Cyclic hexapeptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of polypeptide preparation, in particular to cyclic hexapeptide as well as a preparation method and application thereof. The preparation method comprises the following steps: reacting fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl acyl-L-lysine with 2-chlorotrityl chloride, and removing the protection of trimethylsilylethoxycarbonyl, so that amino is connected to dichloro resin; the preparation method comprises the following steps: taking fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-L-alanine, fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester and fluorenylmethoxycarbonyl-L-leucine as a starting point, and then removing amino protection of terminal amino acid and carboxyl protection of the first amino acid; and carrying out condensation reaction and peptide cutting reaction to obtain the cyclic hexapeptide. The preparation method disclosed by the invention is simple in steps and simple and convenient to operate, the use of tetraphenylphosphine palladium is avoided, and the defects of low efficiency, high cost and low yield of the traditional liquid-phase cyclization method and solid-phase cyclization method are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide preparation, and particularly relates to a cyclohexapeptide, a preparation method thereof and an application thereof. Background Art

[0002] Cyclic peptide drugs are highly praised in the pharmaceutical field for their excellent high affinity, outstanding stability and extremely low cytotoxicity. In the cosmetics industry, cyclic peptides also show unique charm. Cyclic peptides in cosmetics, compared with their linear form before cyclization, generally exhibit the remarkable characteristics of high stability, high affinity and strong skin permeability. Among them, hexapeptide-3 (H-Lys-Leu-Asp-Ala-Pro-Thr-OH), as an outstanding one in cosmetic raw materials, has multiple anti-wrinkle effects such as promoting the regeneration of type I and type II collagen, reducing the depth and length of wrinkles.

[0003] The preparation of traditional macrocyclic peptides, that is, cyclic peptide structures composed of 5 or more amino acids, is mainly achieved through two major approaches: one is the liquid-phase cyclization method. This method first uses solid-phase preparation technology to prepare a linear peptide on a resin, and then cleaves the linear peptide from the resin under the condition of side-chain protection; then, a condensing agent is introduced in an organic solvent to promote the head-to-tail connection of the linear peptide to form a cyclic structure; finally, after removing the side-chain protecting groups, it is purified and freeze-dried to obtain the cyclic peptide. However, this method has cumbersome steps and low efficiency, resulting in a low yield, and at the same time, a large amount of by-products are generated; the other is the solid-phase cyclization method. This method directly constructs a cyclic peptide structure on the resin, but the implementation conditions are relatively harsh: it requires that the starting amino acid must be glutamic acid or aspartic acid with a carboxyl group on the side chain, and these carboxyl groups must be in an unprotected state, while the carboxyl group of the main chain is protected by acrylate. After the amino acids are connected, it also depends on expensive tetraphenylphosphine palladium for deprotection reaction, increasing the production cost and limiting the wide application of solid-phase cyclization in the field of cyclic peptide preparation. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides a cyclohexapeptide, its preparation method and application. The present invention uses fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine as a raw material, reacts it with 2-chlorotrityl chloride in an alkaline environment, and uses tetrabutylammonium fluoride to remove the trimethylsilylethoxycarbonyl protection on the side-chain amino group, so that the amino group is connected to the dichlororesin; starting from this, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-L-alanine, fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester and fluorenylmethoxycarbonyl-L-leucine are successively connected. Then, the amino protection of the terminal amino acid and the carboxyl protection of the first amino acid are removed, so that the carboxyl group of the first amino acid and the amino group of the terminal amino acid are exposed, and a cyclohexapeptide with a side chain connected to the dichlororesin is formed through a condensation reaction; finally, through a peptide cleavage reaction, the cyclohexapeptide is obtained. The entire preparation process of the cyclohexapeptide of the present invention is completed on the dichlororesin. The preparation steps are simple and clear, and the operation is convenient. It not only solves the problems of low efficiency and cumbersome nature of liquid-phase cyclization, but also significantly improves the yield and purity of the cyclohexapeptide; in addition, the preparation method of the present invention avoids the use of tetraphenylphosphine palladium, overcomes the defects of high cost and inability to be widely applied in traditional solid-phase ring preparation, and thus provides a more economical and efficient production route for the application of cyclohexapeptide in cosmetics and other fields.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] The first object of the present invention is to provide a cyclohexapeptide, and the structural formula of the cyclohexapeptide is:

