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 in particular to a cyclohexapeptide and a preparation method and 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 their unique charm. Compared with their linear form before cyclization, cyclic peptides in cosmetics show 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 a leader in cosmetic raw materials, has multiple anti-wrinkle effects such as promoting the regeneration of type I and type II collagen and reducing the depth and length of wrinkles.

[0003] The traditional preparation of macrocyclic peptides, i.e., cyclic peptide structures composed of 5 or more amino acids, is mainly achieved through two major approaches: the first is the liquid phase cyclization method, which first uses solid phase preparation technology to prepare linear peptides on a resin, and then cleaves the linear peptides from the resin under side chain protection; then, a condensing agent is introduced into an organic solvent to cause the linear peptides to be connected head to tail to form a cyclic structure; finally, after removing the side chain protecting groups, the cyclic peptides are purified and freeze-dried to obtain cyclic peptides. However, this method is cumbersome and inefficient, resulting in low yields and the generation of a large number of by-products; the second is the solid phase cyclization method, which directly constructs the 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 groups of the main chain must be protected by propylene esters. After the amino acid connection is completed, it is also necessary to rely on expensive tetraphenylphosphine palladium for deprotection reaction, which increases production costs and limits 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 and a preparation method and application thereof. The present invention uses fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine as a raw material, reacts 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 a dichloro resin; and using this as a starting point, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-L-alanine and fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester and fluorenylmethoxycarbonyl-L-leucine are sequentially 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 cyclohexapeptide with a side chain connected to the dichloro resin is formed through a condensation reaction; and finally, a peptide cleavage reaction is performed to obtain the cyclohexapeptide. The entire preparation process of the cyclohexapeptide of the present invention is completed on dichlororesin, the preparation steps are concise and clear, the operation is simple, not only solves the problems of inefficiency and cumbersomeness 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 non-wide application of traditional solid phase cyclization, thereby providing 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:

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

[0007]

[0008] The second object of the present invention is to provide a method for preparing the above-mentioned cyclohexapeptide, comprising the following steps:

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

[0010] S2. The protecting group trimethylsilylethoxycarbonyl in Fmoc-Lys(Teoc)-O-TRT-2-Cl is removed to obtain Fmoc-Lys-O-TRT-2-Cl. N,N-diisopropylamine is used to provide an alkaline environment. Fmoc-Lys-O-TRT-2-Cl is mixed with dichloro resin in an alkaline environment. A chlorine atom on the dichloro resin reacts with a carboxyl anion on Fmoc-Ly sO-TRT-2-Cl to undergo a nucleophilic substitution reaction to obtain Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl.

[0011] S3. Remove the protective group fluorenylmethoxycarbonyl of Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Lys(2-CTCResin)-O-TRT-2-Cl, and 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. Remove the protective group fluorenylmethoxycarbonyl of Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and 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. Remove the protective group fluorenylmethoxycarbonyl of Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and 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. Remove the protective group fluorenylmethoxycarbonyl 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-4-tert-butyl aspartate to obtain Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0015] S7. Remove the protecting group fluorenylmethoxycarbonyl 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(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0016] S8. Remove the protective group fluorenylmethoxycarbonyl of Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0017] S9. After removing the protecting group 2-chlorotrityl of H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH was obtained.

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

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

[0020] Preferably, in step S1, the molar ratio of fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to 2-chlorotrityl chloride is 1:1-2; the molar ratio of fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to the dichloro resin 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 to S8, the operation for removing the protecting group fluorenylmethoxycarbonyl is the same, specifically:

[0023] Mix Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20℃~25℃ for 20min~40min; Mix Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20℃~25℃ for 20min~40min; Mix Fmoc-Pro-Thr(otbu)-Lys(2-CTCRe sin)-O-TRT-2-Cl was mixed with a piperidine solution and removed at 20°C to 25°C for 20min to 40min; Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl was mixed with a piperidine solution and removed at 20°C to 25°C for 20min to 40min; Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TR T-2-Cl is mixed with a piperidine solution and removed at 20°C to 25°C for 20 min to 40 min; Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl is mixed with a piperidine solution and removed at 20°C to 25°C for 20 min to 40 min; wherein the piperidine solution consists 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 operations of the condensation reaction are the same, specifically:

[0025] Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, H-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, methoxycarbonyl-L-proline, H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and a condensing agent were mixed in N,N-dimethylformamide and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide were used as condensing agents to react fluorenylmethoxycarbonyl-L-alanine, H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TR T-2-Cl and a condensing agent are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide are used as condensing agents, fluorenylmethoxycarbonyl-L-alanine, H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and a condensing agent are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide are used as condensing agents, and fluorenylmethoxycarbonyl-L-alanine, H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, fluorenylmethoxycarbonyl-L-alanine, H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed in N,N-dimethylformamide, and stirred at 20°C to 25°C in a nitrogen atmosphere for 2h to 3h; wherein the molar ratio of 1-hydroxybenzotriazole, N,N-diisopropylcarbodiimide to the dichloro resin 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-chlorotrityl 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 10-15:20-30:55-70.

[0027] Preferably, in step S10, the operation of the condensation reaction is: using benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and N,N-diisopropylethylamine as a condensation agent, H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH and the condensation agent are mixed together in N,N-dimethylformamide, and reacted at 20°C to 25°C in a nitrogen atmosphere for 1h to 2h; wherein the molar ratio of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, N,N-diisopropylethylamine and the dichlororesin in step S2 is 2 to 4:2 to 4:1.

[0028] Preferably, in step S11, the operation of the peptide cleavage reaction is: using trifluoroacetic acid and triisopropylsilane as peptide cleavage liquid, mixing the peptide cleavage liquid and Cyclo (Leu-Asp (otbu) -Ala-Pro-Thr (otbu) -Lys (2-CTCResin)), and reacting at 20 ° C ~ 25 ° C in a nitrogen atmosphere for 1h ~ 2h; 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 use of the above cyclohexapeptide in the preparation of cyclopeptide products.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. The present invention provides a method for preparing a cyclohexapeptide. The present invention uses fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine as a raw material, reacts 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 a dichloro resin; and using this as a starting point, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, fluorenylmethoxycarbonyl-L-proline, fluorenylmethoxycarbonyl-L-alanine, fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester and fluorenylmethoxycarbonyl-L-leucine are connected in sequence, 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 side chain is formed by a condensation reaction and connected to the cyclohexapeptide of the dichloro resin; finally, a peptide cleavage reaction is performed to obtain the cyclohexapeptide. The entire preparation process of the cyclohexapeptide of the present invention is completed on dichlororesin, the preparation steps are concise and clear, the operation is simple, not only solves the problems of inefficiency and cumbersomeness 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 non-wide application of traditional solid phase cyclization preparation, thereby providing a more economical and efficient production route for the application of cyclohexapeptide in cosmetics and other fields.

[0032] First, based on the sequence characteristics of hexapeptide-3 and its head-to-tail cyclized cyclic peptide structure, the present invention changes the original hexapeptide-3 sequence H-Lys-Leu-Asp-Ala-Pro-Thr-OH to H-Leu-Asp-Ala-Pro-Thr-Lys-OH, places lysine at the first position of the sequence, and connects its side chain to dichlororesin, thereby making its solid phase cyclization possible without changing the structure of the cyclohexapeptide. The preparation method of the present invention successfully overcomes the limitation of the 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, thereby 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. 2-chlorotrityl chloride is used to protect the amino and carboxyl groups on the lysine backbone. The carboxyl protection can be selectively removed under weak acid conditions, while the protection state of the amino group is not affected. Compared with the traditional propylene protection method, the use of 2-chlorotrityl chloride for protection not only simplifies the operation process, but also avoids the tedious steps of using expensive tetraphenylphosphine palladium for deprotection, greatly improving production efficiency and economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the HPLC chromatogram of the cyclohexapeptide prepared in Example 1.

