Synthesis method of cyclohexapeptide-9

Abstract

The invention provides a synthetic method of cyclohexapeptide-9, and belongs to the technical field of skin care product raw materials. According to the synthesis method, single amino acid is taken as a starting raw material, and cyclic hexapeptide-9 shown in the formula (I) is efficiently prepared through step-by-step condensation by an activated ester method, orthogonal selective deprotection and coupling reaction. The method overcomes the problems of a solid-phase synthesis method, avoids the use of expensive dipeptide raw materials in an existing liquid-phase synthesis method, and has the advantages of low raw material cost, simple synthesis route, controllable side reaction, high product purity and easiness in process amplification. Formula (I)

Description

A method for synthesizing cyclic hexapeptide-9 Technical Field

[0001] This invention belongs to the field of skin care raw material technology, specifically relating to a method for synthesizing cyclic hexapeptide-9. Background Technology

[0002] With the increasing demand for functional raw materials in the cosmetics and beauty industry, bioactive peptides have received widespread attention due to their ability to regulate skin physiological functions. Studies have shown that bioactive peptides can promote the synthesis of collagen and hyaluronic acid and possess antioxidant activity, thus playing an important role in delaying skin aging (van Walraven N et al. Peptides, 2025: 171440.). Among numerous bioactive peptides, collagen peptide hexapeptide-9 (GPQGPQ), as a signal peptide, can stimulate the synthesis of type I collagen, type IV collagen, laminin-5, and integrins to achieve anti-wrinkle and repair functions. It shows significant effects in reducing facial wrinkles, fading fine lines and crow's feet around the eyes, enhancing skin elasticity, and repairing acne scars (SPEC-CHEM Industry Inc. Technical data sheet SpecPed® H9P(Hexapeptide-9) 2022; Winkey. WKPep Descarin).

[0003] However, linear peptides often suffer from limitations in practical applications, such as poor stability, limited bioavailability, and relatively singular function. In contrast, cyclic peptides exhibit significant advantages in stability, skin affinity, and bioactivity. For example, cyclic hexapeptide-9 not only demonstrates superior anti-wrinkle and firming effects on the skin but also possesses higher chemical stability, promoting the expression of type III collagen and integrin β4, and increasing skin hydration. Furthermore, cyclic hexapeptide-9 exhibits lower cytotoxicity and better skin penetration (CN116162135A; CN116284256A; CN116172894A).

[0004] Currently, the synthesis of cyclic hexapeptide-9 mainly relies on two routes: solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis typically involves first synthesizing a linear peptide, then cleaving it with trifluoroacetic acid to release the peptide chain, followed by cyclization and removal of protecting groups. However, solid-phase synthesis consumes large amounts of raw materials, has complex post-processing steps, and high purification costs. Furthermore, the linear peptide requires cleavage before cyclization, making the process cumbersome. Liquid-phase synthesis uses the protected dipeptide Cbz-Gly-Pro-OH and the customized peptide H-Gln(Trt)-OMe·HCl as starting materials. The peptide chain is progressively extended to obtain Cbz-Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-OMe, followed by removal of the protecting groups at both ends to obtain the linear intermediate H-Gly-Pro-Gln(Trt)-Gly-Pro-Gln(Trt)-OH, which is then cyclized, and finally the Trt protecting group on the side chain is removed. Although liquid-phase synthesis avoids the use of solid-phase supports, it has the following problems: (1) The protected dipeptides Cbz-Gly-Pro-OH and H-Gln(Trt)-OMe·HCl used are expensive. The customized price of Cbz-Gly-Pro-OH exceeds RMB 10,000 / kg, and it is difficult to find manufacturers that can customize H-Gln(Trt)-OMe·HCl; (2) Removing the Cbz protecting group takes a long time; (3) The amount of reaction solvent used is large, a lot of waste liquid is generated, and the post-treatment is troublesome. In summary, there is still room for improvement in the overall efficiency and economy of the existing liquid-phase synthesis method.

[0005] Therefore, developing an efficient, economical, and easy-to-operate synthetic process for cyclic hexapeptide-9 has become an urgent technical problem to be solved in this field. Summary of the Invention

[0006] To address the issues of efficiency, cost, and process complexity in existing methods for synthesizing cyclic hexapeptide-9, the present invention aims to provide a more efficient, economical, and simpler method for synthesizing cyclic hexapeptide-9.