[0007]

[0008] The second object of the present invention is to provide a preparation method of the above cyclohexapeptide, including the following steps:

[0009] S1. Use N,N-diisopropylethylamine to provide an alkaline environment, mix fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine with 2-chlorotrityl chloride in an alkaline environment, and the chlorine atom of 2-chlorotrityl chloride undergoes a substitution reaction with the hydroxyl group on fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to obtain compound I, denoted as Fmoc-Lys(Teoc)-O-TRT-2-Cl.

[0010] S2. Deprotect the trimethylsilylethoxycarbonyl group in Fmoc-Lys(Teoc)-O-TRT-2-Cl to obtain Fmoc-Lys-O-TRT-2-Cl. Use N,N-diisopropylpropylamine to provide an alkaline environment. Mix Fmoc-Lys-O-TRT-2-Cl with dichlororesin in the alkaline environment. A nucleophilic substitution reaction occurs between one chlorine atom on the dichlororesin and the carboxylate anion on Fmoc-Lys-O-TRT-2-Cl to obtain Compound II, denoted as Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl.

[0011] S3. Deprotect the fluorenylmethoxycarbonyl group in Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Lys(2-CTCResin)-O-TRT-2-Cl. Condense H-Lys(2-CTCResin)-O-TRT-2-Cl with fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine to obtain Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0012] S4. Deprotect the fluorenylmethoxycarbonyl group in Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. Condense H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with fluorenylmethoxycarbonyl-L-proline to obtain Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0013] S5. Deprotect the fluorenylmethoxycarbonyl group in Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. Condense H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with fluorenylmethoxycarbonyl-L-alanine to obtain Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0014] S6. Deprotect the fluorenylmethoxycarbonyl group of Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and condense H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with fluorenylmethoxycarbonyl-aspartic acid 4-tert-butyl ester to obtain Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0015] S7. Deprotect the fluorenylmethoxycarbonyl group of Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and condense H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with fluorenylmethoxycarbonyl-L-leucine to obtain Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otb u)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0016] S8. Deprotect the fluorenylmethoxycarbonyl group of Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain Compound III, denoted as H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0017] S9. After deprotecting the 2-chlorotrityl group of H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, obtain Compound IV, denoted as H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH.

[0018] S10. Using N,N - diisopropylethylamine to provide an alkaline environment, condense H - Leu - Asp(otbu) - Ala - Pro - Thr(otbu) - Lys(2 - CTCResin) - OH to obtain Compound V, denoted as Cyclo(Leu - Asp(otbu) - Ala - Pro - Thr(otbu) - Lys(2 - CTCResin)).

[0019] S11. Perform a peptide cleavage reaction on Cyclo(Leu - Asp(otbu) - Ala - Pro - Thr(otbu) - Lys(2 - CTCResin)) to obtain a cyclohexapeptide.

[0020] Preferably, in step S1, the molar ratio of fluorenylmethyloxycarbonyl - N6 - trimethylsilylethoxycarbonyl - L - lysine to 2 - chlorotrityl chloride is 1:1 - 2; the molar ratio of fluorenylmethyloxycarbonyl - N6 - trimethylsilylethoxycarbonyl - L - lysine to the dichlororesin in step S2 is 1 - 1.5:1.

[0021] Preferably, in step S2, a mixed solution of tetrabutylammonium fluoride and N,N - diisopropylethylamine is used to remove the protecting group trimethylsilylethoxycarbonyl in Fmoc - Lys(Teoc) - O - TRT - 2 - Cl, and the molar ratio of tetrabutylammonium fluoride to N,N - diisopropylethylamine is 2 - 3:3 - 4.