[0035] Figure 2 This is the ESI-MS spectrum of the cyclohexapeptide prepared in Example 1. DETAILED DESCRIPTION

[0036] The following will be combined with the data in the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within 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 scope of protection of the present invention. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present invention can be purchased from the market or prepared by existing methods. The information of the raw materials used in the present invention is shown in Table 1, wherein the degree of substitution of dichlororesin is 1.0nmol / g.

[0038] Table 1 Raw material information table 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-CTCResin 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 Fmoc-N6-trimethylsilylethoxycarbonyl-L-lysine Pybop Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Fmoc-Thr(otbu)-OH Fmoc-O-tert-butyl-L-threonine Fmoc-Pro-OH Fmoc-L-proline Fmoc-Ala-OH Fmoc-L-alanine Fmoc-Asp(otbu)-OH Fmoc-4-tert-butyl aspartate Fmoc-Leu-OH Fmoc-L-leucine

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

[0041] The present invention adjusts the sequence of hexapeptide-3, puts lysine in the first place, and uses 2-chlorotrityl chloride to connect to the side chain of lysine, and connects it to dichloro resin, thereby achieving the premise of not changing the structure of the cyclohexapeptide, successfully overcoming the limitation that the starting amino acid must be glutamic acid or aspartic acid with a carboxyl group on the side chain in the traditional solid phase cyclization technology; using 2-chlorotrityl chloride as a protecting group, compared with the traditional propylene 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 carboxyl protection, while the amino protection state is not affected, and this characteristic greatly improves the production efficiency and economy of the cyclohexapeptide. In addition, the whole process of the preparation method of the present invention is completed on dichloro 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 the 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 method for preparing a cyclohexapeptide, comprising the following steps:

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

[0046] 2.8 g of Fmoc-Lys(Teoc)-OH was dissolved in DCM, 1.4 g of DIEA was added, and after stirring evenly, 2.56 g of 2-Cl-TRT-Cl was added. After stirring at room temperature for 2 h, the DCM was evaporated, and then petroleum ether was added and stirred. After the white solid precipitated, it was filtered to obtain Fmoc-Lys(Teoc)-O-TRT-2-Cl.

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

[0048] The Fmoc-Lys(Teoc)-O-TRT-2-Cl in step S1 was added to a mixed solution containing 3.4 g of tetrabutylammonium fluoride and 50 mL of DMF to react for 2 h to remove the amino protecting group Teoc. After the reaction was completed, 2.58 g of DIEA was added, and then 5 g of dichlororesin swollen with DCM was added; the reaction was stirred at 25°C for 2 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of N,N-dimethylformamide in turn to obtain Fmoc-Lys(2-CTCResin)O-TRT-2-Cl.

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

[0050] The Fmoc protecting group on Fmoc-Lys(2-CTCResin)-O-2-CTC in step S2 was removed by using 50 mL of a mixed solution of piperidine and DMF with a volume fraction of 20% at 25°C for 40 min, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Lys(2-CTCResin n)-O-TRT-2-Cl; 4 g of Fmoc-Thr(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 then added to H-Lys(2-CTCResin)-O-TRT-2-Cl, and the mixture was stirred at 25°C for 3 h under nitrogen protection, then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in turn 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] The Fmoc protecting group on Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S3 was removed at 25° C. for 40 min using 50 mL of a 20% by volume piperidine and DMF mixed solution. The mixture was filtered and washed with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. 3.37 g of Fmoc-Pro-OH and 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 then added to H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stirred for reaction at 25 °C for 3 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence 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] The Fmoc protecting group on Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S4 was removed at 25°C with 50 mL of a 20% by volume solution of piperidine and DMF, and then filtered for 40 minutes, and washed with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.1 g of Fmoc-Ala-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 then added to H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stirred for reaction at 25 °C for 3 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence 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] 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 with 50 mL of a 20% by volume solution of piperidine and DMF, and then filtered for 40 minutes, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence 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 1.9 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 the mixture was stirred at 25°C for 3 h under nitrogen protection, then filtered and washed with 100 mL of propanol and 100 mL of DMF in sequence 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] 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 with 50 mL of a 20% by volume piperidine and DMF mixed solution, and then filtered for 40 minutes, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.5 g of Fmoc-Leu-OH and 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, stirred for reaction at 25°C for 3 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

