a peptide synthesis method;

[0117] Perform full protection cleavage on the fully protected peptide resin to obtain a linear fully protected peptide;

[0118] The fully protected peptide is dissolved in an organic solvent and stirred under the conditions of a catalytic system to form a fully protected cyclic peptide;

[0119] The fully protected cyclic peptide is cut to obtain the cyclic peptide;

[0120] The cyclic peptide is dissolved in an organic solvent and oxidized so that the two cysteines are connected by forming a disulfide bond, thereby finally obtaining a bicyclic peptide compound.

[0121] Furthermore, the above polypeptide synthesis method includes solid phase synthesis or liquid phase synthesis.

[0122] Furthermore, the catalytic system includes DIC, HOBt, HATU, DIEA and NMM.

[0123] The bicyclic peptide compound prepared and synthesized by the present invention has good stability, can significantly enhance the production of collagen, and also has a certain ability to inhibit the release of neurotransmitters.

[0124] The present invention uses solid-phase synthesis to prepare a fully protected peptide resin; the fully protected peptide resin is cut and monocyclically cyclized to obtain a monocyclic polypeptide; the monocyclic polypeptide is subjected to a dicyclic cyclization treatment to obtain a bicyclic peptide compound, and the dicyclic cyclization is carried out under the action of iodine methanol and ascorbic acid, thereby having the following beneficial effects: good safety, low cytotoxicity, good stability, slow degradation rate, good effect on enhancing collagen expression, and good effect on inhibiting neurotransmitter release. Therefore, the present invention is a bicyclic peptide compound with good safety, low cytotoxicity, good stability, slow degradation rate, good effect on enhancing collagen expression, good effect on enhancing elastin expression, and good effect on inhibiting neurotransmitter release. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 This is the chromatogram of monocyclic peptide A.

[0126] Figure 2 This is the mass spectrum of monocyclic peptide A.

[0127] Figure 3 This is the chromatogram of bicyclic peptide compound A.

[0128] Figure 4 This is the mass spectrum of bicyclic peptide compound A.

[0129] Figure 5 This is the chromatogram of monocyclic peptide B.

[0130] Figure 6 This is the mass spectrum of monocyclic peptide B.

[0131] Figure 7 This is the chromatogram of bicyclic peptide compound B.

[0132] Figure 8 This is the mass spectrum of bicyclic peptide compound B.

[0133] Figure 9 This is the chromatogram of monocyclic peptide C.

[0134] Figure 10 This is the mass spectrum of monocyclic peptide C.

[0135] Figure 11 This is the chromatogram of bicyclic peptide compound C.

[0136] Figure 12 This is the mass spectrum of bicyclic peptide compound C.

[0137] Figure 13 This is the chromatogram of monocyclic peptide D.

[0138] Figure 14 This is the mass spectrum of monocyclic peptide D.

[0139] Figure 15 This is the chromatogram of bicyclic peptide compound D.

[0140] Figure 16 This is the mass spectrum of the bicyclic peptide compound D. DETAILED DESCRIPTION

[0141] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0142] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0143] The amino acids used in the present invention that do not have a clear conformation are all natural amino acids, that is, in the L conformation.

[0144] Example 1: Preparation of a fully protected peptide resin

[0145] Synthesis of the first amino acid coupling resin: Under a nitrogen atmosphere, CTC resin was swollen in DCM at 25°C for 10 minutes. The DCM was removed by filtration, and Fmoc-Cys(Trt)-OH and DCM were added. DIEA was then added at 10°C and allowed to react at 25°C for 2.5 hours. After completion, methanol was added for end-capping for 30 minutes. The resin was filtered and washed to obtain Fmoc-Cys(Trt)-CTC resin, the first amino acid coupling resin. The molar amount of Fmoc-Cys(Trt)-OH was 200% of the reactive sites on the CTC resin. The ratio of Fmoc-Cys(Trt)-OH to DCM was 1 mol:3000 mL. The molar amount of DIEA was 125% of the molar amount of Fmoc-Gly-OH. Methanol was used for end-capping, and the amount of reactive sites on the CTC resin was 0.1 mol.

[0146] Activation of amino acid reagents: Add the amino acid reagent and HOBT to DMF, then add DIC at 0°C for 5 minutes to obtain the activated amino acid reagent. The amount of amino acid reagent to DMF used is 0.8 mol / L. The molar amount of HOBT used is 100% of the molar amount of the amino acid reagent used. The molar amount of DIC used is 100% of the molar amount of the amino acid reagent used. Amino acid reagents include Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH.

