Bicyclic peptide compound and application thereof

Abstract

The invention discloses a bicyclic peptide compound and application thereof, belongs to the technical field of bicyclic peptide compounds, and particularly relates to full-protection peptide resin prepared through solid-phase synthesis. Carrying out cutting and monocyclic cyclization on the full-protection peptide resin to obtain monocyclic polypeptide; the monocyclic polypeptide is subjected to binary cyclization treatment to obtain a bicyclic peptide compound, and the binary cyclization is cyclized under the action of iodinemethanol and ascorbic acid. The bicyclic peptide compound prepared by the invention has the beneficial effects of good safety, small cytotoxicity, good stability, slow degradation speed, good collagen expression improvement effect, good elastin expression improvement effect and good neurotransmitter release inhibition effect.

Description

Technical Field

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

[0002] In recent years, peptide compounds have been widely used in the fields of medicine and cosmetics due to their biological activity and targeting. Among them, GHK and acetyl hexapeptide-1 are two classic peptide molecules with important application value in the fields of anti-aging and skin repair.

[0003] GHK (Glycyl-L-Histidyl-L-Lysine) is a tripeptide that can activate fibroblasts and promote the synthesis of collagen. Collagen is an important component of skin elasticity, so GHK has a significant effect in the field of 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 can relax facial muscles by inhibiting the release of neurotransmitters, thereby reducing the formation of dynamic wrinkles. Its mechanism of action mainly relies on the blocking of calcium ion channels, and it is a widely recognized anti-wrinkle active ingredient.

[0004] Although GHK and acetyl hexapeptide-1 have shown significant biological activity in their respective fields, traditional peptide compounds usually have the following problems: existing peptide molecules usually only target a single target or a single biological pathway, such as collagen production or neurotransmitter release, and cannot simultaneously meet the dual needs of dynamic wrinkles and static wrinkles in anti-aging treatment. Due to the limitations of a single function, the effect of traditional peptide compounds as cosmetic raw materials is often not ideal, and they need to be used in combination with other active ingredients, which increases the complexity and cost of the formula. In addition, the combination of compounds cannot change the characteristics of linear peptide molecules that are easily degraded under physiological conditions, resulting in a shorter action time and lower bioavailability, which limits 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 which have good safety, low cytotoxicity, good stability, slow degradation rate, good effect of enhancing collagen expression, good effect of enhancing elastin expression and good effect of inhibiting neurotransmitter release.

[0006] In order to solve the above problems, the present invention discloses a dual-target dual-functional cyclic peptide bicyclic peptide compound, which can simultaneously promote collagen production and inhibit neurotransmitter release, meeting the dual needs of dynamic wrinkles and static wrinkles. Compared with the use of GHK or acetyl hexapeptide-1 alone, the bicyclic peptide compound solves the limitations of polypeptide molecules in practical applications due to stability.

[0007] The technical solution adopted by the present invention to achieve the above object is as follows: A method for preparing a monocyclic polypeptide, comprising: mixing a synthetic resin with an amino acid reagent, preparing a fully protected peptide resin by solid-phase synthesis, subjecting the fully protected peptide resin to cleavage treatment and monocyclization to obtain a monocyclic polypeptide, and the structural formula of the monocyclic polypeptide is as follows: , wherein, X1 and X2 are independently selected from any one of Gly, D-Gly, Lys, and D-Lys; Y1 and Y2 are independently selected from any one of Trp, D-Trp, Arg, and D-Arg; R1 and R2 are independently selected from any one of Cys, D-Cys, Cys derivatives, and D-Cys derivatives.

[0008] Preferably, X1 and X2 are independently selected from any one of Gly and Lys; or, Y1 and Y2 are independently selected from any one of Trp and Arg; or, R1 and R2 are independently selected from Cys.

[0009] The present invention discloses a monocyclic polypeptide prepared by the above method.

[0010] Preferably, the monocyclic polypeptide includes 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-).

[0011] The present invention discloses a bicyclic peptide compound, and the structural formula is as follows: , wherein, X1 and X2 are independently selected from any one of Gly, D-Gly, Lys, and D-Lys; Y1 and Y2 are independently selected from any one of Trp, D-Trp, Arg, and D-Arg; R1 and R2 are independently selected from any one of Cys, D-Cys, Cys derivatives, and D-Cys derivatives; L1 is formed by bonding R1 and R2.

[0012] Preferably, L1 is a disulfide bond or other bonding structure.

[0013] Preferably, the bicyclic peptide compound includes any one of the following: Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, disulfide bridge connecting Cys&Cys); Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridge connecting Cys&Cys); Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide bridge connecting Cys&Cys); Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridge connecting Cys&Cys).

[0014] The present invention discloses a preparation method of a bicyclic peptide compound, including: the preparation method of the above-mentioned monocyclic polypeptide.

[0015] Preferably, the monocyclic polypeptide is subjected to bicyclic cyclization treatment to obtain a bicyclic peptide compound, and the bicyclic cyclization is carried out under the action of iodomethanol and ascorbic acid.

[0016] The present invention discloses the use of the above-mentioned monocyclic polypeptide in the preparation of a bicyclic peptide compound.

[0017] The present invention discloses the use of the above-mentioned bicyclic peptide compound in the preparation of a product for increasing collagen expression and / or a product for increasing elastin expression and / or a product for inhibiting neurotransmitter release.

[0018] The present invention discloses the use of the above-mentioned bicyclic peptide compound in the preparation of cosmetics and / or skin care products and / or beauty products.

[0019] The present invention discloses a preparation method of a fully protected peptide resin, including: After mixing Fmoc-Cys(Trt)-OH, DIEA and DCM, CTC resin is added and coupled to obtain the first amino acid coupled 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; The first amino acid coupled resin is subjected to deprotection treatment, and then the coupling of activated amino acid reagents is carried out in sequence according to the peptide sequence to obtain a fully protected peptide resin.

[0020] The present invention discloses a fully protected peptide resin prepared by the above method.

[0021] The present invention discloses a preparation method of a fully protected peptide resin, including: the synthesis of the first amino acid-coupled resin, the activation of amino acid reagents, and the synthesis of the fully protected peptide resin.