[0007] The specific technical solution of the present invention is as follows: The present invention provides a method for synthesizing cyclic hexapeptide-9, wherein the cyclic hexapeptide-9 has the structure shown in formula (I), and the synthesis method is a liquid-phase synthesis method, comprising the following steps: Formula (I) (a) combines organic solvent, R 1 -G-OH, X 1 HP-OR 3 The reaction of a condensing agent and an organic base yields R. 1 -GP-OR 3 (b) R 1 -GP-OR 3 Organic acid X 2 It reacts with an organic solvent to give X. 2·H-GP-OR 3 (c) Dissolve X in an organic solvent 2 ·H-GP-OR 3 And add an organic base to obtain H-GP-OR 3 Solution; dissolve R in an organic solvent 2 -Q(R 4 )-OH and a condensing agent are used to obtain R. 2 -Q(R 4 )-OH solution; the H-GP-OR 3 Solution and R 2 -Q(R 4 The reaction of )-OH solutions yields R 2 -Q(R 4 )GP-OR 3 (d) Add organic solvents, R 2 -Q(R 4 )GP-OR 3 It reacts with an organic base to give HQ(R) 4 )GP-OR 3 (e) R 2 -Q(R 4 )GP-OR 3 The reaction of organic solvent, catalyst and reducing agent yields R. 2 -Q(R 4 (f) GP-OH; (v) organic solvent, R 2 -Q(R 4 GP-OH, activator, and condensing agent react to obtain R. 2 -Q(R 4 )GP-R 5 (g) Add organic solvent, HQ(R) 4 )GP-OR 3 R 2 -Q(R 4 )GP-R 5 It reacts with an organic base to give R. 2 -Q(R 4 )GPQ(R 4 )GP-OR 3 (h) R 2 -Q(R 4 )GPQ(R 4 )GP-OR 3 The reaction of organic solvent, catalyst and reducing agent yields R. 2 -Q(R 4 )GPQ(R 4 )GP-OH; (i) Add organic solvent, R 2 -Q(R 4)GPQ(R 4 GP-OH, activator, and condensing agent react to obtain R. 2 -Q(R 4 )GPQ(R 4 )GP-R 5 (j) Using organic solvents, R 2 -Q(R 4 )GPQ(R 4 )GP-R 5 It reacts with an organic base to give c(Q(R) 4 )GPQ(R 4 )GP);(k) will c(Q(R) 4 )GPQ(R 4 GP undergoes a cyclization reaction with an organic solvent and an organic acid to obtain cyclic hexapeptide-9 as shown in formula (I); wherein, R 1 R 2 Each is independently selected from an amino protecting group; R 3 R 6 Each is independently selected from the carboxyl protecting group; R 4 It is an amide protecting group; X 1 Selected from inorganic or organic acids; R 5 It is used to activate ester groups.

[0008] Further, each of the organic solvents is independently selected from N,N-dimethylformamide, dichloromethane, methanol, ethanol, or ethyl acetate; each of the condensing agents is independently selected from benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate or N,N'-diisopropylcarbodiimide; each of the organic bases is independently selected from N,N-diisopropylethylamine, 4-methylpiperidine, or piperazine; each of the organic acids is independently selected from trifluoroacetic acid or hydrochloric acid; each of the catalysts is independently selected from palladium on carbon or palladium alumina; and the reducing agent is hydrogen.

[0009] Further, each of the organic solvents is independently selected from N,N-dimethylformamide, dichloromethane, or methanol; each of the condensing agents is independently selected from benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate or N,N'-diisopropylcarbodiimide; each of the organic bases is independently selected from N,N-diisopropylethylamine or 4-methylpiperidine; the organic acid is trifluoroacetic acid; and the catalyst is palladium on carbon.

[0010] Further, the organic solvent in steps (a), (c), (d), and (j) is N,N-dimethylformamide; the organic solvent in steps (b), (f), (g), (i), and (k) is dichloromethane; the organic solvent in steps (e) and (h) is methanol; the condensing agent in steps (a) and (c) is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; the condensing agent in steps (f) and (i) is N,N'-diisopropylcarbodiimide; the organic base in steps (a), (c), and (g) is N,N-diisopropylethylamine; and the organic base in steps (d) and (j) is 4-methylpiperidine.

[0011] Furthermore, the R 1 Selected from tert-butyloxycarbonyl or benzyloxycarbonyl; R 2 It is 9-fluorenylmethoxycarbonyl; R 3 Selected from benzyl, methyl ester, or tert-butyl ester; R 4 It is triphenylmethyl; X 1 For hydrochloric acid; X 2 Selected from trifluoroacetate or hydrochloride; R 5 It is selected from pentafluorophenoxycarbonyl or its structural analogues, N-hydroxysuccinimide (NHS) or its structural analogues.

[0012] Furthermore, the R 1 It is tert-butyloxycarbonyl; R 3 It is benzyl; X 2 It is trifluoroacetate; R 5 It is a pentafluorophenoxycarbonyl group.