[0147] Synthesis of fully protected peptide resin: The first amino acid coupling resin is mixed with the deprotection solution for deprotection treatment. The liquid is removed by filtration, and an activated amino acid reagent and NMM are added. The reaction is carried out under a nitrogen atmosphere for 40 minutes. After completion of the reaction, the mixture is filtered and washed. The coupling with the activated amino acid reagent is then repeated. After completion of the coupling, the mixture is washed and dried to obtain H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-CTC resin, i.e., the fully protected peptide resin. The coupling order of the activated amino acid reagents is: Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, and Fmoc-Gly-OH. The molar amount of NMM used was 41.67% of the molar amount of the amino acid reagent used in the activated amino acid reagent. During the deprotection treatment, the resin coupled with the amino acid reagent was immersed in a 20 vol% Pip / DMF solution and deprotected at 25°C for 30 minutes. Repeated couplings of the activated amino acid reagent were followed by deprotection. After coupling, the resin was filtered and washed with DMF. Washing after coupling was sequentially performed with methanol, dichloromethane, and methanol.

[0148] Example 2: A method for preparing a fully protected polypeptide

[0149] Preparation of fully protected peptide: The fully protected peptide resin was mixed with a cutting solution and treated at 30°C for 30 minutes. The resin was filtered to remove the resin, petroleum ether was added to the filtrate for sedimentation, the supernatant was removed by centrifugation, the petroleum ether was washed and centrifuged, and the mixture was vacuum dried to obtain the fully protected peptide H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH. The cutting solution was prepared by mixing TFA and DCM, and the TFA content in the cutting solution was 1 vol%. The cutting solution was immersed in the fully protected peptide resin, and petroleum ether was used for sedimentation and washing in an appropriate amount. The fully protected peptide resin was prepared by the preparation method of Example 1. The yield of the crude fully protected peptide was 83.93%.

[0150] Example 3: Preparation of a fully protected cyclic peptide

[0151] Preparation of a fully protected cyclic peptide: A fully protected peptide was mixed with DMF to obtain a fully protected peptide DMF solution. HOOBT was then added. NMM and DIC were added to the reaction solution at 0°C, and the reaction was stirred at 25°C for 16 h. The reaction was monitored by HPLC. After completion, water was added to precipitate a solid, which was dissolved in ethyl acetate and washed sequentially with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. The solid was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-). The fully protected peptide was mixed with DMF in a mass-to-volume ratio of 1 g:0.5 L. The amount of HOOBT was 8.00 wt % of the fully protected peptide. The amount of NMM was 1 g:0.05 mL. The amount of DIC was 1 g:0.08 mL. The amount of water added to precipitate the solid was appropriate, as was the amount of ethyl acetate used to dissolve the solid. The saturated sodium bicarbonate solution, water, and saturated sodium chloride solution used in washings were all used in appropriate amounts. The yield of the fully protected cyclic peptide was 88.73%, and the purity was 76.53%. The fully protected peptide was prepared using the method described in Example 2.

[0152] Example 4: Preparation of Monocyclic Polypeptide

[0153] Preparation of a monocyclic peptide: A fully protected cyclic peptide was mixed with a cutting solution for 2 hours. After concentration, the mixture was precipitated with glacial ether, washed, centrifuged, and spin-dried to obtain the monocyclic peptide Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-). The cutting solution was a mixture of TFA, Tis, H₂O, and DODT, with TFA, EDT, Tis, PhOH, and H₂O mixed in a volume ratio of 1:0.03:0.03:0.03:0.03. The amount of fully protected cyclic peptide and cutting solution used was 1 g:10 mL. Glacial ether was used in an appropriate amount during the precipitation. The fully protected cyclic peptide was prepared according to the preparation method of Example 3.

[0154] The crude product Cyclo (Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-) prepared by the method of Example 4 was purified by reverse-phase C18 liquid chromatography and lyophilized to obtain pure Cyclo (Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-) with a purity of 95.37%, designated as monocyclic polypeptide A. The reverse-phase C18 liquid chromatogram of monocyclic polypeptide A is shown in FIG. Figure 1 As shown, the mass spectrum of monocyclic peptide A is as follows Figure 2 shown.