[0022] Preferably, in the synthesis of the first amino acid-coupled resin, Fmoc-Gly-OH is coupled to CTC resin. The coupling is carried out by solid-phase synthesis. After the CTC resin is swollen with DCM, it is coupled with Fmoc-Cys(Trt)-OH in a DCM solution containing DIEA.

[0023] 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.

[0024] Preferably, in the synthesis of the first amino acid-coupled resin, under a nitrogen atmosphere, the CTC resin is swollen in DCM at 20 - 40 °C for 5 - 30 min, the DCM is removed by suction filtration, Fmoc-Cys(Trt)-OH and DCM are added, 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 capping for 10 - 60 min, followed by suction filtration and washing to obtain Fmoc-Cys(Trt)-CTC resin, i.e., the first amino acid-coupled resin.

[0025] More preferably, in the synthesis of the first amino acid-coupled resin, the molar amount of Fmoc-Cys(Trt)-OH used is 100 - 300% of the reaction sites on the CTC resin.

[0026] More preferably, in the synthesis of the first amino acid-coupled resin, the usage relationship between Fmoc-Cys(Trt)-OH and DCM is 1 mol: 2000 - 4000 mL.

[0027] More preferably, in the synthesis of the first amino acid-coupled resin, the molar amount of DIEA used is 100 - 200% of the molar amount of Fmoc-Cys(Trt)-OH used. For methanol capping, an appropriate amount can be used.

[0028] Preferably, in the activation of amino acid reagents, the amino acid reagent is mixed with HOBT and DIC in DMF to obtain an activated amino acid reagent.

[0029] Preferably, in the activation of amino acid reagents, the amino acid reagent and HOBT are added to DMF, and DIC is added at 0 - 10 °C, and the activation is carried out for 3 - 20 min to obtain an activated amino acid reagent.

[0030] More preferably, in the activation of amino acid reagents, the usage relationship between the amino acid reagent and DMF is 0.1 - 10 mmol / mL.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] Preferably, in the synthesis of the fully protected peptide resin, the first amino acid-coupled resin is mixed with the deprotection solution for deprotection treatment, the liquid is removed by suction filtration, the activated amino acid reagent and NMM are added, and the reaction is carried out for 10-60 min under a nitrogen atmosphere. After the reaction is completed, suction filtration and washing are carried out; then the coupling of the above-mentioned activated amino acid reagent is repeated. After the coupling is completed, washing and drying are carried out to finally obtain the fully protected peptide resin.

[0035] 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.

[0036] 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 in the activated amino acid reagent.

[0037] More preferably, in the synthesis of the fully protected peptide resin, in the deprotection treatment, the resin coupled with the amino acid reagent is first immersed in a 20 vol% Pip / DMF solution, and the deprotection treatment is carried out at 20-30 °C for 10-60 min.

[0038] More preferably, in the synthesis of the fully protected peptide resin, when repeating the coupling of the activated amino acid reagent, deprotection treatment is carried out in each case. After the coupling is completed, suction filtration and washing are carried out in each case, and the washing is carried out with DMF.

[0039] More preferably, in the synthesis of the fully protected peptide resin, the washing after the coupling is completed is carried out successively with methanol, dichloromethane and methanol.

[0040] The present invention discloses a fully protected polypeptide, including any one of the following: H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH; H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH; H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH; H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH。

[0041] The present disclosure relates to a method for preparing a fully protected polypeptide, comprising: subjecting a fully protected peptide resin to a fully protected cleavage treatment to obtain a fully protected polypeptide.

[0042] Preferably, the fully protected cleavage treatment uses a fully protected cleavage solution, and the fully protected cleavage solution comprises a DCM solution containing TFA.

[0043] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with a cleavage solution, treated at 20 - 40 °C for 10 - 60 min, the resin is filtered off, petroleum ether is added to the filtrate for sedimentation, the supernatant is removed by centrifugation, the residue is washed with petroleum ether by centrifugation, and dried under vacuum to obtain the fully protected polypeptide.

[0044] More preferably, in the preparation of a fully protected polypeptide, the cleavage solution is composed of a mixture of TFA and DCM, and the content of TFA in the cleavage solution is 0.1 - 5 vol%. The fully protected peptide resin is immersed in the cleavage solution, and an appropriate amount of petroleum ether is used for sedimentation and washing.

[0045] Preferably, in the preparation of a fully protected polypeptide, the fully protected peptide resin is mixed with a cleavage solution, treated at 20 - 40 °C for 10 - 60 min, the resin is filtered off, the filtrate is washed successively with a saturated sodium bicarbonate solution and water, the organic phase is finally dried over anhydrous sodium sulfate, filtered, concentrated and dried to obtain the fully protected polypeptide.

[0046] The present invention discloses a fully protected cyclic peptide, including any one 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)-).

[0047] The present invention discloses a method for preparing a fully protected cyclic peptide, comprising: subjecting a fully protected polypeptide to a fully protected cyclization treatment to obtain a fully protected cyclic peptide.

[0048] Preferably, in the fully protected cyclization treatment, a DMF solution containing HOBT, DIC, and NMM is used for the fully protected cyclization.

[0049] 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 HOBT is added, the temperature of the reaction solution is controlled to be 0 - 10°C, NMM and DIC are added, and then the mixture is stirred and reacted at 20 - 40°C for 8 - 24 h, and the reaction is monitored by HPLC. After the reaction is completed, water is added to precipitate a solid, the solid is dissolved in ethyl acetate, and then successively washed with a saturated sodium bicarbonate solution, water, and a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain the fully protected cyclic peptide.

[0050] More preferably, in the preparation of the fully protected cyclic peptide, the fully protected polypeptide and DMF in the fully protected polypeptide DMF solution are mixed at a mass - to - volume ratio of 1 g: 0.2 - 1 L.

[0051] More preferably, in the preparation of the fully protected cyclic peptide, the usage amount of HOBT is 3 - 15 wt% of the fully protected polypeptide.

[0052] More preferably, in the preparation of the fully protected cyclic peptide, the usage amount relationship between the fully protected polypeptide and NMM is 1 g: 0.01 - 0.5 mL.

[0053] More preferably, in the preparation of the fully protected cyclic peptide, the usage amount relationship between the fully protected polypeptide and DIC is 1 g: 0.02 - 0.8 mL.