[0013] Further, in step (a), the organic solvent, R 1 -G-OH, X 1 HP-OR 3 The molar ratio of condensing agent and organic base is (10~20): 1: (1.05~2): (1~2): (3~5); in step (b), the R... 1 -GP-OR 3 Organic solvents and organic acids X 2 The molar ratio is 1 : (4~5) : (10~20); in step (c), X 2 ·H-GP-OR 3 In solution, the organic base and X 2 ·H-GP-OR 3 The molar ratio is (1~5):1; R 2 -Q(R 4 In the )-OH solution, the organic solvent, R 2 -Q(R 4The molar ratio of )-OH and condensing agent is (15~20): 1: (1~2); in step (d), the R 2 -Q(R 4 )GP-OR 3 The molar ratio of organic solvent and organic base is 1:(10~20):(50~100); in step (e), the HQ(R) 4 )GP-OR 3 The molar ratio of organic solvent and catalyst is 1 : (260~450) : (0.02~0.1); in step (f), the organic solvent, R 2 -Q(R 4 The molar ratio of GP-OH, activator, and condensing agent is (150~300): 1 : (1.05~2) : (1.5~3); in step (g), the HQ(R) 4 )GP-OR 3 R 2 -Q(R 4 )GP-R 5 The molar ratio of organic solvent and organic base is 1 : (1.02~1.2) : (100~300) : (1.1~2); in step (h), the R 2 -Q(R 4 )GPQ(R 4 )GP-OR 3 The molar ratio of organic solvent and catalyst is 1 : (260~450) : (0.02~0.1); in step (i), the R 2 -Q(R 4 )GPQ(R 4 The molar ratio of GP-OH, activator, condensing agent, and organic solvent is 1:(1.05~2):(1.5~3):(150~300); in step (j), the R... 2 -Q(R 4 )GPQ(R 4 )GP-R 5 The molar ratio of organic solvent and organic base is 1 : (10~20) : (50~100); in step (k), Q(R) 4 )GPQ(R 4 The molar ratio of GP, organic solvent and organic acid is 1 : (10~15) : (10~20).

[0014] Further, in step (a), the reaction time is 1-2 hours; in step (b), the reaction time is 1-4 hours; in step (c), the mixing reaction time is 1-2 hours; in step (d), the reaction time is 0.5-1 hours; in step (e), the reaction time is 24-48 hours; in step (f), the reaction time is 0.5-2 hours; in step (g), the reaction time is 1-2 hours; in step (i), the reaction time is 1-2 hours; in step (j), the reaction time is 1-2 hours; and in step (k), the reaction time is 1-2 hours.

[0015] Furthermore, after each reaction step is completed, one or more of the following purification steps are also included: extraction, precipitation, concentration, and column chromatography.

[0016] Further, the extraction solution is selected from acidic aqueous solution and / or alkaline aqueous solution; preferably, the acidic aqueous solution is selected from hydrochloric acid aqueous solution, potassium bisulfate aqueous solution or citric acid aqueous solution; the alkaline aqueous solution is saturated sodium bicarbonate aqueous solution.

[0017] The present invention has achieved the following beneficial effects: (1) Significantly improved synthesis efficiency and economy: The method described in the present invention adopts a liquid-phase synthesis route, which avoids the problems of large carrier consumption and lengthy process caused by the stepwise splitting and cyclization in solid-phase synthesis, and the overall operation is simpler and more efficient. Compared with the existing liquid-phase synthesis method that first synthesizes GPQGPQ and then cyclizes it to obtain cyclic hexapeptide-9, the present invention adjusts the polypeptide synthesis order, first synthesizing QGPQGP and then cyclizing it to obtain cyclic hexapeptide-9. The method of this invention avoids the steps of customizing or purchasing protected dipeptides (such as Cbz-Gly-Pro-OH or H-Gln(Trt)-OMe·HCl) in existing liquid-phase synthesis methods. It only requires readily available single amino acids as starting materials, which significantly reduces raw material costs and improves the economy and practicality of the synthesis route. The raw materials used in this invention are all commercially available and common raw materials, which are inexpensive and low in cost: Boc-G-OH 230 yuan / kg, HCl•HP-OBn 600 yuan / kg, and Fmoc-Q(Trt)-OH 1200 yuan / kg.

[0018] (2) Simplified process flow and purification operation: Through reasonable synthetic route design, this invention reduces the number of protecting group removal steps and solvent usage, and shortens the reaction cycle. In terms of purification, the product can be effectively separated by simple column chromatography, which is convenient to operate, has good reproducibility, and is more conducive to subsequent process scale-up and large-scale production.

[0019] (3) As is well known to those skilled in the art, the selection and orthogonality of protecting groups have a crucial impact on the reaction pathway and product purity during peptide synthesis. A suitable combination of protecting groups needs to achieve selective removal under different conditions to avoid side reactions and ensure the effective execution of the cyclization step. This invention achieves orthogonalization and precise control of protecting groups: In key synthetic steps, this invention selects protecting group combinations with orthogonal reaction characteristics (such as Fmoc and Trt), which can achieve selective removal of protecting groups under different conditions. This strategy effectively avoids side reactions, ensures the high-purity preparation of linear precursors and target cyclic peptides, thereby improving reaction specificity and final yield.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 shows the HPLC chromatogram of Boc-GP-OBn.

[0023] Figure 2 shows the HPLC and mass spectra of Fmoc-Q(Trt)GP-OBn.