[0155] Example 5: Preparation of Bicyclic Peptide Compounds

[0156] Preparation of the bicyclic peptide compound: A monocyclic peptide was mixed with an acetic acid solution. Iodine-methanol solution was added at 25°C with stirring and stirred until the reaction solution turned yellow and did not fade, indicating completion of the reaction. Ascorbic acid solution was then added with stirring for reduction until the reaction solution turned from yellow to clear and transparent. The solution was stirred until the color did not change. The reaction was monitored by HPLC. After completion, the reaction solution was filtered through a 0.45 μm filter to obtain a crude bicyclic peptide solution. This was purified by reverse-phase C18 chromatography and lyophilized to obtain the bicyclic peptide compound Cyclo (Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, disulfide-bridged Cys & Cys). The acetic acid solution was prepared by mixing acetic acid and purified water in a volume ratio of 1:2.6. The amount of monocyclic peptide to acetic acid solution used was 1 g:900 mL. The iodine-methanol solution contained 0.01 mol / L iodine-methanol, and the ascorbic acid solution contained 1 wt % ascorbic acid. The iodine-methanol solution and ascorbic acid solution were used in appropriate amounts. The purity of the bicyclic peptide compound was 96.66%. The monocyclic peptide was prepared using the method described in Example 4.

[0157] The bicyclic peptide compound prepared in Example 5 is designated as bicyclic peptide compound A. The reversed phase C18 liquid chromatogram of bicyclic peptide compound A is shown in FIG. Figure 3 As shown, the mass spectrum of bicyclic peptide compound A is as follows Figure 4 shown.

[0158] Example 6: Preparation of a fully protected peptide resin

[0159] The method for preparing the fully protected peptide resin in this example refers to Example 1. The synthesis of the first amino acid coupling resin is the same as in Example 1. In the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagents is: Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH and Fmoc-Gly-OH, to prepare the fully protected peptide resin, i.e., H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-CTC resin.

[0160] Example 7: Preparation method of a fully protected polypeptide

[0161] The method for preparing a fully protected peptide in this example was similar to that in Example 2. The fully protected peptide resin in Example 2 was replaced with the fully protected peptide resin prepared in Example 6 to prepare a fully protected peptide, H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH. The yield of the fully protected peptide was 77.34%.

[0162] Example 8: Preparation of a fully protected cyclic peptide

[0163] The method for preparing a fully protected cyclic peptide in this example was similar to that in Example 3. The fully protected peptide in Example 3 was replaced with the fully protected peptide prepared in Example 7 to prepare a fully protected cyclic peptide, namely, Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-). The yield of the fully protected cyclic peptide was 81.73%, and the purity was 84.53%.

[0164] Example 9: Preparation of Monocyclic Polypeptide

[0165] The method for preparing the monocyclic polypeptide in this example refers to Example 4. The fully protected cyclic peptide in Example 4 was replaced with the fully protected cyclic peptide prepared in Example 8 to prepare a monocyclic polypeptide, namely Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-).

[0166] The crude product Cyclo (Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-) prepared by the method of Example 9 was purified by reverse phase C18 liquid chromatography and lyophilized to obtain pure Cyclo (Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-) with a purity of 97.06%, designated as monocyclic polypeptide B. The reverse phase C18 liquid chromatogram of monocyclic polypeptide B is shown in FIG. Figure 5 As shown, the mass spectrum of monocyclic polypeptide B is as follows Figure 6 shown.

[0167] Example 10: Preparation of Bicyclic Peptide Compounds

[0168] The method for preparing the bicyclic peptide compound in this example refers to Example 5. The monocyclic peptide in Example 5 was replaced with the monocyclic peptide prepared in Example 9 to prepare a bicyclic peptide compound, namely Cyclo (Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys), with a purity of 98.81%.

[0169] The bicyclic peptide compound prepared in Example 10 is designated as bicyclic peptide compound B. The reverse phase C18 liquid chromatogram of bicyclic peptide compound B is shown in FIG. Figure 7 As shown, the mass spectrum of bicyclic peptide compound A is as follows Figure 8 shown.

[0170] Example 11: Preparation of a fully protected peptide resin

[0171] The method for preparing the fully protected peptide resin in this example refers to Example 1. The synthesis of the first amino acid coupling resin is the same as in Example 1. In the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagents is: Fmoc-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH and Fmoc-Lys(Boc)-OH, to prepare the fully protected peptide resin, i.e., H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-CTC resin.

[0172] Example 12: A method for preparing a fully protected polypeptide

[0173] The method for preparing a fully protected peptide in this example was similar to that in Example 2. The fully protected peptide resin in Example 2 was replaced with the fully protected peptide resin prepared in Example 11 to prepare a fully protected peptide, H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH. The yield of the fully protected peptide was 90.16%.

[0174] Example 13: Preparation of a fully protected cyclic peptide

[0175] The method for preparing a fully protected cyclic peptide in this example refers to that in Example 3. The fully protected peptide in Example 3 was replaced with the fully protected peptide prepared in Example 12 to prepare a fully protected cyclic peptide, namely, Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-). The yield of the fully protected cyclic peptide was 78.53%, and the purity was 82.11%.