[0022] Preferably, in steps S3 - S8, the operation of removing the protecting group fluorenylmethyloxycarbonyl is the same, specifically:

[0023] Mix Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and deprotect at 20 °C to 25 °C for 20 min to 40 min; mix Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and deprotect at 20 °C to 25 °C for 20 min to 40 min; mix Fmoc-Pro-Thr(otbu)-Lys(2-CTCRe sin)-O-TRT-2-Cl with piperidine solution and deprotect at 20 °C to 25 °C for 20 min to 40 min; mix Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and deprotect at 20 °C to 25 °C for 20 min to 40 min; mix Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and deprotect at 20 °C to 25 °C for 20 min to 40 min; mix Fmoc-Leu-A sp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and deprotect at 20 °C to 25 °C for 20 min to 40 min; wherein, the piperidine solution is composed of piperidine and N,N-dimethylformamide, and the volume ratio of piperidine to N,N-dimethylformamide is 1:4 to 10.

[0024] Preferably, in steps S3 to S7, the condensation reactions are carried out in the same manner, specifically:

[0025] Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, fluorenylmethyloxycarbonyl-O-tert-butyl-L-threonine, H-Lys(2-CTCResin)-O-TRT-2-Cl and the condensing agents are mixed together in N,N-dimethylformamide, and stirred for 2 h to 3 h at 20 °C to 25 °C under a nitrogen atmosphere; using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, methoxycarbonyl-L-proline, H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensing agents are mixed together in N,N-dimethylformamide, and stirred for 2 h to 3 h at 20 °C to 25 °C under a nitrogen atmosphere; using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, fluorenylmethyloxycarbonyl-L-alanine, H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensing agents are mixed together in N,N-dimethylformamide, and stirred for 2 h to 3 h at 20 °C to 25 °C under a nitrogen atmosphere; using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, fluorenylmethyloxycarbonyl-L-alanine, H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensing agents are mixed together in N,N-dimethylformamide, and stirred for 2 h to 3 h at 20 °C to 25 °C under a nitrogen atmosphere; using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, fluorenylmethyloxycarbonyl-L-alanine, H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensing agents are mixed together in N,N-dimethylformamide, and stirred for 2 h to 3 h at 20 °C to 25 °C under a nitrogen atmosphere; wherein, the molar ratio of 1-hydroxybenzotriazole, N,N-diisopropylcarbodiimide to the dichlororesin in step S2 is 2.5 to 3: 2.5 to 3: 1.

[0026] Preferably, in step S9, a mixed solution of acetic acid, trifluoroethanol and dichloromethane is used to remove the protecting group 2-chlorotriphenylmethyl of H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and the volume ratio of acetic acid, trifluoroethanol and dichloromethane is 10 to 15: 20 to 30: 55 to 70.

[0027] Preferably, in step S10, the condensation reaction is carried out as follows: Using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and N,N-diisopropylethylamine as condensing agents, H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH and the condensing agents are mixed together in N,N-dimethylformamide, and the reaction is carried out at 20 °C to 25 °C for 1 h to 2 h under a nitrogen atmosphere; wherein, the molar ratio of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine to the dichlororesin in step S2 is 2-4:2-4:1.

[0028] Preferably, in step S11, the peptide cleavage reaction is carried out as follows: Using trifluoroacetic acid and triisopropylsilane as the peptide cleavage solution, the peptide cleavage solution and Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)) are mixed, and the reaction is carried out at 20 °C to 25 °C for 1 h to 2 h under a nitrogen atmosphere; wherein, the volume ratio of trifluoroacetic acid to triisopropylsilane is 80-95:20-5.

[0029] The third object of the present invention is to provide the application of the above cyclic hexapeptide in the preparation of cyclic peptide products.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] 1. The present invention provides a preparation method of a cyclic hexapeptide. The present invention uses fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine as a raw material, reacts with 2-chlorotrityl chloride under an alkaline environment, and uses tetrabutylammonium fluoride to remove the trimethylsilylethoxycarbonyl protection on the side-chain amino group, so that the amino group is connected to the dichlororesin; starting from this, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-L-alanine, fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester and fluorenylmethoxycarbonyl-L-leucine are successively connected, and then the amino protection of the terminal amino acid and the carboxyl protection of the first amino acid are removed, so that the carboxyl group of the first amino acid and the amino group of the terminal amino acid are exposed, and a cyclic hexapeptide with a side chain connected to the dichlororesin is formed through a condensation reaction; finally, through a peptide cleavage reaction, a cyclic hexapeptide is obtained. The entire preparation process of the cyclic hexapeptide of the present invention is completed on the dichlororesin. The preparation steps are simple and clear, and the operation is convenient. It not only solves the problems of low efficiency and cumbersome of liquid-phase cyclization, but also significantly improves the yield and purity of the cyclic hexapeptide; in addition, the preparation method of the present invention avoids the use of tetraphenylphosphine palladium, overcomes the defects of high cost and inability to be widely applied in traditional solid-phase cyclization, thereby providing a more economical and efficient production route for the application of cyclic hexapeptide in cosmetics and other fields.