[0024] Figure 3 shows the HPLC and mass spectra of Fmoc-Q(Trt)GPQ(Trt)GP-OBn.

[0025] Figure 4 shows the HPLC chromatograms of Fmoc-Q(Trt)GPQ(Trt)GP-OH and Fmoc-Q(Trt)GPQ(Trt)GP-PFP.

[0026] Figure 5 shows the HPLC and mass spectra of cyclic hexapeptide Q(Trt)GPQ(Trt)GP[Cyclic Q(Trt)GPQ(Trt)GP].

[0027] Figure 6 shows the HPLC and mass spectra of cyclic hexapeptide QGPQGP.

[0028] Figure 7 shows the HPLC chromatograms of Cbz-GP-OH synthesized by different methods.

[0029] Figure 8 shows the HPLC chromatograms of Cbz-G-PFP synthesized by different methods.

[0030] Figure 9 shows the HPLC chromatograms of Cbz-GPQ(Trt)-OH synthesized by different methods.

[0031] Figure 10 shows the HPLC chromatograms of Boc-GPQ-OH synthesized by different methods.

[0032] Figure 11 shows the HPLC chromatograms of Boc-GPQGPQ-OH synthesized by different methods.

[0033] Figure 12 shows the HPLC chromatograms of Fmoc-Q(Trt)GP-OBn synthesized by different methods. Detailed Implementation

[0034] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0035] In the abbreviations used in the embodiments of this invention, G represents glycine residue, P represents proline residue, and Q represents glutamine residue.

[0036] Example 1: Synthesis of Cyclic QGPQGP The structure of cyclic hexapeptide-9 (Cyclic QGPQGP) of this invention is as follows: The specific preparation steps include: (1) Synthesis of dipeptide Boc-GP-OBn: Boc-L-glutamic acid (Boc-G-OH), L-proline benzyl ester hydrochloride (HCl·HP-OBn) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) are dissolved in DMF, and then N,N-diisopropylethylamine (DIEA) is added for reaction. The molar ratio of DMF, Boc-G-OH, HCl·HP-OBn, HBTU and DIEA is 10:1:1.07:1:3. The reaction is carried out for 1.5 hours. After the reaction is completed, the product is extracted with EA, the product is dissolved in the EA layer, and then the EA layer is washed successively with acidic aqueous solution (hydrochloric acid aqueous solution is used in this example, which can also be replaced with potassium bisulfate aqueous solution, citric acid aqueous solution) and alkaline aqueous solution (saturated sodium bicarbonate aqueous solution is used in this example) to remove unreacted raw materials. Then the EA layer is concentrated by rotary evaporation to obtain dipeptide Boc-GP-OBn. The HPLC chromatogram of Boc-GP-OBn is shown in Figure 1. Yield > 95%, purity > 95%.

[0037] (2) Synthesis of dipeptide TFA·H-GP-OBn: The Boc-GP-OBn obtained in step (1) was dissolved in DCM, TFA was added, and the reaction was carried out for 1 hour. The molar ratio of Boc-GP-OBn to DCM and TFA was 1:5:10. After the reaction was completed, the mixture was concentrated by rotary evaporation to obtain TFA·H-GP-OBn. Yield > 95%, purity > 98%.

[0038] (3) Synthesis of tripeptide Fmoc-Q(Trt)GP-OBn: TFA·H-GP-OBn obtained in step (2) was dissolved in DMF, and then three times the molar amount of DIEA was added to adjust the pH to 7-8. Fmoc-N-triphenylmethyl-L-glutamine (Fmoc-Q(Trt)-OH) and HBTU were dissolved in DMF. The molar ratio of DMF, Fmoc-Q(Trt)-OH and HBTU was 15:1:1.05. The above Fmoc-Q(Trt)-OH solution and TFA·H-GP-OBn solution were mixed and reacted for 2 hours. After the reaction was completed, the product was extracted with EA, and the EA layer was washed sequentially with acidic aqueous solution (hydrochloric acid aqueous solution was used in this example, but potassium bisulfate aqueous solution or citric acid aqueous solution could also be used) and alkaline aqueous solution (saturated sodium bicarbonate aqueous solution was used in this example). Subsequent column chromatography elution with PE and EA was performed. EA purified the product from 10% to 60%, and the solvent was concentrated to obtain the tripeptide Fmoc-Q(Trt)GP-OBn. The HPLC and mass spectra of Fmoc-Q(Trt)GP-OBn are shown in Figure 2. Yield > 85%, purity > 98%.

[0039] (4) Synthesis of HQ(Trt)GP-OBn: Fmoc-Q(Trt)GP-OBn was dissolved in DMF, and 4-methylpiperidine was added. The reaction was carried out for 1 hour to remove the Fmoc protecting group. The molar ratio of Fmoc-Q(Trt)GP-OBn to DMF and 4-methylpiperidine was 1:10:50. After the reaction was completed, the product was extracted with EA, and then the EA layer was washed with acidic aqueous solution (hydrochloric acid aqueous solution was used in this example, but potassium hydrogen sulfate aqueous solution or citric acid aqueous solution can also be used). The EA layer was then added dropwise to petroleum ether to precipitate a white solid to obtain HQ(Trt)GP-OBn. Yield > 80%, purity > 95%.