[0176] Example 14: Preparation of Monocyclic Polypeptide

[0177] The method for preparing the monocyclic polypeptide in this example refers to Example 4. The fully protected cyclic peptide in Example 4 was replaced with the fully protected cyclic peptide prepared in Example 13 to prepare a monocyclic polypeptide, namely Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-).

[0178] The crude product Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-) prepared by the method of Example 14 was purified by reverse phase C18 liquid chromatography and lyophilized to obtain pure Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-) with a purity of 98.63%, designated as monocyclic polypeptide C. The reverse phase C18 liquid chromatogram of monocyclic polypeptide C is shown in FIG. Figure 9 As shown, the mass spectrum of monocyclic peptide C is as follows Figure 10 shown.

[0179] Example 15: Preparation of Bicyclic Peptide Compounds

[0180] The method for preparing the bicyclic peptide compound in this example refers to Example 5. The monocyclic peptide in Example 5 was replaced with the monocyclic peptide prepared in Example 14 to prepare a bicyclic peptide compound, namely Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys), with a purity of 95.90%.

[0181] The bicyclic peptide compound prepared in Example 15 is designated as bicyclic peptide compound C. The reverse phase C18 liquid chromatogram of bicyclic peptide compound C is shown in FIG. Figure 11 As shown, the mass spectrum of bicyclic peptide compound C is as follows Figure 12 shown.

[0182] Example 16: Preparation of a fully protected peptide resin

[0183] The method for preparing the fully protected peptide resin in this example refers to Example 1. The synthesis of the first amino acid coupling resin is the same as in Example 1. In the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagents is: Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH and Fmoc-Lys(Boc)-OH, to prepare the fully protected peptide resin, i.e., H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-CTC resin.

[0184] Example 17: Preparation method of a fully protected polypeptide

[0185] The method for preparing a fully protected peptide in this example was similar to that in Example 2. The fully protected peptide resin in Example 2 was replaced with the fully protected peptide resin prepared in Example 16 to prepare a fully protected peptide, H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH. The yield of the fully protected peptide was 89.43%.

[0186] Example 18: Preparation of a fully protected cyclic peptide

[0187] The method for preparing a fully protected cyclic peptide in this example refers to that in Example 3. The fully protected peptide in Example 3 was replaced with the fully protected peptide prepared in Example 17 to prepare a fully protected cyclic peptide, namely, Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-). The yield of the fully protected cyclic peptide was 85.42%, and the purity was 79.64%.

[0188] Example 19: Preparation of Monocyclic Polypeptide

[0189] The method for preparing the monocyclic polypeptide in this example refers to Example 4. The fully protected cyclic peptide in Example 4 was replaced with the fully protected cyclic peptide prepared in Example 18 to prepare a monocyclic polypeptide, namely Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-).

[0190] The crude product Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-) prepared by the method of Example 19 was purified by reverse-phase C18 liquid chromatography and lyophilized to obtain pure Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-) with a purity of 95.84%, designated as monocyclic polypeptide D. The reverse-phase C18 liquid chromatogram of monocyclic polypeptide D is shown in FIG. Figure 13 As shown, the mass spectrum of monocyclic peptide D is as follows Figure 14 shown.

[0191] Example 20: Preparation of Bicyclic Peptide Compounds

[0192] The method for preparing the bicyclic peptide compound in this example refers to that in Example 5. The monocyclic peptide in Example 5 was replaced with the monocyclic peptide prepared in Example 19 to prepare a bicyclic peptide compound, namely Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys), with a purity of 97.73%.

[0193] The bicyclic peptide compound prepared in Example 20 is designated as bicyclic peptide compound D. The reverse phase C18 liquid chromatogram of bicyclic peptide compound D is shown in FIG. Figure 15 As shown, the mass spectrum of the bicyclic peptide compound D is as follows Figure 16 shown.

[0194] Test example:

[0195] In order to verify the physicochemical properties and physiological functions of the monocyclic polypeptide and bicyclic peptide compounds prepared in the present invention, the following tests were performed. The culture medium or cell culture medium used in the tests of the present invention was DMEM medium.

[0196] 1. Cytotoxicity Test

[0197] The present invention evaluates the potential toxicity of monocyclic polypeptides and bicyclic peptide compounds to cells and determines their possible safe concentration ranges.

[0198] In the cytotoxicity test of the present invention, HFF-1 cells were seeded into a 96-well plate, and 200 μL of DMEM culture medium was added to each well, containing about 10,000 cells / well, to ensure consistent cell density. Cultured overnight in an incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall and grow. After 24 hours, the cell culture medium was aspirated, 200 μL of DMEM culture medium containing test samples at different concentrations was added, and the 96-well plate was returned to the incubator for continued culture. After 24 hours, the absorbance value was measured at a wavelength of 490 nm using an enzyme reader by the MTT method to determine whether the bicyclic peptide compound was cytotoxic and its safe concentration range.