[0032] First, based on the sequence characteristics of hexa-peptide-3 and its cyclic peptide structure with head-to-tail cyclization, the original hexa-peptide-3 sequence H-Lys-Leu-Asp-Ala-Pro-Thr-OH was changed to H-Leu-Asp-Ala-Pro-Thr-Lys-OH, with lysine placed at the beginning of the sequence and its side chain connected to dichloro resin, making it possible for solid-phase cyclization without changing the structure of the cyclic hexa-peptide. The preparation method of the present invention successfully overcomes the limitation in traditional solid-phase cyclization technology that the starting amino acid must be glutamic acid or aspartic acid with a carboxyl group on the side chain, thus greatly broadening the sequence range of cyclic peptides that can be prepared.

[0033] Secondly, 2-chlorotrityl chloride and dichloro resin have a high degree of similarity in functional groups. Using 2-chlorotrityl chloride to protect the amino and carboxyl groups on the main chain of lysine, selective deprotection of the carboxyl group can be achieved under weak acid conditions without affecting the protected state of the amino group; compared with the traditional allyl protection method, using 2-chlorotrityl chloride for protection not only simplifies the operation process but also avoids the cumbersome steps of using expensive tetraphenylphosphine palladium for deprotection, greatly improving production efficiency and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 HPLC chromatogram of the cyclic hexa-peptide prepared in Example 1.

[0035] Figure 2 ESI-MS spectrum of the cyclic hexa-peptide prepared in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0036] The technical solutions of the present invention will be clearly and completely described below in conjunction with the data 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 of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0037] It should be noted that the professional terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the following embodiments of the present invention can be obtained through market purchase or prepared by existing methods. The information of the raw materials used in the present invention is shown in Table 1, where the substitution degree of the dichloro resin is 1.0 nmol / g.

[0038] Table 1 Information Table of Raw Materials Used in the Present Invention

[0039] English Abbreviation Chinese Name DCM Dichloromethane DMF N,N-Dimethylformamide HOBT 1-Hydroxybenzotriazole DIC N,N-Diisopropylcarbodiimide PIP Piperidine 2-CTC Resin Dichlororesin 2-Cl-TRT-Cl 2-Chlorotrityl Chloride DIEA N,N-Diisopropylethylamine <![CDATA[CF3COOH]]> Trifluoroacetic Acid <![CDATA[CF3CH2OH]]> Trifluoroethanol <![CDATA[CH3COOH]]> Acetic Acid TIS Triisopropylsilane Fmoc-Lys(teoc)-OH Fluorenylmethyloxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine Pybop Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Fmoc-Thr(otbu)-OH Fluorenylmethyloxycarbonyl-O-tert-butyl-L-threonine Fmoc-Pro-OH Fluorenylmethyloxycarbonyl-L-proline Fmoc-Ala-OH Fluorenylmethyloxycarbonyl-L-alanine Fmoc-Asp(otbu)-OH Fluorenylmethyloxycarbonyl-aspartic acid-4-tert-butyl ester Fmoc-Leu-OH Fluorenylmethyloxycarbonyl-L-leucine

[0040] In the prior art, the preparation of cyclic peptides, especially macrocyclic peptides, faces many challenges. Although traditional liquid-phase cyclization methods and solid-phase cyclization methods can achieve the preparation of cyclic peptides, they each have obvious limitations. The liquid-phase cyclization method has cumbersome steps, low efficiency, and is accompanied by the generation of a large number of by-products, making it difficult to improve the yield. While the solid-phase cyclization method directly constructs the cyclic peptide structure on dichloride resin, its strict requirements for starting amino acids and the use of expensive deprotection reagents greatly limit its wide application in the field of cyclic peptide preparation.