[0054] More preferably, in the preparation of the fully protected cyclic peptide, the water added for precipitating the solid is used in an appropriate amount, the ethyl acetate for dissolving the solid is used in an appropriate amount, and the saturated sodium bicarbonate solution, water, and saturated sodium chloride solution used in the washing are all used in an appropriate amount.

[0055] The present invention discloses monocyclic polypeptides, including 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-).

[0056] The bicyclic peptide compounds in the present invention include the structural formulas shown by any one of the following: , , and .

[0057] The present invention discloses a preparation method of monocyclic polypeptides, including: Preparing a fully protected peptide resin by using solid-phase synthesis; Performing cleavage treatment and monocyclization on the fully protected peptide resin to obtain monocyclic polypeptides.

[0058] Preferably, the polypeptide synthesis method includes solid-phase synthesis and / or liquid-phase synthesis.

[0059] Preferably, in the preparation of monocyclic polypeptides, the fully protected cyclic peptide is mixed with the cleavage solution for 1 - 4 h, concentrated, then precipitated with ice-cold diethyl ether, washed, centrifuged, and dried by rotation to obtain monocyclic polypeptides.

[0060] More preferably, in the preparation of monocyclic polypeptides, the cleavage solution is a mixed solution of TFA, EDT, Tis, PhOH, and H2O, and TFA, EDT, Tis, PhOH, and H2O in the cleavage solution are mixed at a volume ratio of 1:0.01 - 0.1:0.01 - 0.1:0.01 - 0.1:0.01 - 0.1.

[0061] More preferably, in the preparation of monocyclic polypeptides, the usage relationship between the fully protected cyclic peptide and the cleavage solution is 1 g:5 - 20 mL. An appropriate amount of ice-cold diethyl ether is used in the precipitation.

[0062] Preferably, in the preparation of monocyclic polypeptides, the fully protected cyclic peptide is mixed with the cleavage solution for 1 - 4 h, after cleavage is completed, it is precipitated with ice-cold diethyl ether, washed, centrifuged, and dried by rotation to obtain monocyclic polypeptides.

[0063] The present invention discloses bicyclic peptide compounds, including any one of the following: Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, disulfide bridge between Cys & Cys); Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridge between Cys & Cys); Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide bridge between Cys & Cys); Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridge between Cys & Cys).

[0064] The bicyclic peptide compounds in the present invention include the structural formulas shown by any of the following: , , and .

[0065] The present invention discloses a preparation method of the bicyclic peptide compound, including: the preparation method of the above-mentioned monocyclic polypeptide.

[0066] Preferably, the monocyclic polypeptide is subjected to binary cyclization treatment to obtain the bicyclic peptide compound, and the binary cyclization is carried out under the action of iodomethanol and ascorbic acid.

[0067] Preferably, in the preparation of the bicyclic peptide compound, the monocyclic polypeptide is mixed with an acetic acid solution, the insoluble substances are filtered off, the filtrate is diluted with purified water, an iodomethanol solution is added with stirring at 20 - 40 °C, and stirring is continued until the reaction solution turns yellow and does not fade, judging that the reaction is complete; then an ascorbic acid solution is added with stirring for reduction until the reaction solution changes back from yellow to the original milky white and stirring is continued 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 freeze-dried to obtain the bicyclic peptide compound.

[0068] More preferably, in the preparation of the bicyclic peptide compound, the acetic acid solution is composed of acetic acid and pure water mixed at a volume ratio of 1:0.2 - 5, and the usage relationship between the monocyclic polypeptide and the acetic acid solution is 1 g:800 - 1000 mL.

[0069] More preferably, in the preparation of the bicyclic peptide compound, the content of iodomethanol in the iodomethanol 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 iodomethanol solution and the ascorbic acid solution are used in appropriate amounts.

[0070] The present invention discloses the use of the monocyclic polypeptide in the preparation of the bicyclic peptide compound.

[0071] The present invention discloses the use of bicyclic peptide compounds in the preparation of products for enhancing collagen expression and / or inhibiting neurotransmitter release and / or anti-aging products and / or anti-wrinkle products.

[0072] Preferably, the collagen includes at least one of type I collagen, type III collagen, and type XVII collagen.

[0073] The present invention also discloses a preparation method of the above-mentioned bicyclic peptide compound, including: synthesis of a linear polypeptide, cyclization of the linear polypeptide, and secondary cyclization of the monocyclic polypeptide.

[0074] Preferably, the preparation method of the above-mentioned bicyclic peptide compound specifically includes: preparing a fully protected peptide resin including a pair of cysteines by using a polypeptide synthesis method; Performing fully protected cleavage on the fully protected peptide resin to obtain a linear fully protected polypeptide; Dissolving the fully protected polypeptide in an organic solvent and stirring and reacting under a catalytic system condition to form a fully protected cyclic peptide; Performing cleavage on the fully protected cyclic peptide to obtain a cyclic peptide; Dissolving the cyclic peptide in an organic solvent and performing oxidation so that the two cysteines are connected by forming a disulfide bond, and finally obtaining the bicyclic peptide compound.

[0075] Furthermore, the above-mentioned polypeptide synthesis method includes a solid-phase synthesis method or a liquid-phase synthesis method.

[0076] Furthermore, the catalytic system includes DIC, HOBt, HATU, and DIEA, NMM.

[0077] The bicyclic peptide compound prepared and synthesized by the present invention has good stability, can significantly enhance the generation of collagen, and also has a certain ability to inhibit neurotransmitter release.

[0078] Since the present invention uses solid-phase synthesis to prepare a fully protected peptide resin; performing cleavage and monocyclization on the fully protected peptide resin to obtain a monocyclic polypeptide; performing bicyclic cyclization treatment on the monocyclic polypeptide to obtain a bicyclic peptide compound, and the bicyclic cyclization is cyclized under the action of iodine methanol and ascorbic acid, thus 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. Description of the Drawings

[0079] Figure 1 It is a chromatogram of monocyclic polypeptide A.

[0080] Figure 2 It is the mass spectrum of monocyclic polypeptide A.

[0081] Figure 3 It is the chromatogram of bicyclic peptide compound A.