[0199] The test samples were monocyclic peptides and bicyclic peptide compounds. Monocyclic peptides included monocyclic peptide A, monocyclic peptide B, monocyclic peptide C, and monocyclic peptide D, and bicyclic peptide compounds included bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C, and bicyclic peptide compound D. The test sample concentrations ranged from 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM. A negative control group (0 μM) was designated. A separate blank control group containing only culture medium and no cells was designated. Cell viability was calculated as (absorbance of the test group - absorbance of the blank control group / absorbance of the negative control group - absorbance of the blank control group) × 100%.

[0200] Table 1 Cytotoxicity test results

[0201]

[0202] The results of the cytotoxicity experiment are shown in Table 1. After the epithelial cells were treated with different concentrations of the bicyclic peptide compound, the cell viability did not show a significant decrease. When treated with a concentration of 500 μM, the cell viability remained at 95% relative to the control group, confirming that the bicyclic peptide compound had extremely low cytotoxicity.

[0203] 2. Serum stability test

[0204] The present invention evaluates the decomposition of monocyclic polypeptides and bicyclic peptide compounds in a serum environment to predict their metabolism under physiological conditions. The monocyclic polypeptides include monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C and monocyclic polypeptide D. The bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C and bicyclic peptide compound D.

[0205] The lyophilized test sample was directly dissolved in PBS containing 10 vol% fetal bovine serum to a final concentration of 1 mg / mL. Lyophilized GHK was dissolved under the same conditions and served as a GHK control. Lyophilized acetyl hexapeptide-1 was dissolved under the same conditions and served as an acetyl hexapeptide-1 control. The prepared solution was sterile-filtered through a 0.22 μM filter, placed into sterile containers, and incubated in a 37°C incubator. Samples were collected at designated time points (0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h). The residual peptide content was determined using analytical HPLC. The degradation percentage was calculated by comparing the peak areas at different time points, with the content at time 0 being considered 100%. The calculation formula was: Relative content = (test sample peak area / test sample peak area at time 0) × 100%. The residual percentage was plotted against time to calculate the half-life. The test samples included both monocyclic and bicyclic peptide compounds.

[0206] Table 2 Serum degradation level test results

[0207]

[0208] The results of the serum stability experiment are shown in Table 2. The GHK of the control group was almost completely degraded in 4 hours under the condition of 10 vol% fetal bovine serum. According to the degradation curve, its degradation half-life was about 0.7 hours. The acetyl 6-peptide-1 of the control group remained 27% after 8 hours under the condition of 10 vol% fetal bovine serum. According to the degradation curve, its degradation half-life was about 2.8 hours. Under the same conditions, the stability of the monocyclic peptide was significantly improved. The half-lives of monocyclic peptide A, monocyclic peptide B, monocyclic peptide C and monocyclic peptide D were 12.2h, 10.8h, 8.5h and 13.8h respectively. The stability of the bicyclic peptide compound was improved on the basis of the corresponding monocyclic. The half-lives of bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C and bicyclic peptide compound D were further extended to 15.3h, 15.3h, 13.4h and 16.5h.

[0209] 3. Test of protein expression improvement rate

[0210] UVA treatment of cells can significantly reduce the expression levels of various extracellular matrix-related proteins. This study investigated the effects of monocyclic and bicyclic peptide compounds on the protein expression levels of various types of collagen and elastin in irradiated human fibroblast HFF-1 cells. The monocyclic peptides include monocyclic peptide A, monocyclic peptide B, monocyclic peptide C, and monocyclic peptide D. The bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C, and bicyclic peptide compound D.

[0211] Fibroblast HFF-1 cells were seeded into 24-well plates at a seeding density of 10,000 cells / well and cultured in an incubator at 37°C and 5% CO2. They were divided into negative control group, positive control group, sample group, and blank control group. The negative control group, positive control group, and sample group all received a total dose of 9 J / cm 2 UVA radiation, and the blank control group UVA radiation dose was 0 J / cm 2 . The culture medium of the negative control group was DMEM culture medium, 1 mL of culture medium containing 100 μg / mL vitamin C and 7 μg / mL vitamin E was added to the positive control group, and DMEM culture medium of the test sample with a concentration gradient was added to the sample group, and the sample concentrations were 10, 20, and 40 μM. The 24-well plate was then cultured in an incubator at 37°C and 5% CO2. After 24 hours, the culture supernatant was collected and the content of the corresponding protein was determined using an ELISA kit. The graph was drawn with protein content as the vertical axis. The improvement rate was calculated by the formula: Improvement rate = corresponding protein content of the sample group / corresponding protein content of the negative control group × 100% - 100%.