[0041] By adjusting the sequence of hexa-peptide-3 in the present invention, lysine is placed at the first position, and 2-chlorotrityl chloride is connected to the side chain of lysine and then connected to dichloride resin, thereby successfully overcoming the limitation in traditional solid-phase cyclization technology that the starting amino acid must be glutamic acid or aspartic acid with a carboxyl group on the side chain without changing the structure of cyclohexapeptide; using 2-chlorotrityl chloride as a protecting group, compared with the traditional allyl protection method, not only simplifies the operation process but also avoids the use of expensive tetraphenylphosphine palladium for deprotection reaction. Under weak acid conditions, 2-chlorotrityl chloride can selectively remove the carboxyl protection without affecting the amino protection state, and this characteristic greatly improves the production efficiency and economy of cyclohexapeptide. In addition, the whole process of the preparation method of the present invention is completed on dichloride resin, which not only avoids the cumbersome separation and purification steps in the liquid-phase cyclization method but also significantly improves the purity and yield of cyclohexapeptide.

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0043] Example 1

[0044] A preparation method of cyclohexapeptide specifically includes the following steps:

[0045] S1. Preparation of Fmoc-Lys(Teoc)-O-TRT-2-Cl:

[0046] Dissolve 2.8 g of Fmoc-Lys(Teoc)-OH in DCM, add 1.4 g of DIEA, stir evenly, then add 2.56 g of 2-Cl-TRT-Cl, stir at room temperature for 2 h, then evaporate DCM to dryness, and then add petroleum ether for stirring. After white solid is precipitated, filter to obtain compound I, denoted as Fmoc-Lys(Teoc)-O-TRT-2-Cl.

[0047] S2. Preparation of Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl:

[0048] Add Fmoc-Lys(Teoc)-O-TRT-2-Cl from step S1 to a mixed solution containing 3.4 g of tetrabutylammonium fluoride and 50 mL of DMF, and react for 2 h to remove the amino protecting group Teoc. After the reaction is completed, continue to add 2.58 g of DIEA, and then add 5 g of dichlororesin swollen with DCM; stir and react at 25 °C under nitrogen protection for 2 h, then filter, and wash successively with 100 mL of isopropanol and 100 mL of N,N-dimethylformamide to obtain compound II, denoted as Fmoc-Lys(2-CTCResin)O-TRT-2-Cl.

[0049] S3. Preparation of Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0050] Use a mixed solution of 50 mL of piperidine with a volume fraction of 20% and DMF to remove the Fmoc protecting group on Fmoc-Lys(2-CTCResin)-O-2-CTC in step S2 at 25 °C for 40 min, then filter, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Lys(2-CTCResin)-O-TRT-2-Cl; dissolve 4 g of Fmoc-Thr(otbu)-OH, 2 g of 1-hydroxybenzotriazole, and 1.9 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; then add the mixed solution to H-Lys(2-CTCResin)-O-TRT-2-Cl, stir and react at 25 °C under nitrogen protection for 3 h, then filter, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0051] S4. Preparation of Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0052] Deprotect the Fmoc protecting group on Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S3 with a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20% at 25 °C for 40 min, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; dissolve 3.37 g of Fmoc-Pro-OH, 2 g of 1-hydroxybenzotriazole and 1.9 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; then add the mixed solution to H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stir and react at 25 °C for 3 h under nitrogen protection, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0053] S5. Preparation of Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0054] Deprotect the Fmoc protecting group on Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S4 with a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20% at 25 °C for 40 min, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; dissolve 3.1 g of Fmoc-Ala-OH, 2 g of 1-hydroxybenzotriazole and 1.9 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; then add the mixed solution to H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stir and react at 25 °C for 3 h under nitrogen protection, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0055] S6. Preparation of Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0056] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20%, the Fmoc protecting group on Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S5 was removed at 25 °C for 40 min, followed by suction filtration. It was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 4.1 g of Fmoc-Asp(otbu)-OH, 2 g of 1-hydroxybenzotriazole, and 1.9 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution; the mixed solution was added to H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and after stirring and reacting at 25 °C for 3 h under nitrogen protection, suction filtration was carried out, and it was washed successively with 100 mL of propanol and 100 mL of DMF to obtain Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0057] S7. Preparation of Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0058] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20%, the Fmoc protecting group on Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S6 was removed at 25 °C for 40 min, followed by suction filtration. It was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.5 g of Fmoc-Leu-OH, 2 g of 1-hydroxybenzotriazole, and 1.9 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution; the mixed solution was added to H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and after stirring and reacting at 25 °C for 3 h under nitrogen protection, suction filtration was carried out, and it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0059] Preparation of S8, H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0060] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 20%, the Fmoc protecting group on Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S7 was removed at 25 °C for 40 min, followed by suction filtration, and then washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain compound III, denoted as H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTC Resin)-O-TRT-2-Cl.