[0082] Figure 4 It is the mass spectrum of bicyclic peptide compound A.

[0083] Figure 5 It is the chromatogram of monocyclic polypeptide B.

[0084] Figure 6 It is the mass spectrum of monocyclic polypeptide B.

[0085] Figure 7 It is the chromatogram of bicyclic peptide compound B.

[0086] Figure 8 It is the mass spectrum of bicyclic peptide compound B.

[0087] Figure 9 It is the chromatogram of monocyclic polypeptide C.

[0088] Figure 10 It is the mass spectrum of monocyclic polypeptide C.

[0089] Figure 11 It is the chromatogram of bicyclic peptide compound C.

[0090] Figure 12 It is the mass spectrum of bicyclic peptide compound C.

[0091] Figure 13 It is the chromatogram of monocyclic polypeptide D.

[0092] Figure 14 It is the mass spectrum of monocyclic polypeptide D.

[0093] Figure 15 It is the chromatogram of bicyclic peptide compound D.

[0094] Figure 16 It is the mass spectrum of bicyclic peptide compound D. Detailed implementation manners

[0095] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0096] First, the concepts involved in this application will be described in conjunction with the accompanying drawings. It should be noted here that the following descriptions of each concept are only for making the content of this application easier to understand and do not represent a limitation on the protection scope of this application; at the same time, without conflict, the embodiments and features in the embodiments of this application can be combined with each other. This application will be described in detail below with reference to the drawings and in conjunction with the embodiments.

[0097] For the amino acids used in the present invention, those without a specified conformation are natural amino acids, that is, in the L conformation.

[0098] Example 1: A preparation method of a fully protected peptide resin Synthesis of the first amino acid-coupled resin: Under a nitrogen atmosphere, the CTC resin was swollen in DCM at 25°C for 10 min, the DCM was removed by suction filtration, Fmoc-Cys(Trt)-OH and DCM were added, and then DIEA was added at 10°C and reacted at 25°C for 2.5 h. After the reaction was completed, methanol was added for capping for 30 min, suction filtration and washing were carried out to obtain Fmoc-Cys(Trt)-CTC resin, that is, the first amino acid-coupled resin. The molar amount of Fmoc-Cys(Trt)-OH used was 200% of the reaction sites on the CTC resin. The usage relationship between Fmoc-Cys(Trt)-OH and DCM was 1 mol: 3000 mL, and the molar amount of DIEA used was 125% of the molar amount of Fmoc-Gly-OH used. For methanol capping, an appropriate amount can be used. The reaction sites of the CTC resin were 0.1 mol.

[0099] Activation of amino acid reagents: The amino acid reagents and HOBT were added to DMF, and DIC was added at 0°C and activated for 5 min to obtain activated amino acid reagents. The usage relationship between the amino acid reagents and DMF was 0.8 mol / L. The molar amount of HOBT used was 100% of the molar amount of the amino acid reagents used. The molar amount of DIC used was 100% of the molar amount of the amino acid reagents used. The amino acid reagents included 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.

[0100] Synthesis of fully protected peptide resin: The first amino acid-coupled resin was mixed with a deprotection solution for deprotection treatment. The liquid was removed by suction filtration, and an activated amino acid reagent and NMM were added. The reaction was carried out for 40 min under a nitrogen atmosphere. After the reaction was completed, suction filtration and washing were performed; then the coupling of the above-activated amino acid reagent was repeated. After the coupling was completed, washing and drying were carried out, and finally, H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-CTC resin, that is, fully protected peptide resin, was obtained. The coupling order of the activated amino acid reagent was: 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 in the activated amino acid reagent. In the deprotection treatment, the resin coupled with the amino acid reagent was first immersed in a 20 vol% Pip / DMF solution and deprotected at 25 °C for 30 min. When repeating the coupling of the activated amino acid reagent, deprotection treatment was carried out in each case. After the coupling was completed, suction filtration and washing were carried out in each case, and DMF was used for washing. The washing after the coupling was completed was carried out successively with methanol, dichloromethane, and methanol.

[0101] Example 2: A method for preparing a fully protected polypeptide Preparation of fully protected polypeptide: The fully protected peptide resin was mixed with a cleavage solution and treated at 30 °C for 30 min. The resin was removed by filtration, petroleum ether was added to the filtrate for sedimentation, the supernatant was removed by centrifugation, washed with petroleum ether by centrifugation, and dried in vacuo to obtain the fully protected polypeptide H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH. The cleavage solution was composed of TFA and DCM, and the content of TFA in the cleavage solution was 1 vol%. The fully protected peptide resin was immersed in the cleavage solution, and an appropriate amount of petroleum ether was used for sedimentation and washing. The fully protected peptide resin was prepared by the preparation method of Example 1. The crude yield of the fully protected polypeptide was 83.93%.

[0102] Example 3: A method for preparing a fully protected cyclic peptide Preparation of fully protected cyclic peptide: Mix the fully protected polypeptide with DMF to obtain a fully protected polypeptide DMF solution; then add HOBT, control the temperature of the reaction solution at 0 °C, add NMM and DIC, and then stir and react at 25 °C for 16 h. Monitor the reaction by HPLC. After the reaction is completed, add water to precipitate a solid. Dissolve the solid in ethyl acetate, and then wash it successively with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution. Dry it over anhydrous sodium sulfate, filter, and rotary evaporate to obtain the fully protected cyclic peptide Cyclo(Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-). In the fully protected polypeptide DMF solution, the fully protected polypeptide and DMF are mixed at a mass-to-volume ratio of 1 g:0.5 L. The usage amount of HOBT is 8.00 wt% of the fully protected polypeptide. The usage amount relationship between the fully protected polypeptide and NMM is 1 g:0.05 mL. The usage amount relationship between the fully protected polypeptide and DIC is 1 g:0.08 mL. The amount of water added for precipitating the solid is used appropriately, the ethyl acetate for dissolving the solid is used appropriately, and the saturated sodium bicarbonate solution, water, and saturated sodium chloride solution used in the washing are all used appropriately. The yield of the fully protected cyclic peptide is 88.73%, and the purity is 76.53%. The fully protected polypeptide is prepared by the preparation method of Example 2.