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

[0060] Use 50 mL of a mixed solution of piperidine and DMF with a volume fraction of 20% to remove the Fmoc protecting group on Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S7 at 25°C for 40 minutes, then filter and wash with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0061] S9. Mix 10 mL of acetic acid, 20 mL of trifluoroethanol and 70 mL of DCM and add to H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. Stir the reaction at 25 °C for 2 h under nitrogen protection, then filter and wash with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH.

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

[0063] To H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH, 50 mL of DMF, 1.29 g of DIEA and 5.2 g of Pybop were added. The reaction was stirred at 25°C for 3 h under nitrogen protection, and then filtered. The product was washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)).

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

[0065] Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)) was subjected to peptide cleavage reaction for 2 hours at 25°C in a nitrogen atmosphere with a mixture of 95 mL of trifluoroacetic acid and 5 mL of triisopropylsilane solution. After the cyclohexapeptide was detached, the trifluoroacetic acid was evaporated and precipitated in ether to generate a crude product of Cyclo(Ly s-Leu-Asp-Ala-Pro-Thr). The crude product was subjected to reverse preparation and purification using a C18 reverse phase column, and the buffer solution was converted to an acetate solution for separation during the process. After freeze-drying, 1.7 g of cyclohexapeptide was obtained, recorded as Cyclo(Lys-Leu-Asp-Ala-Pro-Thr).

[0066] Example 2

[0067] A method for preparing a cyclohexapeptide, comprising the following steps:

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

[0069] 2.8 g of Fmoc-Lys(Teoc)-OH was dissolved in DCM, 1.4 g of DIEA was added, and after stirring evenly, 5.12 g of 2-Cl-TRT-Cl was added. After stirring at room temperature for 2 h, the DCM was evaporated, and then petroleum ether was added and stirred. After the white solid precipitated, it was filtered to obtain Fmoc-Lys(Teoc)-O-TRT-2-Cl.

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

[0071] The Fmoc-Lys(Teoc)-O-TRT-2-Cl in step S1 was added 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 was completed, 1.9 g of DIEA was added, and then 5 g of dichlororesin swollen with DCM was added; the reaction was stirred at 20°C for 1 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of N,N-dimethylformamide in turn to obtain Fmoc-Lys(2-CTCResin)O-TRT-2-Cl.

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

[0073] The Fmoc protecting group on Fmoc-Lys(2-CTCResin)-O-2-CTC in step S2 was removed by using 50 mL of a mixed solution of piperidine and DMF with a volume fraction of 9% at 20°C for 20 min, and then filtered and washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain H-Lys(2-CTCResin)-O-TRT-2-Cl; 4 g of Fmoc-Thr(otbu)-OH, 1.69 g of 1-hydroxy 1.58 g of benzotriazole and 1.58 g of N,N-diisopropylcarbodiimide were dissolved in 50 mL of DMF to obtain a mixed solution; the mixed solution was then added to H-Lys(2-CTCResin)-O-TRT-2-Cl, stirred for reaction at 20°C for 2 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence 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] The Fmoc protecting group on Fmoc-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S3 was removed by using 50 mL of a mixed solution of piperidine and DMF with a volume fraction of 9% at 20° C. for 20 min, and then filtered and washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. 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; the mixed solution was then added to H-Thr-(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stirred at 20 ° C for 2 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in turn 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] The Fmoc protecting group on Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S4 was removed at 20°C with 50 mL of a 9% by volume solution of piperidine and DMF, and then filtered for 20 minutes, and washed with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.1 g of Fmoc-Ala-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 then added to H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, stirred at 20°C for 2 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence 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] The Fmoc protecting group on Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S5 was removed at 20°C with 50 mL of a mixed solution of piperidine and DMF with a volume fraction of 9% for 20 min, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 4.1 g of Fmoc-Asp(otbu) -OH1.69g of 1-hydroxybenzotriazole and 1.58g of N,N-diisopropylcarbodiimide were dissolved in 50mL 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, stirred and reacted at 20°C for 2h under nitrogen protection, then filtered and washed with 100mL of propanol and 100mL of DMF in sequence to obtain Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCRe sin)-O-TRT-2-Cl.