[0040] (5) Synthesis of Fmoc-Q(Trt)GP-OH: Fmoc-Q(Trt)GP-OBn was dissolved in methanol (MeOH), Pd / C was added, and hydrogen gas was introduced. The reaction was carried out for 24-48 hours to remove the benzyl group (Bn). The molar ratio of Fmoc-Q(Trt)GP-OBn to methanol and Pd / C was 1:260:0.02. After the reaction was completed, the methanol solution was obtained by filtration, and then the methanol solvent was removed by rotary evaporation to obtain Fmoc-Q(Trt)GP-OH. Yield > 95%, purity > 95%.

[0041] (6) Synthesis of intermediate Fmoc-Q(Trt)GP-PFP: Fmoc-Q(Trt)GP-OH from step (5) was dissolved in DCM with pentafluorophenol (PFP) and N,N'-diisopropylcarbodiimide (DIC), and the reaction was carried out for 1 hour. The molar ratio of DCM to Fmoc-Q(Trt)GP-OH, PFP, and DIC was 150:1:1.05:1.5. After the reaction was completed, the reaction solution was concentrated, and the solid was precipitated in diethyl ether. The solid was dried to obtain Fmoc-Q(Trt)GP-PFP. Yield > 90%, purity > 95%.

[0042] (7) Synthesis of hexapeptide Fmoc-Q(Trt)GPQ(Trt)GP-OBn: HQ(Trt)GP-OBn obtained in step (4) and Fmoc-Q(Trt)GP-PFP obtained in step (6) were dissolved in DCM, and DIEA was added and reacted for 1-2 hours. The molar ratio of HQ(Trt)GP-OBn to Fmoc-Q(Trt)GP-PFP, DCM, and DIEA was 1:1.02:100:2. After the reaction was completed, the solution was concentrated and then purified by column chromatography with DCM and MeOH elution. The MeOH concentration was 0.5% to 10%. The purified liquid was dried by rotary evaporation to obtain hexapeptide Fmoc-Q(Trt)GPQ(Trt)GP-OBn. The HPLC and mass spectra of Fmoc-Q(Trt)GPQ(Trt)GP-OBn are shown in Figure 3. Yield > 85%, purity > 95%.

[0043] (8) Synthesis of Fmoc-Q(Trt)GPQ(Trt)GP-OH: The Fmoc-Q(Trt)GPQ(Trt)GP-OBn obtained in step (7) was dissolved in MeOH, and Pd / C was added. Hydrogen gas was then introduced to remove Bn. The molar ratio of Fmoc-Q(Trt)GPQ(Trt)GP-OBn to methanol and Pd / C was 1:260:0.02. After the reaction was completed, the methanol solution was obtained by filtration, and then the methanol solvent was removed by rotary evaporation to obtain Fmoc-Q(Trt)GPQ(Trt)GP-OH. The HPLC analysis chromatogram of Fmoc-Q(Trt)GPQ(Trt)GP-OH is shown in Figure 4. Yield > 95%, purity > 95%.

[0044] (9) Synthesis of intermediate Fmoc-Q(Trt)GPQ(Trt)GP-PFP: Fmoc-Q(Trt)GPQ(Trt)GP-OH was dissolved in DCM, followed by the addition of PFP and DIC, and the reaction was carried out for 1 hour. The molar ratio of Fmoc-Q(Trt)GPQ(Trt)GP-OH to PFP, DIC, and DCM was 1:1.05:1.5:150. After the reaction was completed, the reaction solution was concentrated, and a solid was precipitated in diethyl ether. The solid was dried to obtain Fmoc-Q(Trt)GPQ(Trt)GP-PFP. The HPLC chromatogram of Fmoc-Q(Trt)GPQ(Trt)GP-PFP is shown in Figure 4. Yield > 85%, purity > 95%.

[0045] (10) Synthesis of cyclic hexapeptide Q(Trt)GPQ(Trt)GP: Fmoc-Q(Trt)GPQ(Trt)GP-PFP from step (9) was dissolved in DMF, and then 4-methylpiperidine was added, and the reaction was carried out for 1 hour. The molar ratio of Fmoc-Q(Trt)GPQ(Trt)GP-PFP to DMF and 4-methylpiperidine was 1:10:50. The reaction solvent was then precipitated with diethyl ether to obtain a solid, and the solid was purified by column chromatography by elution with DCM and MeOH. The MeOH content was 2%~10%, and the solvent was removed by rotary evaporation to obtain cyclic hexapeptide Q(Trt)GPQ(Trt)GP. The HPLC analysis chromatogram and mass spectra of cyclic hexapeptide Q(Trt)GPQ(Trt)GP are shown in Figure 5. Yield > 80%, purity > 95%.