[0103] Example 4: Preparation method of monocyclic polypeptide Preparation of monocyclic polypeptide: Mix the fully protected cyclic peptide with the cleavage solution for 2 h, concentrate, precipitate with ice-cold diethyl ether, wash and centrifuge, and rotary dry to obtain the monocyclic polypeptide Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-). The cleavage solution is a mixed solution of TFA, Tis, H2O, and DODT. In the cleavage solution, TFA, EDT, Tis, PhOH, and H2O are mixed at a volume ratio of 1:0.03:0.03:0.03:0.03. The usage amount relationship between the fully protected cyclic peptide and the cleavage solution is 1 g:10 mL. The ice-cold diethyl ether used in the precipitation is used appropriately. The fully protected cyclic peptide is prepared by the preparation method of Example 3.

[0104] Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-) prepared by the method of Example 4 is a crude product. The crude product is purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain the pure product 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 as Figure 1 shown, and the mass spectrum of monocyclic polypeptide A is as Figure 2 shown.

[0105] Example 5: Preparation method of bicyclic peptide compound Preparation of bicyclic peptide compound: Mix the monocyclic polypeptide with acetic acid solution, and add iodine methanol solution with stirring at 25°C. Stir until the reaction solution turns yellow and does not fade, indicating that the reaction is complete. Then add ascorbic acid solution for reduction with stirring until the reaction solution changes from yellow to clear and transparent, and stir until the color does not change. Monitor the reaction by HPLC. After the reaction is completed, filter the reaction solution through a 0.45 μm filter membrane to obtain a crude solution of the bicyclic peptide compound. Purify it by reverse-phase C18 chromatography and lyophilize to obtain the bicyclic peptide compound Cyclo(Gly-His-Lys-Cys-Arg-Phe-Trp-Cys-, with disulfide bridge connecting Cys&Cys). The acetic acid solution is prepared by mixing acetic acid and pure water at a volume ratio of 1:2.6. The usage relationship between the monocyclic polypeptide and the acetic acid solution is 1 g:900 mL. The content of iodine methanol in the iodine methanol solution is 0.01 mol / L, and the content of ascorbic acid in the ascorbic acid solution is 1 wt%. The iodine methanol solution and the ascorbic acid solution are used in appropriate amounts. The purity of the bicyclic peptide compound is 96.66%. The monocyclic polypeptide is prepared by the preparation method of Example 4.

[0106] The bicyclic peptide compound prepared in Example 5 is designated as bicyclic peptide compound A. The reverse-phase C18 liquid chromatogram of bicyclic peptide compound A is as Figure 3 shown, and the mass spectrum of bicyclic peptide compound A is as Figure 4 shown.

[0107] Example 6: A method for preparing a fully protected peptide resin The method for preparing the fully protected peptide resin in this example refers to Example 1. The synthesis of the first amino acid-coupled resin is the same as that 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, namely H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-CTC resin.

[0108] Example 7: A method for preparing a fully protected polypeptide The method for preparing the fully protected polypeptide in this example refers to Example 2. Replace the fully protected peptide resin in Example 2 with the fully protected peptide resin prepared in Example 6 to prepare the fully protected polypeptide, namely H-Gly-His(Trt)-Lys(Boc)-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH. The yield of the fully protected polypeptide is 77.34%.

[0109] Example 8: Preparation Method of a Fully Protected Cyclic Peptide The method for preparing the fully protected cyclic peptide in this example refers to Example 3. Replace the fully protected polypeptide in Example 3 with the fully protected polypeptide prepared in Example 7 to obtain the 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 is 81.73% and the purity is 84.53%.

[0110] Example 9: Preparation Method of a Monocyclic Peptide The method for preparing the monocyclic peptide in this example refers to Example 4. Replace the fully protected cyclic peptide in Example 4 with the fully protected cyclic peptide prepared in Example 8 to obtain the monocyclic peptide, namely Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-).

[0111] Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-) prepared by the method of Example 9 is a crude product. The crude product is purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain the pure product Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-), with a purity of 97.06%, which is designated as monocyclic peptide B. The reverse-phase C18 liquid chromatogram of monocyclic peptide B is as Figure 5 shown, and the mass spectrum of monocyclic peptide B is as Figure 6 shown.

[0112] Example 10: Preparation Method of a Bicyclic Peptide Compound The method for preparing the bicyclic peptide compound in this example refers to Example 5. Replace the monocyclic peptide in Example 5 with the monocyclic peptide prepared in Example 9 to obtain the bicyclic peptide compound, namely Cyclo(Gly-His-Lys-Cys-Trp-Phe-Arg-Cys-, disulfide bridge connecting Cys&Cys), with a purity of 98.81%.

[0113] 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 as Figure 7 shown, and the mass spectrum of bicyclic peptide compound A is as Figure 8 shown.

[0114] Example 11: Preparation Method of a Fully Protected Peptide Resin The method for preparing the fully protected peptide resin in this example refers to Example 1. The synthesis of the first amino acid-coupled resin is the same as that 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, and the fully protected peptide resin, i.e., H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-CTC resin, is prepared.

[0115] Example 12: A method for preparing a fully protected polypeptide The method for preparing the fully protected polypeptide in this example refers to Example 2. Replace the fully protected peptide resin in Example 2 with the fully protected peptide resin prepared in Example 11 to prepare the fully protected polypeptide, i.e., H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-OH. The yield of the fully protected polypeptide is 90.16%.

[0116] Example 13: A method for preparing a fully protected cyclic peptide The method for preparing the fully protected cyclic peptide in this example refers to Example 3. Replace the fully protected polypeptide in Example 3 with the fully protected polypeptide prepared in Example 12 to prepare the fully protected cyclic peptide, i.e., Cyclo(Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Arg(Pbf)-Phe-Trp(Boc)-Cys(Trt)-). The yield of the fully protected cyclic peptide is 78.53%, and the purity is 82.11%.

[0117] Example 14: A method for preparing a monocyclic polypeptide The method for preparing the monocyclic polypeptide in this example refers to Example 4. Replace the fully protected cyclic peptide in Example 4 with the fully protected cyclic peptide prepared in Example 13 to prepare the monocyclic polypeptide, i.e., Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-).