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

[0081] 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 with 50 mL of a 9% by volume solution of piperidine and DMF, and then filtered for 20 min, and washed with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; 3.5 g of Fmoc c-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, stirred and reacted at 20°C for 2 h under nitrogen protection, and then filtered, and washed with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

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

[0083] Use 50 mL of a mixed solution of piperidine and DMF with a volume fraction of 9% to remove the Fmoc protecting group on Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl in step S7 at 20°C for 20 minutes, then filter and wash with 100 mL of isopropanol and 100 mL of DMF in sequence to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl.

[0084] S9. Mix 15 mL of acetic acid, 30 mL of trifluoroethanol and 55 mL of DCM and add to H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl. Stir the reaction at 20°C for 1 h under nitrogen protection, then filter and wash with 100 mL of isopropanol and 100 mL of DMF in turn to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH.

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

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

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

[0088] Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)) was subjected to peptide cleavage reaction for 1 hour at 20°C in a nitrogen atmosphere with a mixture of 80 mL of trifluoroacetic acid and 20 mL of triisopropylsilane solution to remove the cyclohexapeptide. The trifluoroacetic acid was evaporated and the cyclohexapeptide was precipitated in ether to generate a crude product of Cyclo(Ly s-Leu-Asp-Ala-Pro-Thr). The crude product was subjected to reverse preparation and purification using a C18 reverse phase column, and the buffer solution was converted to an acetate solution for separation during the process. After freeze-drying, the cyclohexapeptide was obtained, which was recorded as Cyclo(Lys-Leu-Asp-Ala-Pro-Thr).

[0089] observe Figure 1 It was concluded 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 above 90%.

[0090] observe Figure 2It was concluded that 626 was the mass spectrometry signal of the cyclohexapeptide prepared in Example 1 of the present invention, which was expressed as M+1, and the molecular weight of the product was 625.

[0091] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiment, in order to avoid redundancy, the present invention describes a preferred embodiment. 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 attached claims are intended to be interpreted as including the preferred embodiment and all changes and modifications that fall within the scope of the present invention.

Claims

1. A method for preparing a cyclohexapeptide, characterized in that: The steps include: S1. Using N,N-diisopropylethylamine to provide an alkaline environment, mixing fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine and 2-chlorotrityl chloride in an alkaline environment, and performing a substitution reaction to obtain Fmoc-Lys(Teoc)-O-TRT-2-Cl; S2, removing the protecting group trimethylsilylethoxycarbonyl in Fmoc-Lys(Teoc)-O-TRT-2-Cl to obtain Fmoc-Lys-O-TRT-2-Cl, using N,N-diisopropylamine to provide an alkaline environment, mixing Fmoc-Lys-O-TRT-2-Cl with dichloro resin in an alkaline environment, and performing a nucleophilic substitution reaction to obtain Fmoc-Ly s(2-CTCResin)-O-TRT-2-Cl; S3, removing the protective group fluorenylmethoxycarbonyl of Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Lys(2-CTCResin)-O-TRT-2-Cl, and subjecting H-Lys(2-CTCResin)-O-TRT-2-Cl to a condensation reaction with fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine to obtain Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; S4, removing the protective group fluorenylmethoxycarbonyl of Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and subjecting H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to a condensation reaction with fluorenylmethoxycarbonyl-L-proline to obtain Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; S5, removing the protective group fluorenylmethoxycarbonyl of Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl, and subjecting H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to condensation reaction with fluorenylmethoxycarbonyl-L-alanine to obtain Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; S6, removing the protective group fluorenylmethoxycarbonyl 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 subjecting H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to condensation reaction with fluorenylmethoxycarbonyl-aspartic acid-4-tert-butyl ester to obtain Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; S7, removing the protective group fluorenylmethoxycarbonyl 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 subjecting H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to condensation reaction with fluorenylmethoxycarbonyl-L-leucine to obtain Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl; S8, removing the protective group fluorenylmethyloxycarbonyl of Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTC Resin)-O-TRT-2-Cl; S9, removing the protecting group 2-chlorotrityl of H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl to obtain H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH; S10, using N,N-diisopropylethylamine to provide an alkaline environment, and condensing H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH to obtain Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)); S11. Cyclo(Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)) is subjected to peptide cleavage reaction to obtain a cyclohexapeptide.

2. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: In step S1, the molar ratio of fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to 2-chlorotrityl chloride is 1:1-2; The molar ratio of fluorenylmethoxycarbonyl-N6-trimethylsilylethoxycarbonyl-L-lysine to the dichloro resin in step S2 is 1 to 1.5:

1.

3. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: 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.

4. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: In steps S3 to S8, the operation of removing the protecting group fluorenylmethoxycarbonyl is the same, specifically: Mix Fmoc-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20°C to 25°C for 20min to 40min; Mix Fmoc-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20°C to 25°C for 20min to 40min; Mix Fmoc-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20°C to 25°C for 20min to 40min; Mix Fmoc-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20°C to 25°C for 20min to 40min; Mix Fmoc-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20°C to 25°C for 20min to 40min; Mix Fmoc-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl with piperidine solution and remove at 20°C to 25°C for 20min to 40min; The piperidine solution is composed of piperidine and N,N-dimethylformamide, and the volume ratio of piperidine to N,N-dimethylformamide is 1:4-10.

5. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: In steps S3 to S7, the operation of the condensation reaction is the same, specifically: Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine, H-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, methoxycarbonyl-L-proline, H-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, fluorenylmethoxycarbonyl-L-alanine, H-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, fluorenylmethoxycarbonyl-L-alanine, H-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; Using 1-hydroxybenzotriazole and N,N-diisopropylcarbodiimide as condensation agents, Fmoc-L-alanine, H-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-O-TRT-2-Cl and the condensation agent are mixed together in N,N-dimethylformamide, and stirred at 20°C to 25°C for 2h to 3h in a nitrogen atmosphere; The molar ratio of 1-hydroxybenzotriazole, N,N-diisopropylcarbodiimide and the dichlororesin in step S2 is 2.5-3:2.5-3:

1.

6. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: In step S9, a mixed solution of acetic acid, trifluoroethanol and dichloromethane is used to remove the protecting group 2-chlorotrityl 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 10-15:20-30:55-70.

7. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: In step S10, the operation of the condensation reaction is: using benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate and N,N-diisopropylethylamine as a condensation agent, H-Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)-OH and the condensation agent are mixed in N,N-dimethylformamide, and reacted at 20° C. to 25° C. in a nitrogen atmosphere for 1 h to 2 h; The molar ratio of benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, N,N-diisopropylethylamine and the dichlororesin in step S2 is 2-4:2-4:

1.

8. The method for preparing the cyclohexapeptide according to claim 1, characterized in that: In step S11, the operation of the peptide cleavage reaction is as follows: using trifluoroacetic acid and triisopropylsilane as a peptide cleavage solution, mixing the peptide cleavage solution and Cyclo (Leu-Asp(otbu)-Ala-Pro-Thr(otbu)-Lys(2-CTCResin)), and reacting at 20° C. to 25° C. in a nitrogen atmosphere for 1 h to 2 h; Wherein, the volume ratio of trifluoroacetic acid to triisopropylsilane is 80-95:20-5.

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

10. Use of the cyclohexapeptide according to claim 9 in the preparation of cyclopeptide products.

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