[0212] The test samples include GHK or acetyl hexapeptide-1 or monocyclic polypeptide or bicyclic peptide compounds.

[0213] The experimental results are shown in Tables 3-5. Compared with the blank control group, the expression levels of various collagens and elastin in the negative control group decreased significantly, while the expression levels of various proteins in the positive control group increased significantly, proving that the cell model is working properly.

[0214] Table 3: Type I collagen expression improvement rate / %

[0215]

[0216] The test results of type I collagen content are shown in Table 3. Compared with the negative control group, both the control group GHK and the control group acetyl hexapeptide-1 have the ability to increase the expression of type I collagen. The increase rate of 10μM GHK is 30.2%, the increase rate of 20μM GHK is 131.1%, and the increase rate of 40μM GHK is 263.4%; the increase rate of 10μM acetyl hexapeptide-1 is 2.1%, the increase rate of 20μM acetyl hexapeptide-1 is 45.2%, and the increase rate of 40μM acetyl hexapeptide-1 is 61.3%. Both have the ability to increase the expression of type I collagen.

[0217] Monocyclic peptide A, monocyclic peptide B, monocyclic peptide C and monocyclic peptide D all showed a strong ability to increase the expression of type I collagen. Bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C and bicyclic peptide compound D.

[0218] Compared with monocyclic peptides, bicyclic peptide compounds have a slightly higher rate of enhancing collagen expression based on their single ring.

[0219] Bicyclic peptide compounds A and B significantly increased the expression of type I collagen compared to bicyclic peptide compounds C and D. Structural analysis revealed that X1 = Gly and X2 = Lys in bicyclic peptide compounds A and B, while X1 = Lys and X2 = Gly in bicyclic peptide compounds C and D.

[0220] In general, the ability to promote the expression of type I collagen is ranked as follows: bicyclic peptide compound B > bicyclic peptide compound A > bicyclic peptide compound D > bicyclic peptide compound C.

[0221] Table 4: Improvement rate of type III collagen expression / %

[0222]

[0223] The results of the type III collagen test are shown in Table 4. Compared to the negative control group, GHK only significantly increased type III collagen expression at higher concentrations: 10μM GHK increased by 2%, 20μM GHK increased by 13.1%, and 40μM GHK increased by 64.3%. Acetyl hexapeptide-1 in the control group had almost no significant effect on type III collagen expression: 10μM acetyl hexapeptide-1 increased by 3.1%, 20μM acetyl hexapeptide-1 increased by 5.1%, and 40μM acetyl hexapeptide-1 increased by 4.9%. The four bicyclic peptide compounds and their corresponding monocyclic peptides strongly promoted type III collagen expression. At a concentration of 10μM, the weakest bicyclic peptide compound, compound D, showed a 114% increase. At a concentration of 40μM, the strongest bicyclic peptide compound, compound B, showed a 379% increase. In this test, the bicyclic peptides did not demonstrate a significant improvement over the monocyclic peptides.

[0224] Table 5: Elastin expression improvement rate / %

[0225]

[0226] The results of the elastin test are shown in Table 5: Compared with the negative control group, the control group GHK has a certain ability to increase the expression of elastin, with an increase of 53.1% at 10μM GHK, 86.2% at 20μM GHK, and 115.4% at 40μM GHK; the control group acetyl hexapeptide-1 has almost no ability to significantly increase the expression of elastin, with an increase of 3.1% at 10μM, 2.9% at 20μM, and 4.9% at 40μM. The four bicyclic peptide compounds and their corresponding monocyclic peptides all showed a strong ability to induce the expression of elastin. Even the weakest monocyclic peptide D showed a 194% increase at 10μM. The strongest one was the bicyclic peptide compound B, with an increase of 590.2% at 10μM, 762.9% at 20μM, and 857.2% at 40μM. In the elastin test, compared with the monocyclic peptide, the corresponding bicyclic peptide compound had a significantly increased promotion rate, indicating that the presence of disulfide bonds has the ability to stimulate the promotion rate of elastin production.