[0118] The Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-) prepared by the method of Example 14 was a crude product. The crude product was purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain the pure product Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-), with a purity of 98.63%, which was designated as monocyclic polypeptide C. The reverse-phase C18 liquid chromatogram of monocyclic polypeptide C is as shown in Figure 9 shown, and the mass spectrum of monocyclic polypeptide C is as shown in Figure 10 shown.

[0119] Example 15: Preparation method of bicyclic peptide compound The method for preparing the bicyclic peptide compound in this example refers to Example 5. The monocyclic polypeptide in Example 5 was replaced with the monocyclic polypeptide prepared in Example 14 to prepare the bicyclic peptide compound, namely Cyclo(Lys-His-Gly-Cys-Arg-Phe-Trp-Cys-, disulfide-bridged Cys&Cys), with a purity of 95.90%.

[0120] The bicyclic peptide compound prepared in Example 15 was designated as bicyclic peptide compound C. The reverse-phase C18 liquid chromatogram of bicyclic peptide compound C is as shown in Figure 11 shown, and the mass spectrum of bicyclic peptide compound C is as shown in Figure 12 shown.

[0121] Example 16: Preparation method of a fully protected peptide resin The method for preparing the fully protected peptide resin in this example refers to Example 1. The synthesis of the first amino acid-coupled resin was the same as that in Example 1. In the synthesis of the fully protected peptide resin, the coupling order of the activated amino acid reagents was: 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, namely H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-CTC resin.

[0122] Example 17: Preparation method of a fully protected polypeptide The method for preparing the fully protected polypeptide in this example refers to Example 2. Replace the fully protected peptide resin in Example 2 with the fully protected peptide resin prepared in Example 16 to obtain the fully protected polypeptide, namely H-Lys(Boc)-His(Trt)-Gly-Cys(Trt)-Trp(Boc)-Phe-Arg(Pbf)-Cys(Trt)-OH. The yield of the fully protected polypeptide is 89.43%.

[0123] Example 18: A method for preparing a fully protected cyclic peptide The method for preparing the fully protected cyclic peptide in this example refers to Example 3. Replace the fully protected polypeptide in Example 3 with the fully protected polypeptide prepared in Example 17 to obtain the 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 is 85.42% and the purity is 79.64%.

[0124] Example 19: A method for preparing a monocyclic polypeptide The method for preparing the monocyclic polypeptide in this example refers to Example 4. Replace the fully protected cyclic peptide in Example 4 with the fully protected cyclic peptide prepared in Example 18 to obtain the monocyclic polypeptide, namely Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-).

[0125] Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-) prepared by the method of Example 19 is a crude product. The crude product is purified by reverse-phase C18 liquid chromatography and freeze-dried to obtain the pure product 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 as Figure 13 shown, and the mass spectrum of monocyclic polypeptide D is as Figure 14 shown.

[0126] Example 20: A method for preparing a bicyclic peptide compound The method for preparing the bicyclic peptide compound in this example refers to Example 5. Replace the monocyclic polypeptide in Example 5 with the monocyclic polypeptide prepared in Example 19 to obtain the bicyclic peptide compound, namely Cyclo(Lys-His-Gly-Cys-Trp-Phe-Arg-Cys-, disulfide bridge connecting Cys&Cys), with a purity of 97.73%.

[0127] 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 as Figure 15As shown, the mass spectrum of the bicyclic peptide compound D is as Figure 16 shown.

[0128] Test Example: To verify the physicochemical properties and physiological functions of the monocyclic polypeptides and bicyclic peptide compounds prepared in the present invention, the following tests were carried out. The culture medium or cell culture medium used in the tests of the present invention was DMEM medium.

[0129] 1. Cytotoxicity test The present invention evaluated the potential toxicity of monocyclic polypeptides and bicyclic peptide compounds to cells and determined their possible safe concentration ranges.

[0130] In the cytotoxicity test of the present invention, HFF-1 cells were inoculated into 96-well plates, and 200 μL of DMEM medium containing about 10,000 cells / well was added to each well to ensure consistent cell density. The cells were cultured overnight in an incubator at 37 °C and 5% CO2 to allow the cells to adhere and grow. After 24 hours, the cell culture medium was aspirated, and 200 μL of DMEM medium containing test samples at different concentrations was added. The 96-well plates were returned to the incubator for continued culture. After 24 hours, the absorbance value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader by the MTT method to determine whether the bicyclic peptide compound had cytotoxicity and its safe concentration range.

[0131] The test samples were monocyclic polypeptides and bicyclic peptide compounds. The monocyclic polypeptides included monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C, and monocyclic polypeptide D. The bicyclic peptide compounds included bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C, and bicyclic peptide compound D. The concentrations of the test samples were 0 μM, 1 μM, 5 μM, 10 μM, 50 μM, 100 μM, and 500 μM. 0 μM was the negative control group, and another group containing only the medium without cells was designed as the blank control group. The cell viability was calculated as follows: (absorbance of the test group - absorbance of the blank control group / absorbance of the negative control group - absorbance of the blank control group) × 100%.

[0132] Table 1 Cytotoxicity test results

[0133] The results of the cytotoxicity experiment are shown in Table 1. For the epithelial cells treated with bicyclic peptide compounds at different concentrations, the cell viability did not show a significant decrease. At a concentration of 500 μM, the cell viability remained at 95% relative to the control group, confirming that the bicyclic peptide compound has extremely low cytotoxicity.

[0134] 2. Serum stability detection The present invention evaluates the degradation of monocyclic polypeptide and bicyclic peptide compounds in a serum environment for predicting their metabolism under physiological conditions. The monocyclic polypeptides include monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C, and monocyclic polypeptide D, and the bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C, and bicyclic peptide compound D.

[0135] The freeze-dried test sample was directly dissolved in PBS containing 10 vol% fetal bovine serum at a final concentration of 1 mg / mL. The freeze-dried GHK was dissolved under the same conditions as the GHK control group. The freeze-dried acetyl hexapeptide-1 was dissolved under the same conditions as the acetyl hexapeptide-1 control group. The solutions prepared above were sterile filtered through 0.22 μM and then loaded into sterile containers and incubated in an incubator at 37°C. Sampling was carried out at the set time points (0 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h). The residual amount of the polypeptide was determined using analytical high performance liquid chromatography. Taking the content at time 0 as 100%, the degradation ratio was calculated by comparing the peak areas at different time points. Calculation formula: relative content = (peak area of test sample / peak area of test sample at time 0) × 100%. A curve of residual percentage against time was plotted and the half-life was calculated. The test samples included monocyclic polypeptides and bicyclic peptide compounds.