[0046] (11) Synthesis of cyclic hexapeptide QGP: The cyclic hexapeptide Q(Trt)GP obtained in step (10) was dissolved in DCM, and then TFA was added. The reaction was carried out for 1 hour. The molar ratio of cyclic hexapeptide Q(Trt)GP to DCM and TFA was 1:10:20. After the reaction was completed, the solution was added dropwise to 10 times the volume of THF solution for precipitation to obtain a solid. The solid was dried to obtain cyclic hexapeptide QGP. The HPLC and mass spectra of cyclic hexapeptide QGP are shown in Figure 6. Yield > 85%, purity > 95%.

[0047] The following experimental examples demonstrate the beneficial effects of the present invention.

[0048] Experiment 1: Screening of Preparation Process Experiment 1: Synthesis and Screening of Cbz-GPQ-OH The intermediate Cbz-G-PFP was synthesized using N-benzyloxycarbonyl-glycine (Cbz-G-OH) with pentafluorophenol (PFP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) in DMF solvent. The molar ratio of Cbz-G-OH, PFP, EDC·HCl, and DCM was 1:1.05:1.1:10. After the reaction was complete, impurities were removed by extraction with solvents such as ethyl acetate and water, followed by rotary evaporation and concentration to obtain Cbz-G-PFP.

[0049] Cbz-G-PFP, HP-OH, and triethylamine were reacted in dichloromethane solvent at a molar ratio of 1:2:1:10. After the reaction was complete, the mixture was purified by extraction, and the solution was concentrated to obtain Cbz-GP-OH. The HPLC chromatogram of Cbz-GP-OH is shown in Figure 7.

[0050] Cbz-GP-PFP was obtained using the same method as the synthesis of Cbz-G-PFP. Then, Cbz-GP-PFP, L-glutamine (HQ-OH), and triethylamine were reacted in DCM to synthesize the tripeptide Cbz-GPQ-OH. The molar ratio of Cbz-GP-PFP, HQ-OH, triethylamine, and DCM was 1:2:2:10. The reaction was disordered, producing multiple reaction products, making further reactions impossible. The HPLC monitoring chromatogram of the reaction is shown in Figure 8.

[0051] Experiment 2: Cbz-GPQ(Trt)-OH Synthesis Screening Following the method described in Experiment 1, Cbz-GP-OH or Cbz-GP-PFP was synthesized first. In the synthesis of the tripeptide, glutamine was replaced with triphenylmethyl-protected glutamine to prevent the influence of the glutamine side-chain amide on the reaction. Cbz-GP-PFP, N'-triphenylmethyl-L-glutamine (HQ(Trt)-OH), and triethylamine were reacted in DCM to synthesize the tripeptide Cbz-GPQ(Trt)-OH. The molar ratio of Cbz-GP-PFP, HQ(Trt)-OH, triethylamine, and DCM was 1:2:2:10. However, the reaction remained highly disordered, and even changing the condensing agent (HOBT, etc.) during the reaction of Cbz-GP-OH and HQ(Trt)-OH could not improve the reaction. The HPLC chromatogram of Cbz-GPQ(Trt)-OH is shown in Figure 9.

[0052] Experiment 3: Synthesis and Screening of Boc-GPQ-OH By changing the protecting group of the amino acid, glycine was protected with a tert-butyloxycarbonyl group, and then synthesized according to the method in Experiment 1 to obtain Boc-GPQ-OH with an HPLC purity greater than 94%. The HPLC chromatogram of Boc-GPQ-OH is shown in Figure 10.

[0053] The Boc in Boc-GPQ-OH was removed using dioxane hydrochloride to yield H-GPQ-OH. Boc-GPQ-OH was then reacted with PFP and EDC·HCl to synthesize the intermediate Boc-GPQ-PFP, with impurities removed by extraction. Subsequently, Boc-GPQ-PFP and H-GPQ-OH were reacted with triethylamine to synthesize Boc-GPQGPQ-OH. However, this reaction produced a large amount of impurities, which could not be effectively removed by precipitation and recrystallization, resulting in synthesis failure (Figure 11).

[0054] Experiment 4: Screening of Fmoc-Q(trt)GP-OBn Synthesis Following the synthesis of Fmoc-Q(trt)-OH and H-GP-OBn according to this invention, Fmoc-Q(trt)-PFP was synthesized using PFP and EDC. Subsequently, it reacted with H-GP-OBn under the action of DIEA to synthesize Fmoc-Q(trt)GP-OBn. The reaction generated many impurities, which could not be effectively removed by precipitation and recrystallization, resulting in synthesis failure. However, the synthesis of Fmoc-Q(Trt)GP-OBn mediated by HBTU was relatively cleaner, and the pure product could be obtained in a high yield after simple column chromatography. The HPLC chromatograms of the reactions of Fmoc-Q(Trt)GP-OBn mediated by PFP and HBTU are shown in Figure 12.