[0227] 4. Test of the ability to inhibit neurotransmitter release

[0228] The ability of acetyl hexapeptide-1 to inhibit neurotransmitter release enables it to inhibit dynamic lines caused by excessive skin tension when used as a cosmetic raw material. The present invention tests whether the obtained monocyclic polypeptide and bicyclic peptide compounds have this ability. In the test, neuronal cells HT22 are used to induce high-level expression of the neurotransmitter acetylcholine by KCl stimulation, and are used to detect the inhibitory ability of monocyclic polypeptides and bicyclic peptide compounds on neurotransmitter release after adding different concentrations. Monocyclic polypeptides include monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C and monocyclic polypeptide D, and bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C and bicyclic peptide compound D.

[0229] HT22 cells were seeded at a density of 10,000 cells / well in a 24-well plate and incubated at 37°C, 5% CO₂ for 12 hours. The cells were rinsed three times with PBS and incubated for an additional 24 hours at 37°C, 5% CO₂. The test groups included a blank control group, a model control group, and a sample group. The blank control group contained only culture medium; the model control group contained culture medium supplemented with 50 mM KCl; and the sample group contained culture medium supplemented with 50 mM KCl and a concentration gradient of test samples, including GHK, acetyl hexapeptide-1, or monocyclic or bicyclic peptide compounds, at concentrations of 10, 20, and 40 μM. 1 mM physostigmine was added to all test groups to protect the generated acetylcholine from degradation. After a two-hour incubation, the supernatant containing the released acetylcholine was collected and stored at -80°C for further analysis using a test kit.

[0230] The blank control group was set as 100% and the model control group was set as 0%, and the inhibition rate of each treatment group was normalized and calculated.

[0231] Table 6: Acetylcholine release inhibition rate / %

[0232]

[0233] The results of the neurotransmitter release inhibitory test are shown in Table 6. Compared with the blank control group, the model control group treated with 50 mM KCl showed a significant increase in acetylcholine release, demonstrating the proper function of the cell model system. Compared with acetyl hexapeptide-1, 10 μM acetyl hexapeptide-1 showed an inhibition rate of 19.4%, 20 μM acetyl hexapeptide-1 showed an inhibition rate of 35.4%, and 40 μM acetyl hexapeptide-1 showed an inhibition rate of 54.7%. All four bicyclic peptide compounds exhibited significant inhibition of acetylcholine release: Bicyclic peptide compound B > Bicyclic peptide compound C > Bicyclic peptide compound A, with Bicyclic peptide compound A and Bicyclic peptide compound D showing almost equal inhibitory activity to acetyl hexapeptide-1. In summary, all four bicyclic peptide compounds exhibited significant inhibition of acetylcholine release: at the tested concentrations, the bicyclic peptide compounds exhibited superior inhibitory activity to the corresponding monocyclic peptides. Bicyclic peptide compounds B and C exhibited superior inhibitory activity to GHK and acetyl hexapeptide-1 at the tested concentrations.

[0234] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0235] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing a monocyclic polypeptide, comprising: The synthetic resin is mixed with an amino acid reagent and a fully protected peptide resin is prepared by solid phase synthesis. The fully protected peptide resin is cleaved and monocyclized to obtain a monocyclic polypeptide. The structural formula of the monocyclic polypeptide is as follows: ,in, X1 is Lys, R1 is Cys, Y1 is Arg, Y2 is Trp, R2 is Cys, and X2 is Gly; or, X1 is Lys, R1 is Cys, Y1 is Trp, Y2 is Arg, R2 is Cys, and X2 is Gly; or, X1 is Gly, R1 is Cys, Y1 is Arg, Y2 is Trp, R2 is Cys, and X2 is Lys; or, X1 is Gly, R1 is Cys, Y1 is Trp, Y2 is Arg, R2 is Cys, and X2 is Lys.

2. The method for preparing a monocyclic polypeptide according to claim 1, wherein: In the synthesis of the fully protected peptide resin, the first amino acid coupling resin is mixed with the deprotection solution for deprotection treatment, the liquid is removed by filtration, and the activated amino acid reagent and NMM are added. The reaction is carried out under a nitrogen atmosphere for 10-60 minutes. After the reaction is completed, the resin is filtered and washed. Then, the coupling of the activated amino acid reagent is repeated. After the coupling is completed, the resin is washed and dried to finally obtain the fully protected peptide resin.

3. The method for preparing a monocyclic polypeptide according to claim 1, wherein: The fully protected peptide resin is subjected to a fully protected cleavage treatment to obtain a fully protected polypeptide.

4. The method for preparing the monocyclic polypeptide according to claim 3, wherein: The full protection cutting process uses a full protection cutting liquid, which includes a DCM solution containing TFA. The content of TFA in the full protection cutting liquid is 0.1-5 vol%.