[0136] Table 2 Test results of serum degradation level

[0137] The results of the serum stability experiment are shown in Table 2. The GHK in 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 calculated to be approximately 0.7 hours. The acetyl hexapeptide-1 in 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 calculated to be approximately 2.8 hours. Under the same conditions, the stability of the monocyclic polypeptides was significantly improved. The half-lives of monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C, and monocyclic polypeptide D were 12.2 h, 10.8 h, 8.5 h, and 13.8 h, respectively. The stability of the bicyclic peptide compounds was improved based on the corresponding monocyclic ones, and 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 15.3 h, 15.3 h, 13.4 h, and 16.5 h.

[0138] 3. Test on the protein expression promotion rate Cells treated with UVA can significantly reduce the expression levels of various extracellular matrix-related proteins. This invention studied the effects of monocyclic polypeptides and bicyclic peptide compounds on the protein expression levels of various types of collagen and elastin in human fibroblasts HFF-1 after being added to irradiated human fibroblasts HFF-1. The monocyclic polypeptides include monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C, and monocyclic polypeptide D, and the bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C, and bicyclic peptide compound D.

[0139] Inoculate human fibroblasts HFF-1 into a 24-well plate at an inoculation density of 10,000 cells / well and culture them in an incubator at 37°C and 5% CO2. Divide them into a negative control group, a positive control group, a sample group, and a blank control group. The negative control group, the positive control group, and the sample group all receive UVA radiation with a total dose of 9 J / cm 2 The UVA radiation dose of the blank control group is 0 J / cm 2 . The medium of the negative control group is DMEM medium. The positive control group is added with 1 mL of medium containing 100 μg / mL vitamin C and 7 μg / mL vitamin E. The sample group is added with DMEM medium containing test samples at concentration gradients, and the sample concentrations are 10, 20, and 40 μM. Then continue to culture the 24-well plate in an incubator at 37°C and 5% CO2. After 24 hours, collect the supernatant of the medium and measure the content of the corresponding protein using an ELISA kit. Plot a graph with the protein content as the ordinate. Calculate the promotion rate through the formula: Promotion rate = (corresponding protein content of the sample group / corresponding protein content of the negative control group) × 100% - 100%.

[0140] The test samples include GHK, acetyl hexapeptide-1, monocyclic polypeptides, or bicyclic peptide compounds.

[0141] 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, and the expression levels of various proteins in the positive control group increased significantly, proving that the cell model works normally.

[0142] Table 3: Promotion rate of type I collagen expression / %

[0143] The test results of type I collagen content are shown in Table 3. Compared with the negative control group, both control group GHK and control group acetyl hexapeptide-1 have the ability to increase the expression rate of type I collagen. The increase rate of 10 μM GHK is 30.2%, that of 20 μM GHK is 131.1%, and that of 40 μM GHK is 263.4%; the increase rate of 10 μM acetyl hexapeptide-1 is 2.1%, that of 20 μM acetyl hexapeptide-1 is 45.2%, and that of 40 μM acetyl hexapeptide-1 is 61.3%. They all have the ability to increase the expression rate of type I collagen by themselves.

[0144] Monocyclic polypeptide A, monocyclic polypeptide B, monocyclic polypeptide C, and monocyclic polypeptide D all show a strong ability to increase the expression rate of type I collagen. The bicyclic peptide compounds include bicyclic peptide compound A, bicyclic peptide compound B, bicyclic peptide compound C, and bicyclic peptide compound D.

[0145] Compared with monocyclic polypeptides, the increase rate of collagen expression of bicyclic peptide compounds is slightly higher on the basis of their monocycles.

[0146] The expression of type I collagen by bicyclic peptide compound A and bicyclic peptide compound B is significantly higher than that of bicyclic peptide compound C and bicyclic peptide compound D. By analyzing the structures, it is found that in bicyclic peptide compound A and bicyclic peptide compound B, X1 = Gly and X2 = Lys. While in bicyclic peptide compound C and bicyclic peptide compound D, X1 = Lys and X2 = Gly.

[0147] Generally speaking, the ranking of the ability to promote the expression of type I collagen is: bicyclic peptide compound B > bicyclic peptide compound A > bicyclic peptide compound D > bicyclic peptide compound C.

[0148] Table 4: Increase rate of type III collagen expression / %

[0149] The test results of type III collagen are shown in Table 4: Compared with the negative control group, only at higher concentrations does the control group GHK have the ability to significantly increase the rate of type III collagen. The increase rate of 10 μM GHK is 2%, the increase rate of 20 μM GHK is 13.1%, and the increase rate of 40 μM GHK is 64.3%. The control group acetyl hexapeptide-1 hardly has the ability to significantly increase the expression rate of type III collagen. The increase rate of 10 μM acetyl hexapeptide-1 is 3.1%, the increase rate of 20 μM acetyl hexapeptide-1 is 5.1%, and the increase rate of 40 μM acetyl hexapeptide-1 is 4.9%. The four bicyclic peptide compounds and the corresponding monocyclic polypeptides strongly promoted the expression of type III collagen. At the concentration of 10 μM, the weakest bicyclic peptide compound D showed an increase rate of 114%. At the concentration of 40 μM, the strongest bicyclic peptide compound B showed an increase rate of 379%. In this test, the bicyclic peptides did not show an obvious increase rate in the function of monocyclic peptides.