[0061] S9. Preparation of H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH: 10 mL of acetic acid, 20 mL of trifluoroethanol and 70 mL of DCM were mixed and added to H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. The mixture was stirred and reacted at 25 °C for 2 h under nitrogen protection, followed by suction filtration, and then washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain compound IV, denoted as H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTC Resin)-OH.

[0062] S10. Preparation of Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)):

[0063] 50 mL of DMF, 1.29 g of DIEA and 5.2 g of Pybop were added to H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH. The mixture was stirred and reacted at 25 °C for 3 h under nitrogen protection, followed by suction filtration, and then washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain compound V, denoted as Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)).

[0064] S11. Preparation of Cyclo(Lys-Leu-Asp-Ala-Pro-Thr):

[0065] A peptide cleavage reaction was carried out on Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)) for 2 h at 25 °C in a nitrogen atmosphere using a mixture of 95 mL of trifluoroacetic acid and 5 mL of triisopropylsilane solution. After the cyclohexapeptide was detached, the trifluoroacetic acid was evaporated to dryness, and the crude product of Cyclo(Lys-Leu-Asp-Ala-Pro-Thr) was precipitated in diethyl ether. The crude product was purified by reverse preparative chromatography using a C18 reverse-phase chromatography column, and the buffer was converted to an acetate solution for separation during the process. After lyophilization, 1.7 g of cyclohexapeptide was obtained, denoted as Cyclo(Lys-Leu-Asp-Ala-Pro-Thr).

[0066] Example 2

[0067] A method for preparing a cyclohexapeptide specifically includes the following steps:

[0068] S1. Preparation of Fmoc-Lys(Teoc)-O-TRT-2-Cl:

[0069] Dissolve 2.8 g of Fmoc-Lys(Teoc)-OH in DCM, add 1.4 g of DIEA, stir evenly, then add 5.12 g of 2-Cl-TRT-Cl, stir at room temperature for 2 h, evaporate DCM to dryness, then add petroleum ether and stir. After white solid is precipitated, filter to obtain compound I, denoted as Fmoc-Lys(Teoc)-O-TRT-2-Cl.

[0070] S2. Preparation of Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl:

[0071] Add the Fmoc-Lys(Teoc)-O-TRT-2-Cl obtained in step S1 to a mixed solution containing 3.4 g of tetrabutylammonium fluoride and 50 mL of DCM for reaction for 1 h to remove the amino protecting group Teoc. After the reaction is completed, continue to add 1.9 of DIEA, then add 5 g of dichlororesin swollen with DCM; stir and react at 20 °C under nitrogen protection for 1 h, then filter by suction, and wash successively with 100 mL of isopropanol and 100 mL of N,N-dimethylformamide to obtain compound II, denoted as Fmoc-Lys(2-CTCResin)O-TRT-2-Cl.