5. The method for preparing the monocyclic polypeptide according to claim 3, wherein: The fully protected polypeptide is subjected to a fully protected cyclization treatment to obtain a fully protected cyclic peptide.

6. The method for preparing the monocyclic polypeptide according to claim 5, characterized in that: Fully protected cyclic peptides include any of the following: Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-); Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-); Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-); Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-).

7. The method for preparing a monocyclic polypeptide according to claim 5, wherein: In the fully protective cyclization treatment, a DMF solution containing HOOBT, DIC, and NMM is used for fully protective cyclization.

8. The method for preparing the monocyclic polypeptide according to claim 7, wherein: The usage amount of HOOBT is 3-15wt% of the fully protected peptide. The usage ratio of the fully protected peptide to NMM is 1g: 0.01-0.5mL. The usage ratio of the fully protected peptide to DIC is 1g: 0.02-0.8mL.

9. The method for preparing a monocyclic polypeptide according to claim 5, wherein: The fully protected cyclic peptide is cleaved with a cleavage solution to obtain a monocyclic peptide.

10. The method for preparing a monocyclic polypeptide according to claim 9, characterized in that: The cutting fluid is a mixture of TFA, EDT, Tis, PhOH, and H2O, wherein TFA, EDT, Tis, PhOH, and H2O are mixed in a volume ratio of 1:0.01-0.1:0.01-0.1:0.01-0.1:0.01-0.

1.

11. The monocyclic polypeptide prepared by the preparation method according to any one of claims 1 to 10, wherein the monocyclic polypeptide comprises any one of the following: Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-); Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-); Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-); Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-).

12. Bicyclic peptide compound, the structural formula is as follows: ,in, X1 is Lys, R1 is Cys, Y1 is Arg, Y2 is Trp, R2 is Cys, and X2 is Gly; or, X1 is Lys, R1 is Cys, Y1 is Trp, Y2 is Arg, R2 is Cys, and X2 is Gly; or, X1 is Gly, R1 is Cys, Y1 is Arg, Y2 is Trp, R2 is Cys, and X2 is Lys; or, X1 is Gly, R1 is Cys, Y1 is Trp, Y2 is Arg, R2 is Cys, and X2 is Lys L1 is formed by bonding R1 and R2.

13. The bicyclic peptide compound according to claim 12, characterized in that: L1 is a disulfide bond.

14. The bicyclic peptide compound according to claim 12, characterized in that: The bicyclic peptide compound includes any one of the following: Cyclo (Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys); Cyclo (Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys); Cyclo (Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide bridged Cys & Cys); Cyclo (Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridged Cys & Cys).

15. A method for preparing a bicyclic peptide compound, comprising: A method for preparing the monocyclic polypeptide according to claim 1.

16. The method for preparing the bicyclic peptide compound according to claim 15, characterized in that: The monocyclic polypeptide is subjected to a dicyclic cyclization treatment to obtain a bicyclic peptide compound, and the dicyclic cyclization is carried out under the action of iodine methanol and ascorbic acid.

17. The method for preparing the bicyclic peptide compound according to claim 15, characterized in that: In the preparation of the bicyclic peptide compound, a monocyclic polypeptide is mixed with an acetic acid solution, insoluble matter is removed by filtration, the filtrate is diluted with purified water, an iodine-methanol solution is added at 20-40° C. with stirring, and the reaction is judged to be complete by stirring until the reaction solution turns yellow and does not fade; then, an ascorbic acid solution is added with stirring for reduction until the reaction solution turns from yellow back to the original milky white, and the color is stirred until there is no change in color. The reaction is monitored by HPLC. After the reaction is completed, the reaction solution is filtered through a 0.45 μm filter membrane to obtain a crude bicyclic peptide compound solution, which is purified by reverse-phase C18 chromatography and lyophilized to obtain the bicyclic peptide compound.

18. The method for preparing the bicyclic peptide compound according to claim 17, characterized in that: In the preparation of the bicyclic peptide compound, the acetic acid solution is prepared by mixing acetic acid and pure water in a volume ratio of 1:0.2-5, and the usage ratio of the monocyclic peptide and the acetic acid solution is 1g:800-1000mL.

19. The method for preparing the bicyclic peptide compound according to claim 17, characterized in that: The content of iodine methanol in the iodine methanol solution is 0.001-0.5 mol / L, and the content of ascorbic acid in the ascorbic acid solution is 0.05-5 wt %.

20. Use of the monocyclic polypeptide according to claim 11 in the preparation of a bicyclic peptide compound.

21. Use of the bicyclic peptide compound according to any one of claims 12 to 14 in the preparation of a product for increasing collagen expression and / or increasing elastin expression and / or a product for inhibiting neurotransmitter release.

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