[0055] Conclusion: In Experiment 4, this invention screened a synthetic strategy different from step (3) of Example 1 to prepare the key intermediate Fmoc-Q(Trt)GP-OBn. Specifically, the conditions for the condensation reaction were screened: In Experiment 4, pentafluorophenol (PFP) and EDC•HCl were used to activate the carboxyl group of Fmoc-Q(Trt)-OH to generate an active ester intermediate (Fmoc-Q(trt)-PFP), which was then condensed with H-GP-OBn. The results showed that this method generated more impurities, which could not be effectively purified by precipitation and recrystallization, leading to synthesis failure. In Example 1, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) was used as a condensing agent to directly mediate the coupling reaction between Fmoc-Q(Trt)-OH and H-GP-OBn. The results showed that this method was relatively cleaner, and the product could be purified by simple column chromatography with a high yield. The screening experiment directly compared and confirmed that, in the synthesis of the tripeptide intermediate Fmoc-Q(Trt)GP-OBn, using HBTU as a condensing agent (as described in step (3) of Example 1) is superior to using the PFP active ester method and is a more effective way to obtain high-purity products.

[0056] In summary, this invention provides a method for synthesizing cyclic hexapeptide-9. This method uses a single amino acid as a starting material and efficiently prepares cyclic hexapeptide-9 through stepwise condensation via activated esterification, orthogonal selective deprotection, and coupling reactions. This method overcomes the problems of solid-phase synthesis methods, avoids the use of expensive dipeptide raw materials in existing liquid-phase synthesis methods, and has the advantages of low raw material cost, simple synthetic route, controllable side reactions, high product purity, and easy process scale-up.

Claims

1. A method for synthesizing cyclic hexapeptide-9, characterized in that, The cyclic hexapeptide-9 has the structure shown in formula (I), and the synthesis method is a liquid-phase synthesis method. Includes the following steps: Formula (I) (a) combines organic solvent, R 1 -G-OH, X 1 HP-OR 3 The reaction of a condensing agent and an organic base yields R. 1 -GP-OR 3 (b) R 1 -GP-OR 3 Organic acid X 2 It reacts with an organic solvent to give X. 2 ·H-GP-OR 3 (c) Dissolve X in an organic solvent 2 ·H-GP-OR 3 And add an organic base to obtain H-GP-OR 3 Solution; dissolve R in an organic solvent 2 -Q(R 4 )-OH and a condensing agent are used to obtain R. 2 -Q(R 4 )-OH solution; the H-GP-OR 3 Solution and R 2 -Q(R 4 The reaction of )-OH solutions yields R 2 -Q(R 4 )GP-OR 3 (d) Add organic solvents, R 2 -Q(R 4 )GP-OR 3 It reacts with an organic base to give HQ(R) 4 )GP-OR 3 (e) R 2 -Q(R 4 )GP-OR 3 The reaction of organic solvent, catalyst and reducing agent yields R. 2 -Q(R 4 (f) GP-OH; (v) Organic solvent, R 2 -Q(R 4 GP-OH, activator, and condensing agent react to obtain R. 2 -Q(R 4 )GP-R 5 (g) Add organic solvent, HQ(R) 4 )GP-OR 3 R 2 -Q(R 4 )GP-R 5 It reacts with an organic base to give R. 2 -Q(R 4 )GPQ(R 4 )GP-OR 3 (h) R 2 -Q(R 4 )GPQ(R 4 )GP-OR 3 The reaction of organic solvent, catalyst and reducing agent yields R. 2 -Q(R 4 )GPQ(R 4 )GP-OH; (i) Add organic solvent, R 2 -Q(R 4 )GPQ(R 4 GP-OH, activator, and condensing agent react to obtain R. 2 -Q(R 4 )GPQ(R 4 )GP-R 5 (j) Using organic solvents, R 2 -Q(R 4 )GPQ(R 4 )GP-R 5 It undergoes a cyclization reaction with an organic base to give c(Q(R)). 4 )GPQ(R 4 )GP);(k) will c(Q(R) 4 )GPQ(R 4 GP reacts with an organic solvent and an organic acid to give cyclic hexapeptide-9 as shown in formula (I); wherein, R 1 R 2 Each is independently selected from an amino protecting group; R 3 R 6 Each is independently selected from the carboxyl protecting group; R 4 It is an amide protecting group; X 1 Selected from inorganic or organic acids; R 5 It is used to activate ester groups.

2. The synthesis method according to claim 1, characterized in that, Each of the organic solvents is independently selected from N,N-dimethylformamide, dichloromethane, methanol, ethanol, or ethyl acetate; each of the condensing agents is independently selected from benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate or N,N'-diisopropylcarbodiimide; each of the organic bases is independently selected from N,N-diisopropylethylamine, 4-methylpiperidine, or piperazine; each of the organic acids is independently selected from trifluoroacetic acid or hydrochloric acid; each of the catalysts is independently selected from palladium on carbon or palladium alumina; and the reducing agent is hydrogen.