[0072] S3. Preparation of Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0073] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 9%, the Fmoc protecting group on Fmoc-Lys(2-CTCResin)-O-2-CTC in step S2 was removed at 20 °C for 20 min, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Lys(2-CTCResin)-O-TRT-2-Cl; 4 g of Fmoc-Thr(otbu)-OH, 1.69 g of 1-hydroxybenzotriazole, and 1.58 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution; subsequently, the mixed solution was added to H-Lys(2-CTCResin)-O-TRT-2-Cl, and the mixture was stirred and reacted at 20 °C for 2 h under nitrogen protection, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0074] S4. Preparation of Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0075] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 9%, the Fmoc protecting group on Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S3 was removed at 20 °C for 20 min, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Thr-(o tbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.37 g of Fmoc-Pro-OH, 1.69 g of 1-hydroxybenzotriazole, and 1.58 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution; subsequently, the mixed solution was added to H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and the mixture was stirred and reacted at 20 °C for 2 h under nitrogen protection, followed by suction filtration. Then, it was washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0076] S5. Preparation of Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0077] Deprotect the Fmoc protecting group on Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S4 with a 50 mL mixed solution of piperidine and DMF with a volume fraction of 9% at 20 °C for 20 min, then perform suction filtration, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-P ro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; dissolve 3.1 g of Fmoc-Ala-OH, 1.69 g of 1-hydroxybenzotriazole and 1.58 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; then add the mixed solution to H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stir and react at 20 °C for 2 h under nitrogen protection, then perform suction filtration, and wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0078] S6. Preparation of Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0079] Deprotect the Fmoc protecting group on Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S5 with a 50 mL mixed solution of piperidine and DMF with a volume fraction of 9% at 20 °C for 20 min, then perform suction filtration, wash successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; dissolve 4.1 g of Fmoc-Asp(otbu)-OH, 1.69 g of 1-hydroxybenzotriazole and 1.58 g of N,N-diisopropylcarbodiimide in 50 mL of DMF to obtain a mixed solution; add the mixed solution to H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stir and react at 20 °C for 2 h under nitrogen protection, then perform suction filtration, and wash successively with 100 mL of propanol and 100 mL of DMF to obtain Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCRe sin)-O-TRT-2-Cl.

[0080] Preparation of S7, Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0081] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 9%, the Fmoc protecting group on Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S6 was removed at 20 °C for 20 min, then filtered by suction, and washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.5 g of Fmoc-Leu-OH, 1.69 g of 1-hydroxybenzotriazole and 1.58 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution; the mixed solution was added to H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and stirred and reacted at 20 °C for 2 h under nitrogen protection, then filtered by suction, and washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0082] Preparation of S8, H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl:

[0083] Using a 50 mL mixed solution of piperidine and DMF with a volume fraction of 9%, the Fmoc protecting group on Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S7 was removed at 20 °C for 20 min, then filtered by suction, and washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Compound III, denoted as H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCRe sin)-O-TRT-2-Cl.

[0084] S9. 15 mL of acetic acid, 30 mL of trifluoroethanol and 55 mL of DCM were mixed and added to H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. The mixture was stirred at 20 °C for 1 h under nitrogen protection, then filtered by suction, and washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Compound IV, denoted as H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH.

[0085] S10. Preparation of Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)):

[0086] 50 mL of DMF, 2.58 g of DIEA and 10.4 g of Pybop were added to H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH. The mixture was stirred at 20 °C for 2 h under nitrogen protection, then filtered by suction, and washed successively with 100 mL of isopropanol and 100 mL of DMF to obtain Compound V, denoted as Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)).

[0087] S11. Preparation of Cyclo(Lys-Leu-Asp-Ala-Pro-Thr):

[0088] A mixture of 80 mL of trifluoroacetic acid and 20 mL of triisopropylsilane solution was used to perform a peptide cleavage reaction on Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)) at 20 °C for 1 h under a nitrogen atmosphere. After the cyclohexapeptide was detached, the trifluoroacetic acid was evaporated to dryness, and the crude product of Cyclo(Lys-Leu-Asp-Ala-Pro-Thr) was precipitated in diethyl ether. The crude product was purified by reverse preparative chromatography using a C18 reverse-phase column, and the buffer was converted to an acetate solution for separation during the process. After lyophilization, the cyclohexapeptide was obtained, denoted as Cyclo(Lys-Leu-Asp-Ala-Pro-Thr).

[0089] Observation Figure 1 It was found that the chromatographic peak at 12.501 min was the peak of the cyclohexapeptide prepared in Example 1 of the present invention, and its purity was over 90%.

[0090] Observation Figure 2It is concluded that 626 is the mass spectrometry signal of the cyclohexapeptide prepared in Example 1 of the present invention, showing M+1, and the molecular weight of the product is 625.