[0150] Table 5: Increase rate of elastin expression / %

[0151] The test results of elastin are shown in Table 5: Compared with the negative control group, the control group GHK has a certain ability to increase the rate of elastin. The increase rate of 10 μM GHK is 53.1%, the increase rate of 20 μM GHK is 86.2%, and the increase rate of 40 μM GHK is 115.4%. The control group acetyl hexapeptide-1 hardly has the ability to significantly increase the expression rate of elastin. The increase rate of 10 μM is 3.1%, the increase rate of 20 μM is 2.9%, and the increase rate of 40 μM is 4.9%. The four bicyclic peptide compounds and the corresponding monocyclic polypeptides all showed a strong ability to induce the expression of elastin. Even the weakest monocyclic polypeptide D showed an increase rate of 194% under the action of 10 μM. The strongest is the bicyclic peptide compound B, with an increase rate of 590.2% at 10 μM, 762.9% at 20 μM, and 857.2% at 40 μM. In the elastin test, compared with the monocyclic polypeptides, the increase rate of the corresponding bicyclic peptide compounds was significantly increased, indicating that the presence of disulfide bonds has the ability to increase the rate of stimulating elastin production.

[0152] 4. Test on the ability to inhibit neurotransmitter release The inhibitory ability of acetyl hexapeptide-1 on neurotransmitter release enables it to inhibit dynamic wrinkles caused by excessive skin tension when used as a cosmetic raw material. This invention tests whether the monocyclic polypeptide and bicyclic peptide compounds obtained have this ability. Neuron cells HT22 are used in the test. After being stimulated by KCl, a high level of expression of the neurotransmitter acetylcholine is induced, which is used to detect the inhibitory ability of added monocyclic polypeptides and bicyclic peptide compounds at different concentrations. 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.

[0153] Inoculate HT22 cells into a 24-well plate at an inoculation density of 10,000 cells / well and culture them in an incubator at 37°C and 5% CO2 for 12 h. Rinse the cells three times with PBS and continue to culture them in the incubator at 37°C and 5% CO2 for 24 h. Set up a blank control group, a model control group, and a sample group in the test group. The blank control group only contains the culture medium. The model control group is the culture medium containing 50 mM KCl. The sample group is the culture medium containing 50 mM KCl and test samples with concentration gradients. The test samples are GHK, acetyl hexapeptide-1, monocyclic polypeptides, or bicyclic peptide compounds, and the concentration gradients are 10, 20, and 40 μM. Add 1 mM physostigmine to all test groups to ensure that the generated acetylcholine is not degraded. After incubating for two hours, collect the supernatant containing the released acetylcholine and store it at -80°C, and further test and analyze it using a kit.

[0154] Taking the blank control group as 100% and the model control group as 0%, calculate the inhibition rate of each group's treatment by normalization.

[0155] Table 6: Inhibition rate of acetylcholine release / %

[0156] The results of the test on the ability to inhibit neurotransmitter release are shown in Table 6. Compared with the blank control group, after treatment with 50 mM KCl in the model control group, the significant increase rate of acetylcholine release level proves that the cell model system works normally. Compared with acetyl hexapeptide-1, the inhibition rate of 10 μM acetyl hexapeptide-1 is 19.4%, the inhibition rate of 20 μM acetyl hexapeptide-1 is 35.4%, and the inhibition rate of 40 μM acetyl hexapeptide-1 is 54.7%. All four bicyclic peptide compounds showed a significant ability to inhibit acetylcholine release: among them, bicyclic peptide compound B > bicyclic peptide compound C > bicyclic peptide compound A, and the inhibitory abilities of bicyclic peptide compound A and bicyclic peptide compound D were almost the same as that of acetyl hexapeptide-1. In summary, all four bicyclic peptide compounds showed a significant ability to inhibit acetylcholine release: at the tested concentrations, the ability of bicyclic peptide compounds to inhibit acetylcholine release was better than that of the corresponding monocyclic polypeptides. The abilities of bicyclic peptide compound B and bicyclic peptide compound C to inhibit acetylcholine release at the tested concentrations were better than those of GHK and acetyl hexapeptide-1.

[0157] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for implementing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0158] In this article, specific examples are used to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of language expression and the objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present application, several improvements, refinements or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, refinements, changes or combinations, or directly applying the concept and technical solution of the invention to other occasions without improvement, should all be regarded as the protection scope of the present application.

Claims

1. A method for preparing a monocyclic polypeptide, comprising: A 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 subjected to cleavage treatment and mono-cyclization to obtain a mono-cyclic polypeptide. The structural formula of the mono-cyclic polypeptide is as follows: , wherein, X1 and X2 are independently selected from any one of Gly, D-Gly, Lys, and D-Lys; Y1 and Y2 are independently selected from any one of Trp, D-Trp, Arg, and D-Arg; R1 and R2 are independently selected from any one of Cys, D-Cys, Cys derivatives, and D-Cys derivatives.

2. The method for preparing the mono-cyclic polypeptide according to claim 1, wherein: X1 and X2 are independently selected from any one of Gly and Lys; or, Y1 and Y2 are independently selected from any one of Trp and Arg; or, R1 and R2 are independently selected from Cys.

3. The mono-cyclic polypeptide prepared by the preparation method according to claim 1.

4. The single-ring polypeptide according to claim 3, wherein: The mono-cyclic polypeptide is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-).

5. A bicyclic peptide compound, the structural formula of which is as follows: , wherein, X1 and X2 are independently selected from any one of Gly, D-Gly, Lys, and D-Lys; Y1 and Y2 are independently selected from any one of Trp, D-Trp, Arg, and D-Arg; R1 and R2 are independently selected from any one of Cys, D-Cys, Cys derivatives, and D-Cys derivatives; L1 is formed by bonding R1 and R2.

6. The bicyclic peptide compound according to claim 5, wherein: The bicyclic peptide compound is Cyclo(His-Cys-Lys-Gly-His-Cys-Lys-Gly-, disulfide bridge connecting Cys&Cys).

7. A method for preparing a bicyclic peptide compound, comprising: The method for preparing the mono-cyclic polypeptide according to claim 1.

8. The preparation method of the bicyclic peptide compound according to claim 7, characterized in that: The mono-cyclic polypeptide is subjected to binary cyclization treatment to obtain a bicyclic peptide compound, and the binary cyclization is carried out under the action of iodomethanol and ascorbic acid.

9. Use of the mono-cyclic polypeptide according to any one of claims 3-4 in the preparation of a bicyclic peptide compound.

10. Use of the bicyclic peptide compound according to any one of claims 5-6 in the preparation of a product for increasing collagen expression and / or a product for increasing elastin expression and / or a product for inhibiting neurotransmitter release.

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