3. The synthesis method according to claim 2, characterized in that, Each of the organic solvents is independently selected from N,N-dimethylformamide, dichloromethane, or methanol; each of the condensing agents is independently selected from benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate or N,N'-diisopropylcarbodiimide; each of the organic bases is independently selected from N,N-diisopropylethylamine or 4-methylpiperidine; the organic acid is trifluoroacetic acid; and the catalyst is palladium on carbon.

4. The synthesis method according to claim 3, characterized in that, The organic solvent in steps (a), (c), (d), and (j) is N,N-dimethylformamide; the organic solvent in steps (b), (f), (g), (i), and (k) is dichloromethane; the organic solvent in steps (e) and (h) is methanol; the condensing agent in steps (a) and (c) is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; the condensing agent in steps (f) and (i) is N,N'-diisopropylcarbodiimide; the organic base in steps (a), (c), and (g) is N,N-diisopropylethylamine; and the organic base in steps (d) and (j) is 4-methylpiperidine.

5. The synthesis method according to claim 1, characterized in that, The R 1 Selected from tert-butyloxycarbonyl or benzyloxycarbonyl; R 2 It is 9-fluorenylmethoxycarbonyl; R 3 Selected from benzyl, methyl ester, or tert-butyl ester; R 4 It is triphenylmethyl; X 1 It is hydrochloride; X 2 Selected from trifluoroacetate or hydrochloride; R 5 It is selected from pentafluorophenoxycarbonyl or its structural analogues, N-hydroxysuccinimide or its structural analogues.

6. The synthesis method according to claim 5, characterized in that, The R 1 It is tert-butyloxycarbonyl; R 3 It is benzyl; X 2 It is trifluoroacetate; R 5 It is a pentafluorophenoxycarbonyl group.

7. The synthesis method according to claim 1, characterized in that, In step (a), the organic solvent, R 1 -G-OH, X 1 HP-OR 3 The molar ratio of condensing agent and organic base is (10~20): 1: (1.05~2): (1~2): (3~5); in step (b), the R... 1 -GP-OR 3 Organic solvents and organic acids X 2 The molar ratio is 1 : (4~5) : (10~20); in step (c), X 2 ·H-GP-OR 3 In solution, the organic base and X 2 ·H-GP-OR 3 The molar ratio is (1~5):1; R 2 -Q(R 4 In the )-OH solution, the organic solvent, R 2 -Q(R 4 The molar ratio of )-OH and condensing agent is (15~20): 1: (1~2); in step (d), the R 2 -Q(R 4 )GP-OR 3 The molar ratio of organic solvent and organic base is 1 : (10~20) : (50~100); in step (e), the HQ(R) 4 )GP-OR 3 The molar ratio of organic solvent and catalyst is 1 : (260~450) : (0.02~0.1); in step (f), the organic solvent, R 2 -Q(R 4 The molar ratio of GP-OH, activator, and condensing agent is (150~300): 1: (1.05~2): (1.5~3); in step (g), the HQ(R) 4 )GP-OR 3 R 2 -Q(R 4 )GP-R 5 The molar ratio of organic solvent and organic base is 1 : (1.02~1.2) : (100~300) : (1.1~2); in step (h), the R 2 -Q(R 4 )GPQ(R 4 )GP-OR 3 The molar ratio of organic solvent and catalyst is 1:(260~450):(0.02~0.1); in step (i), the R... 2 -Q(R 4 )GPQ(R 4 The molar ratio of GP-OH, activator, condensing agent, and organic solvent is 1 : (1.05~2) : (1.5~3) : (150~300); in step (j), the R... 2 -Q(R 4 )GPQ(R 4 )GP-R 5 The molar ratio of organic solvent and organic base is 1 : (10~20) : (50~100); in step (k), Q(R) 4 )GPQ(R 4 The molar ratio of GP, organic solvent and organic acid is 1 : (10~15) : (10~20).

8. The synthesis method according to claim 1, characterized in that, In step (a), the reaction time is 1-2 hours; in step (b), the reaction time is 1-4 hours; in step (c), the mixed reaction time is 1-2 hours; in step (d), the reaction time is 0.5-1 hours; in step (e), the reaction time is 24-48 hours; in step (f), the reaction time is 0.5-2 hours; in step (g), the reaction time is 1-2 hours; in step (i), the reaction time is 1-2 hours; in step (j), the reaction time is 1-2 hours; in step (k), the reaction time is 1-2 hours.

9. The synthesis method according to claim 1, characterized in that, After each reaction step is completed, one or more of the following purification steps are also included: extraction, precipitation, concentration, and column chromatography.

10. The synthesis method according to claim 9, characterized in that, The extraction solution is selected from acidic aqueous solutions and / or alkaline aqueous solutions; preferably, the acidic aqueous solution is selected from hydrochloric acid aqueous solution, potassium bisulfate aqueous solution or citric acid aqueous solution; the alkaline aqueous solution is saturated sodium bicarbonate aqueous solution.

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