[0091] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods adopted are the same as those in the embodiments, in order to prevent repetition, the present invention describes preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. A method for preparing a cyclic hexapeptide, characterized in that, It includes the following steps: S1. Under an alkaline environment, mix fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine and 2-chlorotrityl chloride, and carry out a substitution reaction to obtain Compound I; S2. After removing the protecting group trimethylsilylethoxycarbonyl in Compound I, under an alkaline environment, mix it with dichlororesin and carry out a nucleophilic substitution reaction to obtain Compound II; S3. Starting from Compound II, add multiple amino acids, carry out a condensation reaction and remove the protecting group fluorenylmethoxycarbonyl to obtain Compound III; among them, the multiple amino acids are successively fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-L-alanine, fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester, fluorenylmethoxycarbonyl-L-leucine; each time an amino acid is added, a condensation reaction and removal of the protecting group fluorenylmethoxycarbonyl are carried out once; S4. After removing the protecting group 2-chlorotrityl from Compound III, obtain Compound IV; S5. Using N,N-diisopropylethylamine to provide an alkaline environment, carry out a condensation reaction on Compound IV to obtain Compound V; S6. Carry out a peptide cleavage reaction on Compound V to obtain a cyclohexapeptide.

2. The preparation method of the cyclohexapeptide according to claim 1, wherein The molar ratio of fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to 2-chlorotrityl chloride is 1:1 to 2; The molar ratio of fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to dichlororesin is 1 to 1.5:

1.

3. The preparation method of the cyclohexapeptide according to claim 1, wherein, Use a mixed solution of tetrabutylammonium fluoride and N,N-diisopropylethylamine to remove the protecting group trimethylsilylethoxycarbonyl in Compound I, and the molar ratio of tetrabutylammonium fluoride to N,N-diisopropylethylamine is 2 to 3:3 to 4.

4. The preparation method of the cyclohexapeptide according to claim 1, wherein, The conditions for removing the protecting group fluorenylmethoxycarbonyl are: in the presence of a piperidine solution, remove it at 20°C to 25°C for 20 min to 40 min; Among them, the piperidine solution is composed of piperidine and N,N-dimethylformamide, and the volume ratio of piperidine to N,N-dimethylformamide is 1:4 to 10.

5. The preparation method of the cyclohexapeptide according to claim 1, characterized in that, In step S3, the condensation reaction conditions are: using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensing agents, N,N-dimethylformamide as a solvent, in the presence of the condensing agents and the solvent, stir in a nitrogen atmosphere at 20°C to 25°C for 2 h to 3 h; Among them, the molar ratio of 1-hydroxybenzotriazole, N,N-diisopropylcarbodiimide to dichlororesin is 2.5 to 3:2.5 to 3:

1.

6. The preparation method of the cyclohexapeptide according to claim 1, characterized in that, In step S4, use a mixed solution of acetic acid, trifluoroethanol and dichloromethane to remove the protecting group 2-chlorotrityl from Compound III, and the volume ratio of acetic acid, trifluoroethanol and dichloromethane is 10 to 15:20 to 30:55 to 70.

7. The preparation method of the cyclohexapeptide according to claim 1, characterized in that, In step S5, the operation of the condensation reaction is: using benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and N,N-diisopropylethylamine as condensing agents, mix Compound IV and the condensing agents together in N,N-dimethylformamide, and react in a nitrogen atmosphere at 20°C to 25°C for 1 h to 2 h; Among them, the molar ratio of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, N,N-diisopropylethylamine to the dichloro resin in step S2 is 2-4:2-4:

1.

8. The preparation method of the cyclohexapeptide according to claim 1, wherein, In step S6, the operation of the peptide cleavage reaction is as follows: using trifluoroacetic acid and triisopropylsilane as the peptide cleavage solution, mixing the peptide cleavage solution with compound V, and reacting at 20°C - 25°C for 1 h - 2 h under a nitrogen atmosphere; Among them, the volume ratio of trifluoroacetic acid to triisopropylsilane is 80-95:20-5.

9. A cyclohexapeptide prepared by the preparation method according to any one of claims 1 to 8, characterized in that, The structural formula of the cyclic hexapeptide is:

10. Use of the cyclic hexapeptide according to claim 9 in the preparation of a cyclic peptide product.

Images